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VALIDATION REPORT

Validation Report for the Determination of D5 Benzalkonium Chloride (C12 and C14

homologs) in Human Plasma by Liquid Chromatographic Method with MS/MS Detection

REPORT NUMBER

SAI-VR19499

REPORT DATE

13 May, 2019

TESTING FACILITY

Sannova Analytical, Inc.

155 Pierce Street

Somerset, NJ 08873

Phone No: 732 560 0066

Fax: 732 560 0266

Page 1

TABLE OF CONTENTS

VALIDATION REPORT 1

TABLE OF CONTENTS 2

LIST OF TABLES 8

LIST OF CONTRIBUTORS 11

LIST OF ABBREVIATIONS 12

SIGNATURE PAGE 14

QUALITY ASSURANCE STATEMENT 15

COMPLIANCE STATEMENT 16

1.0 INTRODUCTION 17

2.0 SUMMARY 18

2.1 Overall Summary Table for a Method Validation Report: 20

3.0 EXPERIMENTAL MATERIALS AND METHODS 23

3.1 Reference Standard(s) 23

3.2 Summary of Analytical Method 24

3.3 Standard and QC Preparation 25

3.4 Method Description 29

4.0 SUMMARY OF THE BATCHES 30

5.0 RESULTS 32

5.1 Selectivity 32

5.1.1 Blank Check 32

5.1.2 Interference Check: 38

5.1.3 Interference Check in the presence of concomitant drugs (Mixture of

Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine Maleate, Naproxen and

(1R, 2R)-(-)-Pseudoephedrine) 39

5.1.4 Carry over 42

5.2 Matrix Effect 44

5.2.1 Matrix Effect for Method Consistency: 44

5.3 Calibration Standard Curve 46

5.4 Sensitivity 52

5.5 Precision and Accuracy 54

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5.6 Determination of Weighting Factor 62

5.7 Recovery 64

5.7.1 Absolute Recovery of Analyte 64

5.7.2 Absolute Recovery of Internal Standard 66

5.8 Stability 68

5.8.1 Freeze-Thaw Stability 68

5.8.2 Short-Term Stability 70

5.8.3 Autosampler Stability at 4 C 72

5.8.4 Whole Blood Stability 74

5.8.5 Stock Solution Stability 76

5.8.5.1 Stock Solution Stability for D5 Benzalkonium Chloride (C12 and C14

homologs) at Room Temperature 76

5.9 Maximum batch size 78

5.10 Dilution Integrity 80

6.0 MEMO TO FILES 83

7.0 DEVIATIONS 84

8.0 RE-INTEGRATION 84

9.0 ARCHIVAL 84

10.0 REVISION HISTORY 84

11.0 Appendix 85

11.1 Analytical Validation Protocol #SAI-VP19499 85

VALIDATION PROTOCOL 87

TABLE OF CONTENTS 88

1.0 INTRODUCTION: 90

2.0 STUDY PERSONNEL: 90

3.0 ANALYTICAL PROCEDURE: 90

4.0 BIOANALYTICAL METHOD VALIDATION: 90

4.1 STOCK SOLUTION CHECK: 91

4.2 SPIKING SOLUTION CHECK: 91

4.3 STOCK STABILITY AT ROOM TEMPERATURE FOR 6 HRS:

91

Page 3

4.4 SELECTIVITY & INTERFERENCE CHECK: 92

4.4.1 Selectivity 92

4.4.2 Interference Check: 93

4.4.3 Interference Check in the presence of concomitant medication:

93

4.5 CARRY OVER: 94

4.6 MATRIX EFFECT: 95

4.7 TEST BULK SPIKING: 95

4.8 CALIBRATION STANDARD CURVE (Relationship Between Response

And Concentration): 96

4.9 SENSITIVITY OR LIMIT OF QUANTITATION (LOQ): 97

4.10 PRECISION AND ACCURACY: 97

4.11 RECOVERY OF DRUG AND INTERNAL STANDARD: 98

4.11.1 Absolute Recovery of D5 Benzalkonium Chloride: 98

4.11.2 Absolute Recovery of Internal Standard (ISTD): 99

4.12 STABILITY: 99

4.12.1 Freeze-Thaw Stability: 99

4.12.2 Short-Term Stability (Bench Top Stability): 100

4.12.3 Autosampler Stability: 101

4.12.4 Stock Solution Stability: 101

4.12.5 Internal Standard (IS) Stock Solution Stability: 102

4.12.6 Whole Blood Stability: 102

4.12.7 Long-Term Stability: 103

4.13 MAXIMUM BATCH SIZE: 103

4.14 DILUTION INTEGRITY: 104

5.0 ARCHIVING OF RECORDS: 105

6.0 DATA COLLECTION AND COMPUTER SYSTEM: 105

7.0 REGULATORY REFERENCES: 105

8.0 QUALITY ASSURANCE MONITORING: 105

9.0 CHANGES IN THE VALIDATION PROTOCOL: 105

10.0 ATTACHMENTS 105

Page 4

10.1 ATTACHMENTS 1: Analytical Method SAI-BM19499 (DRAFT).

105

11.2 Bioanalytical Method Validation SOP SAI-LP101 107

Title: Bioanalytical Method Validation 108

1.0 PURPOSE: 108

2.0 SCOPE: 108

3.0 RESPONSIBILITIES: 108

4.0 PROCEDURE: 108

4.1 A validation protocol along with a draft bioanalytical method will be

written prior to thestart of the validation. In this case, the validation protocol

overwrites this SOP in regards to all aspects of the validation. 108

4.2 The key criteria for evaluation of the method reliability and performance

are: Selectivity,Calibration range, Precision, Accuracy, Recovery and Stability.

108

4.3 The biological matrix to be employed in the validation will be the same as

that in the intended test samples. 108

4.4 Method validation is performed to verify the following parameters:

108

4.5 Stock Check & Stock Stability (Short Term and Long Term): 109

4.6 Interference Check: 111

4.7 Spiking/Stock solutions check: 112

4.8 Selectivity: 112

4.9 CARRY OVER: 115

4.10 TEST BULK SPIKING: 115

4.11 Calibration Standard Curve: 116

4.12 Precision and Accuracy: 120

4.13 Stability: 121

4.14 Dilution Integrity: 125

4.15 EFFECT OF HEMOLYSIS: 126

4.16 If the run and experiment is acceptable report the results. If not,

investigate and resolve the problem and repeat the experiment. 126

Page 5

4.17 Recovery: 127

4.18 Whole Blood Stability: 128

4.19 Maximum Batch Size: 128

4.20 Omission of QC's during validation: 129

5.0 EXHIBITS: 130

6.0 REFERENCES: 131

7.0 DEFINITIONS: 131

REVISION HISTORY 134

11.3 D5 Benzalkonium Chloride (C12 and C14 homologs) Bio-analytical

method #SAI-BM19499-00 140

Determination of D5 Benzalkonium Chloride(C12 and C14 homologs) in

Human Plasma byLC/MS/MS 141

1.0 PURPOSE: 141

2.0 RESPONSIBILITY: 141

3.0 PROCEDURE: 141

3.1 Summary: 141

3.2 Chemicals: 142

3.3 Chemical structures of D5 Benzalkonium Chloride and Benzalkonium

Chlorided13: 142

3.4 PREPARATION OF REAGENTS 143

3.5 PREPARATION OF MASTER STOCK SOLUTIONS: 143

3.6 PREPARATION OF INTERNAL STANDARD (IS) STOCK

SOLUTIONS: 145

3.7 PREPARATION OF REFERENCE SOLUTION 145

3.8 PREPARATION OF CALIBRATION STANDARDS AND QUALITY

CONTROLSAMPLES: 146

3.9 SAMPLE EXTRACTON PROCEDURE: 147

3.10 LC (LIQUID CHROMATOGRAPHIC) CONDITIONS: 148

3.11 MS/MS CONDITIONS: 149

3.12 RETENTION TIMES: 150

3.13 IDENTIFICATION OF ANALYTES AND INTERNAL STANDARDS:

Page 6

150

3.14 CALIBRATION AND CALCULATION: 151

4.0 EXHIBIT: 151

5.0 TYPICAL MASS SPECTRUM OF D5 BENZALKONIUM CHLORIDE

C12 AND C14 152

5.1 TYPICAL MASS SPECTRUM OF BENZALKONIUM CHLORIDE-dB:

153

REVISION HISTORY 154

11.4 COAs of Deuterated Benzalkonium Chloride, Benzyl dodecyl dimethyl

ammonium chloride D13 (Benzalkonium Chloride-d13) and Over-The-Counter

Multi-Component Mixture-6 157

CoA of Deuterated Benzalkonium Chloride 158

CoA of Benzyl dodecyl dimethyl ammonium chloride D13 159

CoA of Over-The-Counter Multi-Component Mixture-6 160

11.5 Linear Regression Curve 161

11.5.1 Linear Regression Curve for D5 Benzalkonium Chloride-C12 161

11.5.2 Linear Regression Curve for D5 Benzalkonium Chloride-C14 163

11.6 Representative Chromatograms 165

11.6.1 Representative Chromatograms for D5 Benzalkonium Chloride-C12

165


184

Page 7

Sannova Analytical Inc. Report No. SAI-VR19499

Validation Report of the Liquid Chromatographic Method with MS/MS Detection for the

Determination of D5 Benzalkonium Chloride (C12 and C14 homologs) in Human Plasma

LIST OF TABLES Table 1: Preparation of Calibration Curve Standards and Quality Control Samples: ................. 27

Table 2: Summary of all the Batches Run during Validation: ..................................................... 30

Table 3: Selectivity data for D5 Benzalkonium Chloride-C12 and Internal Standard in blank: . 33

Table 4: Selectivity data for D5 Benzalkonium Chloride-C14 and Internal Standard in blank: . 35

Table 5: Interference Check for D5 Benzalkonium Chloride-C12: ............................................. 38

Table 6: Interference Check for D5 Benzalkonium Chloride-C14: ............................................. 38

Table 7: Interference Check for D5 Benzalkonium Chloride-C12 in the presence of

Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine Maleate, Naproxen and (1R, 2R)-(-)-

Pseudoephedrine): ........................................................................................................................ 41

Table 8: Interference Check for D5 Benzalkonium Chloride-C14 in the presence of

Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine Maleate, Naproxen and (1R, 2R)-(-)-

Pseudoephedrine): ........................................................................................................................ 41

Table 9: Carry over for D5 Benzalkonium Chloride-C12: .......................................................... 43

Table 10: Carry over for D5 Benzalkonium Chloride-C14: ........................................................ 43

Table 11: Areas Ratio of Drug (D5 Benzalkonium Chloride-C12) to Internal Standard

(Benzalkonium Chloride-d13) in Extracted Samples: .................................................................. 44

Table 12: Areas Ratio of Drug (D5 Benzalkonium Chloride-C14) to Internal Standard

(Benzalkonium Chloride-d13) in Extracted Samples: .................................................................. 45

Table 13: Summary of calibration curves obtained during method validation for D5

Benzalkonium Chloride-C12: ...................................................................................................... 47

Table 13A: Summary of calibration curves parameters for D5 Benzalkonium Chloride-C12: .. 48

Table 14: Summary of calibration curves obtained during method validation for D5


Table 14A: Summary of calibration curves parameters for D5 Benzalkonium Chloride-C14: .. 50

Table 15: Summary of LLOQ obtained during method validation for D5 Benzalkonium

Chloride-C12: .............................................................................................................................. 52

Table 16: Summary of LLOQ obtained during method validation for D5 Benzalkonium

Chloride-C14: .............................................................................................................................. 53

Page 8




Table 17: Intra-Assay (within) Precision and Accuracy results for Batch 1 for D5 Benzalkonium

Chloride-C12: .............................................................................................................................. 54


Chloride-C14: .............................................................................................................................. 55


Chloride-C12: .............................................................................................................................. 55


Chloride-C14:…………………………………………………………………………… .......... 56


Chloride-C12: .............................................................................................................................. 56


Chloride-C14: .............................................................................................................................. 57

Table 23: Inter-Assay (overall) Precision and Accuracy results for Batches 1, 2 and 3 for D5


Table 24: Inter-Assay (overall) Precision and Accuracy results for Batches 1, 2 and 3 for D5


Table 25: Sum of %RE for the evaluation of weighting factor with linear regression for D5


Table 26: Sum of %RE for the evaluation of weighting factor with linear regression for D5


Table 27: Absolute Recovery data of D5 Benzalkonium Chloride-C12 in human plasma: ........ 64

Table 28: Absolute Recovery data of D5 Benzalkonium Chloride-C14 in human plasma: ........ 65

Table 29: Absolute Recovery of Benzalkonium Chloride-d13 (Internal Standard) in human

plasma: ......................................................................................................................................... 67

Table 30: Stability of D5 Benzalkonium Chloride-C12 in human plasma after 4 freeze-thaw

cycles: .......................................................................................................................................... 69

Table 31: Stability of D5 Benzalkonium Chloride-C14 in human plasma after 4 freeze-thaw

cycles: .......................................................................................................................................... 69

Table 32: Bench Top stability for D5 Benzalkonium Chloride-C12 (6 hours) in human plasma:

...................................................................................................................................................... 71

Page 9





...................................................................................................................................................... 71

Table 34: Autosampler Stability (99 hours) for D5 Benzalkonium Chloride-C12 in human

plasma at 4°C: .............................................................................................................................. 73

Table 35: Autosampler Stability (99 hours) for D5 Benzalkonium Chloride-C14 in human

plasma at 4°C: .............................................................................................................................. 73

Table 36: Human Whole Blood Stability (3 hours) for D5 Benzalkonium Chloride-C12 at room

temperature: ................................................................................................................................. 75

Table 37: Human Whole Blood Stability (3 hours) for D5 Benzalkonium Chloride-C14 at room

temperature: ................................................................................................................................. 75

Table 38: Stock solution stability (22 hours) for D5 Benzalkonium Chloride-C12 at Room

Temperature: ................................................................................................................................ 77

Table 39: Stock solution stability (22 hours) for D5 Benzalkonium Chloride-C14 at Room

Temperature: ................................................................................................................................ 77

Table 40: QC results from Maximum batch size run for D5 Benzalkonium Chloride-C12: ....... 79

Table 41: QC results from Maximum batch size run D5 Benzalkonium Chloride-C14: ............ 79

Table 42: Dilution integrity data for D5 Benzalkonium Chloride-C12 in human plasma: ......... 81

Table 43: Dilution integrity data for D5 Benzalkonium Chloride-C14 in human plasma: ......... 82

Page 10




LIST OF CONTRIBUTORS

This validation was conducted in accordance with the principles of Good Laboratory Practices

(GLPs) and Sannova Analytical Inc. Standard Operating Procedures (SOPs) by the following

personnel.

Ganesh Balakrishnan, M.Sc., Analytical Chemist (Lead)

Uma Maheshwari Gopala Krishnan, M.S., Quality Assurance Lead

Dipa Patel, B.Sc., Chemist

Nageswara Rao Reddy, Ph.D., Analytical Chemist (Lead)

Mayur Surendrakumar Patel., B.Pharm., QC Project Manager

Venkata Naga Swathi Valisetti M.S., QC Associate Reviewer

Sunanda Kethamokkala, M.S., QC Associate Reviewer

Lakshmi Pantangi M.S., Associate Chemist

Srivani Domakuntla, B.Sc., QA Reviewer

Page 11




LIST OF ABBREVIATIONS

The following is a list of the commonly used abbreviations; others are expanded at first use: amu: atomic mass unit

API: Atmospheric Pressure Ionization

BE: Bio-Equivalence

BLQ: Below LOQ (Lower Limit of Quantitation)

CCs: Calibration Curve Standards

Cmax: Maximum observed concentration

CoA: Certificate of Analysis

CV: Coefficient of Variation oC: Degree Celsius

EDTA: EthyleneDiamineTetraAcetic acid

FDA: Food and Drug Administration

FRZ: Freezer

GLP: Good Laboratory Practice

HQC: High Quality Control

Hr: Hour

HPLC: High Performance Liquid Chromatography

ISTD: Internal Standard

IS: Internal Standard

ID: Identity

LC/MS/MS: Liquid Chromatography with tandem Mass Spectrometry

LLOQ: Lower Limit of Quantitation

LP: Laboratory Practice

LQC: Low Quality Control

MQC: Medium Quality Control

MRM: Multiple Reaction Monitoring

MS: Mass Spectrometry

µL: microliter

Page 12




µg: microgram

µg/mL: microgram/milliliters

mg: milligram

mL: milliliter

ng: nanogram

ng/mL: nanogram/milliliters

NAV: Not Available

NA: Not Applicable

NMT: Not More Than

n: number

OCS: Optimization of Chromatographic System

OCCQC: Quality Control with Concentration outside the calibration curve

PA: Precision & Accuracy

pg: picogram

pg/mL: picogram/milliliters

QA: Quality Assurance

QCs: Quality Control Samples

QC: Quality Control

RPM: Revolutions Per Minute

%RE: Percent Relative Errors

SOPs: Standard Operating Procedures

SD: Standard of Deviation

STD: Standard

ULOQ: Upper Limit of Quantitation

UPLC: Ultra Performance Liquid Chromatography

USA: United States of America

USP: United States Pharmacopeia

Page 13

Sannova Analytical Inc. Report No. SAI-VRI 9499


Determination ofD5 Benzalkonium Chloride (Cl2 and C14 homologs) in Human Plasma

SIGNATURE PAGE

AUTHOR:

The following author has compiled and written this report, which accurately reflects the data

generated during the validation.

Usha Ramaka, M.Sc., Date QC Documentation Executive

REVIEWER(S):

The following reviewer(s) have reviewed this report and it accurately reflects the data collected.

Prasa~,M.Pharm. QC Lead, Quality Control Department

SCIENTIFIC REVIEWER(S):

o i:;-~n Lori Date

The following scientist(s) reviewed this report and it accurately reflects the data collected.

Malleswar Kollu, M.Sc. Director, Bioanalytical Department

Venkat Reddy, Ph.D. Vice President

Date

Date

Page 14



Determination ofD5 Benzalkonium Chloride (CI2 and CI4 homologs) in Human Plasma

QUALITY ASSURANCE STATEMENT

All raw data, chromatograms, results and the report have been reviewed by the Quality

Assurance Department for compliance with Good Laboratory Practices (GLPs) and Standard

Operating Procedures (SOPs) of Sannova Analytical Inc. The results reported herein accurately

reflect the raw data.

Phase audited Audit date( s) Date observations reported to

management

In-process audit lO M~ 2-0\1 O..I MU 2-019

Raw data audit (:)I :f U>,\ ~f) 1'1 01 .3 UV\ 1-0 l °I

Report audit 03. -JUV\ ')..0 Ill 01 ::Tu"' r;_.O l °I

J .\Jo.~

Srivani Domakuntla, B.Sc., Date

QA Reviewer, Quality Assurance Department

Uma Maheshwari Gopala Krishnan, M.S. Date

Quality Assurance Lead, Quality Assurance Department

Page 15



Determination ofDS Benzalkonium Chloride (C12 and C14 homologs) in Human Plasma

COMPLIANCE STATEMENT

The method validation results presented in this report were conducted in compliance with the

principles of Good Laboratory Practices (GLPs) and Standard Operating Procedures (SOPs) of

Sannova Analytical Inc. This report accurately reflects the raw data and was inspected by the

Quality Assurance (QA) personnel.

Hanumantha Rao Marepalli, Ph. D.

Chief Scientific Officer

_( o __ --s._0_t-:l_2..D_ t I Date

Page 16




1.0 INTRODUCTION

The purpose of this validation was to validate a LC/MS/MS method for the

determination of D5 Benzalkonium Chloride (C12 and C14 homologs) in human

plasma. The D5 Benzalkonium Chloride (C12 and C14 homologs) bioanalytical method

#SAI-BM19499 was validated as per D5 Benzalkonium Chloride (C12 and C14

homologs) validation protocol provided in Appendix 11.1 #SAI-VP19499, which

complies with the U.S. Food and Drug Administration (FDA) guidance on bioanalytical

method validation. The Bioanalytical Method Validation SOP (SAI-LP101) is provided

in Appendix 11.2. The validation was conducted from 2nd May, 2019 through 12th May,

2019. This validation report provides experimental results conducted to evaluate

sensitivity, selectivity, linearity, precision, accuracy, recovery and various stability

studies. The validated D5 Benzalkonium Chloride (C12 and C14 homologs)

bioanalytical method #SAI-BM19499-00 is provided in Appendix 11.3 will be used

further for clinical plasma sample analysis.

Page 17




2.0 SUMMARY

A LC/MS/MS method, #SAI-BM19499, was validated for the quantification of D5

Benzalkonium Chloride (C12 and C14 homologs) in human plasma over the

concentration range of 106.944 to 13688.831 pg/mL and 32.493 to 4159.090 pg/mL,

respectively. D5 Benzalkonium Chloride (C12 and C14 homologs) were extracted from

human plasma using liquid-liquid extraction procedure. Benzalkonium Chloride-d13 was

used as an Internal Standard (IS) for D5 Benzalkonium Chloride (C12 and C14

homologs).

The extracted plasma samples were analyzed using reverse-phase liquid chromatography

and the analytes were detected using tandem Mass Spectrometry. The validation was

performed using a Triple Quad API 5500 LC/MS/MS system with Turbo Ion spray

interface. The positive ions were measured in Multiple Reaction Monitoring (MRM)

mode. The data was acquired and analyzed by ABSciex “Analyst” software, versions

1.6.2. The Lower Limit of Quantitation (LLOQ) and the Upper Limit of Quantitation

(ULOQ) as mentioned below:

Analyte (s) LLOQ (pg/mL) ULOQ (pg/mL)

D5 Benzalkonium Chloride-C12 106.944 13688.831


The calibration curve consisted of two control blanks, two zero standards and ten non-

zero calibration standards covering a concentration range as mentioned below:

Analyte (s) Concentration range

D5 Benzalkonium Chloride-C12 106.944 to 13688.831 (pg/mL)

D5 Benzalkonium Chloride-C14 32.493 to 4159.090 (pg/mL)

Quality Control (QC) samples at four different concentration levels corresponding to the

Limit of Quantitation Quality Control (LOQQC), Low Quality Control (LQC), Medium

Quality Control (MQC) and High Quality Control (HQC) are mentioned below:

Page 18




Analyte (s) LOQQC (pg/mL)

LQC (pg/mL)

MQC (pg/mL)

HQC (pg/mL)

D5 Benzalkonium Chloride-C12 106.944 307.073 4652.628 9305.256

D5 Benzalkonium Chloride-C14 32.493 93.298 1413.612 2827.224

QC samples were analyzed with every Precision and Accuracy (PA) assay batch.

Analyte to IS peak area ratio values were used to construct the calibration curve and to

determine QC sample concentrations. Linear regression with 1/x2 weighting was used to

obtain the best fit of analyte(s) data for the calibration curve.

Calibration curve standards and quality control samples met the acceptance criteria for

all experiments used in the final data, demonstrating satisfactory performance of the

method during validation.

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2.1 Overall Summary Table for a Method Validation Report:

Results Hyperlink

Methodology LC/MS/MS NA

Method Validation Report Number #SAI-VR19499 NA

Biological matrix Human Plasma NA

Anticoagulant (if applicable) K3EDTA NA

Analytes of interest D5 Benzalkonium Chloride (C12 and C14 homologs) Appendix 11.4

Internal Standards Benzalkonium Chloride-d13 Appendix 11.4

Selectivity No significant interference at the Retention Time of the Analyte and internal standard was observed.

Summary tables: Table 3, Table 4, Report text: Section 5.1, Subsection 5.1.1

Matrix Effect for Method Consistency

Summary tables: Table 11 and Table 12 Report text: Section 5.2, Subsection 5.2.1

Calibration curve range

For D5 Benzalkonium Chloride-C12 : 106.944-13688.831 pg/mL For D5 Benzalkonium Chloride-C14: 32.493-4159.090 pg/mL

Summary tables: Table 13, Table 13A and Table 14, Table 14A Report text: Section 5.3

Inter-run accuracy for quality control samples

D5 Benzalkonium Chloride-C12

LOQQC (106.944 pg/mL): 97.7% LQC (307.073 pg/mL): 98.6% MQC (4652.628 pg/mL): 98.5% HQC (9305.256 pg/mL): 100.3%

Summary tables: Table 23 Report text: Section 5.5




Page 20




Results Hyperlink

Inter-run precision for quality control samples







Absolute Recovery of Analyte & IS

Summary tables: Table 27, Table 28 and Table 29 Report text: Section 5.7, Subsection 5.7.1 and Subsection 5.7.2

Freeze-Thaw Stability

Demonstrated for 4 freeze & thaw cycles at -20°C in LQC & HQC level

Summary tables: Table 30 and Table 31 Report text: Section 5.8 Subsection 5.8.1

Short Term or Bench Top Stability

Samples were stable for 6 hours at LQC & HQC level on bench at room temperature

Summary tables: Table 32 and Table 33 Report text: Section 5.8 Subsection 5.8.2

Stock Solution Stability at Room Temperature

Demonstrated for 22 hours at Room Temperature

Summary tables: Table 38 and Table 39 Report text: Section 5.8, Subsection 5.8.5.1

Maximum batch size

Summary tables: Table 40 and Table 41 Report text: Section 5.9

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Results Hyperlink

Dilution Integrity


Dilution QCs: 18610.512 pg/mL (dilution factor: 2T) Accuracy: 100.0% Precision: 3.0%



Dilution QCs: 5654.449 pg/mL (dilution factor: 2T) Accuracy: 102.3% Precision: 2.4%


Processed Sample Stability:

Autosampler Stability

Demonstrated for 99 hours at 4°C in LQC and HQC Level

Summary tables: Tables 34 and Tables 35 Report text: Section 5.8, Subsection 5.8.3

NA: Not Applicable.

Interference Check, Interference Check with concomitant medication, Carry Over check,

and Whole blood stability experiments were performed and met the acceptance criteria.

Page 22




3.0 EXPERIMENTAL MATERIALS AND METHODS

3.1 Reference Standard(s)

*: For CoAs refer Appendix 11.4

Analyte Name:*

Manufacturer/Supplier:

Lot No.:

Purity:

Retest Date:

Deuterated Benzalkonium Chloride

EAG laboratories

V1JAT100-19485

98.7% (75.7% for C12 + 23.0% for C14)

11 March, 2021.

Internal Standard Name:*

Manufacturer/Supplier :

Batch No.:

Purity:

Retest Date:

Benzyl dodecyl dimethyl ammonium

chloride D13 (Benzalkonium Chloride-

d13)

Clearsynth

CRC-2016-R&D-27

94.5%

04 May, 2024.

Page 23




3.2 Summary of Analytical Method

Matrix Supplier BIOIVT

Species/Matrix Human Plasma

Anticoagulant K3EDTA

Calibration Range

D5 Benzalkonium Chloride-C12: 106.944 to

13688.831 pg/mL

D5 Benzalkonium Chloride-C14: 32.493 to

4159.090 pg/mL

Regression Type Linear 1/(Concentration)2

Data Calculation Peak area ratio

Lower Limit of Quantification

(LLOQ)

D5 Benzalkonium Chloride-C12: 106.944

pg/mL

D5 Benzalkonium Chloride-C14: 32.493

pg/mL

Sample Volume 200 µL

Analytical Instrumentation API 5500 LC/MS/MS

Shimadzu Prominence HPLC

Type of Detection MS/MS

Chromatography Reverse phase

Column Temperature 40°C

Pump Flow Isocratic

Run Time 12.0 minutes

Injection Volume 15 µL

Page 24




3.3 Standard and QC Preparation

Independent stocks were used to prepare calibration standards and quality control

samples.

D5 Benzalkonium Chloride (D5 Benzalkonium Chloride-C12 and D5 Benzalkonium

Chloride-C14 homologs) standards were accurately weighed 6.955 mg & 6.754 mg for

stock 1 (DBKC ST-1) & stock 2 (DBKC ST-1A), respectively and transferred the

standards to a 50 mL centrifuge tube. Using a pipette, 25 mL of methanol was added

followed by sonication with occasional shaking to dissolve the solids. Then 25 mL of

water was added and mixed well. Final concentrations from prepared stocks are given

below:

Final concentration (µg/mL) for stocks Analytes (s)



DBKC ST-1 105.299 31.993

DBKC ST-1A 102.256 31.068

Stock 1 (DBKC ST-1) and Stock 2 (DBKC ST-1A) were prepared on 2nd May, 2019.

These two stocks were used for the preparation of Calibration Curve standards and

Quality Control samples, respectively.

The weighing check (Batch Id: 012-001) was performed for the DBKC ST-1 and DBKC

ST-1A stocks of D5 Benzalkonium Chloride (C12 and C14 homologs). The above

stocks of D5 Benzalkonium Chloride (C12 and C14 homologs) were diluted and verified

(Batch Id: 012-002A) for the dilution accuracy.

Calibration Curve Standards (CCs) were freshly spiked for every batch. Quality Control

samples (QCs) i.e. LQC and HQC were prepared in bulk using human plasma on 6th

May, 2019 as per the method #SAI-BM19499 “Determination of D5 Benzalkonium

Chloride (C12 and C14 homologs) in Human Plasma by LC/MS/MS”, and stored at

-20°C.

Page 25




Bulk spiked QCs were tested (Batch Id: 012-004) for accuracy. These spiked QCs were

used for stability experiments.

The stock dilutions and preparation of CCs (STD-A to H’) and QCs (LQC, MQC &

HQC) were given in Table 1.

Page 26


Validation Report of the Liquid Chromatographic Method with MS/MS Detection for the Determination of D5 Benzalkonium

Chloride (C12 and C14 homologs) in Human Plasma

Table 1: Preparation of Calibration Curve Standards and Quality Control Samples:

NA: Not Applicable.

DBKC ST- D5 Benzalkonium Chloride stock

Stock IDs Volume of stock

(mL)

Final volume

with diluent (mL)

Diluted Stock

Solution IDs

Calculated Concentration Volume of stock solutions

used (mL)

Final volume

with human plasma (mL)

STD IDs

Final plasma conc.


