Topics in Sample Preparation A 5 Part Series…€¦ · Topics in Sample Preparation A 5 Part Series… Part 2 -An Introduction to Pass-Through Sample Preparation ©2015 Waters Corporation

Topics in Sample Preparation A 5 Part Series…

Part 2 - An Introduction to Pass-Through Sample Preparation

©2015 Waters Corporation 1

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worked in Consumables R&D and the Technology Advancement Department.

� She has been focused on evaluating different sample prep techniques such as solid phase extraction(SPE), protein precipitation(PPT), solid supported liquid extraction (SSLE) as well as the Ostro Pass-through sample prepare technique. Recently, she has been working on simplified SPE procedures for the Oasis family. In addition, she also helps customers understand sample preparation by delivering educational seminars and trouble shooting.

Agenda

� Industry overview

– Business & scientific challenges

– Sample preparation techniques

� Ostro Pass-through sample preparation

– What is it?

– How does it work?



– Comparison to traditional techniques (PPT, LLE & SSLE)

� Practical Applications of Ostro

� Summary

� Appendix

– Applications examples

Today’s Industry Challenges

Business Challenges– Increasing revenue

– Decreasing costs

Scientific Challenges– Doing more with less

– Diverse samples/analytes

Meeting the needs of the modern laboratory…

vs.


– Decreasing costs

– Maximizing resources– Diverse samples/analytes

– Speed

How can we address these needs?

vs.

Sample Preparation Techniques

� Examples of various sample preparation techniques

– Dilution followed by injection

– Protein precipitation (PPT)

– Filtration

– Ultrafiltration

– Liquid-liquid extraction (LLE)



– Solid-supported liquid-liquid extraction (SSLE)

– Solid-phase extraction (SPE)

– Immunoaffinity extraction (IA)

– QUECHERS

– ELISA

Selection: Which One?

� How do you chose a technique to remove phospholipids &

proteins from complex sample matrices?

– Protein precipitation (PPT)

– Ultrafiltration


– Solid-supported liquid-liquid extraction (SSLE)


– Solid-phase extraction (SPE)

Objectives:Simple technique

Fast preparation procedureClean, protein-free extracts

Agenda




� Ostro Pass-through sample preparation

– What is it?






– Drug screening

– Direct analysis of opiates

� Conclusions

� Appendix


Sample Preparation Techniques

PPT

Add sample

Ostro

Load sample

LLE

Add sample

Add extract

SLE

Add Sample

SPE

Condition

SPE

(Mixed Mode)

Condition


Add ACN

Mix/vortex

Filter / centrifuge

Add ACN

Mix/vortex

pass-through

Add extract

Mix

Wait

Separate

Evaporate

Reconstitute

Add extraction solvent

Wait 5 -10 mins

Extract

Evaporate

Reconstitute

Equilibrate

Load sample

Wash

Elute

Equilibrate

Load sample

Wash 1

Wash 2

Elute

Fastest Cleanest

Lowest HighestSensitivity

Sample Preparation

A Simple Approach: What is Pass-Through Sample Preparation?

Apply Sample

Retention of Proteins


Analytes of interest

Retention of Phospholipids

OstroSorbent

Proteins

Analytes pass-through free of proteins and phospholipids

What is Ostro?

� The Ostro Pass-through sample preparation plate removes

proteins and phospholipids using a combination of filtration and

sorbent interaction

� Benefits include:

– Cleaner samples in less time

– Minimal-to-no method development


– Minimal-to-no method development

– Simple, generic protocol

� Ostro provides consistent, robust methods and increased

throughput using a simple, easy-to-implement protocol

What is Ostro (cont.)?

� 96-well plate for pass-through sample preparation

– Simply and efficiently removes proteins & phospholipids

– Typical applications areas:

o Works with plasma, serum, whole blood, milk, meat, tissue and

other protein/lipid-containing samples

o Bioanalysis, forensics, clinical research, food, environmental, etc.


