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Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography–electrospray ionization tandem mass spectrometry
Steroids 69 (2004) 101–109
Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography–electrospray ionization tandem mass spectrometry Antti Leinonen a,b , Tiia Kuuranne a,b,c , Tapio Kotiaho b , Risto Kostiainen b,c,∗ a Doping Control Laboratory, United Laboratories Ltd., Helsinki, Finland Department of Pharmacy, Viikki Drug Discovery and Technology Center, University of Helsinki, Helsinki, Finland Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland b
c
Received 14 January 2003; received in revised form 16 June 2003; accepted 21 October 2003
Abstract A qualitative liquid chromatography–electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol–water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1–2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine. © 2003 Elsevier Inc. All rights reserved. Keywords: Anabolic steroids; Metabolism; Liquid chromatography; Mass spectrometry; Electrospray ionization; Doping analysis
1. Introduction Anabolic androgenic steroids (AAS) are still an important class of abused drugs in sports, despite the fact that they have been banned already since the mid-1970s [1]. Furthermore, steroid abuse has become more and more prevalent outside sports. For instance, even teenagers use them as an expression of an improved life style. The abuse of AAS among young people causes significant medical problems. In addition, adolescent steroid users have been found to be engaged in many risk behaviors such as multiple drug use as well as aggressive and suicidal behavior [2]. The fate of AAS in human body is well known [3–5]. These steroids are extensively metabolized, with little or no unchanged steroid being excreted in the urine. The typical metabolic reactions involve oxidation, reduction, hydroxylation and epimerization, followed by conjugation reactions ∗ Corresponding author. Tel.: +358-9-191-59-134; fax: +358-9-191-59-556. E-mail address: [email protected] (R. Kostiainen).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2003.10.007
resulting in the formation of AAS glucuronides or sulfates. As an exception, several 17-alkyl-substituted steroids are excreted in urine mainly or partly as unconjugated metabolites. Alkylation of position C17 reduces androgenic side effects and allows oral administration of the drug [1]. To monitor and prevent the abuse of AAS, efficient and reliable analytical methods are needed. In doping control, the methods are generally divided into two categories: screening procedures and methods of confirmatory analysis [3]. The aim of screening is to find and select suspicious samples for further analysis and an ideal screening method should be sensitive, fast, easy to perform, and have a sufficient sample capacity. Due to the large number of compounds involved, the structural similarity of exogenous and endogenous steroids, the complexity of urine matrix, as well as the low steroid concentrations in urine, analysis of AAS is a challenging task. Because of the large number of target substances, multi-analyte screening methods are superior to single-compound methods. Current testing methods generally rely on gas chromatography–mass spectrometry (GC/MS). In the prevalent procedures, urine
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samples are either directly or after solid phase extraction hydrolyzed enzymatically to cleave the steroid conjugates and then liquid extracted under alkaline conditions. Finally the analytes are trimethylsilylated before measurement by GC/MS with electron ionization and selected ion monitoring [5–10]. Parallel to this, to enhance the detectability of certain steroids, unconjugated metabolites can be measured after alkaline liquid extraction and silylation in a separate method [5–8]. To achieve lower detection limits, negative ion chemical ionization, high-resolution mass spectrometry (HRMS), and tandem mass spectrometry (MS/MS) have been applied in GC-based analysis methods [11–14]. Liquid chromatography–mass spectrometry (LC/MS) has also been used for the analysis of a variety of AAS in human [15–22]. Metabolites of stanozolol have been analyzed in urine by atmospheric pressure chemical ionization (APCI) both prior to and after hydrolysis [15]. Particle beam ionization has been used to determine 28 different AAS or their metabolites in hydrolyzed urine extracts [16]. Electrospray ionization (ESI) has been used to analyze intact glucuronide and sulfate conjugates of testosterone and epitestosterone in urine [17,18] as well as testosterone esters in plasma [19]. An ESI method for simultaneous measurement of 12 different synthetic AAS glucuronides in urine has also been introduced [20]. Metabolites of gestrinone have been characterized by ESI in urine after hydrolysis [21]. Furthermore, our group has recently demonstrated the applicability of ESI, APCI and atmospheric pressure photoionization in the detection of structurally different unconjugated AAS metabolites in urine [22]. LC/MS has the potential to overcome some of the problems associated with GC methods, i.e. derivatization step, adsorption of steroids, and their thermal decomposing during analysis. Therefore, investigation of the suitability of LC/MS assays for multi-analyte screening of AAS is of great interest. This paper describes a qualitative LC/ESI-MS/MS screening method for the direct analysis of nine urinary 17-alkylsubstituted AAS or their metabolites (Fig. 1), all excreted in urine in unconjugated form and used as target compounds in the doping analysis of free steroid fraction [3–8,23]. The method is based on our initial optimization of ionization method for the LC/MS analysis of AAS [22] and is now suitable for the detection of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol abuse.
