sample preparation ostro and oasis prime...

©2017 Waters Corporation 1COMPANY CONFIDENTIAL

Sample Preparation – OSTRO and OASIS PRiME HLB

Willem Joubert

Microsep

Waters Division


Complex Sample Matrices are Challenging...

When creating a new method it is important to consider your analytes of interest

However...

Sample matrix will also have an impact on your sample preparation and chromatographic separations

How do I create methods while managing these differences?

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Developing Methods with Complex Sample

Matrices

In today’s seminar we will examine:

Simplifying Sample Preparation and Solid Phase Extraction (SPE)

Matrix Effects and Matrix Affects:

The Impact of Different Sample Matrices on Sample Preparation and

Chromatographic Analysis

– Case study: THC and it’s metabolites in urine, plasma, whole blood and oral

fluids

– How do these different matrices affect the method?



Matrices







fluids



Ostro Pass-through Sample

Preparation

– A simple, pass though sample preparation product

Simplify Solid Phase Extraction

– Oasis PRiME HLB approach

Sample Preparation Advisor

– Online tool to help customers select the right sorbent for their needs

– www.waters.com/mdtools

SPE Method Development Tool

– Guide the customer though the method development process

Making Sample Preparation Easy!

https://connect.waters.com/profiles/html/profileView.do?userid=89B150D7-C5C2-4FF2-B587-34BFF7BF3F10&entryId=57739bd8-1f1b-4337-95e9-e65fc6c89272



Preparation












Pass through sample preparation

Simple sample preparation

=

A Simple Approach


Pass-Through Protocol

Pass-Through Protocol – Ideal for

high organic (ACN) samples

Analytes not retained by the

sorbent

Rinse step is optional

Load – Collect

Interferences Retained

Analytes Pass-Through

Rinse – Collect

Analytes Pass-Through

Pass-Through Protocol


What is a Pass Through Technique?

Sample

Analyte of interest

Retention of Phospholipids

Proprietary material

Retention of Proteins

Analytes pass through free of most proteins and phospholipids


What is Ostro?

96-well plate for pass through sample preparation

– Removes phospholipids & proteins (PLR)

– Applications :

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

o In the areas of bioanalysis, forensics, clinical research, food, environmental

and others

Utilizes

– Sorbent interaction and filtration

– A proprietary sorbent that retains phospholipids & proteins


Ostro: How does it work?

Methodology Place Ostro onto collection plate

Pipette 50-200µL of plasma into wells

Forcefully add 2% formic acid in 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


Ostro Advantages

Ostro Pass-through Sample Preparation device offers far superior performance when compared to competitive techniques

Generic method

• Little to no method development; rapid implementation

• Simple workflow

•Reproducible, robust methods

Compared to SSLE

• 60 % faster processing time

• Higher analyte recoveries

• Better batch-to-batch reproducibility

•Wide range of sample volumes (50 – 350 µL)

Removes most phospholipids & proteins leading to decreased matrix effects, high sensitivity and greater instrument uptime



Preparation












History – Making SPE Simple

Since 1996, Waters goal was to simplify complex sample preparation

methods with generic sorbent & protocol

– 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 21 years

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

extracts and universal applicability.

– Well understood SPE technology

– 5 step generic protocol


Oasis HLB:

A Water-Wettable Sorbent

Hydrophilic

monomer

Lipophilic

monomer

NO

Reversed-phase Retention

Hydrophilic-Lipophilic Balanced Copolymer

Retention of Polars

Oasis HLB is the backbone of all Oasis sorbents

Stable across pH 1-14 High recoveries for acids, bases and neutrals Water-wettability allows the elimination of condition and equilibration steps Will NOT dry out under vacuum or positive pressure, once wetted


Oasis Reversed Phase SPE:

A Simplified Protocol

Condition

Equilibrate

Wash

5% MeOH in H2O

Elute

100% MeOH

Load

Spiked diluted plasma(1:1 ratio of plasma+ 4% H3PO4)

100% MeOH

100% H2O

5% MeOH in H2O

100% MeOH

Spiked diluted plasma(1:1 ratio of plasma+4% H3PO4)

Traditional Simplified


All Polymeric SPE Sorbents are NOT Created

Equal

0

20

40

60

80

100

120

Oasis HLB Competitor S RP Competitor SX RP Competitor P RP

AZT

7 - Hydroxycoumarin

Phenacetin

Betamethasone

Protriptyline

Alprazolam

Methoxyverapamil

Terfenadine

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

Lower recoveries and higher variability with other sorbents

Comparative separations may not be representative of all applications.


