Analysis of Anabolic Agents in Urine byLC/MS/MS

Application
Forensics/Doping ControlAuthors

Introduction

Michael Zumwalt
Agilent Technologies, Inc.
Englewood, COUSA

The use of anabolic substances for performanceenhancement in sports seems to be a ubiquitous
topic of discussion. While their use is a growing
problem in high school and collegiate athletics,
their use at the professional level is an ongoing
controversy. It is therefore the mandate of agencies like WADA to ensure that fair competition is
being maintained by monitoring the possible use ofbanned substances like anabolic compounds.

John Hughes
Agilent Technologies, Inc.
Pleasanton, CAUSA
Matthew Slawson, Chad Borges, and Dennis Crouch
Sports Medicine Research and Testing Laboratory
University of Utah
Salt Lake City, UTUSA

Abstract
The use of the Agilent 1200 Series liquid chromatography(LC) system coupled to the 6410 Triple Quadrupole Mass
Spectrometer (QQQ) by way of the G1948B electrosprayionization (ESI) source is demonstrated in the analysis ofanabolic substances in urine. The high degree of sensitivity of the QQQ instrument allows for excellent quantitation and linearity for meeting Minimum RequiredPerformance Levels (MRPLs) for each compound as specified by the World Anti-Doping Agency (WADA). For
increased chromatographic resolution and speed, a2.1 mm id C18 column with a 3.5-µm particle size isemployed. The seven compounds, including a designatedinternal standard, all elute within 10 minutes at a flowrate of 0.4 mL/min.

Traditionally, doping control analysis for anabolic
substances, including steroids, in urine includes
screening by derivatization and GC/MS [1], followed by confirmation of the presumptive positive
using high-resolution magnetic sector GC/MS in EI
mode [2]. The high purchase and operational costsof high-resolution magnetic sector instruments
make alternative techniques like LC/MS attractive
for confirming the presence of the banned compounds.
More than 40 anabolic substances are currently
targeted in doping control analysis, many of which
are not easily analyzed using GC/MS but are
amenable to LC/MS. The analysis of some of thesecompounds is very challenging, as they must be
detected and confirmed at MRPLs of 2 ng/mL or
lower in urine.
This work describes the results of using the Agilent
LC/QQQ instrument for detection and confirmation of a number of anabolic substances at theWADA MRPL or, more specifically, covering the1/2 × – 10 × MRPL range. The anabolic compounds
analyzed in this work are listed in Table 1 alongwith their MRPLs.

In this study all compounds are steroids except forclenbuterol.
The structures of the compounds analyzed in thiswork are shown in Figure 1. Based on the resultsof work presented elsewhere [4], a derivatizing
agent is used on these samples, but only reacts
with 19-norandrosterone to improve sensitivity.
The derivatizing agent, known as Girard’s Reagent
P (Sigma Aldrich, St. Louis, MO), reacts withketone groups to form a quaternary amine, which
is more easily ionized by ESI.

Some previous work [3] used the TOF to analyze
these compounds and found that accurate mass
could be used for both screening and confirmation.
However, the QQQ is more specific with MS/MS,
increasing the confidence in confirmation andquantitating compounds of interest.
Table 1.

Minimum Required Performance Levels(ng/mL of urine)

CompoundClenbuterol

MRPL
219-norandrosterone

14β-OH-stanozolol

10Tetrahydrogestrinone (THG)

10

Methyl testosterone metabolite (MeTest metabolite)
or 17α-methyl-5β-androstane-3α,17β-diol

2Epimetendiol

2

Methyl testosterone – Internal Standard

NASample Preparation
The anabolic agents and their metabolites arepurchased from Sigma Aldrich (St. Louis, MO),
Steraloids (Newport, RI), and the National Measurement Institute (Sydney, Australia). Girard’s
Reagant P (GRP) is purchased from Sigma Aldrichand β-glucuronidase is purchased from Roche(Indianapolis, IN).

NH

CH3H3CO

H3 C

CH3H3CCl

ExperimentalHOCH3HH

H2NCl19-norandrosterone

HH

H

HO

N

H

Epimetendiol

Structures of anabolic substances analyzed in this work.

OHCH3H 3CNH

HOMethyltestosterone metabolite(17α-methyl-5β-androstane-3α, 17β-diol)

CH3OH

H
H

H

Tetrahydrogestrinone (THG)

CH3

H

H

H
HOH

OH
CH3

H

2O

HOClenbuterol

Figure 1.

