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Topics in Sample Preparation A 5 Part Series…

Part 1 - Simplifying Solid Phase Extraction

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Today’s Presenter – Xin Zhang

� Xin Zhang, Ph.D - Senior Research Chemist, Waters Corporation

� Today’s Webinar will be presented by Xin Zhang, Senior Research Chemist at Waters Corporation. She received her Ph.D in analytical chemistry from SUNY, Buffalo in 2007. At Waters Corporation, she has worked in Consumables R&D and the Technology


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.

Outline

� Introduction – Industry Trends in Sample Preparation

�Making Solid Phase Extraction (SPE) Easier -

Introducing “Simple Prep”


� Simple Prep Performance Comparisons

� Conclusion

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.

Comparison of Sample Preparation Techniques – Typical

5 µg/mL 5 µg/mL5 µg/mL100 µL Plasma5 µg/mL analytes

Protein Precipitation

Liquid / Liquid Extraction

Solid Supported

Liquid Extraction

Solid Phase Extraction


Fastest Cleanest

~2.5 µg/mL 10 µg/mL~2.5 µg/mL1.25 µg/mL

Lowest HighestSensitivity

Sample Preparation

Sample Preparation Techniques: Drivers



Solid Supported

Liquid Extraction



Simple approach

Cost effective

Fast

Reduces sample complexity

“Good enough”

Sample Preparation Techniques: Perceptions



Solid Supported

Liquid Extraction


Protein precipitation is Solid phase


Protein precipitation is “simple”

LLE & SLE are perceived as “simple”

Solid phase extraction is perceived as “complex”

Sample Preparation Techniques:A New Approach



Solid Supported

Liquid Extraction



SPE is perceived as “complex”:

Too many steps Multiple sorbent options

Need to develop methods

Can we make this easier?

History – Making SPE Simple

� Since 1996, Waters goal was to simplify complex sample

preparation methods with generic sorbent & protocols

– Oasis HLB introduced in 1996 as the first water-wettable polymeric

SPE into the market

– Gold standard SPE sorbent in sample prep market for the last 18

years

– Sample preparation technique of choice for LC-MS due to cleanliness


– Sample preparation technique of choice for LC-MS due to cleanliness

of extracts and universal applicability.

– Well understood SPE technology

– 5 step generic protocol

� How do other reversed-phase solid phase extraction devices

compare?

Recovery (4 Replicates) ComparisonDry Condition

80

100

120

AZT

7 - Hydroxycoumarin

Phenacetin

Oasis AVG Recovery: 100% Oasis AVG RSD: 1%


0

20

40

60

Oasis HLB Competitor S RP Competitor SX RP Competitor P RP

Betamethasone

Protriptyline

Alprazolam

Methoxyverapamil

Terfenadine

Matrix Factors (MF) Comparison

1.20

1.40

1.60

1.80

AZT

7 -Hydroxycoumarin

Phenacetin

Betamethasone

Protriptyline


0.60

0.80

1.00

Oasis HLB Competitor S RP Competitor SX RP Competitor P RP

Alprazolam

Methoxyverapamil

Terfenadine

blankin Response

matrix in ResponseMF =

Oasis HLB exhibits lowest (best) matrix effects performance

Matrix factor:1.0 = no matrix effect (best)

>1.0 = ion enhancement <1.0 = ion suppression

History – Making SPE Simple

�Introduced mixed-mode ion exchangers

–Simplify the complex sample preparation methods

to generic procedures

–Simplified mixed mode ion exchange sample prep

with 2 protocols x 4 sorbents


with 2 protocols x 4 sorbents

………..and now………

�Let’s further simplify SPE protocols

Outline






� Conclusion

Introducing SIMPLE PREP


Make sample preparation simple and easy

Let’s continue with a little more background information…

Oasis HLB: First Water-Wettable Sorbent

Hydrophilic

monomer

Lipophilic

monomer

NO

Hydrophilic-Lipophilic Balanced Copolymer


Reversed-phase retentionRetention of polars

Most C18 silica-based SPE sorbents dewet if allowed to dryOasis sorbents do not exhibit this undesirable behavior

Standard µElution SPE Protocol

Condition

• 200 µL Methanol (MeOH)

Equilibrate

• 200 µL H2O

• 200 µL spiked dilute plasma


Wash

• 200 µL 5% MeOH in H2O

Elute

• 25 µL*2 MeOH

Load

• 200 µL spiked dilute plasma(100 µL plasma+100µL 4% H3PO4)

No evaporation and reconstitution

necessary!