SIMPLER and FASTER

– Easy protocols that produce cleaner

samples

– Condition and equilibration steps

are not required, saving time and

solvent

– Easy to implement into laboratory

workflows without SPE expertise

Load:

Pre-Treated Sample

Wash:

5% MeOH

Elute:

90/10

Acetonitrile/MeOH

Oasis PRiME HLB – Methods

Wash and elute steps can be adjusted to

optimize results

3 Step Protocol


Drug Panel Mixture in Plasma:

3 Step Protocol

Device: Oasis PRiME HLB µElution plate, N=4

Sample pre-treatment: 1:1 dilution of plasma with 4% H3PO4

– Load: 400µL diluted plasma sample

– Wash: 2 x 0.2 mL 5% MeOH

– Elute: 2 x 25 µL 90/10 ACN/MeOH

Dilute eluate with 100µL water and directly inject

Oasis PRiME HLB Protocol

Does it work?


3-Step Protocol:

Recovery and Matrix Effects

-20

0

20

40

60

80

100

120

Luckycat 1cc plasma 500-500 Luckycat 1cc MERecovery Matrix Effects

3-Step Protocol ProducedHIGH analyte recoveries (>80%) and

LOW (<15%) matrix effects


CLEANER Eluates Compared to Oasis HLB:

3 Step Protocol

Phospholipids Remaining in 10 mg Plate Format

N=8 Total %RSD% Remaining

vs. HLB

AVG Oasis HLB 2.3E+07 8

AVG Oasis PRiME HLB

1.5E+06 11 5

Oasis PRiME HLB removed 95% more phospholipids compared to Oasis HLB with the generic 3 step protocol

5.0%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

120%

Oasis HLB Oasis PRiME

HLB

Remaining Phospholipids

Oasis PRiME HLB vs. Oasis HLB


SIMPLER and FASTER

– 2 Step Protocol

o Ideal for high organic containing

samples, such as food or tissue

extracts

– Condition and equilibration steps

are not required

– Fast and easy to implement into

laboratory workflows without SPE

expertise

Load:

Matrix Interferences Retained

Collect:

Analytes Pass Through Unretained

Oasis PRiME HLB - Methods

2 Step Protocol

Pass through solution can be adjusted to

optimize results


Oasis PRiME HLB removes more

than 90% of hexane-extractable

total lipids (determined

gravimetrically).

Oasis PRiME HLB successfully

removes both phospholipids and

fats in pass through method.

The silica C18 sorbent removes

only fats, NOT phospholipids.

Removal of both of these

components results in fewer matrix

effects and less column and/or

instrument contamination

Fatty Meat ACN Extract:

Phospholipids Remaining after 2 Step Protocol

Time1.00 2.00 3.00 4.00 5.00 6.00

%

0

1.00 2.00 3.00 4.00 5.00 6.00

%

0

LC050615_3 1: MRM of 12 Channels ES+ TIC (Phospholipid)

1.87e8

4.58

4.33

LC050615_5 1: MRM of 12 Channels ES+ TIC (Phospholipid)

1.87e8

4.33

Silica C18

Oasis PRiME HLB


FASTER - Flows

Faster, more even sample flows across cartridges and plates with less

plugging

Loading compared to Oasis HLB with 4 inch Hg, N=4

Matrix Device Format Oasis PRiME HLB Speed Increase

1:1 Diluted Plasma µElution Plate 2 - 3X Faster

1:1 Diluted Plasma 1cc / 30mg Cartridge 4X Faster

1:1 Diluted Urine 30 mg Plate 6X Faster

1:1 Diluted Urine 10 mg Plate 2X Faster

1:1 Diluted Milk 3cc / 60 mg Cartridge 1 - 2X Faster

1:1 Diluted Milk 6cc / 200 mg Cartridge 2 - 3X Faster

FASTER flows across multiple devices and sample matrices


Oasis PRiME HLB Summary

Oasis PRiME HLB is the next generation SPE device that

sets the new performance standard for routine analyses

– Best choice for samples that contain proteins, fats, and/or lipids

and can be prepared by reversed-phase ‘catch-and-release’ SPE

or ‘pass-through’ SPE

SIMPLER: Streamlined protocols, no condition and

equilibration steps, easy to implement into laboratory

workflows without SPE expertise

FASTER: Faster flows through device, more even flows

across cartridges and plates with less plugging, faster

overall processing

CLEANER: Reduced matrix effects, phospholipid and fat

removal in the pass-through method, less column and/or

instrument contamination

Load:

