Automated sample preparation for the detection and confirmation of hypoxia-inducible factor stabilizers in urine.

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation.

Efficacy of DRIVEN-FLOW® M7-II, a new on-site drug screening kit in postmortem urine compared with Triage DOA®.

The efficacy of DRIVEN-FLOW® M7-II(DFM7II) for seven drug groups was compared with Triage DOA® (Triage) using 340 autopsy urine samples taken from bodies within 1 month of death based on mass screening analysis of GC/MS and LC-MS/MS. The sensitivity to benzodiazepines was 0.56 in Triage and 0.53 in DFM7II with few false positives, and their accuracy was 0.88. Triage detected triazolo diazepine derivatives more easily than DFM7II. DFM7II detected diazepam and nitro benzodiazepines more easily than Triage. There were nine amphetamine false-positive cases of more than 10 days after death in Triage, but these were absent in DFM7II during this period. The accuracy of amphetamines for Triage was 0.96 and for DFM7II was 1. Tricyclic antidepressant (TCA) was detected in five cases by mass analysis, while there were four false-positive cases using Triage and eight cases using DFM7II. In the TCA false-positive cases of both kits, tricyclic psychotics such as chlorpromazine, carbamazepine, and quetiapine were included as well as the drug poisoning cases. There were no samples containing cocaine or THC. The accuracy of DFM7II for opiate and barbiturates was 1, but those of Triage was less than 1. Based on the above, DFM7II is a more accurate kit with fewer false-positives for target drug groups, other than TCA, than Triage.

Simultaneous voltammetric determination of rizatriptan and acetaminophen using a carbon paste electrode modified with NiFe2O4 nanoparticles.

Nickel-ferrite nanoparticles (NiFe2O4) were synthesized by a hydrothermal method. They were used to modify a carbon paste electrode (CPE) and to prepare an electrochemical sensor for simultaneous determination of rizatriptan benzoate (RZB) and acetaminophen (AC). The structure and morphology of the bare CPE and modified CPE were studied using field emission scanning electron microscopy, while the structural characterization of NiFe2O4 was performed via X-ray diffraction. In the potential range 0.2-1.2 V, AC and RZB were detected at potentials of 0.5 V and 0.88 V (vs. Ag/AgCl saturated KCl 3 M), respectively. Both calibration plots are linear in the 1 to 90 µM concentration range. The limits of detection (at 3σ) of AC and RZB are 0.49 and 0.44 µM, respectively. The performance of the modified CPE was evaluated by quantifying the two drugs in spiked urine and in tablets. Graphical abstract The modified electrode consist of Nickel-ferrite and graphite by differential pulse voltammetry methods are schematically presented for simultaneous detection of acetaminophen (a painkiller drug) and rizatriptan benzoate (an antimigraine drug) in human urine and tablet samples.

Absorption, distribution, metabolism and excretion of molidustat in healthy participants.

The absorption, distribution, metabolism and excretion of molidustat were investigated in healthy male participants. In study 1, a mass balance study, radiolabelled molidustat 25 mg (3.57 MBq) was administered as an oral solution (n = 4). Following rapid absorption, molidustat-related radioactivity was predominantly distributed in plasma rather than in red blood cells. The total recovery of the administered radioactivity was 97.0%, which was mainly excreted renally (90.7%). Metabolite M-1, produced by N-glucuronidation, was the dominant component in plasma (80.2% of the area under the concentration-time curve for total radioactivity) and was primarily excreted via urine (~85% of dose). Only minor amounts of unchanged molidustat were excreted in urine (~4%) and faeces (~6%). Study 2 investigated the absolute bioavailability and pharmacodynamics of molidustat (part 1, n = 12; part 2, n = 16). Orally administered molidustat immediate release tablets had an absolute bioavailability of 59%. Following intravenous administration (1, 5 and 25 mg), total body clearance of molidustat was 28.7-34.5 L/h and volume of distribution at steady state was 39.3-50.0 L. All doses of molidustat transiently elevated endogenous erythropoietin levels, irrespective of the route of administration. Molidustat was considered safe and well tolerated at the administered doses.

Excretion stereoselectivity of triticonazole in rat urine and faeces.

