Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC‐MS/MS and enzyme‐alkaline hydrolysis

A high performance liquid chromatography coupled to tandem mass spectrometry (HPLC‐MS/MS) method for simultaneous quantification of Δ9‐tetrahydrocannabinol (THC), its two metabolites 11‐hydroxy‐Δ9‐tetrahydrocannabinol (11‐OH‐THC) and 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol (THC‐COOH), and four additional cannabinoids (cannabidiol (CBD), cannabigerol (CBG), tetrahydrocannabivarin (THCV), and cannabinol (CBN)) in 1 mL of human urine and plasma was developed and validated. The hydrolysis process was studied to ensure complete hydrolysis of glucuronide conjugates and the extraction of a total amount of analytes. Initially, urine and plasma blank samples were spiked with THC‐COOH‐glucuronide and THC‐glucuronide, and four different pretreatment methods were compared: hydrolysis‐free method, enzymatic hydrolysis with Escherichia Coli β‐glucuronidase, alkaline hydrolysis with 10 M NaOH, and enzyme‐alkaline tandem hydrolysis. The last approach assured the maximum efficiencies (close to 100%) for both urine and plasma matrices. Regarding the figures of merit, the limits of detection were below 1 ng/mL for all analytes, the accuracy ranged from 84% to 115%, and both within‐day and between‐day precision were lower than 12%. Finally, the method was successfully applied to real urine and plasma samples from cannabis users. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of a selection of synthetic cannabinoids and metabolites in urine by UHPSFC‐MS/MS and by UHPLC‐MS/MS

Two different analytical techniques, ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MS/MS) and reversed phase ultra‐high performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS), were used for the determination of two synthetic cannabinoids and eleven metabolites in urine; AM‐2201 N‐4‐OH‐pentyl, AM‐2233, JWH‐018 N‐5‐OH‐pentyl, JWH‐018 N‐pentanoic acid, JWH‐073 N‐4‐OH‐butyl, JWH‐073 N‐butanoic acid, JWH‐122 N‐5‐OH‐pentyl, MAM‐2201, MAM‐2201 N‐4‐OH‐pentyl, RCS‐4 N‐5‐OH‐pentyl, UR‐144 degradant N‐pentanoic acid, UR‐144 N‐4‐OH‐pentyl, and UR‐144 N‐pentanoic acid. Sample preparation included a liquid‐liquid extraction after deconjugation with ß‐glucuronidase. The UHPSFC‐MS/MS method used an Acquity UPC^{2 TM} BEH column with a mobile phase consisting of CO₂ and 0.3% ammonia in methanol, while the UHPLC‐MS/MS method used an Acquity UPLC® BEH C₁₈ column with a mobile phase consisting of 5 mM ammonium formate (pH 10.2) and methanol. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions. Deuterated internal standards were used for six of the compounds. Limits of quantification (LOQs) were between 0.04 and 0.4 µg/L. Between‐day relative standard deviations at concentrations ≥ LOQ were ≤20%, with biases within ±19%. Recoveries ranged from 40 to 90%. Corrected matrix effects were within 100 ± 10%, except for MAM‐2201 with UHPSFC‐MS/MS, and for UR‐144 N‐pentanoic acid and MAM‐2201 N‐4‐OH‐pentyl with UHPLC‐MS/MS. Elution order obtained by UHPSFC‐MS/MS was almost opposite to that obtained by UHPLC‐MS/MS, making this instrument setup an interesting combination for screening and confirmation analyses in forensic cases. The UHPLC‐MS/MS method has, since August 2014, been successfully used for confirmation of synthetic cannabinoids in urine samples revealing a positive immunoassay screening result. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of GHB in human hair by HPLC‐MS/MS: Development and validation of a method and application to a study group and three possible single exposure cases