Ostro: Typical Application Areas and Sample Pre-Treatment

Clinical Research (Forensic)

Whole Blood/Plasma

Bioanalysis

Plasma/Serum

Food

Milk/Tissue


• 1:3 w/Zinc Sulphate – Whole Blood

• Dilute sample

• If required, acidify or basify samples

• Sample is treated with ACN externally

Ostro: How to Use

Clinical Research (Forensics)

Whole Blood/Plasma

Bioanalysis

Plasma/Serum

Food

Milk/Tissue


• 1:3 w/Zinc Sulphate – Whole Blood • Dilute sample

• If required acidify or basify samples

• Sample is treated with ACN externally

Load sample

Add solvent

In-well mixing

Analytes pass-through

Collect & Inject

Analytes pass-through free of proteins, phospholipids, and particulates

Example: Ostro in Bioanalysis

Place Ostro onto collection plate

Pipette 50-200 µL of plasma into wells

Forcefully add 2% formic acid in acetonitrile (3:1 solvent:plasma)

It is possible to work with lower sample volumes (such as 25 µL). When doing so, a higher organic solvent to sample ratio (e.g.,


acetonitrile (3:1 solvent:plasma) (methanol not recommended)

Mix thoroughly by aspirating 3x with pipette

Filter samples using vacuum manifold or positive-pressure manifold

Analyze samples

to sample ratio (e.g., 10:1,20:1) is required.

The well volume is 1.9 mL, however in order to mix by aspiration, the maximum volume is 1.4 mL. This translates to a maximum sample size of 350 µL.

The well volume is 1.9 mL, however in order to pass high sample volumes, multiple

Sample is treated with acetonitrile externally


Example: Ostro in Food Analysis


pass high sample volumes, multiple loading need to be done in the same well.Pass acetonitrile-treated

sample through Ostro

Filter samples using vacuum manifold or

positive pressure manifold

Analyze samples

Example: Forensics (Whole Blood)

Take whole blood and add 1:3 w/zinc


Pipette 50-200 µL of supernant into wells

Forcefully add 2% formic acid in acetonitrile (3:1

solvent:plasma) (methanol


add 1:3 w/zinc sulphate, centrifuge, remove supernatant, and use as sample with Ostro

solvent:plasma) (methanol not recommended)

Mix thoroughly by aspirating 3x with pipette

Filter samples (pass through) using vacuum manifold or positive pressure manifold

Analyze samples

Ostro vs. Other Sample Preparation Techniques

PPT OSTRO LLE SSLE

Method Development NO NO YES YES

Highest Selectivity

Direct Inject √ √

Simple /Ease of Use √ √ √* √*

High Throughput √ √ √* √

Reproducibility √ √ √ √


Reproducibility √ √ √ √

Reduction of Matrix Effects √ √ √

Highest Sensitivity

Sample Concentration √* √

“Clean” Extracts √ √ √

Recovery √ √ √ √

Generic √ √

Protocol Time √ √

Environmentally Friendly √ √

* Perception of being simple

Use of PPT for Bioanalysis

� If PPT works, it is generally the first choice for those developing

sample preparation methods

– Samples can be diluted with water and directly injected to speed up

workflow (“dilute and shoot”)

– Inexpensive to perform in most cases

– Very simple protocol


o Easily transferred from lab to lab

o Inexperienced personnel can perform successfully

� Disadvantages include:

– Variable recoveries

– Not effective for phospholipids

o Doesn’t remove matrix effects

Ostro versus Protein Precipitation

PPT OSTRO LLE SSLE


Highest Selectivity



High Throughput √ √ √ √



Phospholipid Removal √ √

Highest Sensitivity



Generic √ √


Environmentally Friendly √ √


Comparison of Ostro versus PPT Protocols

Ostro (Total processing time: 11 min)

Load sample (3 min)

PPT(Total processing time: 13 min)

Load sample, (3 min)


Add acetonitrile (2 min)

Mix (3 min)

Pass Through (3 min)

Add extraction solvent

(2 min)

Mix (Vortex) (3 min)

Centrifuge (2 min)

Transfer (3 min)

Sample processing time with Ostro is slightly less than PPT

Remaining Phospholipids

2.00 4.00 6.00 8.00 10.00 12.00

%

0

100184.4 > 184.4 (Lipid 184)

1.31e8

7.50

7.43

7.31

7.69

7.76

9.18

8.80

8.319.88

10.08

Traditional PPT


Time

2.00 4.00 6.00 8.00 10.00 12.00

%

0

100

2.00 4.00 6.00 8.00 10.00 12.00

184.4 > 184.4 (Lipid 184)

