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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
8: MRM of 1 Channel ES+ TIC (tripolidine)
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
8: MRM of 1 Channel ES+ TIC (tripolidine)
3.31e65.20
8: MRM of 1 Channel ES+ TIC (tripolidine)
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
C8pH 7 LoadingpH 3 ACN Elution
C8pH 10 LoadingpH 3 ACN Elution
C18pH 3 LoadingpH 3 ACN Elution
C18pH 7 LoadingpH 3 ACN Elution
C18pH 10 LoadingpH 3 ACN Elution
HLBpH 3 LoadingpH 3 ACN Elution
HLBpH 7 LoadingpH 3 ACN Elution
HLBpH 10 LoadingpH 3 ACN Elution
C8pH 3 LoadingpH 10 ACN Elution
C8pH 7 LoadingpH 10 ACN Elution
C8pH 10 LoadingpH 10 ACN Elution
C18pH 3 LoadingpH 10 ACN Elution
C18pH 7 LoadingpH 3 ACN Elution
C18pH 10 LoadingpH 10 ACN Elution
HLBpH 3 LoadingpH10ACN Elution
HLBpH 7 LoadingpH 10 ACN Elution
HLBpH 10 LoadingpH 10 ACN Elution
C8pH 3 LoadingpH 3 MeOH Elution
C8pH 7 LoadingpH 3 MeOH Elution
C8pH 10 LoadingpH 3 MeOH Elution
C18pH 3 LoadingpH 3 MeOH Elution
C18pH 7 LoadingpH 3 MeOH Elution
C18pH 10 LoadingpH 3 MeOH Elution
HLBpH 3 LoadingpH 3 MeOH Elution
HLBpH 7 LoadingpH 3 MeOH Elution
HLBpH 10 LoadingpH 3 MeOH Elution
C8pH 3 LoadingpH 10 MeOH Elution
C8pH 7 LoadingpH 10 MeOH Elution
C8pH 10 LoadingpH 10 MeOH Elution
C18pH 3 LoadingpH 10 MeOH Elution
C18pH 7 LoadingpH 10 meOH Elution
C18pH 10 LoadingpH 10 MeOH Elution
HLBpH 3 LoadingpH 10 MeOH Elution
HLBpH 7 LoadingpH 10 MeOH Elution
HLBpH 10 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
C1 MeOH 10ppb Method 2
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
C1 MeOH 10ppb Method 3
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
Method 4 Method 5 Method 6
Method 7 Method 8 Method 9
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
C7XTMSMSE38 1: TOF MS ES+ 296.4
8.56e5
4.66
C7XTMSMSE38 1: TOF MS ES+ 407.3
8.33e5
5.25
C7XTMSMSE38 1: TOF MS ES+ 317.4
5.27e5
4.48
4.214.10
C7XTMSMSE38 1: TOF MS ES+ 308.4
1.58e6
5.08
C7XTMSMSE38 1: TOF MS ES+ 267.4
3.50e5
4.27
3.916.82
C7XTMSMSE38 1: TOF MS ES+ 268.4
6.88e5
4.55
4.28 6.44
C7XTMSMSE38 1: TOF MS ES+ 260.3
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
C7XTMSMSE22 1: TOF MS ES+ 329.4
2.40e4
6.384.80
4.674.223.59
0.51
5.915.61 6.967.47 8.31 9.08
9.45
C7XTMSMSE22 1: TOF MS ES+ 296.4
1.20e5
4.66
6.90
C7XTMSMSE22 1: TOF MS ES+ 407.3
7.24e5
5.24
C7XTMSMSE22 1: TOF MS ES+ 317.4
6.98e4
4.48
4.214.095.404.78 7.115.61 6.49 9.48
C7XTMSMSE22 1: TOF MS ES+ 308.4
2.72e5
5.08
C7XTMSMSE22 1: TOF MS ES+ 267.4
3.17e5
4.27
3.90 6.82
C7XTMSMSE22 1: TOF MS ES+ 268.4
9.80e4
4.55
4.27
3.63
6.44
6.386.04
6.69
C7XTMSMSE22 1: TOF MS ES+ 260.3
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
C7XTMSMSE97 1: TOF MS ES+ 329.4
4.53e5
5.66
5.124.945.74
6.37
C7XTMSMSE97 1: TOF MS ES+ 296.4
6.77e4
4.66
3.85
3.74
4.626.90
5.27 5.436.03
8.93
C7XTMSMSE97 1: TOF MS ES+ 407.3
2.51e4
5.24
4.844.754.170.51 3.60
5.71 7.576.27
6.35 6.87
7.878.18
C7XTMSMSE97 1: TOF MS ES+ 317.4
1.70e5
5.58
5.50
4.484.095.41
5.75
6.10 7.13
C7XTMSMSE97 1: TOF MS ES+ 308.4
4.21e6
5.00
C7XTMSMSE97 1: TOF MS ES+ 267.4
1.78e5
4.25
3.90
5.224.32
6.825.85 5.97
C7XTMSMSE97 1: TOF MS ES+ 268.4
8.15e4
4.55
4.25
3.62
6.44
5.21 6.376.04
6.69 7.21
C7XTMSMSE97 1: TOF MS ES+ 260.3
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
C7XTMSMSE113 1: TOF MS ES+ 329.4
3.82e5
5.66
4.794.68
5.12 5.746.37
C7XTMSMSE113 1: TOF MS ES+ 296.4
5.30e5
4.66
3.854.11
6.90
C7XTMSMSE113 1: TOF MS ES+ 407.3
1.40e5
5.24
4.83 7.575.70 6.277.87
C7XTMSMSE113 1: TOF MS ES+ 317.4
3.36e5
4.48
4.09
5.585.50 5.75
C7XTMSMSE113 1: TOF MS ES+ 308.4
4.18e6
5.00
C7XTMSMSE113 1: TOF MS ES+ 267.4
1.63e5
4.253.905.21
4.32
6.825.85 5.97
C7XTMSMSE113 1: TOF MS ES+ 268.4
6.60e5
4.55
4.253.90
6.43
C7XTMSMSE113 1: TOF MS ES+ 260.3
3.10e5
4.96
5.94
HLB 2D Protocol Urine extract
HLB 2D Protocol Water extracted standard