Gamma‐hydroxybutyrate (GHB) over the last two decades has generated increased notoriety as a euphoric and disinhibiting drug of abuse in cases of drug‐related sexual assault and for this reason it is considered a ‘date rape’ drug. The first aim of this paper was to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) after liquid‐liquid extraction (LLE). The second aim was the application of the method to hair samples of 30 GHB‐free users in order to determine the basal level. The results obtained showed no significant differences in endogenous concentrations (p = 0.556) between hair samples of the three groups (black, blonde, and dyed hair) and the age and sex of the subjects did not affect the endogenous levels. Another 12 healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) was orally administered to all of them and hair samples were collected before the administration of the single dose and other two samples were collected one month and two months later, respectively. The segmental analysis of the latter two samples allowed us to calculate two ratios: 4.45:1 (95% C.I. 3.52–5.63) and 3.35:1 (95% C.I. 2.14–5.18), respectively, which can be recommended as reasonable values for a positive identification of GHB intake. Finally the method was applied to three real cases where a GHB single exposure probably occurred. Copyright © 2014 John Wiley & Sons, Ltd.     

UPLC‐PDA‐TOF/MS coupled with multivariate statistical analysis to rapidly analyze and evaluate Ginkgo biloba leaves from different origin

In the present study, an ultra performance liquid chromatography coupled with photodiode array detector and time‐of‐flight mass spectrometry (UPLC‐PDA‐TOF/MS) was proposed and validated for rapidly analyzing and evaluating Ginkgo biloba leaves from different origins by using multivariate statistical analysis. Batches of these kinds of G. biloba leaves were subjected to UPLC‐PDA‐TOF/MS analysis, the datasets of retention time (RT)‐m/z pairs, ion intensities and sample codes were further processed with orthogonal partial least squared discriminant analysis (OPLS‐DA) to holistically compare the difference between these G. biloba leaves, and to generate an S‐plot. Those compounds correlated to the points at the two ends of S were regarded as the most differentiating components. By comparing the mass/UV spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae with those of the known compounds published in the literatures, these differentiating components were finally characterized as kaempferol 3‐O‐[2‐O‐(6‐O‐p‐hydroxy‐trans‐cinnamoyl)‐β‐D‐glucosyl)‐α‐L‐rhamnoside], kaempferol 3‐O‐[2‐O,6‐O‐bis(α‐L‐rhamnosyl)‐β‐D‐glucoside], ginkgolide C, kaempferol 3‐O‐[2‐O‐(β‐D‐glucosyl)‐α‐L‐rhamnoside], and bilobetin. These compounds would be the potential chemical markers for the two kinds of leaves. The results suggested that this newly established approach could be used to rapidly evaluate the quality of herbs from different origin, and to provide good strategy for further rectify and standardize the herb market. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantification of THC‐COOH,OH‐THC,and further cannabinoids in human hair by gas chromatography–tandem mass spectrometry with electron ionization applying automated sample preparation

The detection of Δ⁹‐tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair, for the purpose of identifying cannabis consumption, is conducted in many forensic laboratories. Since external contamination of hair with these cannabis components cannot be excluded, even after hair decontamination, only the detection of THC metabolites such as 11‐nor‐9‐carboxy‐Δ⁹‐tetrahydrocannabinol (THC‐COOH) or 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (OH‐THC), is considered to prove cannabis consumption. At present, testing for THC metabolites is not standard practice due to its analytical complexity. For these reasons, we developed a novel method for the detection of THC‐COOH and OH‐THC as well as THC, CBD, and CBN in one single analytical run using gas chromatography–tandem mass spectrometry (GC–MS/MS) with electron ionization. After manual hair washing and grinding, sample preparation was fully automated, by means of a robotic autosampler. The hair extraction took place by digestion with sodium hydroxide. A solid‐phase extraction (SPE) was chosen for sample clean‐up, using a mixed‐mode anion exchange sorbent. Derivatization of all analytes was by silylation. The method has been fully validated according to guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh), with a limit of detection (LOD) of 0.2 pg/mg for THC‐COOH and OH‐THC and 2 pg/mg for THC, CBD and CBN, respectively, thus fulfilling the Society of Hair Testing (SoHT) recommendations. The validated method has been successfully applied to our routine forensic case work and a summary of data from authentic hair samples is given, as well as data from proficiency tests.     