1.31e8

8.30

Ostro

MRM transition 184>184 to demonstrate total remaining PLs

Phospholipid Build-Up: 250 Injections

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100 758.4 > 184.4 (Lipid 758)3.19e6

2.282.131.99

758.4 > 184.4 (Lipid 758)3.19e6

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100 758.4 > 184.4 (Lipid 758)3.19e6

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100 758.4 > 184.4 (Lipid 758)3.19e6

2.282.131.99

758.4 > 184.4 (Lipid 758)3.19e6

2.282.131.99

758.4 > 184.4 (Lipid 758)3.19e6

PPT last

injection

PPT 1st

injection

gradient

end

end

organic hold


Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

Ostro last

injection

Ostro 1st

injection

0.5 min hold at high % organic does not fully elute phospholipids resulting in phospholipid build-up on column and unpredictable

elution in subsequent injections

Advantages of Ostro versus Protein Precipitation

� Simple, universal protocol

– Little-to-no method development

� Removes proteins and phospholipids

– Improved instrument uptime and more robust methods

– PPT requires centrifugation to remove proteins (which takes time)

� Decreased matrix effects


� Decreased matrix effects

� Lower sample variability

� Increased recovery

� Increased method robustness

� “The Ostro plate performed significantly better than protein

precipitation and the marketed plates for removal of all classes

of phospholipids*”

*Brian Hoffman, Advion BiosciencesBioanalysis Journal (2010), 2 (12), 1932-1933

Ostro versus Liquid-Liquid Extraction (LLE) in Bioanalysis

PPT OSTRO LLE SSLE


Highest Selectivity






Phospholipid Removal √ √

Highest Sensitivity



Generic √ √


Environmental Friendly √ √


Using Liquid-Liquid Extraction (LLE)

� LLE: separation of compounds based on solubility in two

immiscible liquids (e.g., water and an organic solvent)

� Advantages:

– Uses existing glassware; no on-going costs other than labor and

solvents


solvents

– Can be more compound specific

� Disadvantages:

– Very time and labor intensive

– Large solvent consumption (purchase and disposal costs)

– Emulsions are possible

– Not readily automatable (poor sample throughput)

Comparison of Ostro versus LLE Protocols

Ostro (Total processing time : 11 min)

Load sample (3 min)

LLE (Total processing time : 20 min)

Load sample (2 min)

Add extraction solvent (2 min)



Mix (3 min)



Mixing (1 min)

Centrifuge (5 min)

Transfer (2 min)

Evaporate (5 min)

Reconstitute (2 min )

Vortex (1 min)

Sample processing time for Ostro is less than LLE

Comparison of Analyte Recovery: Ostro versus LLE

Average Analyte Recovery

80

100

120

Average Analyte Recovery


0

20

40

60

Oxycodone Valethamate Niflumic Acid Ketoprofen Progesterone Hydrocortisone

Ostro

LLE

weak, polarbase

strong base zwitterion acid hydrophobicneutral

hydrophobicneutral

Comparison of Total Remaining Phospholipids: Ostro versus LLE

%

100 184.4 > 184.4 (Lipid 184)1.34e8

1.79 1.45

1.28 1.59 1.85

Ostro Ostro


Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40

%

0

100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 0

184.4 > 184.4 (Lipid 184)1.34e8

2.13 1.88 1.79

1.40

1.95

2.00

2.08

2.21

2.26 2.31

2.70

LLE using

100%

MTBE

LLE using 100% MTBE

Ostro eliminates build-up of PLs on column

Comparison of Total Remaining Phospholipids: Ostro versus LLE

Sum of Remaining Residual Phospholipids

2500000

3000000

3500000

4000000


0

500000

1000000

1500000

2000000

Ostro LLE MTBE - Ostro LLE MTBE

For even cleaner extracts: combination of the Ostro plate and LLE removes>99.9% of residual phospholipids

Advantages of Ostro versus LLE

� Faster run times

� Simple, easy to implement generic method (little-to-no method

development required)

� Superior removal of proteins and phospholipids

� No emulsions with Ostro

� Can be automated to further increase sample throughput


� Can be automated to further increase sample throughput

For additional information, see application note:

720004051EN - Providing a Universal, One-Step Alternative to Liquid-Liquid Extraction in Bioanalysis