2. Experimental 2.1. Chemicals and reagents 9␣-Fluoro-17␣-methyl-androst-4-ene-3␣,6,11,17-tetrol (FLXm), 6-hydroxymetandienone (6MDN), 2-hydroxymethyl-17␣-methyl-androsta-1,4-diene-11␣,17-diol3-one (FMBm), 6-hydroxy-4-chloro-dehydromethyltestosterone (6CDM), 3 -hydroxystanozolol (3STZ), 17-epimetan-
dienone (17MDN), and 17-epioxandrolone (17OXD) were purchased from National Analytical Reference Laboratory (NARL, Pymble, Australia). Oxandrolone (OXD), ethisterone (DNZm), and methyltestosterone (MTS) were from Steraloids (Newport, RI). All other reagents and solvents were either of HPLC or analytical grade. 2.2. Sample work-up Extraction of urine samples was based on the procedure described by Schänzer and Donike [5]. A 2.5-ml aliquot of urine was spiked with 125 ng of methyltestosterone (internal standard). Then 250 mg of a solid mixture of sodium hydrogen carbonate and potassium carbonate (2:1, w/w) was added and the sample extracted with 2.5 ml of diethyl ether. After centrifugation, the organic layer was separated and evaporated to dryness. Finally, the residue was reconstituted in 60 l of 50% methanol (v/v) containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. 2.3. Liquid chromatography The liquid chromatographic system consisted of a Hewlett-Packard (Waldbronn, Germany) HP 1100 Series binary gradient pump and autosampler. Operation conditions were identical to those in our former study [22]. Chromatographic separation was obtained at ambient temperature on a LiChroCART Purospher RP C-18e column (125 mm × 3 mm, 5 m) from Merck (Darmstadt, Germany) equipped with a 4 mm × 4 mm pre-column of the same stationary phase. Injection volume was 5 l. An aqueous solution of 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid was used as solvent A. Solvent B consisted of 90% (v/v) methanol in water with final concentration of 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. A linear gradient was run with a flow rate of 0.5 ml/min from 50 to 100% B in 15 min and held at the final composition for 3 min. Column effluent was finally post-column split 1:100, before introduction to the MS source. 2.4. Mass spectrometry The analyses were carried out on a Perkin-Elmer Sciex API 3000 triple quadrupole mass spectrometer (Sciex, Concord, Canada) equipped with an ion spray source. All experiments were carried out in the positive ion mode. Compressed air (Atlas Copco CD-2 system, Belgium) was used as a nebulizing gas with a flow rate of 0.82 l/min. Nitrogen (Whatman 75-72 nitrogen generator, MA) was used both as collision and curtain gas (0.95 l/min). The ion source was operated in ESI mode. Based on our earlier studies [22], spray needle voltage and orifice (declustering) voltage were set to 5000 V and 20 V, respectively. Flow injection studies were performed using a Harvard Apparatus microsyringe pump (MA, USA) with a flow rate of 5 l/min. In MS and MS/MS studies, mass spectra were collected in a scan
A. Leinonen et al. / Steroids 69 (2004) 101–109
Exogenous source
Compound
103
Structure
MW OH HO
FLXm
9α-Fluoro-17α-methyl-androst-4-ene3α,6β,11β,17β-tetrol
CH 3
Fluoxymesterone
354 F HO OH
OH CH 3
6MDN
6β-Hydoxymetandienone
Metandienone
316 O
OH OH
FMBm
2-Hydroxymethyl-17α-methyl-androsta-1,4diene-11α,17β-diol-3-one
HO
Formebolone
CH 3
346
HO H 2 C
O
OH
6CDM
6β-Hydroxy-4-chloro-dehydromethyltestosterone
CH 3
4-Chlorodehydromethyltestosterone
350 O
OH
Cl
OH CH 3
OXD
Oxandrolone
Oxandrolone
306
O O
H
OH CH 3
HO
3STZ
3'-Hydroxystanozolol
Stanozolol
344 H N N H OH
C CH
DNZm
Ethisterone
Danazol
312 O CH 3 OH
17MDN
17-Epimetandienone
Metandienone
300 O CH 3 OH
17OXD
17-Epioxandrolone
Oxandrolone
306
O O
H OH 17
MTS
1
Methyltestosterone
Internal standard
C
302
2
O
CH 3
D
3
A
B
4
6
Fig. 1. Nomenclature and chemical formulae of the steroids studied.
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A. Leinonen et al. / Steroids 69 (2004) 101–109
range of m/z 250–450 and m/z 50–450. In all measurements, five spectra with scan speed of 5 s were accumulated. In MS/MS experiments, either the protonated molecule or the ammonium adduct was selected as precursor ion and collision offset voltages were optimized over the range of 5–80 V. In the final LC/ESI-MS/MS method, the steroids were detected using multiple reaction monitoring (MRM) of two characteristic product ions per analyte. Dwell time for each ion was 400 ms. Analyst 1.1 software was used for instrument control and data processing. 2.5. Samples and method evaluation In order to evaluate linearity and to calibrate the method, standards were prepared by spiking pooled human drugfree urine with steroids at concentrations of 0, 0.5, 1, 5, 10, 50, 100, 250, 500, and 1000 ng/ml, each sample as a single. Calibration curves were obtained from the analyte to internal standard peak-height ratios using linear regression with 1/x weighing. To estimate inter-day precision and accuracy, quality control samples were prepared in the same way and at the same concentration levels as the standards but from separate stock solutions and analyzed on 5 separate days. Standards were prepared and calibration curves constructed within every analysis batch. Limit of detection (LOD) was verified with spiked samples at estimated concentrations as six replicates. Random interference of biological background was checked from drug-free urine samples of 10 male and 10 female volunteers. The extraction recoveries were measured at a concentration level of 50 ng/ml (six replicates each) by comparing relative intensities of extracted spiked urine samples to those of extracted blank urines spiked with standards; in every case, internal standard was added after extraction. Finally, the method was tested with authentic forensic urine samples, which had in the qualitative GC/MS analysis been found to contain metabolites of danazol, fluoxymesterone and metandienone.
3. Results and discussion 3.1. Mass spectrometry In order to select precursor ions for MRM, full-scan mass spectra of each steroid were measured. Either [M + H]+ or [M + NH4 ]+ was the dominating ion in the mass spectrum (Table 1), the intensity ratio [M + H]+ /[M + NH4 ]+ depending on the proton affinity (PA) and with high PA favoring formation of the protonated molecule as shown previously [22]. The pyrazole ring (3STZ) and the 1,4-diene-3-one structure (17MDN, FMBm, 6MDN) have the highest PA, but exceptionally in the case of 6CDM, PA is decreased due to an electronegative chlorine atom at position C4. PA of the 4-ene-3-one structure (DNZm, MTS) is lower but still significantly higher than that of the 3-one structure without a 4-ene double bond (OXD, 17OXD), whereas hydroxyl
Table 1 Relative abundances (%) of protonated molecule and corresponding ammonium adduct in mass spectra of the steroids Compound
[M + H]+
[M + NH4 ]+
3STZ 17MDN FMBm 6MDN DNZm MTS 6CDM 17OXD OXD FLXm
100 100 100 100 100 100 100 30 29 –
– – – 7 34 35 39 100 100 100
