Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls.

The misuse of the steroid hormone testosterone for performance enhancement has been frequently reported in the past, and its administration is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Testosterone is produced endogenously in human. Endogenous and exogenous testosterone together with their metabolites can be unambiguously distinguished by means of their carbon isotope ratios if compared to endogenous reference compounds. Established isotope ratio mass spectrometry methods for analyzing urinary steroids for doping control purposes consist of up to two time-consuming HPLC purification steps to achieve the required purity of all analytes. In order to accelerate the sample preparation, multidimensional gas chromatography was applied. This technique is known to be suitable for the separation of complex matrices. Multidimensional gas chromatography consists of two gas chromatographs connected by a pressure-controlled heart-cutting device. In the first dimension, a less polar capillary column was installed for peak purification. In the second dimension, separation was achieved employing a column of medium polarity. Retention time stability and transfer windows were monitored by a flame ionization detector. Detection was performed simultaneously by isotope ratio mass spectrometry and a single quadrupole mass spectrometer for analyte identity confirmation and assessment of peak purity. Instead of two working days required for the HPLC-based routine method, the sample preparation is shortened by the herein presented approach to one working day. For glucuronic acid-conjugated steroids, sample pretreatment is based on solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, and derivatization of the target analytes to their corresponding acetates. These steroid acetates are divided according to their polarity into two fractions by solid phase extraction. Further, sulfoconjugated steroids are processed by Pseudomonas aeruginosa arylsulfatase and extracted following a recently established procedure. Following WADA guidelines, the method was validated by determining the parameters linear range, limit of quantification, intra- and interday precision, accuracy and specificity utilizing linear mixing models. Additionally, a reference population (n = 74) was investigated and the obtained data were compared to the established method. An excretion study was also conducted with 4-androstenedione to prove the fit for purpose of the methodology. The results demonstrate that the method is suitable for an application in routine doping control analysis.

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry.

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17ß-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.