Ostro versus SSLE

PPT OSTRO LLE SSLE


Highest Selectivity


Simple / Ease of Use √ √ √* √*




Phospholipid removal √ √

Highest Sensitivity



Generic √ √


Environmental Friendly √ √


Using Solid-Supported Liquid Extraction (SSLE)

� SSLE = SSLE is partition chromatography using buffered water

immobilized on a solid support (diatomaceous earth) and elution

by a water immiscible solvent

� Advantages:

– More compound specific


– More compound specific

� Disadvantages:

– Large solvent consumption (purchase and disposal costs)

– Requires method development

– Multiple dilution buffers required

– Evaporation & reconstitution required

– Processing times

Comparison of Ostro versus SSLE Protocols

Ostro (Total processing time: 11 min)

Load sample (3 min)

SSLE (Total processing time: 28 – 33 min)

Load sample, initiate (3 min)



Mix (3 min)


Wait 5 – 10 min


Wait 5 – 10 min

Extract (1 min)

Evaporate (10 – 15 min)

Reconstitute (2 min )

Sample processing time for Ostro is 2-3 times less than SSLE

Comparison of Analyte Recovery and Matrix Effects from Plasma: Ostro versus SSLE

0

20

40

60

80

100

120

Ostro SSLE

0.60

0.80

1.00

1.20

1.40

Ostro SSLE


0

0.20

0.40

Recovery for Ostro & SSLE Matrix factors for Ostro & SSLE

Ostro showed very good and consistent recoveries across all the tested

analytes with an average recovery of 95±4%

SSLE showed variability in recovery, since the extraction method was selected

for neutral and basic analytes

Comparable matrix factors for Ostro and SSLE

Comparison of Batch to Batch Reproducibility: Ostro versus SSLE

0

20

40

60

80

100

120

Ostro batch 1 Ostro batch 2 Ostro batch 3

Ostro batch to batch recovery &

reproducibility


0

20

40

60

80

100

120

SSLE batch1 SSLE batch2

SSLE batch to batch recovery &

reproducibility

Ostro offers superior recoveries and batch-to-batch reproducibility

Advantages of Ostro versus SSLE

� Simple, easy to implement generic method (little-to-no method

development required)

� 60% in time savings

� Consistently higher and more reproducible recovery across wide

range of analytes

� More environmental friendly methodology


� More environmental friendly methodology

� More consistent batch to batch results

For more information, see application note:

720005199EN Advantages of Ostro Pass-through Sample Preparation Versus Solid Supported Liquid Extraction (SSLE)

Agenda




� Ostro pass-through sample preparation

– What is it?






� Summary

� Appendix: Additional Applications

– Enhanced dried blood spots analysis

– Veterinary drugs from Milk

Ostro in Drug Screening

� Optimizing sample and laboratory workflow is critical in drug

discovery

– Diverse sets of compounds

– Need to achieve acceptable sensitivity

– Clean extracts

– Generic methods


– Generic methods

� Goal: develop a method with high recovery that produces

cleaner extracts and facilitates more robust, shorter run times

– 100 µL of plasma extracted using the generic protocol

Ostro in Drug Screening: Chromatographic Separation

1. 7-aminonitrazepam2. 7-aminoclonazepam3. 7-aminoflunitrazepam4. Clozapine5. Midazolam6. Chlordiazepoxide7. Alpha-Hydroxymidazolam8. Bromazepam9. n-Desmethylflunitrazepam10. Nitrazepam11. Clonazepam d4

110, 11, 12, 13

14, 15, 16, 17, 18, 19, 20, 21

26

100

0.80

1. 7-aminonitrazepam2. 7-aminoclonazepam3. 7-aminoflunitrazepam4. Clozapine5. Midazolam6. Chlordiazepoxide7. Alpha-Hydroxymidazolam8. Bromazepam9. n-Desmethylflunitrazepam10. Nitrazepam11. Clonazepam d4

110, 11, 12, 13

14, 15, 16, 17, 18, 19, 20, 21

26

100

0.80

100100

0.80


11. Clonazepam d412. Clonazepam13. Flunitrazepam14. Triazolam15. 2-Hydroxyethylflurazepam16. Hydroxyalprazolam d517. Alpha-Hydroxyalprazolam18. Alprazolam19. Alprazolam d520. Oxazepam21. Clobazam22. Estazolam23. Desalkylflurazepam24. Temazepam25. Nordiazepam26. Prazepam

