The Practical Non-Existance of Official NMR-Based Test ...process-nmr.com/Presentation/John Edwards - NMR...ASTM D6122 - 15 Standard Practice for Validation of the Performance of Multivariate

The Practical Non-Existance of Official NMR-Based Test Methods - How to Change the Paradigm of NMR Exclusion

The Perspective of a Small NMR Service Company

John Edwards,Process NMR Associates

Validation Workshop – PANIC NMR Conference – February 19, 2016 – Houston Texas

ASTM NMR Test MethodsE2977: Standard Practice for Measuring and Reporting Performance of Fourier Transform Nuclear Magnetic Resonance (FT-NMR) Spectrometers for Liquid Samples

D7171-05: Standard Test Method for Hydrogen Content of Middle Distillate Petroleum Products by Low-Resolution Pulsed Nuclear Magnetic Resonance Spectroscopy

D4273-05: Standard Test Methods for Polyurethane Raw Materials Determination of Primary Hydroxyl Content of Polyether Polyols

D4875-05: Standard Test Methods of Polyurethane Raw Materials: Determination of the Polymerized Ethylene Oxide Content of Polyether Polyols

D5017-96: Standard Test Method for Determination of Linear Low Density Polyethylene (LLDPE) Composition by Carbon-13 Nuclear Magnetic Resonance

D5292-99: Standard Test Method for Aromatic Carbon Contents of Hydrocarbon Oils by High Resolution Nuclear Magnetic Resonance Spectroscopy

F2259-03: Standard Test Method for Determining the Chemical Composition and Sequence in Alginate by Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy

F2260-03: Standard Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy

USP<2251> Adulteration of Dietary Supplements with Drugs and Drug Analogs.Poloxamer Testing

USP <761>: General Chapter – Nuclear Magnetic Resonance SpectroscopyKrill Oil Monograph – 1H and 31P NMR, Beta Glucan Monograph – 1H NMR, Heparin Sodium Monograph – 1H NMR

European Pharmacopoeia Salmon Oil, Farmed 01/2005:1910 13C NMR for distribution of fatty acids.

ISO10565:1998 Oil and Moisture in Seeds by Low Field NMR Spectroscopy8292 Animal and vegetable fats and oils -- Determination of solid fat content -- Pulsed nuclear magnetic resonance method21461:2006 Rubber - Determination of the aromaticity of oil in vulcanized rubber compounds21561:2005 Styrene-butadiene rubber (SBR) - Determination of the microstructure of solution-polymerized SBR11543:2000 Modified starch -- Determination of hydroxypropyl content -- Method using proton nuclear magnetic resonance (NMR) spectrometry10632:2000 Oilseed residues -- Simultaneous determination of oil and water contents -- Method using pulsed nuclear magnetic resonance spectroscopy

AOCSCd 16b-93 : (revised in 2000): Direct Method - Determination of Solid Fat Content -- Pulsed Nuclear Magnetic Resonance Method Cd 16-81 : (revised in 2000): Indirect Method - Determination of Solid Fat Content -- Pulsed Nuclear Magnetic Resonance Method Ak5-01 (01) : Oil Content of Oilseed Residues by NMRAk3-94 (00) : Oil Content in Rapeseed and other Oilseeds by NMRAk4-95 (99):Simultaneous Determination of Oil and Moisture Contents of Oilseeds using Pulsed Nuclear Magnetic Resonance Spectroscopy

American Herbal PharmacopoeiaAloe Vera - 1H NMR Method for Quality Inspection and Identification

Canadian Crude Quality Technical AssociationMaxxam V1.04 11/05 : Determination of Olefin Content of Crude Oils, Condensates and Diluents by 1H NMR

Guide to NMR Method Development and Validation – Part I: Identification and Quantification

1H qNMR is very well referenced in the academic literature but is still not an official standard method validated by a standards organization

Component

Dry Matter Composition

Wt%

Product Composition

Wt%

Product Composition

(mg/L)