The purpose of this study was to study the excretion stereoselectivity of triticonazole enantiomers in rat urine and faeces. Six male Sprague-Dawley (SD) rats were administrated 50 mg/kg rac-triticonazole. Rats urine and faeces were separately and quantitatively collected at the following intervals: 0-3, 3-6, 6-9, 9-12, 12-24, 24-36 and 36-48 h. The faeces samples were homogenized in an aqueous solution containing 0.2% DMSO at the ratio of 1 g: 40 mL. An aliquot of 100 µL rats urine or faeces homogenate was spiked and mixed with 6.0 µL of 1.00 µg/mL flusilazole as an internal standard. The triticonazole enantiomers in urine and faeces were determined by using an HPLC/MS-MS after samples preparation. The excreted amounts of enantiomers in the urine showed a significant difference (P < 0.05) except for 3-6 h. The cumulative excretion rate (Xu0→24) in urine was 26.43 ± 0.08% and 37.58 ± 0.11% for R-(-)- and S-(+)-triticonazole, respectively, indicating high enantioselectivity (P < 0.001). The cumulative excretion rate (Xu0→72) in faeces was 6.93 ± 0.03% and 6.77 ± 0.03% for R-(-)- and S-(+)-triticonazole, respectively, without a difference. The results showed that the total cumulative percentage of triticonazole enantiomers accounted for in urine and faeces was 64.00 ± 0.13% and 13.70 ± 0.32%, the urinary excretion of R-(-)- and S-(+)-triticonazole were significantly different and S-(+)-triticonazole was preferentially excreted. However, the faecal excretion of the enantiomers showed no difference.

15N-labelled nitrite/nitrate tracer analysis by LC-MS/MS: Urinary and fecal excretion of nitrite/nitrate following oral administration to mice.

Determination of the modulation of nitrite and nitrate levels in biological samples usually poses a major challenge, owing to their high background concentrations. To effectively investigate the variation of nitrite/nitrate in vivo, in this study, we developed a15N-labelled nitrite/nitrate tracer analysis using LC-MS/MS following the derivatization with 2,3-diaminonaphthalene. This method allows for the determination of 15N-labelled nitrite/nitrate as 15N-2,3-naphthotriazole (15N-NAT) that can efficiently differentiate newly introduced nitrite/nitrate from the background nitrite/nitrate in biological matrices. We also investigated the contribution of background 14N-NAT isotopomers to 15N-NAT, which has long been overlooked in the literature. Our results indicated that the contribution of background 14N-NAT isotopomers to 15N-NAT is significant. Such contribution is constant (~2.2% under positive ion mode and 1.1% under negative ion mode), and does not depend upon the concentration of 14N-NAT or the sample matrix measured. An equation has been therefore developed, for the first time, to correct the contribution of background 14N-NAT isotopomers to 15N-NAT. With the proposed 15N-labelled nitrite/nitrate tracer analysis, the amount and percentage distribution of 15NO2- and 15NO3-, both in urine and feces, after oral administration of 15N-labelled nitrite/nitrate are clearly demonstrated. The excretions of 15NO2- and 15NO3- were significantly increased with the increasing dose implying that the dietary nitrite/nitrate intake is an important source in urine/feces. The present method allows for the simple, reliable and accurate quantification of 15NO2- and 15NO3-, and it should also be useful to trace the biotransformation of nitrite and nitrate in vivo.

Toxicokinetics of a urinary metabolite of tebuconazole following controlled oral and dermal administration in human volunteers.

Tebuconazole (TEB) is a widely used triazole fungicide, but the toxicokinetics of its human metabolites are not fully described. For proper interpretation of biological monitoring data, knowledge on the metabolism and elimination of the compound is required. A human volunteer study was performed with the aim to describe the time courses of urinary excretion after controlled oral and dermal administration of TEB. Six healthy volunteers (three males and three females) received on separate occasions a single oral dose of 1.5 mg of TEB and a single dermal dose of 2.5 mg during 1 h. In addition to a pre-exposure urine sample, complete urine voids were collected over 48 h post-administration. The main metabolite hydroxy-tebuconazole (TEB-OH) was quantified in each urine sample. Peak excretion rates after oral and dermal administration were reached after 1.4 and 21 h, mean elimination half-lives were 7.8 and 16 h, and recoveries within 48 h were 38% and 1%, respectively. The time courses of excretion were compared to simulations with an established physiologically based toxicokinetic model for TEB that was extended with a parallel model for TEB-OH. Overall, TEB-OH was rapidly excreted into urine after oral exposure, and renal elimination was considerably slower after dermal exposure. Urinary time courses between individuals were similar. The model predictions were in good agreement with the observed time courses of excretion.

