Anabolic Steroids In Urine Research Articles

High-temperature liquid chromatography-isotope ratio mass spectrometry methodology for carbon isotope ratio determination of anabolic steroids in urine

BackgroundGas Chromatography Isotope Ratio Mass Spectrometry (GC-C-IRMS) has long been used in routine laboratories to determine the δ13C values of anabolic steroids in urine, differentiating between, e.g., endogenous and synthetic testosterone (T) in sports doping control. Until now, liquid chromatography (LC-IRMS) has not been used. The LC-IRMS setup doesn't allow organic solvents or modifiers in the mobile phase for δ13C determinations. Mid-to non-polar analytes such as steroids can be analysed in water heated to High Temperatures (HT, up to 200 °C) because at 200 °C has a similar polarity as 80/20 methanol/water at ambient temperature. In this work, we developed a method for steroids in urine, extending the application of the LC-IRMS to non-polar analytes in complex matrices. ResultAn HT-LC-IRMS method capable of determining the δ13C values of four steroids (i.e., testosterone (T), 5α-androstane-3α,17β-diol (ααβ), 5β-androstane-3α,17β-diol (βαβ) and pregnanetriol (PT)) in urine was developed and validated. Accuracy ranged from 0.23 ‰ (ααβ and βαβ) to 0.49 ‰ (T), and the detection limit was set at 10 ng mL−1 (T, ααβ+βαβ). The validation data and a comparison of authentic urine samples analysed with HT-LC-IRMS and GC-C-IRMS indicated a comparable performance between HT-LC-IRMS and GC-C-IRMS. SignificanceHT-LC-IRMS can be used to determine δ13C values of anabolic steroids, extending the applicability of both HT-LC and LC-IRMS to non-polar substances determined in a complex matrix in routine laboratory practice.

Screening for anabolic steroids in urine of forensic cases using fully automated solid phase extraction and LC-MS-MS.

A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, suggesting that only a small number of common steroids are likely to occur in a forensic context. The steroids were often in high concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse were seen in the majority of cases. The method presented serves as a fast and automated screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids.

Direct analysis of anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption-dielectric barrier discharge ionization mass spectrometry

Rapid detection of trace level anabolic steroids in urine is highly desirable to monitor the consumption of performance enhancing anabolic steroids by athletes. The present article describes a novel strategy for identifying the trace anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption (LPTD) coupled to dielectric barrier discharge (DBD) ionization mass spectrometry. Using this method the steroid molecules are enriched within a liquid droplet during the thermal desorption process and desorbed all-together at the last moment of droplet evaporation in a short time domain. The desorbed molecules were ionized using a dielectric barrier discharge ion-source in front of the mass spectrometer inlet at open atmosphere. This process facilitates the sensitivity enhancement with several orders of magnitude compared to the thermal desorption at a lower temperature. The limits of detection (LODs) of various steroid molecules were found to be in the range of 0.05–0.1ngmL−1 for standard solutions and around two orders of magnitude higher for synthetic urine samples. The detection limits of urinary anabolic steroids could be lowered by using a simple and rapid dichloromethane extraction technique. The analytical figures of merit of this technique were evaluated at open atmosphere using suitable internal standards. The technique is simple and rapid for high sensitivity and high throughput screening of anabolic steroids in urine.

Analysis of anabolic steroids in urine by gas chromatography–microchip atmospheric pressure photoionization-mass spectrometry with chlorobenzene as dopant

A gas chromatography–microchip atmospheric pressure photoionization-tandem mass spectrometry (GC–μAPPI-MS/MS) method was developed for the analysis of anabolic androgenic steroids in urine as their trimethylsilyl derivatives. The method utilizes a heated nebulizer microchip in atmospheric pressure photoionization mode (μAPPI) with chlorobenzene as dopant, which provides high ionization efficiency by producing abundant radical cations with minimal fragmentation. The performance of GC–μAPPI-MS/MS was evaluated with respect to repeatability, linearity, linear range, and limit of detection (LOD). The results confirmed the potential of the method for doping control analysis of anabolic steroids. Repeatability (RSD<10%), linearity (R2≥0.996) and sensitivity (LODs 0.05–0.1ng/mL) were acceptable. Quantitative performance of the method was tested and compared with that of conventional GC–electron ionization-MS, and the results were in good agreement.

