Sport Doping Screening in Biological Matrices Trap ... · TO DOWNLOAD A COPY OF THIS POSTER, VISIT ©2018Waters Corporation v1 Robert Walsh2, Claude R Mallet1, Sabra Botch-Jones2

TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2018Waters Corporation v1

Robert Walsh2, Claude R Mallet1, Sabra Botch-Jones2

Separation Technologies1, Workflow Integration Group, Waters Corporation, 34 Maple St., Milford, MA 01757-3696 corresponding author: [email protected]

Boston University School of Medicine2, Boston, MA, USA, [email protected]

Abstract

As seen in the past few decades, the reputations of many

professional sports leagues and players have been tarnished

due to cheating in the form of sports doping. As such, it is

commonplace to have organizations, protocols and methods

dedicated to detecting performance enhancing agents in

biological samples such as urine. While doping commonly

refers to steroids, the World Anti-Doping Agency (WADA)’s

list of prohibited substances contains a wide variety of com-

pounds with anabolic agents being only one of many

classes. The wide variety of compound, low levels and the

short time frame in which results are expected all contrib-

ute to the challenges of developing a comprehensive

screening method for sports doping agents.

All target drugs in this screening study were classified in 9

classes based on their chemical structure (79 total). Once

in solution, target analytes were individually infused to ac-

quire their respective precursor and product ions. Once the

quantitative and qualitative transitions for all drugs were

identified, the chromatography conditions were optimized

for each class using a 6x6 automated methods development

protocol. Each target analyte was subjected to a total of

108 LC/MS/MS methods which were carried out within 72

hours. The multi-dimensional LC was configured for "Trap &

Elute" with At-Column Dilution. The total run time was 10

minutes. The mass spectrometer was a XEVO G2 QToF op-

erated in positive ionization mode.

Current analytical techniques use a combination of extrac-

tion procedures, often requiring an enrichment process and

accurate detection for any given target analyte. As such,

new analytical strategies are needed to reach those goals.

This work evaluated the performance of 2D LC variant using

a QToF setup instead of a triple quadrupole mass spec-

trometer for the analysis of drug of abuse in urine targeting

low and sub ppb level. From a previous study1 focusing on

the analysis illicits drugs in urine by 2D LC/MS/MS, target

LOD at 100 ppt was reported from a 1 mL urine sample.

Considering the level of complexity of urine samples and

also ensuring the retention of all target analyte in a clean

extract, several strategic extraction protocols were evalu-

ated to determine which one can reach a target detection

level between 0.1 ppb and 1 pbb.

1-Mallet, C.R, Botch-Jones, S.R., Ilicit Drug Analysis Using

Two-Dimension Liquid Chromatography/Tandem Mass Spec-

trometry, J. Anal. Tox., 2016:1-11

Results

Conclusions

- Low level detection (ppb)

- Fast sample preparation

- Rapid method development

- High level of reproducibility and robustness

- Good precision & accuracy (< 5 %)

Time5.05 5.10 5.15 5.20 5.25 5.30 5.35

%

0

100

5.05 5.10 5.15 5.20 5.25 5.30 5.35

%

0

100

8: MRM of 1 Channel ES+ TIC (tripolidine)