groups with a single double bond (FLXm) have the lowest PA. The PA order of the steroids was estimated using PAs of their structural analogs [24]. The protonated molecule was chosen as a precursor ion for 6MDN, FMBm, 6CDM, 3STZ, DNZm, 17MDN and MTS, and the ammonium adduct was selected for FLXm, OXD and 17OXD. It should be noted that because of its lowest PA, FLXm did not form any protonated molecule. This means that the use of adduct-forming additives may be essential for its ionization also with other atmospheric pressure ionization techniques than ESI. Product ion spectra were measured with different collision offset voltages to optimize fragmentation for MRM. The most abundant and specific product ions were selected, excluding unspecific dehydrated fragments. In general, the optimum collision offset voltages varied between 15 and 30 V. However, due to its stable pyrazole ring, breakdown of 3STZ required significantly higher collision energy (60 V) [22]. Optimized MRM parameters are listed in Table 2. Fragmentation of the steroids was not studied in detail in this work. However, as shown elsewhere, a rational correlation between steroid structures and fragmentation patterns can be found [25,26]. For instance, MTS and DNZm formed intensive ions at m/z 97 and 109, typical for steroids having the 4-ene-3-one structure [25]. The fragmentation patterns of OXD and 17OXD were similar, as reported for C17 epimers Table 2 Compound-specific MRM parameters of the steroids listed in elution order Compound
Collision offset voltage (V)
Segment 1 (0–12.5 min) FLXm 30 6MDN 30 FMBm 20 6CDM 15 Segment 2 (12.5–18 min) OXD 30 3STZ 60 DNZm 30 17MDN 25 17OXD 30 MTS 30
Precursor ion (m/z)
Product ions (m/z)
372 317 347 351
95,337 147,121 147,281 147,209
324 345 313 301 324 303
121,229 97,121 97,109 121,149 121,229 97,109
A. Leinonen et al. / Steroids 69 (2004) 101–109
of other AAS [26]. Loss of water molecules was characteristic for almost all steroids, but sensitivity for dehydration is structure dependent [26]. In general, fragmentation of all compounds was efficient producing several specific ions, which is essential for the reliable verification of the suspective samples in doping control. Product ion spectra of FLXm, 6MDN, FMBm, DNZm, and 17OXD are presented in Fig. 2. The fragmentation patterns of 6CDM, OXD, 3STZ, 17MDN,
105
and MTS have been published elsewhere [15,22,27,28] and are in good agreement with the data obtained in this study. 3.2. Chromatography The retention behavior of AAS in reversed-phase C18 column has been reported to depend greatly on the organic modifier of the mobile phase, on column temperature and
95
100.0
FLXm
80.0
[M+NH4–NH3–H2O]+
60.0
337
40.0
161
20.0
195
299
275
317
0.0 50
100
150
121
100.0
200
250
300
350
400
147
6MDN
171
80.0 60.0
225
107
40.0
281
241
20.0
299 [M+H–H2O]+
0.0 50
100
150
200
250
147
100.0
300
350
400
281
FMBm
80.0 60.0
173
40.0
329 311 347 [M+H]+
121
293
20.0 0.0 50
100
97
100.0
150
200
250
300
350
109
400
DNZm
80.0 60.0 40.0
123 145
20.0
171
277 295
313 [M+H]+
0.0 50
100
150
135
100.0
200
149
250
229
300
350
289
400
17OXD
80.0 60.0
95
175
121
193
40.0
253 271
20.0
324 [M+NH4]+
0.0 50
100
150
200
250
300
350
400
Fig. 2. Product ion mass spectra (x-axis: m/z; y-axis: relative abundance) of FLXm, 6MDN, FMBm, DNZm, and 17OXD. Precursor ions and collision offset voltages are listed in Table 2.
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on the type of reversed-phase material [29]. In this method, the steroids were separated in an endcapped LiChroCART Purospher RP C-18e column at ambient temperature using a linear methanol–water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. The separation of the substances is presented in Fig. 3. The steroids were identified based on their retention times using compound-specific summed product ion profiles. All compounds were eluted between 4.5 and 15.8 min. Although several analytes (6MDN/FMBm, 3STZ/DNZm, and 17MDN/17OXD/MTS) were completely or partly overlapping, their unique MS properties allowed distinguishing between them. In general, the elution order was proportional to the number of polar moieties in the respective chemical structures. FLXm, with four hydroxyl groups, eluted first and MTS with only one hydroxyl group eluted last. As shown previously, the dienone structure in the A ring (6MDN, FMBm, 6CDM, 17MDN) increases polarity and shortens retention [30]. Isomerism at position C17 apparently has great effect on retention: OXD with 17␣-methyl, 17-hydroxyl configuration had a clearly shorter retention time than 17OXD with 17-methyl, 17␣-hydroxyl configuration. Although it is well known that the separation power of LC is significantly weaker than that of GC, it was fully compensated by the excellent selectivity of MS/MS detection in this application. LC generally avoids many problems associated with GC. For instance, GC/MS analysis of compounds such as OXD and stanozolol is difficult owing to their thermal instability and sensitivity towards active sites of the column [7,9]. 3.3. Analytical performance of the method The method was linear up to 250 ng/ml for all analytes, except up to 100 ng/ml for 17MDN. At higher concentrations, the responses were curvelinear, which is consistent with our previous observations [22]. Narrow dynamic range is typical for both ESI and ion spray ionization and can be explained by the ionization mechanism [31]. Limitations in the linearity should be taken into account when using the method in quantitative work, but they do not restrict its use in a wide concentration range in qualitative screening in doping control. Statistics of the calibration curves for each steroid are presented in Table 3. Correlation coefficients of all curves were higher than 0.9947. Precision and accuracy of the method were calculated for all steroids at concentration levels of 5 and 250 ng/ml, except for 17MDN at 1 and 100 ng/ml (Table 4). Inter-day precision coefficients of variation (CV) varied between 3.3 and 18.9% depending on concentration level and analyte. The largest variations were observed for FLXm and FMBm, a phenomenon that can be derived from the poor extraction recoveries of these compounds, as shown below. LOD, with the criterion of a signal to noise ratio of 3, was calculated from the summed product ion profiles and
Fig. 3. Summed product ion profiles (x-axis: time in minutes; y-axis: absolute abundance) of the steroids obtained from blank urine and from 5 ng/ml spiked urine sample. MRM parameters are listed in Table 1. MTS at 50 ng/ml was used as an internal standard.
varied from 0.1 to 2.0 ng/ml. The lowest LOD was achieved for 17MDN and the highest for 3STZ (Table 4). The excellent detectability of 17MDN is most probably due to its hydrophobic nature and high PA, which cause efficient
A. Leinonen et al. / Steroids 69 (2004) 101–109
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Table 3 Statistics of the calibration curves obtained from method validation Compound
Calibration range (ng/ml)
Slope (mean ± S.D.)