A validated,sensitive HPLC‐MS/MS method for quantification of cis‐para‐methyl‐4‐methylaminorex (cis‐4,4'‐DMAR) in rat and human plasma: application to pharmacokinetic studies in rats

4,4′‐DMAR is an analogue of the known psychostimulants 4‐methylaminorex and aminorex. In the light of reports of deaths associated with its abuse, and the easy access from Internet vendors, the EU Council recently decided on control measures across member states. Here we describe a validated method for measuring plasma levels of cis ‐4,4′‐DMAR, crucial for preclinical studies and analysis in human plasma. Chromatographic separation was done by gradient elution on a Kinetex C18 column with 0.1% formic acid in water and 0.1% formic acid in acetonitrile at 0.2 mL/min. Detection was by positive electrospray ionization (ESI+) in multiple reaction monitoring mode monitoring the quantifier transitions m /z 191.4 → m /z 148.3 for cis ‐4,4'‐DMAR and m /z 259.3 → m /z 194.2 for carbamazepine (internal standard). Protein precipitation with 1% of formic acid in acetonitrile was used in cis ‐4,4'‐DMAR extraction from plasma; recovery was high (>93%) with a negligible matrix effect. This method provides an accurate, precise, and sensitive method for cis ‐4,4’‐DMAR quantification in human and rat plasma, following European Medicine Agency guidelines for bioanalytical method validation. Pharmacokinetic studies were conducted in rats. After an intravenous dose of 1 mg/kg, plasma levels declined rapidly (≥80% in 4 h), followed by a slow elimination phase (t_1/2 of 5.14 ± 0.65 h). Absorption was rapid after intraperitoneal injection (t_max = 15 min) with a rapid decline thereafter; C_max and AUC_0‐240min showed dose‐proportionality over the dose range 1–10 mg/kg. This method was successfully applied to investigate pharmacokinetic properties in rats and could be used to quantify cis ‐4,4’‐DMAR levels in human plasma. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of DP‐VPA and its active metabolite,VPA, in human plasma,urine, and feces by UPLC–MS/MS: A clinical pharmacokinetics and excretion study

DP‐VPA is a phospholipid prodrug of valproic acid (VPA) that is developed as a potential treatment for epilepsy. To characterize the pharmacokinetics and excretion of DP‐VPA, four reliable ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) methods were validated for quantitation of DP‐VPA and its metabolite, VPA, in human plasma, urine, and feces. Protein precipitation and solid‐phase extraction (SPE) were used for extraction of C16, C18 homologs of DP‐VPA and VPA, respectively, from plasma. Urine and fecal homogenate involving the three analytes were efficiently prepared by methanol precipitation. The determinations of C16 DP‐VPA, C18 DP‐VPA, and VPA were performed using the positive multiple reaction monitoring (MRM) mode and the negative single ion monitoring (SIM) mode, respectively. The analytes were separated using gradient elution on C8 or phenyl column. Satisfactory results pertaining to selectivity, linearity, matrix effect, accuracy and precision, recovery, stability, dilution integrity, carryover, and incurred sample analysis (ISR) were obtained. The calibration ranges in human plasma were as follows: 0.00200–1.00 μg/mL for C16 DP‐VPA, 0.0100–5.00 μg/mL for C18 DP‐VPA, and 0.0500–20.0 μg/mL for VPA. The linear ranges in urine and fecal homogenate were 0.00500–2.00 μg/mL and 0.00200–0.800 μg/mL for C16 DP‐VPA, 0.00500–2.00 μg/mL and 0.0100–4.00 μg/mL for C18 DP‐VPA, and 0.200–80.0 μg/mL for VPA, respectively. The intra‐ and inter‐batch coefficients of variation in three matrices ranged from 1.7% to 12.4% while the accuracy values ranged from 85.4% to 111.7%. The developed methods were successfully applied to determine pharmacokinetics of DP‐VPA tablet after a single oral dose of 1200 mg in 12 healthy Chinese subjects under fed condition.     

Method development and validation of montelukast in human plasma by HPLC coupled with ESI-MS/MS: application to a bioequivalence study

A simple, sensitive, and specific LC-ESI–MS/MS method for quantification of Montelukast (MO) in human plasma using Montelukast-d₆ (MOD6) as an internal standard (IS) is discussed here. Chromatographic separation was performed on YMC-pack pro C₁₈, 50 x 4.6 mm, S-3 μm column with an isocratic mobile phase composed of 10mM ammonium formate (pH 4.0):acetonitrile (20:80 v/v), at a flow-rate of 0.8 mL min⁻¹. MO and MOD6 were detected with proton adducts at m/z 586.2→568.2 and 592.3→574.2 in multiple reaction monitoring (MRM) positive mode respectively. MO and MOD6 were extracted using acetonitrile as precipitating agent. The method was validated over a linear concentration range of 1.0–800.0 ng mL⁻¹ with correlation coefficient (r²) ≥ 0.9996. The intraday precision and accuracy were within 1.91–7.10 and 98.32–99.17. The inter-day precision and accuracy were within 3.42–4.41% and 98.14–99.27% for MO. Both analytes were found to be stable throughout three freeze-thawing cycles, bench top, and autosampler stability studies. This method was utilized successfully for the analysis of plasma samples following oral administration of MO (5 mg) in 31 healthy Indian male human volunteers under fasting conditions.     

Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA,LC‐MS/MS and western blotting: a comparative study

The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol‐epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC‐MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC‐MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of a selection of anti‐epileptic drugs and two active metabolites in whole blood by reversed phase UPLC‐MS/MS and some examples of application of the method in forensic toxicology cases

Quantitative determination of anti‐epileptic drug concentrations is of great importance in forensic toxicology cases. Although the drugs are not usually abused, they are important post‐mortem cases where the question of both lack of compliance and accidental or deliberate poisoning might be raised. In addition these drugs can be relevant for driving under the influence cases. A reversed phase ultra‐performance liquid chromatography‐tandem mass spectrometry method has been developed for the quantitative analysis of the anti‐epileptic compounds carbamazepine, carbamazepine‐10,11‐epoxide, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, 10‐OH‐carbazepine, phenobarbital, phenytoin, pregabalin, and topiramate in whole blood, using 0.1 mL sample volume with methaqualone as internal standard. Sample preparation was a simple protein precipitation with acetonitrile and methanol. The diluted supernatant was directly injected into the chromatographic system. Separation was performed on an Acquity UPLC® BEH Phenyl column with gradient elution and a mildly alkaline mobile phase. The mass spectrometric detection was performed in positive ion mode, except for phenobarbital, and multiple reaction monitoring was used for drug quantification. The limits of quantification for the different anti‐epileptic drugs varied from 0.064 to 1.26 mg/L in blood, within‐day and day‐to‐day relative standard deviations from 2.2 to 14.7% except for phenobarbital. Between‐day variation for phenobarbital was 20.4% at the concentration level of 3.5 mg/L. The biases for all compounds were within ±17.5%. The recoveries ranged between 85 and 120%. The corrected matrix effects were 88–106% and 84–110% in ante‐mortem and post‐mortem whole blood samples, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Diphenidine,a new psychoactive substance: metabolic fate elucidated with rat urine and human liver preparations and detectability in urine using GC‐MS,LC‐MSn,and LC‐HR‐MSn

Diphenidine is a new psychoactive substance (NPS) sold as a ‘legal high’ since 2013. Case reports from Sweden and Japan demonstrate its current use and the necessity of applying analytical procedures in clinical and forensic toxicology. Therefore, the phase I and II metabolites of diphenidine should be identified and based on these results, the detectability using standard urine screening approaches (SUSAs) be elucidated. Urine samples were collected after administration of diphenidine to rats and analyzed using different sample workup procedures with gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐(high resolution)‐mass spectrometry (LC‐(HR)‐MS). With the same approaches incubates of diphenidine with pooled human liver microsomes (pHLM) and cytosol (pHLC) were analyzed. According to the identified metabolites, the following biotransformation steps were proposed in rats: mono‐ and bis‐hydroxylation at different positions, partly followed by dehydrogenation, N,N‐bis‐dealkylation, and combinations of them followed by glucuronidation and/or methylation of one of the bis‐hydroxy‐aryl groups. Mono‐ and bis‐hydroxylation followed by dehydrogenation could also be detected in pHLM or pHLC. Cytochrome‐P450 (CYP) isozymes CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were all capable of forming the three initial metabolites, namely hydroxy‐aryl, hydroxy‐piperidine, and bis‐hydroxy‐piperidine. In incubations with CYP2D6 hydroxy‐aryl and hydroxy‐piperidine metabolites were detected. After application of a common users’ dose, diphenidine metabolites could be detected in rat urine by the authors’ GC‐MS as well as LC‐MSⁿ SUSA. Copyright © 2016 John Wiley & Sons, Ltd.     

