Analysis for higenamine in urine by means of ultra‐high‐performance liquid chromatography–tandem mass spectrometry: Interpretation of results

Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine and is present in many plants. The substance may be also found in supplements or nutrients, consumption of which may result in violation of anti‐doping rules. Higenamine is prohibited in sport at all times and included in Class S3 (β‐2‐agonists) of the World Anti‐Doping Agency (WADA) 2017 Prohibited List. The presence of higenamine in urine samples at concentrations greater than or equal to 10 ng/mL constitutes an adverse analytical finding (AAF). This work presents a new metabolite of higenamine in urine sample which was identified by means of ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Samples were prepared according to 2 protocols – a Dilute and Shoot (DaS) approach and a method involving acid hydrolysis and double liquid‐liquid extraction (LLE). To meet the requirements typical for a confirmatory analysis, the screening procedure was further developed. In samples prepared by the DaS method, 2 peaks were observed; the earlier one was specific for higenamine and the later one unknown. MS scan analysis showed mass about 80 Da higher than that of higenamine. In turn, in samples prepared in accordance with the protocol involving hydrolysis, an increase in the area under peak for higenamine was observed, while the second peak was absent. It seems that the described strategy of detection of higenamine in urine avoids false negative results.     

Use of liquid chromatography-tandem mass spectrometry for determination of higenamine in urine following oral administration of traditional Chinese medicine

Since higenamine (HG) was first included in the World Anti-doping Agency (WADA) 2017 Prohibited List, an increasing number of plants have been found to contain this ingredient. As a result, doctors are hesitant to prescribe traditional Chinese medicine (TCM) to athletes. Thus, it is very important to assess the risks of doping violations due to HG following the oral administration of TCM. We determined the drug concentration-time curves for HG in urine by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after single or multiple administrations of lotus seed powder on volunteers, the single dose was equivalent to 750 μg of HG, and the multiple doses were equivalent to 90 μg of HG each, 3 times daily for 5 consecutive days. For the single-dose group, the HG could be detected in urine 0.5 h after administration and reached a maximum concentration of 16.5 ng/mL 1 h after administration. For the multiple-dose group, the HG concentrations in urine showed two peaks at 29 and 77 h post-administration with 22.6 and 23.1 ng/mL, respectively. At the dosage used in this study, the maximum concentration of HG in some urine samples exceeded the WADA limit of 10.0 ng/mL; the risk was still very high, so athletes must avoid this amount of HG when using TCM. In addition, our study provided further data supporting the presence of sulfonated metabolites of HG in urine samples.     

Presence of higenamine in beetroot containing ‘foodstuffs’ and the implication for WADA-relevant anti-doping testing

Higenamine is an alkaloid found within plant species including some that are used in traditional Asian and Chinese herbal medicines. Identified as having mixed mode adrenergic receptor activity, higenamine is present within some nutritional supplements marketed for stimulant and/or weight loss. Its inclusion within nutritional supplements can be via its natural presence within botanical ingredients or as a synthetic additive, often added in mg amounts. The World Anti-doping Agency (WADA) prohibited list has contained higenamine since 2017 as banned at all times in the beta-2 agonist (S3) category, with a reporting level of 10 ng/ml for the free parent form in urine. In this study, an investigation into the content of beetroot or beetroot-containing foodstuffs and supplement products was conducted. Higenamine was confirmed as present within the majority of foodstuffs and supplements, with experimental evidence that higenamine can arise within beetroot extracts through heating. The results in this paper demonstrate the first reported evidence of a link between beetroot and this WADA prohibited substance. To investigate the link between intake and excretion, concentrated beetroot drinks were consumed by six individuals and higenamine quantified in their urine. Free higenamine was detected in the urine of all individuals, with maximum measured concentration in samples of less than 1% of the current WADA reporting limit. Although the risk of an inadvertent doping violation by consumption of the foodstuffs and products investigated in this study is low, beetroot as a source of higenamine should be considered by athletes.     