FLXm 6MDN FMBm 6CDM OXD 3STZ DNZm 17MDN 17OXD
5–250 1–250 1–250 1–250 1–250 5–250 1–250 0.5–100 1–250
0.0020 0.0090 0.0036 0.0024 0.0077 0.0021 0.0048 0.0537 0.0094
± ± ± ± ± ± ± ± ±
0.0011 0.0015 0.0004 0.0006 0.0022 0.0003 0.0049 0.0107 0.0033
Intercept (mean ± S.D.) 0.0000 0.0027 0.0012 0.0007 0.0003 −0.0014 0.0012 0.0263 0.0068
± ± ± ± ± ± ± ± ±
0.0011 0.0030 0.0013 0.0006 0.0022 0.0023 0.0015 0.0207 0.0065
Correlation coefficient 0.9947 0.9979 0.9953 0.9993 0.9985 0.9984 0.9980 0.9972 0.9980
Results are expressed as mean value ± S.D. of five independent calibrations. Table 4 Method validation data Compound
LOD (ng/ml)
FLXm
1.8
6MDN
Extraction recovery (%)
Added (ng/ml)
Precision CV (%)
Accuracy bias (%)
28
5 250
18.9 13.0
16.8 3.5
0.5
92
5 250
5.8 8.3
2.2 −3.7
FMBm
0.4
26
5 250
18.9 14.6
12.0 −3.4
6CDM
0.4
100
5 250
3.9 4.8
4.7 −7.3
OXD
0.6
98
5 250
10.0 6.2
2.7 −2.2
3STZ
2.0
103
5 250
12.9 12.3
14.7 1.1
DNZm
0.5
97
5 250
8.9 3.6
4.1 −6.9
17MDN
0.1
96
1 100
18.3 8.8
−9.1 1.0
17OXD
0.5
98
5 250
7.7 6.9
5.1 −4.5
Extraction recoveries were determined at 50 ng/ml with 6 replicates. Precision and accuracy were calculated from spiked urine samples at two different concentration levels on 5 separate days.
formation of the MRM precursor ion [M + H]+ . Higher hydrophilicity of 3STZ obviously explains its slightly higher detection limit, despite of its high PA. LODs for all compounds were, however, sufficient for doping control and enabled real trace-level measurements. Similar sensitivity cannot be reached by the generally used GC/MS methods; this would instead require the use of either MS/MS or HRMS [12–14]. Several aliquots of blank human urine were analyzed to check the selectivity of the assay. Based on the data obtained, chemical and biological background was repeatedly extremely low with no interfering peaks (Fig. 3). This makes the interpretation of the data easier and more reliable than in many GC/MS methods, in which disturbing peaks often occur in the extracted ion profiles of the analytes [6,8]. High specificity of the method might enable to decrease analysis time by changing chromatographic operation conditions.
For most of the steroids, the extraction recoveries were higher than 92% (Table 4). However, a poor yield (26–28%) was observed for FLXm and FMBm, which were also the most polar substances. Extraction of these two steroids could probably be enhanced by optimization of salting-out. On the other hand, LODs sufficient for doping control were achieved without modifying the procedure. Overall, the sample pretreatment was easier, faster and safer than in GC/MS-based methods, which require derivatization of the steroids with environmentally harmful reagents. Authentic urine samples, which had previously been found to contain metabolites of fluoxymesterone, metandienone and danazol in a qualitative GC/MS analysis, yielded all positive results when screened by LC/ESI-MS/MS. Fig. 4 shows the product ion profiles of the urine samples. Good applicability of the procedure for authentic samples containing metabolites of
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FLUOXYMESTERONE URINE
METANDIENONE URINE
FLXm
2,5E+04
DANAZOL URINE
6MDN
4,0E+04
4.56
1,5E+05
DNZm
9.63 14.55
0,0E+00
0,0E+00 2
3
4
5
6
8,5
1,5E+05
9,5
10,5
11,5
12,5
0,0E+00 12,5
13,5
14,5
15,5
16,5
17MDN 15.49
0,0E+00 12,5
13,5
14,5
15,5
16,5
Fig. 4. Summed product ion profiles (x-axis: time in minutes; y-axis: absolute abundance) obtained from authentic urine samples indicating abuse of fluoxymesterone, metandienone, and danazol. Estimated concentrations of FLXm, 6MDN, 17MDN, and DNZm are 61, 17, 12, and 33 ng/ml, respectively. MRM parameters are listed in Table 1. MTS at 50 ng/ml was used as an internal standard.
4-chlorodehydromethyltestosterone, oxandrolone and stanozolol has been showed previously [22]. In conclusion, the developed positive ion LC/ESI-MS/MS method enabled the measurement of nine 17-alkyl-substituted AAS or their unconjugated metabolites in human urine at the low nanogram per milliliter level. With simple and fast sample preparation, low limits of detection, high degree of selectivity and precision, the introduced method is thus a potential alternative for GC/MS-based procedures in routine multi-analyte screening of unconjugated AAS in human urine.
Acknowledgements We wish to acknowledge Dr. Kimmo Kuoppasalmi for his scientific contribution and Mrs. Teija Moisander for her technical assistance. The study was funded in part by National Technology Agency (TEKES), Research Foundation of Clinical Chemistry (Kliinisen Kemian Tutkimussäätiö) and University Pharmacy (Yliopiston Apteekki). References [1] Wadler GI, Hainline B. Anabolic steroids. In: Ryan AJ, editor. Drugs and the athlete. Philadelphia: FA Davis Company; 1989. p. 55–69. [2] Middleman AB, DuRant RH. Anabolic steroid use and associated health risk behaviours. Sports Med 1996;21:251–5. [3] Gower DB, Houghton E, Kicman AT. Anabolic steroids: metabolism, doping and detection in equestrian and human sports. In: Makin HLJ, Gower DB, Kirk DN, editors. Steroid analysis. London: Blackie Academic and Professional; 1995. p. 468–518. [4] Sample RHB, Baenziger JC. Gas chromatography/mass spectrometry screening for anabolic steroids. In: Deutsch DG, editor. Chemical analysis: analytical aspects of drug testing. New York: Wiley; 1989. p. 247–72.