1.65e65.21


1.97e65.19

Time5.00 5.10 5.20 5.30 5.40 5.50

%

0

100

5.00 5.10 5.20 5.30 5.40 5.50

%

0

100


3.31e65.20


8.08e55.20

Aqueous StdACD off

Aqueous StdACD on

Acetonitrile StdACD off

Acetonitrile StdACD on

A

B

C

D

EXPERIMENTAL

Pump

A

Pump

C

Injector

Loop

MS

waste

Elution

Loader

Loading Conditions Eluting Conditions

Trapping Chemistries

Separation Chemistries

Resolving

Dimension

Trapping

Dimension

D1

D2

Pump

B

Dilutor

C8pH 3 LoadingpH 3 ACN Elution






HLBpH 3 LoadingpH 3 ACN Elution









HLBpH 3 LoadingpH10ACN Elution



C8pH 3 LoadingpH 3 MeOH Elution






HLBpH 3 LoadingpH 3 MeOH Elution







C18pH 7 LoadingpH 10 meOH Elution





Load

ing

pH

Load

ing

pH

3x3 Method Optimization (9 permutations)10 min LC Run Time / 4.5 hours

3x6 Method Optimization (18 permutations)10 min LC Run Time / 9 hours

6x6 Method Optimization (36 permutations)10 min LC Run Time / 18 hours

Trap Retention Strength Elution pH

Load

ing

pH

Elution Polarity

1

4

7

2

5

8

3

6

9

10

13

16

11

14

17

12

15

18

19

22

25

20

23

26

21

24

27

28

31

34

29

32

35

30

33

36

INSTRUMENTATION Method 1 2 3 4 5 6 7 8 9

A - H2O

A - MeoH lead e5 lead e3 MP e5 e4 MP shoulder

A - ACN lead lead lead lead e5 shoulder shoulder

B - H2O e5 MP e6 MP e4 tail e4 e5 e5 MP e8

B - MeoH e5 MP e6 MP e5 lead e5 e5 e5 e8

B - ACN e5 MP e6 MP e5 lead e5 e5 e5 e8

C- H2O e5 e4 e4 e5

C - MeoH e5 e4 MP e5

C - ACN e5 e4 MP e4 MP e5

D - H2O e5 e6 e5 e5 e6

D - MeoH e5 e7 e4 e5 e7 e4 e5 e7 e7

D - ACN e5 e7 e4 e4 e7 e4 e4 e7 e7

E - H2O e4 MP e6 MP e4 e5 e4 MP e5 e6

E - MeoH e5 MP e6 e4 e4 e6 e4 e4 MP e6 e7

E - ACN e4 MP e6 e4 e4 e6 e4 e4 MP e6 e6

F - H2O

F - MeoH e4 MP e6 MP e4 MP e4 MP e6 e4 MP

F - ACN e4 MP e6 MP e4 MP e4 MP e6 e4 MP

G - H2O e4 MP e5 MP e4 MP e5 MP e5 MP

G - MeoH e5 MP e6 e5 e5 e6 e4 e4 MP e5 MP e6 MP

G - ACN e5 MP e6 e4 e4 e6 e4 e4 MP e5 MP e6

H - H2O e5 MP e7 MP e4 MP e4 MP e6 MP e4 MP e4 MP e6 MP e7 MP

H - MeoH e5 MP e7 MP e5 MP e5 MP e7 MP e5 MP e5 MP e7 MP e7 MP

H - ACN e5 MP e7 MP e5 MP e5 MP e7 MP tail e5 MP e7 MP e7 MP

A- 5α-Dihydrotestosterone B- Clenbuterol C- Epitestosterone D- Danazol E- Androstenedione F- Tibolone

G- Trans-Androsterone H- Testosterone

Methods 1 2 3 4 5 6 7 8 9

Highest Signal 0 5 0 0 4 0 0 2 6

10 11 12 13 14 15 16 17 18

3 3 3 4 0 2 2 0 2

19 20 21 22 23 24 25 26 27

8 7 1 5 8 1 7 7 1

28 29 30 31 32 33 34 35 36

4 2 0 4 2 0 3 2 0

C1 MeOH 10ppb Method 1

Time5.00 6.00 7.00

%

0

100

5.00 6.00 7.00

%

0

100

5.00 6.00 7.00

%

0

100

6.57

6.54

6.54


Time5.00 6.00 7.00

%

0

100

5.00 6.00 7.00

%

0

100

5.00 6.00 7.00

%

0

1006.52

6.52

6.52


Time5.00 6.00 7.00 8.00

%

0

100

5.00 6.00 7.00 8.00

%

0

100

5.00 6.00 7.00 8.00

%

0

1006.57

Method 1 Method 2 Method 3



Danazol (MeOH)