Pre-Treated Sample

Wash:

5% MeOH

Elute:

90/10

Acetonitrile/MeOH

Load

Matrix Interferences

Retained

Collect

Analytes Pass Through

Unretained

2-Step Pass-Through

3-Step Catch and Release


Ostro

Pass-Through

Load sample

Add ACN

Mix

Pass through

Oasis HLB

Load sample

Wash

Elute

Oasis PRiME HLB

Load Sample

Wash

Elute

Oasis Mixed Mode IEX

Condition

Equilibrate

Load sample

Wash 1

Wash 2

Elute

Cleanest

Where should I start?


Ostro

Pass-Through

CUSTOMER:

NO need to concentrate

samples, remove salts, or remove matrix components

besides phospholipids

Oasis HLB

SPE

CUSTOMER:

Requires high capacity of

polar compounds or high pH (>pH 10) stability

START HERE

Oasis PRiME HLB

SPE

CUSTOMER:

First choice for reversed-phase SPE cleanup of

samples in routine analysis

Oasis Mixed Mode IEX

SPE

CUSTOMER:

More analyte specificity or

higher sensitivity

and/or cleanliness

needed

Cleanest

Where should I start?



Matrices







fluids



Case Study Analytes

Δ-9-tetrahydrocannabinol (THC) is the principal psychoactive constituent

of cannabis.

Major metabolites are inactive THC-COOH and active THC-OH

A highly abused recreational drug with the increasing number of states

legalizing it for both medical and recreational purposes

Target analytes are neutral and acidic

THC THC-OH“Hydroxy-THC”

THC-COOH“Carboxy-THC”


Solid Phase Extraction (SPE)

SPE

Target analytes Sample matrices

Drug and its metabolites

Whole drug panel

Urine Whole blood Oral fluidPlasma

Oasis Mixed Mode SPE (MCX)

Oasis (PRiME) HLB

How do you treat different matrices?









SPE



Whole drug panel



Oasis (PRiME) HLB









THCs in Plasma:

What to Consider Prior LC/MS Analysis?

Plasma: A very common matrix, widely used

Sample pre-treatment:

– Plasma:0.1% FA in ACN (1:2) PPT first, then dilute with water (To be discussed

in detail in a few slides...)

SPE procedure with Oasis PRiME HLB µElution plate:

Eluate was diluted with 50µL water then directly injected into the LC-MS

Wash2 x 250 µL 25% MeOH

Elute2 x 25 µL (90:10 ACN:MeOH)

LoadPretreated Plasma Sample



Approximately 80% recovery for all analytes with %RSDs <6%

All Matrix Effects were less than 20%

-40

-20

0

20

40

60

80

100

THC-OH THC-COOH THC

Rec

ME


Different Plasma Sample Pretreatment

-Dilute with H3PO4 vs. PPT

1:1 dilution with 4% H3PO4 was tried first but yields very low recovery,

indicating stronger protein binding with this sample

Protein Precipitation did a better job of releasing the target drugs vs. 4%

H3PO4 dilution

0

20

40

60

80

100

THC-OH THC-COOH THC

SPE Recovery with different sample pre-treatments

Dilute with 4% H3PO4 1:2 PPT


0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

Phospholipids in different elution solutions

Phospholipids Profile:

The Elution Solution Matters

ACN does not elute as many phospholipids as MeOH and is a better

elution solvent

>80% ACN in Water or >75/25 ACN/MeOH as elution solutions are

recommended


Time1.00 2.00 3.00 4.00 5.00 6.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00

%

0

100

20160401_BEHC18_03 2: MRM of 11 Channels ES+ TIC

7.58e8

2.322.23 3.04

20160401_BEHC18_29 2: MRM of 11 Channels ES+ TIC

7.58e85.60

2.582.33 5.344.992.67 4.662.943.03

Phospholipids Remaining:

Oasis PRiME HLB Elution vs. PPT only

Removes over 99% phospholipids, resulting in a much cleaner extraction

Eliminated the co-elution of analytes of interest with the phospholipids

Orange trace: Chromatogram of 3 target compounds

Black trace: Plasma SPE extract

Black trace: Plasma PPT supernatant


Method Validations and QCs

Accuracy and Precision

N=6 THC-OH THC-COOH THC

QC Level (ng/mL)

