Application of electrospray ionization product ion spectra for identification with atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry – a case study with seized drugs

Product ion spectra obtained with liquid chromatography‐electrospray ionization tandem mass spectrometry (LC‐ESI/MS/MS) were applied to the identification of seized drug samples from atmospheric pressure matrix‐assisted laser desorption/ionization product ion spectra (AP‐MALDI‐MS/MS spectra). Data acquisition was performed in the information‐dependent acquisition (IDA) mode, and the substance identification was based on a spectral library previously created with LC‐ESI/MS/MS using protonated molecules as precursor ions. A total of 39 seized drug samples were analyzed with both AP‐MALDI and LC‐ESI techniques using the same triple‐quadrupole instrument (AB Sciex 4000QTRAP). The study shows that ESI‐MS/MS spectra can be directly utilized in AP‐MALDI‐MS/MS measurements as the average fit and purity score percentages with AP‐MALDI were 90% and 85%, respectively, being similar to or even better than those obtained with the reference LC/ESI‐MS/MS method. This fact enables the possibility to use large ESI spectral libraries, not only to ESI analyses but also to analyses with other ionization techniques which produce protonated molecules as the base peak. The data obtained shows that spectral library search works also for analytical techniques which produce multi‐component mass spectra, such as AP‐MALDI, unless isobaric compounds are encountered. The spectral library search was successfully applied to rapid identification of confiscated drugs by AP‐MALDI‐IDA‐MS/MS. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography‐tandem mass spectrometry method for simultaneous detection of 10 illicit drugs in oral fluid collected with FLOQSwabs™ and application to real samples

According to French law, the roadside testing for drugs of abuse (DOA) should be performed in oral fluid (OF) using an immunological screening kit. If the screening is positive, confirmation has to be done in OF collected by a special swab, called the FLOQSwab? (FS). Unlike other sampling kits, this device was not designed to collect OF since it does not contain an elution buffer. An analytical method was developed for the simultaneous detection of 10 DOA under control in France: tetrahydrocannabinol (THC) at 1 ng/mL, and cocaine, benzoylecgonine (BZE), morphine, 6‐monoacetylmorphine (6‐MAM), amphetamine, methamphetamine, 3,4‐methylenedioxy‐N‐ethylamphetamine (MDEA), 3,4‐methylenedioxyamphetamine (MDA), and 3,4‐methylenedioxy‐N‐methylamphetamine (MDMA) at 10 ng/mL. Samples were eluted using the Quantisal^® buffer and extracted by liquid–liquid extraction for THC and by solid‐phase extraction for the remaining analytes. Analyses were performed by ultra‐high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). The validated method made it possible to detect the concentrations required by law and was successfully applied to samples from drivers who screened positive. The main limitations of this kit are the large variability of the collected OF volume and the poor stability of DOA in OF, requiring the use of a conservation buffer.     

Sulfate metabolites as alternative markers for the detection of 4‐chlorometandienone misuse in doping control

Sulfate metabolites have been described as long‐term metabolites for some anabolic androgenic steroids (AAS). 4‐chlorometandienone (4Cl‐MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl‐MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl‐MTD metabolism by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to establish potential long‐term metabolites valuable for doping control purposes. 4Cl‐MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and gas chromatography‐tandem mass spectrometry (GC‐MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. 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.     

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.     

Method validation and preliminary pharmacokinetic studies on the new designer stimulant 3‐fluorophenmetrazine (3‐FPM)

Pharmaceutical research not only provides the basis for the development of new medicinal products but also for the synthesis of new drugs of abuse. 3‐Fluorophenmetrazine (3‐FPM), a fluorinated derivative of the anorectic phenmetrazine, was first patented in 2011 and appeared on the drug market in 2014. Though invented for potential medical purposes, pharmacokinetic data on this compound, crucial for interpreting forensic as well as clinical cases, are not available. Therefore, a liquid chromatography?electrospray ionization?tandem mass spectrometry (LC?ESI?MS/MS) method for the detection of 3‐FPM in serum, urine, and oral fluid was developed, validated for urine and serum, and used to quantify 3‐FPM in samples obtained during a controlled self‐experiment. The method proved to be linear, selective and sufficiently sensitive. The limits of detection (LODs) were 0.1 ng/mL, 0.2 ng/mL, and 0.05 ng/mL in serum, urine, and oral fluid. Inter‐day precision and intra‐day precision (RSD) in serum samples were below 6.3% and below 8.5%, respectively. The highest serum concentration (c_max) of 210 ng/mL was reached 2.5 hours (t_max) after ingestion. The elimination half‐life and the volume of distribution were calculated to be approx. 8.8 hours and 400 L (5.3 L/kg). 3‐FPM could be detected in serum and urine up to 82 hours and 116 hours, respectively. It was still detected in the last oral fluid sample taken 55 hours after ingestion. 3‐FPM was mainly excreted unchanged. Main metabolic reactions were aryl‐hydroxylation and N‐hydroxylation. Interestingly, the product of oxidative ring opening (2‐amino‐1‐(3‐fluorophenyl)propan‐1‐ol) showed the largest window of detection in the self‐experiment.     