Acemannan Wt% 13.96 0.158 1549

Glucose Wt% 16.00 0.181 1776

Malic Acid Wt% 27.89 0.315 3096

Lactic Acid 0.00 0.000 0

Citric Acid 22.07 0.249 2449

Pyruvic Acid 0.16 0.002 18

Sorbate 0.00 0.000 0

Benzoate 0.00 0.000 0

Acetoin 0.00 0.000 0

2,3-butandiol 0.00 0.000 0

Whole Leaf Marker (WLM) Not Observed Not Observed Trace Observed

Isocitrate (WLM-1) 0.00 0.000 0

Isocitrate Lactone (WLM-2) 0.00 0.000 0

Acetic Acid 0.00 0.002 17

Succinic Acid 0.00 0.000 0

Formic Acid 0.00 0.000 0

Fumaric Acid 0.00 0.000 0

Ethanol 0.00 0.000 0

Glycine 0.00 0.000 0

Propylene Glycol 0.00 0.000 0

Sucrose 0.00 0.000 0

Dry Matter Weight (%)

Organic Aloe Vera Juice Hand Fillet with Pulp Batch OAVWP0116

1.13

•Serving Size 8 fl oz.•Servings Per Container 2•Amount Per Serving•Calories 30•Calories from Fat 0•% Daily Value*•Total Fat 0g 0%•Cholesterol 0mg 0%•Sodium 10mg 1%•Total Carbohydrate 7g (2%)•Sugar 2g•Protein 0g•Probiotic Content•Bacillus coagulans GBI-30 6086: 1 billion organisms•S. Boulardii: 1 billion organisms•Antioxidants and Organic Acids•EGCG 100mg•Glucuronic Acid 10mg•L(+) Lactic Acid 25mg•Acetic Acid 30 mg

Alcohol and Tobacco Tax and Trade Bureau (TTB)

USP General Chapter 2251 Adulteration of Dietary Supplements with Drugs and Drug Analogs

Typical Herbal Extracts

300 MHz NMR – PDE5i Standards

Component Wt% = 100 x (Component Molar Integral / Std Molar Integral) x (Wt Std / Wt Sample) x (Component Mw / Std Mw)

Component Wt% = 100 x (Component Molar Integral / Std Molar Integral) x (Wt Std / Wt Sample) x (Component Mw / Std Mw)

ASTM D6122 - 15

Standard Practice for Validation of the Performance of Multivariate

Online, At-Line, and Laboratory Infrared Spectrophotometer Based

Analyzer Systems

ASTM E1655 - 05(2012)

Standard Practices for Infrared Multivariate Quantitative Analysis

Correlative (Chemometric Methods)

NMR Spectrometer Specification42 MHz, 60 MHz, 80 MHz, 300 MHz, 400 MHz, 700 MHz?

Experimental Varian Mercury - 300 MHz NMR Supercon Spectrometer4 pulse on pure sample in 5 mm tube, Run UnlockedIn MNova 8.1.2 SPC Files Imported, Stacked, Binned at 3 Hz interval, Area Normalized to 100, Saved as Transposed Ascii MatrixPLS Regression Performed Thermo Grams IQ and Eigenvector Solo

300 MHz 1H NMR

DHA

EPA-a

Olefins

=C-CH2-C=

In chain

CH2

EtOOC-CH2-R

DHA

EPA

CH3-CH2-O-OC-CH2-R

CH3

EthylCH3

R-CH2-C=CH3

w6/ w9

w3EPA-b

www.process-nmr.com www.nmrblog.com Tel: 203-744-5905

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0.5 Hz binning interval

3 Hz binning interval







Spinsolve 1H NMR data at 42.5 MHz of a series of fish oils and esterified omega-3 fatty acids

0 10 20 30 400

1

2

3

4

5

6

Hotelling T 2̂ (99.91%)

Q R

esid

uals

(0.0

9%

)

0 0.2 0.4 0.6 0.8

-3

-2

-1

0

1

2

3

Leverage

Y S

tdnt

Resid

ual 1

0 20 40 60 80-20

0

20

40

60

80

Y Measured 1

Y C

V P

redic

ted 1

-100 -50 0 50-20

-15

-10

-5

0

5

10

15

Scores on LV 1 (95.78%)S

core

s o

n L

V 2

(2.4

8%

)