Mean

%Acc. %RSD

Mean

% Acc. %RSD

Mean

% Acc. %RSD(ng/mL) (ng/mL) (ng/mL)

0.375 0.34 90 9% 0.36 97 6% 0.40 107 3%

1.75 1.62 93 5% 1.77 101 5% 1.82 104 2%

7.5 7.35 98 3% 7.65 102 2% 7.62 102 2%

37.5 37.32 100 4% 39.62 106 2% 38.09 102 2%Mean 97 5% 103 4% 102 2%

All compounds linearity range from 0.1-100 ng/mL with R2>0.99

Accurate and precise QCs (accuracy 90-110%, AVG %RSDs <5%)

R2

(0.1-100ng/mL)Mean % Dev.

THC-OH 0.997 2.5%

THC-COOH 0.998 4.7%

THC 0.999 2.3%

Compound name: THC-COOHCorrelation coefficient: r = 0.998752, r̂ 2 = 0.997506Calibration curve: 19.0439 * x + 6.98613Response type: Internal Std ( Ref 5 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

1000



SPE



Whole drug panel



Oasis (PRiME) HLB









THCs in Urine:


Analyte conjugation

– THC and major metabolites are highly glucuronidated, requiring efficient de-

conjugation using enzyme hydrolysis

– THC-COOH requires extra alkaline hydrolysis to fully de-conjugate

SPE Method on Oasis PRiME HLB µElution Plate

*T.T.Abraham, R.H.Lowe, S.O. Pirany, W.D. Darwin, M.A. Huestis, J. of Analytical Toxicology, Vol 31, 2007, P477-485

*Recovery in urine with THC THC-OH THC-COOH

No hydrolysis No No Partial

Enzyme hydrolysis only Full Full Partial

Alkaline hydrolysis only No No Partial

Both Hydrolysis Full Full Full


Elute2 x 25 µL (60:40 ACN:IPA)

LoadPretreated Urine Sample

Dilute eluate with 50µL water, directly inject to LC/MS/MS



Consistent recoveries were obtained

– 90% with THC-OH and THC-COOH with %RSD within 8%

Minimal matrix effects

– <15% for all compounds

-20.0

0.0

20.0

40.0

60.0

80.0

100.0

THC-OH THC-COOH THC

Recovery

Matrix Effect




N=6 THC-OH (R2=0.997) THC-COOH (R2=0.998) THC (R2=0.998)

QC Level

(ng/mL)

Mean

(ng/mL)%Acc. %RSD

Mean

(ng/mL)% Acc. %RSD

Mean

(ng/mL)% Acc. %RSD

0.75 0.66 88.6 1.7 0.76 100.8 1.4 0.72 96.3 0.4

7.5 6.70 89.3 1.3 7.37 98.3 1.3 7.15 95.3 1.2

75 73.4 97.9 1.8 73.6 98.2 0.7 75.5 100.7 0.8

Mean 91.7 99.7 97.4

Waters Application Note PN#: 720005556EN

Calibration ranges:0.1-100 ng/mL for THC-COOH and THC-OH and 0.2-100 ng/mL for THC

Linear responses over the entire calibration range with R2>0.99

All QC results were within 15% of expected values and %RSDs <2%

All FDA recommendations for accuracy, precision, linearity and analytical sensitivity were met for validated methods.



SPE



Whole drug panel



Oasis (PRiME) HLB









THCs in Whole Blood:


Why do blood sample analysis?

Reliability of results: It can provide more accurate information about an

individual’s state of impairment at the time of sample collection (forensic

overdose)

What to consider with Whole blood sample?

Cell lysis (0.1M ZnSO4/0.1 NH4OAc)

Protein precipitation (ACN, with acid or base if necessary)

Dilute with water to a lower organic percentage prior to loading

SPE Method on Oasis PRiME HLB µElution plate:

*Optimized for consistent recoveries


Elute *2 x 25 µL (90:10 ACN:IPA)

LoadPretreated Blood Sample

Dilute eluate with 50 µL Water for direct LC-MS injection



All compounds over 80% recovery with <10% in %RSD

All ME within 20%

-20

0

20

40

60

80

100

THC-OH THC-COOH THC

Recovery

Matrix effects


Precipitation Solvent:

Additive Choice Matters

PPT, (blood+lysis buffer):organic at 1:3

Formic Acid (FA) in ACN was selected as PPT solvent

– Both 100% ACN and 5% NH4OH in ACN showed low recovery with THC-

COOH

– 2% H3PO4 in ACN PPT showed darkest color and clogged the well (see next

slide for more detail about color!)