Direct injection of eluate

µElution plate simplified SPE protocol by

eliminating evaporation and reconstitution

Simplified µElution SPE Protocol

Condition


Equilibrate

• 200 µL H2O

• 200 µL spiked dilute plasma

Eliminate these steps


Wash


Elute

• 25 µL*2 MeOH

Load


Oasis uElution Protocol Comparison: Standard vs. Simplified

Condition

Equilibrate


• 200 µL H2O

Standard Simplified


Wash


Elute

• 25 µL*2 MeOH

Load



• 25 µL*2 MeOH


� Direct inject eluate

� Evaluate analyte recoveries, variability, matrix factors and cleanliness.

Outline





�Simple Prep Performance Comparisons

� Conclusion

Recovery with Plasma Samples :Standard vs. Simplified Protocol

20

40

60

80

100

120

Simplified protocolStandard protocol


5-step protocol vs. 3-step protocol:

Comparable recoveries and standard deviations

Average recoveries 90% and RSDs <5%

)samplepostspikedResponse

sampleextractedResponse(100Recovery ×=

0AZT Propra 7HC Phen Protrip Amitrip BetaMeth Alpraz Naprox AVG

Matrix Factors (MF):Standard vs. Simplified Protocol

Standard protocol Simplified protocol

Matrix factor

1.00

1.20

1.40

-


*Bioanalytical method validation from European Medicines Agency 2012

blankin Response

matrix in ResponseMF =

*

0.60

0.80Procain N

-

acety AZT Propra 7HC Phen Protrip Amitrip BetaMeth Alpraz Naprox

Equivalent matrix factors were obtained with both protocols (1.00±0.15 matrix factors obtained with simplified 3-step (Load-

Wash-Elute) protocol)Matrix factor:

1.0 = no matrix effect (best) >1.0 = ion enhancement <1.0 = ion suppression

Simplified Protocol:Sample Loading Capacity

40

60

80

100

120

Different plasma loading volume

25µL 50µL 100µL 250µL 375µL


Loading capacity remains consistent and comparable at different

sample volumes (loads)

The sample loading capacity tested was 25 to 375 µL plasma

Recoveries ranged from 80 - 110% and RSDs were within 5% for all drugs

0

20

40

Simplified Protocol: Batch-to-Batch Reproducibility

40

60

80

100

120

Batch 1 Batch 2 Batch 3 Batch 4


Very consistent recoveries across all batches

Four different batches of Oasis HLB were selected from 2005 - 2013

Average recovery across all batches was 90±6%

0

20

Now let’s look at how


Now let’s look at how

urine samples perform with simple prep

Simplified vs. Standard Protocol: Evaluation of Drugs in Urine

0.8

1.0

1.2

1.4

Propra 7HC Phen BetaMeth Alpraz Naprox20

40

60

80

100

Standard protocol Simplified protocol

Recovery Matrix factorStandard protocol Simplified protocol


Comparable recoveries and matrix factors obtained

with simplified 3-step (Load-Wash-Elute) protocol

0.6

0.8

0

20

Propra 7HC Phen BetaMeth Alpraz Naprox

Urine sample diluted 1:1 with 100 mM pH 5.0 sodium acetate

Matrix factors of 1±0.2 is considered comparable

Can we apply simplified protocol with

other sorbents?


Silica based sorbent?

Oasis HLB like Competitor’s sorbent?

Oasis HLB vs. Silica-Based C18

Recovery: Simplified Protocol

40

60

80

100

Plasma sampleOasis HLB Silica-based C18

Urine sample0

5

Oasis HLB Silica-based C18


HLB performed well with simplified 3-step (Load-Wash-Elute) protocol

Both plasma and urine sample were evaluated

Silica-based C18 SPE showed poor (very low) recoveries

0

20

40

Silica-Based C18 sorbent

Oasis HLB vs. Competitor Recovery: Simplified Protocol

20

30

40

50

60

70

80

90

100

µElution plate 3 step SOLAµ plate 3 stepCompetitor S µ plate 3 step


Oasis HLB works well with simplified 3-step (Load-Wash-Elute) protocol

Average %REC = 83 and %RSD = 2

Competitor Sµ plate does not work with simplified 3-step (Load-Wash-Elute)

protocol

0

10

20

Competitor: Polymeric sorbent

Particle Shedding Comparison

� Procedure

– Pass 200 µL MeOH in each well, combine 48 well together

– Evaporate and send the residue for SEM (scanning electron microscope)

Oasis µElution plate shedding Competitor Sµ plate shedding


Shedding particles can cause premature column failure if injected

Oasis µElution plate has much less particle shedding

Can we do the simplified protocol in Oasis


Can we do the simplified protocol in Oasis

other configurations such as cartridges?

Simplified Protocol Plasma Load: Oasis HLB 1cc/30 mg Cartridges

0

20

40

60

80

100

120

Simplified protocolStandard protocol Simplified protocolStandard protocol

Recovery Matrix factor

0.80

1.00

1.20

1.40

AZT Propra 7HC Phen Protrip Amitrip Beta

MethAlprazN-actryl


Comparable (high) recoveries and (low) matrix effects

with plasma when comparing 5-step protocol versus

simplified 3-step (Load-Wash-Elute) protocol

0

0.60

0.80

AZT Propra 7HC Phen Protrip AmitripBeta

MethAlprazN-actryl

Meth

Simplified Protocol Urine Load: Oasis HLB 1cc/30 mg Cartridges

0

20

40

60

80

100

120

1cc, 30mg, 5 step urine 1mL Load, rec 1cc, 30mg, 3 step urine 1mL Load, rec

0.80

1.00

1.20

1.40

5 step HLB 1cc urine ME 3 step HLB 1cc urine ME

Recovery Matrix factor


Comparable (high) recoveries and (low) matrix effects

with urine when comparing 5-step protocol versus

simplified 3-step (Load-Wash-Elute) protocol

0

0.60

0.80

Summary for Simplified Protocol:

� Simplified 3-step (Load-Wash-Elute) protocol works with Oasis HLB (a water wettable sorbent).

– Excellent analyte recoveries, matrix factor and capacity

– Consistent batch-to-batch reproducibility

– Compatible with both Oasis HLB plates and cartridges

– Compatible with plasma and urine samples


� Low (very poor) recoveries were observed with silica based sorbent

� Low (very poor) recoveries were observed with competitor polymeric sorbents

� Particle shedding was observed when working with competitor polymeric sorbents

Outline






�Conclusion

Conclusion

� Simplified 3-step (Load-Wash-Elute) protocol for

Oasis HLB enables:

– Improved workflow by reducing sample processing steps from

5 steps to 3 steps

– A 40% reduction in sample processing time and an average of

70% solvent savings with the 3 step protocol


70% solvent savings with the 3 step protocol

– High analyte recovery, low variability, and low matrix factor

– Benefits realized when using Oasis plates and cartridges

– µElution plate is designed to work with sample volumes of 25

– 375 µL and can improve sensitivity by concentrating

samples up to 15x

Acknowledgement

� Frank Marszalkowski

� Doug McCabe

� Chris Poz

� Kim Haynes

� Pamela Iraneta

� Euan Ross


� Euan Ross

� Erin Chambers

� Kenneth Fountain

� Gregory Lawless

Thank You for Attending!

� Post-Event Home Page http://www.waters.com/Feb12

� 25% Offer On Oasis, Ostro, SepPak Products

� 30% Product Specific Offer on Oasis Syringe Barrels

– 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]

LC Test Conditions

InstrumentACQUITY UPLC I class -

Xevo TQ-SUPLC Column 1.7 µm CSH C18, 2.1 x 100mm

Column Temperature 40°C

Sample Temperature 10°C

Run Time (Flow Rate) 7 minutes (0.5 mL/min)

Mobile Phase A: Water with 0.1% formic acid

Mobile Phase B: ACN with 0.1% formic acid

Wash solvent 70/30 ACN/water with 2%


Wash solvent (both strong and weak)

70/30 ACN/water with 2% formic acid

Seal wash 10% MeOH

Detection MS: Xevo TQ-S

MS Condition ESI+ MRM

CSH: Charged Surface HybridUPLC : Ultra Performance Liquid chromatography

Tested Drugs

Name pKa Log P Comments

1B Azidothymidine (AZT) 9.68 0.05 Antiretroviral drug for HIV/AIDS

2B 7-Hydroxycoumarin 7.8 1.6 Gradient in sunscreen, absorb UV

3A Phenacetin 2.2 1.6 Pain, fever reducer

4N Betamethasone -- 1.1Anti-inflammatory and immunosuppressive


immunosuppressive

5B Protriptyline 8.2 4.4 Antidepressant

6A Alprazolam 2.4 4.9 Panic and anxiety disorders

7B Amitriptyline 9.7 4.8 Antidepressant

8A Naproxen 4.2 3.2 Pain, fever reducer

9B Propranolol 9.5 2.5 Hypertension

� Drugs with different therapeutics

� Wide range of pKas and Log Ps (indication of hydrophobicity)

MS Cleanliness:HLB Method Comparison

Reference: MeOH

ESI+ MS scan50-650


Comparable MS cleanliness in 5-step vs. simplified 3-step (Load-Wash-Elute) protocols

LC/MS chromatograms obtained using Xevo TQ-S

Extracts with simplified protocol

Extracts with standard protocol

50-650

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