Effects of Food and Antacids on Pharmacokinetics and Pharmacodynamics of Lesinurad, a Selective Urate Reabsorption Inhibitor.

Two clinical studies were performed in healthy volunteers to investigate food and antacid effects on lesinurad, a novel selective uric acid reabsorption inhibitor approved for treatment of hyperuricemia associated with gout in combination with xanthine oxidase inhibitors. Study 1 evaluated a high-fat, high-calorie meal or high doses of antacids (3000 mg calcium carbonate or 1600 mg magnesium hydroxide/1600 mg aluminum hydroxide) on the pharmacokinetics (PK) and pharmacodynamics (PD) of 400 mg oral lesinurad. Study 2 evaluated low doses of antacids (1250 mg calcium carbonate or 800 mg magnesium hydroxide/800 mg aluminum hydroxide) on the PK and PD of 400 mg lesinurad. Food did not alter the plasma AUC of lesinurad and only reduced its Cmax by 18%. In the fasted conditions, high-dose calcium carbonate reduced the Cmax and AUC of lesinurad by 54% and 38%, respectively, whereas high-dose magnesium hydroxide/aluminum hydroxide reduced Cmax and AUC by 36% and 31%, respectively. Food enhanced the maximum serum urate (sUA)-lowering effect of lesinurad by approximately 20% despite reducing the Cmax of lesinurad. High-dose calcium carbonate decreased the urate-lowering effect approximately 20% in the first 6 hours, whereas high-dose magnesium hydroxide/aluminum hydroxide reduced the effect by 26%. Low-dose calcium carbonate or magnesium hydroxide/aluminum hydroxide in the presence of food did not significantly affect plasma lesinurad Cmax and AUC or the sUA lowering and renal handling of uric acid. In summary, study results suggest food did not meaningfully alter lesinurad PK and PD. High doses of antacids reduced lesinurad AUC up to 40% and reduced the lesinurad uric acid-lowering effect.

Characterisation of the absorption, distribution, metabolism, excretion and mass balance of doravirine, a non-nucleoside reverse transcriptase inhibitor in humans.

Absorption, distribution, metabolism and elimination of doravirine (MK-1439), a novel non-nucleoside reverse transcriptase inhibitor, were investigated. Two clinical trials were conducted in healthy subjects: an oral single dose [14 C]doravirine (350 mg, ∼200 µCi) trial (n = 6) and an intravenous (IV) single-dose doravirine (100 µg) trial (n = 12). In vitro metabolism, protein binding, apparent permeability and P-glycoprotein (P-gp) transport studies were conducted to complement the clinical trials. Following oral [14 C]doravirine administration, all of the administered dose was recovered. The absorbed dose was eliminated primarily via metabolism. An oxidative metabolite (M9) was the predominant metabolite in excreta and was the primary circulating metabolite (12.9% of circulating radioactivity). Following IV administration, doravirine clearance and volume of distribution were 3.73 L/h (95% confidence intervals (CI) 3.09, 4.49) and 60.5 L (95% CI 53.7, 68.4), respectively. In vitro, doravirine is not highly bound to plasma proteins (unbound fraction 0.24) and has good passive permeability. The metabolite M9 was generated by cytochrome P450 3A (CYP3A)4/5-mediated oxidation. Doravirine was a P-gp substrate but P-gp efflux is not expected to play a significant role in limiting doravirine absorption or to be involved in the elimination of doravirine. In conclusion, doravirine is a low clearance drug, primarily eliminated by CYP3A-mediated metabolism.

UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets.