Analysis of anabolic androgenic steroids in urine by full-capillary sample injection combined with a sweeping CE stacking method

This study describes an on-line stacking CE approach by sweeping with whole capillary sample filling for analyzing five anabolic androgenic steroids in urine samples. The five anabolic steroids for detection were androstenedione, testosterone, epitestosterone, boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because they can promote muscle growth. Therefore, a sensitive detection method is imperatively required for monitoring the urine samples of athletes. In this research, an interesting and reliable stacking capillary electrophoresis method was established for analysis of anabolic steroids in urine. After liquid-liquid extraction by n-hexane, the supernatant was dried and reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously accomplished at -20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium dodecyl sulfate and 40 % methanol. During the method validation, calibration curves were linear (r≥0.990) over a range of 50-1,000 ng/mL for the five analytes. In the evaluation of precision and accuracy for this method, the absolute values of the RSD and the RE in the intra-day (n=3) and inter-day (n=5) analyses were all less than 6.6 %. The limit of detection for the five analytes was 30 ng/mL (S/N=5, sampling 99.9 s at 10 psi). Compared with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be used as a powerful tool for monitoring the illegal use of doping.

Feasibility of gas chromatography–microchip atmospheric pressure photoionization-mass spectrometry in analysis of anabolic steroids

Mass spectrometers equipped with atmospheric pressure ion sources (API-MS) have been designed to be interfaced with liquid chromatographs (LC) and have rarely been connected to gas chromatographs (GC). Recently, we introduced a heated nebulizer microchip and showed its potential to interface liquid microseparation techniques and GC with API-MS. This study demonstrates the feasibility of GC–microchip atmospheric pressure photoionization-tandem mass spectrometry (GC–μAPPI-MS/MS) in the analysis of underivatized anabolic steroids in urine. The APPI microchip provides high ionization efficiency and produces abundant protonated molecules or molecular ions with minimal fragmentation. The feasibility of GC–μAPPI-MS/MS in the analysis of six selected anabolic steroids in urine samples was studied with respect to intra-batch repeatability, linearity, linear range, and limit of detection (LOD). The method showed good sensitivity (LODs 0.2–1 ng/mL), repeatability (relative standard deviation < 10%), and linearity (regression coefficient ≥ 0.9995) and, therefore, high potential for the analysis of anabolic steroids. Quantitative performance of the method was tested with two authentic urine samples, and the results were in good agreement with those obtained with conventional GC–electron ionization-MS after derivatization.

Feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization mass spectrometry in analyzing anabolic steroids in urine samples

We examined the feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization tandem mass spectrometry (capLC/microAPPI-MS/MS) for the analysis of anabolic steroids in human urine. The urine samples were pretreated by enzymatic hydrolysis (with beta-glucuronidase from Helix pomatia), and the compounds were liquid-liquid extracted with diethyl ether. After separation the compounds were vaporized by microchip APPI, photoionized by a 10 eV krypton discharge lamp, and detected by selected reaction monitoring. The capLC/microAPPI-MS/MS method showed good sensitivity with detection limits at the level of 1.0 ng mL(-1), good linearity with correlation coefficients between 0.9954 and 0.9990, and good repeatability with relative standard deviations below 10%. These results demonstrate that microchip APPI combined with capLC/MS/MS provides a new potential method for analyzing non-polar and neutral compounds in biological samples.