Detection and phase I metabolism of the 7‐azaindole‐derived synthetic cannabinoid 5F‐AB‐P7AICA including a preliminary pharmacokinetic evaluation

In June 2018, a 'research chemica'l labeled 'AB‐FUB7AICA' was purchased online and analytically identified as 5F‐AB‐P7AICA, the 7‐azaindole analog of 5F‐AB‐PINACA. Here we present data on structural characterization, suitable urinary consumption markers, and preliminary pharmacokinetic data. Structure characterization was performed by nuclear magnetic resonance spectroscopy, gas chromatography–mass spectrometry, infrared and Raman spectroscopy. Phase I metabolites were generated by applying a pooled human liver microsome assay (pHLM) to confirm the analysis results of authentic urine samples collected after oral self‐administration of 2.5 mg 5F‐AB‐P7AICA. Analyses of pHLM and urine samples were performed by liquid chromatography?time‐of‐flight mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS/MS). An LC–MS/MS method for the quantification of 5F‐AB‐P7AICA in serum was validated. Ten phase I metabolites were detected in human urine samples and confirmed in vitro. The main metabolites were formed by hydroxylation, amide hydrolysis, and hydrolytic defluorination, though – in contrast with most other synthetic cannabinoids – the parent compound showed the highest signals in most urine samples. The compound detection window was more than 45 hours in serum. The concentration‐time profile was best explained by a two‐phase pharmacokinetic model. 5F‐AB‐P7AICA was detected in urine samples until 65 hours post ingestion. Monitoring of metabolite M07, hydroxylated at the alkyl chain, next to parent 5F‐AB‐P7AICA, is recommended to confirm the uptake of 5F‐AB‐P7AICA in urinalysis. It seems plausible that the shift of the nitrogen atom from position 2 to 7 (e.g. 5F‐AB‐PINACA to 5F‐AB‐P7AICA) leads to a lower metabolic reactivity, which might be of general interest in medicinal chemistry.     

Identification of five substituted phenethylamine derivatives 5‐MAPDB, 5‐AEDB,MDMA methylene homolog, 6‐Br‐MDMA,and 5‐APB‐NBOMe

This paper reports analytical properties of five substituted phenethylamine derivatives seized from a clandestine laboratory. These five derivatives include 5‐(2‐methylaminopropyl)‐2,3‐dihydrobenzofuran (5‐MAPDB, 1 ), 5‐(2‐aminoethyl)‐2,3‐dihydrobenzofuran (5‐AEDB, 2 ), N ,2‐dimethyl‐3‐(3,4‐methylenedioxyphenyl)propan‐1‐amine (MDMA methylene homolog, 3 ), 6‐bromo‐3,4‐methylenedioxymethamphetamine (6‐Br‐MDMA, 4 ), and 1‐(benzofuran‐5‐yl)‐N ‐(2‐methoxybenzyl)propan‐2‐amine (5‐APB‐NBOMe, 5 ). These compounds were identified by liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS), gas chromatography‐mass spectrometry (GC‐MS), and nuclear magnetic resonance spectroscopy (NMR). No analytical properties about compounds 1‐4 have appeared until now, making this the first report on these compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

MALDI MSI and LC‐MS/MS: Towards preclinical determination of the neurotoxic potential of fluoroquinolones

Fluoroquinolones are broad‐spectrum antibiotics with efficacy against a wide range of pathogenic microbes associated with respiratory and meningeal infections. The potential toxicity of this class of chemical agents is a source of major concern and is becoming a global issue. The aim of this study was to develop a method for the brain distribution and the pharmacokinetic profile of gatifloxacin in healthy Sprague‐Dawley rats, via Multicenter matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and quantitative liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). We developed a sensitive LC‐MS/MS method to quantify gatifloxacin in plasma, lung, and brain homogenates. A pharmacokinetic profile was observed where there is a double peak pattern; a sharp initial increase in the concentration soon after dosing followed by a steady decline until another increase in concentration after a longer period post dosing in all three biological samples was observed. The imaging results showed the drug gradually entering the brain via the blood brain barrier and into the cortical regions from 15 to 240 min post dose. As time elapses, the drug leaves the brain following the same path as it followed on its entry and finally concentrates at the cortex. Copyright © 2015 John Wiley & Sons, Ltd.     

Non‐targeted acquisition strategy for screening doping compounds based on GC‐EI‐hybrid quadrupole‐Orbitrap mass spectrometry: A focus on exogenous anabolic steroids

This is a first look at a non‐targeted screening method based on Orbitrap gas chromatography–mass spectrometry (GC–MS) technology for a large number of banned compounds in sports found in urine, including exogenous anabolic steroids, β‐agonists, narcotics, stimulants, hormone modulators, and diuretics. A simple sample preparation was processed in four steps: an enzymatic hydrolysis, liquid–liquid extraction, evaporation, and trimethylsilylation. All compounds were able to meet the World Anti‐Doping Agency's sensitivity criteria with mass accuracies less than 1 ppm and with sufficient points across the peak by running the Orbitrap GC–MS in full‐scan mode. In addition, we discuss our initial findings of using a full‐scan selected ion monitoring‐tandem mass spectrometry (SIM‐MS/MS) approach as a way to obtain lower detection limits and reach desirable selectivity for some exogenous anabolic steroids.     

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) – a group of new psychoactive substances (NPS) – is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography—tandem mass spectrometry (LC?MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography?high resolution tandem mass spectrometry (LC?HRMS/MS) and finally included to the MS/MS spectra in‐house library. Sample preparation was performed using a cheap‐and‐easy salting‐out liquid–liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05 ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.     

Enantiomeric separation and quantification of R/S‐amphetamine in serum using semi‐automated liquid‐liquid extraction and ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry

The amphetamine molecule contains a chiral center and its enantiomers exhibit differences in pharmacological effects, with the S‐enantiomer mediating most of the central nervous system stimulating activity. The majority of prescribed amphetamine consists of the pure S‐enantiomer, but therapeutic formulations containing the R‐enantiomer in various proportions are also available. Illegal amphetamine remains available mainly as a racemic mixture of the R‐ and S‐enantiomers. To distinguish between legal and illegal consumption of amphetamine a method for enantiomeric separation and quantification of R/S‐amphetamine in serum was developed and validated using ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MS/MS). Sample preparation prior to UHPSFC‐MS/MS analysis was performed by a semi‐automated liquid–liquid extraction method. The UHPSFC‐MS/MS method used a Chiralpak AD‐3 column with a mobile phase consisting of CO₂ and 0.1% ammonium hydroxide in 2‐propanol/methanol (50/50, v/v). The injection volume was 2 μL and run time was 4 minutes. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 12.5–1,000 nM for each analyte. The between‐assay relative standard deviations were in the range of 1.3–3.0%. Recovery was 73% and matrix effects ranged from 95 to 100% when corrected with internal standard. After development and validation, the method has been successfully implemented in our laboratory for both separation and quantification of R/S‐amphetamine and has proved to be a reliable and useful tool for distinguishing intake of R‐ and S‐amphetamine in authentic patient samples.     

UHPLC‐MS/MS and UHPLC‐HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination

Zolpidem and zopiclone (Z‐compounds) are non‐benzodiazepine hypnotics of new generation that can be used in drug‐facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z‐compounds and their metabolites by ultra‐high‐pressure liquid chromatography/tandem mass spectrometry (UHPLC‐MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC‐HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy‐ and three hydroxy‐ metabolites, that could be also detected by gas chromatography/mass spectrometry (GC‐MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N‐demethyl and N‐oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC‐MS/MS method for the determination of Z‐compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC‐MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacokinetic properties of the synthetic cannabinoid JWH‐018 in oral fluid after inhalation