2

3

4 6

5

7

8

9 22, 23, 24

25

26

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

11. Clonazepam d412. Clonazepam13. Flunitrazepam14. Triazolam15. 2-Hydroxyethylflurazepam16. Hydroxyalprazolam d517. Alpha-Hydroxyalprazolam18. Alprazolam19. Alprazolam d520. Oxazepam21. Clobazam22. Estazolam23. Desalkylflurazepam24. Temazepam25. Nordiazepam26. Prazepam

2

3

4 6

5

7

8

9 22, 23, 24

25

26

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0 Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0 Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

Ostro in Drug Screening:Average Analyte Recovery

60

80

100

Average Recovery ~86%


0

20

40

Triazo

lam

(T-9

10)

alph

a-Hyd

roxy

mid

azola

m (H

-902)

2-hyd

roxy

ethylf

lura

zepam

(F-9

01)

Hyd

roxy

alpr

azol

am d

5 (A

-904)

Clo

zapin

e (C-0

59)

Mid

azol

am (M

-908)

Praze

pam

(P-9

06)

alph

a-Hyd

roxy

alpr

azola

m (A

-905)

Clo

nazep

am d

4 (C

-905

)

Brom

azep

am (B

-903

)

Clo

nazep

am (C

-907)

Fluni

traze

pam

(F-9

07)

Alpra

zola

m d

5 (A-9

02)

Alpra

zola

m (A

-903)

Temaz

epam

(T-9

07)

Clo

bazam

(C-9

09)

n-D

esm

ethyl

fluni

traze

pam

(D-9

19)

Chl

ordia

zepo

xide

(C-0

22)

Estaz

olam

(E-9

01)

Des

alky

lflur

azepa

m (D

-915

)

Oxa

zepam

(O-9

02)

7-am

inoc

lonaz

epam

(A-9

15)

7-am

inof

luni

traze

pam

(A-9

12)

Nitr

azepam

(N-9

06)

Nor

diaz

epam

(N-9

05)

7-am

inon

itraz

epam

(A-9

14)

Ostro in Drug Screening: Sum of Phospholipids

5000000

6000000

7000000

8000000

9000000

sum of 8 phospholipids


0

1000000

2000000

3000000

4000000

Ostro PPT

sum of 8 phospholipids

Ostro removes >99% of phospholipids relative to traditional PPT

Ostro in Drug Screening: Remaining Phospholipids

2.00 4.00 6.00 8.00 10.00 12.00

%

0

100184.4 > 184.4 (Lipid 184)

1.31e8

7.50

7.43

7.31

7.69

7.76

9.18

8.80

8.319.88

10.08

Traditional PPT


Time

2.00 4.00 6.00 8.00 10.00 12.00

%

0

100

2.00 4.00 6.00 8.00 10.00 12.00

184.4 > 184.4 (Lipid 184)

1.31e8

8.30

Ostro

MRM transition 184>184 to demonstrate total remaining PLs

Ostro in Drug Screening: Phospholipid Build-up

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100 758.4 > 184.4 (Lipid 758)3.19e6

2.282.131.99

758.4 > 184.4 (Lipid 758)3.19e6

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100 758.4 > 184.4 (Lipid 758)3.19e6

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100 758.4 > 184.4 (Lipid 758)3.19e6

2.282.131.99

758.4 > 184.4 (Lipid 758)3.19e6

2.282.131.99

758.4 > 184.4 (Lipid 758)3.19e6

PPT last

injection

PPT 1st

injection

gradient

end

end

organic hold


Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

758.4 > 184.4 (Lipid 758)3.19e6

Ostro last

injection

Ostro 1st

injection

0.5 min hold at high % organic does not fully elute phospholipids resulting in phospholipid build-up on column and unpredictable

elution in subsequent injections

Ostro in Drug Screening: Phospholipid Elution in Shortened Gradient

%

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100 184.4 > 184.4 (Lipid 184)1.34e8

0.55

0.02

0.65 0.73 0.87

184.4 > 184.4 (Lipid 184)1.34e8

%

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100 184.4 > 184.4 (Lipid 184)1.34e8

%

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100 184.4 > 184.4 (Lipid 184)1.34e8

