Testing for clomiphene in keratinous matrices using LC-MS/MS in doping purpose: Is a single intake of clomiphene detectable in hair and nail clippings?

Clomiphene is a selective estrogen receptor modulator. It is indicated for the treatment of female infertility issues but in sport, it can be misused to stimulate endogenous testosterone secretion in men. Therefore, it has been prohibited at all times by the World Anti-doping Agency. The aim of this study was to get data to be able to interpret concentrations in athletes. A healthy volunteer (male, 62 years-old) ingested a single therapeutic dose of clomiphene (Clomid™, 50 mg). Strands of hair (blond, 4 cm) were collected one month after the ingestion. Body hair (beard, axillary, pubic and chest hair), and finger and toenails were collected over 4-5 months. A previous method was modified to identify and quantify clomiphene in keratinous matrices. 30 mg of specimen were sonicated and incubated in 1 mL of methanol, in presence of 200 pg of clomiphene-D5 (internal standard). After centrifugation and evaporation of the organic phase, the samples were analyzed using LC-MS/MS. Linearity was verified in hair and nail clippings between 1 and 500 pg/mg. The limits of detection and quantification were determined at 0.3 and 1 pg/mg respectively. The study demonstrated that clomiphene tested positive in all the analyzed specimens at 9 pg/mg in head hair, from 28 to 486 pg/mg (body hair) and from 4 to 57 pg/mg (nails). Clomiphene was identified for the first time in multiple keratinous matrices. This study demonstrated that a single oral therapeutic dose is detectable in keratinous matrices over a long period of time.

Testing for clomiphene in keratinous matrices (hair and nail).

Clomiphene or clomifene is a selective estrogen receptor modulator used to treat female fertility in case of ovulatory dysfunction. In sport, clomiphene is prohibited at all times for use by athletes and is listed in the section S4.2 (hormone and metabolic modulators) by the World Anti-Doping Agency. Indeed, clomiphene can indirectly increase testosterone levels in the body and can mitigate some side effects of synthetic steroid abuse. Despite its prescription to millions of subjects, its detection in human hair or nail clippings has never been reported. The aim of this study was to develop a specific method to identify clomiphene in hair and nail clippings by liquid chromatography-quadrupole tandem mass spectrometry. The procedure was then applied in a case of challenged doping results. The method involves sonication/incubation for 1 h of 30 mg of pulverized material in 1 mL of methanol in the presence of 2 ng diazepam-d5 used as internal standard. The chromatographic separation was performed using a HSS C18 column with a 15 min gradient elution. After spiking blank hair and nail with the corresponding amounts of clomiphene, linearity was verified from 1 to 500 pg/mg (r2 = 0.9994 and 0.9995 for hair and nail, respectively). The limit of detection was estimated at 0.3 pg/mg for both matrices. No interference was noted from endogenous compounds, particularly steroids. Clomiphene was identified at 85 and 20 pg/mg in the pubic hair and the fingernail clippings, respectively, of a male athlete challenging an adverse analytical finding.

Fingerprints: A New Specimen for Innovative Applications for the Detection of Xenobiotics.

Fingerprints are invisible traces that result from a deposition of sweat and sebum present on the papillary ridges. As sweat and sebum contain drugs, fingerprints are promising since collection is rapid, non-invasive and difficult to falsify. Very limited data are available in the literature, and therefore, it seems opportune to study the transfer of xenobiotics onto the items taken in hand via the fingerprints. Two studies were implemented using the ballpoint pen as a model. The objective of the first study was to compare the nicotine concentrations found on the pens of three smokers and three non-smokers. Five pens, belonging to each subject and used regularly, were rubbed with a cotton swab dipped in methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). The second study was to analyze the transfer via fingerprints of four volunteers, after administration of 30 mg of codeine. The objective was to determine the feasibility of this study and the time corresponding to the highest concentration of codeine. Over a 24-h period, new pens were handled for 5 min by the four volunteers, rubbed with a cotton swab dipped in methanol, and then analyzed by LC-MS-MS. The nicotine study showed a major difference between the nicotine concentrations obtained from smokers (between 6 and 276 ng/pen) and non-smokers (between 2 and 4 ng/pen). After administration of 30 mg of codeine, the analysis of the pens of the four volunteers allowed to demonstrate the presence of codeine up to 24 h between 9 and 544 pg/pen. Normal hygiene practices did not influence the final result. The highest concentration was observed after 2 h. Morphine was also detected (between 19 and 33 pg/pen). These preliminary results should be considered a demonstration of the interest of fingerprints testing to document drug exposure.

Characterization of letrozole in human hair using LC-MS/MS and confirmation by LC-HRMS: Application to a doping case.

Letrozole is a reversible aromatase inhibitor, used in the treatment of hormone-dependent woman cancer. No indication for medical use is available for men. In recent years, several cases of doping with letrozole have been observed, especially among high level athletes. Aromatase inhibitors reverse the harmful effects (feminizing) of the abuse of anabolic androgenic steroids. Letrozole is included on the list of products prohibited in- and out-competition by the World Anti-Doping Agency, under section S4.1. The aim of the present work was to develop a specific method to identify letrozole in human hair of a male amateur athlete by LC-MS/MS and confirmation by LC-HRMS, after incubation of 20 mg of matrix in 1 mL of methanol. The chromatographic separation was performed using a reverse phase column HSS C18 with a gradient elution of 15 min (from 87% to 5% of formate buffer adjusted to pH 3). Linearity was observed from 1 to 1000 pg/mg (r2 = 0.9999), after spiking blank hair with the corresponding amounts of letrozole. The limit of detection was estimated at 0.5 pg/mg and the lower limit of quantification was the first point of the calibration curve, i.e. 1 pg/mg. The precision was lower than 20% and there was no interference with the analytes by chemicals or any extractable endogenous materials present in hair. Letrozole was identified in the male amateur athlete hair at 310 pg/mg (segment 0-2 cm) and 245 pg/mg (segment 2-4 cm).

Detection of the designer benzodiazepine metizolam in urine and preliminary data on its metabolism.

Designer benzodiazepines provide an attractive alternative to prescribed benzodiazepines for abuse purposes as they are readily available via the Internet without control. Metizolam was ordered via the Internet and a 2 mg blue tablet was orally administered to a 54-year-old man. Urine samples were collected over 6 days in polypropylene tubes. After liquid/liquid extraction at pH 9.5, metizolam was analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) using a standard method devoted to benzodiazepines, and ions transitions, at m/z 328.9 > 275.0 and 328.9 > 300.0. Metizolam was detectable in hydrolyzed urine during the 46-h period, with concentrations always lower than 11 ng/mL. About 0.3% of the initial dose was excreted in urines as total unchanged metizolam during the first 24 h. The most relevant potential CYP- and UGT-dependent metabolites of metizolam were investigated in vitro using human liver microsome incubation and, subsequently, liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. Three mono-hydroxylated metabolites were produced including a hydroxylation compound at the 2-ethyl moiety of metizolam (M1) as quantitatively main metabolite, and a N-hydroxymetiazolam (M2). The structure of the third metabolite (M3) could not be elucidated because of a too low experimental production rate. Two authentic urine samples were analyzed using the same analytical method to search for metabolites of metizolam. M1, together with its glucuronide (M1-Glu), and M2 were observed in urine at the 8 h mark, whereas only M1 and M1-Glu were still detected in urine at 30 h post administration. Copyright © 2016 John Wiley & Sons, Ltd.