0

20

40

60

80

100

120

100% ACN 5% NH4OH in ACN 2% FA in ACN 2% H3PO4 in ACN

% Recovery with Different PPT Solvent

THC-OH

THC-COOH

THC


Precipitation Solvent:

Concentration Matters

0.1% FA in ACN has the least color

– further optimize as PPT solvent

Use lowest possible concentration of additive

0.1 1.00.2 0.5 2.0

% of FA in ACN for PPT




N=6 THC-OH (0.1-100ng/mL) THC-COOH (0.1-100ng/mL) THC (0.05-100ng/mL)

QC Level

(ng/mL)

Mean%Acc. %RSD

Mean% Acc. %RSD

Mean% Acc. %RSD

(ng/mL) (ng/mL) (ng/mL)

0.375 0.33 97.9 0.6% 0.40 105.8 8.1% 0.41 108.2 3.0%

2 1.92 96.0 3.7% 1.89 94.7 2.3% 2.01 100.5 3.7%

7.5 7.50 100.0 2.7% 7.34 98.9 2.8% 7.42 98.9 1.4%

20 19.87 99.3 3.2% 20.04 100.2 2.1% 19.56 97.8 1.2%

37.5 36.19 96.5 2.2% 37.95 101.2 3.0% 35.32 94.2 0.7%

Mean 98 2% 100 4% 99.9 2%

Compound name: THC-COOHCorrelation coefficient: r = 0.999661, r^2 = 0.999322Calibration curve: 21.2358 * x + 1.5691Response type: Internal Std ( Ref 5 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 10 20 30 40 50 60 70 80 90 100

Re

sp

on

se

-0

500

1000

1500

2000

Compound name: THCCorrelation coefficient: r = 0.998930, r^2 = 0.997861Calibration curve: 27.8363 * x + 0.165145Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 10 20 30 40 50 60 70 80 90 100

Re

sp

on

se

-0

1000

2000

Compound name: THC-OHCorrelation coefficient: r = 0.999214, r^2 = 0.998428Calibration curve: 27.1578 * x + 4.8392Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 10 20 30 40 50 60 70 80 90 100

Re

sp

on

se

-0

500

1000

1500

2000

2500

R2>0.99 Linearity ranged in 0.05/0.1-100 ng/mL

Accurate and precise QCs


Oasis PRiME HLB removes over 99% phospholipids resulting in a much cleaner

extraction

The overlapped analytes chromatograms of combined PRiME extract eliminated

the co-elution of analytes of interest with the phospholipids

Phospholipids Remaining:

Oasis PRiME HLB Elution vs. PPT

B: Blood sample SPE (black trace)

C: Blood sample PPT

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

100

2: MRM of 11 Channels ES+ TIC

4.84e8

2: MRM of 11 Channels ES+ TIC

4.84e82.97

2.652.14

1.981.88

2.59

3.19

5.45

4.84

3.62

A: Chromatogram of 3 target compounds (red)



SPE



Whole drug panel



Oasis (PRiME) HLB









THCs in Oral Fluid:


Why do oral fluid sample analysis?

– Ease of sample collection: no special training, no privacy required

What to consider with oral fluid sample?

Oral fluid device extraction

– Add 1mL ACN in the collection device vial to help extraction

– Wait for 24 hours for complete extraction

– Add 2 mL 4% H3PO4 to dilute

SPE Method with Oasis PRiME HLB µElution plate

Pad to collect oral fluid

Extraction buffer

Indicator

Wash2 x 250 µL 5% NH4OH 25% MeOH

Elute2 x 25 µL (90:10 ACN:MeOH)

LoadPretreated Oral Fulid Sample

Dilute eluate with 50 µL water for direct LC-MS analysis



Consistent recoveries with low %RSDs

Minimal Matrix Effects across all tested analytes

-20

0

20

40

60

80

100

120

THC-OH, REC THC-COOH, REC THC, REC

REC

ME


Both devices extracted for 24 hours in the refrigerator

ACN helps the extraction, especially with the most hydrophobic compound THC

Maximizing the Oral Fluid Extraction

0

20

40

60

80

100

120

THC-OH THC-COOH THC

% r

eco

very

With ACN Without ACN

Device Extraction Efficiency


How to Decrease Matrix Effects?