Budesonide use and misuse in sports: Elimination profiles of budesonide and metabolites after intranasal,high‐dose inhaled and oral administrations

Budesonide (BUD) is a glucocorticoid (GC) widely used in therapeutics. In sports, the World Anti‐doping Agency (WADA) controls the use of GCs, and WADA‐accredited laboratories use a reporting level of 30 ng/mL for 6β‐hydroxy‐budesonide (6βOHBUD) to detect the systemic administration of BUD. In the present work, we examined the urinary excretion profile of 6βOHBUD, BUD, and 16α‐hydroxy‐prednisolone (16αOHPRED) after intranasal (INT), inhaled (INH) (at high doses) and oral administrations in male and female volunteers. BUD was administered to healthy volunteers using INT route (256 μg/day for three days, n = 4 males and 4 females), INH route (800 μg/day for three days, n = 4 males and 4 females, and 1600 μg/day for three days, n = 4 males) or oral route (3 mg, n = 8 females). Urine samples were collected before and after administration at different time periods, and were analyzed by liquid chromatography–tandem mass spectrometry. 6βOHBUD and BUD concentrations were very low after INT treatment (0.0–7.1 and 0.0–8.1 ng/mL, respectively), and higher after INH treatments (0.0–35.4 and 0.0–48.3 ng/mL, respectively). For 16αOHPRED, elevated concentrations were detected after INT and INH treatments (2.6–66.4 and 3.4–426.5 ng/mL, respectively). Concentrations obtained following oral administration were higher than after therapeutic administrations (2.8–80.6, 1.5–36.1, and 10.4–532.2 ng/mL for 6βOHBUD, BUD, and 16αOHPRED, respectively). After all administrations, concentrations were higher in males than in females. Results demonstrated that 6βOHBUD is the best discriminatory marker and a reporting level of 40 ng/mL was found to be the best criterion to distinguish allowed from forbidden administrations of BUD.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

Advances in the detection of growth hormone releasing hormone synthetic analogs

The administration of growth hormone releasing hormone (GHRH) and its synthetic analogs is prohibited by the World Anti-Doping Agency (WADA). Although there is evidence of their use, based on admissions and intelligence, they do not appear to have been found in anti-doping samples by WADA accredited laboratories. This might be due to their small concentration in urine and limited knowledge about their metabolism, especially for unapproved synthetic analogs. This study investigates the in vitro metabolism and detection of four of the larger GHRH synthetic analogs (sermorelin, tesamorelin, CJC-1295, and CJC-1295 with drug affinity complex) in fortified urine. Nineteen major in vitro metabolites were identified, selected for synthesis, purified, and characterized in house. These were used as reference materials to spike into urine together with commercially available parent peptides and a metabolite of sermorelin (sermorelin(3-29)-NH₂) to develop a sensitive liquid chromatography-tandem mass spectrometry method for their detection to help prove GHRH administration. Limits of detection of the target peptides were generally 1 ng/ml (WADA required performance limit) or less.     

Study of the in vitro and in vivo metabolism of the tryptamine 5‐MeO‐MiPT using human liver microsomes and real case samples

The synthetic tryptamine 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (5‐MeO‐MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5‐MeO‐MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5‐MeO‐MiPT using liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC–MS), LC‐HRMS/MS, and nuclear magnetic resonance (NMR). 5‐MeO‐MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5‐MeO‐MiPT in urine. Seven different in vitro phase I metabolites of 5‐MeO‐MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5‐MeO‐MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5‐methoxy‐N‐isopropyltryptamine (5‐MeO‐NiPT), 5‐hydroxy‐N‐methyl‐N‐isopropyltryptamine (5‐OH‐MiPT), 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine‐N‐oxide (5‐MeO‐MiPT‐N‐oxide), and hydroxy‐5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (OH‐5‐MeO‐MiPT) as biomarkers for the development of new methods for the detection of 5‐MeO‐MiPT consumption, as they have been present in both blood and urine samples.     

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction–tandem mass spectrometry to detect hCG use

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti‐Doping Agency (WADA) list of prohibited substances. The majority of WADA‐accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography–tandem mass spectrometry (LC–MS/MS). In this study we measured the concentration of intact hCG, free β‐subunit (hCGβ) and β‐subunit core fragment (hCGβcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC–MS/MS method. Mean concentrations of intact hCG, hCGβ and hCGβcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5^th percentile) for intact hCG, hCGβ and hCGβcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.     

