Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography–electrospray ionization tandem mass spectrometry (original) (raw)
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Journal of Chromatography A
An isotope dilution mass spectrometry (IDMS) method for the determination of selected endogenous anabolic androgenic steroids (EAAS) in urine by UHPLC-MS/MS has been developed using the isotope pattern deconvolution (IPD) mathematical tool. The method has been successfully validated for testosterone, epitestosterone, androsterone and etiocholanolone, employing their respective deuterated analogs using two certified reference materials (CRM). Accuracy was evaluated as recovery of the certified values and ranged from 75% to 108%. Precision was assessed in intraday (n=5) and interday (n=4) experiments, with RSDs below 5% and 10% respectively. The method was also found suitable for real urine samples, with limits of detection (LOD) and quantification (LOQ) below the normal urinary levels. The developed method meets the requirements established by the World Anti-Doping Agency for the selected steroids for Athlete Biological Passport (ABP) measurements, except in the case of androsterone, which is currently under study.
Journal of Chromatography B, 2002
Detection of anabolic steroids in animal urine samples is currently performed with GC-MS in our lab. However we found that the detection of 17a-trenbolone (17a-TbOH), 4-chloroandrost-4-ene-3,17-dion (CLAD), 16-b-OH-stanozolol (16OHstan) and aand b-boldenone (a-Bol, b-Bol) was very difficult, if not impossible. Therefore a sensitive, specific and selective qualitative multi-analyte LC-MS-MS method was developed. The LC separation was achieved by using a ® Symmetry C column and methanol-water-formic acid (54.7-44.7-0.6) as a mobile phase at a flow-rate of 0.3 ml / min. 18 The mass spectrometer was operated in multiple reaction monitoring mode with positive electrospray interface. Validation of the method was done according to draft SANCO / 1805 / 2000 Rev.1 and a CCb smaller then 1 ng / ml was obtained for each compound.
Molecules
Testing and monitoring anabolic androgenic steroids in biological fluids is a key activity in anti-doping practices. In this study, a novel approach is proposed, based on dried urine microsampling through two different workflows: dried urine spots (DUS) and volumetric absorptive microsampling (VAMS). Both techniques can overcome some common drawbacks of urine sampling, such as analyte instability and storage and transportation problems. Using an original, validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) method, exogenous and endogenous unconjugated steroids were analysed. Despite the limitations of microsampling volume, good sensitivity was obtained (limit of quantitation ≤1.5 ng/mL for all analytes), with satisfactory precision (relative standard deviation <7.6%) and absolute recovery (>70.3%). Both microsampling platforms provide reliable results, in good agreement with those obtained from urine.
Journal of Chromatography B: Biomedical Sciences and Applications, 1997
A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated from normal human urine was achieved by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. TIE ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted TIE threshold of 6:1, Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urine.
Indian Journal of Pharmaceutical Education and Research, 2019
Background: Androgenic Anabolic Steroids (AAS) are also synthetic derivatives of testosterone, modified to improve its anabolic actions. The misuse of AAS is of particular concern in sports and society. Gas chromatography-mass spectrometry had some limitations and allows identification and characterization of steroids and their metabolites in the urine but may not be able to distinguish between pharmaceutical (Exogenous) and endogenous origin. Thus, it is of great importance to discriminate endogenous steroids such as testosterone or testosterone prohormones from their chemically identical synthetic copies. The abuse of Androgenic Anabolic Steroids (AAS) by sports person is banned by World Anti-doping Agency (WADA) as per the WADA Prohibited list 2019. Methodology: The gas chromatography-combustion/isotope ratio mass spectrometry (GC/C/IRMS) technique differentiates between natural and synthetic endogenous steroids by comparing compounds specific 13 C/ 12 C ratio. However, the analytes have to be efficiently isolated and purified prior to GC/C/IRMS analysis. Results and Discussion: HPLC Cleanup method prior to analysis by GC−C/IRMS needs to be developed and validated for discriminating the origin of anabolic androgenic steroids. These methods involves the solid-phase extraction, enzymatic hydrolysis with β-glucuronidase, HPLCfractionation for the cleanup and analysis by GC−C/IRMS. The difference (Δ 13 C) of urinary δ 13 C values between synthetic analogues and Endogenous Reference Compounds (ERC) by GC-C/IRMS would be used to elucidate the origin of steroids. The present perspective gives an overview of the use of anabolic-androgenic steroids in sport and methods used in anti-doping laboratories for their detection in urine, with special emphasis on GC−C/ IRMS technique after twofold HPLC cleanup.