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spectrometry and gas chromatography-high-resolution mass spectrometry for determining metandienone metabolites in urine. J Chromatogr B 1999;734:179–89. Mück WM, Henion JD. High-performance liquid chromatography/ tandem mass spectrometry: its use for the identification of stanozolol and its major metabolites in human and equine urine. Biomed Environ Mass Spectrom 1990;19:37–51. Barrón D, Barbosa J, Pascual JA, Segura J. Direct determination of anabolic steroids in human urine by on-line solid-phase extraction liquid chromatography/mass spectrometry. J Mass Spectrom 1996;31:309–19. Bowers LD, Sanaullah . Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry. J Chromatogr B 1996;687:61–8. Bean KA, Henion JD. Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry. J Chromatogr B 1997;690:65–75. Shackleton CHL, Chuang H, Kim J, de la Torre X, Segura J. Electrospray mass spectrometry of testosterone esters: potential for use in doping control. Steroids 1997;62:523–9. Kuuranne T, Kotiaho T, Pedersen-Bjergaard S, Rasmussen KE, Leinonen A, Westwood S, et al. Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples. J Mass Spectrom 2003;38:16– 26. Kim Y, Lee Y, Kim M, Yim Y, Lee W. Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 2000;14:1717–26. Leinonen A, Kuuranne T, Kostiainen R. Liquid chromatography/mass spectrometry in anabolic steroid analysis—optimization and comparison of three ionization techniques: electrospray ionization,
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atmospheric pressure chemical ionization and atmospheric pressure photoionization. J Mass Spectrom 2002;37:693–8. de Boer D, de Jong EG, Maes RA. The detection of danazol and its significance in doping analysis. J Anal Toxicol 1992;16:14–8. National Institute of Standards and Technology. NIST Chemistry WebBook—NIST Standard Reference Database Number 69, July 2001 Release. Available at: http://webbook.nist.gov/chemistry/ (accessed July 2002). Williams TM, Kind AJ, Houghton E, Hill DW. Electrospray collisioninduced dissociation of testosterone and testosterone hydroxy analogs. J Mass Spectrom 1999;34:206–16. Schaff JE, Bowers LD. Structure-function relationships in the ESIMS-MS fragmentation of free and conjugated steroids: a tool for qualitative steroid analysis. In: Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, 31 May–4 June 1998. Abstracts; 1998. p. 83. Draisci R, Giannetti L, Lucentini L, Palleschi L, Purificato I, Moretti G. Confirmation of anabolic hormone in bovine blood by micro-HPLC-ion spray-tandem mass spectrometry. J High Resol Chromatogr 1997;20:421–6. Edlund PO, Bowers L, Henion J. Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultaraviolet detection and confirmation by tandem mass spectrometry. J Chromatogr 1989;487:341–56. Gonzalo-Lumbreras R, Izquierdo-Hornillos R. High-performance liquid chromatographic optimization study for the separation of natural and synthetic anabolic steroids. Application to urine and pharmaceutical samples. J Chromatogr B 2000;742:1–11. Noggle FT, Clark CR, DeRuiter J. Liquid chromatographic and spectral analysis of the 17-hydroxy anabolic steroids. J Chromatogr Sci 1990;28:162–6. Kostiainen K, Bruins AP. Effect of multiple sprayers on the dynamic range and flow rate limitations in electrospray and ionspray mass spectrometry. Rapid Commun Mass Spectrom 1994;8:549–58.
Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography–electrospray ionization tandem mass spectrometry Antti Leinonen a,b , Tiia Kuuranne a,b,c , Tapio Kotiaho b , Risto Kostiainen b,c,∗ a Doping Control Laboratory, United Laboratories Ltd., Helsinki, Finland Department of Pharmacy, Viikki Drug Discovery and Technology Center, University of Helsinki, Helsinki, Finland Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland b
c
Received 14 January 2003; received in revised form 16 June 2003; accepted 21 October 2003
Abstract A qualitative liquid chromatography–electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol–water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1–2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine. © 2003 Elsevier Inc. All rights reserved. Keywords: Anabolic steroids; Metabolism; Liquid chromatography; Mass spectrometry; Electrospray ionization; Doping analysis
1. Introduction Anabolic androgenic steroids (AAS) are still an important class of abused drugs in sports, despite the fact that they have been banned already since the mid-1970s [1]. Furthermore, steroid abuse has become more and more prevalent outside sports. For instance, even teenagers use them as an expression of an improved life style. The abuse of AAS among young people causes significant medical problems. In addition, adolescent steroid users have been found to be engaged in many risk behaviors such as multiple drug use as well as aggressive and suicidal behavior [2]. The fate of AAS in human body is well known [3–5]. These steroids are extensively metabolized, with little or no unchanged steroid being excreted in the urine. The typical metabolic reactions involve oxidation, reduction, hydroxylation and epimerization, followed by conjugation reactions ∗ Corresponding author. Tel.: +358-9-191-59-134; fax: +358-9-191-59-556. E-mail address: [email protected] (R. Kostiainen).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2003.10.007
resulting in the formation of AAS glucuronides or sulfates. As an exception, several 17-alkyl-substituted steroids are excreted in urine mainly or partly as unconjugated metabolites. Alkylation of position C17 reduces androgenic side effects and allows oral administration of the drug [1]. To monitor and prevent the abuse of AAS, efficient and reliable analytical methods are needed. In doping control, the methods are generally divided into two categories: screening procedures and methods of confirmatory analysis [3]. The aim of screening is to find and select suspicious samples for further analysis and an ideal screening method should be sensitive, fast, easy to perform, and have a sufficient sample capacity. Due to the large number of compounds involved, the structural similarity of exogenous and endogenous steroids, the complexity of urine matrix, as well as the low steroid concentrations in urine, analysis of AAS is a challenging task. Because of the large number of target substances, multi-analyte screening methods are superior to single-compound methods. Current testing methods generally rely on gas chromatography–mass spectrometry (GC/MS). In the prevalent procedures, urine
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samples are either directly or after solid phase extraction hydrolyzed enzymatically to cleave the steroid conjugates and then liquid extracted under alkaline conditions. Finally the analytes are trimethylsilylated before measurement by GC/MS with electron ionization and selected ion monitoring [5–10]. Parallel to this, to enhance the detectability of certain steroids, unconjugated metabolites can be measured after alkaline liquid extraction and silylation in a separate method [5–8]. To achieve lower detection limits, negative ion chemical ionization, high-resolution mass spectrometry (HRMS), and tandem mass spectrometry (MS/MS) have been applied in GC-based analysis methods [11–14]. Liquid chromatography–mass spectrometry (LC/MS) has also been used for the analysis of a variety of AAS in human [15–22]. Metabolites of stanozolol have been analyzed in urine by atmospheric pressure chemical ionization (APCI) both prior to and after hydrolysis [15]. Particle beam ionization has been used to determine 28 different AAS or their metabolites in hydrolyzed urine extracts [16]. Electrospray ionization (ESI) has been used to analyze intact glucuronide and sulfate conjugates of testosterone and epitestosterone in urine [17,18] as well as testosterone esters in plasma [19]. An ESI method for simultaneous measurement of 12 different synthetic AAS glucuronides in urine has also been introduced [20]. Metabolites of gestrinone have been characterized by ESI in urine after hydrolysis [21]. Furthermore, our group has recently demonstrated the applicability of ESI, APCI and atmospheric pressure photoionization in the detection of structurally different unconjugated AAS metabolites in urine [22]. LC/MS has the potential to overcome some of the problems associated with GC methods, i.e. derivatization step, adsorption of steroids, and their thermal decomposing during analysis. Therefore, investigation of the suitability of LC/MS assays for multi-analyte screening of AAS is of great interest. This paper describes a qualitative LC/ESI-MS/MS screening method for the direct analysis of nine urinary 17-alkylsubstituted AAS or their metabolites (Fig. 1), all excreted in urine in unconjugated form and used as target compounds in the doping analysis of free steroid fraction [3–8,23]. The method is based on our initial optimization of ionization method for the LC/MS analysis of AAS [22] and is now suitable for the detection of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol abuse.