Flubromazolam – Basic pharmacokinetic evaluation of a highly potent designer benzodiazepine

Since their first appearance on the Internet in 2012, designer benzodiazepines established as an additional, quickly growing compound class among new psychoactive substances. Data regarding pharmacokinetic parameters, metabolism, and detectability for new compounds are limited or often not available. One of these compounds, flubromazolam (8‐bromo‐6‐(2‐fluorophenyl)‐1‐methyl‐4H‐[1,2,4]triazolo[4,3‐a][1,4]benzodiazepine), the triazolo‐analogue of flubromazepam, has been offered on the Internet from 2014 on. The purpose of the present study was to assess the period of detectability in biological samples along with preliminary basic pharmacokinetic parameters of the designer benzodiazepine flubromazolam. To investigate these, one of the authors ingested a capsule containing 0.5 mg of the drug. Metabolism studies and suitability tests for the detection with immunochemical assays were performed with the samples obtained from the self‐experiment and five authentic case samples. Flubromazolam and its mono‐hydroxylated metabolite were detectable by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in urine for up to 6.5 and 8 days, respectively (lower limit of quantification (LLOQ) flubromazolam: 0.1 ng/mL). Peak serum concentrations were as low as 8 ng/mL (8 h post ingestion). Glucuronides were also detected. The terminal elimination half‐life could be estimated in the range of 10–20 h. Immunochemical assays yielded negative results for serum samples and positive results for urine samples for up to five days post ingestion. The presented data demonstrate the detectability of a single uptake of 0.5 mg of flubromazolam in hair samples collected two weeks after drug uptake by LC–MS³ (c_max 0.6 pg/mg; LOD 0.01 pg/mg). The detected metabolites were in good agreement with those described in other studies. Copyright © 2017 John Wiley & Sons, Ltd.     

The development and validation of a method for quantifying olanzapine in human plasma by liquid chromatography tandem mass spectrometry and its application in a pharmacokinetic study

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Intracerebral microdialysis coupled to LC–MS/MS for the determination tramadol and its major pharmacologically active metabolite O‐desmethyltramadol in rat brain microdialysates

A rapid and sensitive method involving liquid chromatography electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) coupled to an intracerebral microdialysis technique was developed for the determination and pharmacokinetic investigation of tramadol and its major active metabolite O ‐desmethyltramadol (ODT) in rat brain. The microdialysis samples were separated on a C18 column and eluted with a mobile phase of acetonitrile‐water‐formic acid (50:50:0.1; v/v/v ) at a flow rate of 0.3 mL/min. The ESI‐MS/MS spectra were performed in electrospray positive ion mode, and the analytes were detected by multiple reaction monitoring (MRM) of the transitions m/z [M + H]⁺ 264.3 → 58.2 for tramadol, m/z [M + H]⁺ 250.3 → 58.3 for ODT, and m/z [M + H]⁺ 379.4 → 264.0 for ambroxol (internal standard; IS). The total run time was 4.0 min. A lower limit of quantitation (LLOQ) was achieved as 1 ng/mL for tramadol and 0.5 ng/mL for ODT, with excellent linearity over a concentration range of 1 ~ 500 ng/mL (r  > 0.99) for tramadol and 0.5 ~ 50 ng/mL for ODT (r  > 0.99), respectively. The proposed method was successfully applied to the pharmacokinetic studies of tramadol and ODT in rat brain. Copyright © 2017 John Wiley & Sons, Ltd.     

Enantioselective disposition of (R,R)‐formoterol, (S,S)‐formoterol and their respective glucuronides in urine following single inhaled dosing and application to doping control