Journal of Chromatography A, 2012
Liquid chromatography/mass spectrometry (LC/MS) has been successfully applied to the detection of anabolic steroids in biological samples. However, the sensitive detection of saturated hydroxysteroids, such as androstanediols, by electrospray ionisation (ESI) is difficult because of their poor ability to ionise. In view of this, chemical derivatisation has been used to enhance the detection sensitivity of hydroxysteroids by LC/MS. This paper describes the development of a sensitive ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) method for the screening of anabolic steroids in horse urine by incorporating a chemical derivatisation step, using picolinic acid as the derivatisation reagent. The method involved solid-phase extraction (SPE) of both free and conjugated anabolic steroids in horse urine using a polymer-based SPE cartridge (Abs Elut Nexus). The conjugated steroids in the eluate were hydrolysed by methanolysis and the resulting extract was further cleaned up by liquidliquid extraction. The resulting free steroids in the extract were derivatised with picolinic acid to form the corresponding picolinoyl esters and analysed by UHPLC/MS/MS in the positive ESI mode with selectedreaction-monitoring. Separation of the targeted steroids was performed on a C18 UHPLC column. The instrument turnaround time was 10.5 min inclusive of post-run equilibration. A total of thirty-three anabolic steroids (including 17-estradiol, 5(10)-estrene-3,17␣-diol, 5␣-estrane-3,17␣-diol, 17␣ethyl-5␣-estran-3␣,17-diol, 17␣-methyl-5␣-androstan-3,17-diols, androstanediols, nandrolone and testosterone) spiked in negative horse urine at the QC levels (ranging from 0.75 to 30 ng/mL) could be consistently detected. The intra-day and inter-day precisions (% RSD) for the peak area ratios were around 7-51% and around 1-72%, respectively. The intra-day and inter-day precisions (% RSD) for the relative retention times were both less than 1% for all analytes, except the inter-day precision for boldione at 1.2%. The extraction recoveries for all targets were not less than 48%. With exceptional separation achieved by the UHPLC system, matrix interferences were minimal at the expected retention times of the selected transitions. As detection was performed with an UHPLC system coupled to a fast-scanning triple quadrupole mass spectrometer, the method could easily be expanded to accommodate additional steroid targets. This method has been validated for recovery and precision, and could be used regularly for doping control testing of anabolic steroids in horse urine samples.
High-temperature liquid chromatography-isotope ratio mass spectrometry methodology for carbon isotope ratio determination of anabolic steroids in urine, 2024
Gas Chromatography Isotope Ratio Mass Spectrometry (GC-C-IRMS) has traditionally been used in routine laboratories to measure the δ13C values of anabolic steroids in urine, enabling differentiation between endogenous and synthetic testosterone (T) in sports doping control. Liquid Chromatography Isotope Ratio Mass Spectrometry (LC-IRMS) has not been applied due to its limitation of not allowing organic solvents or modifiers in the mobile phase for δ13C analysis. Mid-to non-polar analytes like steroids can be analyzed in water heated to high temperatures (up to 200°C), where water's polarity becomes comparable to an 80/20 methanol/water mixture at ambient temperature. This study developed a method for analyzing steroids in urine, extending the application of LC-IRMS to non-polar analytes in complex matrices. An HT-LC-IRMS method was successfully developed and validated to determine the δ13C values of four steroids: testosterone (T), 5α-androstane-3α,17β-diol (ααβ), 5β-androstane-3α,17β-diol (βαβ), and pregnanetriol (PT) in urine. Accuracy ranged from 0.23‰ (ααβ and βαβ) to 0.49‰ (T), with a detection limit of 10 ng/mL for T and ααβ+βαβ. Validation data and comparisons of authentic urine samples analyzed with HT-LC-IRMS and GC-C-IRMS showed comparable performance between the two methods. HT-LC-IRMS is suitable for determining the δ13C values of anabolic steroids, expanding the applications of both HT-LC and LC-IRMS to non-polar substances in complex matrices in routine laboratory settings.
British Journal of Sports Medicine, 1975
Identification of anabolic steroids in urine from athletes: Urine samples were collected from athletes attending the 1974 Commonwealth Games in Christchurch and the European Games held in Rome, 1974. These specimens were analysed initially by Professor Brooks using radioimmunoassays and then submitted to us for independent assessment. Steroid extraction and fractionation The urine specimens were poured on to columns of Amberlite XAD-2 resin to extract free and conjugated *Permission to carry out these drug studies was granted by the Northwick Park Hospital Ethical Committee.
Rapid Communications in Mass Spectrometry, 2001
Findings of illegal hormone preparations such as syringes, bottles, cocktails, and so on, are an important information source for the nature of the current abuse of anabolic steroids and related compounds as growth-promoting agents in cattle. A new screening method for steroids in cocktails is presented based on liquid chromatography (LC) with diode-array UV-absorbance detection and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS). Accurate mass measurements were performed at a mass resolution of 4000 using continuous introduction of a lock mass through a second (electro)sprayer. Similar experiments were carried out using dual-sprayer quadrupole time-of-flight mass spectrometry (ESI-QTOFMS/MS) at a mass resolution of 10 000 with data-dependent MS/MS acquisition; i.e. beyond an intensity threshold for the [M + H](+) ions, MS/MS spectra were automatically acquired at three different collision energies. Elemental compositions were calculated for precursor and product ions and it is shown that the combined information from LC retention behavior, UV spectra, elemental compositions, and accurate mass MS/MS spectra yield a fast impression of the steroids present in the complex mixture. Using a new software tool for structure elucidation of MS/MS spectra, an additional non-steroidal additive was identified as well.