0.55

0.02

0.65 0.73 0.87

184.4 > 184.4 (Lipid 184)1.34e8

0.55

0.02

0.65 0.73 0.87

184.4 > 184.4 (Lipid 184)1.34e8

PPT

200 injections

PPT

1 injection

end

gradient


Time0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

00.73

184.4 > 184.4 (Lipid 184)1.34e8

0.33

184.4 > 184.4 (Lipid 184)1.34e8

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40

%

00.20 0.40 0.60 0.80 1.00 1.20 1.40

%

00.73

184.4 > 184.4 (Lipid 184)1.34e8

0.73

184.4 > 184.4 (Lipid 184)1.34e8

0.33

184.4 > 184.4 (Lipid 184)1.34e8

Ostro

200 injections

Ostro

1 injection

Gradient cannot be shortened using PPT since PLs continue to elute after gradient has ended

Ostro in Drug Screening:Summary

� Ostro generates high recoveries for a discovery screen with no

method development

� Simple, universal protocol

� Direct injection of Ostro-processed samples

� Removes >99% of PLs




� Reduces sample variability

� Facilitates use of shorter runtimes, improving throughput

Ostro in Forensics:Direct Analysis of Opiates

� Forensic laboratories often need to analyze whole blood

specimens for the presence of different drugs to determine the

precise cause of death, in cases of driving under the influence of

drugs, or other criminal or research purposes

� Many sample preparation strategies have been used for whole


� Many sample preparation strategies have been used for whole

blood analysis

� With Ostro the complete sample preparation workflow for whole

blood is done within the wells, including sample pre-treatment

Ostro in Forensics:Sample Preparation Workflow

Add 150 µL of aqueous 0.1 M ZnSO4 /0.1 M NH4CH3COOH to the wells

Add 50 µL of Whole blood

Mix (to Lyse the cells)

Add 600 µL of ACN


Vortex

Pass through (vacuum or positive pressure)

Evaporate (N2)

Reconstitute with 50 µL of 0.1% formic acid in 2% acetonitrile

Analyze

Ostro in Forensics: Chromatographic Separation of Opiates and Synthetic Analgesic Compounds


Ostro in Forensics: Recoveries of Opiates from Whole Blood

Mean recovery from whole blood samples (n=4)


Ostro provides reproducible recoveries

Direct Analysis of Opiates:Summary

� The Ostro pass-through sample preparation plate allows for

rapid, in-well cell lysis and protein precipitation

� Provides additional benefit of phospholipid removal

� Ostro simplifies the forensic laboratory’s workflow by

eliminating multiple transfers & lab wares

� Cost savings & throughput achieved


� Cost savings & throughput achieved

Ostro With Dried Blood Spots (DBS)

�Dried blood spot (DBS) analysis is being evaluated in

the pharmaceutical industry because it facilitates the

use of smaller sample sizes and provides easier

handling and long term storage of samples


� The goal was to improve current DBS methods and

provide cleaner DBS extracts, as well as offer a

single-step DBS method with no extract transfer or

centrifugation

Ostro With Dried Blood Spots

O N

F N

N

N

O

CH3

Risperidone

MW 410.48

pKa 8.24

API


O N

NH

N

N

N

CH3

O N

F N

N

N

O

CH3

OH

Clozapine

MW 326.82

pKa 7.6

9-OH Risperidone

MW 426.48

pKa 7.86

Internal Standard

Metabolite

�Ostro in-well extraction

� 3.0 mm DBS punch

extracted in-well using

250 µL of 95/5

� Traditional DBS

extraction

� 3.0 mm DBS punch

extracted in centrifuge

tube using 250 µL of

Ostro With Dried Blood Spots


250 µL of 95/5

methanol/water 95/5 methanol/water

20 µL whole blood was spotted onto Whatman DMPK-C cards and dried for 2 hrs

Ostro With Dried Blood Spots:Sum of Phospholipids

Sum of Phospholipids

30000

35000

40000

45000

50000

Sum of Phospholipids


0

5000

10000

15000

20000

25000

30000

Ostro in-well Traditional In-tube

Ostro removes 99.9% of residual PLs relative to the traditional DBS extraction

Ostro With Dried Blood Spots:Phospholipids Remaining

0.50 1.00 1.50 2.00 2.50 3.00

%

0

100

TIC1.36e8

2.84

2.19

Traditional DBS


Time0.50 1.00 1.50 2.00 2.50 3.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.000

