Determination of anabolic steroids in human urine by automated in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry

https://doi.org/10.1016/j.jpba.2010.02.027Get rights and content

Abstract

A simple, rapid and sensitive method was developed for determining the presence of seven anabolic steroids (boldenone, nandrolone, testosterone, methyltestosterone, epiandrosterone, androsterone, and atnozolol) in human urine. Glucuronide-conjugates of these compounds were hydrolyzed with β-glucuronidase. The anabolic steroids were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–mass spectrometry (LC–MS). The steroids were separated within 14 min by high performance liquid chromatography using a Chromolith RP-18e column and 5 mM ammonium formate/methanol (35/65, v/v) as a mobile phase at a flow rate of 1.0 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for the MS detection of these compounds. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 μL using a Supel-Q PLOT capillary column for the extraction. The extracted compounds could be desorbed readily from the capillary column by flow of the mobile phase, and no carryover was observed. Using the in-tube SPME LC–MS with SIM mode detection, good linearity of the calibration curve (r > 0.995) was obtained in the concentration range of 0.5–20 ng/mL, except for stanozolol. The detection limits (S/N = 3) of anabolic steroids were in the range 9–182 pg/mL and the proposed method showed 20–33-fold higher sensitivity than the direct injection method. The within-day and between-day precisions were below 4.0% and 7.3% (n = 5), respectively. This method was applied successfully to the analysis of urine samples without the interference peaks. The recovery rates of anabolic steroids spiked into urine samples were above 85%. This method is useful to analyze the urinary levels of these compounds in anti-doping tests.

Introduction

The misuse of drugs to enhance performance in human and animal sports, usually referred to as doping, is unfortunately widespread and has a long history. This unacceptable practice violates the spirit of fair play in sports, affects medical ethics and potentially puts the health of the athlete at risk. In particular, synthetic anabolic steroids, structurally related to testosterone, belong to a pharmacological group that has a great impact on sport due to its use in doping, and have been used to enhance anabolic effects, such as improving skeletal muscle performance and recovery by controlling catabolism after stress [1], [2], [3]. Other features of these compounds, often referred to as side effects, are their androgenic effects, such as cardiovascular and hepatic disorders. Although the use of anabolic steroids by athletes has been prohibited since 1976, doping using these substances remains a problem for sporting authorities. Therefore, the control of anabolic steroid abuse is a demanding task, and requires high speed, high sensitivity, and specific analytical methods [3], [4], [5].

Doping control analyses of anabolic steroids have been mainly carried out on urine because in general it contains relatively high concentrations of the drugs and/or their metabolites. Testing for anabolic steroids in urine samples is mainly carried out by gas chromatography with mass spectrometry (GC–MS) [5], [6], [7], [8], [9], [10], [11] and liquid chromatography with tandem mass spectrometry (LC–MS–MS) [12], [13], [14], [15], [16], [17]. Although the GC–MS methods are robust and sensitive, they always require a laborious derivatization step and therefore sample throughput is quite low with long turn-around times. However, LC–MS–MS methods can provide a sensitive and selective way of comprehensively measuring anabolic steroid concentrations. Gas or liquid chromatography coupled with mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization coupled with the linear ion trap mass spectrometry technique has been tested for its applicability as a rapid screening technique [18]. However, most of the above methods generally require time-consuming sample preparation procedures, such as liquid–liquid extraction or solid-phase extraction, to remove coexisting substances in urine samples prior to analysis.

In-tube solid-phase microextraction (SPME), using an open tubular fused-silica capillary with an inner surface coating as the SPME device, is simple and can be coupled easily on-line with HPLC and LC–MS. In-tube SPME allows convenient automation of the extraction process, which not only reduces analysis time, but also provides better precision and sensitivity than manual off-line techniques. We recently developed an in-tube SPME method for the determination of urinary drugs [19], cortisol [20], and nicotine and cotinine [21] by coupling the methods with LC–MS. The details of the in-tube SPME technique and its applications have also been summarized in a number of reviews [22], [23], [24], [25]. Here we report an automated on-line in-tube SPME LC–MS method for the simultaneous determination of anabolic steroids in urine samples.

Section snippets

Materials

Boldenone, nandrolone, testosterone, methyltestosterone, epiandrosterone, androsterone, and stanozolol were purchased from Sigma–Aldrich, Japan (Tokyo, Japan). Δ-Methyltestosterone as an internal standard (IS) was purchased from Sigma–Aldrich. The structures of these compounds are shown in Fig. 1. Each compound was dissolved in methanol to make a stock solution at a concentration of 1 mg/mL. Each solution was stored at 4 °C and diluted to the required concentrations with pure water prior to use.

LC–MS analysis of anabolic steroids

For MS operation, ESI positive ion mode was evaluated for the determination of anabolic steroids. To select the monitoring ion for these compounds, the ESI mass spectra were initially analyzed by LC–MS with direct liquid injection into the column. As shown in Fig. 2, each compound gave a very simple spectrum in scan mode for the mass range m/z 100–400. Most anabolic steroids had protonated molecules ([M+H]+) as base ions. The ammonium adducts ([M+NH4]+) were also observed in epiandrosterone and

Conclusions

The on-line in-tube SPME/LC–MS method developed in the present study can continuously perform extraction and concentration of anabolic steroids from urine samples, and then allow analysis by LC–MS. This method is automated, simple, rapid, selective, and sensitive, and can be readily applied to the analysis of urine samples. This method is a useful tool for anti-doping analysis.

Acknowledgements

This work was supported by a Grant-in-Aid for Basic Scientific Research (C, No. 19590049) and a Grant-in-Aid for Exploratory Research (No. 16659014).

References (25)

  • C.G. Georgakopoulos et al.

    Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

    Rapid Commun. Mass Spectrom.

    (2007)
  • J. Marcos et al.

    Fast screening of anabolic steroids and other banned doping substances in human urine by gas chromatography/tandem mass spectrometry

    J. Mass Spectrom.

    (2002)
  • Cited by (68)

    • Quantification of steroid hormones in human urine by DLLME and UHPLC-HRMS detection

      2020, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
    View all citing articles on Scopus
    View full text