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.

In-house validation of a liquid chromatography-tandem mass spectrometry method for the determination of selective androgen receptor modulators (SARMS) in bovine urine.

A sensitive and robust LC-MS/MS method allowing the rapid screening and confirmation of selective androgen receptor modulators in bovine urine was developed and successfully validated according to Commission Decision 2002/657/EC, chapter 3.1.3 'alternative validation', by applying a matrix-comprehensive in-house validation concept. The confirmation of the analytes in the validation samples was achieved both on the basis of the MRM ion ratios as laid down in Commission Decision 2002/657/EC and by comparison of their enhanced product ion (EPI) spectra with a reference mass spectral library by making use of the QTRAP technology. Here, in addition to the MRM survey scan, EPI spectra were generated in a data-dependent way according to an information-dependent acquisition criterion. Moreover, stability studies of the analytes in solution and in matrix according to an isochronous approach proved the stability of the analytes in solution and in matrix for at least the duration of the validation study. To identify factors that have a significant influence on the test method in routine analysis, a factorial effect analysis was performed. To this end, factors considered to be relevant for the method in routine analysis (e.g. operator, storage duration of the extracts before measurement, different cartridge lots and different hydrolysis conditions) were systematically varied on two levels. The examination of the extent to which these factors influence the measurement results of the individual analytes showed that none of the validation factors exerts a significant influence on the measurement results.

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).

Determination and confirmation of selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors in bovine and porcine urine using UHPLC-MS/MS.

Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry.

So far, in sports drug testing compounds of different classes are processed and measured using different screening procedures. The constantly increasing number of samples in doping analysis, as well as the large number of substances with doping related, pharmacological effects require the development of even more powerful assays than those already employed in sports drug testing, indispensably with reduced sample preparation procedures. The analysis of native urine samples after direct injection provides a promising analytical approach, which thereby possesses a broad applicability to many different compounds and their metabolites, without a time-consuming sample preparation. In this study, a novel multi-target approach based on liquid chromatography and high resolution/high accuracy mass spectrometry is presented to screen for more than 200 analytes of various classes of doping agents far below the required detection limits in sports drug testing. Here, classic groups of drugs as diuretics, stimulants, ß2-agonists, narcotics and anabolic androgenic steroids as well as various newer target compounds like hypoxia-inducible factor (HIF) stabilizers, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), plasma volume expanders and other doping related compounds, listed in the 2016 WADA prohibited list were implemented. As a main achievement, growth hormone releasing peptides could be implemented, which chemically belong to the group of small peptides (<2kDa) and are commonly determined by laborious and time-consuming stand-alone assays. The assay was fully validated for qualitative purposes considering the parameters specificity, robustness (rRT: <2%), intra- (CV: 1.7-18.4 %) and inter-day precision (CV: 2.3-18.3%) at three concentration levels, linearity (R2>0.99), limit of detection (0.1-25ng/mL; 3'OH-stanozolol glucuronide: 50pg/mL; dextran/HES: 10µg/mL) and matrix effects.

Mass spectrometric characterization of urinary toremifene metabolites for doping control analyses.

Toremifene is a selective estrogen receptor modulator included in the list of prohibited substances in sport by the World Anti-doping Agency. The aim of the present study was to investigate toremifene metabolism in humans in order to elucidate the structures of the most abundant urinary metabolites and to define the best marker to detect toremifene administration through the analysis of urine samples. Toremifene (Fareston) was administered to healthy volunteers and the urine samples were subjected to different preparation methods to detect free metabolites as well as metabolites conjugated with glucuronic acid or sulphate. Urinary extracts were analyzed by LC-MS/MS with triple quadrupole analyzer using selected reaction monitoring mode. Transitions for potential metabolites were selected by using the theoretical [M+H](+) as precursor ion and m/z 72 or m/z 58 as product ions for N,N-dimethyl and N-desmethyl metabolites, respectively. Toremifene and 20 metabolites were detected in excretion study samples, excreted free or conjugated with glucuronic acid or sulphate. Structures for most abundant phase I metabolites were proposed using accurate mass measurements performed by QTOF MS, based on fragmentation pattern observed for those metabolites available as reference standards. Several metabolic pathways including mono- and di-hydroxylation, N-desmethylation, hydroxymethylation, oxidation, dehalogenation and combinations were proposed. All metabolites were detected up to one month after toremifene administration; the most abundant metabolites were detected in the free fraction and they were metabolites resulting from dehalogenation. Several of the metabolites elucidated in this work have not been reported until now in the scientific literature.

Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes.

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.

Mass spectrometric characterization of tamoxifene metabolites in human urine utilizing different scan parameters on liquid chromatography/tandem mass spectrometry.

Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were considered for the characterization of tamoxifene metabolites in human urine for anti-doping purpose. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 166, 152 and 129) and neutral loss scan (neutral loss of 72 Da and 58 Da) in positive ion mode were assessed to recognize common ions or common losses of tamoxifene metabolites. The applicability of these methods was checked first by infusion and then by the injection of solution of a mixture of reference standards of four tamoxifene metabolites available in our laboratory. The data obtained by the analyses of the mixture of the reference standards showed that the five methods used exhibited satisfactory results for all tamoxifene metabolites considered at a concentration level of 100 ng/mL, whereas the analysis of blank urine samples spiked with the same tamoxifene metabolites at the same concentration showed that the neutral loss scan of 58 Da lacked sufficient specificity and sensitivity. The limit of detection in urine of the compounds studied was in the concentration range 10-100 ng/mL, depending on the compound structure and on the selected product ion. The suitability of these approaches was checked by the analysis of urine samples collected after the administration of a single dose of 20 mg of tamoxifene. Six metabolites were detected: 4-hydroxytamoxifene, 3,4-dihydroxytamoxifene, 3-hydroxy-4-methoxytamoxifene, N-demethyl-4-hydroxytamoxifene, tamoxifene-N-oxide and N-demethyl-3-hydroxy-4-methoxytamoxifene, which is in conformity to our previous work using a time-of-flight (TOF) mass spectrometer in full scan acquisition mode.

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).

Pharmacokinetic parameters of tibolone and metabolites in plasma, urine, feces, and bile from ovariectomized cynomolgus monkeys after a single dose or multiple doses of tibolone.

Levels of nonsulfated and sulfated tibolone metabolites were determined in plasma, urine, and feces from six ovariectomized, mature female cynomolgus monkeys after a single dose and multiple p.o. doses (including bile) of tibolone using validated gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry assays. In plasma, the predominant nonsulfated metabolite after single and multiple dosing was the estrogenic 3alpha-hydroxytibolone; levels of the estrogenic 3beta-hydroxytibolone were 10-fold lower and of progestagenic/androgenic Delta(4)-tibolone, 5-fold lower. Tibolone was undetectable. The predominant sulfated metabolite was 3alphaS,17betaS-tibolone; levels of 3betaS,17betaS-tibolone were about 2-fold lower, and monosulfated 3-hydroxymetabolites were about 10-fold lower. After multiple doses, areas under the curve of nonsulfated metabolites were lower (2-fold), and those of sulfated metabolites were 25% higher. In plasma, >95% metabolites were disulfated. In urine, levels of all the metabolites after single and multiple doses were low. After a single dose, high levels of 3beta-hydroxytibolone and the 3-monosulfated metabolites (3betaS,17betaOH-tibolone and 3alphaS,17betaOH-tibolone) were found in feces. After multiple dosing, 3alpha-hydroxytibolone increased, and the ratio of 3alpha/3beta-hydroxytibolone became about 1. The predominant sulfated metabolite was 3alphaS,17betaS-tibolone. Levels of all the metabolites in feces were higher after multiple doses than after a single dose. Levels of nonsulfated and 3-monosulfated metabolites were higher in feces than in plasma. Bile contained very high metabolite levels, except monosulfates. This may contribute to the metabolite content of the feces after multiple doses. 3beta-Hydroxytibolone and 3alphaS,17betaS-tibolone predominated. In conclusion, tibolone had different metabolite patterns in plasma, urine, feces, and bile in monkeys. The bile contributed to the metabolite pattern in feces after multiple doses. The major excretion route was in feces.