Rapid Detection of Anabolic Steroids in Urine by Protein Arrays

Rapid Detection of Anabolic Steroids in Urine by Protein Arrays

Rapid Screening of Anabolic Steroids in Urine by Reactive Desorption Electrospray Ionization

Fast screening for anabolic steroids in whole urine is achieved by combining reactive desorption electrospray ionization (reactive DESI) and tandem mass spectrometry. Spray solutions containing hydroxylamine allow heterogeneous reactions of hydroxylamine with the carbonyl group of the steroids during the ionization process. Seven steroids, including a glycosteroid, were examined. The ion/molecule reaction adduct and the oxime formed via its dehydration were observed using reactive DESI; the protonated and sodiated forms of the ionized steroid were also observed both in reactive DESI and in DESI performed without the added hydroxylamine reagent. Paper, glass, and polytetrafluoroethylene were tested as sample substrates, but the glycosteroid was ionized intact without hydrolysis only from polytetrafluoroethylene. Limits of detection for the pure compounds were less than 1 ng, dynamic ranges were typically 2 orders of magnitude, and analysis times were just a few seconds. Concentration levels of ketosteroids in raw urine relevant to screening for sports doping (approximately 20 ng/mL) can be reached using a simple solid-phase microextraction (SPME) preconcentration step. Reactive DESI provided significant improvements in ionization efficiency of these steroids in raw undiluted urine as compared to conventional DESI; suppression effects due to the sample matrix were minimal and the urine matrix had no deleterious effect on steroid detection limits. Tandem mass spectrometry provided confirmation of analyte identification in this rapid screening process.

Development and validation of a qualitative screening method for the detection of exogenous anabolic steroids in urine by liquid chromatography-tandem mass spectrometry

A screening method for the urinary detection of 34 exogenous anabolic steroids has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography-tandem mass spectrometry. The use of some adducts such as [M+NH(4)](+), [M+CH(3)COO](-) and [M+H+MeOH](+) was necessary in order to detect some analytes at the required level (lower than 10 ng/ml). Two transitions were selected for each analyte. Different concentration factors have been studied in order to increase the sensitivity. A concentration factor of 50 was selected for the screening method although the high ion suppression observed under these conditions can hamper its application as a quantitative method. The method was validated and limits of detection were obtained by spiking ten different blank urine samples at five different concentration levels. Up to 29 analytes were detected in all spiked urines at the required level. Limits of detection between 1 and 10 ng/ml were obtained for most analytes which fulfil current requirements. The applicability of the method was shown by analysing positive samples.

Multi-residue screening of a minimum package of anabolic steroids in urine with GC–MS

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography–mass spectrometry (GC–MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCα and CCβ values are based on the detection of the most abundant ion. Results of validation experiments are presented.The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC–MS3)

Within the scope of the European Community member states’ residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC–MSn analysis was no longer required. Analytical results could be obtained within 1 day and only 5mL urine was needed tot carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved.A higher GC–MS technique's specificity was achieved by detecting the steroids using GC–MS3. Nevertheless, it was decided to screen routinely sampled urine with GC–MS2 whereas GC–MS3 was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.

Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine

The applicability of in-vial two-phase liquid-phase microextraction (LPME) in porous hollow polypropylene fiber was studied for the sample preparation of unconjugated anabolic steroids in urine. Four different anabolic steroids – metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol – were used as test compounds and methyltestosterone as an internal standard. A standard two-phase LPME method for use with liquid chromatography/mass spectrometry (LC/MS) was set up and the influence of different parameters, including the nature of organic solvent, extraction time, salting-out and temperature, on the LPME process was investigated. Taking advantage of the preliminary studies, a novel two-phase LPME method utilizing simultaneous in-fiber silylation was developed and validated for gas chromatographic/mass spectrometric (GC/MS) analysis of a danazol metabolite in urine. In all, LPME allowed a very straightforward, simple and selective way to prepare urine samples for steroid analysis, being most suitable for hydrophobic steroids. The LPME method with in-fiber derivatization for GC/MS analysis exhibited high sensitivity, repeatability and linearity and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine matrix without any other steps in sample pretreatment.