Suvorexant is a novel sedative/hypnotic drug approved for treatment of insomnia. It has significant forensic importance due to its hypnotic and depressant effects on central nervous system. In this study, a highly sensitive UPLC-MS/MS assay was developed and validated for the determination of suvorexant in urine sample. A simplified dispersive liquid-liquid microextraction followed by ultrasound assisted back extraction from solidified floating organic droplets was employed for sample preparation. The 20 µL of 1-undecanol and 200 µL of acetonitrile were used as extraction solvent and dispersive solvent, respectively. An ultrasound assisted back extraction step was employed to enable the cleanup procedure compatible with mass spectrometric detection. Acquity CSH™C18 column with mobile phase composition of 15 mM ammonium acetate: acetonitrile: formic acid (15:85:0.1%; v/v/v) were used for chromatographic separation. The multiple reaction monitoring transition of 451.12 →104.01 and 451.12→186.04 were used for identification and quantification of suvorexant, respectively, whereas 237.06→194.1 was used for IS in positive mode. The assay demonstrated good linearity in the range of 0.27-1000 ng mL-1 with limit of detection (LOD) and quantification (LOQ) of 0.10 and 0.27 ng mL-1, respectively. Assay validation was performed by following SWGTOX guidelines and all validation results were found to be within acceptable limits. This is the first report of dispersive liquid-liquid microextraction based on solidification of floating organic droplets employed to UPLC-MS/MS for application in biological fluids.

A sensitive and accurate vortex-assisted liquid-liquid microextraction-gas chromatography-mass spectrometry method for urinary triazoles.

The development and validation of an innovative, simple, rapid, selective and sensitive vortex-assisted liquid-liquid microextraction-gas chromatography- mass spectrometry (VALLME-GC/MS) method to seven triazole fungicides in urine is reported. The confidence parameters were exhaustive evaluated and linearity (r > 0.99), precision (below 15%, to quality controls, and 20%, to lower limit of quantification), accuracy and robustness were adequate for the analysis of cyproconazole, epoxiconazole, metconazole, tebuconazole, triadimenol, propiconazole and triadimefon. To assess the applicability of the method, analyses were performed in volunteers exposed to triazoles in the last 30 days. The method demonstrated satisfactory analytical performance, with sufficient detectability to be applied as a potential tool to biomonitoring individuals exposed to the seven evaluated fungicides.

Profiles, variability, and predictors of urinary benzotriazoles and benzothiazoles in pregnant women from Wuhan, China.

BACKGROUND: Benzotriazoles (BTRs) and benzothiazoles (BTHs) are emerging contaminants with high production volume worldwide, which exhibit potential health risk to human. To date, little is known about the exposure of BTRs and BTHs (BTs) on human, especially in the context of pregnancy. OBJECTIVES: We aimed to characterize the exposure profiles, temporal variability, and potential predictors of urinary BTs during pregnancy. METHODS: Between 2014 and 2015, we recruited 856 pregnant women in Wuhan who provided urine samples at three trimesters (13.1 ±â€¯1.1, 23.7 ±â€¯3.2, and 35.7 ±â€¯3.4 gestational weeks). We measured the urinary concentrations of five BTRs (1­H­benzotriazole, 1­hydroxy­benzotriazole, xylyltriazole, tolyltriazole, 5­chloro­1­H­benzotriazole) and five BTHs (benzothiazole, 2­hydroxy­benzothiazole, 2­methylthio­benzothiazole, 2­amino­benzothiazole, 2­thiocyanomethylthio­benzothiazole) to characterize the exposure profiles of BTs. We calculated the intra-class correlation coefficients (ICCs) to assess the temporal variability and investigated potential predictors of urinary BTs by using the mixed models. RESULTS: Most of the targeted BTs were detected in over 50% of urine samples, except for 5­chloro­1­H­benzotriazole (9.3%) and 2­thiocyanomethylthio-benzothiazole (1.4%). The predominant BTRs in urine was 1­hydroxy­benzotriazole [Geometric Mean (GM): 0.77 ng/mL]. Benzothiazole was the major derivative in urine samples with a GM concentration of 1.6 ng/mL. Correlations among BTHs (r = 0.04-0.39) were higher than that among BTRs (r = 0.02-0.14). The exposure pattern was constant at low level and co-exposure to all the targeted compounds was infrequent during pregnancy. Urinary concentrations of BTRs exhibited considerable within-subject variation (ICCs: 0.12-0.56) during pregnancy. Relatively high temporal reliability was observed for urinary concentrations of BTHs with ICCs ranging from 0.42 to 0.85. It was found that parity, household income, pregnancy occupational status, sampling season and menstrual cycle were associated with urinary concentrations of BTs in pregnant women (P < 0.05). CONCLUSIONS: To the best of our knowledge, this is the first study to report the exposure profiles, variability and predictors of urinary BTs among pregnant women. Exposure assessment using multiple samples is essential in reducing measurement errors and identifying susceptible window of exposure in etiological studies. The potential predictors of urinary BTs raised concerns on tracing exposure routes and eliminating confounding variables in future studies.

