1

AP PLICAT ION BENEFITSn Simple, universal sample prep protocol

n Speeds up workflow through direct

injection of eluates

n Eliminates the vast majority (>99%)

of plasma phospholipids

n Reduces sample variability, eliminating

a major source of suppression

n Facilitates use of shorter runtimes,

improving throughput

WAT ErS SOLuT IONSn ACQUITY UltraPerformance LC® technology

n Xevo® TQ MS system

n Ostro™ sample preparation products

n IntelliStart™ software

k Ey WOrdSPhospholipid removal, LC/MS/MS, Ostro, matrix

effects, drug discovery, screening

Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up MethodJessalynn P. Wheaton, Erin E. Chambers, John Martin and Kenneth J. Fountain

INT rOduCT IONDrug discovery is a vital segment of pharmaceutical research where vast numbers

of compounds are screened to determine therapeutic efficacy, activity, and ADME

properties. This process helps identify the handful of drug candidates that will

progress further. Many closely related drug compounds must be rapidly analyzed

and quick decisions must be made as to which drugs will continue into development

and eventually clinical trials. During the drug discovery stage, speed, time, ease of

use, and high throughput are key aspects of everyday work. There is little time for

method development, making simple and universal sample prep methods such as

protein precipitation (PPT) an attractive choice. Crude techniques such as PPT are

often quite efficient in terms of generating high analyte recovery but result in

relatively dirty samples. In particular, PPT does little to eliminate phospholipids

(PLs), a major source of concern in bioanalysis. PLs build up in LC/MS/MS systems

and are one of the major sources of matrix effects in plasma-based assays. Amongst

other problems, matrix effects also alter mass spectrometry response in an unpre-

dictable manner, decrease method robustness, and add to method variability. In this

publication, Ostro™ 96-well sample preparation plates are used to eliminate both

proteins and the vast majority of PLs while maintaining high analyte recovery, all

with a simple single step method. A screening method for a group of 26 structural

analogs and metabolites (see Figure 1 for representative structures) in plasma was

developed using this technique.

N

NO

Cl

N

HNO

Cl

diazepam

nordazepam

N

NO

Cl

OH

N

HNO

Cl

temazepam

oxazepam

N

NO

Cl

OH

Cl

N

HNO

Cl

OH

Cl

lormetazepam

lorazepam

Figure 1. Representative structures of 6 of the 26 analogs utilized in this study.

2 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method

Table 1. MRM transitions and MS conditions for the 26 structural analogs in plasma, generated using IntelliStart1

Compound NamePrecursor Ion

Product Ion

Cone Voltage (V)

Collision Energy (eV)

Triazolam 343.1 238.9 42 40

Alpha-hydroxymidazolam 342.0 203.0 38 28

2-hydroxyethylflurazepam 333.1 108.9 36 26

Clozapine 327.2 269.9 50 24

Midazolam 326.1 290.9 42 26

Prazepam 325.2 270.9 32 22

Alpha-hydroxyalprazolam 325.1 296.9 40 24

Bromazepam 318.1 181.9 38 32

Clonazepam 316.1 269.9 38 24

Flunitrazepam 314.1 268.0 42 24

Alprazolam 309.1 280.9 42 26

Temazepam 301.1 254.9 26 24

Clobazam 301.1 223.9 32 30

n-Desmethylflunitrazepam 300.2 253.9 38 22

Chlordiazepoxide 300.2 226.9 28 22

Estazolam 295.1 266.9 34 22

Desalkylflurazepam 289.1 139.9 42 26

Oxazepam 287.1 241.0 28 32

7-aminoflunitrazepam 284.2 135.0 46 26

Nitrazepam 282.2 207.0 38 34

Nordiazepam 271.1 139.9 32 30

7-aminonitrazepam 252.1 121.0 40 26

7-aminoclonazepam 286.1 120.9 38 28

EX PErIMENTAL

ACQuITy uPLC Conditions

Column: ACQUITY UPLC® BEH C18, 2.1 x 50 mm, 1.7 µm

Mobile Phase A: 0.1% HCOOH in H2O

Mobile Phase B: Methanol

Flow Rate: 0.6 mL/min

Gradient:

Injection Volume: 18.0 µL

Injection Mode: Partial Loop

Column Temperature: 35 °C

Sample Temperature: 15 °C

Strong Needle Wash: 60:40 ACN:IPA + 0.2% conc. HCOOH (600 µL)

Weak Needle Wash: 80/20 H2O/MeOH (200 µL)

Waters Xevo™ TQ MS Conditions, ESI+

Capillary Voltage: 1.0 V

Desolvation Temp: 400 °C

Cone Gas Flow: Not used

Desolvation Gas Flow: 1000 L/Hr

Collision Cell Pressure: 2.6 x 10(-3) mbar

MRM transition monitored, ESI+: See Table 1

Time Profile Curve(min) %A %B

0.0 98 2 6

2.0 1 99 6

2.5 1 99 6

2.6 98 2 6

3.0 98 2 6

3Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method

SAMPLE PrEPArATION PrOTOCOL

Ostro 96-well sample preparation platesSample preparation was performed using the standard protocol

included with the Ostro 96-well plate (see Figure 2). Sample

volume was 100 µL of plasma. This was extracted in the wells

of the Ostro plate using 300 µL of 1% HCOOH in acetonitrile.