Formoterol is a long‐acting beta2‐adrenoceptor agonist (LABA) used for the treatment of asthma and exercise‐induced bronchoconstriction. Formoterol is usually administered as a racemic (rac‐) mixture of (R,R)‐ and (S,S)‐enantiomers. While formoterol is restricted by the World Anti‐Doping Agency (WADA), inhalation of formoterol is permitted to a predetermined dose (54 μg/24 hours) and a urine threshold of 40 ng/mL. However, chiral switch enantiopure (R,R)‐formoterol is available, effectively doubling the therapeutic advantage for the same threshold. The aim of this study was to investigate whether formoterol exhibits enantioselective urinary pharmacokinetics following inhalation. Six healthy volunteers were administered a 12 μg inhaled dose of rac‐formoterol. Urine was collected over 24 hours and analyzed by enantioselective ultra‐performance liquid chromatography?tandem mass spectrometry (UPLC?MS/MS) assay. Total (free drug plus conjugated metabolite) median (min‐max) rac‐formoterol urine levels following inhalation were 1.96 (1.05–13.4) ng/mL, 1.67 (0.16–9.67) ng/mL, 0.45 (0.16–1.51) ng/mL, 0.61 (0.33–0.78) ng/mL, and 0.17 (0.08–1.06) ng/mL at 2, 4, 8, 12, and 24 hours, respectively, well below the 2019 urine threshold. The proportion of conjugation differed between enantiomers with glucuronide conjugation much greater for (R,R)‐formoterol (around 30%–60% of total) compared to (S,S)‐formoterol (0%–30%). There was clear evidence of inter‐individual enantioselectivity observed in the ratios of (R,R):(S,S)‐formoterol, where (S,S)‐ was predominant in free formoterol, and (R,R)‐ predominant in the conjugated metabolite. In conclusion, rac‐formoterol delivered by inhalation exhibits enantioselective elimination in urine following single‐dose administration. Enantioselective assays should be employed in doping control to screen for banned beta2‐agonist chiral switch products such as (R,R)‐formoterol, and total hydrolyzed rac‐formoterol is warranted to account for inter‐individual differences in enantioselective glucuronidation.     

Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC‐C‐IRMS

Prednisone and prednisolone are two anti‐inflammatory steroidal drugs listed by the World Anti‐Doping Agency (WADA) within the class of glucocorticoids, which are prohibited “in competition” and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC‐C‐IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ¹³C value reference range (?28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC‐C‐IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro‐11‐deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC‐C‐IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples.     

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements

Diuretic agents are prohibited in sports in‐ and out‐of‐competition according to the regulations of the World Anti‐Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (μg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA‐accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (μg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ‐contaminated powder (6.4 μg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid–liquid extraction and liquid chromatography–tandem mass spectrometry determination. Post‐administration specimens were found to contain HCTZ at concentrations of 5–230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.     

Determination of GnRH and its synthetic analogues' abuse in doping control: Small bioactive peptide UPLC–MS/MS method extension by addition of in vitro and in vivo metabolism data; evaluation of LH and steroid profile parameter fluctuations as suitable biomarkers

Gonadotropin‐releasing hormone (GnRH) and its small peptide synthetic analogues are included in Section S2 of the World Anti‐Doping Agency (WADA) Prohibited List as they stimulate pituitary luteinizing hormone (LH) and testicular testosterone (T) secretion. Both the following approaches can be applied for determination of abuse of these peptides: direct identification of intact compounds and their metabolites in athletes' biofluids and evaluation of LH and T concentrations as mediate markers of drug intake. To develop an effective concept for GnRH and its analogues determination in anti‐doping control, in vitro and in vivo studies were conducted. A new method was applied to the evaluation of the slow‐release profile of buserelin, goserelin, and leuprolide biodegradable microspheres after the intramuscular injection in male volunteers. Eight metabolites of 10 GnRH analogues were identified after incubation with human kidney microsomes, most of them were leuprolide degradation products. Obtained data were added into ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for GnRH analogues determination. The detection time windows for administered peptides and their metabolites in urine samples were evaluated with 2 sample preparation techniques: dilute‐and‐shoot and solid‐phase extraction. To support the second hypothesis, the measurement of LH and the main parameters of the steroid profile were performed in urine samples. Just 1 compound among those investigated resulted in the LH concentration dropping to non‐physiological levels. Thus, for doping‐control purposes, monitoring of hormone levels fluctuations could be applied only together with longitudinal passport steroid profile data.     

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB‐CHMINACA in an authentic human urine specimen

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB‐CHMINACA, was investigated. Although unchanged MAB‐CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB‐CHMINACA metabolites from a commercial source. The MAB‐CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid‐phase extraction, and analyzed by liquid chromatography–tandem mass spectrometry with or without hydrolysis with β‐glucuronidase. Among the six MAB‐CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β‐glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4‐monohydroxycyclohexylmethyl metabolite M1 (N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide) and a dihydroxyl (4‐hydroxycyclohexylmethyl and tert‐butylhydroxyl) metabolite M11 (N‐(1‐amino‐4‐hydroxy‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB‐CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB‐CHMINACA metabolites in an authentic human urine specimen.     