ModelLinear regression model usingPartial Least Squares calculated with the SIMPLS algorithmDeveloped 27-Jul-2014 15:03:05.984Author: John@JCEPNAX-block: Final Fish Oil Spectra - 43MHz.csv 55 by 830 (John@[email protected] m:20140727140753.619)Included: [ 1-55 ] [ 1-830 ] Preprocessing: Mean CenterY-block: EPA-GC Data.xlsx 55 by 1 (John@[email protected] m:20140727141758.529)Included: [ 1-55 ] [ 1 ] Preprocessing: Mean CenterNum. LVs: 8Cross validation: venetian blinds w/ 7 splitsRMSEC: 1.40566 RMSECV: 2.20172 Bias: 0 CV Bias: -0.00054578 R^2 Cal: 0.995028 R^2 CV: 0.987838

SSQ TablePercent Variance Captured by Regression Model

-----X-Block----- -----Y-Block-----Comp This Total This Total ---- ------- ------- ------- -------

1 95.78 95.78 84.51 84.512 2.48 98.26 3.47 87.983 0.29 98.55 9.21 97.194 0.76 99.31 0.59 97.785 0.43 99.73 0.60 98.386 0.07 99.80 0.77 99.157 0.06 99.86 0.17 99.338 0.04 99.91 0.18 99.50

Omega-3 EPA Content (wt%) in Fish Oil and Pharmaceutical Grade Supplements

Correlation between 40 second 1H NMR at 42.5 MHzand primary analysis method – 50 minute GC analysis

0 10 20 30 400

1

2

3

4


Q R

esid

uals

(0.0

9%

)

0 0.2 0.4 0.6 0.8-4

-2

0

2

4

Leverage

Y S

tdnt

Resid

ual 1

0 10 20 30 40 50 60-20

0

20

40

60

80

Y Measured 1

Y C

V P

redic

ted 1

-100 -50 0 50-20

-10

0

10

20

Scores on LV 1 (95.78%)

Score

s o

n L

V 2

(2.2

3%

)

R 2̂ = 0.989

8 Latent Variables

RMSEC = 0.75363

RMSECV = 1.2463

Calibration Bias = 0

CV Bias = 0.098796

ModelLinear regression model usingPartial Least Squares calculated with the SIMPLS algorithmDeveloped 27-Jul-2014 14:50:021.58Author: John@JCEPNAX-block: Final Fish Oil Spectra - 43MHz.csv 55 by 830 (John@[email protected] m:20140727140753.619)Included: [ 1-55 ] [ 1-830 ] Preprocessing: Mean CenterY-block: DHA-GC Data.xlsx 55 by 1 (John@[email protected] m:20140727140259.401)Included: [ 1-55 ] [ 1 ] Preprocessing: Mean CenterNum. LVs: 8Cross validation: venetian blinds w/ 7 splitsRMSEC: 0.753632 RMSECV: 1.2463 Bias: 0 CV Bias: 0.0987959 R^2 Cal: 0.99605 R^2 CV: 0.989345



1 95.78 95.78 73.08 73.082 2.23 98.01 6.53 79.613 0.55 98.56 15.91 95.534 0.50 99.06 1.63 97.165 0.67 99.73 0.78 97.936 0.07 99.80 1.42 99.357 0.07 99.87 0.15 99.508 0.04 99.91 0.10 99.61

Omega-3 DHA Content (wt%) in Fish Oil and Pharmaceutical Grade Supplements


Picospin-80 NMR Data – 82.3 MHz

0 10 20 30 400

1

2

3

4

5

6


Q R

esid

uals

(0.0

9%

)

0 0.2 0.4 0.6 0.8

-3

-2

-1

0

1

2

3

Leverage

Y S

tdnt

Resid

ual 1

0 20 40 60 80-20

0

20

40

60

80

Y Measured 1

Y C

V P

redic

ted 1

-100 -50 0 50-20

-15

-10

-5

0

5

10

15

Scores on LV 1 (95.78%)S

core

s o

n L

V 2

(2.4

8%

)