Samples were mixed by aspiration. Vacuum was then applied

to collect the eluates. Eluates were diluted 1:1 with water to

ensure compatibility with the LC mobile phase and directly

injected onto the LC/MS/MS system.

rESuLTS ANd dISCuSSIONSeparation of the 26 structural analogs (Figure 3) was achieved

using a two-minute gradient at low pH with methanol. MS was

performed in positive ion mode. Precursor and product ions

were automatically optimized and an MS method automatically

generated using IntelliStart software. A previous application

note (720002569EN) by Rainville et al describes the

capabilities of IntelliStart in detail.

1. 7-aminonitrazepam2. 7-aminoclonazepam3. 7-aminoflunitrazepam4. Clozapine5. Midazolam6. Chlordiazepoxide7. Alpha -Hydroxymidazolam8. Bromazepam9. n-Desmethylflunitrazepam

10. Nitrazepam11. Clonazepam d412. Clonazepam13. Flunitrazepam14. Triazolam15. 2-Hydroxyethylflurazepam16. Hydroxyalprazolam d517. Alpha -Hydroxyalprazolam18. Alprazolam19. Alprazolam d520. Oxazepam21. Clobazam22. Estazolam23. Desalkylflurazepam24. Temazepam25. Nordiazepam

Prazepam26.

9.

1

2

3

4 6

5

7

8

9

10, 11, 12, 13

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

22, 23, 24

25

26

1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 min0.20 0.40 0.60 0.80 1.00

%

0

100

0.80

Figure 3. Representative ESI+ MRM chromatogram of a mixture of 26 structural analogs and metabolites.

Basic Protocol

Add plasma

Add organic

Aspirate to mix

Apply vacuum

Analyze

Figure 2. Ostro 96-well plate protocol.

4 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method

The Ostro 96-well plate was used to remove residual

PLs prior to LC/MS/MS analysis. Utilizing the generic,

simple protocol provided by the manufacturer (Figure

2), a group of analogous compounds were extracted.

The resulting analysis demonstrated an average recovery

of 86% for the group of structural analogs in plasma

(Figure 4), which is equal to or better than traditional

PPT. To assess PL removal, eight individual PLs were

summed. Results showed that Ostro plates remove

>99% of the 8 PLs relative to traditional PPT (Figure

5). In addition, the MRM transition, 184>184, was

monitored to visually demonstrate the significant

decrease in residual PLs using Ostro plates compared to

traditional PPT (Figure 6). One of the primary reasons

to eliminate PLs is to improve method robustness.

Overnight runs of both Ostro plates and traditional PPT

samples were carried out using the generic gradient

and PLs were monitored continuously. Figure 7 shows

the LC/MS trace for a representative PL at the beginning

and end of the runs. When Ostro plates are used, the

PL levels are negligible and no build-up occurs. When

PPT is used, a significant amount of PLs are present

and accumulate throughout the run. The result of this

undesirable build-up is a continuous downward trend in

area counts throughout the duration of the run (Figure

8). This in turn results in high signal variability relative

to samples prepared with Ostro plates, 33% using

PPT vs. 9% for Ostro samples. In addition, area counts

decrease by 57% from the first injection to the final

injection when PPT is used. In discovery bioanalysis,

high throughput is of utmost importance. If one tries

to increase throughput by shortening gradient time,

the impact of residual PLs is further magnified. To

demonstrate the negative effect PLs have on analytical

throughput, the gradient time was decreased by half.

Flow rate was increased and organic content was

ramped from 50 to 98% in 0.5 minutes. 200 Ostro

samples and 200 PPT samples were injected using the

shorter gradient. The MRM transition 184>184 was

monitored to reveal overall PL build-up and elution in

the shortened gradient window (Figure 9). Using the

2 minute gradient, PLs elute within 0.2 minutes of

the end of the gradient. Under the truncated gradient

conditions PLs continue to elute for more than 1 minute

after the end of the gradient and well into the re-

equilibration phase and beginning of the next

injection. These resultant chromatograms demonstrate the inability to shorten

gradient time with PPT due to PLs which continue to elute significantly after the

gradient ends at 0.5 minutes. Overall, the Ostro plate allows for increased method

robustness and reduced variability. Additionally both improved instrument uptime

and the ability to significantly shorten run times are realized through the elimination

of PLs, all of which are highly desirable in a discovery setting. Calibration curves from

1-500 ng/mL for each of the 26 structural analogs had a resulting average r2 value

of 0.925, sufficient for discovery screens.