Urine collection methods for non-toilet-trained children in biological monitoring studies: Validation of a disposable diaper for characterization of tebuconazole exposure.

Young children differ from adults in their exposure and susceptibility to environmental chemicals (e.g. pesticides) because of various factors such as behavior, diet and physiology. Their heightened vulnerability to environmental stressors makes it important to obtain appropriate urine samples for exposure characterization. However, collecting urine from non-toilet-trained children has been shown to be methodologically and practically challenging. Four urine collection approaches were tested: a disposable diaper, a urine bag, a collection pad and the clean catch. The success rate and the user rating of each method was evaluated. The success rates were 67%, 21%, 17% and 4% for the disposable diaper, urine bag, collection pad and clean catch, respectively. The average user ratings on a 0-10 (0 = inconvenient, 10 = convenient) scale were 9.0, 4.7, 7.3 and 2.5, respectively. Subsequently, the best rated method, the disposable polyacrylate diaper was tested with hydroxy-tebuconazole as an exposure biomarker for the fungicide tebuconazole and creatinine for urine density adjustment. After LC-MS/MS analysis, the recoveries of hydroxy-tebuconazole in the range of 0.05-25 ng/mL were on average 106%, and for creatinine 87%. Precisions (relative standard deviation) were for both 3%. The overall procedure including collection and extraction was assessed, resulting in three out of seven positive samples. Based on this study, the disposable diaper is a suitable method for urine collection of non-toilet-trained children for biomonitoring of tebuconazole. This method can serve as a basis for extension to other substances of interest.

Benzotriazoles and benzothiazoles in paired maternal urine and amniotic fluid samples from Tianjin, China.

Benzotriazoles (BTRs) and benzothiazoles (BTHs) are two groups of heterocyclic compounds that are widely detected in the environment. In this study, the levels of BTRs and BTHs in 79 paired maternal urine and amniotic fluid samples from Tianjin were investigated. BTRs were detected in most maternal urine samples, with a median concentration of ∑BTRs of 0.88 ng/mL. BTH was detected in all maternal urine samples, with a median concentration of 1.35 ng/mL. Tolyltriazole (TTR, i.e., the sum of 4-methyl-1H-benzotriazole and 5-methyl-1H-benzotriazole) and BTH were detected in amniotic fluid with detection rates (DRs) > 50% and median concentrations of 0.026 and 0.61 ng/mL, respectively. The median concentrations of ∑BTRs and ∑BTHs (0.026 and 0.72 ng/mL) in amniotic fluid were lower than those in maternal urine. The median ratio of the ∑BTRs concentrations in amniotic fluid to those in maternal urine was 0.030, with a range of 0.017-1.82, while the median value for TTR, BTH and 5-Cl-1H-BTR were 0.12, 0.46, and 1.43, respectively. This indicates greater distribution in fetal excretion to 5-Cl-1H-BTR than BTH and TTR. The concentrations of ∑BTRs in maternal urine exhibited significant distribution differences (p < 0.05) with respect to some parameters, including maternal age, gestational week, gravidity, parity, and fetal weight. However, no significant correlations (p > 0.05) were observed in target compounds in amniotic fluid for the epidemiological factors assessed herein. The geometric means of the estimated daily intakes were 1.15 (0.052-7.66) µg/day and 1.92 (0.027-6.64) µg/day for ∑BTRs and ∑BTHs in present study, which are lower than those reported in previous study.

Determination of benzotriazoles and benzothiazoles in human urine by UHPLC-TQMS.