Rapid Detection of Anabolic Steroids in Urine by Protein Arrays

The purpose of this study was to develop a rapid and sensitive method utilizing the state-of-the-art protein arrays technique to detect urinary anabolic steroids (ASs) in athletes. Three experiments were designed to investigate the feasibility of the protein arrays for ASs testing. Firstly, androgen receptor (AR) and estrogen receptor (ER) protein arrays were prepared on polysaccharide-coated slides to investigate whether they can bind to ASs (affinity tests). Secondly, in comparison to adrenergic receptor (the receptor of beta-blockers) and opioid receptor (the receptor of narcotic analgesics) arrays, AR and ER protein arrays were used to test whether they can determine the ASs positive urine sample specifically (specific binding tests). At last protein arrays were used to estimate qualitatively the ASs in positive urine samples (qualitative tests). From the results of the affinity tests the shape of the dose-dependence curve suggested a positive cooperative binding of ASs with the protein arrays. The AR and ER protein arrays showed affinities for fluorescence labelled testosterone and estradiol that were similar to those of literatures (0.65 vs. 0.89 nM, 5.96 vs. 10.3 nM, respectively). Based on the data, the sensitivity of testing can reach 0.1 nM that was much better than the World Anti-Doping Code (WADA) standard. Specific binding tests showed that the prohibited substance in positive urine samples belonged to the anabolic estrogenic inhibitor of ASs. From the results of qualitative tests, we could estimate that there were anabolic androgenic steroids in the positive urine samples and their concentration was lower than 50 microM Methyltestosterone. The total time of the test process for ASs in urine needed less than 1 h. In summary, the present study showed that the protein arrays method provided a highly sensitive and rapid alternative to screen urine samples for the detection of the misuse of ASs in athletes and was suitable for testing in both weekly training sessions as well as large-scale competition events.

Monitoring Anabolic Steroids in Meat-Producing Animals. Review of Current Hyphenated Mass Spectrometric Techniques

Monitoring anabolic steroids in meat-producing animals is a challenging task. It implies very specific and sensitive analytical methods able to detect and identify sub-μg kg−1 residue levels in complex biological matrices such as meat, urine, or hair. Gas and liquid chromatography coupled to mass spectrometry are the most efficient means of achieving these objectives. In this paper we review how developments in mass spectrometry have been rapidly applied to this problem, how efficient analysis of anabolic steroids in urine, edible tissue, and hair has been achieved, and, later, how measurement of conjugate steroids and determination of the origin of natural steroid hormones has been achieved. The performance characteristics of different mass spectrometers (quadrupole, ion-trap, electromagnetic, isotope-ratio, tandem, and hybrid instruments), the efficiency of different acquisition techniques (LR-SIM, HR-SIM, MRM), and, finally, sample introduction (gas chromatography and liquid chromatography, with discussion of alternative interfaces) are discussed, with numerous applications.

Gas chromatographic and mass spectrometric analysis of conjugated steroids in urine.

This study was carried out qualitatively and quantitatively to investigate the presence and the concentrations of anabolic steroids in urine collected from orally administered humans. Microanalysis of conjugated steroids by gas chromatography and mass spectrometry (GC/MS) has been carried out. Following oral administration three major metabolites of anabolic steroid drugs have been detected and partially characterized. The six steroids can be analysed at the same time in 17 min. The lower detection limit was 10 ng/ml in 5 ml of urine. The conjugated steroids from urine were centrifuged to 2,430 g for 10 min, the supernatant solution passed through Amberlite XAD-2 column and the steroids eluted fraction esterified by using MSTFA and TMSI. The rate of metabolism and urinary excretion seem to be reasonably fast.

GC-MS determination of anabolic steroids after multi-immunoaffinity purification.