Triazo

lam (T

-910)

alpha

-Hydro

xymida

zolam

(H-9

02)

2-hydro

xyeth

ylflur

azep

am (F

-901)

Hydrox

yalpr

azola

m d5 (A

-904)

Cloza

pine (

C-059)

Midazo

lam (M

-908)

Praze

pam (P

-906)

alpha

-Hydro

xyalp

razola

m (A-9

05)

Clonaz

epam d4

(C-9

05)

Bromaz

epam (B

-903)

Clonaz

epam (C

-907)

Flunit

razep

am (F

-907)

Alprazo

lam d5

(A-9

02)

Alprazo

lam (A

-903)

Temaz

epam (T

-907)

Cloba

zam (C

-909)

n-Desm

ethylf

lunitr

azepa

m (D-9

19)

Chlor

diaze

poxid

e (C-0

22)

Estaz

olam (E

-901)

Desalky

lflura

zepam

(D-9

15)

Oxazep

am (O

-902)

7-aminoc

lonaze

pam (A

-915)

7-aminofl

unitr

azepa

m (A-9

12)

Nitrazep

am (N

-906)

Nordiaz

epam (N

-905)

7-aminon

itraz

epam (A

-914)

0

20

40

60

80

100

Figure 4. Average analyte recovery data for 26 structural analogs and metabolites in plasma using an Ostro 96-well plate. Overall average recovery was 86%.

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

Ostro PPT

sum of 8 phospholipids

Figure 5. Comparison of phospholipids remaining after traditional PPT and PPT using Ostro 96-well plate.

5Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method

0

100

%%

7.50

7.43

7.31

7.69

7.76

9.188.80

8.31 9.8810.08

Traditional PPT

2 4 6 8 10 12 min0

100

8.30

Ostro Sample Preparation Products

2 4 6 8 10 12 min

184.4 > 184.4 (Lipid 184)1.31e8

184.4 > 184.4 (Lipid 184)1.31e8

Figure 6. Representative chromatograms of MRM transition 184>184 to demonstrate total remaining residual phospholipids from traditional PPT and Ostro eluates.

2.282.131.99

PPT lastinjection

PPT 1st injection

Ostro lastinjection

Ostro 1stinjection

gradientend

endorganic hold

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min

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

Figure 7. Representative chromatograms of MRM transition 758>184 to demonstrate build-up of an individual PL over subsequent injections using traditional PPT. A gradient from 2-98% B in 2 minutes was used followed by a 0.5 minute hold at high organic.

6 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method

0

10000

20000

30000

40000

50000

60000

70000

Ostro9% RSD

PPT33% RSD

time

PPTOstro

Figure 8. Variability of area counts over subsequent injections using Ostro 96-well plate vs. traditional PPT.

0.02

Ostro 200injections

endgradient

Ostro 1stinjection

%

0

100

0.2 0.4 0.6 0.8 1.0 1.2 1.4 min

PPT 1 injection

%

0

100

0.2 0.4 0.6 0.8 1.0 1.2 1.4 min

%

0

100

0.2 0.4 0.6 0.8 1.0 1.2 1.4 min

0.65 0.73 0.87

0.73

0.33

0.55

PPT 200 injections

%

0

100

0.2 0.4 0.6 0.8 1.0 1.2 1.4 min

184.4 > 184.4 (Lipid 184)1.34e8

184.4 > 184.4 (Lipid 184)1.34e8

184.4 > 184.4 (Lipid 184)1.34e8

184.4 > 184.4 (Lipid 184)1.34e8

Figure 9. Representative chromatograms of the MRM transition 184>184 to demonstrate the overall build-up of PLs over subsequent injections using traditional PPT vs. Ostro 96-well plate. A fast gradient from 50-98% in 0.5 minutes was used. When PPT is used, PLs continue to elute well after the end of the gradient and when the Ostro plate is used, the short gradient can readily be implemented without concern over PL build-up.

7 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method

CONCLuSIONSn Simple, universal sample preparation protocol

n Direct injection of the eluate streamlines workflow

n Removes >99% of phospholipids from plasma resulting in improved

instrument uptime and more robust methods

n High recovery for analogs and metabolites with no method

development

n Reduces sample variability

n Facilitates use of shorter runtimes, improving throughput

rEFErENCES1 Rainville, P.D., Mather J, Waters Application Note; 2008, 720002569EN

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

© 2011 Waters Corporation. Waters, T he Science of W hat’s Possible, ACQUITY UltraPerformance LC, ACQUITY UPLC, Xevo, IntelliStart, and Ostro are trademarks of Waters Corporation.

©2011 Waters Corporation. Produced in the U.S.A.July 2011 720004046EN KK-PDF

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