Quantitation of R-(-)- and S-(+)-amphetamine in hair and blood by gas chromatography-mass spectrometry: an application to compliance monitoring in adult-attention deficit hyperactivity disorder treatment

Amphetamine has been used in Attention Deficit Hyperactivity Disorder (ADHD), narcolepsy, and as an appetite suppressant either as the racemate or in different proportions of its enantiomers. In Link?ping, Sweden, the Department for Drug Dependence has successfully treated drug abusers also diagnosed with Adult ADHD with Metamina [S-(+)-amphetamine]. Because of the high risk of relapse into drug abuse, a strategy involving the analysis of amphetamine enantiomers in blood and hair was investigated for the assessment of compliance as well as abstinence from street amphetamine. Four patients were included: one patient was treated with racemic amphetamine, and three with Metamina. Blood and hair samples were obtained as a part of the treatment. A basic extraction of the analytes into iso-octane was used. Hair was dissolved in sodium hydroxide before extraction. Chiral derivatization was performed by reaction with S-(-)-N-(trifluoroacetyl)prolyl chloride. Quantitation of R-(-)- and S-(+)-amphetamine was performed by gas chromatography-mass spectrometry in selected ion monitoring. Both blood and hair sample results showed good compliance for patients 1 and 2. Patient 3 and 4 showed different percentages of S-(+)-amphetamine in hair together with varying total concentrations, suggesting intake of additional racemic illicit amphetamine. During treatment, these patients also showed other signs of noncompliance, and one was temporarily withdrawn from treatment. We conclude that the method is suitable to detect therapeutic concentrations of R-(-)- and S-(+)-amphetamine in both blood and hair and that hair reveals noncompliance not shown by concentrations or enantiomer ratios in blood.     

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.     

Cocaine adulteration with the anthelminthic tetramisole (levamisole/dexamisole): Long‐term monitoring of its intake by chiral LC–MS/MS analysis of cocaine‐positive hair samples

Recent studies indicate that not only the anthelminthic levamisole but also the racemate tetramisole (R‐/S‐phenyltetraimidazothiazole, PTHIT) was found as an adulterant for cocaine. We herein report on the investigation of the prevalence of PTHIT among cocaine‐positive hair samples and the discrimination of the presence of its stereoisomers levamisole and dexamisole. Cocaine‐positive hair samples were collected in a forensic context in 2015 and mainly 2017 (n = 724). Cocaine and PTHIT concentrations have been determined by achiral liquid chromatography–tandem mass spectrometry (LC–MS/MS). For distinction of levamisole/dexamisole chiral LC–MS/MS was performed. Cocaine hair concentrations ranged from 500 (cut‐off) to approximately 800 000 pg/mg. The study demonstrates a strong prevalence of PTHIT in cocaine users' hair (87%, n = 627). PTHIT hair concentrations ranged from below LLOQ 3.5 to approximately 61 000 pg/mg (median: 260 pg/mg). Surprisingly, enantiomeric ratios of levamisole/dexamisole ranged from 0.17 to 1.34 (median: 0.63). Therefore, PTHIT‐adulterated street cocaine samples (n = 24) seized between 2013 and 2016 were tested. Samples mainly contained racemic tetramisole (87.5%), only one sample contained levamisole only and two samples contained non‐racemic PTHIT. Our experiments suggest that the presence of tetramisole in biological samples may have hitherto been underestimated. Most probably higher dexamisole than levamisole concentrations in hair specimens arise from stereoselective metabolism and/or elimination. This is particularly important in light of the different pharmacological activities of the two enantiomers and potentially different adverse effects. Toxicological interpretations in intoxication cases with adulterated cocaine should not only consider levamisole but also tetramisole and terminology in scientific contributions should be used accordingly.     