TIC

1.36e8

DBS in-well using Ostro

TIC of 5 individual PLsNo PL build-up on column with Ostro

Ostro With Dried Blood Spots:Phospholipid Build-up

DBS in-well Ostro1st injection

DBS in-well Ostro

Last injection

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100758.4 > 184.4 (Lipid 758)

4.88e6

758.4 > 184.4 (Lipid 758)4.88e6

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100758.4 > 184.4 (Lipid 758)

4.88e6

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100758.4 > 184.4 (Lipid 758)

4.88e6

758.4 > 184.4 (Lipid 758)4.88e6

758.4 > 184.4 (Lipid 758)4.88e6


Traditional DBS

1st injection

Traditional DBSLast injection

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

758.4 > 184.4 (Lipid 758)4.88e6

2.20

758.4 > 184.4 (Lipid 758)4.88e6

2.212.16

3.37Time

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

758.4 > 184.4 (Lipid 758)4.88e6

758.4 > 184.4 (Lipid 758)4.88e6

2.20

758.4 > 184.4 (Lipid 758)4.88e6

2.20

758.4 > 184.4 (Lipid 758)4.88e6

2.212.16

3.37

No PL build-up on column with Ostro

Ostro With Dried Blood Spots:High Sensitivity

%

0

100 427.1 > 109.8 (9-OH Risperidone)

4.71e4

2.232.041.871.29

0.54

0.300.030.78 0.81 1.16

1.851.68 1.58

2.30

3.442.39

2.522.63 3.292.95 3.14

Blank Whole Blood Spot


Time0.50 1.00 1.50 2.00 2.50 3.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 0

427.1 > 109.85 (9-OH Risperidone) 4.71e4

1.30984

Whole Blood Spiked at 50 pg/mL with 9-OH

risperidone

Excellent sensitivity for dilute DBS extracts using Xevo TQ-S

Ostro With Dried Blood Spots:Summary

� Simple, single step method for DBS samples

� Removes 99.9% of PLs relative to tradition DBS

extraction

– More robust LC methods

� Achieve lower LODs with high sensitivity analyses


� Achieve lower LODs with high sensitivity analyses

� Eliminates build-up of PLs on columns allowing for

the use of shorter gradients

– Improved laboratory throughput

Ostro for Veterinary Drug Analyses in Milk

�Multiple sample preparation methods are available

today for this application

– PPT, LLE, SPE, etc.

�Goal is to compare Ostro performance with protein

precipitation


precipitation

Ostro for Veterinary Drug Analyses in Milk: Chromatographic Separation


Removal of phospholipids from whole milk using Ostro

Ostro for Veterinary Drug Analyses in Milk: Summary

– Rapid & Simple

– Clean extracts

– Removal of potentially interfering phospholipids from samples

– High recovery for analyte of interest

– Increased throughput


Agenda




� Ostro pass-through sample preparation

– What is it?






� Summary

� Appendix


Summary

� Ostro pass-through sample preparation device offers far

superior performance when compared to competitive techniques

� Filters samples while removing proteins & phospholipids

� Lower matrix effects, high sensitivity and increased instrument

uptime

� Generic method


� Generic method

– Little-to-no method development; rapid implementation

– Simple workflow

� 96-well format

– Automatable for increased sample throughput

� Reproducible, robust methods

Simple protocol Cleaner extracts Consistent results

Thank You for Attending!

� Post-Event Home Page: http://www.waters.com/Feb26

� 30% Product Specific Offer on Ostro Plates

� 25% Offer On Oasis, SepPak Products

– Full Webinar Recording of Today’s Session w/PDF Slide

Deck

– Compilation of TODAY’S KEY Literature, Brochures etc…


– Compilation of TODAY’S KEY Literature, Brochures etc…

� For Questions and to Submit your Ideas for our Next Topic

– Please eMail - [email protected]