Benzotriazole (BTR) and benzothiazole (BTH) derivatives are extensively applied in industrial processes and consumer products, and are thus frequently detected in the environmental matrices. Due to their potential estrogenic effects reported in animal studies, the assessment of human exposure to BTRs and BTHs is important. In this study, a method was developed and validated to determine six BTRs and five BTHs in human urine by using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-TQMS) in positive electrospray ionization mode. After de-conjugation by ß-glucuronidase, urine samples were liquid-liquid extracted for the measurement of total concentrations of BTRs and BTHs. The linearity, detection limit, precision, recovery, accuracy and matrix effect were evaluated. The limits of detection were less than 0.5ng/mL. The validated method demonstrated good precision (RSD%<15%), linearity (R2>0.99), recovery (>80%) and accuracy (80-100%). The method was successfully applied for the analysis of 22 human urine samples. Four out of eleven targeted compounds were detected in more than half of participants at ng/mL levels.

Evaluation of single and multiple doses of a novel mGlu2 agonist, a potential antipsychotic therapy, in healthy subjects.

AIMS: The safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of single and multiple doses of a novel mGlu2 agonist were assessed in healthy males. METHODS: In two, Phase 1 investigator- and subject-blind, placebo-controlled studies, oral doses of prodrug LY2979165 were evaluated: single doses (20-150 mg, N = 30) and multiple once-daily (QD) doses (20-400 mg; N = 84), using a titration regimen. The plasma and urine PK of LY2979165 and active moiety, 2812223, were measured. Cerebrospinal fluid (CSF) was collected to determine PK and neurotransmitter levels. Safety parameters were assessed throughout. RESULTS: Nausea and vomiting were dose limiting following single doses; dose titration allowed higher doses to be tested over 14 days. The most common adverse events related to LY2979165 were dizziness, vomiting, nausea, somnolence and headache. The plasma PK of 2812223 were approximately linear with minimal accumulation with QD dosing. Conversion of LY2979165 to 2812223 was extensive, with minimal LY2979165 measurable in plasma. There was no effect of food on the PK of LY2979165 and 2812223. After 60 mg LY2979165 single-dose, 2812223 exposure in CSF was approximately 2-6% and plasma exposure and peak concentrations were approximately four-fold higher than the mGlu2 agonist in vitro EC50 value. No consistent effects were observed on CSF neurotransmitter levels. CONCLUSIONS: Oral doses of LY2979165 up to 60 mg as a single dose and up to 400 mg given as multiple QD doses, using a titration regimen, were well tolerated with linear PK. Overall, these data support further clinical evaluation of LY2979165.

Magnetic iron oxide nanoparticles grafted N-isopropylacrylamide/chitosan copolymer for the extraction and determination of letrozole in human biological samples.

Magnetic iron oxide nanoparticles are used for the extraction of a drug from an aqueous solution. In the current study, the magnetic iron oxide nanoparticles were synthesized via a facile coprecipitation approach, and then modified by (3-mercaptopropyl)trimethoxysilane followed by grafting thermosensitive polymer N-isopropylacrylamide and biopolymer chitosan. Structure, morphology, size, thermal resistance, specific surface area, and magnetic properties of the grafted nanosorbent were characterized by using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, elemental analysis, thermogravimetric analysis, specific surface area analysis and vibrating sample magnetometry. The effective parameters on sorption/desorption of letrozole on grafted magnetic nanosorbent were evaluated. The best sorption of letrozole via the grafted nanosorbent occurred at 20°C at an optimum pH of 7. The extraction of trace letrozole in human biological fluids is investigated and revealed 89.1 and 97.8% recovery in plasma and urine, respectively.

Mass spectrometric characterization of the hypoxia-inducible factor (HIF) stabilizer drug candidate BAY 85-3934 (molidustat) and its glucuronidated metabolite BAY-348, and their implementation into routine doping controls.

