High-throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction.

In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high-throughput assay based on liquid chromatography-triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96-well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5-ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96-well collection plate. With a 10-min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti-Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02-0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

New approach based on immunochemical techniques for monitoring of selective estrogen receptor modulators (SERMs) in human urine.

Antiestrogenic compounds such as tamoxifen, toremifen and chlomifen are used illegally by athletes to minimize physical impacts such as gynecomastia resulting from the secondary effects of anabolic androgenic steroids, used to increase athletic efficiency unlawfully. The use of these compounds is banned by the World Anti-Doping Agency (WADA) and controls are made through analytical methodologies such as HPLC-MS/MS, which do not fulfil the sample throughput requirements. Moreover, compounds such as tamoxifen are also used to treat hormone receptor-positive breast cancer (ER + ).Therapeutic drug monitoring (TDM) of tamoxifen may also be clinically useful for guiding treatment decisions. An accurate determination of these drugs requires a solid phase extraction of patient serum followed by HPLC-MS/MS. In the context of an unmet need of high-throughput screening (HTS) and quantitative methods for antiestrogenic substances we have approached the development of antibodies and an immunochemical assay for the determination of these antiestrogenic compounds. The strategy applied has taken into consideration that these drugs are metabolized and excreted in urine as the corresponding 4-hydroxylated compounds. A microplate-based ELISA procedure has been developed for the analysis of these metabolites in urine with a LOD of 0.15, 0.16 and 0.63 µg/L for 4OH-tamoxifen, 4OH-toremifen and 4OH-clomifen, respectively, much lower than the MRPL established by WADA (20 µg/L).

Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies.

The use of selective oestrogen receptor modulators has been prohibited since 2005 by the World Anti-Doping Agency regulations. As they are extensively cleared by hepatic and intestinal metabolism via oxidative and conjugating enzymes, a complete investigation of their biotransformation pathways and kinetics of excretion is essential for the anti-doping laboratories to select the right marker(s) of misuse. This work was designed to characterize the chemical reactions and the metabolizing enzymes involved in the metabolic routes of clomiphene, tamoxifen and toremifene. To determine the biotransformation pathways of the substrates under investigation, urine samples were collected from six subjects (three females and three males) after oral administration of 50 mg of clomiphene citrate or 40 mg of tamoxifen or 60 mg of toremifene, whereas the metabolizing enzymes were characterized in vitro, using expressed cytochrome P450s and uridine diphosphoglucuronosyltransferases. The separation, identification and determination of the compounds formed in the in vivo and in vitro experiments were carried out by liquid chromatography coupled with mass spectrometry techniques using different acquisition modes. Clomiphene, tamoxifen and toremifene were biotransformed to 22, 23 and 18 metabolites respectively, these phase I reactions being catalyzed mainly by CYP3A4 and CYP2D6 isoforms and, to a lesser degree, by CYP3A5, CYP2B6, CYP2C9, CYP2C19 isoforms. The phase I metabolic reactions include hydroxylation in different positions, N-oxidation, dehalogenation, carboxylation, hydrogenation, methoxylation, N-dealkylation and combinations of them. In turn, most of the phase I metabolites underwent conjugation reaction to form the corresponding glucuro-conjugated mainly by UGT1A1, UGT1A3, UGT1A4, UGT2B7, UGT2B15 and UGT2B17 isoenzymes.

Mass spectrometric identification and characterization of new clomiphene metabolites in human urine by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry.

Clomiphene, a selective estrogen receptor modulator, is prohibited by World Anti Doping Agency (WADA) out-of-competition and in-competition. As it is extensively metabolized, further investigation of clomiphene metabolic profile will be essential to routine anti-doping analysis. The metabolic pathway and the different metabolites of clomiphene in human urine collected from three healthy volunteers during 1 week were studied by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOFMS) based on accurate mass measurement. Seven unreported metabolites were identified and characterized, and all of the newly found urinary metabolites belonged to a new metabolic pathway (hydrogenation). An approach for the metabolism study of clomiphene and its analogs by LC-QTOFMS was presented. Two metabolites, 3,4-dihydroxy-dihydro-clomiphene (m/z 440.1991) and 3,4-dihydroxy-dihydro-deethyl-clomiphne (m/z 412.1674), are the potential biomarkers for monitoring oral administration of clomiphene in doping control.

A mass spectrometric approach for the study of the metabolism of clomiphene, tamoxifen and toremifene by liquid chromatography time-of-flight spectroscopy.

In this paper, we discuss the capabilities of liquid chromatography coupled to mass spectrometry with a time-of flight system with accurate mass measurement for the detection and characterisation of drug metabolites in biological samples for anti-doping purpose. Urinary excretion samples of three selective oestrogen receptor modulators (SERMs) with a common triphenylethylene structure: clomiphene, toremifene, and tamoxifen, obtained after oral administration of a single dose of each drug, were analysed using a time-of-flight system, after automatic tuning and calibration of the equipment, in positive full scan mode using an electrospray ionisation source. Following this approach we detected most of all significant metabolites reported by others and postulated new metabolites, especially for toremifene, have been characterised: N-demethyl-3-hydroxy-4-methoxy-toremifene and 3- hydroxy-4-methoxy-toremifene; in addtiona to this, in the urinary excretion samples of toremifene some metabolites, without the characteristic chlorine isotope pattern, discarded in previous studies, that are also metabolites of tamoxifen, were identified. The lack of certified reference materials does not allow an accurate determination of the limit of detection (LODs) of all metabolites; however an estimation taking into account the response factor of similar compounds allows to estimate that all metabolites are clearly detectable in a range of concentration comprised between 10 ng mL(-1) and 30 ng mL(-1).