Lisdexamfetamine

Lisdexamfetamine is an amphetamine prodrug, comprising an l-lysine amino acid covalently bonded to dextroamphetamine (d-amphetamine). Lisdexamfetamine is approved in the US for the treatment of attention-deficit hyperactivity disorder in children aged 6-12 years. Lisdexamfetamine is a therapeutically inactive molecule. After oral ingestion, lisdexamfetamine is hydrolyzed to l-lysine, a naturally occurring essential amino acid, and active d-amphetamine, which is responsible for the activity of the drug. In a well designed pharmacodynamic study in adult stimulant abusers, 50 or 100 mg doses of oral lisdexamfetamine had less likability than d-amphetamine 40 mg, suggesting a reduced abuse potential. Through rate-limited hydrolysis in the body, l-lysine is cleaved, gradually releasing pharmacologically active d-amphetamine. The pharmacokinetics of lisdexamfetamine suggest a reduced potential for abuse. In two well designed trials in children aged 6-12 years with attention-deficit hyperactivity disorder (ADHD), the efficacy of lisdexamfetamine was superior to that of placebo in improving symptoms associated with ADHD. Adverse events with lisdexamfetamine were, in general, mild to moderate in severity and consistent with those commonly reported with amphetamine.     

In vitro metabolism of the synthetic cannabinoid 3,5‐AB‐CHMFUPPYCA and its 5,3‐regioisomer and investigation of their thermal stability

Recently, the pyrazole‐containing synthetic cannabinoid N ‐(1‐amino‐3‐methyl‐1‐oxobutan‐2‐yl)‐1‐(cyclohexylmethyl)‐3‐(4‐fluorophenyl)‐1H ‐pyrazole‐5‐carboxamide (3,5‐AB‐CHMFUPPYCA) has been identified as a ‘research chemical’ both in powdered form and as an adulterant present in herbal preparations. Urine is the most common matrix used for abstinence control and the extensive metabolism of synthetic cannabinoids requires implementation of targeted analysis. The present study describes the investigation of the in vitro phase I metabolism of 3,5‐AB‐CHMFUPPYCA and its regioisomer 5,3‐AB‐CHMFUPPYCA using pooled human liver microsomes. Metabolic patterns of both AB‐CHMFUPPYCA isomers were qualitatively similar and dominated by oxidation of the cyclohexylmethyl side chain. Biotransformation to monohydroxylated metabolites of high abundance confirmed that these species might serve as suitable targets for urine analysis. Furthermore, since synthetic cannabinoids are commonly administered by smoking and because some metabolites can also be formed as thermolytic artefacts, the stability of both isomers was assessed under smoking conditions. Under these conditions, pyrolytic cleavage of the amide bond occurred that led to approximately 3 % conversion to heat‐induced degradation products that were also detected during metabolism. These artefactual ‘metabolites’ could potentially bias in vivo metabolic profiles after smoking and might have to be considered for interpretation of metabolite findings during hair analysis. This might be relevant to the analysis of hair samples where detection of metabolites is generally accepted as a strong indication of drug use rather than a potential external contamination. Copyright © 2016 John Wiley & Sons, Ltd.     

Oral fluid with three modes of collection and plasma methamphetamine and amphetamine enantiomer concentrations after controlled intranasal l‐methamphetamine administration

Methamphetamine is included in drug testing programmes due to its high abuse potential. d‐Methamphetamine is a scheduled potent central nervous system stimulant, while l‐methamphetamine is the unscheduled active ingredient in the over‐the‐counter nasal decongestant Vicks® VapoInhaler?. No data are available in oral fluid (OF) and few in plasma after controlled Vicks® VapoInhaler? administration. We quantified methamphetamine and amphetamine enantiomers in OF collected with two different devices and plasma via a fully validated liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method. Additionally, OF were analyzed with an on‐site screening device. Sixteen participants received 7 Vicks® VapoInhaler? doses according to manufacturer's recommendations. Specimens were collected before and up to 32 h after the first dose. No d‐methamphetamine or d‐amphetamine was detected in any sample. All participants had measurable OF l‐methamphetamine with median maximum concentrations 14.8 and 16.1 μg/L in Quantisal? and Oral‐Eze® devices, respectively, after a median of 5 doses. One participant had measurable OF l‐amphetamine with maximum concentrations 3.7 and 5.5 μg/L after 6 doses with the Quantisal? and Oral‐Eze® devices, respectively. There were no positive DrugTest® 5000 results. In the cutoff range 20–50 μg/L methamphetamine with amphetamine ≥limit of detection, 3.1–10.1% of specimens were positive; first positive results were observed after 1–4 doses. Two participants had detectable plasma l‐methamphetamine, with maximum observed concentrations 6.3 and 10.0 μg/L after 2 and 5 doses, respectively. Positive OF and plasma methamphetamine results are possible after Vicks® VapoInhaler? administration. Chiral confirmatory analyses are necessary to rule out VapoInhaler? intake. Implementing a selective d‐methamphetamine screening assay can help eliminate false‐positive OF results. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.     