2. Experimental 2.1. Chemicals and reagents 9␣-Fluoro-17␣-methyl-androst-4-ene-3␣,6,11,17-tetrol (FLXm), 6-hydroxymetandienone (6MDN), 2-hydroxymethyl-17␣-methyl-androsta-1,4-diene-11␣,17-diol3-one (FMBm), 6-hydroxy-4-chloro-dehydromethyltestosterone (6CDM), 3 -hydroxystanozolol (3STZ), 17-epimetan-
dienone (17MDN), and 17-epioxandrolone (17OXD) were purchased from National Analytical Reference Laboratory (NARL, Pymble, Australia). Oxandrolone (OXD), ethisterone (DNZm), and methyltestosterone (MTS) were from Steraloids (Newport, RI). All other reagents and solvents were either of HPLC or analytical grade. 2.2. Sample work-up Extraction of urine samples was based on the procedure described by Schänzer and Donike [5]. A 2.5-ml aliquot of urine was spiked with 125 ng of methyltestosterone (internal standard). Then 250 mg of a solid mixture of sodium hydrogen carbonate and potassium carbonate (2:1, w/w) was added and the sample extracted with 2.5 ml of diethyl ether. After centrifugation, the organic layer was separated and evaporated to dryness. Finally, the residue was reconstituted in 60 l of 50% methanol (v/v) containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. 2.3. Liquid chromatography The liquid chromatographic system consisted of a Hewlett-Packard (Waldbronn, Germany) HP 1100 Series binary gradient pump and autosampler. Operation conditions were identical to those in our former study [22]. Chromatographic separation was obtained at ambient temperature on a LiChroCART Purospher RP C-18e column (125 mm × 3 mm, 5 m) from Merck (Darmstadt, Germany) equipped with a 4 mm × 4 mm pre-column of the same stationary phase. Injection volume was 5 l. An aqueous solution of 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid was used as solvent A. Solvent B consisted of 90% (v/v) methanol in water with final concentration of 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. A linear gradient was run with a flow rate of 0.5 ml/min from 50 to 100% B in 15 min and held at the final composition for 3 min. Column effluent was finally post-column split 1:100, before introduction to the MS source. 2.4. Mass spectrometry The analyses were carried out on a Perkin-Elmer Sciex API 3000 triple quadrupole mass spectrometer (Sciex, Concord, Canada) equipped with an ion spray source. All experiments were carried out in the positive ion mode. Compressed air (Atlas Copco CD-2 system, Belgium) was used as a nebulizing gas with a flow rate of 0.82 l/min. Nitrogen (Whatman 75-72 nitrogen generator, MA) was used both as collision and curtain gas (0.95 l/min). The ion source was operated in ESI mode. Based on our earlier studies [22], spray needle voltage and orifice (declustering) voltage were set to 5000 V and 20 V, respectively. Flow injection studies were performed using a Harvard Apparatus microsyringe pump (MA, USA) with a flow rate of 5 l/min. In MS and MS/MS studies, mass spectra were collected in a scan
A. Leinonen et al. / Steroids 69 (2004) 101–109
Exogenous source
Compound
103
Structure
MW OH HO
FLXm
9α-Fluoro-17α-methyl-androst-4-ene3α,6β,11β,17β-tetrol
CH 3
Fluoxymesterone
354 F HO OH
OH CH 3
6MDN
6β-Hydoxymetandienone
Metandienone
316 O
OH OH
FMBm
2-Hydroxymethyl-17α-methyl-androsta-1,4diene-11α,17β-diol-3-one
HO
Formebolone
CH 3
346
HO H 2 C
O
OH
6CDM
6β-Hydroxy-4-chloro-dehydromethyltestosterone
CH 3
4-Chlorodehydromethyltestosterone
350 O
OH
Cl
OH CH 3
OXD
Oxandrolone
Oxandrolone
306
O O
H
OH CH 3
HO
3STZ
3'-Hydroxystanozolol
Stanozolol
344 H N N H OH
C CH
DNZm
Ethisterone
Danazol
312 O CH 3 OH
17MDN
17-Epimetandienone
Metandienone
300 O CH 3 OH
17OXD
17-Epioxandrolone
Oxandrolone
306
O O
H OH 17
MTS
1
Methyltestosterone
Internal standard
C
302
2
O
CH 3
D
3
A
B
4
6
Fig. 1. Nomenclature and chemical formulae of the steroids studied.
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range of m/z 250–450 and m/z 50–450. In all measurements, five spectra with scan speed of 5 s were accumulated. In MS/MS experiments, either the protonated molecule or the ammonium adduct was selected as precursor ion and collision offset voltages were optimized over the range of 5–80 V. In the final LC/ESI-MS/MS method, the steroids were detected using multiple reaction monitoring (MRM) of two characteristic product ions per analyte. Dwell time for each ion was 400 ms. Analyst 1.1 software was used for instrument control and data processing. 2.5. Samples and method evaluation In order to evaluate linearity and to calibrate the method, standards were prepared by spiking pooled human drugfree urine with steroids at concentrations of 0, 0.5, 1, 5, 10, 50, 100, 250, 500, and 1000 ng/ml, each sample as a single. Calibration curves were obtained from the analyte to internal standard peak-height ratios using linear regression with 1/x weighing. To estimate inter-day precision and accuracy, quality control samples were prepared in the same way and at the same concentration levels as the standards but from separate stock solutions and analyzed on 5 separate days. Standards were prepared and calibration curves constructed within every analysis batch. Limit of detection (LOD) was verified with spiked samples at estimated concentrations as six replicates. Random interference of biological background was checked from drug-free urine samples of 10 male and 10 female volunteers. The extraction recoveries were measured at a concentration level of 50 ng/ml (six replicates each) by comparing relative intensities of extracted spiked urine samples to those of extracted blank urines spiked with standards; in every case, internal standard was added after extraction. Finally, the method was tested with authentic forensic urine samples, which had in the qualitative GC/MS analysis been found to contain metabolites of danazol, fluoxymesterone and metandienone.