(ng/mL)


(ng/mL)


(pg/mL)


(pg/mL)

Calibration standard samples:

DBKC ST-1 1.300 50.000 DBKC ST-2 2737.766 831.818 NA NA NA NA NA

DBKC ST-2 5.000 50.000 DBKC ST-H 273.777 83.182 0.500 10.000 STD H 13688.831 4159.090

0.500 10.000 STD H’ 13688.831 4159.090

DBKC ST-H 5.000 10.000 DBKC ST-G 136.888 41.591 0.500 10.000 STD-G 6844.416 2079.545

DBKC ST-G 5.000 10.000 DBKC ST-F 68.444 20.795 0.500 10.000 STD-F 3422.208 1039.773

DBKC ST-F 5.000 10.000 DBKC ST-E 34.222 10.398 0.500 10.000 STD-E 1711.104 519.886

DBKC ST-E 5.000 10.000 DBKC ST-D 17.111 5.199 0.500 10.000 STD-D 855.552 259.943

DBKC ST-D 5.000 10.000 DBKC ST-C 8.556 2.599 0.500 10.000 STD-C 427.776 129.972

DBKC ST-C 5.000 10.000 DBKC ST-B 4.278 1.300 0.500 10.000 STD-B 213.888 64.986

DBKC ST-B 5.000 10.000 DBKC ST-A 2.139 0.650 0.500 10.000 STD-A’ 106.944 32.493

0.500 10.000 STD-A 106.944 32.493

Page 27




Table 1 (Continued):

Stock IDs Volume of stock

(mL)

Final volume

with diluent (mL)

Diluted Stock Solution IDs

Calculated Concentration Volume of stock solutions

used (mL)

Final volume

with plasma (mL)

QC IDs

Final plasma conc.


(ng/mL)


(ng/mL)


(pg/mL)


(pg/mL)

Quality control samples:

DBKC ST-1A 1.300 50.000 DBKC ST-2A 2658.645 807.778 NA NA NA NA NA

DBKC ST-2A 3.500 50.000 DBKC ST-HQC 186.105 56.544 1.250 25.000 HQC 9305.256 2827.224

DBKC ST-HQC 5.000 10.000 DBKC ST-MQC 93.053 28.272 1.250 25.000 MQC 4652.628 1413.612

DBKC ST-MQC 3.300 50.000 DBKC ST-LQC 6.141 1.866 1.250 25.000 LQC 307.073 93.298

NA: Not Applicable.

DBKC ST- D5 Benzalkonium Chloride stock.

Page 28




3.4 Method Description

The extraction procedure described here was to be applied for calibration standards and

QCs. These samples were allowed to thaw at room temperature prior to extraction. The

following steps were followed during sample extraction.

• 200 µL of samples was aliquoted into appropriate tubes.

• Using a pipette, 50 µL of IS was added to each sample tube (except for blank

samples to which 50 µL of diluent was added) and vortexed for 10 seconds.

• Using a pipette, to each sample tube 3.0 mL of ethyl acetate was added followed

by shaking for 10 minutes at 1000 rpm on a Micro plate shaker.

• After flash freezing, the organic layer was completely transferred into fresh

disposable glass tubes and dried in a nitrogen evaporator at 45°C.

• Then the dried residue was reconstituted with 0.6 mL of mobile phase followed

by shaking for 5 minutes at 1000 rpm on a Micro plate shaker.

• Reconstituted samples were transferred to respective HPLC vials and injected into

autosampler of LC/MS/MS system for analysis.

Page 29




4.0 SUMMARY OF THE BATCHES A summary of all the batches conducted under this validation is given in Table 2.

Table 2: Summary of all the Batches Run during Validation:

Run # Batch ID Purpose of the Batch Extraction Date

Run Accepted/ Rejected

Experiment Accepted/ Rejected

01 012-001 Stock Check 03-May-19 NA Accepted

02 012-002 Spiking solutions check

03-May-19 NA Rejected①

012-002A (Reinjected) Spiking solutions check NA Accepted①

03 012-003 Blank Check (Selectivity) and

Stock Stability at Room Temperature for 22 Hours

04-May-19 Accepted Accepted

04 012-004 Test Bulk Spiking 06-May-19 Accepted Accepted

05 012-005 Precision and Accuracy 1,

Interference Check, Interference Check with concomitant Check

07-May-19 Accepted Accepted

06 012-006 Precision and Accuracy 2 08-May-19 Accepted Accepted

07 012-007 Carry Over Check 07-May-19 NA Accepted

08 012-008 Maximum Batch Size 07-May-19 Accepted Accepted

09

009-001 (as Extracted)* 014-001 (as

Injected)

Precision and Accuracy 3 09-May-19 Accepted Accepted

10* 014-002$ Matrix Effect for method consistency 09-May-19 NA Accepted

11 014-003 Whole Blood Stability for 3 Hours 10-May-19 NA Accepted

12 014-004 Freeze Thaw Stability, Bench Top stability for 6 Hours 10-May-19 Accepted Accepted

13 014-005 Dilution Integrity 11-May-19

Accepted Accepted Autosampler Stability for 99 Hours 07-May-19

Page 30




Note: MS instrument ID # is the first three digits of the Batch Id # (e.g., 012-001 refers to MS#12) NA: Not Applicable. *: Run #09 Precision and Accuracy 3 samples were extracted to inject as Batch Id: 009-001 using SAI-

MS-009. These samples were not injected in the same instrument SAI-MS-009, as low sensitivity was

observed in Optimization of Chromatographic System (OCS). Hence, these samples were moved and

injected in to a different instrument SAI-MS-014 as Batch Id: 014-001. Additionally, for Run #10 Matrix

Effect samples, sequence was created as Batch Id: 009-002. However, due to low sensitivity in the SAI-

MS-009, the Matrix Effect batch was processed and injected using SAI-MS-014 as Batch Id: 014-002.

$: Absolute Recovery results were calculated from Matrix Effect Method Consistency Experiment Batch

Id: 014-002.

①: Refer Memo to file-01 in section 6.0, A

Page 31




5.0 RESULTS

5.1 Selectivity

5.1.1 Blank Check

Selectivity (blank check) refers to the ability of an analytical method to differentiate and

quantify the analyte in the presence of other components in the sample. Nine independent

blank lots were screened for possible interference at the retention time of the analyte and

Internal Standard (IS). The interference at the retention time of analyte in each blank

plasma lot was calculated using respective peak area of the analyte with average area of

accepted STD A and STD A’. The interference at the retention time of IS in each blank

plasma lot was calculated using respective peak area of the IS with average area of all

accepted calibration standards and QCs. Three different solutions containing Blank,

Blank+IS and LOQ are prepared for each matrix lot. All the samples were analyzed along

with the freshly prepared and extracted CCs and QCs (in duplicate). The results are given

in Table 3 and Table 4.

Page 32




Table 3: Selectivity data for D5 Benzalkonium Chloride-C12 and Internal Standard in blank:

Batch ID Matrix ID Area of Analyte

in Blank

% Interference (Limit NMT 20%)

Area of IS in Blank


Area of Analyte

in Blank+IS


% Accuracy (Limit 80.0-120.0%)

Accepted/ Rejected

012-003

BRH 1637978 0 0 0 0 0 0 102.9 Accepted

BRH 1637979 0 0 0 0 0 0 92.6 Accepted

BRH 1637980 0 0 0 0 0 0 92.1 Accepted

BRH 1637981 0 0 0 0 0 0 99.3 Accepted

BRH 1637982 0 0 0 0 0 0 91.2 Accepted

BRH 1637983 0 0 0 0 0 0 92.1 Accepted

BRH 1637984 0 0 13925 15 0 0 92.0 Rejected

BRH 1637985 0 0 0 0 0 0 105.1 Accepted

BRH 1637986 0 0 5847 6 0 0 101.1 Rejected Note: BRH1637978 to BRH1637986 blank lots were received on 19th February, 2019.

Page 33




Table 3 (Continued): Batch ID Sample ID Area of

Analyte Area of IS

012-003

STD A 36260 92726 STD A' 35789 89003 STD B

NA

96507 STD C 88987 STD D 80377 STD E 86652 STD F 89069 STD G 98103 STD H 95926 STD H ' 92813 FR-LQC 99347 FR-LQC' 90649 FR-MQC 93356 FR-MQC' 97776 FR-HQC 93566 FR-HQC' 93878

Average Area 36025 92421

NA: Not Applicable.

Page 34




Table 4: Selectivity data for D5 Benzalkonium Chloride-C14 and Internal Standard in blank:

Batch ID Matrix ID Area of Analyte

in Blank


Area of IS in Blank


Area of Analyte

in Blank+IS


% Accuracy (Limit 80.0-120.0%)

Accepted/ Rejected

012-003

BRH 1637978 0 0 0 0 0 0 101.2 Accepted BRH 1637979 0 0 0 0 0 0 91.8 Accepted BRH 1637980 0 0 0 0 0 0 87.5 Accepted BRH 1637981 0 0 0 0 0 0 95.2 Accepted BRH 1637982 0 0 0 0 0 0 79.7 Rejected BRH 1637983 0 0 0 0 0 0 88.9 Accepted BRH 1637984 0 0 13925 15 0 0 86.6 Rejected BRH 1637985 0 0 0 0 0 0 101.8 Accepted BRH 1637986 0 0 5847 6 0 0 94.4 Rejected

Note: BRH1637978 to BRH1637986 blank lots were received on 19th February, 2019.

Page 35




Table 4 (Continued): Batch ID Sample ID Area of Analyte Area of IS

012-003

STD A 34451 92726 STD A' 33396 89003 STD B

NA

96507 STD C 88987 STD D 80377 STD E 86652 STD F 89069 STD G 98103 STD H 95926 STD H ' 92813 FR-LQC 99347 FR-LQC' 90649 FR-MQC 93356 FR-MQC' 97776 FR-HQC 93566 FR-HQC' 93878

Average Area 33924 92421 NA: Not Applicable.

Page 36




Run Acceptance Criteria:

Interference in the blank, if any, at the retention time of analyte must not exceed 20% of

the average response of accepted LLOQ (STD-A and STD-A’).

Interference in the blank, if any, at the retention time of IS must not exceed 5% of the

average response of IS from accepted standards and QCs.

At least one each of Blank & Blank+IS sample must meet the acceptance criteria to

accept the batch.

The % deviation of standards must be ±15%, except for the LLOQ standard where it must

be within ±20% from the nominal or expected concentration.

The analyte response at LLOQ should be at least 5 times the response compared to blank

response. At least 75% of calibration standards, when back calculated including upper

LOQ ULOQ should fall within ±15%, except for LLOQ where it should be ±20% of the

nominal value. Values falling outside these limits should not be used to calculate

regression analysis.

Also at least four out of the six freshly prepared QCs and one QC at each level must be

within ±15% of the nominal value.

Experiment Acceptance Criteria:

Any interference arising from blanks at the retention time of analyte(s) should not be

more than 20% of the average response of accepted LLOQ (STD-A and STD-A’) of

calibration curve.

Any interference arising from blanks at the retention time of IS should not be more than

5% of the average response of IS from all accepted standards and QCs.

Percent Accuracy of LLOQQC of each matrix must be within ±20%.

Conclusion

Nine blank lots were screened for the selectivity, seven out of nine blank lots for D5

Benzalkonium Chloride-C12 and six out of nine blank lots D5 Benzalkonium Chloride-

C14 met the acceptance criteria. Four of them (Lot BRH1637978, BRH1637979,

BRH1637980 and BRH1637985) were pooled and renamed as SAIVP19499BL and

Page 37




which was used for bulk spiking of Quality Control samples and fresh preparation of the

Calibration Curve samples.

5.1.2 Interference Check:

An extracted sample of the analyte (s) at approximately the highest standard

concentration was prepared without the IS in duplicate and injected to observe any

possible interference at the retention time of the IS.

An extracted sample of the IS at the approximate concentration equivalent to the IS

concentration was prepared without the analyte in duplicate and injected to observe any

possible interference at the retention time of the analyte (s). Additionally, blank samples

in duplicate were injected.

The results for the interference due to analyte(s) and IS are given in Table 5 and Table 6.

Table 5: Interference Check for D5 Benzalkonium Chloride-C12:

Batch ID Extraction Date

Area of Analyte in

STD A

Area of IS in STD A

Area of analyte

Interference due to IS

%Interference of analyte due

to IS

Area of IS Interference

due to analyte

%Interference of IS due to

analyte

012-005 07-May-19 33218 86804 0 0 0 0 36082 90255 0 0 0 0

Mean 34650 88530

Table 6: Interference Check for D5 Benzalkonium Chloride-C14:


Area of Analyte in

STD A

Area of IS in STD A

Area of analyte

Interference due to IS


to IS


due to analyte

%Interference of IS due to

analyte

012-005 07-May-19 30377 86804 0 0 0 0

31924 90255 0 0 0 0

Mean 31151 88530


Any interference at the retention time of analyte (s) should not be more than 20% of the

average analyte response of the extracted LLOQs and any interference at the retention

time of IS should not be more than 5% of the average IS response of the extracted LLOQ

Page 38




solutions. At least one of the each Standard H without IS, Blank and Blank+IS should

meet the acceptance criteria.

Conclusion

The Interference check met the acceptance criteria since there was no significant

interference observed at the retention times of analyte (s) due to the IS and at the

retention times of the IS due to the analyte (s).

5.1.3 Interference Check in the presence of concomitant drugs (Mixture of Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine Maleate, Naproxen and (1R, 2R)-(-)-Pseudoephedrine)

Concomitant drugs ((mixture of Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine

Maleate, Naproxen and (1R,2R)-(-)-Pseudoephedrine) and analyte (s) of interest (D5

Benzalkonium Chloride-C12 and D5 Benzalkonium Chloride-C14) molecular weights are

given in the Table A and Table B, respectively.

Table A:

Compound Drug Molecular Weight (AMU)

Difference between Molecular weight of D5 Benzalkonium

Chloride-C12 and Concomitant drugs


345.0 NA

Acetaminophen 151.16 193.84

Ibuprofen 206.29 138.71

Caffeine 194.19 150.81

Chlorpheniramine Maleate

390.86 45.86

Naproxen 230.26 114.74

(1R, 2R)-(-)-Pseudoephedrine)

165.24 179.76

NA: Not Applicable.

Page 39




Table B:

Compound Drug Molecular Weight (AMU)

Difference between Molecular weight of D5 Benzalkonium

Chloride-C14 and Concomitant drugs


373.1 NA

Acetaminophen 151.16 221.94

Ibuprofen 206.29 166.81

Caffeine 194.19 178.91

Chlorpheniramine Maleate

390.86 17.76

Naproxen 230.26 142.84

(1R, 2R)-(-)-Pseudoephedrine)

165.24 207.86

NA: Not Applicable.

D5 Benzalkonium Chloride (C12 and C14 homologs) was conducted using LC/MS/MS.

All LC/MS/MS used for the estimation of D5 Benzalkonium Chloride (C12 and C14

homologs) are unit mass resolution instruments. Hence, mass difference between

concomitant drugs and D5 Benzalkonium Chloride-C12 and D5 Benzalkonium Chloride

C14 were more than 45 amu and 17 amu, respectively. Hence, interference would not be

an issue. However, it was decided to conduct concomitant drugs interference check

experiment.

Blank, Blank+IS and LLOQQCs in the presence of generally administered Over the

Counter medication drugs (mixture of Acetaminophen, Ibuprofen, Caffeine,

Chlorpheniramine Maleate, Naproxen and (1R,2R)-(-)-Pseudoephedrine) were prepared

in duplicate and injected to observe any possible interference at the retention time of the

analyte and internal standard.

The spiked concentrations for Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine

Maleate, Naproxen and (1R, 2R)-(-)-Pseudoephedrine) was 2500.000 ng/mL each,

respectively. (For COA refer Appendix 11.4). All the samples were analyzed along with

the freshly spiked and extracted calibration curve standards and quality control samples

(in duplicate). The results are given in Table 7 and Table 8.

Page 40




Table 7: Interference Check for D5 Benzalkonium Chloride-C12 in the presence of Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine Maleate, Naproxen and (1R, 2R)-(-)-Pseudoephedrine):


Area of Analyte in

STD A

Area of IS in

STD A

Area of analyte

Interference due to

concomitant medication

in Blank


to concomitant

medication in Blank


due to concomitant medication

in Blank

%Interference of IS due to concomitant

medication in Blank

Area of analyte

Interference due to

concomitant medication in BL+IS


to concomitant

medication in BL+IS

% Accuracy of LLOQ

with concomitant medication

012-005 07-May-19 33218 86804 0 0 0 0 0 0 93.3

36082 90255 0 0 0 0 0 0 98.2 Mean 34650 88530

Table 8: Interference Check for D5 Benzalkonium Chloride-C14 in the presence of Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine Maleate, Naproxen and (1R, 2R)-(-)-Pseudoephedrine):

Batch ID

Extraction Date

Area of Analyte

in STD A

Area of IS in

STD A

Area of analyte

Interference due to

concomitant medication

in Blank


to concomitant

medication in Blank


due to concomitant medication

in Blank

%Interference of IS due to concomitant

medication in Blank

Area of analyte Interference

due to concomitant

medication in BL+IS

%Interference of analyte due to concomitant medication in

BL+IS

% Accuracy of LLOQ

with concomitant medication

012-005

07-May-19

30377 86804 0 0 0 0 0 0 95.2 31924 90255 0 0 0 0 0 0 93.3

Mean 31151 88530

Page 41




Run Acceptance Criteria

Run Acceptance was based on acceptability of the calibration curve only as described in

Section 5.1 (Acceptance criteria) of this validation report.

Experiment Acceptance Criteria

Any interference at the retention time of analyte must not be more than 20% of the

average response of the extracted LLOQs and any interference at the retention time of

internal standard should not be more than 5% of the average internal standard response of

the extracted LLOQ solutions. Percent Accuracy of LOQQC must be within ±20%. At

least one of the each Blank, Blank+IS and LLOQ should meet the acceptance criteria.

Conclusion

Run and Experiment met the acceptance criteria. The interference check met the

acceptance criteria since there was no significant interference at the retention times of the

analyte in the presence of concomitant drugs (Mixture of Acetaminophen, Ibuprofen,

Caffeine, Chlorpheniramine Maleate, Naproxen and (1R, 2R)-(-)-Pseudoephedrine).

5.1.4 Carry over

Carry over experiment was assessed by injecting extracted highest standard followed by

extracted blank to quantitate any carry over. Extracted blank, extracted low standard

(LLOQ) and highest standard (ULOQ) followed by extracted blank samples were

injected in duplicate to verify the carry over. The results are given in Table 9 and Table

10.

Page 42




Table 9: Carry over for D5 Benzalkonium Chloride-C12:

Batch ID 012-007 Extraction Date 07-May-19

Sequence of injections ↓ Analyte Internal Standard Area Mean area Area Mean area

Extracted Blank in duplicate at beginning

0 NA

0 NA

0 0

LLOQ in duplicate 32280 33790 100582 102388 35299 104193

ULOQ in duplicate 4481788 4593029 92448 93367 4704269 94285

Extracted Blank in duplicate after ULOQ

0 NA

0 NA

0 0

Carry over calculation

% carry over in blank compared with LLOQ

mean area

0.0 0.0

0.0 0.0

NA: Not Applicable.

Table 10: Carry over for D5 Benzalkonium Chloride-C14:

Batch ID 012-007 Extraction Date 07-May-19

Sequence of injections ↓ Analyte Internal Standard

Area Mean area Area Mean area

Extracted Blank in duplicate at beginning

0 NA

0 NA

0 0

LLOQ in duplicate 29563 27752 100582 102388 25941 104193

ULOQ in duplicate 4290825 4364053 92448 93367 4437280 94285

Extracted Blank in duplicate after ULOQ

0 NA

0 NA

0 0

Carry over calculation

% carry over in blank compared with LLOQ

mean area

0.0 0.0

0.0 0.0

NA: Not Applicable.

Page 43





Response from the extracted blank should not be more than 20% compared to average of

LLOQ (low standard). Response from the IS should not be more than 5% of the average

IS response of the LLOQ.

Conclusion

No significant carry over was observed at the retention time of the analyte and IS.

5.2 Matrix Effect

Matrix effect is defined as inconsistent response for the analyte(s) of interest, IS or both

(all) when the same concentration of compounds are extracted from different sources of

biological blank matrices.

5.2.1 Matrix Effect for Method Consistency:

Six independent sources of plasma matrices screened for selectivity were used for the

matrix effect. Six replicates of LQC, MQC and HQC for each matrix (analytes were

prespiked) were prepared and extracted containing all the analyte of interest and IS as per

method of analysis to check method consistency. The results are given in Table 11 and

Table 12.

Table 11: Areas Ratio of Drug (D5 Benzalkonium Chloride-C12) to Internal Standard (Benzalkonium Chloride-d13) in Extracted Samples:

Batch ID Matrix ID

LQC MQC HQC Mean Areas

ratio of drug to internal standard

%CV

Mean Areas ratio of drug to

internal standard

%CV

Mean Areas ratio of drug to

internal standard

%CV

014-002

BRH1637978 1.246 7.9 17.741 5.5 35.538 3.7 BRH1637979 1.198 2.8 17.032 2.4 34.519 4.0 BRH1637980 1.200 4.0 17.439 4.9 34.502 2.1 BRH1637981 1.208 0.7 17.083 2.7 33.771 3.2 BRH1637983 1.183 2.0 17.583 5.2 34.466 1.8 BRH1637985 1.197 2.4 16.559 2.6 33.535 3.8

Mean 1.206 17.239 34.388 %CV 1.8 2.5 2.0

Page 44




Table 12: Areas Ratio of Drug (D5 Benzalkonium Chloride-C14) to Internal Standard (Benzalkonium Chloride-d13) in Extracted Samples:

Batch ID Matrix ID

LQC MQC HQC

Mean Areas ratio of drug to internal standard

%CV


%CV


%CV

014-002

BRH1637978 1.240 8.8 17.778 5.2 34.919 5.3 BRH1637979 1.174 3.0 16.357 1.2 32.511 3.5 BRH1637980 1.195 4.5 17.316 5.5 32.811 3.0 BRH1637981 1.220 2.1 17.199 2.0 33.343 1.6 BRH1637983 1.185 2.7 17.632 5.0 33.071 2.6 BRH1637985 1.165 2.8 15.713 2.2 30.758 3.5

Mean 1.196 16.999 32.902 %CV 2.4 4.7 4.1


At each level, the %CV of the peak area ratio of the analyte (s) to the IS from each matrix

must be within ±15% (for each lot at least 4 out of 6 replicates must be used for statistical

calculations) and %CV of the mean peak area ratios from 5 out of 6 matrix lots must be

within ±15% to assume that no major matrix effect is present.

Conclusion

For D5 Benzalkonium Chloride-C12:

Results LQC MQC HQC

%CV for mean area ratios 1.8 2.5 2.0


Results LQC MQC HQC

%CV for mean area ratios 2.4 4.7 4.1

These results indicate that there was no major matrix effect.

Page 45




5.3 Calibration Standard Curve

Calibration Curve (CC) is the relationship between the instrument response and the

known concentration of the analyte(s) in the solution. A linear weighted regression (1/x2)

is chosen (based upon smallest % RE, refer section 5.6) to represent the peak area ratio

response of D5 Benzalkonium Chloride (C12 and C14 homologs) to D5 Benzalkonium

Chloride-d13, respectively. A linear regression describing the calibration curve is then

calculated using the reciprocal of the square of the drug concentrations (1/

(Concentration)2 or 1/x²) versus peak area ratio.

The regression equation is y=mx+c

y= peak area ratio of Analyte/IS

x= concentration of Analyte

m= slope of the calibration curve

c= y-intercept of the calibration curve.

The linear regression curve and Representative chromatograms data of Precision and

Accuracy batch are provided in Appendix 11.5 and Appendix 11.6, respectively. The CCs

back-calculated concentrations and calibration curve parameters are given in Table 13,

Table 13A for D5 Benzalkonium Chloride-C12 and Table 14, Table 14A for D5

Benzalkonium Chloride-C14, respectively for all accepted runs.

Page 46




Table 13: Summary of calibration curves obtained during method validation for D5 Benzalkonium Chloride-C12:

Run # Batch ID Date of batch

Injected

STD A STD A' STD B STD C STD D STD E STD F STD G STD H STD H'

106.944 106.944 213.888 427.776 855.552 1711.104 3422.208 6844.416 13688.831 13688.831

pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL

03 012-003 04-May-19 106.597 109.503 203.517 425.039 902.395 1647.440 3546.018 6544.760 13828.280 13881.412

04 012-004 06-May-19 107.696 109.092 204.935 418.753 848.890 1729.673 3291.180 6867.949 14254.981 14048.359

05 012-005 07-May-19 105.586 110.070 213.723 408.713 826.033 1677.230 3349.778 7186.671 13941.202 14179.228

06 012-006 08-May-19 115.631 100.941 207.341 398.835 889.364 1718.550 3542.067 7592.355 11668.564 14134.733

08 012-008 09-May-19 106.188 111.586 194.549 443.445 856.405 1712.636 3322.907 6923.186 13296.522 14533.720

09 014-001 09-May-19 107.818 106.230 211.139 432.279 860.353 1742.549 3511.738 6858.206 13020.719 13654.214

12 014-004 11-May-19 102.209 112.351 212.530 421.276 842.968 1765.959 3512.829 6777.415 13394.238 13726.405

13 014-005 11-May-19 109.312 106.843 204.941 423.907 851.422 1768.768 3425.914 7237.730 13235.357 13351.913

Mean 107.630 108.327 206.584 421.531 859.729 1720.351 3437.804 6998.534 13329.983 13938.748

SD 3.847 3.644 6.180 13.632 24.822 41.820 104.380 325.677 788.096 365.662

%CV 3.6 3.4 3.0 3.2 2.9 2.4 3.0 4.7 5.9 2.6

% ACCURACY 100.6 101.3 96.6 98.5 100.5 100.5 100.5 102.3 97.4 101.8

n 8 8 8 8 8 8 8 8 8 8

Page 47




Table 13A: Summary of calibration curves parameters for D5 Benzalkonium Chloride-C12:


Injected Slope Intercept r

03 012-003 04-May-19 0.0038100 -0.0149000 0.9991

04 012-004 06-May-19 0.0037200 -0.0528000 0.9995

05 012-005 07-May-19 0.0038200 -0.0202000 0.9993

06 012-006 08-May-19 0.0027400 0.0045000 0.9957

08 012-008 09-May-19 0.0033200 -0.0308000 0.9986

09 014-001 09-May-19 0.0036500 0.0110000 0.9997

12 014-004 11-May-19 0.0039400 -0.0176000 0.9994

13 014-005 11-May-19 0.0039000 -0.0107000 0.9993

Mean 0.0036130 -0.0164380 0.9988

Page 48




Table 14: Summary of calibration curves obtained during method validation for D5 Benzalkonium Chloride-C14:


Injected

STD A STD A' STD B STD C STD D STD E STD F STD G STD H STD H'

32.493 32.493 64.986 129.972 259.943 519.886 1039.773 2079.545 4159.090 4159.090

pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL

03 012-003 04-May-19 33.115 33.417 59.628 125.226 268.710 496.182 1085.164 2005.123 4251.684 4379.966

04 012-004 06-May-19 33.510 33.026 60.760 123.897 251.884 526.592 990.185 2086.211 4406.006 4438.881

05 012-005 07-May-19 32.933 33.243 62.768 123.064 249.007 503.198 1026.547 2159.641 4306.592 4423.386

06 012-006 08-May-19 34.856 31.737 59.826 120.338 271.304 533.318 1071.195 2269.107 3617.665 4339.276

08 012-008 09-May-19 31.026 34.580 61.173 135.059 265.098 510.552 1001.245 2083.308 4076.453 4382.472

09 014-001 09-May-19 32.719 32.630 64.401 127.371 253.249 520.284 1087.416 2103.611 4038.851 4218.731

12 014-004 11-May-19 31.335 34.434 63.031 125.848 250.729 540.755 1066.702 2064.462 4129.654 4248.369

13 014-005 11-May-19 33.588 31.956 64.275 123.862 258.509 520.890 1043.794 2130.356 4301.956 4083.058

Mean 32.885 33.128 61.983 125.583 258.561 518.971 1046.531 2112.727 4141.108 4314.267

SD 1.237 1.034 1.896 4.356 8.730 15.037 37.439 77.943 246.233 121.864

%CV 3.8 3.1 3.1 3.5 3.4 2.9 3.6 3.7 5.9 2.8

% ACCURACY 101.2 102.0 95.4 96.6 99.5 99.8 100.6 101.6 99.6 103.7

n 8 8 8 8 8 8 8 8 8 8

Page 49




Table 14A: Summary of calibration curves parameters for D5 Benzalkonium Chloride-C14:


Injected Slope Intercept r

03 012-003 04-May-19 0.0122000 -0.0330000 0.9985

04 012-004 06-May-19 0.0117000 -0.0814000 0.9984

05 012-005 07-May-19 0.0121000 -0.0498000 0.9988

06 012-006 08-May-19 0.0087400 -0.0503000 0.9961

08 012-008 09-May-19 0.0104000 -0.0789000 0.9987

09 014-001 09-May-19 0.0118000 0.0022400 0.9996

12 014-004 11-May-19 0.0123000 -0.0381000 0.9991

13 014-005 11-May-19 0.0123000 -0.0308000 0.9995

Mean 0.0114430 -0.0450080 0.9986

Page 50




Run Acceptance criteria:



Interference in the blank, if any, at the retention time of IS must not exceed 5% of the

average response of IS from accepted standards and quality control samples.


accept the batch.





LOQ should fall within ±15%, except for LLOQ where it should be ±20% of the nominal

value. Values falling outside these limits should not be used to calculate regression

analysis.

Conclusion

During the validation, eight calibration curves were executed and all met the acceptance

criteria which prove the ruggedness of the method. The average accuracy for the

calibration standards is between:

96.6-102.3% for D5 Benzalkonium Chloride-C12

95.4-103.7% for D5 Benzalkonium Chloride-C14.

Page 51




5.4 Sensitivity

The lowest standard on the calibration curve is accepted as the Lower Limit of

Quantification (LLOQ). A summary of all LLOQ (STD A and STD A’) obtained during

validation is given in Table 15 and Table 16.