Radiosynthesis and in vivo evaluation of [11C]Ro‐647312: a novel NR1/2B subtype selective NMDA receptor radioligand

[2‐(3,4‐Dihydro‐1H‐isoquinolin‐2‐yl)‐pyridin‐4‐yl]‐dimethylamine, Ro‐647312 ( 1 ) represents a new novel class of NR1/2B subtype selective NMDA ligand. Ro‐647312 has been radiolabelled with carbon‐11 using [¹¹C]methyl triflate from the nor‐methyl compound 2 . The reaction was performed in acetone as solvent using aqueous NaOH as base. Following HPLC purification [¹¹C]Ro‐647312 ([¹¹C]‐ 1 ) was obtained in 6.9–9.2% (n = 3) radiochemical yield decay‐corrected based on starting [¹¹C]CO₂, with specific radioactivity measured at the end of the radiosynthesis ranging from 1.0 to 3.5 Ci/µmol (37–129 GBq/µmol). Radiochemical and chemical purities were assessed as >99 and >95%, respectively. Following i.v. injection of [¹¹C]‐ 1 in rat, the distribution of radioactivity was homogeneous in all brain structures and did not correlate with the known distribution of NR2B subunits. The radioactivity observed in plasma was also higher than any brain structure throughout the time course of the experiment. [¹¹C]‐ 1 does not possess the required properties for imaging NMDA receptors using positron emission tomography. Copyright © 2004 John Wiley & Sons, Ltd.     

Molecular interactions of chlorpyrifos and its environmental degradation products with human sex hormone‐binding globulin: an in silico study

In recent years, there has been a widespread interest and awareness about health issues posed by endocrine‐disrupting chemicals present in the environment. These chemicals, often present in food and many consumer products, can interfere with hormone biosynthesis and metabolism and may result in deviation from normal homeostatic control. Chlorpyrifos (CPF), a major endocrine‐disrupting chemical is used worldwide as an agricultural insecticide against a broad spectrum of insect pests in rice cultivation and to control termites. The insecticide mostly undergoes environmental degradation to chlorpyrifos‐oxon (CPYO), des‐ethyl chlorpyrifos (DEC), 3,5,6‐trichloro‐2‐methoxypyridine (TMP) and 3,5,6‐trichloro‐2‐pyridinol (TCP). Results from several epidemiological studies suggest that exposure to CPF can result in reproductive disorders, including infertility in male and female. Sex hormone‐binding globulin (SHBG) is a circulatory protein that binds sex steroids and is a potential target for endocrine disruptors in the human body. The objective of the present study involved computational approaches to apprehend the mechanism of molecular interaction of CPF and its four degradation products (CPYO, DEC, TMP, TCP) with human SHBG using molecular docking simulation. All five compounds (CPF, CPYO, DEC, TMP, TCP) showed high binding affinity with SHBG; however, the binding affinity values were higher (more negative) for CPF, CPYO, DEC and TMP than for TCP indicating that CPF, CPYO, DEC and TMP formed a tight interaction with SHBG. From the results obtained with the docking analysis, it can be opined that CPF, CPYO, DEC and TMP could possibly act as potential endocrine disruptors for androgen signaling.     

Development and validation of a DESI‐HRMS/MS method for the fast profiling of esomeprazole and its metabolites in rat plasma: a pharmacokinetic study