ModelLinear regression model usingPartial Least Squares calculated with the SIMPLS algorithmDeveloped 27-Jul-2014 15:03:05.984Author: John@JCEPNAX-block: Final Fish Oil Spectra - 43MHz.csv 55 by 830 (John@[email protected] m:20140727140753.619)Included: [ 1-55 ] [ 1-830 ] Preprocessing: Mean CenterY-block: EPA-GC Data.xlsx 55 by 1 (John@[email protected] m:20140727141758.529)Included: [ 1-55 ] [ 1 ] Preprocessing: Mean CenterNum. LVs: 8Cross validation: venetian blinds w/ 7 splitsRMSEC: 1.40566 RMSECV: 2.20172 Bias: 0 CV Bias: -0.00054578 R^2 Cal: 0.995028 R^2 CV: 0.987838



1 95.78 95.78 84.51 84.512 2.48 98.26 3.47 87.983 0.29 98.55 9.21 97.194 0.76 99.31 0.59 97.785 0.43 99.73 0.60 98.386 0.07 99.80 0.77 99.157 0.06 99.86 0.17 99.338 0.04 99.91 0.18 99.50

Omega-3 EPA Content (wt%) in Fish Oil and Pharmaceutical Grade Supplements


Model

Linear regression model usingPartial Least Squares calculated with the SIMPLS algorithmDeveloped 15-Nov-2014 23:10:039.19Author: John@JCEPNAX-block: Fish Oils - 40 Sample - X Matrix.xlsx 37 by 57 (John@[email protected] m:20141115231022.946)Included: [ 1-11 13-27 29-32 34-40 ] [ 1-2 71-125 ] Preprocessing: Mean CenterY-block: DHA.xlsx 37 by 1 (John@[email protected] m:20141115231022.953)Included: [ 1-11 13-27 29-32 34-40 ] [ 1 ] Preprocessing: Mean CenterNum. LVs: 3Cross validation: venetian blinds w/ 6 splitsRMSEC: 1.09711 RMSECV: 1.20325 Bias: 3.55271e-015 CV Bias: 0.0624194 R^2 Cal: 0.992002 R^2 CV: 0.99041



1 79.58 79.58 77.43 77.432 11.40 90.97 20.16 97.593 7.91 98.88 1.61 99.20

0 5 10 15 20 25 300

0.5

1

1.5

2

2.5

3


Q R

esid

uals

(1.1

2%

)

0 0.2 0.4 0.6 0.8

-3

-2

-1

0

1

2

3

4

Leverage

Y S

tdnt

Resid

ual 1

0 10 20 30 40 50 60-20

0

20

40

60

Y Measured 1

Y C

V P

redic

ted 1

-20 -10 0 10 20-10

-5

0

5

10

15

20

Scores on LV 1 (79.58%)

Score

s o

n L

V 2

(11.4

0%

)

DHA model – 0.02 ppm binPicospin 80.3 MHz 1H NMR Data

PLS Regression DataEPA Content (Wt%)

R2, SECV (Wt%)DHA Content (Wt%)

R2, SECV (Wt%)

42 MHz NMR 0.988, 2.20 0.989, 1.25

60 MHz NMR 0.993, 2.13 0.992, 1.13

82 MHz NMR 0.984, 2.53 0.990, 1.20

300 MHz NMR 0.988, 1.68 0.991, 1.09

Peak Integral Data 0.981, 2.71 0.964, 2.24

Fused - NMR-FTIR 0.993, 1.62 0.992, 1.08

Regression Results for Various NMR Approaches




1. Submission and Validation of a generic qNMR method within an official standards organization.

i) Internal Standard Approachii) Electronic Standard Approachiii) Spectral Deconvolution Approach

2. Validation of Correlative NMR methods on superconducting and benchtop NMR systems

3. How and With Whom?

AOAC, AOCS, ISO, ASTM, US Pharmacopoia, European Pharmacopoeia ?

Personal Objective:

Documents

The Practical Non-Existance of Official NMR-Based Test ...process-nmr.com/Presentation/John Edwards - NMR...ASTM D6122 - 15 Standard Practice for Validation of the Performance of Multivariate