3. Results and discussion 3.1. Mass spectrometry In order to select precursor ions for MRM, full-scan mass spectra of each steroid were measured. Either [M + H]+ or [M + NH4 ]+ was the dominating ion in the mass spectrum (Table 1), the intensity ratio [M + H]+ /[M + NH4 ]+ depending on the proton affinity (PA) and with high PA favoring formation of the protonated molecule as shown previously [22]. The pyrazole ring (3STZ) and the 1,4-diene-3-one structure (17MDN, FMBm, 6MDN) have the highest PA, but exceptionally in the case of 6CDM, PA is decreased due to an electronegative chlorine atom at position C4. PA of the 4-ene-3-one structure (DNZm, MTS) is lower but still significantly higher than that of the 3-one structure without a 4-ene double bond (OXD, 17OXD), whereas hydroxyl
Table 1 Relative abundances (%) of protonated molecule and corresponding ammonium adduct in mass spectra of the steroids Compound
[M + H]+
[M + NH4 ]+
3STZ 17MDN FMBm 6MDN DNZm MTS 6CDM 17OXD OXD FLXm
100 100 100 100 100 100 100 30 29 –
– – – 7 34 35 39 100 100 100
groups with a single double bond (FLXm) have the lowest PA. The PA order of the steroids was estimated using PAs of their structural analogs [24]. The protonated molecule was chosen as a precursor ion for 6MDN, FMBm, 6CDM, 3STZ, DNZm, 17MDN and MTS, and the ammonium adduct was selected for FLXm, OXD and 17OXD. It should be noted that because of its lowest PA, FLXm did not form any protonated molecule. This means that the use of adduct-forming additives may be essential for its ionization also with other atmospheric pressure ionization techniques than ESI. Product ion spectra were measured with different collision offset voltages to optimize fragmentation for MRM. The most abundant and specific product ions were selected, excluding unspecific dehydrated fragments. In general, the optimum collision offset voltages varied between 15 and 30 V. However, due to its stable pyrazole ring, breakdown of 3STZ required significantly higher collision energy (60 V) [22]. Optimized MRM parameters are listed in Table 2. Fragmentation of the steroids was not studied in detail in this work. However, as shown elsewhere, a rational correlation between steroid structures and fragmentation patterns can be found [25,26]. For instance, MTS and DNZm formed intensive ions at m/z 97 and 109, typical for steroids having the 4-ene-3-one structure [25]. The fragmentation patterns of OXD and 17OXD were similar, as reported for C17 epimers Table 2 Compound-specific MRM parameters of the steroids listed in elution order Compound
Collision offset voltage (V)
Segment 1 (0–12.5 min) FLXm 30 6MDN 30 FMBm 20 6CDM 15 Segment 2 (12.5–18 min) OXD 30 3STZ 60 DNZm 30 17MDN 25 17OXD 30 MTS 30
Precursor ion (m/z)
Product ions (m/z)
372 317 347 351
95,337 147,121 147,281 147,209
324 345 313 301 324 303
121,229 97,121 97,109 121,149 121,229 97,109
A. Leinonen et al. / Steroids 69 (2004) 101–109
of other AAS [26]. Loss of water molecules was characteristic for almost all steroids, but sensitivity for dehydration is structure dependent [26]. In general, fragmentation of all compounds was efficient producing several specific ions, which is essential for the reliable verification of the suspective samples in doping control. Product ion spectra of FLXm, 6MDN, FMBm, DNZm, and 17OXD are presented in Fig. 2. The fragmentation patterns of 6CDM, OXD, 3STZ, 17MDN,
105
and MTS have been published elsewhere [15,22,27,28] and are in good agreement with the data obtained in this study. 3.2. Chromatography The retention behavior of AAS in reversed-phase C18 column has been reported to depend greatly on the organic modifier of the mobile phase, on column temperature and
95
100.0
FLXm
80.0
[M+NH4–NH3–H2O]+
60.0
337
40.0
161
20.0
195
299
275
317
0.0 50
100
150
121
100.0
200
250
300
350
400
147
6MDN
171
80.0 60.0
225
107
40.0
281
241
20.0
299 [M+H–H2O]+
0.0 50
100
150
200
250
147
100.0
300
350
400
281
FMBm
80.0 60.0
173
40.0
329 311 347 [M+H]+
121
293
20.0 0.0 50
100
97
100.0
150
200
250
300
350
109
400
DNZm
80.0 60.0 40.0
123 145
20.0
171
277 295
313 [M+H]+
0.0 50
100
150
135
100.0
200
149
250
229
300
350
289
400
17OXD
80.0 60.0
95
175
121
193
40.0
253 271
20.0
324 [M+NH4]+
0.0 50
100
150
200
250
300
350
400
Fig. 2. Product ion mass spectra (x-axis: m/z; y-axis: relative abundance) of FLXm, 6MDN, FMBm, DNZm, and 17OXD. Precursor ions and collision offset voltages are listed in Table 2.
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on the type of reversed-phase material [29]. In this method, the steroids were separated in an endcapped LiChroCART Purospher RP C-18e column at ambient temperature using a linear methanol–water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. The separation of the substances is presented in Fig. 3. The steroids were identified based on their retention times using compound-specific summed product ion profiles. All compounds were eluted between 4.5 and 15.8 min. Although several analytes (6MDN/FMBm, 3STZ/DNZm, and 17MDN/17OXD/MTS) were completely or partly overlapping, their unique MS properties allowed distinguishing between them. In general, the elution order was proportional to the number of polar moieties in the respective chemical structures. FLXm, with four hydroxyl groups, eluted first and MTS with only one hydroxyl group eluted last. As shown previously, the dienone structure in the A ring (6MDN, FMBm, 6CDM, 17MDN) increases polarity and shortens retention [30]. Isomerism at position C17 apparently has great effect on retention: OXD with 17␣-methyl, 17-hydroxyl configuration had a clearly shorter retention time than 17OXD with 17-methyl, 17␣-hydroxyl configuration. Although it is well known that the separation power of LC is significantly weaker than that of GC, it was fully compensated by the excellent selectivity of MS/MS detection in this application. LC generally avoids many problems associated with GC. For instance, GC/MS analysis of compounds such as OXD and stanozolol is difficult owing to their thermal instability and sensitivity towards active sites of the column [7,9]. 3.3. Analytical performance of the method The method was linear up to 250 ng/ml for all analytes, except up to 100 ng/ml for 17MDN. At higher concentrations, the responses were curvelinear, which is consistent with our previous observations [22]. Narrow dynamic range is typical for both ESI and ion spray ionization and can be explained by the ionization mechanism [31]. Limitations in the linearity should be taken into account when using the method in quantitative work, but they do not restrict its use in a wide concentration range in qualitative screening in doping control. Statistics of the calibration curves for each steroid are presented in Table 3. Correlation coefficients of all curves were higher than 0.9947. Precision and accuracy of the method were calculated for all steroids at concentration levels of 5 and 250 ng/ml, except for 17MDN at 1 and 100 ng/ml (Table 4). Inter-day precision coefficients of variation (CV) varied between 3.3 and 18.9% depending on concentration level and analyte. The largest variations were observed for FLXm and FMBm, a phenomenon that can be derived from the poor extraction recoveries of these compounds, as shown below. LOD, with the criterion of a signal to noise ratio of 3, was calculated from the summed product ion profiles and
Fig. 3. Summed product ion profiles (x-axis: time in minutes; y-axis: absolute abundance) of the steroids obtained from blank urine and from 5 ng/ml spiked urine sample. MRM parameters are listed in Table 1. MTS at 50 ng/ml was used as an internal standard.