Testing for AB‐PINACA in human hair: Distribution in head hair versus pubic hair

Synthetic cannabinoid receptor agonists were first identified in herbal products in 2008 advertised as a legal replacement for cannabis. These herbal incense are usually called “spice” and among these, one product in particular has gained popularity: AB‐PINACA (N‐[(2S)‐1‐Amino‐3‐methyl‐1‐oxobutan‐2‐yl]‐1‐pentyl‐1H‐indazole‐3‐carboxamide). This drug has been discovered to have a stronger binding to human cannabinoid CB₁ and CB₂ receptors than ?⁹‐THC.While some articles have been published regarding the presence of AB‐PINACA in biological fluids such as blood and urine, none reports the presence of AB‐PINACA in hair. We have developed and validated a method for detection of AB‐PINACA in hair using a liquid chromatography?tandem mass spectrometry system and applied it to head and pubic hair obtained in a case of intoxication. The validation procedure demonstrated a limit of detection and a limit of quantification of 0.5 and 1 pg/mg, respectively and acceptable linearity, repeatability, and reproducibility. AB‐PINACA tested positive in the blood (5.7 ng/mL) and less than 1 ng/mL was found in urine. The analysis of the hair specimens resulted in an unusual distribution of the drug between head and pubic hair. AB‐PINACA was identified at a higher concentration in head hair (195 pg/mg) versus in pubic hair (5 pg/mg). The very low concentration of AB‐PINACA in the urine after consumption, due to rapid metabolism, could explain this infrequent distribution, as pubic hair can be contaminated by urine. In any case, it cannot be excluded that the high concentration in head hair may be due to environmental contamination.     

The metabolic fate of two new psychoactive substances − 2‐aminoindane and N‐methyl‐2‐aminoindane − studied in vitro and in vivo to support drug testing

The aim of this study was to characterize the in vitro and in vivo metabolism of 2‐aminoindane (2,3‐dihydro‐1H‐inden‐2‐amine, 2‐AI), and N‐methyl‐2‐aminoindane (N‐methyl‐2,3‐dihydro‐1H‐inden‐2‐amine, NM‐2‐AI) after incubations using pooled human liver microsomes (pHLMs), pooled human liver S9 fraction (pS9), and rat urine after oral administration. After analysis using liquid chromatography coupled to high‐resolution mass spectrometry, pHLM incubations revealed that 2‐AI was left unmetabolized, while NM‐2‐AI formed a hydroxylamine and diastereomers of a metabolite formed after hydroxylation in beta position. Incubations using pS9 led to the formation of an acetyl conjugation in the case of 2‐AI and merely a hydroxylamine for NM‐2‐AI. Investigations on rat urine showed that 2‐AI was hydroxylated also forming diasteromers as described for NM‐2‐AI or acetylated similar to incubations using pS9. All hydroxylated metabolites of NM‐2‐AI except the hydroxylamine were found in rat urine as additional sulfates. Assuming similar patterns in humans, urine screening procedures might be focused on the parent compounds but should also include their metabolites. An activity screening using human recombinant N‐acetyl transferase (NAT) isoforms 1 and 2 revealed that 2‐AI was acetylated exclusively by NAT2, which is polymorphically expressed.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis of (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐ 3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate‐1‐14C

γ‐Cyhalothrin ( 1a ), (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate, is a single‐isomer, synthetic pyrethroid insecticide marketed by Pytech Chemicals GmbH, a joint venture between Dow AgroSciences and Cheminova A/S. As a part of the registration process there was a need to incorporate a carbon‐14 label into the cyclopropyl ring of this molecule. A high yielding radiochemical synthesis of γ‐cyhalothrin was developed from readily available carbon‐14 labeled N‐t‐Boc protected glycine. This seven step synthesis, followed by a preparative normal phase HPLC separation of diastereomers, provided 21.8 mCi of γ‐cyhalothrin‐1‐¹⁴C ( 1b ) with >98% radiochemical purity. Copyright © 2007 John Wiley & Sons, Ltd.     

