Development of an extraction method for anabolic androgenic steroids in dietary supplements and analysis by gas chromatography-mass spectrometry: Application for doping-control (original) (raw)
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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, 1991
A method was developed for the detection of anabolic steroid residues in edible muscle tissues. After enzymic digestion of the tissue and purification on disposable C18 solid-phase extraction columns, the extract was injected onto a C18 reversed-phase high-performance liquid chromatographic column. Three fractions or windows were collected, each containing specific analytes. After evaporation to dryness, the residues were subjected to a derivatization procedure which yielded suitable derivatives. After gas chromatographic-mass spectrometric analysis, both gas chromatographic retention data and mass spectral data were used for the detection and identification of nortestosterone, testosterone, estradiol, ethinylestradiol, trenbolone, methyltestosterone, chlormadinone acetate, medroxyprogesterone acetate and megestrol acetate.
Enhancement of chemical derivatization of steroids by gas chromatography/mass spectrometry (GC/MS)
Journal of Chromatography B, 2009
Steroid derivatization was investigated by varying the experimental parameters (reagent, reaction time, and reaction temperature) to determine the optimal conditions for individual steroids, and for larger subsets. Three methods of derivatization enhancement were also investigated: the use of sonication, the use of a microwave heating, and the addition of solvents to the reaction mixture. On a comprehensive level, derivatization using N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) was most efficient, while the application of solvent addition and microwave heating, in several cases, provided a clear enhancement. In addition, generalized rules for steroid derivatization are described.
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.
A uniform sample preparation procedure for gas chromatography combustion isotope ratio mass spectrometry for all human doping control relevant anabolic steroids using online 2/3-dimensional liquid chromatography fraction collection, 2021
Androgenic anabolic steroids are the most misused substances in sports due to their performance-enhancing effects. Synthetic analogues of endogenously present steroids are often administered. To determine their endogenous or exogenous origin, gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) is used in doping control. Compounds analyzed by IRMS must be free from interferences, with liquid chromatography fraction collection (HPLC-FC) being a crucial clean-up step. However, this process is challenging, especially for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interest for IRMS analyses in doping control include testosterone and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol), epitestosterone, 19-norandrosterone, boldenone and its main metabolite, formestane, and 6αOH-androstenedione. Current methods only address a selection of these compounds, with some (e.g., 19-norandrosterone, boldenone, its metabolite, and 6αOH-androstenedione) present in very low concentrations, requiring extensive and dedicated clean-up procedures. This makes developing a universal clean-up method challenging. Many existing methods rely on different and multiple offline HPLC-FC setups, which are labor-intensive and time-consuming, posing a problem during large sports events where rapid reporting is necessary. In this work, a uniform online 2D/3D HPLC-FC method was developed, capable of purifying all relevant target compounds in a single run. This method achieves the fastest clean-up procedure to date, taking 31 minutes for testosterone and its main metabolites, 46 minutes for 19-norandrosterone, formestane, and 6αOH-androstenedione, and 48 minutes for boldenone and its metabolite.
Analysis of estrogens and anabolic steroids by SPME with on‐fiber derivatization and GC/MS
Journal of Microcolumn Separations, 1998
The solid-phase micro-extraction SPME , Bis trimethylsilyl trifluo-Ž. roacetamide BSTFA headspace derivatization on the SPME fiber, and gas chro-Ž. matography᎐mass spectrometry GCrMS analysis of steroids from aqueous and biological solutions are shown. The effects of extraction and derivatization parameters have been shown. The extraction time of several steroids was shown to be approximately 30 min., which is typical of SPME methods. The analyte solution pH and ionic strength may have dramatic effects on the extraction recovery and should be optimized as part of method development. The incubation temperature may have several influences on the recovery from the derivatization reaction. Each analyte has a separate optimum temperature for derivatization. Thus, for mixtures, a compromise will be necessary. The analysis of anabolic steroids of abuse is shown. A detection limit of less than 200 pgrmL for testosterone from water is seen. Calibration for testosterone from water indicates a linear range to at least 1000 ngrmL, or three to four orders of magnitude. Several other steroids were extracted from urine at 10᎐100 ngrmL levels, and calibration for these compounds also exhibited linear behavior. The SPME with on-fiber headspace derivatization is a promising technique for the analysis of polar or nonvolatile compounds. ᮊ 1998
Journal of Separation Science, 2019
For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid-phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 μg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10-0.48 and 0.17-0.95 μg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.
Drug Testing and Analysis, 2010
The method of high sensitive gas chromatographic/time-of-flight mass-spectrometric (GC/TOF-MS) analysis of steroids was developed. Low-resolution TOF-MS instrument (with fast spectral acquisition rate) was used. This method is based on the formation of the silyl derivatives of steroids; exchange of the reagent mixture (pyridine and N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA)) for tert-butylmethylether; offline large sample volume injection of this solution based on sorption concentration of the respective derivatives from the vapour-gas mixture flow formed from the solution and inert gas flows; and entire analytes solvent-free concentrate transfer into the injector of the gas chromatograph. Detection limits for 100 µl sample solution volume were 0.5-2 pg/µl (depending on the component). Application of TOF-MS model 'TruTOF' (Leco, St Joseph, MO, USA) coupled with gas chromatograph and ChromaTOF software (Leco, St Joseph, MO, USA) allowed extraction of the full mass spectra and resolving coeluted peaks. Due to use of the proposed method (10 µl sample aliquot) and GC/TOF-MS, two times more steroid-like compounds were registered in the urine extract in comparison with the injection of 1 µl of the same sample solution.
Analysis of conjugated steroid androgens: Deconjugation, derivatisation and associated issues
Journal of Pharmaceutical and Biomedical Analysis, 2009
Gas chromatography/mass spectrometry (GC/MS) is the preferred technique for the detection of urinary steroid androgens for drug testing in athletics. Excreted in either the glucuronide or sulfated conjugated form, steroids must first undergo deconjugation followed by derivatisation to render them suitable for GC analysis. Discussed herein are the deconjugation and the derivatisation preparative options. The analytical challenges surrounding these preparatory approaches, in particular the inability to cleave the sulfate moiety have led to a focus on testing protocols that reply on glucuronide conjugates. Other approaches which alleviate the need for deconjugation and derivatisation are also highlighted.
Journal of Chromatography A, 1978
High-performance liquid chromatography (HPLC) diode-array UV-spectrophotometric detection is used for estimating impurity profiles of steroid drugs. It is shown to be a very useful first screening method for the identification of UV-active impurities and degradation products, giving a rapid answer to many questions or at least providing important initial information to complement the results obtained by other spectroscopic techniques.