Table 15: Summary of LLOQ obtained during method validation for D5 Benzalkonium Chloride-C12:


Injected

STD A STD A'

106.944 106.944

pg/mL pg/mL

03 012-003 04-May-19 106.597 109.503

04 012-004 06-May-19 107.696 109.092

05 012-005 07-May-19 105.586 110.070

06 012-006 08-May-19 115.631 100.941

08 012-008 09-May-19 106.188 111.586

09 014-001 09-May-19 107.818 106.230

12 014-004 11-May-19 102.209 112.351

13 014-005 11-May-19 109.312 106.843

Mean 107.630 108.327

SD 3.847 3.644

%CV 3.6 3.4

% ACCURACY 100.6 101.3

n 8 8

Page 52




Table 16: Summary of LLOQ obtained during method validation for D5 Benzalkonium Chloride-C14:


Injected

STD A STD A'

32.493 32.493

pg/mL pg/mL

03 012-003 04-May-19 33.115 33.417

04 012-004 06-May-19 33.510 33.026

05 012-005 07-May-19 32.933 33.243

06 012-006 08-May-19 34.856 31.737

08 012-008 09-May-19 31.026 34.580

09 014-001 09-May-19 32.719 32.630

12 014-004 11-May-19 31.335 34.434

13 014-005 11-May-19 33.588 31.956

Mean 32.885 33.128

SD 1.237 1.034

%CV 3.8 3.1

% ACCURACY 101.2 102.0

n 8 8

Acceptance criteria

The response peak of analyte(s) must be identifiable, discrete and reproducible with a

precision of ≤ 20% and an accuracy of 80-120%.

Conclusion

The response peak of D5 Benzalkonium Chloride (C12 and C14 homologs) in LLOQ

solution (STD A and STD A’) was identifiable, discrete and reproducible with average

accuracy and %CV given below:

Analyte (s) %Accuracy %CV

D5 Benzalkonium Chloride-C12 100.6-101.3 3.4-3.6


Page 53




5.5 Precision and Accuracy

Precision of an assay is defined as the closeness of the results when replicate assays of a

test sample are carried out. The precision is expressed as %CV. The accuracy of a

method is defined as the proximity of the determined values to the true or actual

concentrations of the samples. Accuracy is expressed as %Accuracy. During validation,

both the intra-assay and inter-assay precision and accuracy were assessed by replicate

analysis of samples containing known amounts of the analytes (QCs). Intra-assay and

inter-assay precision and accuracy results of six replicate control samples at four QC

concentration levels for three separate batches are shown below in Table 17, Table 18,

Table 19, Table 20, Table 21, Table 22, Table 23 and Table 24.

Table 17: Intra-Assay (within) Precision and Accuracy results for Batch 1 for D5 Benzalkonium Chloride-C12:


LOQQC LQC MQC HQC

106.944 307.073 4652.628 9305.256

pg/mL pg/mL pg/mL pg/mL

012-005 07-May-19

101.682 266.349 4360.549 8914.661

96.445 282.384 4024.905 8772.668

107.256 295.806 4228.560 8591.449

104.738 282.099 4266.596 8506.816

98.244 284.730 4218.073 8852.234

103.018 291.881 4324.805 8707.004

Mean 101.897 283.875 4237.248 8724.139

SD 4.029 10.195 117.697 155.043

%CV 4.0 3.6 2.8 1.8

% Accuracy 95.3 92.4 91.1 93.8

n 6 6 6 6

Page 54






LOQQC LQC MQC HQC

32.493 93.298 1413.612 2827.224


012-005 07-May-19

32.899 84.121 1320.461 2626.054

29.559 88.498 1180.760 2661.169

31.509 91.207 1299.758 2558.828

31.937 88.899 1276.209 2519.444

30.028 86.761 1266.002 2632.925

30.745 89.510 1261.685 2577.971

Mean 31.113 88.166 1267.479 2596.065

SD 1.245 2.451 47.919 53.068

%CV 4.0 2.8 3.8 2.0

% Accuracy 95.8 94.5 89.7 91.8

n 6 6 6 6



LOQQC LQC MQC HQC 106.944 307.073 4652.628 9305.256 pg/mL pg/mL pg/mL pg/mL

012-006 08-May-19

101.871 305.837 4767.912 10549.487 109.222 323.039 5175.080 9728.738 107.392 333.091 4565.349 9346.849 112.598 296.905 4770.383 9344.619 101.465 317.450 4978.720 9047.690 107.891 317.323 4744.064 10373.470

Mean 106.740 315.608 4833.585 9731.809 SD 4.330 12.747 212.582 607.695

%CV 4.1 4.0 4.4 6.2 % Accuracy 99.8 102.8 103.9 104.6

n 6 6 6 6

Page 55







012-006 08-May-19

31.754 97.794 1409.306 3116.969 32.814 101.591 1555.365 2901.168 32.544 101.902 1374.965 2751.067 34.696 90.645 1417.239 2747.872 31.551 100.417 1496.504 2694.782 30.972 99.123 1410.992 3090.745

Mean 32.389 98.579 1444.062 2883.767 SD 1.315 4.180 67.726 184.021

%CV 4.1 4.2 4.7 6.4 % Accuracy 99.7 105.7 102.2 102.0

n 6 6 6 6



LOQQC LQC MQC HQC 106.944 307.073 4652.628 9305.256


014-001 09-May-19

107.384 314.142 4502.678 9847.220 105.020 301.188 4482.941 9588.169

107.999 310.882 4936.788 9426.407

105.381 303.266 4625.074 9438.824 102.276 309.487 4776.627 9129.554

101.342 314.355 4748.600 9851.864

Mean 104.900 308.887 4678.785 9547.006 SD 2.667 5.527 175.081 277.588

%CV 2.5 1.8 3.7 2.9

% Accuracy 98.1 100.6 100.6 102.6 n 6 6 6 6

Page 56






LOQQC LQC MQC HQC

32.493 93.298 1413.612 2827.224


014-001 09-May-19

32.870 95.909 1383.456 3031.445

31.997 93.083 1395.472 2904.737

31.842 98.901 1491.348 2945.682

30.859 92.931 1377.066 2888.730

32.940 99.345 1462.278 2820.807

30.388 96.710 1497.326 3000.009

Mean 31.816 96.147 1434.491 2931.902

SD 1.036 2.754 55.458 77.047

%CV 3.3 2.9 3.9 2.6

% Accuracy 97.9 103.1 101.5 103.7

n 6 6 6 6

Page 57




Table 23: Inter-Assay (overall) Precision and Accuracy results for Batches 1, 2 and 3 for D5 Benzalkonium Chloride-C12:



012-005 07-May-19

101.682 266.349 4360.549 8914.661 96.445 282.384 4024.905 8772.668

107.256 295.806 4228.560 8591.449 104.738 282.099 4266.596 8506.816 98.244 284.730 4218.073 8852.234

103.018 291.881 4324.805 8707.004

012-006 08-May-19

101.871 305.837 4767.912 10549.487 109.222 323.039 5175.080 9728.738 107.392 333.091 4565.349 9346.849 112.598 296.905 4770.383 9344.619 101.465 317.450 4978.720 9047.690 107.891 317.323 4744.064 10373.470

014-001 09-May-19

107.384 314.142 4502.678 9847.220 105.020 301.188 4482.941 9588.169 107.999 310.882 4936.788 9426.407 105.381 303.266 4625.074 9438.824 102.276 309.487 4776.627 9129.554 101.342 314.355 4748.600 9851.864

Mean 104.512 302.790 4583.206 9334.318 SD 4.074 16.874 306.551 584.368

%CV 3.9 5.6 6.7 6.3 % Accuracy 97.7 98.6 98.5 100.3

n 18 18 18 18

Page 58




Table 24: Inter-Assay (overall) Precision and Accuracy results for Batches 1, 2 and 3 for D5 Benzalkonium Chloride-C14:


LOQQC LQC MQC HQC

32.493 93.298 1413.612 2827.224 pg/mL pg/mL pg/mL pg/mL

012-005 07-May-19

32.899 84.121 1320.461 2626.054 29.559 88.498 1180.760 2661.169

31.509 91.207 1299.758 2558.828 31.937 88.899 1276.209 2519.444 30.028 86.761 1266.002 2632.925 30.745 89.510 1261.685 2577.971

012-006 08-May-19

31.754 97.794 1409.306 3116.969 32.814 101.591 1555.365 2901.168 32.544 101.902 1374.965 2751.067 34.696 90.645 1417.239 2747.872

31.551 100.417 1496.504 2694.782 30.972 99.123 1410.992 3090.745

014-001 09-May-19

32.870 95.909 1383.456 3031.445 31.997 93.083 1395.472 2904.737

31.842 98.901 1491.348 2945.682 30.859 92.931 1377.066 2888.730 32.940 99.345 1462.278 2820.807 30.388 96.710 1497.326 3000.009

Mean 31.772 94.297 1382.011 2803.911 SD 1.252 5.485 99.447 189.245

%CV 3.9 5.8 7.2 6.7 % Accuracy 97.8 101.1 97.8 99.2

n 18 18 18 18


Run Acceptance was based on acceptability of the calibration curve as described in

Section 5.3 (Acceptance criteria) of this validation report.

Page 59




Experiment Acceptance criteria:

The criteria for acceptable accuracy for both within (intra) and overall (inter) batches; the

mean value should be within ±15% of the nominal value at each level, except for

LOQQC where it should be within ±20%. The criteria for acceptable precision for both

within (intra) and overall (inter) batches; the %CV should be within ±15% at each level,

except for LOQQC where it should be within 20%. Additionally, at least 67% of the

overall QCs and at least 50% at each level should be within ±15% (for LQC, MQC and

HQC) and ±20% (for the LOQQC) of their nominal value.

A minimum of three and maximum of six Precision and Accuracy batches should be

performed in a validation. At least 67% of the overall Precision and Accuracy batches

(CC & QC both) must meet the acceptance criteria.

Conclusion

All the three PAs run and experiment met the acceptance criteria. These three different

PA batches were run on three different days by three different analysts. All these PA

batches were run using a minimum of two different instruments and two different

columns. Additionally, different in-house solutions were used for ruggedness PA batch.


QC Ids Intra batch Inter batch

Precision (%) Accuracy (%) Precision (%) Accuracy (%)

LOQQC 2.5-4.1 95.3-99.8 3.9 97.7

LQC,MQC & HQC 1.8-6.2 91.1-104.6 5.6-6.7 98.5-100.3

Page 60





QC Id’s Intra batch Inter batch

Precision (%) Accuracy (%) Precision (%) Accuracy (%)

LOQQC 3.3-4.1 95.8-99.7 3.9 97.8

LQC,MQC & HQC 2.0-6.4 89.7-105.7 5.8-7.2 97.8-101.1

Thus, precision and accuracy at each level (intraday and inter day) in all the batches met

the acceptance criteria.

Page 61




5.6 Determination of Weighting Factor

Calibration standard accuracy results were calculated for three PA batches with Linear regression using weighting factors as

1/x2, 1/x and with no weighting. The percent Relative Errors (%RE) were calculated for each standard level as the percent

difference between the observed value and nominal value. For each weighting factor, the absolute values of all percent relative

errors are summed for the three calibration curves. The weighting factor that gives the smallest value of the sum of the %RE

was taken as the simplest model that gives the best results and adequately describes the concentration-response relationship for

the D5 Benzalkonium Chloride (C12 and C14 homologs) method. The results are given in Table 25 and Table 26.

Table 25: Sum of %RE for the evaluation of weighting factor with linear regression for D5 Benzalkonium Chloride-C12:

Regression Linear Linear Linear

Weighting None 1/x 1/x2 Batch ID 012-005 012-006 014-001 012-005 012-006 014-001 012-005 012-006 014-001

Standard ID %RE %RE %RE %RE %RE %RE %RE %RE %RE STD A 27.0 100.0 43.3 4.4 3.9 2.7 1.3 8.1 0.8 STD A' 31.0 100.0 44.8 8.5 10.1 4.2 2.9 5.6 0.7 STD B 12.5 62.4 22.1 1.5 4.4 2.3 0.1 3.1 1.3 STD C 0.5 34.1 8.0 4.8 6.6 1.3 4.5 6.8 1.1 STD D 2.5 6.8 2.6 4.8 5.1 1.5 3.5 4.0 0.6 STD E 3.0 2.4 1.6 3.9 1.9 3.1 2.0 0.4 1.8 STD F 4.1 4.8 3.8 4.2 5.3 4.0 2.1 3.5 2.6 STD G 2.3 14.6 2.1 2.6 12.9 1.6 5.0 10.9 0.2 STD H 1.0 11.4 2.8 0.6 13.2 3.5 1.8 14.8 4.9 STD H' 0.7 7.6 2.0 1.1 5.2 1.2 3.6 3.3 0.3 ∑ %RE 561.8 130.4 101.6

Page 62




Table 26: Sum of %RE for the evaluation of weighting factor with linear regression for D5 Benzalkonium Chloride-C14:

Regression Linear Linear Linear

Weighting None 1/x 1/x2 Batch ID 012-005 012-006 014-001 012-005 012-006 014-001 012-005 012-006 014-001

Standard ID %RE %RE %RE %RE %RE %RE %RE %RE %RE STD A 58.0 93.3 23.7 10.0 4.7 0.7 1.4 7.3 0.7 STD A' 59.0 100.0 24.0 11.0 5.0 0.5 2.3 2.3 0.4 STD B 22.6 56.8 12.8 0.8 8.8 0.9 3.4 7.9 0.9 STD C 5.4 30.1 7.6 5.7 7.3 2.0 5.3 7.4 2.0 STD D 1.3 4.7 5.1 6.2 5.1 2.6 4.2 4.4 2.6 STD E 4.2 0.1 0.9 6.1 3.5 0.1 3.2 2.6 0.1 STD F 4.3 3.7 4.5 4.6 4.1 4.6 1.3 3.0 4.6 STD G 0.4 11.6 1.4 0.2 10.3 1.1 3.9 9.1 1.2 STD H 1.1 10.5 2.5 0.2 12.1 2.9 3.5 13.0 2.9 STD H' 1.5 7.5 1.9 2.5 5.5 1.4 6.4 4.3 1.4

∑ %RE 560.5 130.5 113.0

Conclusion

The sum of the % RE was smallest for 1/x2 weighting with linear regression. Hence, it will be used for the D5 Benzalkonium

Chloride (C12 and C14 homologs) method.

Page 63




5.7 Recovery

5.7.1 Absolute Recovery of Analyte

The recovery of D5 Benzalkonium Chloride (C12 and C14 homologs) from human plasma was determined by comparing the

analyte area for extracted analyte samples at three concentrations (low, medium and high) with unextracted (reference)

samples of same concentration levels which represents 100% recovery. The recovery data is given in Table 27 and Table 28.

Table 27: Absolute Recovery data of D5 Benzalkonium Chloride-C12 in human plasma:

Absolute Recovery of Analyte

Batch ID(s) Extraction Date

LQC MQC HQC % Recovery Unextracted (Absolute)

Analyte area

Extracted Analyte area

Unextracted (Absolute)

Analyte area



Analyte area

Extracted Analyte area LQC level MQC

level HQC level

014-002 9-May-19

43869 40472 687224 568982 1298808 1097365 91.0 82.8 84.6

44517 37621 685423 568958 1294772 1185805 84.6 82.8 91.5

44726 36024 685254 562450 1291844 1129994 81.0 81.8 87.1

44804 38395 676211 517983 1298208 1107585 86.3 75.4 85.4

45249 35811 698746 541203 1296459 1105147 80.5 78.7 85.2

43806 33787 690916 538966 1299785 1125722 75.9 78.4 86.8 Mean 44495.2 Mean 687295.7 Mean 1296646.0 Mean 83.2 80.0 86.8

STDEV 5.3 3.0 2.5

% CV 6.4 3.8 2.9

Overall Mean of % Recovery 83.3

Overall SD of % Recovery 4.5

% CV of Overall % Recovery 5.4

Page 64




Table 28: Absolute Recovery data of D5 Benzalkonium Chloride-C14 in human plasma:

Absolute Recovery of Analyte

Batch ID(s) Extraction Date

LQC MQC HQC % Recovery


Analyte area



Analyte area



Analyte area

Extracted Analyte area LQC level MQC

level HQC level

014-002 9-May-19

44351 39914 697048 573063 1337643 1133924 91.3 82.7 85.7

43376 38509 695227 571649 1330944 1139167 88.1 82.5 86.1

43600 36014 688577 556367 1310907 1116151 82.4 80.2 84.3

44296 38566 683083 520865 1310738 1101139 88.2 75.1 83.2

44280 34907 695717 534421 1336090 1070271 79.8 77.1 80.9

42443 33051 700171 549667 1314898 1069662 75.6 79.3 80.8 Mean 43724.3 Mean 693303.8 Mean 1323536.7 Mean 84.2 79.5 83.5

STDEV 6.0 3.0 2.3

% CV 7.1 3.8 2.8

Overall Mean of % Recovery 82.4

Overall SD of % Recovery 4.4

% CV of Overall % Recovery 5.3

Experiment Acceptance criteria

The %CV of Recovery at each QC level should be within 15% (4 out of 6 replicates must be used for statistical calculations)

and recovery should not be more than 115%. The overall %CV should be within 15%.

Page 65




Conclusion

Analyte (s) % Recovery % CV Overall %CV

Overall mean of %Recovery

D5 Benzalkonium Chloride-C12 80.0-86.8 2.9-6.4 5.4 83.3

D5 Benzalkonium Chloride-C14 79.5-84.2 2.8-7.1 5.3 82.4

5.7.2 Absolute Recovery of Internal Standard The recovery of the internal standard (Benzalkonium Chloride-d13) from human plasma

was determined by comparing the internal standard area for extracted internal standard

samples with unextracted samples of same concentration which represents 100%

recovery. The recovery data for internal standard is given in Table 29.

Page 66




Table 29: Absolute Recovery of Benzalkonium Chloride-d13 (Internal Standard) in human plasma:

Absolute Recovery of Internal Standard

Batch Id Extraction Date QC Levels


Internal standard area

Extracted Internal

standard area % Recovery

014-002 09-May-19

LQC

36337 29269 92.5

34467 29647 93.7

36585 29635 93.6

35945 29113 92.0

36056 30161 95.3

35073 30599 96.7

MQC

29935 29750 94.0

29366 31662 100.1

29516 31131 98.4

30027 31336 99.0

31029 29950 94.6

29808 32310 102.1

HQC

29013 29567 93.4

28775 32063 101.3

29870 32813 103.7

29212 32120 101.5

29745 32185 101.7

28847 31382 99.2 Mean 31644.8 Mean 97.4

SD 3.8 %CV 3.9


The %CV of recovery of Internal Standard should be within 15% (4 out of 6 replicates at

each level must be used for statistical calculations) and recovery should not be more than

115%.

The difference between drug and stable labeled internal standard recovery should not be

greater than ±15%.

Page 67




Conclusion

The % CV for Benzalkonium Chloride-d13 was 3.9% and % recovery was 97.4%.

The difference between drug and stable labeled internal standard recovery was -14.1%.

5.8 Stability

Drug stability in a biological matrix is a function of the storage conditions and the

chemical properties of the drug and matrix. Stability must be demonstrated at various

handling and processing steps (bench top as well as freeze and thaw during the initial

validation and long term stability will be added at a later date as an addendum to the

report).

5.8.1 Freeze-Thaw Stability The freeze and thaw stability (minimum 3 cycles) evaluation of D5 Benzalkonium

Chloride (C12 and C14 homologs) in human plasma was determined by analyzing six

replicates of low and high QCs which were frozen at -20°C for at least 12 hours for every

freeze-thaw cycle. The samples were then thawed on bench at room temperature for

approximately 3 hours and refrozen. This procedure (thaw and freeze) was performed for

3 times and the samples were retrieved for fourth freeze thaw cycle. These samples were

analyzed (after 3 hours thawing) along with the freshly spiked calibration curve standards

and quality control samples (in duplicate). The results are given in Table 30 and Table

31.

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Table 30: Stability of D5 Benzalkonium Chloride-C12 in human plasma after 4 freeze-thaw cycles:

Batch ID Extraction Date LQC HQC

307.073 9305.256 pg/mL pg/mL

014-004 10-May-19

295.919 8823.588 301.096 8872.949 312.461 8673.234 300.415 9067.420 303.874 8436.894 309.414 8956.541

Mean 303.863 8805.104 SD 6.122 223.453

%CV 2.0 2.5 % Accuracy 99.0 94.6

n 6 6

Table 31: Stability of D5 Benzalkonium Chloride-C14 in human plasma after 4 freeze-thaw cycles:


LQC HQC 93.298 2827.224 pg/mL pg/mL

014-004 10-May-19

91.110 2690.985 90.108 2737.701 93.725 2616.247 91.362 2778.336 89.129 2572.418 92.511 2674.178

Mean 91.324 2678.311 SD 1.644 75.862

%CV 1.8 2.8 % Accuracy 97.9 94.7

n 6 6

Page 69





Run acceptance will be based on acceptability of the calibration curve as described in

section 5.3 (Acceptance criteria) of this validation report. Also at least four out of the six

freshly prepared QCs and one QC at each level must be within ±15% of the nominal

value.


The mean value of the stability sample at each level should be within ±15% of the

nominal value and the %CV at each level should be within ±15%. Additionally, at least

67% of the overall stability QCs and at least 50% at each level should be within ±15% of

their nominal value.

Conclusion

Run and Experiment met the acceptance criteria. The mean value of the stability sample

at each level was within ±15% from the nominal value and the %CV at each level was

within ±15%. Thus, D5 Benzalkonium Chloride (C12 and C14 homologs) freeze and

thaw stability determined in human plasma met the acceptance criteria. The %Accuracy

and %CV were found to be between:




Thus, D5 Benzalkonium Chloride (C12 and C14 homologs) samples in human plasma are

stable after 4 freeze thaw cycles at room temperature.

5.8.2 Short-Term Stability The bench top stability (short-term stability) of D5 Benzalkonium Chloride (C12 and C14

homologs) in human plasma was determined by analyzing six replicates of low and high

QCs kept on bench at room temperature for a period of approximately 6 hours prior to

extraction. After 6 hours, all the samples were extracted and analyzed along with the

Page 70




freshly prepared and extracted CCs and QCs (in duplicate). Results for bench top stability

are given in Table 32 and Table 33.



LQC HQC 307.073 9305.256 pg/mL pg/mL

014-004 10-May-19

299.186 9026.065 298.591 8820.547 304.280 8488.914 300.506 8878.229 303.552 8840.710 305.053 8901.591

Mean 301.861 8826.009 SD 2.778 180.189

%CV 0.9 2.0 % Accuracy 98.3 94.8

n 6 6




014-004 10-May-19

94.055 2728.136 93.343 2692.965 92.722 2636.838 95.231 2580.990 92.928 2694.232 94.847 2738.316

Mean 93.854 2678.580 SD 1.032 59.569

%CV 1.1 2.2 % Accuracy 100.6 94.7

n 6 6

Page 71








value.






Conclusion

Run and Experiment met the acceptance criteria. The mean value of the stability sample

at each level was within ±15% of the nominal value and the %CV at each level was also

within ±15%. The %Accuracy and %CV were found to be between:




Thus, the D5 Benzalkonium Chloride (C12 and C14 homologs) samples in human plasma

are stable for 6 hours at room temperature conditions.

5.8.3 Autosampler Stability at 4°C

Autosampler stability of the D5 Benzalkonium Chloride (C12 and C14 homologs)

samples were taken from Precision and Accuracy Batch #012-005. Auto sampler stability

of the D5 Benzalkonium Chloride (C12 and C14 homologs) samples were evaluated by

analyzing six replicates of low and high QCs which were stored in autosampler at 4°C

after LC/MS/MS analysis for 99 hours and were analyzed with a freshly spiked and

prepared calibration curve and QCs (in duplicate). The interpolated concentrations of the

Page 72




autosampler stability QCs were compared with their nominal values and are given in

Table 34 and Table 35.

Table 34: Autosampler Stability (99 hours) for D5 Benzalkonium Chloride-C12 in human plasma at 4°C:

Batch ID Stability samples Extraction date

LQC HQC

307.073 9305.256

pg/mL pg/mL

014-005 07-May-19

284.942 9117.720

323.297 9251.222

310.669 8677.183

300.822 9149.960

290.081 9108.846

313.536 9164.106

Mean 303.891 9078.173

SD 14.661 202.870

%CV 4.8 2.2

% Accuracy 99.0 97.6

n 6 6

Table 35: Autosampler Stability (99 hours) for D5 Benzalkonium Chloride-C14 in human plasma at 4°C:

Batch ID Stability samples Extraction date


014-005 07-May-19

91.258 2811.182 102.018 2811.611 99.689 2633.100 92.065 2741.289 93.426 2756.432 96.674 2747.729

Mean 95.855 2750.224 SD 4.353 65.310

%CV 4.5 2.4 % Accuracy 102.7 97.3

n 6 6

Page 73





Run acceptance was based on acceptability of the calibration curve as described in



value.






Conclusion

Run and Experiment met the acceptance criteria. The mean value of the autosampler

stability samples at each level was within ±15% of nominal value and the %CV at each

level was within ±15%. The %Accuracy and %CV were found to be between:




Hence, it has been determined for D5 Benzalkonium Chloride (C12 and C14 homologs)

samples total batch run time on LC/MS/MS should not exceed the autosampler stability

for 99 hours from the end time of the batch processing.

5.8.4 Whole Blood Stability

D5 Benzalkonium Chloride (C12 and C14 homologs) stability in human whole blood was

determined by spiking six replicates of low (LQC) and high QC (HQC) sample in whole

Page 74




blood and kept on bench at room temperature conditions for a period of 3 hours. After 3

hours, stability QC samples were analyzed with six freshly prepared whole blood low

QCs (LQC level) and high QCs (HQC level). The results are given in Table 36 and Table

37.

Table 36: Human Whole Blood Stability (3 hours) for D5 Benzalkonium Chloride-C12 at room temperature:


Area Ratio of Stability LQC

Area Ratio of fresh LQC

Area Ratio of Stability HQC

Area Ratio of fresh HQC

014-003 10-May-19

1.090 1.203 34.457 35.581 1.124 1.163 30.049 32.220 1.098 1.205 31.964 35.289 1.127 1.065 34.012 33.808 1.142 1.116 35.098 35.179 1.085 1.208 33.164 33.107

Mean area ratio 1.111 1.160 33.124 34.197 SD 0.023 0.059 1.858 1.365

%CV 2.1 5.1 5.6 4.0 % difference: -4.2 -3.1

Table 37: Human Whole Blood Stability (3 hours) for D5 Benzalkonium Chloride-C14 at room temperature:

Batch ID Extraction Date Area Ratio of Stability LQC

Area Ratio of fresh LQC

Area Ratio of Stability HQC

Area Ratio of fresh HQC

014-003 10-May-19

0.821 0.901 26.168 26.718 0.865 0.888 23.014 24.941 0.797 0.890 24.919 26.912 0.843 0.837 26.139 26.085 0.880 0.871 26.909 25.808 0.787 0.900 26.078 25.240

Mean area ratio 0.832 0.881 25.538 25.951 SD 0.037 0.024 1.392 0.784

%CV 4.4 2.7 5.5 3.0 % difference: -5.6 -1.6

Page 75





The % difference of the stability LQC and HQC samples should be within ± 15%

compared to freshly prepared LQC and HQC samples and the %CV of LQC and HQC

samples response should be within ±15%.

Conclusion

The %CV of LQC and HQC samples were within ±15%. The %difference of the stability

LQC and HQC samples compared to freshly prepared LQC and HQC samples were

within ±15%. The %CV and %difference were found to be:


QC Level %CV %difference

LQC 2.1-5.1 -4.2

HQC 4.0-5.6 -3.1

For D5 Benzalkonium Chloride-C 14:

QC Level %CV %difference

LQC 2.7-4.4 -5.6

HQC 3.0-5.5 -1.6

Consequently, D5 Benzalkonium Chloride (C12 and C14 homologs) samples in human

whole blood are considered stable for a period of 3 hours, when kept at room temperature

conditions.

5.8.5 Stock Solution Stability

5.8.5.1 Stock Solution Stability for D5 Benzalkonium Chloride (C12 and C14

homologs) at Room Temperature

Stock solution stability for D5 Benzalkonium Chloride (C12 and C14 homologs) at Room

Temperature was performed by preparing the stock solutions. Stability stocks of LQC

and HQC were split into three aliquots which were left at room temperature for a period

Page 76




of 22 hours. After 22 hours, two replicates of LQC and HQC from each aliquot in human

plasma were prepared and analyzed against freshly prepared and extracted calibration

curve and QCs (in duplicate). These results are given in Table 38 and Table 39.

Table 38: Stock solution stability (22 hours) for D5 Benzalkonium Chloride-C12 at Room Temperature:

Batch ID Extraction Date LQC HQC

307.073 9305.256 pg/mL pg/mL

012-003 04-May-19

292.048 8963.641 296.022 9020.756 274.419 8340.567 283.749 8902.787 276.501 8602.828 293.863 9001.241

Mean 286.100 8805.303 SD 9.254 274.358

%CV 3.2 3.1 % Accuracy 93.2 94.6

n 6 6

Table 39: Stock solution stability (22 hours) for D5 Benzalkonium Chloride-C14 at Room Temperature:



012-003 04-May-19

88.025 2788.236 87.639 2690.358 84.102 2518.909 87.425 2732.328 82.848 2617.002 90.004 2767.734

Mean 86.674 2685.761 SD 2.671 101.959

%CV 3.1 3.8 % Accuracy 92.9 95.0

n 6 6

Page 77








value.


The mean value of the stability sample at each level should be within ±15% of nominal

value and the %CV at each level should be within ±15%. Additionally, at least 67% of

the overall stored QCs and at least 50% at each level should be within ±15% of their

nominal value.

Conclusion

Run and Experiment met the acceptance criteria. The mean value of the stability samples

at each level was within ±15% of nominal value and the %CV at each level was within

±15%. The %Accuracy and %CV were found to be between:




Thus, stock solutions (D5 Benzalkonium Chloride (C12 and C14 homologs)) are

considered stable for a period of 22 hours when stored at room temperature conditions.

5.9 Maximum batch size

The maximum batch size was determined by analyzing a batch containing a CCs and QC

samples interspersed among reference blank samples. A total of 215 samples were

analyzed in the batch and the results are given in Table 40 and Table 41.