Maximum Coverage for all 79 Pharmaceuticals

Method 6, 9 and 19

Sport Doping Screening in Biological Matrices

by Multi-Dimensional LC-QToF

HLB 2D Protocol

Single Methods Multiple Methods

Class 1 20 23 9 19

Class 2 1 2 3 6

Class 3 NA

Class 4 2 6

Class 5 8 9 17 24 25 27 35

Class 6 8 9 15 26 35

Class 7 8 9 16 17 19 25 32 35

Class 8 NA

Class 9 6 7 8 9 14 17 26 31 32

Class 10 1 2 5 9 10 11 13 15 16 18 19 20 22 23 25 26 28 29 31 32 34 35

1- 10 % ACN2- 20 % ACN3- 30 % ACN4- 40 % ACN5- 50 % ACN6- 60 % ACN7- 70 % ACN8- 80 % ACN9- 90 % ACN10- 100 % ACN

All elution with2% FA

Repeatwith MeOH

1- 10 % ACN2- 20 % ACN3- 30 % ACN4- 40 % ACN5- 50 % ACN6- 60 % ACN7- 70 % ACN8- 80 % ACN9- 90 % ACN10- 100 % ACN

All elution with 2% NH4OH

Repeatwith MeOH

Step 1Load 2 mL Water

Spike @1 ppb

Step 2Collect Load

Step 3

Sequential

Elution

Step 4

Sequential

Elution

SPE 1 SPE 2

0 10 20 30 40 50 60 70 80 90 100Propanolol Acn Hi pH 0.09 0.00 1.42 1.04 55.06 37.47 3.37 1.01 0.05 0.27 0.2