varied from 0.1 to 2.0 ng/ml. The lowest LOD was achieved for 17MDN and the highest for 3STZ (Table 4). The excellent detectability of 17MDN is most probably due to its hydrophobic nature and high PA, which cause efficient
A. Leinonen et al. / Steroids 69 (2004) 101–109
107
Table 3 Statistics of the calibration curves obtained from method validation Compound
Calibration range (ng/ml)
Slope (mean ± S.D.)
FLXm 6MDN FMBm 6CDM OXD 3STZ DNZm 17MDN 17OXD
5–250 1–250 1–250 1–250 1–250 5–250 1–250 0.5–100 1–250
0.0020 0.0090 0.0036 0.0024 0.0077 0.0021 0.0048 0.0537 0.0094
± ± ± ± ± ± ± ± ±
0.0011 0.0015 0.0004 0.0006 0.0022 0.0003 0.0049 0.0107 0.0033
Intercept (mean ± S.D.) 0.0000 0.0027 0.0012 0.0007 0.0003 −0.0014 0.0012 0.0263 0.0068
± ± ± ± ± ± ± ± ±
0.0011 0.0030 0.0013 0.0006 0.0022 0.0023 0.0015 0.0207 0.0065
Correlation coefficient 0.9947 0.9979 0.9953 0.9993 0.9985 0.9984 0.9980 0.9972 0.9980
Results are expressed as mean value ± S.D. of five independent calibrations. Table 4 Method validation data Compound
LOD (ng/ml)
FLXm
1.8
6MDN
Extraction recovery (%)
Added (ng/ml)
Precision CV (%)
Accuracy bias (%)
28
5 250
18.9 13.0
16.8 3.5
0.5
92
5 250
5.8 8.3
2.2 −3.7
FMBm
0.4
26
5 250
18.9 14.6
12.0 −3.4
6CDM
0.4
100
5 250
3.9 4.8
4.7 −7.3
OXD
0.6
98
5 250
10.0 6.2
2.7 −2.2
3STZ
2.0
103
5 250
12.9 12.3
14.7 1.1
DNZm
0.5
97
5 250
8.9 3.6
4.1 −6.9
17MDN
0.1
96
1 100
18.3 8.8
−9.1 1.0
17OXD
0.5
98
5 250
7.7 6.9
5.1 −4.5
Extraction recoveries were determined at 50 ng/ml with 6 replicates. Precision and accuracy were calculated from spiked urine samples at two different concentration levels on 5 separate days.
formation of the MRM precursor ion [M + H]+ . Higher hydrophilicity of 3STZ obviously explains its slightly higher detection limit, despite of its high PA. LODs for all compounds were, however, sufficient for doping control and enabled real trace-level measurements. Similar sensitivity cannot be reached by the generally used GC/MS methods; this would instead require the use of either MS/MS or HRMS [12–14]. Several aliquots of blank human urine were analyzed to check the selectivity of the assay. Based on the data obtained, chemical and biological background was repeatedly extremely low with no interfering peaks (Fig. 3). This makes the interpretation of the data easier and more reliable than in many GC/MS methods, in which disturbing peaks often occur in the extracted ion profiles of the analytes [6,8]. High specificity of the method might enable to decrease analysis time by changing chromatographic operation conditions.
For most of the steroids, the extraction recoveries were higher than 92% (Table 4). However, a poor yield (26–28%) was observed for FLXm and FMBm, which were also the most polar substances. Extraction of these two steroids could probably be enhanced by optimization of salting-out. On the other hand, LODs sufficient for doping control were achieved without modifying the procedure. Overall, the sample pretreatment was easier, faster and safer than in GC/MS-based methods, which require derivatization of the steroids with environmentally harmful reagents. Authentic urine samples, which had previously been found to contain metabolites of fluoxymesterone, metandienone and danazol in a qualitative GC/MS analysis, yielded all positive results when screened by LC/ESI-MS/MS. Fig. 4 shows the product ion profiles of the urine samples. Good applicability of the procedure for authentic samples containing metabolites of
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FLUOXYMESTERONE URINE
METANDIENONE URINE
FLXm
2,5E+04
DANAZOL URINE
6MDN
4,0E+04
4.56
1,5E+05
DNZm
9.63 14.55
0,0E+00
0,0E+00 2
3
4
5
6
8,5
1,5E+05
9,5
10,5
11,5
12,5
0,0E+00 12,5
13,5
14,5
15,5
16,5
17MDN 15.49
0,0E+00 12,5
13,5
14,5
15,5
16,5
Fig. 4. Summed product ion profiles (x-axis: time in minutes; y-axis: absolute abundance) obtained from authentic urine samples indicating abuse of fluoxymesterone, metandienone, and danazol. Estimated concentrations of FLXm, 6MDN, 17MDN, and DNZm are 61, 17, 12, and 33 ng/ml, respectively. MRM parameters are listed in Table 1. MTS at 50 ng/ml was used as an internal standard.
4-chlorodehydromethyltestosterone, oxandrolone and stanozolol has been showed previously [22]. In conclusion, the developed positive ion LC/ESI-MS/MS method enabled the measurement of nine 17-alkyl-substituted AAS or their unconjugated metabolites in human urine at the low nanogram per milliliter level. With simple and fast sample preparation, low limits of detection, high degree of selectivity and precision, the introduced method is thus a potential alternative for GC/MS-based procedures in routine multi-analyte screening of unconjugated AAS in human urine.
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