H

H

OH

H
H

H

4β-OH-stanozololH

To 3 mL urine negative control sample, 1 mL 0.8 M
potassium phosphate buffer, pH 7.0, is added. A
further 25 µL β-glucuronidase is added and thenthe mixture is incubated at 50 °C for one hour. A
750-µL mixture of 20% (w/v) K2CO3/KHCO3 (1:1)
mixture is then added. Extract with methyl-t-butyl
ether and then remove and dry the organic extract.
The same extraction procedure used for GC/MS
screening is employed except that the compounds
are not derivatized as usual for GC/MS analysis.
Rather, the samples are dried and then reconstituted in 100 µL of LC mobile phase.
As part of the reconstitution step for LC/MS/MS
analysis, 20 µL methanol, followed by 8 µL of 1M
GRP in 50 mM ammonium acetate buffer, pH 4.2, isadded. Incubation at room temperature for one
hour is then followed by LC/MS/MS analysis.
Of the compounds analyzed, only the 19-norandrosterone is reactive with the GRP derivative.
This compound has been problematic in
LC/MS/MS analysis and the GRP improves sensitivity.
The MeTest internal standard has a fixed concentration of 10 ng/mL.

Mobile phase:A = 0.1% formic acid in water
B = 0.1% formic acid in methanol
0.4 mL/min; injection vol: 2 µL
Time (min)%B
0–15315
3.01
40
1250
1595

Flow rate:
Gradient:

Stop time = 15 min; Post-run time = 3 min.
MS Conditions
Mode:

Positive ESI using the Agilent G1948B
ionization source
Nebulizer:
40 psig
Drying gas flow:
9 L/min
Drying gas temp: 350 °C
Vcap:
4000 VQ1 resolution:
0.7 amuQ2 resolution:
0.7 amu
MRM transitions shown in Table 2. Chromatographic retention
times (RTs), fragmentor (Frag), collision energy (CE), and dwell
times are included. Time segments in which the MRM transitions
are implemented are also noted.

Results and Discussion
The chromatographic elution profile of all compounds at their equivalent 10 × MRPL is shown inFigure 2. The responses vary quite significantly
among the compounds and the background interference from the matrix is evident.

LC/MS Method Details
LC Conditions
Agilent 1200 Series binary pump SL, wellplate sampler, thermostatted column compartment, inline filter 0.5 µm between
needle seat and injector valve.
Column:Column temp:

Table 2.

Concentration levels ranging from 1/2 × to
10 × MRPL are run in triplicate injections. The
results for clenbuterol are shown in Figures 3a to3c. Linearity over this range has a correlation coefficient of R2 > 0.999 using the most conservative

Agilent ZORBAX XDB-CN
2.1 × 100 mm, 3.5 µm (p/n 961764-905)
50 °C

Data Acquisition Parameters for MRM Transitions

CompoundSegment 1 (0–4.0 min)Clenbuterol

RT (min)

MRM

Frag (V)

CE (V)

Dwell (msec)

2.74

277.0 > 203.1

100

15200

Segment 2 (4.0–6.3 min)
19-norandrosterone

5.82

410.3 > 331.3

1303075

Segment 3 (6.3– 6.93 min)4β-OH-stanozolol

6.64345.2 > 327.2

14015200Segment 4 (6.93–7.55 min)MeTest (IStd)

7.19303.2 > 97.1

1402575

Segment 5 (7.55–8.8 min)
THG
MeTest metabolite

7.88
8.08

313.2 > 295.1
271.2 > 161.2

150
110

1520100
100

Segment 6 (8.8–12.0 min)Epimetendiol

9.47

269.2 > 105.1

90

20200
3

curve fit settings of linear, ignored origin, and noweighting. A closer look at the reproducibility ofthe lowest three level replicates is included inFigure 3a. The limit of detection (LOD), which isdefined here as being a peak-to-peak signal-tonoise (S/N) ratio of 3:1, the S/N of the lowest level(1/2 × MRPL) is measured first. Then the same
factor that is applied to this S/N, in order to obtaina S/N of 3:1, is also applied to the lowest level.
For example, in Figure 3b the S/N is nearly 60:1
for all three injections at the 1/2 × MRPL. A factorof 20 is applied to achieve 3:1 so that the LOD is
1/20th the concentration of this level, or1/40 × MRPL.
To determine the on-column injection amount itshould be noted that the original sample corresponds to 3 mL of urine. Since the MRPL of clenbuterol is 2 ng/mL, according to Table 1, then the1/2 × MRPL contains 3 ng clenbuterol in the 3 mLurine sample. Following extraction and evaporating to dryness, this 3 ng of clenbuterol is reconstituted in 100 µL of LC mobile phase. Of this volume,
2 µL is injected. Therefore, the on-column injection
amount of clenbuterol at the 1/2 × MRPL corre-

