Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison. 2008

Laura Hintikka, and Tiia Kuuranne, and Antti Leinonen, and Mario Thevis, and Wilhelm Schänzer, and John Halket, and David Cowan, and Joachim Grosse, and Peter Hemmersbach, and Michel W F Nielen, and Risto Kostiainen
Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Helsinki, Viikinkaari 5E, FIN-00014 Helsinki, Finland.

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.

UI MeSH Term Description Entries
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004300 Doping in Sports Illegitimate use of substances for a desired effect in competitive sports. It includes humans and animals. Blood Doping,Sports Blood Doping,Blood Doping, Sports,Blood Dopings,Blood Dopings, Sports,Doping in Sport,Doping, Blood,Dopings, Blood,Dopings, Sports Blood,Sport, Doping in,Sports Blood Dopings,Sports, Doping in,in Sport, Doping,in Sports, Doping
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D013256 Steroids A group of polycyclic compounds closely related biochemically to TERPENES. They include cholesterol, numerous hormones, precursors of certain vitamins, bile acids, alcohols (STEROLS), and certain natural drugs and poisons. Steroids have a common nucleus, a fused, reduced 17-carbon atom ring system, cyclopentanoperhydrophenanthrene. Most steroids also have two methyl groups and an aliphatic side-chain attached to the nucleus. (From Hawley's Condensed Chemical Dictionary, 11th ed) Steroid,Catatoxic Steroids,Steroids, Catatoxic
D015203 Reproducibility of Results The statistical reproducibility of measurements (often in a clinical context), including the testing of instrumentation or techniques to obtain reproducible results. The concept includes reproducibility of physiological measurements, which may be used to develop rules to assess probability or prognosis, or response to a stimulus; reproducibility of occurrence of a condition; and reproducibility of experimental results. Reliability and Validity,Reliability of Result,Reproducibility Of Result,Reproducibility of Finding,Validity of Result,Validity of Results,Face Validity,Reliability (Epidemiology),Reliability of Results,Reproducibility of Findings,Test-Retest Reliability,Validity (Epidemiology),Finding Reproducibilities,Finding Reproducibility,Of Result, Reproducibility,Of Results, Reproducibility,Reliabilities, Test-Retest,Reliability, Test-Retest,Result Reliabilities,Result Reliability,Result Validities,Result Validity,Result, Reproducibility Of,Results, Reproducibility Of,Test Retest Reliability,Validity and Reliability,Validity, Face
D015813 Substance Abuse Detection Detection of drugs that have been abused, overused, or misused, including legal and illegal drugs. Urine screening is the usual method of detection. Drug Abuse Testing,Illicit Drug Testing,Street Drug Testing,Substance Abuse Testing,Drug Abuse Detection,Drug Abuse Screening,Illicit Drug Detection,Street Drug Detection,Detection, Drug Abuse,Detection, Illicit Drug,Detection, Street Drug,Detection, Substance Abuse,Detections, Drug Abuse,Detections, Illicit Drug,Detections, Street Drug,Detections, Substance Abuse,Drug Abuse Detections,Drug Abuse Screenings,Drug Abuse Testings,Drug Testing, Illicit,Drug Testings, Illicit,Illicit Drug Detections,Illicit Drug Testings,Screening, Drug Abuse,Screenings, Drug Abuse,Street Drug Detections,Street Drug Testings,Substance Abuse Detections,Substance Abuse Testings,Testing, Drug Abuse,Testing, Illicit Drug,Testing, Street Drug,Testing, Substance Abuse,Testings, Drug Abuse,Testings, Illicit Drug,Testings, Street Drug,Testings, Substance Abuse
D045930 Anabolic Agents These compounds stimulate anabolism and inhibit catabolism. They stimulate the development of muscle mass, strength, and power. Anabolic Effect,Anabolic Effects,Agents, Anabolic,Effect, Anabolic,Effects, Anabolic
D053719 Tandem Mass Spectrometry A mass spectrometry technique using two (MS/MS) or more mass analyzers. With two in tandem, the precursor ions are mass-selected by a first mass analyzer, and focused into a collision region where they are then fragmented into product ions which are then characterized by a second mass analyzer. A variety of techniques are used to separate the compounds, ionize them, and introduce them to the first mass analyzer. For example, for in GC-MS/MS, GAS CHROMATOGRAPHY-MASS SPECTROMETRY is involved in separating relatively small compounds by GAS CHROMATOGRAPHY prior to injecting them into an ionization chamber for the mass selection. Mass Spectrometry-Mass Spectrometry,Mass Spectrometry Mass Spectrometry,Mass Spectrometry, Tandem
D020719 Glucuronides Glycosides of GLUCURONIC ACID formed by the reaction of URIDINE DIPHOSPHATE GLUCURONIC ACID with certain endogenous and exogenous substances. Their formation is important for the detoxification of drugs, steroid excretion and BILIRUBIN metabolism to a more water-soluble compound that can be eliminated in the URINE and BILE. Glucuronide
D021241 Spectrometry, Mass, Electrospray Ionization A mass spectrometry technique used for analysis of nonvolatile compounds such as proteins and macromolecules. The technique involves preparing electrically charged droplets from analyte molecules dissolved in solvent. The electrically charged droplets enter a vacuum chamber where the solvent is evaporated. Evaporation of solvent reduces the droplet size, thereby increasing the coulombic repulsion within the droplet. As the charged droplets get smaller, the excess charge within them causes them to disintegrate and release analyte molecules. The volatilized analyte molecules are then analyzed by mass spectrometry. ESI Mass Spectrometry,Electrospray Ionization Mass Spectrometry,Mass Spectrometry, ESI,Spectrometry, ESI Mass

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