The advances in pharmaceutical development and drug discovery impose the availability of reliable high‐throughput screening methods for the rapid evaluation of drug metabolism and pharmacokinetic (PK) in biological samples. Here, a desorption electrospray mass spectrometry (DESI‐MS) method has been developed and validated for the PK profiling of esomeprazole and its metabolites (5‐hydroxyomeprazole and omeprazole sulfone) in rat plasma. Rats were treated with an esomeprazole solution (2.5 mg/mL) for endovenous administration and the analyte levels were profiled over 2 h after liquid‐liquid extraction from plasma. MS and tandem mass spectrometry (MS/MS) experiments were performed by using a DESI‐LTQ‐Orbitrap XL instrument and an on‐spot fixed time analysis on PMMA surfaces. Validation was performed for the esomeprazole. The DESI‐MS/MS method exhibited for the esomepazole excellent sensitivity (limit of detection (LOD)=60 ng/mL), linearity (0.2‐20 µg/mL concentration range; y=23848(±361)X, n=15; r²=0.987) and precision (RSD<9%) by using an internal standard method. The PK results were discussed in terms of Area Under the Curve, C_max and T_max. Data reliability was demonstrated by comparison with a liquid chromatography‐tandem mass spectrometry method (p>0.05). The data achieved demonstrated that the DESI‐MS method is suitable for sensitive and fast profiling of a drug and its metabolites at the therapeutic concentration levels. Copyright © 2015 John Wiley & Sons, Ltd.     

Significant enhancement of 11‐Hydroxy‐THC detection by formation of picolinic acid esters and application of liquid chromatography/multi stage mass spectrometry (LC‐MS3): Application to hair and oral fluid analysis

Formation of picolinic acid esters of hydroxylated drugs or their biotransformation products is a promising tool to improve their mass spectrometric ionization efficiency, alter their fragmentation behaviour and enhance sensitivity and specificity of their detection. The procedure was optimized and tested for the detection of cannabinoids, which proved to be most challenging when dealing with alternative specimens, for example hair and oral fluid. In particular, the detection of the THC metabolites hydroxyl‐THC and carboxy‐THC requires ultimate sensitivity because of their poor incorporation into hair or saliva. Both biotransformation products are widely accepted as incorporation markers to distinguish drug consumption from passive contamination. The derivatization procedure was carried out by adding a mixture of picolinic acid, 4‐(dimethylamino)pyridine and 2‐methyl‐6‐nitrobenzoic anhydride in tetrahydrofuran/triethylamine to the dry extraction residues. Resulting derivatives were found to be very stable and could be reconstituted in aqueous or organic buffers and subsequently analyzed by liquid chromatography‐mass spectrometry (LC‐MS). Owing to the complex consecutive fragmentation patterns, the application of multistage MS3 proved to be extremely useful for a sensitive identification of doubly picolinated hydroxy‐THC in complex matrices. The detection limits – estimated by comparison of corresponding signal‐to‐noise ratios – increased by a factor of 100 following picolination. All other species examined, like cannabinol, THC, cannabidiol, and carboxy‐THC, could also be derivatized exhibiting only moderate sensitivity improvements. The assay was systematically tested using hair samples and exemplarily applied to oral fluid. Concentrations of OH‐THC identified in THC‐positive hair samples ranged from 0.02 to 0.29pg/mg. Copyright © 2014 John Wiley & Sons, Ltd.     

HLA‐A*24:02 is associated with metronidazole‐induced cutaneous adverse drug reactions in Han Chinese individuals: A pilot case‐control study with both HLA gene and T cell receptor repertoire analysis

Metronidazole, a widely used drug for the treatment of infections with anaerobic and facultative anaerobic bacteria and protozoa, can frequently cause metronidazole‐induced cutaneous adverse reactions (McADRs). The aim of the present study was to investigate the association between human leucocyte antigen (HLA) alleles and McADRs in a Chinese Han population. The frequency of HLA‐B*24:02 carriers among the McADR patients was 73.3%, which was significantly higher than that of the population controls (32.16%, OR = 5.80, 95% CI = [1.80‐18.72], Pc = 0.004) and of the metronidazole‐tolerant patients (26.67%, OR = 7.56, 95% CI = [2.02‐28.35], Pc = 0.004). Molecular docking showed that metronidazole and one of its major metabolites had the potential to bind in the HLA groove and that there was a relatively stable binding state of the HLA‐B*24:02‐metronidazole/the metabolite complex. The CDR3 repertoires of both T cell receptor (TCR)Vα and Vβ of the patients showed a significantly skewed or an oligoclonal distribution. The TCRVβ CDR3 of the patients shared a similar motif, “CASSxxxxxxQxF.” The current study demonstrated that both the HLA‐A*24:02 allele and TCR are involved in the pathogenesis of McADRs.