Effect of Food on Early Drug Exposure from Extended-Release Stimulants: Results from the Concerta®, Adderall XR™ Food Evaluation (CAFÉ) Study

Summary

Stimulant therapy is the mainstay of treatment for children, adolescents and adults with attention-deficit/hyperactivity disorder (ADHD). Once-daily, extended-release oral formulations offer long acting control of symptoms by modifying drug delivery and absorption. In particular, consistency in early drug exposure is important for symptom control during school or work hours. Because these once-daily formulations are usually taken in the morning, the timing of the doses with breakfast is important. This study compared the effect of a high-fat breakfast on early drug exposure from a morning dose of two extended-release stimulant formulations: the osmotic-controlled OROS® tablet of methylphenidate HCl (Concerta®) and the capsule containing extended-release beads of mixed amphetamine salts (Adderall XR?). The study had a single-dose, open-label, randomised, four-treatment, crossover design in which healthy subjects received either 36?mg Concerta® or 20?mg Adderall XR? in the morning after an overnight fast or a high-fat breakfast. Serial blood samples were collected over 28?h to determine plasma concentrations of methylphenidate and amphetamine. The food effect on early drug exposure and the pharmacokinetic profiles up to 8?h after dosing of the two extended-release stimulants were directly compared using partial area (AUCp4h, AUCp6h and AUCp8h) fed/fasted ratios. Amphetamine concentrations were markedly lower when the subjects had eaten breakfast, resulting in lower early drug exposures (p?<?0.0001). By contrast, methylphenidate concentrations over the same 8?h were unaffected by breakfast, providing consistent levels of early drug exposure. Therefore, as a child's or adult's eating pattern varies, methylphenidate exposure over the first 8?h would be expected to have less day-to-day variation compared with amphetamine exposure. The osmotic-controlled OROS® tablet provides a reliable and consistent delivery of methylphenidate HCl, independent of food, for patients with ADHD.     

Application of a chiral high‐performance liquid chromatography‐tandem mass spectrometry method for the determination of 13 related amphetamine‐type stimulants to forensic samples: Interpretative hypotheses

Interpretation of amphetamine‐type stimulant (ATS) findings in urine samples can be challenging without chiral information. We present a sensitive enantioselective high‐performance liquid chromatography–tandem mass spectrometry method for the quantification of (R)‐amphetamine, (S)‐amphetamine, (R)‐methamphetamine, (S)‐methamphetamine, (1R,2R)‐pseudoephedrine, (1S,2S)‐pseudoephedrine, (1R,2S)‐ephedrine, (1S,2R)‐ephedrine, (1R,2S)‐norephedrine, (1S,2R)‐norephedrine, (R)‐cathinone, (S)‐cathinone, and (1S,2S)‐norpseudoephedrine (cathine) in urine. The method was successfully applied to more than 100 authentic urine samples from forensic casework. In addition, samples from a controlled self‐administration of (1S,2S)‐pseudoephedrine (Rinoral, 1200 mg within 6 days) were analyzed. The results strengthen the hypothesis that (1R,2S)‐norephedrine is a minor metabolite of amphetamine and methamphetamine. We suggest cathine and (1S,2R)‐norephedrine as minor metabolites of amphetamine racemate in humans. Small methamphetamine concentrations detected in samples with high concentrations of amphetamine could result from a metabolic formation by methylation of amphetamine although in samples with an (R)/(S) ratio for methamphetamine < 1 an additional (previous) (S)‐methamphetamine consumption seems likely. Our data suggest that even amphetamine concentrations exceeding methamphetamine concentrations in urine can be caused by the biotransformation of methamphetamine to amphetamine as long as no (R)‐amphetamine is detected. However, without chiral information, such findings might be (falsely) assumed as a co‐consumption of both substances. Cathinone enantiomers detected in urine samples with high amphetamine concentrations can be interpreted as metabolites of amphetamine. In addition, the results of the self‐administration study revealed that both cathinone enantiomers are minor metabolites of (1S,2S)‐pseudoephedrine, which is the active ingredient of various medicines used for cold. The enantioselective analysis is a powerful tool to avoid the misinterpretation of ATS findings in urine samples.     