For many years, EC regulations have prohibited the use of anabolic agents in food-producing animals. Multiple screening methods have been published, but some lack specificity and some are difficult to apply when screening for unknowns in surveillance programmes. This paper presents a new and powerful technique, combining multiresidue immunoaffinity chromatography and GC-MS, for the simultaneous identification and semi-quantification of various anabolic steroids in urine and faeces samples of bovine origin. It should reduce the cost, time and effort of screening by limiting the number of tedious clean-up steps and analyses required. A preliminary extraction step is applied to the individual biological specimens: solid-phase extraction followed by enzymatic digestion in the case of urine samples and a single liquid extraction step for faeces. This step is followed by a first clean-up step involving both a solid-phase column and a rapid RP-HPLC separation. The individual biological fractions (urine or faeces) are further purified on a multiresidue immunoaffinity chromatographic gel (MIAC-steroids-CER) so as to decrease interferences due mainly to background signals. A final trimethylsilyl derivatization is followed by the analysis of the biological samples by a sensitive and specific GC-MS procedure.

Validation of multi-residue methods for the detection of anabolic steroids by GC-MS in muscle tissues and urine samples from cattle†

The use of anabolic steroids is prohibited in the European Community by Directive 96/22/EC and the control of compliance is regulated by Directive 96/23/EC. Multi-residue methods are necessary for screening for the use of forbidden substances. Because accreditation is gaining more and more importance, validation of the methods used and of the results obtained has become indispensable. The developed GC-MS methods for the detection of anabolic steroids in urine and muscle tissue were validated with regard to the following parameters: specificity, recovery at the 2 micrograms kg-1 level and limit of detection. For urine the recoveries ranged from 17 to 81% and for muscle tissue from 26 to 65%. The limit of detection ranged from 0.1 to 2.6 micrograms kg-1 for urine and from 0.3 to 4.6 micrograms kg-1 for muscle tissue. Specificity was guaranteed in both matrices by the selection of four specific ions. Blank samples were evaluated for interferences and it could be concluded that in no case did the four selected ions appear simultaneously at the correct retention time. The practicability of the criteria for low resolution mass spectrometry set in Decision 93/256 in the low micrograms kg-1 range is also briefly discussed.

Separation and confirmation of anabolic steroids with quadrupole ion trap tandem mass spectrometry.

Gas chromatography-mass spectrometry (GC-MS) is the method of choice for separation and detection of anabolic steroids in urine. Recently, there have been advances in the areas of gas chromatography columns, tandem mass spectrometry using ion traps, and large volume sample injection that have promise for lowering detection limits and extending the utility of GC-MS for steroid analysis. In this work, a Varian Saturn III GC-MS system has been used in its tandem mass spectrometry mode to detect low picogram levels of model steroids in standard solution and the urine matrix. Application of MS-MS-MS provided structurally informative spectra for 3'-hydroxystanozolol at concentrations of 1 ng/ml. In addition, four polysilphenylene-polydimethylsiloxane capillary columns were examined for background and selectivity. The columns had bleed several-fold lower than conventional polysiloxane columns. The columns also exhibited significant differences in selectivity for structurally similar steroids. Finally, a new temperature-programmed split-splitless injector was used to inject as much as 25 microliters on column. The resulting limits of detection were 5 pg/ml for norandrosterone.

Multi-residue screening and confirmatory analysis of anabolic steroids in urine by gas chromatography coupled with tandem mass spectrometry

The diversity of substances used illegally as growth promoters in meat production requires the development of multi-analyte methods of analysis involving a sample pretreatment step that is as rapid and as easy as possible, followed by a specific and sensitive determination of several residues within the same run. A general strategy for the screening and confirmatory analysis of fifteen artificial anabolic compounds in urine samples is described. It is based on solid-phase extraction on C 18 Empore discs and amino-bonded columns followed, after derivatization (trimethylsilyl) or methyloxime-trimethylsilyl derivatives), by gas chromatography coupled with collisionally activated dissociation tandem mass spectrometry.