Propanolol Acn Lo pH 0.75 0.08 74.23 23.04 0.54 1.01 0.05 0.03 0.18 0.04 0.06

Propanolol MeOH Hi pH 0.68 0.21 0.01 0.87 0.00 0.05 1.02 8.97 64.13 22.51 1.54

Propanolol MeOH lo pH 0.16 0.04 0.03 1.47 89.08 7.38 1.36 0.02 0.01 0.41 0.04

Atenolol Acn Hi pH 0.57 62.12 17.7 1.25 2.68 3.9 3.26 2.08 3.46 2.42 0.56

Atenolol Acn Lo pH 0.33 52.33 43.27 0.68 0.69 0.42 0.95 0.20 0.51 0.4 0.23

Atenolol MeOH Hi pH 1.19 0.30 0.57 93.8 0.87 0.75 0.71 0.65 0.47 0.49 0..4

Atenolol MeOH lo pH 0.62 88.26 6.26 0.69 1.01 1.54 0.23 0.36 0.26 0.43 0.19

Metaprolol Acn Hi pH 6.06 0.76 45.32 30.05 3.16 1.72 4.64 3.75 1.73 1.48 1.34

Metaprolol Acn Lo pH 1.54 40.21 54.46 1.68 0.43 0.28 0.05 0.71 0.11 0.02 0.54

Metaprolol MeOH Hi pH 0.2 0.00 0.58 60.35 5.1 13.8 7.52 10.34 0.11 0.95 1.05

Metaprolol MeOH lo pH 0.57 0.66 38.61 50.27 3.42 0.31 1.25 0.22 1.49 1.20 2.00

Esmolol Acn Hi pH 0.05 0.01 23.29 72.48 3.24 0.13 0.11 0.24 0.00 0.37 0.07

Esmolol Acn Lo pH 0.00 9.55 84.3 5.11 0.07 0.71 0.13 0.00 0.07 0.05 0.00

Esmololl MeOH Hi pH 0.03 0.00 0.02 25.42 1.07 9.48 14.79 38.97 8.47 1.67 0.10

Esmolol MeOH lo pH 0.00 0.03 2.41 79.54 17.38 0.08 0.02 0.00 0.47 0.00 0.08

Betaxolol Acn Hi pH 0.27 0.22 0.26 34.33 57.09 5.29 0.59 0.44 0.66 0.28 0.57

Betaxolol Acn Lo pH 0.21 0.12 86.23 10.74 1.48 0.36 0.53 0.11 0.00 0.11 0.12

Betaxolol MeOH Hi pH 0.37 0.02 0.19 0.79 0.22 0.94 1.9 23.58 58.97 11.81 1.21

Betaxolol MeOH lo pH 0.00 0.11 0.18 8.48 82.75 6.56 0.85 0.29 0.50 0 0.29

Timolol Acn Hi pH 0.58 0.08 55.58 32.61 4.89 0.93 0.76 0.25 1.49 2.55 0.27

Timolol Acn Lo pH 0.00 41.46 55.68 2.23 0.37 0.05 0.01 0.05 0.00 0.05 0.09

Timolol MeOH Hi pH 0.37 0.02 0.19 0.79 0.22 0.94 1.9 23.58 58.97 11.81 1.21

Timolol MeOH lo pH 0.01 0.11 50.88 46.56 1.44 0.31 0.03 0.12 0.23 0.27 0.04

Carvedilol Acn Hi pH 2.35 2.44 2.56 2.69 2.95 54.12 17.65 6.17 3.81 3.06 2.14

Carvedilol Acn Lo pH 1.83 1.62 2.18 66.85 13.97 3.97 2.61 1.46 2.15 1.77 1.58

Carvedilol MeOH Hi pH 2.11 2.33 2.06 2.21 1.6 1.82 2.6 1.52 4.97 47.58 31.19

Carvedilol MeOH lo pH 1.27 1.6 1.75 1.43 1.77 19.69 37.71 24.27 5.6 2.78 2.12

Labetalol Acn Hi pH 0.73 13.33 77.89 1.06 1.34 0.25 1.19 0.96 1.85 0.46 0.95

Labetalol Acn Lo pH 0.74 1.24 87.65 4.5 0.76 1.12 0.9 0.88 1.37 0.29 0.55

Labetalol MeOH Hi pH 0.00 1.57 0.56 90.82 1.09 1.34 1.96 0.54 0.96 0.89 0.27

LabetalolMeOH lo pH 1.26 0.71 0.03 8.76 81.26 3.26 1.36 1.01 0.81 0.74 0.8

Elution - 40 % ACN Lo pH

Wash - 20 % MeOH Hi pH

Class 7

Water Extract 10 ppb A Elute MS

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

C7XTMSMSE38 1: TOF MS ES+ 329.4

1.11e5

4.81

4.68 6.396.115.69 6.97


8.56e5

4.66


8.33e5

5.25


5.27e5

4.48

4.214.10


1.58e6

5.08


3.50e5

4.27

3.916.82


6.88e5

4.55

4.28 6.44


7.92e5

4.97

Water Extract 1 ppb A Elute MSE

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100


2.40e4

6.384.80

4.674.223.59

0.51

5.915.61 6.967.47 8.31 9.08

9.45


1.20e5

4.66

6.90


7.24e5

5.24


6.98e4

4.48

4.214.095.404.78 7.115.61 6.49 9.48


2.72e5

5.08


3.17e5

4.27

3.90 6.82


9.80e4

4.55

4.27

3.63

6.44

6.386.04

6.69


1.52e5

4.97

3.61

Urine Extract 1 ppb A Elute MSE

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100


4.53e5

5.66

5.124.945.74

6.37


6.77e4

4.66

3.85

3.74

4.626.90

5.27 5.436.03

8.93


2.51e4

5.24

4.844.754.170.51 3.60

5.71 7.576.27

6.35 6.87

7.878.18


1.70e5

5.58

5.50

4.484.095.41

5.75

6.10 7.13


4.21e6

5.00


1.78e5

4.25

3.90

5.224.32

6.825.85 5.97


8.15e4

4.55

4.25

3.62

6.44

5.21 6.376.04

6.69 7.21


4.22e4

5.954.96

4.333.61 5.40

5.98

6.55 9.448.57

Urine Extract 10 ppb A Elute MS

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100


3.82e5

5.66

4.794.68

5.12 5.746.37


5.30e5

4.66

3.854.11

6.90


1.40e5

5.24

4.83 7.575.70 6.277.87


3.36e5

4.48

4.09

5.585.50 5.75


4.18e6

5.00


1.63e5

4.253.905.21

4.32

6.825.85 5.97


6.60e5

4.55

4.253.90

6.43


3.10e5

4.96

5.94

HLB 2D Protocol Urine extract

HLB 2D Protocol Water extracted standard

Documents

Sport Doping Screening in Biological Matrices Trap ... · TO DOWNLOAD A COPY OF THIS POSTER, VISIT ©2018Waters Corporation v1 Robert Walsh2, Claude R Mallet1, Sabra Botch-Jones2