The development of new therapeutics potentially exhibiting performance-enhancing properties implicates the risk of their misuse by athletes in amateur and elite sports. Such drugs necessitate preventive anti-doping research for consideration in sports drug testing programmes. Hypoxia-inducible factor (HIF) stabilizers represent an emerging class of therapeutics that allows for increasing erythropoiesis in patients. BAY 85-3934 is a novel HIF stabilizer, which is currently undergoing phase-2 clinical trials. Consequently, the comprehensive characterization of BAY 85-3934 and human urinary metabolites as well as the implementation of these analytes into routine doping controls is of great importance. The mass spectrometric behaviour of the HIF stabilizer drug candidate BAY 85-3934 and a glucuronidated metabolite (BAY-348) were characterized by electrospray ionization-(tandem) mass spectrometry (ESI-MS(/MS)) and multiple-stage mass spectrometry (MSn ). Subsequently, two different laboratories established different analytical approaches (one each) enabling urine sample analyses by employing either direct urine injection or solid-phase extraction. The methods were cross-validated for the metabolite BAY-348 that is expected to represent an appropriate target analyte for human urine analysis. Two test methods allowing for the detection of BAY-348 in human urine were applied and cross-validated concerning the validation parameters specificity, linearity, lower limit of detection (LLOD; 1-5 ng/mL), ion suppression/enhancement (up to 78%), intra- and inter-day precision (3-21%), recovery (29-48%), and carryover. By means of ten spiked test urine samples sent blinded to one of the participating laboratories, the fitness-for-purpose of both assays was provided as all specimens were correctly identified applying both testing methods. As no post-administration study samples were available, analyses of authentic urine specimens remain desirable. Copyright © 2016 John Wiley & Sons, Ltd.

Quantification of suvorexant in urine using gas chromatography/mass spectrometry.

Suvorexant (Belsomra®) is a novel sedative hypnotic drug that is prescribed to promote sleep in patients with insomnia. It is the first of a new class of drugs classified as dual orexin receptor antagonists (DORAs). Sedative hypnotics with central nervous system depressant effects feature prominently in forensic toxicology investigations. For this reason, a new analytical method was developed to identify suvorexant in urine using liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). Due to the absence of a commercially available isotopically labeled internal standard, estazolam-D5 was used due to its azepine, triazole and chlorinated functionality. The limit of detection and limit of quantitation was 10ng/mL and the linear range of the assay was 10-1000ng/mL. Accuracy and precision (%CV) were 98-101% and <11% at 30, 250 and 800ng/mL. Interferences from matrix and fifty common drugs were not present and processed samples were stable for 24h at room temperature. Suvorexant is a new drug of significant forensic interest due to its hypnotic and central nervous system depressant effects. The absence of commercially available metabolites and its chromatographic properties present some challenges in terms of identification. Nevertheless, a robust, reliable and sensitive assay was developed to identify suvorexant using GC/MS analysis.

Identification of Suvorexant in Urine Using Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry (LC-Q/TOF-MS).

Suvorexant (Belsomra®) is a new hypnotic drug with a novel mechanism of action. In prescribed doses of 10 mg before bedtime, the drug produces rapid onset of sleep by inhibiting the orexin neurons of the arousal system, promoting decreased wakefulness. Suvorexant is a potent and highly selective dual orexin receptor antagonist. Sedative hypnotics are of forensic importance due to their widespread use, potential for additive effects with other central nervous system depressants, impairing effects and potential for misuse. In this report we describe a highly sensitive assay for the identification and quantification of suvorexant in urine. Suvorexant was isolated using liquid/liquid extraction (LLE) and identified using liquid chromatography-quadrupole/time-of-flight mass spectrometry. Suvorexant was quantified using a quadratic calibration model between 5 and 250 ng/mL (R2 = 1.000, n = 6). Processed sample stability was demonstrated for up to 24 h. The limit of detection was 0.5 ng/mL and the limit of quantification (LOQ) was 5 ng/mL. The accuracy, bias and precision of the assay at the LOQ were 99% (81-117%), -1% and 12% (n = 18). Intraassay (n = 5) and interassay (n = 15) precision (% CV) at 10, 50 and 200 ng/mL were ≤8%, and bias ranged from -2% to 4% (98-104% accuracy). No qualitative interferences were detected from matrix, internal standard or 50 common drugs. Matrix effects evaluated at low and high concentrations were -16% and -9%, respectively, and produced CVs of 11% and 5% (n = 20). Suvorexant is a new drug of forensic importance. In this report we describe how a simple acidic/neutral LLE can be used to isolate this lipophilic drug with high recoveries and sound analytical performance.