Appendix


Appendix

Ostro in Clinical Study

� Antipsychotic drug

marketed as Risperdal®

� 9-OH Risperidone is the

primary circulating species N

F

N

O

N CH3

OH9-Hydroxyrisperidone

M.W. 426.2

Risperidone and 9-OH Metabolite in Human Plasma


NO

F

N

NH

N

N

Cl

CH3

NO

N

F

N

O

N CH3

Risperidone

M.W. 410.2

pKa = 8.24

Clozapine, ISTD

M.W. 326.1

pKa’s = 7.1, 5.3

Method Requirements

� Wide linear dynamic range

– 0.025 – 500 ng/mL

� Meets accuracy and precision regulatory requirements for

standards and QC samples

� Reproducible

� Achieve required detection limit


� Achieve required detection limit

� Easy

� Fast

� No method development

Wide Linear Dynamic Range:Standard Curve for Risperidone

Compound name: Risperidone

Correlation coefficient: r = 0.997938, r 2̂ = 0.995879

Calibration curve: 0.466273 * x + 0.00860374

Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None

Conc

Re

sid

ua

l

-10.0

-5.0

0.0

5.0

10.0

Compound Name: RisperidoneCorrelation coefficient r = 0.997938, r^2 = 0.995879Calibration curve: 0.466273*x + 0.00860374Response type: Internal Std (Ref1), Area*(IS Conc./IS Area)Curve Type: Linear, Origin:Exclude, Weighting 1/x^2, Axis trans: None


Conc0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Re

sp

on

se

0

50

100

150

200

Calibration curve is linear over 4.5 orders of magnitude

Calibration curve range: 0.025 (25 pg/mL) to 500 ng/mL in human plasma

Wide Linear Dynamic Range: Standard Curve for 9-OH Risperidone

Compound name: 9-OH Risperidone

Correlation coefficient: r = 0.998454, r^2 = 0.996910

Calibration curve: 0.321752 * x + 0.00172858

Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None

Conc

Re

sid

ua

l

-5.0

0.0

5.0

10.0

15.0

20.0

Compound Name: 9-OH RisperidoneCorrelation coefficient r = 0.998454, r^2 = 0.996910Calibration curve: 0.321752*x + 0.00172858Response type: Internal Std (Ref1), Area*(IS Conc./IS Area)Curve Type: Linear, Origin:Exclude, Weighting 1/x^2, Axis trans: None


Conc0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

Re

sp

on

se

0

25

50

75

100

125

150

Curve is linear over 4.5 orders of magnitude

Curve range is 0.025 (25 pg/mL) to 500 ng/mL in human plasma

Method Meets Regulatory Criteria for Accuracy and Precision

Human Plasma Type Area IS Area Conc. %Dev

25 pg/mL Standard 401.8 19390.7 0.026 3.9

50 pg/mL Standard 579.5 18436.2 0.049 -2.1

100 pg/mL Standard 909.5 18751.3 0.086 -14.4

250 pg/mL Standard 2304.7 17667.0 0.261 4.5

500 pg/mL Standard 3135.0 12261.9 0.530 6

2.5 ng/mL Standard 20772.9 18459.5 2.395 -4.2

12.5 ng/mL Standard 112761.8 19344.0 12.483 -0.1

25 ng/mL Standard 218078.2 18115.3 25.800 3.2


Meets Regulatory Criteria: all standards within 15% deviation of expected

25 ng/mL Standard 218078.2 18115.3 25.800 3.2

50 ng/mL Standard 428895.1 17760.3 51.773 3.5

250 ng/mL Standard 2180640.3 19283.9 242.503 -3

500 ng/mL Standard 3702304.5 15468.5 513.297 2.7

75 pg/mL QC 847.8 17836.0 0.083 11.3

750 pg/mL QC 5868.9 16136.6 0.762 1.5

7.5 ng/mL QC 48311.8 13565.3 7.620 1.6

75 ng/mL QC 558263.4 15764.1 75.932 1.2

Std Conc(ng/mL)

Average Calculated

Concentration (ng/mL) %RSD

0.075 0.083 11.3

Std Conc(ng/mL)

Average Calculated

Concentration (ng/mL) %RSD

0.075 0.09 20.3

Meets Regulatory Criteria: Accuracy and Precision

Risperidone 9-OH Risperidone


0.75 0.762 1.5

7.5 7.62 1.6

75 75.932 1.2

0.75 0.787 5

7.5 8.262 10.2

75 74.686 -0.4

All QC samples meet accuracy and precision regulatory criteria

Documents

Topics in Sample Preparation A 5 Part Series…€¦ · Topics in Sample Preparation A 5 Part Series… Part 2 -An Introduction to Pass-Through Sample Preparation ©2015 Waters Corporation