Analysis of ethyl glucuronide in hair samples by liquid chromatography‐electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS)

Many different biomarkers can be used to evaluate ethanol intake. Ethyl glucuronide (EtG) is a direct phase II and minor metabolite of ethanol formed through the UDP‐glucuronosyl transferase‐catalyzed conjugation of ethanol with glucuronic acid. Its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. A sensitive LC‐MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology for the analysis of EtG in hair. Sample preparation and chromatographic separation were thoroughly optimized. The analysis was performed in the multiple reaction monitoring mode using the transitions m/z 221 → 203 (for the quantification) and 221 → 85 or 75 (for the qualification) for EtG, and m/z 226 → 208 (for quantification) and 226 → 75 or 85 (for qualification) for EtG‐D5, used as the internal standard. Analyses were carried out using an Inertsil ODS‐3 column (100 × 3 mm i.d., 3 µm particle size) and a mobile phase composed of formic acid and acetonitrile. Various SPE cartridges and solvents were tested in order to obtain the highest recoveries and cleanest extracts. The assay linearity of EtG was confirmed over the range from 20 to 2500 pg mg^?1, with a coefficient of determination (R²) above 0.99. The lower limit of quantitation (LLOQ) was 20 pg mg^?1 and the limit of detection was 10 pg mg^?1. Intra‐ and inter‐day assays were less than 15% except at the LLOQ (20%). The analytical method was applied to 72 post‐mortem hair samples. EtG concentration in the hair ranged from 0 to 653 pg mg^?1 hair. Copyright © 2012 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.     

Surveillance of drug abuse in Hong Kong by hair analysis using LC‐MS/MS

The aim of this study is to reveal the habits of drug abusers in hair samples from drug rehabilitation units in Hong Kong. With the application of liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology, a total of 1771 hair samples were analyzed during the period of hair testing service (January 2012 to March 2016) provided to 14 drug rehabilitation units including non‐governmental organizations (NGOs), rehabilitation centers, and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously, including ketamine, norketamine, cocaine, benzoylecgonine, cocaethylene, norcocaine, codeine, MDMA, MDA, MDEA, amphetamine, methamphetamine, morphine, 6‐acetylmorphine, phencyclidine, and methadone. The results showed that ketamine (77.2%), cocaine (21.3%), and methamphetamine (16.5%) were the frequently detected drugs among those drug abusers, which is consistent with the reported data. In addition, the usage of multiple drugs was also observed in the hair samples. About 29% of drug‐positive samples were detected with multiple drug use. Our studies prove that our locally developed hair drug‐testing method and service can be a valid tool to monitor the use of abused drugs, and which could facilitate rehabilitation program management.     

Determination of hydroxy metabolites of cocaine in hair samples for proof of consumption

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC‐positive samples for these compounds was performed by liquid chromatography–quadrupole time of flight–mass spectrometry (LC–QTOF–MS). Once identified, mass transitions for o‐, p‐ and m‐isomers of hydroxy COC as well as p‐ and m‐isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography–tandem mass spectrometry (LC–MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC‐positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p‐OH‐BE, m‐OH‐BE as well as p‐OH‐NCOC and m‐OH‐NCOC have been identified for the first time in COC‐positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC‐positive hair samples.     

Design,syntheses, and evaluation of novel 1,1‐dihalo‐2,3‐diphenylcyclopropanes as potential cyclooxygenase‐2 (COX‐2) inhibitors with analgesic‐antiinflammatory activity