Clenbuterol(offscale)
2.7

19-norandrosterone(offscale)
5.8

sponds to 2/100 × 3 ng = 60 pg. The LOD is therefore 1/20 × 60 pg, or about 3 pg on-column.
The LOD for clenbuterol is given in Figure 3b.
Note that the negative quality control (NQC) is also
shown as evidence that the calculated S/N isjustifiable.
Figure 3c shows the replicate injections at thelowest three levels.
The results for THG, MeTest metabolite, epimetendiol, and 4β-OH-stanozolol are shown in Figures
4, 5, 6, and 7, respectively.
As can be seen from Figure 5a, the 1/2 x MRPL
does not appear to be a limit of detection because
an S/N of 3:1 does not seem possible. However, incomparison to the matrix blank (NegQC) this levelis certainly detectable. For this reason, including
the fact that the 1/2 × MRPL replicate injections
are at the lowest end of the range investigated andlinear with the curve fit, the 1/2 × MRPL of theMeTest metabolite is considered the LOD.

β-OH-stanozolol
4β(offscale)
6.6
10 × MRPL

Epimetendiol
9.4

MeTest (IStd)
7.2

THG
7.8

Figure 2.

4

Chromatographic profile of 10 × MRPL extract in urine.

MeTestmetabolite
8.1

Clenbuterol
Excellent linearity and reproducibility(½ × – 10 × MRPL)
R2 > 0.999

Three replicate
injections each

Figure 3a. Linearity of clenbuterol.

NQC (negative quality control, blank)

1/2 × MRPLLOD (S/N = 3) ~ 1/40 × MRPL or 3 pg on-column

Figure 3b. Estimate of LOD for clenbuterol.
5

1/2 × MRPLArea RSD = 2.7%

MRPL =2 ng/mL

2 × MRPL

Area RSD = 1.4%

Area RSD = 0.7%

Figure 3c. Triplicate injections of the lowest three levels of clenbuterol.

6

THG
Good linearity and reproducibility(½ × – 10 × MRPL)
R2 > 0.992

Three replicate
injections each

Figure 4a. Linearity of THG.

NegQC

1/2 × MRPLLOD (S/N = 3) ~ 1/20 × MRPL or 30 pg on-column

Figure 4b. Estimate of LOD for THG.
7

1/2 × MRPLMRPL =10 ng/mL

2 × MRPL

Area RSD = 1.1%

Area RSD = 0.8%

Area RSD = 0.1%

Figure 4c. Triplicate injections of the lowest three levels of THG.

8

Methyltestosterone metabolite
Excellent linearity and reproducibility(½ × – 10 × MRPL)
R2 > 0.997

Three replicateinjections each

Figure 5a. Linearity of methyltestosterone metabolite.

NegQC1/2 × MRPLMRPLLOD (S/N = 3) ~ 1/2 × MRPL or 60 pg on-column

Figure 5b. Estimate of LOD for methyltestosterone metabolite.

91/2 × MRPLMRPL =2 ng/mL

2 × MRPL

Area RSD = 4.2%

Area RSD = 2.2%

Area RSD = 0.5%

Figure 5c. Triplicate injections of the lowest three levels of methyltestosterone metabolite.

10Epimetendiol
Good linearity and reproducibility(½ × – 10 × MRPL)
R2 > 0.997

Three replicate
injections each

Figure 6a. Linearity of epimetendiol.

NegQC

1/2 × MRPLLOD (S/N = 3) ~ 1/10 × MRPL or 12 pg on-column

Figure 6b. Estimate of LOD for epimetendiol.
11

1/2 × MRPLMRPL =2 ng/mL

2 × MRPL

Area RSD = 12.0%

Area RSD = 1.1%

Area RSD = 0.8%

Figure 6c. Triplicate injections of the lowest three levels of epimetendiol.