Page 78




Table 40: QC results from Maximum batch size run for D5 Benzalkonium Chloride-C12:

Batch ID Extraction Date LQC MQC HQC

307.073 4652.628 9305.256 pg/mL pg/mL pg/mL

012-008 07-May-19

270.799 4477.141 9317.205 301.349 4401.529 9683.013 297.844 4575.840 9583.906 325.087 4758.924 9256.926 312.746 4768.729 10052.299 322.798 4404.016 8909.231

Mean 305.104 4564.363 9467.097 SD 20.072 167.084 395.115

%CV 6.6 3.7 4.2 % Accuracy 99.4 98.1 101.7

n 6 6 6

Table 41: QC results from Maximum batch size run D5 Benzalkonium Chloride-C14:

Batch ID Extraction Date LQC MQC HQC

93.298 1413.612 2827.224 pg/mL pg/mL pg/mL

012-008 07-May-19

86.787 1381.990 2794.353 94.227 1353.917 2962.368 95.177 1417.105 2855.528 98.879 1451.142 2783.875 98.105 1480.646 3131.974

106.783 1368.216 2702.849 Mean 96.660 1408.836 2871.825

SD 6.559 49.803 153.903 %CV 6.8 3.5 5.4

% Accuracy 103.6 99.7 101.6 n 6 6 6

Run Acceptance criteria

Run acceptance was based on acceptability of the calibration curve as described in

section 5.3 (Acceptance criteria) of this validation report. At least 75% of the calibration

Page 79




standards should be within ±15% of nominal (±20% for STD A). Additionally, at least

67% of the overall QCs and at least 50% at each level should be within ±15% of their

nominal value and the %CV at each level should be within 15%.


At least 67% of the reference blank samples must meet the below criteria;


the average response of accepted LLOQ (STD-A and STD-A’). Interference in the blank,

if any, at the retention time of internal standard (IS) must not exceed 5% of the average

response of IS from accepted standards and quality control samples.

Conclusion

The %CV and %Accuracy of all the QCs in the maximum batch size experiment were

found to be between:

Analyte (s) %CV %Accuracy



The %CV of all the QCs in the maximum batch size experiment was less than 15% and

the %Accuracy was within ±15%. No significant interference was observed in reference

blank samples. This indicates that there was no drift in the instrument, analyte and IS

response. This experiment was performed to determine the drift (instrument and the

analytical method) during the run. However, the number of samples in a batch had no

material impact on the maximum batch size, since the QCs was intermittently spread

throughout the batch to monitor the drift.

5.10 Dilution Integrity

Dilution integrity was validated by preparing at least six replicates from OCC-QC (these

samples were diluted two times with plasma before processing) in blank biological matrix

Page 80




using a dilution factor of 1 in 2 (2T), such that the final concentration falls within the

calibration curve range. All diluted QCs were processed with freshly prepared calibration

curve and quality control samples in duplicate. The data is given in Table 42 and Table

43.

Table 42: Dilution integrity data for D5 Benzalkonium Chloride-C12 in human plasma:


OCCQC (dilution 2-fold)

18610.512 pg/mL

014-005 11-May-19

18282.707 19472.208 18608.340 17909.319 18426.377 19004.052

Mean 18617.167 SD 553.203

%CV 3.0 % Accuracy 100.0

n 6

Page 81




Table 43: Dilution integrity data for D5 Benzalkonium Chloride-C14 in human plasma:


OCCQC (dilution 2-fold)

5654.449 pg/mL

014-005 11-May-19

5802.229 5984.109 5827.021 5595.742 5658.436 5835.595

Mean 5783.855 SD 138.633

%CV 2.4 % Accuracy 102.3

n 6





value.

Experiment Acceptance criteria for dilution samples:

The mean value of diluted samples should be within ±15% of the nominal value and the

%CV should be within 15%. Additionally, 67% of the stability QCs should be within

±15% of their nominal value.

Conclusion

Run and Experiment met the acceptance criteria. The mean value of the diluted samples

at each level was within ±15% and %CV was within ±15%. The %CV and %Accuracy

was found to be between:

Page 82




Diluted samples for Analyte (s) %CV %Accuracy

Dilution Integrity D5 Benzalkonium Chloride-C12 100.0 3.0


Thus, over the calibration curve samples can be diluted up to 2 times, if necessary, to fit

within the calibration curve.

6.0 MEMO TO FILES

A: Refer Memo to file-01; Failure of Spiking solutions check experiment

Detailed Explanation and Reason for Failure:

The Spiking solutions check experiment was performed on 3rd May, 2019 as Batch Id:

012-002 (Run #02) and injected using SAI-MS-012. Upon review of the data, the spiking

solutions check did not meet the acceptance criteria for both D5 Benzalkonium Chloride-

C12 and D5 Benzalkonium Chloride-C14. In D5 Benzalkonium Chloride-C12, the

accuracies of REF STD H and REF STD H’ were found to be 89.3 and 89.8%,

respectively (Acceptance Limit: 92.0-108.0%). In D5 Benzalkonium Chloride-C14, the

accuracies of REF STD G and REF STD G’ were found to be 110.6% and 108.3 %,

respectively (Acceptance Limit: 92.0-108.0%). Hence, spike check experiment was

rejected for both D5Benzalkonium Chloride-C12 and D5 Benzalkonium Chloride-C14.

Evaluation of the data revealed a very high analyte peak area (~133000) for the lowest

standard (STD A). Therefore, the low accuracy at higher concentration (STD H and STD

H’) in D5 Benzalkonium Chloride-C12 was might be due to system saturation issue.

Hence, it was decided to re-inject the batch after reducing the analyte response by

optimizing collision energy.

Corrective Action:

The spiking solutions check experiment was re-injected as Batch Id: 012-002A (Run #02)

on 4th May, 2019 after optimizing analyte response.

Page 83




Impact Statement:

Re-injected spiking solution check experiment met the acceptance criteria. The accepted

stock solutions were used further in the validation. Therefore, the initial failure of the

spiking solutions check experiment due to system saturation did not affect the integrity of

data.

7.0 DEVIATIONS

There are no deviations in this validation.

8.0 RE-INTEGRATION

There is no chromatographic peak re-integration in this validation.

9.0 ARCHIVAL

All raw data, analyst files, chromatograms along with this report will be archived at

Sannova Analytical Inc.

10.0 REVISION HISTORY

None.

Page 84




11.0 APPENDIX

11.1 Analytical Validation Protocol #SAI-VP19499

Page 85

Note to File-01

Project Name: D5 Benzalkonium Chloride Validation No.: SAI-VP19499

In the Validation protocol (SAI-VP19499) Section 4.2 Spike Solution Check experiment

acceptance criteria mentioned as "Percent Accuracy of each spiking calibration standards and

quality control samples solutions must be within ±12% and 75% of the calibration standards must

meet the above criteria". This is a typo error. Acceptance criteria: Percent Accuracy of each

spiking calibration standards and quality control samples solutions must be within ±8% and 75%

of the calibration standards. This will be followed in the validation and the same will be updated

in final method after validation.

~S::~; .t<l\'I Reviewed by/Date:~

, n ~ Approved by/Date:-~-·___,~-~-· -'---'~~-=-~

Prepared by/Date:

l S"' M°"'i-~\ \

Page 1

Page 86

•

•

•

ORIGINAL Validation Protocol No. SAI-VP19499

VALIDATION PROTOCOL

TITLE: VALIDATION OF LC/MS/MS BIOANAL YTICAL METHOD FOR THE DETERMINATION OF D5 BENZALKONIUM CHLORIDE (C12 AND C14 HOMOLOGS) IN HUMAN PLASMA

Prepared by:

Reviewed by:

Reviewed by:

Approved by:

VALIDATION PROTOCOL NO.: SAI-VP19499

Name Department

f,N_ ec)v00.,rq_ ~ 1bfo AvoJ-cf1((ilf

tJ ~at' 16~~~ , V--l \.\\M.Mf Jl ~~t1y JM'\iYMVe

r, '\') ~"' « ~ B ·,all V\L\ \ffet_cA

~t.e<isw 6-c te,\ l'"'

(~San~ova ~/Analytical Inc. Quality at its Finest

SANNOVA ANALYTICAL INC

155 PIERCE STREET

SOMERSET

NJ-08873, USA .

Page 1 of 19

Signature & Date

tic\~

Page 87

Validation Protocol No. SAI-VP19499

Page 2 of 19

TABLE OF CONTENTS CONTENT PAGE#

VALIDATION PROTOCOL.......................................................................................................... 1

TABLE OF CONTENTS ................................................................................................................ 2

1.0 INTRODUCTION:........................................................................................................... 4

2.0 STUDY PERSONNEL: ................................................................................................... 4

3.0 ANALYTICAL PROCEDURE: ...................................................................................... 4

4.0 BIOANALYTICAL METHOD VALIDATION: ............................................................ 4

4.1 STOCK SOLUTION CHECK: .................................................................................... 5

4.2 SPIKING SOLUTION CHECK: .................................................................................. 5

4.3 STOCK STABILITY AT ROOM TEMPERATURE FOR 6 HRS: ............................ 5

4.4 SELECTIVITY & INTERFERENCE CHECK: ....................................................... 6

4.4.1 Selectivity ................................................................................................................. 6

4.4.2 Interference Check: ................................................................................................... 7

4.4.3 Interference Check in the presence of concomitant medication: .............................. 7

4.5 CARRY OVER: ........................................................................................................... 8

4.6 MATRIX EFFECT: ...................................................................................................... 9

4.7 TEST BULK SPIKING: ............................................................................................... 9

4.8 CALIBRATION STANDARD CURVE (Relationship Between Response And

Concentration): ..................................................................................................................... 10

4.9 SENSITIVITY OR LIMIT OF QUANTITATION (LOQ): ....................................... 11

4.10 PRECISION AND ACCURACY: .......................................................................... 11

4.11 RECOVERY OF DRUG AND INTERNAL STANDARD: .................................. 12

4.11.1 Absolute Recovery of D5 Benzalkonium Chloride: ............................................... 12

4.11.2 Absolute Recovery of Internal Standard (ISTD): ................................................... 13

4.12 STABILITY: ........................................................................................................... 13

4.12.1 Freeze-Thaw Stability: ............................................................................................ 13

4.12.2 Short-Term Stability (Bench Top Stability): .......................................................... 14

4.12.3 Autosampler Stability: ............................................................................................ 15

4.12.4 Stock Solution Stability: ......................................................................................... 15

4.12.5 Internal Standard (IS) Stock Solution Stability: ..................................................... 16

Page 88


Page 3 of 19

4.12.6 Whole Blood Stability: ........................................................................................... 16

4.12.7 Long-Term Stability: .............................................................................................. 17

4.13 MAXIMUM BATCH SIZE: ................................................................................... 17

4.14 DILUTION INTEGRITY: ...................................................................................... 18

5.0 ARCHIVING OF RECORDS: ....................................................................................... 19

6.0 DATA COLLECTION AND COMPUTER SYSTEM: ................................................ 19

7.0 REGULATORY REFERENCES: ................................................................................. 19

8.0 QUALITY ASSURANCE MONITORING: ................................................................. 19

9.0 CHANGES IN THE VALIDATION PROTOCOL: ...................................................... 19

10.0 ATTACHMENTS ...................................................................................................... 19

10.1 ATTACHMENTS 1: Analytical Method SAI-BM19499 (DRAFT). ..................... 19

Page 89


Page 4 of 19

1.0 INTRODUCTION:

Title Validation of a LC/MS/MS Bioanalytical Method for the

analysis of D5 Benzalkonium Chloride (C12 and C14

homologs) in Human Plasma.

Testing Facility Sannova Analytical Inc.

155 Pierce Street

Somerset, NJ-08873

Purpose The purpose of this validation is to establish and to validate

LC/MS/MS based bioanalytical method for the

determination of D5 Benzalkonium Chloride (C12 and C14

homologs) in Human Plasma i.e. SAI-BM19499 (Draft).

2.0 STUDY PERSONNEL:

Analytical Chemist Lead Nageswara Rao, Ph.D.

Sr. Lab Manager Pinakin Patel, B.Pharm

Director Malleswar Kollu, M.Sc.

Additional personnel, if any, will be documented in the study file.

3.0 ANALYTICAL PROCEDURE:

A draft Bio-analytical method describing analytical procedure in detail has been attached

for complete details regarding analytical procedure.

4.0 BIOANALYTICAL METHOD VALIDATION:

The bioanalytical method used for the determination of D5 Benzalkonium Chloride (C12

and C14 homologs) in Human Plasma using LC/MS/MS has been developed at Sannova

Analytical Inc. This method will be validated as outlined in this document but not limited

to these experiments. All the experiments performed under this validation must be

documented in Validation Batch Summary Form.

Page 90


Page 5 of 19

4.1 STOCK SOLUTION CHECK:

Prepare two stock solutions for D5 Benzalkonium Chloride (C12 and C14 homologs).

Prepare six reference solutions from both stocks and analyze as per Bio-analytical

method.


Stock check results are based on the comparison of the mean response of the two stock

solutions. The %CV of each stock response should be within 8% and % difference

between two stock solutions should not be more than ±5%.

4.2 SPIKING SOLUTION CHECK:

D5 Benzalkonium Chloride spiking solutions are used to spike Human Plasma calibration

standards and quality control samples. Prepare a series of spiking solutions as per the bio-

analytical method and check the accuracy before spiking into biological matrix. Use

separate stock solutions for preparing calibration standards and quality control samples.




calibration curve.

Any interference arising from blanks at the retention time of internal standard should not

be more than 5% of the average response of IS from all accepted standards and quality

control samples.

Percent Accuracy of each spiking calibration standards and quality control samples

solutions must be within ±12% and 75% of the calibration standards must meet the above

criteria.

4.3 STOCK STABILITY AT ROOM TEMPERATURE FOR 6 HRS:

Prepare stock solutions of analyte and split stability stock of each LQC and HQC in three

aliquots and store at specified conditions (at room temperature). After six hours, prepare

two replicates of LQC and HQC from each aliquot in Human Plasma and analyze

Page 91


Page 6 of 19

against freshly prepared and extracted calibration curve. Also run freshly prepared QCs

in duplicate.



section 4.8 of this validation protocol. Also at least four out of the six freshly prepared

QCs and one QC at each level must be within ±15% of the nominal value.



value and the %CV at each level should be within 15%. Additionally, at least 67% of the

overall stored QCs and at least 50% at each level should be within ±15% of their nominal

value.

4.4 SELECTIVITY & INTERFERENCE CHECK:

4.4.1 Selectivity

Selectivity is the ability of an analytical method to differentiate and quantify the analyte

in the presence of other components in the sample.

A minimum of nine independent sources of blank biological matrix should be checked

for the presence of any interfering compound that affects the selectivity of the method.

Prepare three different solutions containing LLOQ+ISTD, ISTD and Blank in each

matrix. Selectivity should be ensured at the lower limit of quantitation (LLOQ).

All the above samples will be analyzed against freshly spiked and prepared calibration

curve. Also run freshly prepared QCs in duplicate.





Page 92


Page 7 of 19




calibration curve.

Any interference arising from blanks at the retention time of internal standard should not

be more than 5% of the average response of IS from all accepted standards and quality

control samples.

Percent Accuracy of LLOQ QC of each matrix must be within ±20%.

4.4.2 Interference Check:

Prepare two samples of extracted LLOQ+IS and inject.

Prepare two samples of extracted solution of analyte at approx. highest standard

concentration without IS (Standard H without IS) and inject to observe any possible

interference at the retention time of internal standard.

Prepare two samples of extracted solution of internal standard at approx. the

concentration equivalent to the internal standard concentration in the samples without

analyte (Blank+IS) and inject to observe any possible interference at the retention time of

the analyte. Also, run blank samples in duplicate.


Any interference at the retention time of analyte should not be more than 20% of the



the extracted LLOQ solutions. At least one of the each Standard H without IS, Blank and

Blank+IS should meet the acceptance criteria.

4.4.3 Interference Check in the presence of concomitant medication:

Selectivity demonstration of an analyte in the presence of concomitant medications

(Acetaminophen, Ibuprofen, Caffeine, Chlorpheniramine maleate, Naproxen and

Pseudoephedrine) or of specific metabolites will be performed by analyzing Blank

Page 93


Page 8 of 19

(including concomitant medication), Blank with internal standard (including concomitant

medication) and LLOQ samples (including concomitant medication) in duplicate and

inject to observe any possible interference at the retention time of the analyte and internal

standard. For each of Blank, Blank+IS and LLOQ samples, spike 25µL/mL of over the

counter multicomponent mixture-6 solution (Approximate concentration 2.5 µg/mL in

plasma) in pooled matrix/screened matrix before extraction.

All the above samples will be analyzed against freshly spiked and prepared calibration


Over The Counter Multi Component Mixture-6 Composition Drug Approximate Concentration Molecular weight Acetaminophen 100.1+ 0.5µg/mL 151.16 g/mole Ibuprofen 100.1+ 0.5µg/mL 206.29 g/mole Caffeine 99.93+ 0.46µg/mL 194.19 g/mole Chlorpheniramine Maleate 100.1+ 0.5µg/mL (as free base) 390.86 g/mole Naproxen 99.93+ 0.46µg/mL 230.26 g/mole R,R(-)-Pseudoephedrine 100.0+ 0.5µg/mL 165.24 g/mole


Acceptance of the run will be based on acceptability of the calibration curve only as

described in Section 4.8 of this validation protocol. Also at least four out of the six


value.





the extracted LLOQ solutions.

Percent Accuracy of LLOQ QC must be within ±20%.

At least one of the each Blank, Blank+IS and LLOQ should meet the acceptance criteria.

4.5 CARRY OVER:

Carry over will be assessed by injecting extracted highest standard followed by extracted

blank to quantitate any carry over. Extracted blank, Extracted low standard (LLOQ)

and highest standard (ULOQ) followed by extracted blank samples will be injected in

duplicate to verify the carry over.

Page 94


Page 9 of 19


Response from the extracted blank should not be more than 20% compared to average of

STD A (low standard). Response from the internal standard should not be more than 5%

of the average internal standard response of the STD A.

4.6 MATRIX EFFECT:

Matrix effect is defined as inconsistent response for the analyte(s) of interest, internal

standards(s) or both (all) when the same concentration of compounds are extracted from

different sources of biological blank matrices.

Six independent sources of matrices screened for selectivity will be checked for the

matrix effect. Prepare six replicates of LQC, MQC and HQC for each matrix containing

all the analyte(s) of interest and internal standard; extract these samples as per method of

analysis to check Matrix Effect for Method Consistency of the method.

Prepare six replicates of reference LQC, MQC and HQC containing all the analyte(s) of

interest and internal standard and inject.


At each level, the %CV of the peak area ratio of the analyte to the internal standard from

each matrix must be within 15% (for each lot at least 4 out of 6 replicates must be

used for statistical calculations) and %CV of the mean peak area ratios from 5 out of 6

matrix lots must be within 15% to assume that no major matrix effect is present.

4.7 TEST BULK SPIKING:

After bulk spiking of the Quality Control (QC’s) samples, check the accuracy of the bulk

spiked QCs to further proceed with validation experiments.

Analyze three (3) replicates of all bulk spiked QC’s against freshly prepared calibration


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described in Section 4.8 of this validation protocol. Also at least four out of the six


value.


Percent Accuracy of each QC must be within ±10%. At least two of three QCs at each

level must be within ±10% of the nominal value.

NOTE: Values falling outside these limits will not be used to calculate the regression

analysis. However, if only CC didn’t meet acceptance criteria in the two first

continuous runs, it is recommended to re-prepare the standards

4.8 CALIBRATION STANDARD CURVE (Relationship Between Response And

Concentration):

A calibration curve is the relationship between instrument response and known

concentration of the analyte(s) in the solution.

A calibration curve should consist of blank sample (matrix sample processed without

internal standard), zero standard sample (matrix sample processed with internal standard)

and non-zero standard samples covering the expected range, including LLOQ. The

Calibration curve standards shall be prepared in fresh for the Precision and

accuracy experiments. The simplest model that adequately describes the concentration-

response relationship (linear regression) should be used.

During Validation, at least six extracted calibration curves must be performed to verify

the method ruggedness.




Interference in the blank, if any, at the retention time of internal standard (IS) must not

exceed 5% of the average response of IS from accepted standards and quality control

samples.

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accept the batch.





LOQ should fall within 15%, except for LLOQ where it should be ±20% of the nominal

value. Values falling outside these limits should not be used to calculate regression

analysis.

4.9 SENSITIVITY OR LIMIT OF QUANTITATION (LOQ):

The lowest standard on the calibration curve is accepted as the limit of quantitation.

Acceptance criteria:

The response peak of D5 Benzalkonium Chloride must be identifiable, discrete and

reproducible with a precision of ≤ 20% and an accuracy of 80-120%.

4.10 PRECISION AND ACCURACY:

Accuracy and Precision are determined by replicate analysis of samples containing

known amounts of the analyte, i.e. QC samples. Intra assay precision and accuracy

evaluations will be performed using freshly prepared and extracted calibration curve

standards covering the entire calibration range, plus six (6) replicates of QC samples at

each concentration level of LOQ (LOQQC), Low (LQC), Medium (MQC) and High

(HQC) QC samples. For Inter-assay precision and accuracy evaluations, the procedure

for intra assay will be repeated a minimum of two (2) more times, giving a total of three

(3) batches (in 3 different days by 3 different analysts using a minimum of 2 different

columns and 2 different instruments) for determination of the inter-assay statistics. A

minimum of three and maximum of six Precision and Accuracy batches shall be

performed.

Note: A minimum of one PA batch for ruggedness should use different lot of Mobile

Phase and Diluent.

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described in Section 4.8 of this validation protocol.


The criteria for acceptable accuracy for both within (intra) and overall (inter) batches; the

mean value should be within ±15% of the nominal value at each level, except for the

LOQQC where it should be within ±20%.

The criteria for acceptable precision for both within (intra) and overall (inter) batches; the

%CV should be within 15% at each level, except for the LOQQC where it should be

within 20%. Additionally, at least 67% of the overall QCs and at least 50% at each level

should be within ±15% (for LQC, MQC and HQC) and ±20% (for the LOQQC) of their

nominal value.

If the calibration curve is acceptable, the QC data will be presented in the validation

report. The data will be used for the calculation of between run precision and accuracy.

At least 67% of the overall Precision and Accuracy batches (CC & QC both) must meet

the acceptance criteria. If these criteria are not met, investigate and resolve the problem.

4.11 RECOVERY OF DRUG AND INTERNAL STANDARD:

Note: If Recovery is calculated from the Matrix effect samples, the Recovery needs to be

calculated from the first accepted Matrix effect lot.

4.11.1 Absolute Recovery of D5 Benzalkonium Chloride:

The recovery of D5 Benzalkonium Chloride from human plasma will be determined by

comparing the analyte area for extracted analyte samples at three concentrations (low,

medium, and high) with un-extracted standards of same concentration levels that

represent 100% recovery.


The %CV of Recovery at each QC level should be within 15% (4 out of 6 replicates

must be used for statistical calculations) and recovery should not be more than 115%.

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The overall %CV should be within 15%. If an absolute difference of greater than 15%

between LQC, MQC and HQC is observed, an investigation will be performed to

determine the source of difference.

4.11.2 Absolute Recovery of Internal Standard (ISTD):

The recovery of internal standard from human plasma will be determined by comparing

the internal standard area for extracted internal standard samples with un-extracted

standards of same concentration that represent 100% recovery.


The %CV of Recovery of Internal standard should be within 15% (4 out of 6 replicates

at each level must be used for statistical calculations) and recovery should not be more

than 115%.

If a difference of greater than ±15% in drug and stable labeled internal standard

recovery is observed, an investigation will be performed to determine the source of

the difference.

4.12 STABILITY:

Drug stability in a biological matrix is a function of the storage conditions and the

chemical properties of the drug and matrix. For D5 Benzalkonium Chloride, the

following stability studies will be undertaken.

4.12.1 Freeze-Thaw Stability:

Analyte stability should be determined after a minimum of three freeze and thaw cycles

at concentrations corresponding to low and high QC samples in the matrix. Freeze all

aliquots at appropriate temperature for at least 12 hours.

Remove at least six low and high frozen QCs and allow them to thaw on bench at room

temperature for a minimum of 3 hours and maximum of 4 hours. Freeze all aliquots at the

appropriate temperature for at least 12 hours.

Repeat the above step for two more times (minimum three cycles) using the same

aliquots previously thawed and refrozen.

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After completion of freeze and thaw cycles, analyze all twelve aliquots of the frozen-

thaw QCs with freshly prepared and extracted calibration curve. Also run freshly

prepared QCs in duplicate.









value.

4.12.2 Short-Term Stability (Bench Top Stability):

Six aliquots of each of the low and high QCs samples should be kept on bench at room

temperature for a minimum of 6 hours (based on the expected duration that samples will

be maintained at room temperature in the intended study) and analyzed with freshly

prepared and extracted calibration curve. Also run freshly prepared QCs in duplicate.







nominal value and the %CV at each level should be within 15%. Additionally, at least



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4.12.3 Autosampler Stability:

The stability of processed samples, the resident time in the auto sampler should be

determined. The aim of the experiment is to demonstrate the samples are stable over the

time interval required to analyze a full study batch run (batch time). For auto sampler

stability, six replicates of extracted LQC and HQC after analysis will be stored in the auto

sampler or refrigerator for the maximum anticipated period of time to run a batch. Then

the stored QC samples will be analyzed with freshly spiked and prepared calibration








nominal value and the %CV at each level should be within 15%. Additionally, at least



4.12.4 Stock Solution Stability:

Split stability stock of each LQC and HQC in three aliquots and store at specified

conditions (in refrigerator). After a minimum of 7 days, prepare two replicates of LQC

and HQC from each aliquot in Human Plasma and analyze against freshly prepared and

extracted calibration curve. Also run freshly prepared QCs in duplicate.





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value.

Note: The long term stability will be performed at a later date as an Addendum.

4.12.5 Internal Standard (IS) Stock Solution Stability:

If the stability of D5 Benzalkonium Chloride stock solution is established, it indicates

Benzalkonium Chloride-d13 stock solution is stable. However, absence of analyte in the

stored Benzalkonium Chloride-d13 (Isotope exchange) stock solution needs to be

evaluated. Isotope exchange reaction will be evaluated by preparing reference IS samples

from the stored Benzalkonium Chloride-d13 stock solution (in duplicate) and compared

with reference LLOQ+IS samples (in duplicate).



average response of the Reference LLOQs. At least one of the Reference IS should meet


Note: The long term internal standard stock stability will be performed at a later

date as an Addendum.

4.12.6 Whole Blood Stability:

Analyte stability in whole blood should be determined by spiking six replicates of low

and high QCs (LQC and HQC level) sample in whole blood and leave on bench at room

temperature for minimum of 3 hours. After minimum hours, analyze stability QCs with

six replicates of freshly prepared low and high QCs in whole blood using extraction

procedure.

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The % difference of the stability LQC and HQC samples should be within ±15%

compared to freshly prepared LQC and HQC samples and the %CV of each LQC and

HQC samples response should be within 15%.

i.e. [(Mean area ratio of stability LQC and HQC samples / Mean area ratio of freshly

prepared LQC and HQC samples *100)-100].

4.12.7 Long-Term Stability:

Stability of D5 Benzalkonium Chloride in Human Plasma will be determined for a

minimum of 14 days. Additional stability may be undertaken outside of this validation

protocol. Evaluation of long-term stability will be performed by analysis of six aliquots

of the low and high QCs samples that have been stored at a temperature of -20°C for the

required period of time. The QC samples will be analyzed with freshly prepared and

extracted calibration curve. Also run freshly prepared QCs in duplicate.









value.

Note: The long term stability will be performed at a later date as an Addendum.

4.13 MAXIMUM BATCH SIZE:

The Maximum batch size will be determined by running a minimum of 200 samples

including reference blank samples with QCs interspaced throughout the batch and

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Page 18 of 19

analyzed with a calibration curve. This experiment is used to determine the drift in the

instrument response when large numbers of samples are analyzed.



section 4.8 of this validation protocol. Additionally, at least 67% of the overall QCs and

at least 50% at each level should be within ±15% of their nominal value and the %CV at

each level should be within 15%.


At least 67% of the reference blank samples must meet the below criteria;


the average response of accepted LLOQ (STD-A and STD-A’). Interference in the blank,

if any, at the retention time of internal standard (IS) must not exceed 5% of the average

response of IS from accepted standards and quality control samples.

4.14 DILUTION INTEGRITY:

Dilution accuracy and precision must be validated prior to diluting any samples. Prepare

a QC sample over calibration curve (OCC-QC) in biological matrix at two times the

concentration of the highest quality control (HQC) sample. Make at least six replicates

from OCC-QCs (these samples will be diluted two times with blank plasma before

processing) in blank biological matrix using dilution factor 1 in 2 (2T) such that the final

concentration falls within the calibration curve range. Process all diluted QCs with a

freshly prepared calibration curve. Also run freshly prepared QC’s in duplicate for

freshly prepared calibration curve.





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The mean value of diluted samples should be within ±15% of the nominal value and the

%CV should be within 15%. Additionally, 67% of the stability QCs should be within

±15% of their nominal value.

5.0 ARCHIVING OF RECORDS:

All data documenting experimental details, validation procedures and observations will

be recorded and maintained as raw data in appropriate laboratory binders. After

completion of the validation, all reports, raw data (including electronic records),

validation protocols and amendments (if any) and a copy of the final report will be

maintained in the archives of Sannova Analytical Inc.

6.0 DATA COLLECTION AND COMPUTER SYSTEM:

Computer application programs used to acquire and derive data by Analyst software.

7.0 REGULATORY REFERENCES:

This validation design is based on the United States Food and Drug Administration’s

Guidance for Industry: Bio-analytical Method Validation.

8.0 QUALITY ASSURANCE MONITORING:

The Sannova Analytical Inc., Quality Assurance Unit will audit raw data and final report.

9.0 CHANGES IN THE VALIDATION PROTOCOL:

Change in this validation protocol may be made as the validation progresses.

Amendments to the validation protocol must be approved by the Lab Manager or above.

10.0 ATTACHMENTS

10.1 ATTACHMENTS 1: Analytical Method SAI-BM19499 (DRAFT).

Page 105

D5 Benzalkonium Chloride Method (Draft) was successfully validated. Hence, this method was

finalized as SAI-BM19499-00 provided in Appendix 11.3.

Page 106




11.2 Bioanalytical Method Validation SOP SAI-LP101

Page 107

.,

Title: Bioanalytical Method Validation

SOP #SAI-LP101 Revision: 12 Effective Date: 1 3 AUG 2018 1 of ·32

1.0 PURPOSE:

The purpose of this procedure is to describe a general approach for validation of .

bioanalytical methods based on FDA guidelines.

2.0 SCOPE:

This procedure applies to all bioanalytical method validations performed at Sannova

Analytical Inc.

3.0 RESPONSIBILITIES:

3.1 All staff members in the Bioana_lytical department are responsible to understand and

follow this SOP.