Ephedra alkaloid contents of Chinese herbal formulae sold in Taiwan

Consumption of Ephedra alkaloids is prohibited in‐competition by the World Anti‐Doping Agency (WADA). In Taiwan, colds are often treated with Chinese herbal formulae containing Herba Ephedrae. We screened products sold in Taiwan and preliminarily assessed their relationships with WADA threshold violations. Fifty‐six concentrated powder products, including 19 Chinese herbal formulae that contained Herba Ephedrae, were collected. The content of Ephedra alkaloids, namely ephedrine (E), methylephedrine (ME), norpseudoephedrine (NPE; cathine), pseudoephedrine (PE), and norephedrine (NE; phenylpropanolamine), was determined using a validated high‐performance liquid chromatography method. The results revealed that the phenotypic indicators of the collected products, E/PE and E/total ratios, were 1.52–4.70 and 0.49–0.72, respectively, indicating that the Herba Ephedrae species in these products was probably E. sinica or E. equisetina, but not E. intermedia. The contents of E, ME, NPE, PE, and NE and the total alkaloid contents in the daily doses of the products were 0.45–34.97, 0.05–4.87, 0.04–3.61, 0.15–12.09, and 0.01–2.00 mg and 0.68–53.64 mg, respectively. The alkaloid contents followed a relatively consistent order (E > PE > ME ≈ NPE > NE), even for products from different manufacturers. We calculated that single doses of 50.0% and 3.6% of the products would result in the WADA thresholds of E and NPE being exceeded, respectively. Our data provide critical information for athletes and medical personnel, who should be wary of using complex Chinese herbal formulae in addition to over‐the‐counter products.     

Metabolism and pharmacokinetics in rats of ganoderiol F,a highly cytotoxic and antitumor triterpene from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

The metabolism of ganoderiol F (GF), a cytotoxic and antitumor triterpene from Ganoderma lucidum, by intestinal bacteria and its pharmacokinetics in rats were investigated by using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). GF was converted to ganodermatriol by anaerobic incubation with bacterial mixtures from rats and humans. This metabolite was detected in rat feces, but not in plasma and urine, after oral administration of GF. The fate of GF after oral (p.o.) and intravenous (i.v.) administration to rats was examined in pharmacokinetics studies. Plasma samples pretreated by solid-phase extraction were quantified by HPLC/MS/MS over a GF concentration range of 1.25–100 ng/ml (S/N = 5). The intra- and interday precision (CV%) was below 8% and accuracy was within the range of 95.9–103.6% for all samples. The range of recovery ratios was 89.2–98.2%. After the administration of GF at 0.5 mg/kg i.v., the plasma concentrations of GF quickly declined and the elimination half-life values (t _1/2α and t _1/2β) were about 2.4 and 34.8 min. On the other hand, the elimination half-life values (t _1/2α) after p.o. administration of GF at doses of 20 and 50 mg/kg were 14.4 and 143.3 min for the former, and 18.6 and 114.6 min for the latter. The AUC_0–t value was 11.17 (ng/ml) h at a GF dose of 0.5 mg/kg i.v., but 49.4 and 111.6 (ng/ml) h at GF doses of 20 and 50 mg/kg p.o., respectively, indicating that the AUC_0–t value is proportional to the administered oral doses. The estimated absolute bioavailability of GF in rats was F = 0.105.     

Doping control analysis of four JWH‐250 metabolites in equine urine by liquid chromatography–tandem mass spectrometry

JWH‐250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH‐250 metabolites (JWH‐250 4‐OH‐pentyl, JWH‐250 5‐OH‐pentyl, JWH‐250 5‐OH‐indole, and JWH‐250 N‐pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The chromatographic separation was carried out using a Phenomenex Lux^® 3 μm AMP column (150 x 3.0 mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50 pg/mL, respectively. The linear dynamic range of quantification was 50–10000 pg/mL. Enzymatic hydrolysis indicated that JWH‐250 4‐OH‐pentyl, JWH‐250 5‐OH‐pentyl, and JWH‐250 5‐OH indole are highly conjugated whereas JWH‐250 N‐pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post‐race urine samples collected from horses suspected of being exposed to JWH‐250. All four JWH‐250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.