12β-OH-stanozolol
4βGood reproducibility(½ × – 10 × MRPL)
R2 > 0.982

Three replicate
injections each

β-OH-stanozolol.
Figure 7a. Linearity of 4β

1/2 × MRPLNegQC

LOD (S/N = 3) ~ 1/40 × MRPL or 15 pg on-column

β-OH-stanozolol.
Figure 7b. Estimate of LOD for 4β13

1/2 × MRPLArea RSD= 4.3%MRPL =2 ng/mL

Area RSD= 1.3%

β-OH-stanozolol.
Figure 7c. Triplicate injections of the lowest three levels of 4β

142 × MRPLArea RSD= 0.6%

In Figure 8 the reason for using the GRP derivative
is shown by comparing the sensitivity of analyzing
the 19-norandrosterone with and without thederivative.

19-norandrosterone. In Figure 9b we see noticeable
signal in the negative quality control. However, thissignal definitely comes from the matrix itself as it
is not seen in the solvent blank.

Figures 9a to 9c show the linearity, LOD, andthe lowest three level replicate injections for

The results for all compounds are summarized inTable 3.

Derivatized

Figure 8.

MRPL

NonderivatizedComparison of signal response for the derivatized (left) versus nonderivatized forms of19-norandrosterone.

1519-norandrosterone
Nice linearity and reproducibility(1/2 × – 10 × MRPL)
R2 > 0.998

Three replicate
injections each

Figure 9a. Linearity of 19-norandrosterone.

NegQCLOD (S/N = 3) ~ 1/2 × MRPL or 30 pg on-column

Figure 9b. Estimate of LOD for 19-norandrosterone.
16

1/2 × MRPL

1/2 × MRPLMRPL =1 ng/mL

2 × MRPL

Area RSD= 0.5%

Area RSD= 1.4%

Area RSD= 2.4%

Figure 9c. Triplicate injections of the lowest three levels of 19-norandrosterone.

Table 3.

Linearity, Reproducibility, and Calculated Sensitivity for All Compounds Analyzed

CompoundLinearity R2% RSD at
1/2 × MRPL

Clenbuterol
THG
MeTest metabolite
Epimetendiol
4β-OH-stanozolol
19-norandrosterone> 0.999> 0.992> 0.997> 0.997> 0.982> 0.998

1.1
1.1
4.2
12.0
4.3
0.5

LOD
on-column(pg)330
60121530LODMRPL
(×)
1/40
1/20
1/2
1/10
1/40
1/2

17

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Conclusions
The analysis of anabolic substances in urine can bedifficult and may require the sensitivity of a triple
quadrupole mass spectrometer as seen in thiswork. Linearity over the range of 1/2 × to
10 × MRPL for each compound is demonstrated
and shown to be very good, especially for clenbuterol, which has a correlation coefficient of
more than 0.999. The liquid chromatography inthis work only uses solvents of water andmethanol, with the addition of formic acid for a
simple gradient. Limits of detection at levels lower
than the minimum required performance levels aredemonstrated with percent relative standard deviations of peak areas ranging from 12.0% to as low
as 0.5%. The addition of Girard’s Reagent P solution shows a marked improvement in sensitivity
for the 19-norandrosterone compound.

References
1. H. Pereira, M. Marques, I. Talhas, and F. Neto,Analysis of Androgenic Steroids, Beta-2-Agonists and Other Substances by GC-MS-ITD. In
W. Schanzer, H. Geyer, A. Gotzmann, andU. Mareck, Recent Advances in Doping Analysis. (2003) Sport und Buch Straub, Koln, 11, 259.

2. G. Trout, S. Soo, and R. Kazlauskas, Single
Screen for Steroids Using HRMS. In RecentAdvances in Doping Analysis. (2003) Sportund Buch Straub, Koln, 11, 249.
3. C. Borges, M. Slawson, J. Taccogno, D. Crouch,and J. Hughes, Screening and Confirmation ofAnabolic Steroids Using Accurate Mass LC/MS.
(2006) Agilent Technologies publication
5989-4738EN.
4. C. Borges, N. Miller, M. Shelby, M. Hansen, C.
White, M. Slawson, K. Mont, and D. Crouch,Analysis of a Challenging World Anti-Doping
Agency-Banned Steroids and Antiestrogens by
LC-MS-MS, Journal of Analytical Toxicology(2007), 31, 125.

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