A group of (Z)‐ and (E)‐1,1‐dihalo‐2‐(4‐substituted‐phenyl)‐3‐phenylcyclopropane [ (Z)‐10 , (E)‐11 ] stereoisomers having a variety of substituents (H, Br, Cl, F, NO₂, SO₂Me) at the para‐position of the C‐2 phenyl ring in conjunction with either two chloro or bromo substituents at C‐1 were synthesized for in vivo evaluation as analgesic and antiinflammatory (AI) agents, and as potential selective cyclooxygenase‐2 (COX‐2) inhibitors. This group of compounds ( 10‐11 ) exhibited significant analgesic activity since 4% NaCl‐induced abdominal constriction was reduced by 44–73% at 30 min, and 48–77% at 60 min, post‐drug administration relative to the reference drugs aspirin and celecoxib (58 and 32% inhibition at 30 min post‐drug administration) for a 50 mg/kg intraperitoneal dose. In the 1,1‐dichloro group of compounds, a Cl or MeSO₂ substituent at the para‐position of the C‐2 phenyl ring generally provided superior analgesic activity. The most active analgesic compound, (E)‐1,1‐dichloro‐2‐(4‐methanesufonylphenyl)‐3‐phenylcyclopropane ( 11h ) inhibited abdominal constriction by 72 and 77% at 30 and 60 min post‐drug administration, respectively. AI activities, determined using the carrageenan‐induced rat paw edema assay, showed that this class of ( Z)‐10 and ( E)‐11 compounds exhibited AI activities in the inactive‐to‐moderate activity range (1.5–45% inhibition) for a 50 mg/kg oral dose. The AI potency order, with respect to the para‐substitutent on the C‐2 phenyl ring, for the ( Z)‐10 compounds was NO₂ > MeSO₂ ≈ H ≥ Cl, and for the ( E)‐11 compounds was H ≥ MeSO₂ > Cl ≈ Br. (E)‐1,1‐dibromo‐2‐(4‐methanesufonylphenyl)‐3‐phenylcyclopropane ( 11l ), which was the most active AI compound, reduced inflammation by 45 and 37% at 3 and 5 h post‐drug administration, respectively. The ( E)‐11 stereoisomer was generally a more potent AI agent than the corresponding ( Z)‐10 stereoisomer. In vitro COX‐1 and COX‐2 inhibition studies showed that (E)‐1,1‐dichloro‐2‐(4‐nitrophenyl)‐3‐phenylcyclopropane ( 11c ) inhibited COX‐1 (IC₅₀ = 278.8 μM) and COX‐2 (IC₅₀ = 80.5 μM) for a COX‐2 selectivity index of 3.5, whereas (E)‐1,1‐dichloro‐2‐(4‐methanesulfonylphenyl)‐3‐phenylcyclopropane ( 11h ) was a more potent inhibitor of COX‐1 and COX‐2, but it was more selective for COX‐1 (COX‐1 IC₅₀ = 0.59 μM, COX‐2 IC₅₀ = 3.04 μM). A molecular modeling (docking) study for (E)‐1,1‐dichloro‐2‐(4‐methanesulfonylphenyl)‐3‐phenylcyclopropane ( 11h ) on the active site of the human COX‐2 isozyme shows it binds in the center of the active site with the 1,1‐dichloro substituents oriented in the direction of the mouth of the channel towards Arg¹²⁰, and the C‐2 MeSO₂ moiety oriented towards the apex of the active site with the S‐atom of the MeSO₂ substituent positioned about 6.56 Å inside the entrance to the secondary pocket (Val⁵²³) of COX‐2. In contrast, the corresponding (Z)‐10h stereoisomer assumes a different position in the COX‐2 binding site where the S‐atom of the MeSO₂ moiety is near (4.02 Å) the Ser⁵³⁰ OH, but a much greater distance from the COX‐2 secondary pocket (Val⁵²³). The results from these docking studies are consistent with the observation that (E)‐11h is an inhibitor of both COX isozymes, whereas the (Z)‐10h stereoisomer is an inactive COX inhibitor (COX‐1 IC₅₀ > 100 μM, COX‐2 IC₅₀ > 200 μM). Drug Dev. Res. 55:79–90, 2002. © 2002 Wiley‐Liss, Inc.     

Phase I metabolism of the highly potent synthetic cannabinoid MDMB‐CHMICA and detection in human urine samples

Among the recently emerged synthetic cannabinoids, MDMB‐CHMICA (methyl N ‐{[1‐(cyclohexylmethyl)‐1H ‐indol‐3‐yl]carbonyl}‐3‐methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB‐CHMICA was investigated using liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS) and liquid chromatography‐electrospray ionization‐quadrupole time‐of‐flight‐mass spectrometry (LC‐ESI‐Q ToF‐MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC‐ESI‐MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB‐CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB‐CHMICA at the CB₁ receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd.