Ultra‐fast LC–MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma

A novel approach to high‐throughput, targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96‐well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC–MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow‐injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences.     

Identification of non‐reported bupropion metabolites in human plasma

Bupropion and its three active metabolites exhibit clinical efficacy in the treatment of major depression, seasonal depression and smoking cessation. The pharmacokinetics of bupropion in humans is highly variable. It is not known if there are any non‐reported metabolites formed in humans in addition to the three known active metabolites. This paper reports newly identified and non‐reported metabolites of bupropion in human plasma samples. Human subjects were dosed with a single oral dose of 75 mg of an immediate release bupropion HCl tablet. Plasma samples were collected and analysed by LC–MS/MS at 0, 6 and 24 h. Two non‐reported metabolites (M1 and M3) were identified with mass‐to‐charge (m/z) ratios of 276 (M1, hydration of bupropion) and 258 (M3, hydroxylation of threo/erythrohydrobupropion) from human plasma in addition to the known hydroxybupropion, threo/erythrohydrobupropion and the glucuronidation products of the major metabolites (M2 and M4–M7). These new metabolites may provide new insight and broaden the understanding of bupropion's variability in clinical pharmacokinetics. © 2016 The Authors Biopharmaceutics & Drug Disposition Published by John Wiley & Sons Ltd.     

Detection of in utero cannabis exposure by umbilical cord analysis

According to the 2014 National Survey on Drug Use and Health, 5.3% of pregnant women smoked marijuana in the past month. This prevalence is expected to increase as a growing number of states and countries are now considering legalization. Although the umbilical cord is becoming a useful objective tool to detect in utero drug exposure, currently data about analytical methods and its utility to detect cannabis exposure are scarce. The objective of this work was to develop a method for the determination of Δ⁹‐tetrahydrocannabinol (THC), 11‐hydroxyTHC (THC‐OH), 11‐nor‐9‐carboxy‐THC (THCCOOH), 8‐β‐11‐dihydroxyTHC (THC‐diOH), THC and THCCOOH glucuronides, and cannabidiol (CBD) in the umbilical cord by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with dual ionization source. Umbilical cord samples (0.5 g) were homogenized in methanol and extracted by solid‐phase extraction. Reversed‐phase chromatographic separation was performed in 14 minutes, and 2 transitions per analyte were monitored in multiple reaction monitoring mode. Method validation included linearity (1–10 to 20–200 ng/g), precision (4.1%–23.4%), accuracy (87.5%–111.4%), matrix effect (‐54.8% to ‐5.8%), extraction efficiency (25%–45.6%), limits of detection and quantification (1–10 ng/g), and endogenous (n = 5) or exogenous interferences (not detected). The method was applied to 13 authentic samples from cannabis‐exposed newborns, which meconium samples had tested positive for cannabis. Twelve cord specimens tested positive for THCCOOH‐glucuronide (1.6–19.1 ng/g). We developed and validated a specific and sensitive method for the simultaneous determination of THC, its metabolites, including THC and THCCOOH glucuronides, and CBD in umbilical cord samples by LC–MS/MS. The analysis of authentic samples showed the usefulness of umbilical cord to detect cannabis in utero exposure.     

Valproic acid determination by liquid chromatography coupled to mass spectrometry (LC–MS/MS) in whole blood for forensic purposes

Valproic acid (VPA) is a well-known drug prescribed as anti-epileptic. It has a narrow therapeutic range and shows great individual differences in pharmacodynamics and pharmacokinetics. Consequently, the therapeutical drug monitoring (TDM) in patient's plasma is of crucial importance. Liquid chromatography coupled to mass spectrometry (LC–MS/MS) has gained importance in TDM applications for its features of sensitivity, selectivity and rapidity. However, in case of VPA, the LC–MS/MS selectivity could be hampered by the lack of a sufficient number of multiple reaction monitoring (MRM) transitions describing the molecule. In fact, the product ion scan of deprotonated molecules of VPA does not produce any ion and thus most LC–MS/MS methods are based on the detection of the unique MRM transition m/z 143➔143. In this way, the advantages of selectivity in LC–MS cannot be effectively exploited. In the present method, stable analyte adducts were exploited for the determination of VPA in blood. An Acquity HSS C18 column and mobile phases consisting of 5-mM ammonium formate and acetonitrile both added 0.1% formic acid were used. Source worked in negative acquisition mode and parameters were optimized to increase the adduct (m/z 189) and dimer (m/z 287) stability, and their fragmentation were used to increase the selectivity of MRM detection. The method has been validated according to the toxicological forensic guidelines and successfully applied to 10 real blood samples. Finally, the present method showed suitable for the rapid LC–MS/MS detection of VPA in whole blood, demonstrating the possibility to increase specificity by exploiting stable in-source adducts. This should be considered of utmost importance in the case of forensic applications.     

Enantioselective analysis of citalopram and demethylcitalopram in human whole blood by chiral LC–MS/MS and application in forensic cases

Citalopram is one of the most frequently used antidepressants in Denmark. It is marketed as a racemic mixture (50:50) of S‐ and R‐enantiomers as well as of the S‐enantiomer alone, which is the active enantiomer named escitalopram that processes the inhibitory effects. In this study, a chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS) method is developed for the measurement of citalopram and demethylcitalopram enantiomers in whole blood and is applied to forensic cases. Whole blood samples (0.10 g) were extracted with butyl acetate after adjusting the pH with 2 M NaOH. The analytes were separated on a 250 × 4.6 mm Chirobiotic V, 5 μm column by isocratic elution with methanol:ammonia:acetic acid (1000:1:1) using an ultra‐high‐pressure liquid chromatography (UHPLC) system. Quantification was performed by tandem mass spectrometry (MS/MS) using multiple reaction monitoring (MRM) in the positive mode. The total chromatographic run time was 20 min. The limit of detection (LOD) and quantification (LOQ) were 0.001 and 0.005 mg/kg of all four enantiomers, respectively. Linear behaviour was obtained for all four enantiomers from LOQ to 0.50 mg/kg blood with absolute recoveries from 71 to 80%. The method showed an acceptable precision and accuracy as the obtained coefficient of variation, and bias values were ≤16% for all enantiomers. After the validation of the method, a correlation with the racemic method was assessed and found to be acceptable. Then, the method was successfully applied to authentic blood samples from forensic investigations demonstrating that escitalopram was less frequent than citalopram among all forensic cases. Copyright © 2017 John Wiley & Sons, Ltd.     

A multi‐analyte approach to help in assessing the severity of acute poisonings – Development and validation of a fast LC–MS/MS quantification approach for 45 drugs and their relevant metabolites with one‐point calibration

Diagnosis, monitoring of the efficiency of detoxification, and estimating the prognosis of acute poisonings are important tasks in emergency toxicology. Comprehensive screening and quantification of relevant substances by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) help in assessing the severity of most acute poisonings. Turnaround time for such analyses must be short enough to impact on clinical decisions. Therefore, a multi‐analyte LC–MS/MS approach with a 5‐minute gradient was developed and validated for 45 drugs and their active metabolites as a complement to an existing GC–MS approach using the same liquid–liquid extraction. The determination ranges were defined by quality control samples of low and high, representing concentrations from low therapeutic to highly toxic levels. To shorten the turnaround time, one‐point calibration was used. Validation showed low matrix effects and ionization effects of co‐eluting analytes thanks to APCI source as well as sufficient recoveries, precisions, and selectivities. For accuracy, 32 of the 45 compounds fulfilled the criteria for quantification in lower therapeutic and 41 in overdosed and toxic concentrations, considering limits of ±30% deviation. The reuse of the processed calibrator for a period of 30 days was possible for 32 compounds, showing sufficient stability at 8°C. In addition, analysis of authentic blood samples showed the applicability and yielded drug levels, which were comparable to those determined by fully validated therapeutic drug monitoring methods. In conclusion, the present approach in combination with the GC–MS approach should provide sufficient support for clinical assessment of the severity of poisonings with 68 compounds in an acceptable turnaround time.     

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.     

Direct and efficient liquid chromatographic‐tandem mass spectrometric method for opiates in urine drug testing – importance of 6‐acetylmorphine and reduction of analytes

Opiates comprise a class of abused drugs that is of primary interest in clinical and forensic urine drug testing. Determination of heroin, codeine, or a multi‐drug ingestion is complicated since both heroin and codeine can lead to urinary excretion of free and conjugated morphine. Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) offers advantage over gas chromatography‐mass spectrometry by simplifying sample preparation but increases the number of analytes. A method based on direct injection of five‐fold diluted urine for confirmation of morphine, morphine‐3‐glucuronide, morphine‐6‐glucuronide, codeine, codeine‐6‐glucuronide and 6‐acetylmorphine was validated using LC‐MS/MS in positive electrospray mode monitoring two transitions using selected reaction monitoring. The method was applied for the analysis of 3155 unknown urine samples which were positive for opiates in immunochemical screening. A linear response was observed for all compounds in the calibration curves covering more than three orders of magnitude. Cut off was set to 2 ng/ml for 6‐acetylmorphine and 150 ng/ml for the other analytes. 6‐Acetylmorphine was found to be effective (sensitivity 82%) in detecting samples as heroin intake. Morphine‐3‐glucuronide and codeine‐6‐glucuronide was the predominant components of total morphine and codeine, 84% and 93%, respectively. The authors have validated a robust LC‐MS/MS method for rapid qualitative and quantitative analysis of opiates in urine. 6‐Acetylmorphine has been demonstrated as a sensitive and important parameter for a heroin intake. A possible interpretation strategy to conclude the source of detected analytes was proposed. The method might be further developed by reducing the number of analytes to morphine‐3‐glucuronide, codeine‐6‐glucuronide and 6‐acetylmorphine without compromising test performance. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of cocaine and cocaine metabolites in dried blood spots from a controlled administration study using liquid chromatography–tandem mass spectrometry

Cocaine is a common illicit stimulant and is mainly metabolized by hydrolysis to benzoylecgonine (BE) and ecgonine methyl ester (EME), but also to minor metabolites like norcocaine, or hydroxy‐BE. When ethanol is present, cocaethylene is formed. Dried blood spot (DBS) sampling is a minimally invasive microsampling technique with possible advantages for analyte stability and ease of storage, making it an attractive matrix in forensic and clinical settings. We developed a liquid chromatography–tandem mass spectrometry‐based (LC–MS/MS) method for quantifying cocaine, BE, EME, norcocaine, hydroxy‐BE, and cocaethylene in DBS. Six‐mm punches were extracted with aqueous buffer followed by protein precipitation, evaporation and reconstitution in mobile phase. Separation was achieved on a Polar‐RP column (Phenomenex) in a 6‐minute gradient including baseline‐separation of norcocaine and BE. For MS detection, a QTRAP 5500 (Sciex) was used in positive electrospray ionization (ESI) multiple reaction monitoring (MRM) mode. The method was validated for selectivity, sensitivity [lower limited of quantification (LLOQ) 1.0–5.0 ng/mL], imprecision (≤13.4%, ≤19.6% at LLOQ), accuracy (≤ ± 14.9%), matrix effects, extraction efficiency (≥20.9%), hematocrit effect, volume spotted, punch location, long‐term and autosampler stability. Concentrations in DBS from a controlled cocaine administration study in healthy volunteers were compared to whole blood and plasma. Although concentrations correlated moderately to strongly (Spearman's ρ 0.603–0.958), agreement between paired samples was poor, with overestimation of DBS concentrations and wide confidence intervals in Bland–Altman analysis. A possible cause are differences in capillary and venous blood concentrations, with the underlying mechanism requiring further research before DBS analysis for cocaine and its metabolites can be considered equivalent to whole blood or plasma analysis.     

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.     

Development and validation of an LC‐MS/MS method after chiral derivatization for the simultaneous stereoselective determination of methylenedioxy‐methamphetamine (MDMA) and its phase I and II metabolites in human blood plasma

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) is a racemic drug of abuse and its two enantiomers are known to differ in their dose‐response curves. The S‐enantiomer was shown to be eliminated at a higher rate than the R‐enantiomer. The most likely explanation for this is a stereoselective metabolism also claimed in in vitro studies. Urinary excretion studies showed that the main metabolites in humans are 4‐hydroxy 3‐methoxymethamphetamine (HMMA) 4‐sulfate, HMMA 4‐glucuronide and 3,4‐dihydroxymethamphetamine (DHMA) 3‐sulfate. For stereoselective pharmacokinetic analysis of phase I and phase II metabolites in human blood plasma useful analytical methods are needed. Therefore the aim of the presented study was the development and validation of a stereoselective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the simultaneous quantification of MDMA, 3,4‐methylenedioxyamphetamine, DHMA, DHMA 3‐sulfate, HMMA, HMMA 4‐glucuronide, HMMA 4‐sulfate, and 4‐hydroxy 3‐methoxyamphetamine in blood plasma for evaluation of the stereoselective pharmacokinetics in humans. Blood plasma samples were prepared by simple protein precipitation and afterwards all analytes were derivatized using N‐(2,4‐dinitro‐5‐fluorophenyl) L‐valinamide resulting in the formation of diastereomers which were easily separable on standard reverse phase stationary phases. This simple and fast method was validated according to international guidelines including specificity, recovery, matrix effects, accuracy and precision, stabilities, and limits of quantification. The method proved to be selective, sensitive, accurate and precise for all tested analytes except for DHMA. Copyright © 2014 John Wiley & Sons, Ltd.     

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

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

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography–high resolution mass spectrometry

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography–high‐resolution tandem mass spectrometry (LC–HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC–HRMS/MS system consisted of a YMC‐UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β‐glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono‐hydroxy metabolites, and mono‐hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono‐hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2–19‐fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.     

Quantitative urine confirmatory testing for synthetic cannabinoids in randomly collected urine specimens

Synthetic cannabinoid intake is an ongoing health issue worldwide, with new compounds continually emerging, making drug testing complex. Parent synthetic cannabinoids are rarely detected in urine, the most common matrix employed in workplace drug testing. Optimal identification of synthetic cannabinoid markers in authentic urine specimens and correlation of metabolite concentrations and toxicities would improve synthetic cannabinoid result interpretation. We screened 20 017 randomly collected US military urine specimens between July 2011 and June 2012 with a synthetic cannabinoid immunoassay yielding 1432 presumptive positive specimens. We analyzed all presumptive positive and 1069 negative specimens with our qualitative synthetic cannabinoid liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method, which confirmed 290 positive specimens. All 290 positive and 487 randomly selected negative specimens were quantified with the most comprehensive urine quantitative LC‐MS/MS method published to date; 290 specimens confirmed positive for 22 metabolites from 11 parent synthetic cannabinoids. The five most predominant metabolites were JWH‐018 pentanoic acid (93%), JWH‐N‐hydroxypentyl (84%), AM2201 N‐hydroxypentyl (69%), JWH‐073 butanoic acid (69%), and JWH‐122 N‐hydroxypentyl (45%) with 11.1 (0.1‐2,434), 5.1 (0.1‐1,239), 2.0 (0.1‐321), 1.1 (0.1‐48.6), and 1.1 (0.1‐250) µg/L median (range) concentrations, respectively. Alkyl hydroxy and carboxy metabolites provided suitable biomarkers for 11 parent synthetic cannabinoids; although hydroxyindoles were also observed. This is by far the largest data set of synthetic cannabinoid metabolites urine concentrations from randomly collected workplace drug testing specimens rather than acute intoxications or driving under the influence of drugs. These data improve the interpretation of synthetic cannabinoid urine test results and suggest suitable urine markers of synthetic cannabinoid intake. This article is a U.S. Government work and is in the public domain in the USA.     

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

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

Flubromazolam – Basic pharmacokinetic evaluation of a highly potent designer benzodiazepine

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

Chiral analysis of amphetamines in hair by liquid chromatography–tandem mass spectrometry: compliance‐monitoring of attention deficit hyperactivity disorder (ADHD) patients under Elvanse® therapy and identification after controlled low‐dose application

Amphetamine (AMP) is used as an illicit drug and also for the treatment of attention deficit hyperactivity disorder (ADHD). Respective drugs most often contain the enantiomer (S)‐AMP as active compound or (S)‐AMP is formed from the prodrug lisdexamfetamine (Elvanse®) whereas the illicit drug is usually traded as racemate ((R/S)‐AMP). A differentiation between the use of the medically prescribed drug and the abuse of illicit street amphetamine is of great importance, for example in retrospective consumption monitoring by hair analysis. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the chiral separation and quantitation of (S)‐ and (R)‐AMP in hair was developed. For this purpose, 20 mg hair was extracted and derivatized with N‐(2,4‐dinitro‐5‐fluorophenyl)‐L(S)‐valinamide L(S)‐(DNPV) to yield amphetamine diastereomers. Baseline separation of the resulting diastereomers was achieved on a high‐pressure liquid‐chromatography system (HPLC) coupled to a Sciex QTRAP® 5500 linear ion trap quadrupole mass spectrometer. The method was successfully validated. Analysis of hair samples from nine Elvanse® patients revealed only (S)‐AMP in eight cases; one subject showed both enantiomers indicating a (side‐) consumption of street amphetamine. The analysis of the 16 amphetamine users' samples showed only racemic amphetamine. Furthermore, it could be shown in a controlled study that (S)‐AMP can be detected after administration of even very low doses of lisdexamfetamine and dexamphetamine, which can be of interest in forensic toxicology and especially in drug‐facilitated crime (DFC). The method now enables the retrospective compliance‐monitoring of ADHD patients and the differentiation between medically prescribed intake of (S)‐amphetamine and abuse of illicit street amphetamine.     

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

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

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.     

Post‐marketing assessment of generic tamoxifen in Brazilian breast cancer patients

Generic formulations of tamoxifen are commonly prescribed to oestrogen receptor‐positive breast cancer patients at the Brazilian National Cancer Institute (INCA). We carried out a post‐marketing surveillance of the generic tamoxifen formulation in current use at INCA, by comparing plasma concentrations of the parent drug and metabolites obtained with the generic vs the reference formulation. Thirty patients participated in an open‐label, bracketed protocol, comprising 3 successive phases of 30‐32 days each: the generic formulation was used in phases 1 and 3 and the reference formulation in phase 2. Two blood samples were collected in the last 4 days of each phase, for LC‐MS/MS quantification of tamoxifen and metabolites in plasma. The median plasma concentrations (ng/mL) for the reference formulation were as follows: tamoxifen, 135.0 (CI 95% 114.2‐155.8); endoxifen, 35.3 (30.0‐40.8); and 4‐hydroxytamoxifen, 4.8 (4.2‐5.4). The endoxifen/tamoxifen plasma concentration ratio was 0.27 (0.21‐0.25). ANOVA detected no statistically significant difference in plasma concentrations of tamoxifen, metabolites or the endoxifen/tamoxifen ratio among the three phases. The genetic component (rGC) of the CYP2D6‐mediated conversion of tamoxifen into endoxifen, estimated using the repeated drug administration procedure across the three phases, was 0.87, pointing to an important component of genetic variability. In conclusion, this first post‐marketing surveillance trial of oncologic generic drugs carried out in Brazilian patients verified the switchability between the reference and the generic tamoxifen formulation currently used at our institution. The adopted bracketed protocol adds confidence to this conclusion and may serve as a frame for future trials of post‐marketing assessment of other generic drug products.