Each year, synthetic cannabinoids occur in high numbers on the illicit drug market, but data on their detectability are rarely available. A pilot study was performed to assess adverse effects of JWH‐018, which is one of the oldest and best known synthetic cannabinoids. Oral fluid has been evaluated as a specimen for drug monitoring. Six subjects inhaled smoke derived from 2 and 3 mg JWH‐018. The drug and 10 of its metabolites were analyzed in oral fluid samples collected during the following 12 hours using the Quantisal collection device by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Maximum concentrations of JWH‐018 reached 2.2–2036 (median 25.7) ng/mL after inhalation and decreased during the next hour to only 0.08–8.42 (median 0.89) ng/mL. Metabolites were not found. During the elimination phase (median half‐life 1.69 hours), detection of the drug over 6–12 hours (median 8 hours) after inhalation was achieved (0.024 ng/mL limit of quantification). Oral fluid/serum ratios varied considerably intra‐ and inter‐individually in a range of 0.05–555 (median 1.38). The detection of JWH‐018 in oral fluid requires high analytical sensitivity even 1 hour after inhalation. The pharmacokinetic properties of inhaled JWH‐018 are similar to those of THC. Times for detection are typically less than 12 hours. High variability of the oral fluid/serum ratio precludes extrapolation of oral fluid concentrations to blood.     

Methylhexanamine is not detectable in Pelargonium or Geranium species and their essential oils: A multi‐centre investigation

In an earlier study, we developed two sensitive and reliable procedures for gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis of methylhexaneamine (MHA) in P. graveolens plant materials and volatile oils. None of the analyzed plant materials or oils showed any detectable levels of MHA which was further substantiated by high resolution liquid chromatography‐quantum time of flight‐mass spectrometry (LC‐QTOF‐MS) analysis with a limit of detection of 10 ppb. However, other laboratories (two studies) reported the presence of MHA in some samples of P. graveolens and pelargonium oil acquired by the investigators from China. Because of the controversy of whether Pelargonium species or pelargonium oil contains MHA, it was recommended that splits of multiple samples be analyzed by different laboratories. In this investigation, multiple plant materials and oil samples were collected from around the world. These samples were submitted to four different sites for analysis. All sites adopted a similar extraction method. All the analysis sites used LC‐MS/MS or LC‐QTOF‐MS and detection limit was set close to the 10 ng/mL as previously reported. A total of 18 plant samples belonging to 6 different Pelargonium species and 9 oils from different locations around the world were split among 4 different analytical laboratories for analysis (each lab received the same samples). None of the laboratories detected MHA in any of the samples at or around the 10 ppb detection level of the procedure used. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of the designer steroid Androsta‐3,5‐diene‐7,17‐dione in a dietary supplement

A liquid chromatography‐mass spectrometry (LC–MS) screen for known anabolic‐androgenic steroids in a dietary supplement product marketed for “performance enhancement” detected an unknown compound having steroid‐like spectral characteristics. The compound was isolated using high performance liquid chromatography with ultraviolet detection (HPLC–UV) coupled with an analytical scale fraction collector. After the compound was isolated, it was then characterized using gas chromatography with simultaneous Fourier Transform infrared detection and mass spectrometry (GC–FT–IR–MS), liquid chromatography–high resolution accurate mass–mass spectrometry (LC–HRAM–MS) and nuclear magnetic resonance (NMR). The steroid had an accurate mass of m/z 285.1847 (error?0.57 ppm) for the protonated species [M + H]⁺, corresponding to a molecular formula of C₁₉H₂₄O₂. Based on the GC–FT–IR–MS data, NMR data, and accurate mass, the compound was identified as androsta‐3,5‐diene‐7,17‐dione. Although this is not the first reported identification of this designer steroid in a dietary supplement, the data provided adds information for identification of this compound not previously reported. This compound was subsequently detected in another dietary supplement product, which contained three additional active ingredients.     

Small‐scale purification of butyrylcholinesterase from human plasma and implementation of a μLC‐UV/ESI MS/MS method to detect its organophosphorus adducts