3.2 Management is responsible to ensure compliance.

4.0 PROCEDURE:

4.1 A validation protocol along with a draft bioanalytical method will be written prior to the

start of the validation. In this case, the validation protocol overwrites this SOP in regards

to all aspects of the validation.

4.2 The key criteria for evaluation of the method reliability and performance are: Selectivity,

Calibration range, Precision, Ac~uracy, Recovery and Stability.

4.3 The biological matrix to be employed in the validation will be the same as that in the

intended test samples.

4.4 Method validation is performed to verify the following parameters:

1. Stock Check, Interference Check and Spiking Solution Check

2. Selectivity (Blank check) and Mat;rix Effect

3. Calibration curve (Linearity)

4. Precision and Accuracy (Intra-assay and Inter-Assay)

5. Stability

Page 108

.)

T_itle: Bioanalytical Method Validation

SOP #SAI-LP101 Revision: 12 Effective Date: 13 AUG ?QW Page 2 of 32

Written By/Date

6.

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Dilution Integrity

7. Recovery

4.5 Stock Check & Stock Stability (Short Term and Long Term):

1. At least two stock solutions per analyte must be prepared and stored at -80°C or -I

20°C or refrigerator. Dilute the stock solutions as per method of analysis. For <"

short term stability experiment, aliquot three stocks of each LQC and HQC and

store at specified conditions (such as room temperature). Following the storage of

the room temperature stock for a minimum of six hours, prepare two replicates of

LQC and HQC from each aliquot in matrix and analyze against freshly prepared

and extracted calibration curve.Also run freshly prepared QCs in duplicate.

2. If the internal standard used is a stable labeled compound, the stability established

for the analyte will be co~sidered for the stable labeled internal standard as _well.

3. In case of analog (non-stable labeled) Internal standard short term stability

experim~nt, aliquot three stocks and store at room temperature. Following the

storage of the room temperature stock for a minimum of six hours, retrieve from

the stock stored at -80°C or -20°C or Refrigerator. Prepare two replicate samples

from each aliquot by spiking in to matrix from each of the stock solutions

(stability stock and stored reference stock) and analyze.

4. If the stock solutions are refrigerated or frozen for the relevant period, the.stability

will be evaluat~d. After completion of the desired storage time, the stability stock

solution will be tested by preparing two replicates of LQC and HQC from each

aliquot in matrix and analyze against freshly prepared and extracted calibration


Page 109

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Title: Bioanalytical Method. Validation

SOP #SAI-LP101 Revision: 12 Page 3 of 32

Written By/Date Q ~ ,~ ~-~<:Ae\ \oA~ a.,,&-- \..0

5. Experiment Acceptance Criteria:

• Stock check results are based on the comparison of the mean response (area or

area ratio of analyte / IS) of the two stock solutions. The %CV of each stock

response must be within 8% and the % difference between two stock solutions

must not be more than ±5%.

• · For Analyte Short term and Long term stability results


Run acceptance will be based on acceptability of the calibration curve as

described in section 4.11.6 of this SOP. Also at least four out of the six freshly

prepared QCs and one QC at each level must be within ±15% of the nominal

value.


The mean value of the stability sample at each level should be within ±15% of

nominal value and the %CV at each level should be within 15%. Additionally,

at least 67% of the overall stored QCs and at least 50% at each level should be

within ±15% of their nominal value.

• For Internal standard stability results

Stability results are based on the comparison of the mean response from the

stability stock versus the mean response from the comparison/ fresh stock

stored at -20°C or -80°C or refrigerator. The %CV of eac;h stock response

must be within 15% and_,% difference between two stock solutions must not

be more than ±15%.

Page 110

ORIGINAlL


SOP #SAI-LPlOl Revision: 12 EffectiveDate: 13 AUG 2018 Page 4 of 32

Written By/Date

4.6 Interference Check:

1. Prepare the extracted Lower Limit of Quantitation (LLOQ) solution in duplicate

and inject.

2. Prepare a solution of analyte at approx. the highest standard concentration without

addition of the internal standard in duplicate ·and inject to observe any possible

interference at the retention time of the internal standard. The interference must.

not be more than 5% of the average internal standard response of the extracted

LLOQ solutions (Analyte interference Check).

3. Prepare a solution of the internal standard at approx. the concentration equivalent

to the internal standard concentration in the samples in duplicate and inject to

observe any possible interference at the retention time of. the analyte. The

interference must not be more than 20% of the average response of the extracted

LLOQs solution (Internal standard interference check).

4. Selectivity demonstration of an analyte in the presence of concomitant

medications or of specific metabolites will be performed by analyzing Blank, BL

(including concomitant medication), Blank with internal standard (including

concomitant medication) and extracted LLOQ samples in duplicate and inject to

observe any possible interference at the retention time of the analyte and internal

standard.

5. Specificity demonstradon in between analyte(Cross talk between Drug(s)) and

internal standards will be performed by extracting and injecting STD H of each

~alyte(s) individually without internal standard, Blank, Blank+IS and extracted

LLOQ in duplicate. Any response at the retention time of the analyte due to·

internal . standard and _vice versa will indicate possible interference from each

other.

Page 111

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SOP #SAI-LPlOl Revision: 12

Written By/Date

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5 of 32

• Any interference at the retention time of respective rui.alyte(s) must not be

more than 20% of the average response of the extracted LLOQs and any

interference at the retention time of internal standard must not be more than

5% of the average internal standard response of the extracted LLOQ solutions.

At least one of the each BL, BL + IS and extracted highest standard

concentration without IS samples prepared for interference check should meet


4.7 Spiking/Stock solutions check:

1. Spiking solutions are used to spike biological calibration standards and QCs.

Prepare a series of spiking solutions at concentrations, e.g. 20-100 times greater

. than those intended for biological calibration standards in a suitable diluent as per

method of analysis. Spiking volume of organic solvent must not be more than

2.5% of the total biological media.

2. The purpose of th~ spiking solution check is to allow _the user to identify and

replace inaccurately prepared spiking solutions before they are used for spiking of

biological matrix.


Percent Accuracy of each spiking solution must be within ±8% for spike check

experiment. At least 75% of the calibration standards must meet the above

criteria.

4.8 Selectivity:

Selectivity is the ability of an analytical method to differentiate and quantify the analyte

. in the presence of other co.mponents in the sample.

Page 112


SOP #SAI-LP101 Revision: 12 Effective Date: Page 6 of 32

Written By/Date

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1. Blank Check:

A. A minimum of nine independent sources of blank biological matri.ces will

be screened for the presence of any interfering compound that may affect

the selectivity of the method. Additionally, 2 each of hemolyzed, Lipemic

biological matrices must be screened for the presence of any interfering

compound. Prepare three different solutions containing LLOQ+ISTD, -

ISTD and Blank in each matrix. Selectivity will be ensured at the lower

limit of quantitation (LLOQ). All these samples will be analyzed against

freshly spiked and prepared calibration curve. Also run freshly prepared

QCs in duplicate.

B. Run Acceptance Criteria

• Run acceptance will be based on acceptability of the calibration

curve as described in section 4.1L6 of this SOP. Also at least four

out of the six freshly prepared QCs and one QC at each level must

be within ±15% of the nominal value.

·. C. Experiment Acceptance Criteria:

@ Any interference arising from blanks at the retention time of

analyte(s) should not be more than 20% of the average response of

accepted LLOQ (STD-A and STD-A').

8 · Any interference arising from blanks at the retention time of

internal standard should not be more than 5% of the average

response of IS from all accepted standards and quality control

samples.

- • Percent Accuracy of LLOQ QC of each matrix must be within

±20%.

Page 113

SOP #SAI-LP101 Revision: 12

\, - \'l ~ ~c..+:\ \ OA'"'\l-t·'' r


Effective Date: Page 7 of 32

.• At least 67% of the biological matrices (non-hemolysed and non

lipemic) screened should meet the above stated acceptance criteria.

• 50% each of the hemolysed and lipemic matrices screened ·should

meet the above stated acceptance criteria.

2. Matrix· Effect:

A. . Matrix effect is defined as inconsistent response for the analyte(s) of

interest, the internal standards(s) or both (all) when the same concentration

of compounds are extracted from different sources of biological blank

matrices.

B. Six independent sources of matrices screened for interference will be used

for the matrix effect. Prepare six. replicates of LQC, MQC and HQC for

each matrix, containing all the analyte(s) of interest and internal standard,

extract these samples as per method of analysis and also prepare post

spiked sample for all of these sample ( extracted blank matrix samples

spiked with drug and internal standard post extraction).

C. Experiment Acceptance criteria for extracted and post spiked samples:

• At each level, the %CV of the peak area of the analyte and internal

standard from each matrix must be within. 15% (for each lot at ~east 4

out. of .6 replicates must be used for statistical calculations) and %CY

of the mean response from 5 out of 6 matrix lots must be within 15%

to assume that no major matrix effect is present.

• · At each level, the %CV of the peak area ratio of the analyte to the

internal standard from each matrix must be within 15% (for each lot at

. least 4 out of 6 replicates must be used for statistical calculations) and

Page 114

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Page 8 of 32

%CV of the mean peak area ratios from 5 out of 6 matrix lots must be

within 15% to assume that no major matrix effect is present.

s At each level, normalized (relative) matrix factor for at least 4 out of 6

lots must be within 0.85 to 1.15 and %CV of normalized matrix factor

obtained for the six lots must be within 15%.

4.9 . . CARRY OVER:

Carry over is assessed by injecting extracted highest standard followed by extracted blank

to quantitate any carry over. Extracted blank, extracted low standard and highest standard

followed by extracted blank samples were injected in duplicate to verify the carry over.


Response from the extracted blank after highest standard must not be more than 20%

compared to average of STD A (low standard). Response from the internal standard must

not be more than 5% of the average internal standard response of the STD A.

4.10 TEST BULK SPIKING:

After bulk spiking of the quality control samples (QCs), check the accuracy of the bulk

spiked QCs to further proceed with validation experiments.

Analyze two replicates of bulk spiked QC's along with freshly spiked calibration curve

also run two freshly prepared QC's along with this.



section 4.11.6 of this SOP. Also at least four out of the six freshly prepared QCs and one

QC at each level must be within ±15% of the nominal value.

Page 115

SOP #SAI-LPtOl Revision: 12

Written By/Date ·

,~-~-~~\ \ Q A-~J cl.As--


Effective Date: 1 Page 9 of 32

Experiment Acceptance Criterja:

Percent Accuracy of each QC must be within ±10%. At least one QC at each level must

be within ±10% of the nominal value, except for the LOQQC where it must be within

±15% ..

4.11 Calibration Standard Curve:

1. A calibration curve is the relationship between instrument response and the

known concentration of the analyte(s) in the solution. A sufficient number of

calibration standards must be used to adequately define the relationsliip between

the concentration and the response. A calibration curve· will be prepared in the

same biological matrix as the samples in the intended study by spiking the matrix

with known concentrations of the analyte wheri.ever possible. The number of

calibration standards used in a calibration curve will be a function of- the

anticipated range of the analytical values and the nature of the analyte/response

relationship. Concentrations of standards must be chosen on the basis of the

concentration range expected in the particular study.

2. . A calibration curve must consist of two blank samples (matrix sample processed

without the internal standard), two zero samples (matrix sample processed with

the internal standard), and a minimum of six non-zero samples ·covering the

expected range, including the LLOQ. Additionally, Calibration standards consist

of STD A and STD H in duplicate and both the standards will be included in the

construction of the calibration curve. The Calibration curve standards shall be

prepared in fresh for the Precision and accuracy experiments.

3. The simplest model that adequately describes the concentration-response

relationship must be use~. Selection of weighting and the use of a complex

regression equation must be justified. Calibration standards' accuracy results for

Page 116



Written By/Date

~--?-~~~ , o A4\. -t ., r


all accepted PA batches with· linear or quadratic regression using weighting

factors as 1/x2, 1/x and with no weighting will be calculated. The percent relative

errors (%RE) will be calculated for each standard level as the percent difference

between the observed value and nominal value. For each weighting factor, the

absolute values of all the percent relative errors are summed for all the calibration

curves (from all accepted PA batches). The weighting factor that gives the

smallest value of the sum of the relative errors (%RE) will be taken as the

simplest model that gives the best results and adequately describes the

concentration-response relation~hip for the method. The model which gives the

lowest sum of the %RE will be used in the final method. If the alternative ' .

simplest model may be used with proper justification.

4. During Validation, at least six extracted calibration curves must be performed to

verify the method ruggedness.

5. Omission of Standards:

1. Depending upon the initial number of non-zero calibration standards, one or

more standards may be removed from the calibration curve if the percentage

deviation of more than 75% of standards or a minimum of 6 standards

(whichever is greater) is within ±15% of nominal (±20% for STD A) value. In

addition, at least one of the criteria described in section 4.9.5.4 must be met

for every omitted standard.

2. The table below specifies allowable numbers of standards that may be

omitted:'

Number of Non-Zero Standards Maximum number permissible

for each Calibration curve number of Omitted-Standards

6 0

7 1

Page 117


SOP #SA1-LP101 Revision: 12 Effective Date: Page 11 of 32

Written By/Date

8-11 2

12-15 3

3. In all cases, a mi:q.imum of six (6) non-zero standards must remain in each

curve and no more than two consecutive standards may be removed. ,,

4. Any Standard failed due to any of the following analytical reason should not

be included in the construction of the curve:

a. Loss of sensitivity

b. Poor chromatography

c. Loss during sample processing

d. Suspected Processing error

e. Internal Standard variation

5. Internal Standard variations: If the Internal Standard (IS) peak area in the

· standard is less than 5 0% or more than 15 0% of the average IS peak area of all

calibration standards and quality control samples (in a run), it will be

considered as failed.

6. No extrapolations are allowed outside the low and high standards (at nominal

values) of the calibration curve.

7. If any calibration standard did not meet the acceptance criteria, result table

must be printed before omitting the failed calibration standard. This is for

information purpose only. ··

8. If two standards did not meet the acceptance .criteria, calibration standard with

greater deviation (% accuracy deviating from 100) must be removed first and

then followed by second failed calibration standard and the data must be

printed. This is for information purpose only. The following are few examples

A. If STD-B has an accuracy of 115.4 and STD-F has an accuracy of

120.3. STD-F must be removed first and then followed by STD-B.

Page 118

Q :RJI%GI1\:JAJL ( -~ Sanno~t~t ~') Analytical Inc.


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SOP #SAI-LP101 Revision: 12 Effective Date: 1 3 AUG 20'18 Page 12 of 32

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B. If two standards did not meet the acceptance criteria with same

deviation, then the lowest standard must be removed. If STD-B has

an accuracy of 115.4_ and STD-F has an accuracy of 84.6. STD-B

must be removed first and then followed by STD-F.

C. If a standard and its duplicate both did not meet the acceptance

criteria with same deviation. The duplicate standard must be

removed first.

6. Calibration Standard Curve / Run Acceptance criteria:

• Interference in the blank, if any, at the retention time of analyte must not

exceed 20% of the average response of accepted LLOQ (STD-A and STO

A').

• Interference in the blank, if any, at the retention time of internal standard (IS)

must not exceed 5% of the average response of accepted calibration standards

and quality control samples.

• The% deviation of standards must be ±15%, except for the LLOQ standard

where it must be within ±20% fr.om the theoretical or expected concentratic:m.

• At least 75% of the calibration standards, when back calculated including

upper LOQ must fall within 15%, except for the LLOQ where it must be

within ±20% of the value. Values fall~ng outside these limits will not be used

to calculate the regression analysis.

NOTE: Blank and Blank+IS are used to determine whether the

contamination is randoin or consistent. Therefore, at least one each of Blank

& Blank+IS sample must meet the acceptance criteria to accept the batch.

Page 119



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4.12 Precision and Accuracy:

1. The precision of an analytical method describes the closeness of the individual

measurement of an analyte when the procedure is applied repeatedly to multiple

aliquots of single homogenous volume of biological matrix.

2. The accuracy of an analytical method describes the closeness of the mean test

results obtained by the method to the true value of the analyte.

3. Precision of the bioanalytical assays will be evaluated by analyzing freshly

prepared QC samples and determining the Coefficient of Variation (%CV) for

within (intra) and for overall (inter) batches of analyses.

4. The accuracy of the bioanalytical method will be determined by measuring the

concentration of the analyte(s) in QC samples and comparing them with the

theoretical concentration of the analyte(s) in these samples .

. 5. Six samples of each LOQQC (Lower limit of quantitation QC), LQC (Low QC),

MQC (Medium QC) and HQC (High QC) will be analyzed along with bulk

spiked or freshly spiked calibration standards on a minimum of three separate

precision and accuracy batches on different days.

NOTE: For ruggedness check, three different PA's will be performed h)' 3

different analysts on 2 different systems with at least 2 different columns and 2

different lots of critical reagents.

6. The interday precision is expressed as %CV of the mean concentrations obtained

at each level (LOQQC, LQC, MQC and HQC) from all accepted PA batches and

the interday accuracy is expressed as % accuracy of the mean concentrations

obtained at each level (LOQQC, LQC, MQC and HQC) from all accepted PA

batches.

7. Experi~ent Acceptance Criteria:

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SOP #SAI-LP101 Revision: 12 Effective Date: 1 3 AUG ' . · Page 14 of 32

Written By/Date

~-\Q~~~ \u A'J l a,r

• For precision and accuracy batches, acceptance of the run will be ·based on

acceptability of the calibration curve only as described in Sec. 4.11.6 of this

SOP.

• The criteria. for acceptable precision for both ;within (intra) and overall (inter)

batches; the %CV must be within 15% at each_ level, except for the LOQQC

where it must be within 20%. Additionally, at least 67% of the overall QCs

and at least 50% at each level must be within ±15% (for LQC, MQC and

HQC) and ±20% (for the LOQQC) of their nominal value.

• If the calibration curve is acceptable, · the QC data will be presented in the

validation report. The data will be used for the calculation· of between run

precision and accuracy.

• In a validation, a · minimum of three and maximum of six Precision and

Accuracy batches shall be performed. At least 67% of the overall Precision

and Accuracy batches (CC & QC both) must meet the acceptance criteria. If

these criteria are not met, investigate and resolve the problem.

4.13 Stability:

Stability of analyte in biologi_cal matrices and in the extracted samples · must be

determined for the intended period under the conditions of storage.

1. Short-Term Temperature Stability (Bench Top stability):

A. Six aliquots of each of the low and high QCs must be _kept on bench at ,.,

appropriate conditions for a minimum of 6 hours (based on the expected

duration that samples will be maintained at appropriate conditions in the

intended study) and analyzed with freshly spiked calibration standards and

two replicates each of Low, Medium and High QCs.

B. Run Acceptance criteria:

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SOP #SA1-LP101 Revision: 12 Effective Date: 1 3 AUG 2018. 15 of 32

Written By/Date te


described in Sec. 4.11.6 of this SOP. Also at least four out of the six

freshly prepared, QCs and one QC at each level must be within ±15% of

the nominal value.

C. Experiment Acceptance criteria:

The mean value of the stability sample at each level must be within ±15%

of the nominal value and the %CV at each level must be within 15%.

Additionally, at least 67% of the overall stability QCs and at least 50% at

each level must be within ±15% of their nominal value.

2. Freeze and Thaw Stability:

A. Analyte stability will be determined after a minimum three . freeze and

thaw cycles at the concentrations corresponding to high and low QC

samples in the matrix. Freeze all aliquots at the appropriate temperature

for at least 12 hours prior to first thaw cycle.

B. Remove at least six low and high frozen QCs and allow them to thaw at

appropriate condition for at least 3 hours. Freeze all of the aliquots at the

appropriate temperature for at least 12 hours.

C. Repeat the above step two or more times (minimum three cycles) using the

same aliquots that were previously thawed and refrozen.

D. After the completion of the freeze and thaw cycles, analyze all .twelve

aliquots of the frozen-thawed QCs witp. freshly spiked calibration

standards and two replicates each of Low, Medium and High QCs;

E. Run Acceptance criteria:


described in Sec. 4.11.6 of this SOP. Also at least four out of the six.

Page 122


SOP #SAI-LPlOl Revision: 12 EffectiveDate: 13 AUG 2018 Page 16 of 32

te

freshly prepared QCs and one QC at each level must be within ±15% of

the nominal value.

F. Experiment Acceptance_ criteria:

The mean value of the stability sample at each leveI"must be within ±15%


Additionally, at least 67% of the overall stability QCs and at least 50% at


3. Long-Term Stability:

A. The storage time in a long-term stability evaluation must exceed the time

between the date of first sample collection and the date of last sample

analysis. Long-term stability will be determined by storing six aliquots of

each low and high QCs samples under the same conditions as the study

· sample.

B. Analyze these stability sa~ples with freshly spiked calibration standards

and two replicates of each Low, Medium and High QCs.

C. Run Acceptance criteria:


described· in Sec. 4.11.6 of this SOP. Also at least four out of the six


the nominal value.

D. Experiment Acceptance criteria:



Additionally, at least 67% of the overall stabili!Y QCs and at least 50% at


Page 123


SOP #SAI-LP101 Revision: 12 Effective .. Date: 1 3 AUG 20"\8 Page 17 of 32

Written By/Date

E. In studies involving separate analytical methods for the determination of

co-administrated analyte(s), long term stability studies must be performed

in the presence of all analytes. ·

4. Autosampler Stability:

A. The evaluation of the extract stability must be performed with a freshly

prepared standard curve. The stability of processed samples, including the

resident time in the auto sampler will be determined. The aim of

experiment is to demonstrate that the samples are stable over the time

interval required to analyze a full study batch run (batch time).

B. For post-preparative (auto sampler) stability, six replicates of extracted

LQC and HQCs after analysis will be stored in the

refrigerator/autosampler for the maximum anticipated period of time

(auto-sampler stability samples batch processing end time to fresh CC/QC

processing end time) to run a batch. The stored_ QC samples will be

analyzed with freshly spiked calibration standards and two replicates each

of Low, Medium and High QCs.

C. Run Acceptance criteria: Run acceptance will be based on acceptability of the calibration curve as

described in Sec. 4.11.6 of this SOP. Also at least four out of the six


the nominal value.

D. Experiment Acceptance criteria:



Additionally, at least.67% of the overall stability QCs and at least ?0% at


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l- i~ ~ v~\44 \ (} ·""':1 ·{ ',g-

4.14 Dilution Integrity:


Effective Date: 'J 3 t\UG 2018 Page 18 of 3 2

p:roved By/Date

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1.. Dilution accuracy and precision must be validated prior to diluting any samples.

Prepare a QC sample Over Calibration Curve-QC (OCC-QC) in biological matrix

at least two times the concentration of the highest quality control sample.

2. Make at least six replicates from OCC-QC in blank biological matrix using an

appropriate dilution factor such as 2T (1 in 2) and/or 5T (1 in 5) so that the final

concentration falls within the calibration curve range. Process all of the diluted

QCs along with freshly spiked calibration standards. Whenever calibration

standard is freshly spiked include two replicates of each Low, Medium and High

QCs.

3. Also FT, BT, LT stability using six aliquots of OCC QC (ST) will be performed

as per section 4.13.1, 4.13.2 and 4.13.3. For the stability experiments process all

of the diluted QCs along with freshly spiked calibration standards and two

replicates each of Low, Medium and High QCs.

4. Run Acceptance criteria:


described in Sec. 4.11.6 of this SOP. Also at least four out of the six freshly

prepared QCs and one QC at each level must be within ±15% of the nominal

value.

5. Experiment Acceptance criteria:

• The mean value of the dilution integrity sample at each level must be within

±15%·of the nominal value and the %CV at each level must be within 15%.

Additionally, at least 67% of the overall stability QCs and at least 50% at each

level must be within ±15% of their nominal value.

8 For each dilution sample stability experiment (FT, BT, LT), the mean value of

the stability sample must be. within ±15% of nominal value and the %CV must

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SOP #SAI-LP101 Revision: 12 Effective Date: · ·1 3 AUG 20'i8 Written By/Date

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be within 15%. Additionally, at least 67% of the stability QCs for each

experiment must be within ±15% of their nominal Value.

4ol5 EFFECT OF HEMOLYSIS:

The aim of the experiment is to evaluate the effect of hemolysis in plasma samples are

not affected by hemolysis.

Prepare six replicates of LQC and HQC in 2% hemolyzed plasma and analyze against

freshly prepared and extracted calibration curve. Also run freshly prepared QCs (prepared

in pooled plasma) in duplicate.

• Run Acceptance criteria:

Run acceptance will be based on acceptability of the calibration curve only as described

in Sec. 4.11.6 ofthis SOP. At least four out of the six freshly prepared QCs and one QC

at each level must be within ±15% of the nominal value.

• Experiment Acceptance criteria:

The mean value ofhemolyzed QC's must be within ±15% of the nominal value at each

level. The %CV at each level must be within 15%. Additionally, at least 67% of the

overall stability QCs and at least 50% at each level must be within ±15% of their nominal

value.

Note: If 2% hemolysis experiment did not meet the acceptance criteria, hemolysis can be

verified at 1 % or 0.5%.

4.16 If the run and experiment is acceptable report the results. If not, investigate and resolve

the problem and repeat the experiment.

Page 126

Title: Bioanalyticai Method Validation


4.17 Recovery:

The recovery of the analyte(s) in an assay is the detector area response obtained from an

amount of the analyte(s) added to and extracted from the biological matrix, compared to

the detector area response obtained for the true concentration of the pure authentic

standard in neat solution.

1. Analyte Recovery:

A. Analyte recovery experiment must be performed by comparing the analyte ·

area for extracted analyte samples at three concentrations (low, medium, and

high) with post spiked analyte samples of same concentration levels. to

which known amounts of analyte is added. The amounts of analyte added to

the extracted blank plasma samples will correspond to 100% recovery ..

B. Experiment Acceptance criteria:

The %CV of Recovery at each QC level should be within 15% (4 out of 6

replicates must be used for statistical calculations) and recovery should not

be more than 115%. The overall %CV should be within 15%.

2. Internal Standard Recovery:

A. Internal standard recovery experiment must be performed by comparing

the Internal standard area for extracted internal standard samples with post spiked

internal standards of same concentration to which known amount of Internal

standard is added that represent 100% recovery. ·

B. Experiment Acceptance criteria:

The %CV of Recovery of Internal standard should be within 15% (4 out of 6

replicates at each level must be used for statistical calculations) and

recovery should not be more than 115%.

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~·~--------------------------------------------

( -~ s al ffiJ fJ1J ([j) W' aJ ·'---- ) Analytical Inc. Quality at it;s Finest


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Page 21 of 32

Approved By/Date Fcft fl&_tf~.,.,....._.~ 1'3 fl ;Lo lg

3. If an absolute difference of greater than ±15% in drug and stable labeled internal

standard recovery is observed, an investigation will be performed to determine the

source of the difference. The difference will be calculated by subtracting the

percent internal standard recovery from the percent drug recovery. If that

difference is more than ±15%, an investigation will be conducted.

4. If an absolute difference of greater than ±15% between LQC, MQC and HQC is

observed, an investigation will be performed to determine the source of the

difference. If that difference is more than ±15%, an investigation will be

conducted.

4.18 Whole Blood Stability:

Analyte stability in whole blood should be determined by spiking six replicates of Low

and High QCs (LQC and HQC level) sample in whole blood and leaving on bench at

specified conditions for a minimum of 3 hours. Then the stability QCs should be

analyzed with six replicates of freshly prepared Low and High QCs in whole blood using

extraction procedure.


The% difference of the stability QC samples should be within ±15% compared to freshly

prepared QC samples and the mean %CV of QC samples response should be within 15%.

i.e. [(Mean area ratio of stability QC samples/ Mean area ratio of freshly prepared QC

samples *100)-100].

4.19 Maximum Batch Size:

The Maximum batch size will be determined by running a minimum of 200 samples

including reference blank samples with QCs interspaced throughout the batch and

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SOP #SA1-LP101 Revision: 12 Effective Date: 1 3 AUG Z Page 22 of 32

Written By/Date

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analyzed with a calibration curve. This experiment is used to determine the drift in the

instrument response when large numbers of samples are analyzed.

• Run Acceptance criteria:

At least 75% of the calibration standards must be within+ 15%"ofnominal (+20% for

STD A). Additionally, at least 67% of the overall QCs and at least 50% at each level

must be within ±15% of their nominal value.

• · Experiment Acceptance criteria:

At least 67% of the Reference Blank samples must meet the below criteria;

Interference in the blank, if any, at the retention time of analyte must not exceed

20% of the average response of accepted LLOQ (STD-A and STD-A'). Interference

in the blank, if any, at the retention time of internal standard (IS) must not exceed

5% of the average response ofIS from all acceptable non-zero standards and QC's.

4.20 Omission of QC's during validation:

For any QC removed from the statistical calculations, one of the following· valid reasons

must exist. For any experiment only one out of six QCs at each level must be omitted.

Any Omission of QC must be authorized by Project manager or higher level.

A. Lost In Process (LIP): QC sample for which no final extract is obtained and loss duriI?-g

preparation.

B. Sample Processing Error (PE):

1. If the analyte peak area in the sample is less than 50% or more than 150% of

the average analyte peak area of other quality control samples at that level.

2. No analyte area or internal standard area is observed.

C, Poor Chromatography (PC).

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Written Byillate

~-\lrV <:A-e.\ \ (T }\J ,li'\y


Effective Date: J 3 AUG 2018 Page 23 of 32

D. Internal Standard variations: If the Internal Standard (IS) peak area in the QC is less

than 50% or more than 150% of the average IS peak area of all calibration standards

and quality control samples (in a run), it will be considered as failed.

E. Documented Assignable Cause: Acquisition error due to analyst software or etc.

Note: All the above mentioned criteria will be applicable for stock check and stock

stability samples also.

4.21 System sensitivity and carry over check will be evaluated as part of the analytical

run/batch and documented.

4.22 All of the analytical batches run during validation will be tabulated in the validation

report. The list must include the batch ID, run #, extraction date, purpose of the

experiment and its outcome (accepted/rejected).

4.23 A validation report will be prepared after a successful execution of the validation

protocol and review of validation data. The report will be reviewed by QA prior to its

final approval.

4.24 Analytical validation report may be issued in absence of long term stability data, which

can be added later as an addendum to the report. However, vaJidation report must be

signed off prior to analyzing any pivotal study samples.

5.0 EXHIBITS: None

Page 130

-~ r:-


SOP #SAI-LP101 Revision: 12 Effective Date: 13 AUG ZQi8 Page 24 of 32

6.0 REFERENCES:

· 6.1 Guidance for Industry: Bioanalytical Method validation, Food and Drug Administration

May 2018

7.0 DEFINITIONS:

Accuracy: The degree of closeness of the determined value to the or known true value under

prescribed conditions. This is sometimes termed trueness.

Analyte: A specific chemical moiety being measured, which can be intact drug, biomolecule or

its derivative, metabolite, and/or degradation product in a biologic matrix.

Analytical run (or batch): A complete set of analytical and study samples with appropriate.'

. number of standards and QCs for their validation. Several runs ( or batches) may be completed in

one day, or one run (or b_atch) may take several days to complete.

Biological matrix: A discrete material of biological origin that can be sampled and processed in

a reproducible manner. Examples are blood, serum, plasma, urine, feces, saliva, sputum, and

various discrete tissues.

Blank: A sample of a biological matrix to which no analytes have been added that is used to

assess the specificity of the· bioanalytical method.

Calibration standard: A biological matrix to which a known amount of analyte has been added

or spiked. Calibration standards are used to construct calibration curves :from which the

concentrations of analytes in QCs and in unknown study samples are determined.

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SOP #SAI-LP101 Revision: 12 Effective Date: ·1 3 AUG . 2 0 i · Page 25 of 32

~-~~,~

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Internal standard: Test compound(s) (e.g. structurally similar analog, stable labeled compound)

added to both calibration standards and samples at known and constant concentration to facilitate

quantification of the target analyte(s).

Lower limit of quantification (LLOQ): The lowest amount of an analyte in a sample that can

be quantitatively determined with suitable precision and accuracy.

Matrix effect: The direct or indirect alteration or interference in response due to the presence of

· unintended analytes (for analysis) or other interfering substances in the sample.

Method: A comprehensive description of all procedures used in sample analysis.

Precision: The closeness of agreement (degree of scatter) between a series of measurements

obtained from multiple sampling of the same homogenous sample under the prescribed

conditions.

Processed: The final extract (prior to instrumental analysis) of a sample that has been subjected

to various manipulations ( e.g., extraction, dilution, concentration).

Quality control sample (QC): A spiked sample used to monitor the · performance of a·

bioanalytical method and to assess the integrity and vafidity of the results of the unknown

samples analyzed in an individual batch.

Recovery: The extraction efficiency of an analytical pro~ess, reported as a percentage of the

known amount of an analyte carried through the sample extraction and processing steps of the

method.

Sample: A generic term encompassing controls, blanks, unknowns, and processed samples~ as

described below

Page 132


SOP #SAI-LP101 Revision: 12 G 2018 Page 26 of 32

. Selectivity: The ability of the bioanalytical method to measure and differentiate the analytes in

the presence of components that may be expected to be present. These could include metabolites,

impurities, degradants, or matrix components.

Stability: The chemical stability of an analyte in a given matrix under specific conditions for

given time intervals.

Standard curve: The relationship between the experimental response value and the analytical

concentration (also called a calibration curve)

Upper limit of quantification (ULOQ): The highest amount of an analyte in a sample that can

be quantitatively determined with precision and accuracy.

Page 133



REVISION NUMBER

00

01

02

03

REVISION ffiSTORY

EFFECTIVE DATE

29 JAN2007

10MAR2008

04APR2009

16NOV2009

DESCRIPTION OF CHANGES

New SOP

Deleted "nominal" before

freezer temperatures, elaborated

experimental procedures in 4.7 &

4.1 l.4B sections and added sections

4.15 (validation report) and 4.16

(system suitability).

Details CCN#: 08-018

Calibration standards were replaced

with freshly spiked CCs wherever

quantitation is required. .

Autosampler stability, Autosampler

re-injection reproducibility and

processed samples stability are

defined in detail in section 4.11.4,

4.11.5 and 4.11.6. Added maximum

batch size ·experiment in section

4.15.

Details: CCN#: 09-027

· Added freshly prepared QCs in .

BT, FT & LT experil_Ilents.

Deleted partial validation section

in this SOP and created a new

SOP for partial validation.

Page 134


SOP #SA1-LP101 Revision: 12 Effective Date:

Written By/Date

04 09DEC 2009

05 09 Aug2010

06 06 SEP 2012

Page 28 of 32


Added freshly prepared medium

QC to BT, FT & LT experiment.

Also added freshly prepared

QC's to autosampler stability

experiment.


Included validation of long term

stability studies in presence of

co-analytes in case of separate

analytical methods and added

summary of all batches run during

· validation. Added issuance of

validation report prior to pivotal

study sample analysis.

Details: CCN#: CCNl0-030

Sections 4.5 to 4.12 were Explained

in detail. Additionally, hemolyzed

matrix screening for selectivity was

included in section 4.8, calculation of

interday precision and accuracy was

explained in section 4.10, stability of

OCCQCs was included in section 4.11.

Section 4.11.6 was deleted. Percent

Recovery acceptance criteria was

included in section 4.13.

Page 135



Written By/Date

Details: CCN#: CCN12-056.

07 12 NOV 2014 Selectivity acceptance criteria were

modified and Lipemic matrix screening

for selectivity was included in section

4.8.1. Design and acceptance criteria of

matrix effect were modified in Section

4.8.2. Maximum batch size experiment

was modified in section 4.14.

Interference check in presence of

concomitant drugs experiment was

added to section 4.6. Section 4.9.4 was

added. Omission of standard and QC

samples were included in section 4.9.5

and 4.15 respectively. Stability of

OCCQC was included · along with

Dilution Integrity ~xperiment. Overall %

CV for analyte recovery was included in

section 4.13.1. Sections 4.9.1, 4.9.4.A,

4.10.5, 4.11.1, 4.11.2, 4.11.4, 4.16 were

modified and explained in detail.

08 01 Jun 2015

Details: CCN#CCN14-068.

Included Carry over, Test bulk spiking

and Hemolysis effect experiments in the

SOP. The acceptance criteria for stock

check, stock stability and spike check

was made stringent. Matrix effect and

Page 136


SOP #SAI-LP101 Revision: 12 Effective Date:

Written By/Date

~-i-t~ ·,oA~ ~o'k'°

09 03 JUN2015

10 14 SEP 2015

Page 30 of 32

recovery experiments design were

modified. Sections 4.11.2 and 4.12.5

were modified for use of bulk spiked

Calibration curve for PA batches.

Criteria for overall number of accepted

PA batches were included in section

4.12.7. Acceptance criteria was

separated · into Run and experiment

acceptance criteria in sections 4.12, 4.13,

4.14 and 4.15. Also added freshly

prepared QC's to dilution integrity

experiment. Section 4.20 was updated as

per current practices.

Details: CCN#CCNl 5-055

Sections 4.17.3 was explained

in detail.

Details: CCN#CCN15-057

Included Whole blood stability

experiment in the SOP as section 4.18.

Section 4.5.1 was modifiecl. The

acceptance criteria for stock check, short

term and Jong term stock stability,

Interference check and Matrix effect

were modified under section 4.5.4, 4.6.4

and respectively. The

demonstration of selectivity of an

Page 137

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~ . Analytical Inc. C-}ua.lit:y at; it:s Finest:


Written By/Date

~-~~\c4-c\ , oA-... J..,,y


Effective Date:. 1 3 AUG zo·w Page 31 of 32

analyte in presence of the metabolites

was included under section 4.6.4.

Details: CCN# CCN15-091

1r 23 AUG 2017 In section 4.7, 4.10 and 4.18 acceptance

criterion was updated. Experiment

design was updated in Section 4.9. IS

variation criteria was included in Section

4.11.5.4, 4.11.5.5, and 4.20.D. Blank

interference calculation was updated in

Section 4.11.6. Calculation of stability

duration was updated in Section

4.13.4.B. Section 4.14 was modified for

use of bulk spiked Calibration curve for

dilution integrity. Acceptance criterion

was included for recovery difference

between the QC's in section 4.17.4.

Details: CCN# CCNl 7-092

12 13 AUG 2018 The following changes were made in

align with FDA Bioanalytical Method

validation guidelines, May · 2018.

Experiment design was updated in

Section 4.5.1, 4.5.3, 4.5.4, 4.8.1 and

4.10, accordingly acceptance criteria was

updated. Section 4.6.5 was updated.

Section 4.11.3 was updated for clarity . . .

Precision and accuracy executed by

Page 138

Analytical Inc.


Written By/Date

Title: Bioan.alytical Method Validation .


\)-\'' ~ ,\·

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using freshly prepared QC' s was

updated in section 4.12.3. First freeze

cycle was changed to 12 hrs. Auto

sampler re-injection reproducibility

experiment was · removed. Recovery I

experiment was updated to relative

instead of absolute. Whole blood

stability will be executed at LQC and

HQC instead ofMQC level.

Details: CCN# CCNl 8-046

Page 139




11.3 D5 Benzalkonium Chloride (C12 and C14 homologs) Bio-analytical method #SAI-BM19499-00

Page 140

( ~ Sarnurn@va1 ~- ,.1 Analytical Inc. Quality at its Finest:

Title: Determination of D5 Benzalkonium Chloride (C12 and C14 homologs) in Human Plasma by LC/MS/MS

Method#: SAI-BM19499 Revision:00 Effective Date:1 5 MAY 2019 Page: 1 of 14

Written By/Date Reviewed By/Date

1.0 PURPOSE:

To describe a liquid chromatographic method with mass spectrometry detection for the

determination ofD5 Benzalkonium Chloride (Cl2 and C14 homologs) in Human Plasma.

2.0 RESPONSIBILITY:

All analysts are responsible to follow all applicable GLP guidelines and Sannova

Analytical Inc. SOP's,

3~0 PROCEDURE:

Unless otherwise indicated in the method, procedure for preparation of solutions,

reagents, stock solutions, standards and QC samples in this method are given only

as examples. Alternate weights and concentrations may be used, provided the final

composition of the critical end product remains the same as specified in this niet~od.

When alternate weights or volumes from those specified herein are used, they must

be properly documented in the raw data.

3.1 Summary:

Matrix

Anticoagulant

Sample Volume

Method of Extraction

Internal standard

Concentration Range

Human Plasma

EDTAK3

200µL

Liquid-Liquid Extraction

Benzalkonium Chloride-dl3

About 101.563 pg/mL to 13000.000 pg/mL for


About 30.469 pg/mL to 3900.000 pg/mL for

D5 Benzalkonium Chloride-CM

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Method#: SAI-BM19499 Revision:00 Effective Date: 1 5 MAY 2019 Page: 2 of 14


Detection Mode MRM

Quantitation Peak area ratio

Regression Linear

Weighting Factor l/Concentration2 (1/X:2)

Compound MW Free base Exact Mass

D5 Benzalkonium Chloride-C12 345.0 345.0 344.3

D5 Benzalkonium Chloride-C14 373.1 373.1 372.3

Benzalkonium Chloride-d 13 353.1 353.1 352.3

3.2 Chemicals:

Name Grade

D5 Benzalkonium Chloride

(C12 and C14 homologs) Certified Reference standard

Benzalkonium Chloride-dl3 Certified Reference standard

Acetonitrile HPLC Grade

Methanol HPLC Grade

Formic acid Analytical Grade

Water HPLC Grade

3.3 Chemical structures of D5 Benzalkonium Chloride and Benzalkonium Chloride

d13:

D D R\ +'CH3 N

.o 'cHa

er a°'\~

D D

D D D D

Where R is C12H2J for the C!2 homolog or CHHll for the Cl4 homolog. D

DS Benzalkonium Chloride Benzalkonium Chloride-dl3

Page 142

R~~:!---A--!-r-------------.---------------~ Title: Determination of D5 Benzalkonium Chloride ( ~ Satnnn(rv21

~ / Analytical Inc. Qu,ali-t.y at; its Finest

(C12 and C14 homologs) in Human Plasma by LC/MS/MS

Method#: SAI-BM19499 Revision:00 Effective Date: 15 MAY 2019 Page: 3 of 14

Written By/Date Reviewed ~y/Date

3.4 PREPARATION OF REAGENTS

3.4.1 Preparation of Mobile phase (0.2% Formic acid in Water: Acetonitrile 50:50):

Transfer 1000 mL of water and 1000 mL of acetonitrile to suitable container and mix

well add 4.0 mL of formic acid. Mix well and degas for 5 minutes.

3.4.2 Preparation of Rinsing Solution:

Transfer 500 mL of_Acetonitrile and 500 mL of water to suitable container and mix well.

3.4.3 Preparation of Diluent (50:50 Methanol: Water):

Transfer 500 mL of Methanol and 500 mL of water to suitable container and mix well.

3.5 PREPARATION OF MASTER STOCK SOLUTIONS:

3.5.1 Preparation D5 Benzalkonium Chloride (Cl2 and C14 homologs) Stock Solution:

DBKC ST-1

Accurately weigh about 6.65(±0.33) mg ofD5 Benzalkonium Chloride (Cl2 and C14

homo logs) certified reference standard and transfer the pan along with the standard to a

50mL centrifuge tube. Add 25 mL of Methanol with pipette, sonicate with occasional

shaking to dissolve solids and add 25 mL of Water and mix well. This is D5

Benzalkonium Chloride Cl2 stock solution containing about 100.000 µg/mL and D5

Benzalkonium Chloride C14 stock solution containing about 30.000 µg/mL.

Note: Separate stock solutions will be used to prepare calibration standards and quality

control (LQC, MQC and HQC) samples prepared by two different Analysts

Note: Store all the stock solution in refrigerator (2-8"C).

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3.5.2 Stock dilution for Spiking Solutions

Prepare all serial dilutions of Stock solution using Diluent.

Final Approx. Cone.of Approx. Cone.of

Diluted Stock Volume of Taken from Volume

D5 Benzalkonium D5 Benzalkonium ID Stock(mL) Stock (ID)

(mL) Chloride-C 12 Chloride-C 14 (ng/mL) (ng/mL)

DBKCST-2 1.3

DBKC ST-1 50 2600.000 780.000

DBKC ST-H 5.0

DBKC ST-2 50 260.000 78.000

DBKC ST-G 5.0

DBKC ST-H 10 130.000 39.000

DBKC ST-F 5.0

DBKC ST-G 10 65.000 19.500

DBKC ST-E 5.0

DBKCST-F 10 32.500 9.750

DBKCST-D 5.0

DBKCST-E 10 16.250 4.875

DBKC ST-C 5.0

DBKCST-D 10 8.125 2.438

DBKCST-B 5.0

DBKCST-C 10 4.063 1.219

DBKC ST-A 5.0

DBKCST-B 10 2.031 0.609

DBKCST-HQC 3.5

DBKCST-2 50 182.000 54.600

DBKCST-MQC 5.0

DBKCST-HQC 10 91.000 27.300

DBKCST-LQC 3.3

DBKCST-MQC 50 6.006 1.802

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3.6 PREPARATION OF INTERNAL STANDARD (IS) STOCK SOLUTIONS:

3.6.1 Preparation Benzalkonium Chloride-d13 (IS) Stock (dBKC ST-1):

Add 1.0 ml of methanol and 1.0 mL of water with pipette to the reference standard vial

containing about 1.0 mg of Benzalkonium Chloride-d13. Pipette out the above 2.0 mL

diluted stock in a glass tube and add 8.0 mL of Diluent and mix well. This is

Benzalkonium Chloride-d13 stock solution containing about 100.000 µg/mL of

Benzalkonium Chloride-cl 13.

Note: Reference standard vial concentration will be varied from one lot to another lot. Hence, adjust the final stock concentration (100.000µg/mL).

3.6.2 Stock dilution for IS Solutions

Prepare all serial dilution ofIS stock solution using diluent.

Diluted Volume of Taken from Final Volume Stock Stock (mL) Stock ID

ID

dBKCST-2 2.0 dBKCST-1

3.7 PREPARATION OF REFERENCE SOLUTION

3.7.1 PREPARATION OF REFERENCE SOLUTION

(mL)

200

Approx. Concentration

D5 Benzalkonium Chloride (ug/mL)

1.000

Pipette lOOµL of analyte (Diluted stock) and 500 µL of internal standard (dBKCST-2)

into a test tube containing 5.400 mL of Mobile Phase.

3.7.2 PREPARATION OF REFERENCE IS SOLUTION

Pipette 500 mL of internal standard (dBKCST-2) in to a test tube containing 5.500 mL of

Mobile Phase.

3.7.3 PREPARATION OF REFERENCE BLK SOLUTION

Use Mobile Phase )

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Method#: SA1-BM19499 Revision:00 Effective Date: 1 5 MAY 2019 Page: 6 of 14


3.8 PREPARATION OF CALIBRATION STANDARDS AND QUALITY CONTROL

SAMPLES:

Prepare all calibration standards in and quality control samples in the same biological

matrix as the samples to be analyzed (i.e. human plasma).

3.8.1 Preparation of Calibration Standards

Calibration standards samples are prepared in bulk or fresh by spiking stock solutions in

human plasma. Allow blank plasma ( screened for absence of interference at the retention

time of the drug and internal standard) to thaw at room temperature prior to spiking.

Prepare calibration standards as per table below and mix well. Transfer 300µL bulk

prepared standards into individual Polypropylene tubes and store at -20°C.

Stock Apprx: Cone.of Apprx: Cone.of

Matrix Total D5 D5 STD ID

Stock Solution Solution Volume Volume Benzalkonium Benzalkonium

ID Volume (mL)

(mL) (mL) Chloride-C 12 Chloride-Cl4 (pg/mL) (pg/mL)

STD-H DBKCST-H 0.500 9.500 10 13000.000 3900.000

STD-G DBKCST-G 0.500 9.500 10 6500.000 1950.000

STD-F DBKC ST-F 0.500 9.500 10 3250.000 975.000

STD-E DBKCST-E 0.500 9.500 10 1625.000 487.500

STD-D DBKC ST-D 0.500 9.500 10 812.500 243.750

STD-C DBKC ST-C 0.500 9.500 10 406.250 121.875

STD-B DBKCST-B 0.500 9.500 10 203.125 60.938

STD-A DBKC ST-A 0.500 9.500 10 101.563 30.469

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Method#: SA1-BM19499 Revision:00 Effective Date: 15 MAY 2019 Page: 7 of 14


3.8.2 Preparation of Quality control samples

Quality Control samples are prepared in bulk by spiking stock solutions in human

plasma. Allow blank plasma to thaw at room temperature (use plasma screened for

absence of interference at the retention time of the drug and internal standard). Prepare

quality control samples as per table below and mix well. Transfer 300µL bulk prepared

quality control samples immediately into individual Polypropylene tubes and store at -

20°C.

Stock Apprx Cone.of Apprx Cone.of

Solution Matrix Total D5 D5

STD ID Stock Solution ID Volume Volume Benzalkonium Benzalkonium Volume

added(mL) (mL) Chloride-C12 Chloride-C 14 (mL)

(pg/mL) ( g/mL)

QC-LQC DBKC ST-LQC 1.25 23.75 25 300.300 90.090

QC-MQC DBKCST-MQC 1.25 23.75 25 4550.000 1365.000

QC-HQC DBKC ST-HQC 1.25 23.75 25 9100.000 2730.000

3.9 SAMPLE EXTRACTON PROCEDURE:

The extraction procedure described here has to be applied for calibration standards,

subject samples and quality control samples. Allow samples to thaw on Room

temperature prior to extraction. In order to perform sample extraction the following steps

should be followed.

3.9.1 Place sufficient number of disposable glass tubes in a rack labeled according to analytical

batch file.

3.9.2 Vortex all thawed samples for a few seconds, then aliquot 200 µL of sample into

appropriate tubes.

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3.9.3 To each sample tube (except for blank) add 50µL of internal standard (dBKCST-2)

using a pipette and vortex for about 10 seconds. To blank add 50µL of Diluent.

3.9.4 To each sample add 3.0mL of Ethyl acetate

3.9.5 Shake all the samples for 10 minutes at 1000 rpm on a Micro plate shaker.

3.9.6 Flash freeze all samples using cold methanol or dry ice in methanol for approx.

5min(Methanol should be cooled in the freezer at -80°C at least for 2 hours prior to use)

Transfer the organic layer completely into fresh disposable glass tubes and dry in a

nitrogen evaporator at 45°C.

3.9.7 Reconstitute the dried residue with 0.6 mL of mobile phase and shake all the samples for

5 minutes at 1000 rpm on a Micro plate shaker.

3.9.8 Transfer the supernatant of each tube into labeled respective HPLC vials and load into

LC-MS/MS system for analysis.

3.10 LC (LIQUID CHROMATOGRAPHIC) CONDITIONS:

Analytical column

Mobile Phase

Flow rate

Column Temperature

: X Select CSH C18 3.5 µm 4.6X150mm

Part No.:186005270

: 0.2% Formic acid in (Water: Acetonitrile (50:50))

: 1.0 mL/min

: 40°c

Auto sampler Cooler Temperature : 4 °C

Injection Volume

*Run time

Rinsing Solution

Rinsing Volume

Needle Stroke (Rack changer)

: 15 µL

: 12.0 min

: Water: Acetonitile 50:50

: 200 µL

: 48 mm ( 46 - 48mm)

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Method#: SAI-BM19499 Revision:00 Effective Date3 5 MAY 2019 Page: 9 of 14


Rinse Dip time : 2 Sec.

Rinse Mode : Before and after aspiration

*Variations in these conditions may be applied in order to optimize chromatography.

3.11 MS/MS CONDITIONS:

Ion Source

Mode

** Acquisition Time

Compound


D5 Benzalkonium Chloride-C 14

Benzalkonium Chloride-dl3

**CAD

**CUR

**GSl

**GS2

IS

**TEM

lhe

**EP

Turbo Ion Spray

Positive Ion

12.0 min

Ql Mass Q3 Mass

(amu) (amu)

309.3 212.2

337.3 240.3

317.2 217.2

8

40

65

60

5500

550

ON

10

Dwell DP CE

(msec) ** **

600 100 25

600 100 30

600 100 28

** In order to optimize chromatography and MS conditions parameters may vary.

CXP

**

10

10

10

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Method#: SAI-BM19499 Revision:00 Effective Date: 1 ~ MAY io,. ! Page: 10 of 14


3.12 RETENTION TIMES:

* D5 Benzalkonium Chloride-C12: About 4.20 min (RT should be within 3.4 min to 5.00 min)

* D5 Benzalkonium Chloride-C14: About 9.6 min (RT should be within 7.7 min to l l.5min)

* Benzalkonium Chloride-dB: About 4.20 min (RT should be within 3.4 min to 5.00 min)

3.13 IDENTIFICATION OF ANALYTES AND INTERNAL STANDARDS:

The identification of D5 Benzalkonium Chloride-C12, D5 Benzalkonium Chloride-Cl 4,

and Benzalkonium Chloride-d13 is based on m/z ratio and the retention times of each

compound in reference samples.

3.13.1 Quantitation Parameters for Analyte & Internal Standard:

Bunching Factor: 1 *Noise Threshold: 10.00

*Num. Smooths: 5

Separation Width: 0.20

RT Window: 30 sec

* Area Threshold: 50.00

Separation Height: 1.00

Regression: Linear 1/x2

*: The above parameters can be modified to optimize chromatography

3.13.2 Sensitivity threshold for Analyte & Internal Standard

Drug Sensitivity Threshold for

API-5500

D5 Benzalkonium Chloride-Cl2 4000

D5 Benzalkonium Chloride-C14 1000

Benzalkonium Chloride-d13(IS) 10000

Note: It is recommended to optimize the instrument if the analyte and internal standard

are not meeting the above minimum sensitivity.

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Title: Determination of D5 Benzalkonium Chloride (C12 and Cl4 homologs) in Human Plasma by LC/MS/MS

Method#: SAI-BM19499 Revision:00 Effective Date: 15 MAY 201 Page: 11 of 14

Written Byillate Reviewed Byillate

3.14 CALIBRATION AND CALCULATION:

A set of ten calibration standards (STD A and STD H in duplicate), two zero standards,

two blanks and QCs during validation and biostudy are analyzed with every batch of D5

Benzalkonium Chloride (C12 and C14 homolog) assay. All ten standards should be used

to plot calibration curve. For each calibration standard, the peak area ratios for each

Analyte/IS are determined. A linear regression describing the calibration curve is then

calculated using the reciprocal of the square of the drug concentrations (1/Concentration2

or l/x2) versus peak area ratio. Data acquisition and integration will be performed using

AB Sciex Triple Quad (LC/MS/MS) system and Analyst software.

The regression equation is y=mx+c

y= peak area ratio of Analyte(s)/IS

x= concentration of Analyte(s)

m= slope of the calibration curve

c= y-intercept of the calibration curve.

4.0 EXHIBIT:

Batch Processing form for D5 Benzalkonium Chloride.

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ENCE COP


Method#: SA1-BM19499 Revision:00 Effective Date15 MAY 2019 Page: 12 of 14

Written By/Date Reviewed By/Date Approved By/Date

5.0 TYPICAL MASS SPECTRUM OF D5 BENZALKONIUM CHLORIDE C12 AND C14

tll +MS2 (309.aO) CE (25)': 0.439 ITlin fron1 Samplet 1 (.TunaSan,plalD) of PRODUCT ION 05 BKZ C12 309.2 rrn:.wiff (TudJo Spr .•• M:nc...4.De7cp:3,

13-;Ii,

I

4..0e7

3.8e7

3.Ge7

3-407

32c7

3.0a7

2.S..7

2.607

2.4c7

1.6o7

1.4e7

1..2o7

1.0o7

8.0e.5

6.0e6

4.0e6

2.0o&

<-----Q3rnfz 05BKC C12

58.0

.2

212.4

309.2

<----Q1 nt/z 05 BKC C'l2

<----Q3 m/z 05 BKC C1

3·1 .3

I

o.o,.._~6"'0---sa;;,;;a;----'-;,=--,,=o--,1=0----:;,, °"0--:;:1 <ao:-----c;:=--'-'-:;,;;s-----c;r;;---=::-----c=---=;;;--''-'-=----,;,=--=:---,;=---=4 o

+MS2 (337.70) CE (30): 0,410 min from Sample 1 {TuneSnrnplolD) of PRODUCT ION 05 BKZ C14 337.4 mz.wiff (Turl,o SPf" •.• Max. 2.7e7 cps

2.Go7

T 2.5o7 2Aa7

=o7

2.2o7

2-1a7

2.0e7 I 1.9o7 I 1.Bo7

1.7c7 I 1-6a7

337.5

<------- Q3 m/z DB BKC C14 Q1 mlz. OS BKC C14

240.3

1,5o7 i 1.41117

fl 1.3o7

1.2~7

1.1-o7 Ii 1.0o7

11 9.0e6

<---- Q;J m/z D5 BKC C14

8.0c6 I 7.0eS

Ii

G.Oo6

S.Oe,6

4.0oG 58.1 3.0&G

2.0e6

1.0e6

0.0 60 80 100

I

11

I 140 1:$0 ,iio 200 220 :i6o 2.80 300 :.do

ll 3fu 380 400 240 340 120

nilZ',Oa

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Metbod#:SA1-BM19499 Revision:00 EffectiveDate: 1§ l'IAY 2015 Page: 13 of 14

Written By/Date \.

Reviewed By/Date Approved By/Date

5.1 TYPICAL MASS SPECTRUM OF BENZALKONIUM CHLORIDE-dB:

m +MS2.(317 .20) CE (30): 10 MCA scans from Sample 1 (TuneSamplelO) of 03 SCAN_d13 BKC.wiff (Turbo Spray).. Centroide- Max. 9.6e6 C

98.1 9.5e6

9.0e6

8.5e6

e_oe6

7.Se6

7.0e6

6.Se6 317.3

6.0e6

5.5e6 §

5.0e6 ~ j 4.5e6

4.0e6 Q1 MASS d13 BKC-->

3.5e6 217.3

3.0e6

2.5e6 Q3 MASS d13 BKC---> 2.0e6

85.0

1.5e6 57.0 .D 95.

1.De6

0.0 340

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Method#: SAI-BM19499 Revision:00 Effecfi:ve Date: 1 5 MAY 2019 Page: 14 of 14

Written By/Date

REVISION NUMBER

00

Reviewed By/Date

REVISION HISTORY

EFFECTIVE DATE

1 5 MAY 2019

DESCRIPTION OF CHANGES

New method

CCN#CCN19-055

Page 154

0 ~~~Jli1)1fa} ~ 1 Ana1yUcaf ln~. ' Quality at its Fiitest

Project Name:

Run#:

Pipette(s) ID: PIP-

Batch ID:

Batch Processing Start Time:

Purpose of the Batch:

PIP-

Reagents, Solvents and Solutions:

Reagent/Solvent/Solution

DILUENT

INTERNAL STANDARD

ETHYL ACETATE

MOBILE P AHSE

REFLLOQ

REF BLANK

S.No.: REFERENCE COPY

Issued by/Date: ______ _

Batch Processing

Validation/Study No.:

Method ID: SAI-BM19499-00

PIP-

End Time:

Mfg. Lot No . ... IN HOUSE -s~\>

-~Q. IN HOUSE

-Ill...-~ ~~.\~ ~

'-l.: .·. _'f7) '\,P \ \ ' .. \..\

IN HOUSE ,~ ,- f ....,.,~ \

_.),.. \ '> IN HOUSE -IN HOUSE

Calibration Standards, QCs and Subject Samples:

Sample Sample ID Date STDs & QCs prepared

Calibration Standards

QC samples

Subject Samples

Sample Extraction Procedure:

Place labeled disposable glass tubes in a rack according to analytical batch file.

Vortex thawed samples for a few seconds and then aliquot 200 µL of the sample into

appropriate tubes.

LPl 10-EOl-02 Page 1 of2

Page 155

OR.~~-Y@ \E= ) Analytical Inc.

REFERENCE COPY S.No.:

:.Quality at its Finest Issued by/Date: ______ _

___ To each sample tube, add 50 µL of internal standard (dBKC ST-2)( Lot ID ___ ~)

Exp Date: using pipette and vortex for 10 secs except for BLANK samples

in which add 50µL of Diluent.

___ Add 3.0mL of Ethyl acetate to each sample

___ Shake all the samples for 10 minutes at 1000 rpm on a Micro plate shaker.

Microplate Shaker ID(s): _________________ _

___ Flash freeze all samples using cold methanol or dry ice in methanol for approx.5min

(Methanol should be cooled in the freezer at -80°C at least for 2 hours prior to use)

___ Transfer the organic layer completely into fresh disposable glass tubes and dry in a

nitrogen evaporator at 45°C.

Evaporator ID(s): ----------------------

Reconstitute the dried residue with 0.6 mL of Mobile Phase and shake all the samples for 5 ---

minutes at 1000 rpm on a Micro plate shaker.

Microplate Shaker ID(s): -~e~ . Transfer the supernatant of each tube into HPLC Vials and~~1Alo autosampler of LC-

MS/MS system for analysis. {l~· \ h ~rl'J ~\~

-~'~ ~ Storage of extraction samples: NO o YES o irr < vN:

\ "':)

If Yes Storage Temperature: _______ °C

Notes:

Prepared by/Date: ______ _ Verified by/Date: _____ _

LPl 10-EOI-02 Page 2 of2

Page 156




11.4 COAs of Deuterated Benzalkonium Chloride, Benzyl dodecyl dimethyl ammonium chloride D13 (Benzalkonium Chloride-d13) and Over-The-Counter Multi-Component Mixture-6

Page 157

STUDY NUMBER 88523 ---------CERTIFICATE OF ANALYSIS FOR TEST/REFERENCE/CONTROL SUBSTANCES

.__ __ T_I_T_L_E_IO_B_.m_;C_.T_I_V_E_• : __ __.l Deuterated Benzalkonium Chloride: Determination of Purity and Identity

Test Substance: Lot Number: Structure:

TEST/REFERENCE/CONTROL SUBSTANCE: I Deuterated Benzalkonium Chloride VIJATl00-19485

-

er

I

Molecular Weight (amu): 345.02 (Cl2 homolog) 373.07 (C14 homolog)

I

*D D R\+'CH3

N, D CH3

I Molecular Formula: C21HnDsClN (Cl 2 homoli°g) C23H37DsCIN (C14 homolog)

D D I

Recommended Storage Conditions: Ambient I - -Where R is C 12H25 for the Cl2 homolog or C 14H27 for the Cl4 homolog.

Reference Substance: Lot No. LRAB78I7; CAS No. 63449-41-2; Purity 8.98% (w/v)

INITIATION DATE: 26 February 2019 EXPERIMENTAL COMPLETION DA TE: 20 March 2019

METHODS USED: PURITY: !DENTIFICA TION:

HPLC-DAD LC-MS/MS and 13C-NMR

RESULTS and CONCLUSIONS: X INITIAL DETERMINATION:

Result (Purity± Standard Deviation): 98.7 ± 1.1 % (w/w) Total Purity; 75.7% as the C12 homolog, 23.0% as the C14 homolog

X I IDENTITY: The LC-MS/MS analytical data supports the proposed stmcture ofdeuterated benzalkonium chloride (C12 and C14 homologs). 13C-NMR data indicate the spectrum is consistent with the structure. Identity confirmed.

X !ACCELERATED STORAGE: I After storage for two weeks at 54°C, the total purity of the test substance was 96.3%, which was within 3% of the initial urity and justified the re-certification date assigned.

RE-CERTIFICATION DATE: 11 March 2021

I CALCULATIONS: Area Normalized: N.A. Internal Standard: N.A.

STUDY DIRECTOR SIGNATURE:

j {1u;\_ v"'//vr:zs;---, <:.::.:.:?

QUALITY ASSURANCE REVIEWER.SIGNATURE:

·JZltMa!W (aivwr TESTING FACILITY (HPLC-UV AND LC-MS/MS) & STUDY DIRECTOR ADDRESS:

DATE:

DATE:

SPONSOR: Lonza Inc.

External Standard:

Eurofins EAG Agroscience, LLC 7200 East ABC Lane Columbia, Missouri 65202

412 Mount Kemble Ave Morristown, New Jersey 07960

ADDITIONAL TESTING FACILITY (NMR): Spectral Data Services, Inc. 818 Pioneer St. Champaign, Illinois 61820

X

Only descriptive statistics were used unless otherwise noted in the results. This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practices 21 CFR Part 58.

Page 158

CS-O-33730 CRC-2016-R&D-27C21H25D13ClN 353.07

04/05/2019 04/05/2024

Storage conditions Keep in a tightly closed vial, store in refrigerator

Test Specification Result

CAS #

Molecular formula Molecular weight

319-07-1 (Unlabeled)

Retest DateDate of Analysis

CERTIFICATE OF ANALYSISProduct Name Benzyl dodecyl dimethyl ammonium chloride D13

Synonyms Not available

N-benzyl-N,N-dimethyldodecan-1-aminium chloride-D13Chemical Name

Catalogue # Batch #

06/05/2019 06/05/2019

Purity by HPLC Not less than 90%

1H NMR spectrum Conform Structure Confirms

Isotopic enrichment (by MS) Not specified 93.90%Confirms

This material complies.

AppearanceColorless to yellowish transparentliquid

Colorless to yellowish transparent liquid

Solubility Soluble in DMSO, in chloroform In DMSO, in chloroform

Test Specification Result

The re-test data is assigned based on the literature available. The balances used are calibrated with weights traceable to the National standards NIST

Reviewed & Approved by( Quality Assurance )

Reviewed & Approved by( QC Analytical )

94.53%

Mass Spectrum Conform molecular weight

Page 1 of 1Page 159

O-034FE04051601Revision 00Page 1 of 4

Catalog Number: O-034Solution Lot: FE04051601Expiration Date:Description: Over-The-Counter Multi-Component Mixture-6 in Acetonitrile.Packaging: Solution in 2 mL amber USP Type I glass ampoule

containing not less than 1 mL of certified solution.Storage: Store unopened in freezer (-10 °C to -25 °C).Shipping: Ambient. See Stability Section.Intended Use: This Certified Reference Material is suitable for the in vitro identification, calibration, and quantification of the

analyte(s) in analytical and R&D applications. Not suitable for human or animal consumption.Instructions for Use: Users should quantitatively transfer desired volume using established good laboratory practices to

spike into matrix or to dilute to the desired concentration. Each ampoule is intended for one-time use.Safety: Danger. See Safety Data Sheet

Expiration date has been established through real time stability studies. Ampoules are overfilled to ensure a minimum 1 mL volume can be transferred when using a 1 mL Class A volumetric pipette. For quantitative applications, the minimum sample size for intended use is 1 µL.

Component

A t i h

Certificate of Analysis

> 99 9%

ConcentrationChromatographic Purity

Over-The-Counter Multi-Component Mixture-6

100 0 ± 0 5 / L

April 2021

Cerilliant Corporation 811 Paloma Drive, Suite A, Round Rock, TX 78665 800-848-7837 / 512-238-9974

AcetaminophenIbuprofenCaffeineChlorpheniramine maleateNaproxen(1R,2R)-(-)-Pseudoephedrine

Uncertainty of the concentration is expressed as an expanded uncertainty in accordance with ISO 17025 and Guide 34 at the approximate 95% confidence interval using a coverage factor of k = 2 and has been calculated by statistical analysis of our production system andincorporates uncertainty of the purity factor, material density, and balance and weighing technique.

Concentration is corrected for chromatographic purity, residual water, residual solvents and residual inorganics.

Traceability

Gravimetrically prepared using qualified balances calibrated semi-annually by Mettler Toledo using NIST traceable weights. Calibrationverification performed weekly and prior to each use utilizing NIST traceable weights. Each balance has been assigned a minimum weighingby Mettler Toledo taking into consideration the balance and installed environmental conditions to ensure weighing complies with USP tolerances of no more than 0.1% relative error.

In addition, each neat material utilized has been identified and thoroughly characterized through the use of multiple analytical techniques.Spectral data is provided on subsequent pages of the COA.

Darron Ellsworth, Quality Assurance Manager Date

> 99.9%100.0 ± 0.5 µg/mL100.0 ± 0.5 µg/mL

May 04, 2016

100.0 ± 0.5 µg/mL

Cerilliant certifies that this standard meets the specifications stated in this certificate and warrants this product to meet the statedacceptance criteria through the expiration/retest date when stored unopened as recommended. Product should be used shortlyafter opening to avoid concentration changes due to evaporation. Warranty does not apply to ampoules stored after opening.

> 99.9%99.6%

Concentration is verified against an independently prepared 4-point calibration curve gravimetrically prepared using balances calibrated to NIST.

This standard is prepared gravimetrically and mass results are reported on the conventional basis for weighing in air. Concentration is calculatedbased on: the actual measured mass; Purity Factor of the analyte(s); measured mass of the solution; and the density of the pure diluent at 20 °C.

100.0 ± 0.5 µg/mL99.6%

100.0 ± 0.5 µg/mL> 99.9%

100.0 ± 0.5 µg/mL (as free base)> 99.9%

Cerilliant Corporation 811 Paloma Drive, Suite A, Round Rock, TX 78665 800-848-7837 / 512-238-9974Page 160




11.5 Linear Regression Curve

11.5.1 Linear Regression Curve for D5 Benzalkonium Chloride-C12

Page 161

012_005_PA1_INT CHK_CONC CHK_C12.rdb (d5 BKC C12): "Linear" Regression ("1 / (x * x)" weighting): y = 0.00382 x + -0.0202 (r = 0.9993)

1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4Analyte Conc. / IS Conc.

0

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Results Path: C:\Analyst Data\Projects\SAI_VP19499\D5 BENZALKONIUM CHLORIDE\Results\012_005_PA1_INT CHK_CONC CHK C12.rdb

Project: SAI_VP19499

Results Name: 012_005_PA1_INT CHK_CONC CHK_C12.rdb

Printing Date: Wednesday, May 15, 2019

Printing Time: 3:31:34 PM

Page 1 of 1

Page 162




11.5.2 Linear Regression Curve for D5 Benzalkonium Chloride-C14

Page 163

012_005_PA1_INT CHK_CONC CHK_C14.rdb (d5 BKC C14): "Linear" Regression ("1 / (x * x)" weighting): y = 0.0121 x + -0.0498 (r = 0.9988)

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000Analyte Conc. / IS Conc.

0

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Page 1 of 1

Page 164




11.6 Representative Chromatograms


Page 165

Sample Name Sample ID Sample Type Acquisition Date Vial Position

Plate Position File Name Dilution

FactorAnalyte Peak Area (counts)

Analyte Concentration

(pg/mL)Area Ratio

IS Peak Area

(counts)

Use Record

Record Modified

Calculated Concentration

(pg/mL)Accuracy (%)

1 REF LLOQ RUN 05_001 Unknown 5/7/2019 4:01:46 PM 1 1 012_005.wiff 1.000 48532 N/A 0.410 118393 112.728 N/A2 REF LLOQ RUN 05_002 Unknown 5/7/2019 4:15:45 PM 1 1 012_005.wiff 1.000 47667 N/A 0.402 118650 110.585 N/A3 PA1-STD BL RUN 05_003 Blank 5/7/2019 4:28:21 PM 3 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A4 PA1-STD BL' RUN 05_004 Blank 5/7/2019 4:40:53 PM 4 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A5 PA1-STD BL+IS RUN 05_005 Blank 5/7/2019 4:53:33 PM 5 1 012_005.wiff 1.000 0 0.000 0.000 93740 N/A N/A6 PA1-STD BL+IS' RUN 05_006 Blank 5/7/2019 5:06:08 PM 6 1 012_005.wiff 1.000 0 0.000 0.000 88548 N/A N/A7 PA1-STD A RUN 05_007 Standard 5/7/2019 5:18:44 PM 7 1 012_005.wiff 1.000 33218 106.944 0.383 86804 105.586 98.78 PA1-STD A' RUN 05_008 Standard 5/7/2019 5:31:21 PM 8 1 012_005.wiff 1.000 36082 106.944 0.400 90255 110.070 102.99 PA1-STD B RUN 05_009 Standard 5/7/2019 5:43:57 PM 9 1 012_005.wiff 1.000 74797 213.888 0.795 94049 213.723 99.910 PA1-STD C RUN 05_010 Standard 5/7/2019 5:56:33 PM 10 1 012_005.wiff 1.000 139765 427.776 1.539 90796 408.713 95.511 PA1-STD D RUN 05_011 Standard 5/7/2019 6:09:10 PM 11 1 012_005.wiff 1.000 292581 855.552 3.132 93425 826.033 96.512 PA1-STD E RUN 05_012 Standard 5/7/2019 6:21:46 PM 12 1 012_005.wiff 1.000 582789 1711.104 6.380 91350 1677.230 98.013 PA1-STD F RUN 05_013 Standard 5/7/2019 6:34:19 PM 13 1 012_005.wiff 1.000 1185870 3422.208 12.762 92924 3349.778 97.914 PA1-STD G RUN 05_014 Standard 5/7/2019 6:46:58 PM 14 1 012_005.wiff 1.000 2351228 6844.416 27.403 85803 7186.671 105.015 PA1-STD H RUN 05_015 Standard 5/7/2019 6:59:35 PM 15 1 012_005.wiff 1.000 4459997 13688.831 53.176 83872 13941.202 101.816 PA1-STD H' RUN 05_016 Standard 5/7/2019 7:12:12 PM 16 1 012_005.wiff 1.000 4634055 13688.831 54.085 85682 14179.228 103.617 REF BLANK RUN 05_017 Blank 5/7/2019 7:24:44 PM 2 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A18 STD H NO IS RUN 05_018 Unknown 5/7/2019 7:37:23 PM 17 1 012_005.wiff 1.000 4836295 N/A #DIV/0! 0 #DIV/0! N/A19 STD H NO IS' RUN 05_019 Unknown 5/7/2019 7:49:58 PM 18 1 012_005.wiff 1.000 4727642 N/A #DIV/0! 0 #DIV/0! N/A20 CONCO - BLK RUN 05_020 Blank 5/7/2019 8:02:34 PM 19 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A21 CONCO - BLK' RUN 05_021 Blank 5/7/2019 8:15:11 PM 20 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A22 CONCO - BLK + IS RUN 05_022 Blank 5/7/2019 8:27:45 PM 21 1 012_005.wiff 1.000 0 0.000 0.000 99780 N/A N/A23 CONCO - BLK + IS' RUN 05_023 Blank 5/7/2019 8:40:22 PM 22 1 012_005.wiff 1.000 0 0.000 0.000 94023 N/A N/A24 CONCO -LLOQ-01 RUN 05_024 Quality Control 5/7/2019 8:52:58 PM 23 1 012_005.wiff 1.000 35424 106.944 0.360 98285 99.757 93.325 CONCO -LLOQ-02 RUN 05_025 Quality Control 5/7/2019 9:05:33 PM 24 1 012_005.wiff 1.000 35870 106.944 0.380 94315 104.971 98.226 PA1-LOQQC-01 RUN 05_026 Quality Control 5/7/2019 9:18:11 PM 25 1 012_005.wiff 1.000 32496 106.944 0.368 88357 101.682 95.127 PA1-LOQQC-02 RUN 05_027 Quality Control 5/7/2019 9:30:46 PM 26 1 012_005.wiff 1.000 31568 106.944 0.348 90767 96.445 90.228 PA1-LOQQC-03 RUN 05_028 Quality Control 5/7/2019 9:43:19 PM 27 1 012_005.wiff 1.000 36232 106.944 0.389 93132 107.256 100.329 PA1-LOQQC-04 RUN 05_029 Quality Control 5/7/2019 9:55:58 PM 28 1 012_005.wiff 1.000 33345 106.944 0.379 87881 104.738 97.930 PA1-LOQQC-05 RUN 05_030 Quality Control 5/7/2019 10:08:32 PM 29 1 012_005.wiff 1.000 33281 106.944 0.355 93839 98.244 91.931 PA1-LOQQC-06 RUN 05_031 Quality Control 5/7/2019 10:21:09 PM 30 1 012_005.wiff 1.000 34627 106.944 0.373 92867 103.018 96.332 PA1-LQC-01 RUN 05_032 Quality Control 5/7/2019 10:33:45 PM 31 1 012_005.wiff 1.000 91588 307.073 0.996 91945 266.349 86.733 PA1-LQC-02 RUN 05_033 Quality Control 5/7/2019 10:46:20 PM 32 1 012_005.wiff 1.000 99243 307.073 1.057 93865 282.384 92.034 PA1-LQC-03 RUN 05_034 Quality Control 5/7/2019 10:58:59 PM 33 1 012_005.wiff 1.000 100775 307.073 1.109 90911 295.806 96.335 PA1-LQC-04 RUN 05_035 Quality Control 5/7/2019 11:11:36 PM 34 1 012_005.wiff 1.000 96420 307.073 1.056 91289 282.099 91.936 PA1-LQC-05 RUN 05_036 Quality Control 5/7/2019 11:24:11 PM 35 1 012_005.wiff 1.000 99194 307.073 1.066 93032 284.730 92.737 PA1-LQC-06 RUN 05_037 Quality Control 5/7/2019 11:36:46 PM 36 1 012_005.wiff 1.000 99261 307.073 1.094 90771 291.881 95.138 PA1-MQC-01 RUN 05_038 Quality Control 5/7/2019 11:49:19 PM 37 1 012_005.wiff 1.000 1629560 4652.628 16.619 98056 4360.549 93.739 PA1-MQC-02 RUN 05_039 Quality Control 5/8/2019 12:01:55 AM 38 1 012_005.wiff 1.000 1439750 4652.628 15.338 93869 4024.905 86.540 PA1-MQC-03 RUN 05_040 Quality Control 5/8/2019 12:14:34 AM 39 1 012_005.wiff 1.000 1482360 4652.628 16.115 91986 4228.560 90.941 PA1-MQC-04 RUN 05_041 Quality Control 5/8/2019 12:27:09 AM 40 1 012_005.wiff 1.000 1477207 4652.628 16.260 90848 4266.596 91.742 PA1-MQC-05 RUN 05_042 Quality Control 5/8/2019 12:39:48 AM 41 1 012_005.wiff 1.000 1494408 4652.628 16.075 92965 4218.073 90.743 PA1-MQC-06 RUN 05_043 Quality Control 5/8/2019 12:52:23 AM 42 1 012_005.wiff 1.000 1534367 4652.628 16.482 93092 4324.805 93.044 PA1-HQC-01 RUN 05_044 Quality Control 5/8/2019 1:05:01 AM 43 1 012_005.wiff 1.000 2848125 9305.256 33.996 83778 8914.661 95.845 PA1-HQC-02 RUN 05_045 Quality Control 5/8/2019 1:17:36 AM 44 1 012_005.wiff 1.000 2951549 9305.256 33.454 88226 8772.668 94.346 PA1-HQC-03 RUN 05_046 Quality Control 5/8/2019 1:30:10 AM 45 1 012_005.wiff 1.000 3019082 9305.256 32.763 92150 8591.449 92.347 PA1-HQC-04 RUN 05_047 Quality Control 5/8/2019 1:42:46 AM 46 1 012_005.wiff 1.000 2961750 9305.256 32.440 91300 8506.816 91.448 PA1-HQC-05 RUN 05_048 Quality Control 5/8/2019 1:55:20 AM 47 1 012_005.wiff 1.000 3062811 9305.256 33.758 90729 8852.234 95.149 PA1-HQC-06 RUN 05_049 Quality Control 5/8/2019 2:07:59 AM 48 1 012_005.wiff 1.000 3053415 9305.256 33.204 91960 8707.004 93.6






Page 1 of 1

Page 166

Sample Name: "REF LLOQ" Sample ID: "RUN 05_001" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""

Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 112.728 pg/mL Acq. Date: 5/7/2019 Acq. Time: 4:01:46 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.30 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.19 minArea: 48532 countsHeight: 5.42e+003 cpsStart Time: 3.93 minEnd Time: 4.45 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Inte

nsity

, cps

4.19

0.96

1.37

Sample Name: "REF LLOQ" Sample ID: "RUN 05_001" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""

Sample Index: 1 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 5/7/2019 Acq. Time: 4:01:46 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.17 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.16 minArea: 118393 countsHeight: 1.32e+004 cpsStart Time: 3.93 minEnd Time: 4.45 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

Inte

nsity

, cps

4.16

0.92

3.753.23 6.262.28 8.657.13 10.221.620.26 6.02 10.679.508.245.20



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Inte

nsity

, cps

4.19

0.96



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

Inte

nsity

, cps

4.16

0.92

9.477.381.18 5.20 7.875.392.10 10.596.02 9.080.24 9.962.38 3.58

Sample Name: "PA1-STD BL" Sample ID: "RUN 05_003" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""

Sample Index: 3 Sample Type: Blank Concentration: 0.000 pg/mL Calculated Conc: N/A Acq. Date: 5/7/2019 Acq. Time: 4:28:21 PM Modified: No

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

300

350

400

450

Inte

nsity

, cps

0.98

1.42

5.009.14 10.238.163.45 3.962.18 6.28 6.715.93 10.552.480.46

Sample Name: "PA1-STD BL" Sample ID: "RUN 05_003" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""

Sample Index: 3 Sample Type: Blank Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 5/7/2019 Acq. Time: 4:28:21 PM Modified: No

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

Inte

nsity

, cps

2.21

2.93

0.93

1.325.785.28 5.99 7.15 7.454.20 9.868.97 10.20 11.580.20 1.68






Page 1 of 17

Page 167

Sample Name: "PA1-STD BL'" Sample ID: "RUN 05_004" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

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250

300

350

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450

Inte

nsity

, cps

0.98

1.40

3.222.15 7.604.96 11.343.41 9.456.65 11.016.190.48 9.148.55

Sample Name: "PA1-STD BL'" Sample ID: "RUN 05_004" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

Inte

nsity

, cps

2.93

2.21

0.93

1.32

5.77 6.00 7.235.234.19 7.615.01 9.08 9.418.86 11.2110.810.20 1.66

Sample Name: "PA1-STD BL+IS" Sample ID: "RUN 05_005" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

300

350

400

450

Inte

nsity

, cps

0.99

1.42

3.161.87 10.274.59 8.310.10 9.314.984.20 11.707.377.846.975.82

Sample Name: "PA1-STD BL+IS" Sample ID: "RUN 05_005" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""

Sample Index: 5 Sample Type: Blank Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 5/7/2019 Acq. Time: 4:53:33 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.17 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.17 minArea: 93740 countsHeight: 1.00e+004 cpsStart Time: 3.81 minEnd Time: 4.39 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.17

2.93

0.94

1.33

5.80 6.95 7.276.284.96 10.710.28 9.799.59

Sample Name: "PA1-STD BL+IS'" Sample ID: "RUN 05_006" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

300

350

400

Inte

nsity

, cps

0.98

1.40

1.84 2.52 3.43 11.694.85 6.164.03 7.23 10.400.21 7.43 9.875.30 8.20

Sample Name: "PA1-STD BL+IS'" Sample ID: "RUN 05_006" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.162.93

2.22

0.93

1.32

5.79 6.904.82 6.09 7.46 7.65 9.80 10.10 10.779.100.38






Page 2 of 17

Page 168

Sample Name: "PA1-STD A" Sample ID: "RUN 05_007" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""

Sample Index: 7 Sample Type: Standard Concentration: 106.944 pg/mL Calculated Conc: 105.586 pg/mL Acq. Date: 5/7/2019 Acq. Time: 5:18:44 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.30 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.19 minArea: 33218 countsHeight: 3.70e+003 cpsStart Time: 3.93 minEnd Time: 4.45 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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Inte

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4.19

0.981.40

Sample Name: "PA1-STD A" Sample ID: "RUN 05_007" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""

Sample Index: 7 Sample Type: Standard Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 5/7/2019 Acq. Time: 5:18:44 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.17 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.16 minArea: 86804 countsHeight: 9.50e+003 cpsStart Time: 3.81 minEnd Time: 4.42 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.93

2.22

0.93

1.32

5.80 6.32 6.935.27 7.27 11.369.989.59 11.158.641.68

Sample Name: "PA1-STD A'" Sample ID: "RUN 05_008" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

4.19

0.981.41

Sample Name: "PA1-STD A'" Sample ID: "RUN 05_008" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.212.95

0.93

1.326.966.355.25 5.79 7.20 11.3110.9710.319.249.030.40 1.72

Sample Name: "PA1-STD B" Sample ID: "RUN 05_009" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

8000

Inte

nsity

, cps

4.19

0.98

Sample Name: "PA1-STD B" Sample ID: "RUN 05_009" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93 2.94

2.22

1.325.795.25 6.944.96 7.236.30 9.27 9.91 11.297.770.35






Page 3 of 17

Page 169

Sample Name: "PA1-STD C" Sample ID: "RUN 05_010" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

Inte

nsity

, cps

4.19

Sample Name: "PA1-STD C" Sample ID: "RUN 05_010" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.21

2.940.93

1.325.79 6.904.82 7.236.29 11.5010.359.118.730.10

Sample Name: "PA1-STD D" Sample ID: "RUN 05_011" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

Inte

nsity

, cps

4.19

Sample Name: "PA1-STD D" Sample ID: "RUN 05_011" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

2.940.93 2.21

1.325.795.25 7.006.34 10.647.47 7.74 8.96 11.8210.040.37

Sample Name: "PA1-STD E" Sample ID: "RUN 05_012" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

Inte

nsity

, cps

4.19

Sample Name: "PA1-STD E" Sample ID: "RUN 05_012" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

2.21

2.94

0.93

1.325.25 5.79 6.946.32 7.21 10.019.02 11.157.740.08 1.79






Page 4 of 17

Page 170

Sample Name: "PA1-STD F" Sample ID: "RUN 05_013" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

1.10e5

1.20e5

Inte

nsity

, cps

4.20

Sample Name: "PA1-STD F" Sample ID: "RUN 05_013" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

2.93

2.220.93

1.33

5.785.26 6.33 6.99 7.72 9.118.80 9.69 10.040.24 1.69

Sample Name: "PA1-STD G" Sample ID: "RUN 05_014" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

Inte

nsity

, cps

4.19

Sample Name: "PA1-STD G" Sample ID: "RUN 05_014" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.21

2.93

0.93

1.325.79 6.964.81 7.29 10.23 11.788.889.388.510.61

Sample Name: "PA1-STD H" Sample ID: "RUN 05_015" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

Inte

nsity

, cps

4.19

Sample Name: "PA1-STD H" Sample ID: "RUN 05_015" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.932.21

0.93

1.32

5.815.25 6.986.31 7.18 9.17 10.57 11.2710.198.610.42






Page 5 of 17

Page 171

Sample Name: "PA1-STD H'" Sample ID: "RUN 05_016" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

Inte

nsity

, cps

4.19

Sample Name: "PA1-STD H'" Sample ID: "RUN 05_016" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.210.93 2.94

1.325.78 6.955.23 7.336.274.77 9.13 11.510.40 8.65

Sample Name: "REF BLANK" Sample ID: "RUN 05_017" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

100

200

300

400

500

600

700

Inte

nsity

, cps

0.96

1.37

3.392.852.480.31 7.08 11.584.56 4.82 6.72 10.827.89 9.08 9.448.17

Sample Name: "REF BLANK" Sample ID: "RUN 05_017" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

0.92

11.079.716.716.51 7.17 10.478.665.19 9.124.02 4.320.22 1.18 2.762.29

Sample Name: "STD H NO IS" Sample ID: "RUN 05_018" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""

Sample Index: 18 Sample Type: Unknown Concentration: N/A Calculated Conc: No Intercept Acq. Date: 5/7/2019 Acq. Time: 7:37:23 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.30 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.19 minArea: 4836295 countsHeight: 5.33e+005 cpsStart Time: 3.90 minEnd Time: 4.75 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

Inte

nsity

, cps

4.19

Sample Name: "STD H NO IS" Sample ID: "RUN 05_018" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""

Sample Index: 18 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 5/7/2019 Acq. Time: 7:37:23 PM Modified: No

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

0.93

2.952.21

1.32

5.786.975.274.773.51 6.41 7.16 8.48 8.93 10.71 11.850.36






Page 6 of 17

Page 172

Sample Name: "STD H NO IS'" Sample ID: "RUN 05_019" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

Inte

nsity

, cps

4.19

Sample Name: "STD H NO IS'" Sample ID: "RUN 05_019" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Inte

nsity

, cps

2.21

2.950.93

1.32

5.81 6.973.48 5.26 7.186.334.97 7.74 11.309.789.16 9.980.37 1.69

Sample Name: "CONCO - BLK" Sample ID: "RUN 05_020" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

300

350

400

450

500

Inte

nsity

, cps

0.98

1.40

3.422.62 4.980.33 3.78 7.07 9.85 11.257.98 8.555.47 10.996.60

Sample Name: "CONCO - BLK" Sample ID: "RUN 05_020" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

2.210.93

2.94

1.32

5.77 6.975.26 7.244.814.00 6.03 9.24 10.818.360.14

Sample Name: "CONCO - BLK'" Sample ID: "RUN 05_021" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

300

350

400

450

Inte

nsity

, cps

0.98

1.40

1.620.68 6.513.35 4.88 6.09 9.272.53 9.988.43 10.314.11 11.607.60

Sample Name: "CONCO - BLK'" Sample ID: "RUN 05_021" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

Inte

nsity

, cps

2.942.21

0.93

1.32

5.77 6.59 6.894.804.21 7.45 10.06 11.6511.339.548.290.42 1.82






Page 7 of 17

Page 173

Sample Name: "CONCO - BLK + IS" Sample ID: "RUN 05_022" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

300

350

400

Inte

nsity

, cps

0.97

1.40

0.33 1.652.14 6.212.753.60 4.993.91 11.196.47 7.42 7.77 10.809.085.86

Sample Name: "CONCO - BLK + IS" Sample ID: "RUN 05_022" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

1.20e4

Inte

nsity

, cps

4.16

0.93

2.952.211.32

5.805.24 6.976.07 7.465.01 9.07 9.59 11.4010.597.820.36

Sample Name: "CONCO - BLK + IS'" Sample ID: "RUN 05_023" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

300

350

400

450

Inte

nsity

, cps

0.98

1.40

2.932.390.40 3.41 7.60 10.274.89 6.16 7.22 9.674.34 8.365.75 10.99

Sample Name: "CONCO - BLK + IS'" Sample ID: "RUN 05_023" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93

2.952.221.33

5.82 7.285.263.50 6.96 11.497.75 9.188.87 10.19

Sample Name: "CONCO -LLOQ-01" Sample ID: "RUN 05_024" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""

Sample Index: 24 Sample Type: QC Concentration: 106.944 pg/mL Calculated Conc: 99.757 pg/mL Acq. Date: 5/7/2019 Acq. Time: 8:52:58 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.30 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.19 minArea: 35424 countsHeight: 3.93e+003 cpsStart Time: 3.96 minEnd Time: 4.45 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

, cps

4.19

0.971.40

Sample Name: "CONCO -LLOQ-01" Sample ID: "RUN 05_024" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""

Sample Index: 24 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 5/7/2019 Acq. Time: 8:52:58 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.17 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.16 minArea: 98285 countsHeight: 1.08e+004 cpsStart Time: 3.81 minEnd Time: 4.45 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93

1.32 2.22 2.99 4.81 6.075.78 6.94 7.18 11.5810.399.618.780.20






Page 8 of 17

Page 174



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

, cps

4.19

0.971.40



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93

1.322.922.21 5.79 6.984.953.48 6.31 7.44 10.32 10.538.96 10.050.24

Sample Name: "PA1-LOQQC-01" Sample ID: "RUN 05_026" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

, cps

4.19

0.98

1.41

Sample Name: "PA1-LOQQC-01" Sample ID: "RUN 05_026" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.210.93

2.94

1.325.80 6.944.95 7.43 11.649.70 10.009.251.700.30



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

, cps

4.19

0.971.40



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

0.932.21 2.94

1.32

5.785.26 6.946.013.49 4.78 7.40 9.15 9.97 11.4510.668.750.59






Page 9 of 17

Page 175



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

4.19

0.981.40



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93 2.942.21

1.32

5.795.23 6.31 7.496.95 11.1310.479.218.710.32



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

, cps

4.19

0.971.40



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.930.932.21

1.32

5.78 6.945.264.99 6.32 7.25 9.608.56 9.09 10.74 11.420.15 1.70



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

, cps

4.18

0.981.40



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.932.94

2.211.32

5.785.25 6.32 6.90 7.26 11.429.26 10.649.619.02






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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

, cps

4.19

0.971.40



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93 2.21

2.941.32

5.80 6.995.26 6.013.50 7.234.78 10.088.87 11.820.40 8.53

Sample Name: "PA1-LQC-01" Sample ID: "RUN 05_032" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.19

Sample Name: "PA1-LQC-01" Sample ID: "RUN 05_032" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.940.93

2.22

1.32

5.79 6.965.26 6.01 7.37 10.59 11.4710.148.860.37



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.19



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.21

0.932.94

1.32

5.79 6.956.324.923.48 7.32 9.21 9.48 10.04 10.747.890.38 1.69






Page 11 of 17

Page 177



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.20



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.17

0.93 2.952.22

1.33

5.79 6.935.28 6.34 7.28 11.327.76 11.0910.289.500.31



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.950.93

2.221.32

5.804.94 6.92 10.447.32 10.149.25 11.160.17



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.19



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93

2.942.211.32

6.956.335.803.49 5.254.96 7.19 8.87 9.33 10.700.15






Page 12 of 17

Page 178



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.15

0.93

2.211.32

2.945.815.27 6.936.32 7.213.54 11.7910.449.448.87

Sample Name: "PA1-MQC-01" Sample ID: "RUN 05_038" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Inte

nsity

, cps

4.19

Sample Name: "PA1-MQC-01" Sample ID: "RUN 05_038" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.210.93 2.94

1.32

5.795.254.96 6.94 7.226.33 9.42 11.7611.039.018.420.23


Sample Index: 39 Sample Type: QC Concentration: 4652.628 pg/mL Calculated Conc: 4024.905 pg/mL Acq. Date: 5/8/2019 Acq. Time: 12:01:55 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.30 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.19 minArea: 1439750 countsHeight: 1.59e+005 cpsStart Time: 3.90 minEnd Time: 4.66 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

Inte

nsity

, cps

4.19


Sample Index: 39 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 5/8/2019 Acq. Time: 12:01:55 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 50.00 cpsArea Threshold: 500.00 cps,Num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.17 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.16 minArea: 93869 countsHeight: 1.04e+004 cpsStart Time: 3.87 minEnd Time: 4.42 min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

2.21

0.932.94

1.325.78 6.974.82 6.333.48 7.277.70 10.80 11.619.619.391.720.41






Page 13 of 17

Page 179



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Inte

nsity

, cps

4.19



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.210.93

2.94

1.32

5.78 6.975.09 6.28 7.25 11.7110.628.90 9.170.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Inte

nsity

, cps

4.19



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.15

0.93

1.32 2.952.215.795.25 6.973.50 7.284.76 6.32 10.059.57 10.818.820.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Inte

nsity

, cps

4.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93

2.951.32 2.21

5.79 6.955.985.253.50 7.19 9.074.78 9.38 11.208.560.10 1.70






Page 14 of 17

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Inte

nsity

, cps

4.19



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.940.93 2.21

1.32

5.795.23 6.92 7.216.29 10.668.80 9.470.08

Sample Name: "PA1-HQC-01" Sample ID: "RUN 05_044" File: "012_005.wiff"Peak Name: "d5 BKC C12" Mass(es): "309.300/212.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

4.19

Sample Name: "PA1-HQC-01" Sample ID: "RUN 05_044" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

0.93

1.33

5.783.16 5.27 7.00 7.192.72 9.492.26 11.2310.678.83



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

4.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.15

0.92

1.322.21 5.773.13 5.994.91 6.93 7.43 9.74 10.409.47 11.008.500.32






Page 15 of 17

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

4.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.92

1.32

5.792.21 6.963.15 5.24 6.31 7.23 7.79 9.60 10.679.141.71



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

4.19



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

0.93

1.335.813.15 6.37 6.944.952.21 7.40 11.4411.198.98 10.158.620.42



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

4.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93

1.322.21 2.95 5.805.25 6.893.52 6.04 7.224.77 7.72 9.368.85 10.560.11






Page 16 of 17

Page 182



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

4.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.92

1.32

2.21 5.773.17 7.206.954.765.26 7.69 10.10 11.5511.228.630.28 1.66






Page 17 of 17

Page 183





Page 184

Sample Name Sample ID Sample Type Acquisition Date Vial Position

Plate Position File Name Dilution

FactorAnalyte Peak Area (counts)

Analyte Concentration

(pg/mL)

Area Ratio

IS Peak Area (counts) Use Record Record

ModifiedCalculated

Concentration (pg/mL)

Accuracy (%)

1 REF LLOQ RUN 05_001 Unknown 5/7/2019 4:01:46 PM 1 1 012_005.wiff 1.000 45441 N/A 0.384 118393 35.724 N/A2 REF LLOQ RUN 05_002 Unknown 5/7/2019 4:15:45 PM 1 1 012_005.wiff 1.000 45732 N/A 0.385 118650 35.857 N/A3 PA1-STD BL RUN 05_003 Blank 5/7/2019 4:28:21 PM 3 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A4 PA1-STD BL' RUN 05_004 Blank 5/7/2019 4:40:53 PM 4 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A5 PA1-STD BL+IS RUN 05_005 Blank 5/7/2019 4:53:33 PM 5 1 012_005.wiff 1.000 0 0.000 0.000 93740 N/A N/A6 PA1-STD BL+IS' RUN 05_006 Blank 5/7/2019 5:06:08 PM 6 1 012_005.wiff 1.000 0 0.000 0.000 88548 N/A N/A7 PA1-STD A RUN 05_007 Standard 5/7/2019 5:18:44 PM 7 1 012_005.wiff 1.000 30377 32.493 0.350 86804 32.933 101.48 PA1-STD A' RUN 05_008 Standard 5/7/2019 5:31:21 PM 8 1 012_005.wiff 1.000 31924 32.493 0.354 90255 33.243 102.39 PA1-STD B RUN 05_009 Standard 5/7/2019 5:43:57 PM 9 1 012_005.wiff 1.000 66969 64.986 0.712 94049 62.768 96.610 PA1-STD C RUN 05_010 Standard 5/7/2019 5:56:33 PM 10 1 012_005.wiff 1.000 131097 129.972 1.444 90796 123.064 94.711 PA1-STD D RUN 05_011 Standard 5/7/2019 6:09:10 PM 11 1 012_005.wiff 1.000 277702 259.943 2.972 93425 249.007 95.812 PA1-STD E RUN 05_012 Standard 5/7/2019 6:21:46 PM 12 1 012_005.wiff 1.000 553365 519.886 6.058 91350 503.198 96.813 PA1-STD F RUN 05_013 Standard 5/7/2019 6:34:19 PM 13 1 012_005.wiff 1.000 1153142 1039.773 12.410 92924 1026.547 98.714 PA1-STD G RUN 05_014 Standard 5/7/2019 6:46:58 PM 14 1 012_005.wiff 1.000 2244796 2079.545 26.162 85803 2159.641 103.915 PA1-STD H RUN 05_015 Standard 5/7/2019 6:59:35 PM 15 1 012_005.wiff 1.000 4379786 4159.090 52.220 83872 4306.592 103.516 PA1-STD H' RUN 05_016 Standard 5/7/2019 7:12:12 PM 16 1 012_005.wiff 1.000 4595751 4159.090 53.638 85682 4423.386 106.417 REF BLANK RUN 05_017 Blank 5/7/2019 7:24:44 PM 2 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A18 STD H NO IS RUN 05_018 Unknown 5/7/2019 7:37:23 PM 17 1 012_005.wiff 1.000 4405226 N/A #DIV/0! 0 #DIV/0! N/A19 STD H NO IS' RUN 05_019 Unknown 5/7/2019 7:49:58 PM 18 1 012_005.wiff 1.000 4370215 N/A #DIV/0! 0 #DIV/0! N/A20 CONCO - BLK RUN 05_020 Blank 5/7/2019 8:02:34 PM 19 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A21 CONCO - BLK' RUN 05_021 Blank 5/7/2019 8:15:11 PM 20 1 012_005.wiff 1.000 0 0.000 #DIV/0! 0 N/A N/A22 CONCO - BLK + IS RUN 05_022 Blank 5/7/2019 8:27:45 PM 21 1 012_005.wiff 1.000 0 0.000 0.000 99780 N/A N/A23 CONCO - BLK + IS' RUN 05_023 Blank 5/7/2019 8:40:22 PM 22 1 012_005.wiff 1.000 0 0.000 0.000 94023 N/A N/A24 CONCO -LLOQ-01 RUN 05_024 Quality Control 5/7/2019 8:52:58 PM 23 1 012_005.wiff 1.000 32007 32.493 0.326 98285 30.932 95.225 CONCO -LLOQ-02 RUN 05_025 Quality Control 5/7/2019 9:05:33 PM 24 1 012_005.wiff 1.000 30015 32.493 0.318 94315 30.322 93.326 PA1-LOQQC-01 RUN 05_026 Quality Control 5/7/2019 9:18:11 PM 25 1 012_005.wiff 1.000 30883 32.493 0.350 88357 32.899 101.227 PA1-LOQQC-02 RUN 05_027 Quality Control 5/7/2019 9:30:46 PM 26 1 012_005.wiff 1.000 28046 32.493 0.309 90767 29.559 91.028 PA1-LOQQC-03 RUN 05_028 Quality Control 5/7/2019 9:43:19 PM 27 1 012_005.wiff 1.000 30982 32.493 0.333 93132 31.509 97.029 PA1-LOQQC-04 RUN 05_029 Quality Control 5/7/2019 9:55:58 PM 28 1 012_005.wiff 1.000 29690 32.493 0.338 87881 31.937 98.330 PA1-LOQQC-05 RUN 05_030 Quality Control 5/7/2019 10:08:32 PM 29 1 012_005.wiff 1.000 29530 32.493 0.315 93839 30.028 92.431 PA1-LOQQC-06 RUN 05_031 Quality Control 5/7/2019 10:21:09 PM 30 1 012_005.wiff 1.000 30032 32.493 0.323 92867 30.745 94.632 PA1-LQC-01 RUN 05_032 Quality Control 5/7/2019 10:33:45 PM 31 1 012_005.wiff 1.000 89299 93.298 0.971 91945 84.121 90.233 PA1-LQC-02 RUN 05_033 Quality Control 5/7/2019 10:46:20 PM 32 1 012_005.wiff 1.000 96149 93.298 1.024 93865 88.498 94.934 PA1-LQC-03 RUN 05_034 Quality Control 5/7/2019 10:58:59 PM 33 1 012_005.wiff 1.000 96113 93.298 1.057 90911 91.207 97.835 PA1-LQC-04 RUN 05_035 Quality Control 5/7/2019 11:11:36 PM 34 1 012_005.wiff 1.000 93955 93.298 1.029 91289 88.899 95.336 PA1-LQC-05 RUN 05_036 Quality Control 5/7/2019 11:24:11 PM 35 1 012_005.wiff 1.000 93335 93.298 1.003 93032 86.761 93.037 PA1-LQC-06 RUN 05_037 Quality Control 5/7/2019 11:36:46 PM 36 1 012_005.wiff 1.000 94095 93.298 1.037 90771 89.510 95.938 PA1-MQC-01 RUN 05_038 Quality Control 5/7/2019 11:49:19 PM 37 1 012_005.wiff 1.000 1566627 1413.612 15.977 98056 1320.461 93.439 PA1-MQC-02 RUN 05_039 Quality Control 5/8/2019 12:01:55 AM 38 1 012_005.wiff 1.000 1340565 1413.612 14.281 93869 1180.760 83.540 PA1-MQC-03 RUN 05_040 Quality Control 5/8/2019 12:14:34 AM 39 1 012_005.wiff 1.000 1446537 1413.612 15.726 91986 1299.758 91.941 PA1-MQC-04 RUN 05_041 Quality Control 5/8/2019 12:27:09 AM 40 1 012_005.wiff 1.000 1402675 1413.612 15.440 90848 1276.209 90.342 PA1-MQC-05 RUN 05_042 Quality Control 5/8/2019 12:39:48 AM 41 1 012_005.wiff 1.000 1423836 1413.612 15.316 92965 1266.002 89.643 PA1-MQC-06 RUN 05_043 Quality Control 5/8/2019 12:52:23 AM 42 1 012_005.wiff 1.000 1420908 1413.612 15.263 93092 1261.685 89.344 PA1-HQC-01 RUN 05_044 Quality Control 5/8/2019 1:05:01 AM 43 1 012_005.wiff 1.000 2666065 2827.224 31.823 83778 2626.054 92.945 PA1-HQC-02 RUN 05_045 Quality Control 5/8/2019 1:17:36 AM 44 1 012_005.wiff 1.000 2845226 2827.224 32.249 88226 2661.169 94.146 PA1-HQC-03 RUN 05_046 Quality Control 5/8/2019 1:30:10 AM 45 1 012_005.wiff 1.000 2857291 2827.224 31.007 92150 2558.828 90.547 PA1-HQC-04 RUN 05_047 Quality Control 5/8/2019 1:42:46 AM 46 1 012_005.wiff 1.000 2787293 2827.224 30.529 91300 2519.444 89.148 PA1-HQC-05 RUN 05_048 Quality Control 5/8/2019 1:55:20 AM 47 1 012_005.wiff 1.000 2894824 2827.224 31.906 90729 2632.925 93.149 PA1-HQC-06 RUN 05_049 Quality Control 5/8/2019 2:07:59 AM 48 1 012_005.wiff 1.000 2872776 2827.224 31.239 91960 2577.971 91.2






Page 1 of 1

Page 185



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

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3000

Inte

nsity

, cps

9.58

0.95



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

Inte

nsity

, cps

4.16

0.92

3.753.23 6.262.28 8.657.13 10.221.620.26 6.02 10.679.508.245.20



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

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Inte

nsity

, cps

9.59

0.96



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

Inte

nsity

, cps

4.16

0.92

9.477.381.18 5.20 7.875.392.10 10.596.02 9.080.24 9.962.38 3.58

Sample Name: "PA1-STD BL" Sample ID: "RUN 05_003" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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1000

Inte

nsity

, cps

0.98

1.41

4.51 7.421.94 2.26 8.29 8.96 11.852.743.21 11.0010.257.085.450.43

Sample Name: "PA1-STD BL" Sample ID: "RUN 05_003" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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1000

2000

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8000

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Inte

nsity

, cps

2.21

2.93

0.93

1.325.785.28 5.99 7.15 7.454.20 9.868.97 10.20 11.580.20 1.68






Page 1 of 17

Page 186

Sample Name: "PA1-STD BL'" Sample ID: "RUN 05_004" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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100

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500

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700

800

900

1000

Inte

nsity

, cps

0.97

1.41

2.517.41

8.848.21 9.314.520.31 7.014.971.84 3.08 6.043.97 11.17

Sample Name: "PA1-STD BL'" Sample ID: "RUN 05_004" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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1000

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4000

5000

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7000

8000

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Inte

nsity

, cps

2.93

2.21

0.93

1.32

5.77 6.00 7.235.234.19 7.615.01 9.08 9.418.86 11.2110.810.20 1.66

Sample Name: "PA1-STD BL+IS" Sample ID: "RUN 05_005" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

100

200

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500

600

700

800

900

1000

Inte

nsity

, cps

0.99

1.40

2.66 4.53 8.707.422.488.083.55 10.85 11.096.495.310.11

Sample Name: "PA1-STD BL+IS" Sample ID: "RUN 05_005" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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3000.00

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5000.00

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7000.00

8000.00

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1.10e4

Inte

nsity

, cps

4.17

2.93

0.94

1.33

5.80 6.95 7.276.284.96 10.710.28 9.799.59

Sample Name: "PA1-STD BL+IS'" Sample ID: "RUN 05_006" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

100

200

300

400

500

600

700

800

Inte

nsity

, cps

0.97

1.402.51

4.538.26 9.127.361.840.27 9.353.04 9.946.07 11.44

Sample Name: "PA1-STD BL+IS'" Sample ID: "RUN 05_006" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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1000.00

2000.00

3000.00

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5000.00

6000.00

7000.00

8000.00

9000.00

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Inte

nsity

, cps

4.162.93

2.22

0.93

1.32

5.79 6.904.82 6.09 7.46 7.65 9.80 10.10 10.779.100.38






Page 2 of 17

Page 187

Sample Name: "PA1-STD A" Sample ID: "RUN 05_007" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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2400

Inte

nsity

, cps

9.58

0.97

1.41 2.531.67 9.154.53 8.097.350.27 3.87 5.09 11.346.08 11.01

Sample Name: "PA1-STD A" Sample ID: "RUN 05_007" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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Inte

nsity

, cps

4.16

2.93

2.22

0.93

1.32

5.80 6.32 6.935.27 7.27 11.369.989.59 11.158.641.68

Sample Name: "PA1-STD A'" Sample ID: "RUN 05_008" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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Inte

nsity

, cps

9.58

0.98

1.41 2.538.144.51 8.817.450.28 4.813.14 7.21 10.126.72 11.314.02

Sample Name: "PA1-STD A'" Sample ID: "RUN 05_008" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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2000.00

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4000.00

5000.00

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7000.00

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9000.00

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Inte

nsity

, cps

4.16

2.212.95

0.93

1.326.966.355.25 5.79 7.20 11.3110.9710.319.249.030.40 1.72

Sample Name: "PA1-STD B" Sample ID: "RUN 05_009" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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500

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1500

2000

2500

3000

3500

4000

4500

5000

Inte

nsity

, cps

9.59

0.98

1.412.48

Sample Name: "PA1-STD B" Sample ID: "RUN 05_009" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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4000.00

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6000.00

7000.00

8000.00

9000.00

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1.10e4

Inte

nsity

, cps

4.16

0.93 2.94

2.22

1.325.795.25 6.944.96 7.236.30 9.27 9.91 11.297.770.35






Page 3 of 17

Page 188

Sample Name: "PA1-STD C" Sample ID: "RUN 05_010" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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Inte

nsity

, cps

9.58

0.98

Sample Name: "PA1-STD C" Sample ID: "RUN 05_010" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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2000.00

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Inte

nsity

, cps

4.16

2.21

2.940.93

1.325.79 6.904.82 7.236.29 11.5010.359.118.730.10

Sample Name: "PA1-STD D" Sample ID: "RUN 05_011" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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Inte

nsity

, cps

9.58

0.98

Sample Name: "PA1-STD D" Sample ID: "RUN 05_011" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

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6000.00

7000.00

8000.00

9000.00

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1.10e4

Inte

nsity

, cps

4.16

2.940.93 2.21

1.325.795.25 7.006.34 10.647.47 7.74 8.96 11.8210.040.37

Sample Name: "PA1-STD E" Sample ID: "RUN 05_012" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

Inte

nsity

, cps

9.59

Sample Name: "PA1-STD E" Sample ID: "RUN 05_012" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

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8000.00

9000.00

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1.10e4

Inte

nsity

, cps

4.16

2.21

2.94

0.93

1.325.25 5.79 6.946.32 7.21 10.019.02 11.157.740.08 1.79






Page 4 of 17

Page 189

Sample Name: "PA1-STD F" Sample ID: "RUN 05_013" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

Inte

nsity

, cps

9.58

Sample Name: "PA1-STD F" Sample ID: "RUN 05_013" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

2.93

2.220.93

1.33

5.785.26 6.33 6.99 7.72 9.118.80 9.69 10.040.24 1.69

Sample Name: "PA1-STD G" Sample ID: "RUN 05_014" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Inte

nsity

, cps

9.58

Sample Name: "PA1-STD G" Sample ID: "RUN 05_014" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.21

2.93

0.93

1.325.79 6.964.81 7.29 10.23 11.788.889.388.510.61

Sample Name: "PA1-STD H" Sample ID: "RUN 05_015" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

9.58

Sample Name: "PA1-STD H" Sample ID: "RUN 05_015" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.932.21

0.93

1.32

5.815.25 6.986.31 7.18 9.17 10.57 11.2710.198.610.42






Page 5 of 17

Page 190

Sample Name: "PA1-STD H'" Sample ID: "RUN 05_016" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

9.58

Sample Name: "PA1-STD H'" Sample ID: "RUN 05_016" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.210.93 2.94

1.325.78 6.955.23 7.336.274.77 9.13 11.510.40 8.65

Sample Name: "REF BLANK" Sample ID: "RUN 05_017" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

Inte

nsity

, cps

0.96

2.54

1.850.16

9.198.03 8.953.49 11.873.77 4.52 5.87 10.426.42

Sample Name: "REF BLANK" Sample ID: "RUN 05_017" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

0.92

11.079.716.716.51 7.17 10.478.665.19 9.124.02 4.320.22 1.18 2.762.29

Sample Name: "STD H NO IS" Sample ID: "RUN 05_018" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

9.58

Sample Name: "STD H NO IS" Sample ID: "RUN 05_018" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

0.93

2.952.21

1.32

5.786.975.274.773.51 6.41 7.16 8.48 8.93 10.71 11.850.36






Page 6 of 17

Page 191

Sample Name: "STD H NO IS'" Sample ID: "RUN 05_019" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

Inte

nsity

, cps

9.58

Sample Name: "STD H NO IS'" Sample ID: "RUN 05_019" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Inte

nsity

, cps

2.21

2.950.93

1.32

5.81 6.973.48 5.26 7.186.334.97 7.74 11.309.789.16 9.980.37 1.69

Sample Name: "CONCO - BLK" Sample ID: "RUN 05_020" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

1200

Inte

nsity

, cps

0.98

1.41

2.481.862.780.58 4.523.36 9.158.117.41 9.314.86 11.3911.085.97 6.37

Sample Name: "CONCO - BLK" Sample ID: "RUN 05_020" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, cps

2.210.93

2.94

1.32

5.77 6.975.26 7.244.814.00 6.03 9.24 10.818.360.14

Sample Name: "CONCO - BLK'" Sample ID: "RUN 05_021" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

Inte

nsity

, cps

0.97

1.42

2.440.22 4.493.58 11.799.377.45 8.965.70 7.215.45 10.83

Sample Name: "CONCO - BLK'" Sample ID: "RUN 05_021" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

Inte

nsity

, cps

2.942.21

0.93

1.32

5.77 6.59 6.894.804.21 7.45 10.06 11.6511.339.548.290.42 1.82






Page 7 of 17

Page 192

Sample Name: "CONCO - BLK + IS" Sample ID: "RUN 05_022" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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200

400

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1200

Inte

nsity

, cps

0.98

1.40

1.88 2.39 4.500.61 3.12 3.47 11.787.49 8.93 10.149.504.96 6.00 6.89

Sample Name: "CONCO - BLK + IS" Sample ID: "RUN 05_022" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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5000.00

6000.00

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1.10e4

1.20e4

Inte

nsity

, cps

4.16

0.93

2.952.211.32

5.805.24 6.976.07 7.465.01 9.07 9.59 11.4010.597.820.36

Sample Name: "CONCO - BLK + IS'" Sample ID: "RUN 05_023" File: "012_005.wiff"Peak Name: "d5 BKC C14" Mass(es): "337.300/240.300 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

1200

Inte

nsity

, cps

0.98

1.41

2.51

0.28 3.06 4.538.77 9.348.103.57 7.44 11.7810.046.01 7.155.31

Sample Name: "CONCO - BLK + IS'" Sample ID: "RUN 05_023" File: "012_005.wiff"Peak Name: "d13 BKC(IS)" Mass(es): "317.200/217.200 Da"Comment: "" Annotation: ""


0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93

2.952.221.33

5.82 7.285.263.50 6.96 11.497.75 9.188.87 10.19



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

Inte

nsity

, cps

9.57

0.98

1.41

2.602.03 3.060.29 4.52 8.898.724.93 7.40 11.595.54 6.33



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93

1.32 2.22 2.99 4.81 6.075.78 6.94 7.18 11.5810.399.618.780.20






Page 8 of 17

Page 193



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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2200

2400

Inte

nsity

, cps

9.57

0.98

1.412.50

1.65 4.530.284.02 7.42 7.99 8.986.08 6.685.72



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93

1.322.922.21 5.79 6.984.953.48 6.31 7.44 10.32 10.538.96 10.050.24



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

Inte

nsity

, cps

9.57

0.98

1.412.47

2.770.27 4.54 7.38 8.123.67 9.12 11.866.26 10.38



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.210.93

2.94

1.325.80 6.944.95 7.43 11.649.70 10.009.251.700.30



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Inte

nsity

, cps

9.57

0.98

1.412.52

1.890.39 4.513.64 7.43 8.898.73 11.6710.976.38



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

0.932.21 2.94

1.32

5.785.26 6.946.013.49 4.78 7.40 9.15 9.97 11.4510.668.750.59






Page 9 of 17

Page 194



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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1400

1600

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2400

Inte

nsity

, cps

9.57

0.98

1.412.51

1.640.23 4.51 7.413.36 9.173.70 8.03 11.4410.345.18 5.70 6.06



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

0.93 2.942.21

1.32

5.795.23 6.31 7.496.95 11.1310.479.218.710.32



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Inte

nsity

, cps

9.58

0.98

1.412.52

2.78 11.681.780.28 10.714.54 8.908.257.913.58 5.30 5.93



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.930.932.21

1.32

5.78 6.945.264.99 6.32 7.25 9.608.56 9.09 10.74 11.420.15 1.70



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

Inte

nsity

, cps

9.57

0.98

1.41 2.52

4.490.24 3.11 4.05 5.243.57 7.50 8.708.065.82 6.68 10.96



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.932.94

2.211.32

5.785.25 6.32 6.90 7.26 11.429.26 10.649.619.02






Page 10 of 17

Page 195



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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1200

1400

1600

1800

2000

2200

2400

Inte

nsity

, cps

9.57

0.97

1.41 2.532.81 4.52 8.990.31 7.414.71 6.043.45 6.62 8.06 11.2110.69



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93 2.21

2.941.32

5.80 6.995.26 6.013.50 7.234.78 10.088.87 11.820.40 8.53



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

Inte

nsity

, cps

9.58

0.98

1.42 2.43 4.520.19 3.06 3.52



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

2.940.93

2.22

1.32

5.79 6.965.26 6.01 7.37 10.59 11.4710.148.860.37



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

Inte

nsity

, cps

9.57

0.97

2.481.420.61 4.51 4.82



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.21

0.932.94

1.32

5.79 6.956.324.923.48 7.32 9.21 9.48 10.04 10.747.890.38 1.69






Page 11 of 17

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

Inte

nsity

, cps

9.58

0.991.41 2.500.24 4.50 4.90



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.17

0.93 2.952.22

1.33

5.79 6.935.28 6.34 7.28 11.327.76 11.0910.289.500.31



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

Inte

nsity

, cps

9.57

0.98

1.40 2.531.66 4.570.46 3.743.40



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.950.93

2.221.32

5.804.94 6.92 10.447.32 10.149.25 11.160.17



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

Inte

nsity

, cps

9.57

0.98

1.42 2.501.70 4.530.63 3.08 3.63



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93

2.942.211.32

6.956.335.803.49 5.254.96 7.19 8.87 9.33 10.700.15






Page 12 of 17

Page 197



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

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3000

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5000

6000

7000

Inte

nsity

, cps

9.57

0.97

1.42 2.560.34 4.703.10 7.483.83



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.15

0.93

2.211.32

2.945.815.27 6.936.32 7.213.54 11.7910.449.448.87



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

1.10e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.210.93 2.94

1.32

5.795.254.96 6.94 7.226.33 9.42 11.7611.039.018.420.23



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.16

2.21

0.932.94

1.325.78 6.974.82 6.333.48 7.277.70 10.80 11.619.619.391.720.41






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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.210.93

2.94

1.32

5.78 6.975.09 6.28 7.25 11.7110.628.90 9.170.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.15

0.93

1.32 2.952.215.795.25 6.973.50 7.284.76 6.32 10.059.57 10.818.820.18



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93

2.951.32 2.21

5.79 6.955.985.253.50 7.19 9.074.78 9.38 11.208.560.10 1.70






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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

2.940.93 2.21

1.32

5.795.23 6.92 7.216.29 10.668.80 9.470.08



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

0.93

1.33

5.783.16 5.27 7.00 7.192.72 9.492.26 11.2310.678.83



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

Inte

nsity

, cps

9.56



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.15

0.92

1.322.21 5.773.13 5.994.91 6.93 7.43 9.74 10.409.47 11.008.500.32






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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

Inte

nsity

, cps

9.56



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.92

1.32

5.792.21 6.963.15 5.24 6.31 7.23 7.79 9.60 10.679.141.71



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

Inte

nsity

, cps

4.16

0.93

1.335.813.15 6.37 6.944.952.21 7.40 11.4411.198.98 10.158.620.42



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

Inte

nsity

, cps

9.57



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.93

1.322.21 2.95 5.805.25 6.893.52 6.04 7.224.77 7.72 9.368.85 10.560.11






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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

Inte

nsity

, cps

9.56



0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Inte

nsity

, cps

4.15

0.92

1.32

2.21 5.773.17 7.206.954.765.26 7.69 10.10 11.5511.228.630.28 1.66






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