Optimize the Wash Step

– Scan different additives with similar % organic in the wash solution

-60

-40

-20

0

20

25/75 MeOH/water 2%FA in 25/75

MeOH/water

5% NH4OH in 25%

MeOH

15/15/70

ACN/MeOH/water

40/60 MeOH/water 15/15/70

ACN/IPA/Water

Matrix Effects with different wash solution

THC-OH, ME THC-COOH, ME THC, ME

The optimized wash is 5% NH4OH in 25% MeOH for minimal Matrix Effects


How to Decrease Matrix Effects?

Different Elution Solutions

90/10 ACN/MeOH results in best Matrix Effects with comparable recovery

-100

-80

-60

-40

-20

0

20

ME, 90/10 ACN/MeOH ME, 60/40 ACN/MeOH ME, 60/40 ACN/IPA ME, 90/10 ACN/IPA ME, 90/10 ACN/water

Matrix Effects with different elution solutions

THC-OH THC-COOH THC

Urine BloodPlasma/OF



Conc-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

500

1000

1500

2000

2500

THC-OH, R2=0.9992

Conc-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

500

1000

1500

THC-COOH, R2=0.9994

Conc-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

500

1000

1500

2000

2500THC, R2=0.9995

Accuracy and Precision for QCs

N=6 THC-OH THC-COOH THC

QC Level

(ng/mL)

Mean%Acc. %RSD

Mean% Acc. %RSD

Mean% Acc. %RSD

(ng/mL) (ng/mL) (ng/mL)

0.375 0.36 96.6 8.3% 0.35 93.8 7.1% 0.39 105.2 5.7%

1.75 1.77 101.1 3.4% 1.65 94.3 2.7% 1.69 96.6 3.2%

7.5 7.57 101.0 2.7% 6.94 92.5 3.9% 7.12 94.9 2.4%

37.5 36.88 98.3 1.9% 37.77 100.7 1.4% 36.34 96.9 0.8%

Mean 100 3% 96 3% 96.1 2%

R2>0.999 Linearity ranged in 0.05/0.1-100 ng/mL

Accurate and precise QCs


Conclusion

Urine Whole blood Oral fluidPlasma (Serum)

De-conjugation? Cell lysis

Appropriate PPT

Collection device

(extraction efficiency)First try to dilute with 4% H3PO4/

PPT to disrupt protein binding

Oasis PRiME HLB: Load-Wash-Elute (Start with µElution plate)

Buffer dilutionBuffer/acid/base

dilution

Load: Pre-treated sample

Wash: Highest organic % with acid/base to wash away impurities

Elute: Organic w/acid/base for best REC and ME

Different sample matrices

Prior to method validation, a REC and ME test are recommended to prove true method robustness


Extracted Sample

(with analyte(s))

Sample Matrix

(with analyte(s))

Spike Standards into

Blank Matrix

SPE Extraction Procedure: Calculating

Recovery

RESPONSEExtracted Sample(with analyte(s))

RESPONSEPost-Extracted SPIKED Sample

%RE =100 x

*Matuszewski, B.K., Constanzer, M.L., Chavez-Eng, C.M. Anal. Chem. 2003, 75, 3019-3030.

Recovery of the Extraction Procedure (RE)*(or, SPE Recovery)

Post-Extracted

SPIKED Sample

Blank Sample Matrix

(No analyte(s))

Spike

Standards

into

Extracted

Matrix


Post-Extracted

SPIKED Sample

Blank Sample Matrix

(No analyte(s))

Spike

Standards

into

Extracted

Matrix

SPE Extraction Procedure: Calculating Recovery



% RE = 100 x


Both extracted samples should be in the same solution


Extracted Sample

(with analyte(s))

Sample Matrix

(with analyte(s))


Blank Matrix


Quantitative Assessment of Matrix Effects:

Post-Extracted Spiked Sample

Both samples should be in the same composition

solution

MF Value <1, negative % ME = suppression

MF Value >1, positive % ME = enhancement

100*1)-)Response

Response(((ME) EffectsMatrix%

StandardSolvent

Sample Spiked Extracted-Post

)Response

Response((MF)Factor Matrix

Matrix of Absence

Matrix of Presence

Standard Solution

(Analyte(s))

Post-Extracted

SPIKED Sample

Blank Sample Matrix

(No analyte(s))

Spike

Standards

into

Extracted

Matrix

sample preparation ostro and oasis prime...

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