Human butyrylcholinesterase (hBChE) is a serine hydrolase (EC 3.1.1.8) present in all mammalian tissues and the bloodstream. Similar to acetylcholinesterase, the enzyme reacts with organophosphorus compounds (OP) like nerve agents or pesticides that cause enzyme inhibition (BChE adducts). These adducts represent valuable biomarkers for analytical verification of OP exposure. For establishment of these mass spectrometry based methods sufficient amounts of hBChE in high purity are required. Unfortunately, commercial lots are of inappropriate purity thus favouring in‐house isolation. Therefore, we developed a small scale procedure to isolate hBChE from citrate plasma. After precipitation by polyethylene glycol (8% w/v and 20% w/v PEG 6000) hBChE was purified from plasma by four consecutive chromatographic steps including anion exchange, affinity extraction and size exclusion. Protein elution was monitored on‐line by UV‐absorbance (280 nm) followed by continuous fractionation for off‐line analysis of (1) hBChE enzyme activity by Ellman assay, (2) protein purity by gel electrophoresis, and (3) protein identity by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI MS). Numerous major impurities separated from hBChE were identified. The purified material was used for in vitro incubation with diverse OP to establish a μ‐liquid chromatography‐ultra violet detection/electrospray ionization tandem‐mass spectrometric method (μLC‐UV/ESI MS/MS) for detection of hBChE adducts suitable for verification analysis. Analytical data for diverse OP pesticides including deuterated analogues as well as G‐ and V‐type nerve agents and their precursor are summarized. This method was successfully applied to plasma samples provided by the Organisation for the Prohibition of Chemical Weapons (OPCW) for the 4th Biomedical Exercise. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous detection and quantitation of organic impurities in methamphetamine by ultra‐high‐performance liquid chromatography–tandem mass spectrometry,a complementary technique for methamphetamine profiling

The analysis of organic impurities plays an important role in the impurity profiling of methamphetamine, which in turn provides valuable information about methamphetamine manufacturing, in particular its synthetic route, chemicals, and precursors used. Ultra‐high‐performance liquid chromatography – tandem mass spectrometry (UHPLC – MS/MS) is ideally suited for this purpose due to its excellent sensitivity, selectivity, and wide linear range in multiple reaction monitoring (MRM) mode. In this study, a dilute‐and‐shoot UHPLC – MS/MS method was developed for the simultaneous identification and quantitation of 23 organic manufacturing impurities in illicit methamphetamine. The developed method was validated in terms of stability, limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, and precision. More than 100 illicitly prepared methamphetamine samples were analyzed. Due to its ability to detect ephedrine/pseudoephedrine and its high sensitivity for critical target markers (eg, chloro‐pseudoephedrine, N‐cyclohexylamphetamine, and compounds B and P), more impurities and precursor/pre‐precursors were identified and quantified versus the current procedure by gas chromatography – mass spectrometry (GC – MS). Consequently, more samples could be classified by their synthetic routes. However, the UHPLC – MS/MS method has difficulty in detecting neutral and untargeted emerging manufacturing impurities and can therefore only serve as a complement to the current method. Despite this deficiency, the quantitative information acquired by the presented UHPLC – MS/MS methodology increased the sample discrimination power, thereby enhancing the capacity of methamphetamine profiling program (MPP) to conduct sample‐sample comparisons.     

A Supercritical Fluid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Quantification of Metformin and Gliclazide in Human Plasma

Present study reports the development and validation of a simultaneous estimation of metformin and gliclazide in human plasma using supercritical fluid chromatography followed by tandem mass spectrometry. Acetonitrile:water (80:20) mixture was used as a mobile phase along with liquid CO₂ in supercritical fluid chromatography and phenformin as an internal standard. The modified plasma samples were analyzed by electro-spray ionization method in selective reaction monitoring mode in tandem mass spectrometry. Supercritical fluid chromatographic separation was performed using nucleosil C₁₈ containing column as a stationary phase. The separated products were identified by characteristic peaks and specific fragments peaks in tandem mass spectrometry as m/z 130 to 86 for metformin, m/z 324 to 110 for gliclazide and m/z 206 to 105 for phenformin. The present method was found linear in the concentration ranges of 6.0-3550 ng/ml and 7.5-7500 ng/ml for metformin and gliclazide, respectively. Pharmacokinetic study was performed after an oral administration of dispersible tablets containing 500 mg of metformin and 80 mg of gliclazide using same techniques.     

Post‐Injection Delirium/Sedation Syndrome after Olanzapine Long‐Acting Intramuscular Injection – Who is at Risk?

The post‐injection olanzapine delirium/sedation syndrome (PDSS) was observed in a 60‐year‐old Caucasian, schizophrenic, non‐smoker and underweight [body mass index (BMI), 18.2 kg/m²] women after the fourth intramuscular injection of 405 mg olanzapine pamoate. Clinical symptoms of PDSS were similar to those of acute oral olanzapine intoxication. The patient received supportive treatment and recovered fully. High olanzapine concentrations in serum, with maximum level of 698 ng/mL, were confirmed by liquid chromatography with tandem mass spectrometry (LC‐MS/MS). The authors wonder whether a low BMI and advanced age may predispose patients to PDSS occurrence.