Showing papers in "Forensic Toxicology in 2014"

Chemical analysis of a benzofuran derivative, 2-(2-ethylaminopropyl)benzofuran (2-EAPB), eight synthetic cannabinoids, five cathinone derivatives, and five other designer drugs newly detected in illegal products

Abstract: From November 2013 to May 2014, 19 newly distributed designer drugs were identified in 104 products in our ongoing survey of illegal products in Japan. The identified compounds included 8 synthetic cannabinoids, FUB-PB-22 (1), 5-fluoro-NNEI indazole analog (5-fluoro-MN-18, 2), AM-2201 indazole analog (THJ-2201, 3), XLR-12 (4), 5-fluoro-AB-PINACA (5), 5-chloro-AB-PINACA (6), AB-CHMINACA (7), and 5-fluoro-AMB (8); 5 cathinone derivatives, DL-4662 (9), α-PHP (10), 4-methoxy-α-POP (11), 4-methoxy-α-PHPP (12), and 4-fluoro-α-PHPP (13); and 6 other substances, namely, the benzofuran derivative 2-(2-ethylaminopropyl)benzofuran (2-EAPB, 14), nitracaine (15), diclofensine (16), diphenidine (17), 1-benzylpiperidine (18), and acetylfentanyl (19). To our knowledge, this is the first report on the chemical properties of compounds 9–11 and 14. A total of 33 designer drugs, including compounds 1–19, were detected in the 104 illegal products, in 60 different combination patterns. The numbers of detected compounds per product ranged from 1 to 7. In addition, several products contained three different types of compounds, such as synthetic cannabinoids, cathinone derivatives, and phenethylamine derivatives per product. It is apparent that the types of compounds emerging as illegal products are becoming more diverse, as are their combinations.

Postmortem distribution of α-pyrrolidinovalerophenone and its metabolite in body fluids and solid tissues in a fatal poisoning case measured by LC–MS–MS with the standard addition method

Abstract: We experienced an autopsy case, in which the cause of death was judged as α-pyrrolidinovalerophenone (α-PVP) poisoning. Other drugs or poisons that could have caused the death were not detected by our screening using gas chromatography–mass spectrometry. The deceased was a 41-year-old man. The postmortem interval was about 40 h. Cardiac blood, femoral vein blood, urine, stomach contents, and seven solid tissues were collected and frozen until analysis to investigate the distribution of α-PVP and its metabolite 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol (OH-α-PVP) in the body of the cadaver. After sample pretreatment, they were subjected to solid-phase extraction with Oasis HLB cartridges and analysis by liquid chromatography–tandem mass spectrometry. Because the distribution study dealt with different matrices, we used the standard addition method to overcome the matrix effects. The concentration of α-PVP in urine was much higher than in other specimens; the concentrations of α-PVP in solid tissues except for the spleen were about twofold those in blood specimens. Among the solid tissues, the highest α-PVP concentration was found in the pancreas, followed by the kidney. The extremely high concentration of the drug in urine and the relatively high concentration in the kidney suggested that α-PVP is rapidly excreted into urine via the kidney. The distribution profile of OH-α-PVP was generally similar to that of α-PVP in body fluids and solid tissues. The concentration of OH-α-PVP in urine was also much higher than those in other specimens. Among the solid tissues, the OH-α-PVP concentration was highest in the liver, followed by the kidney. The high concentration of OH-α-PVP in the liver was expected, because the metabolism of α-PVP was probably most active in the liver. The high levels of OH-α-PVP in urine and kidney also suggested that the metabolite was also rapidly excreted into urine via the kidney. To test whether conjugated metabolites were present in urine and solid tissues, urine and homogenates of the liver and spleen were incubated with β-glucuronidase/sulfatase at 37 °C for 5 h. Concentrations of OH-α-PVP were measured before and after the incubation, but differences in the concentrations before and after the incubation were within 10 % for the urine, liver, and spleen samples. To date, data on the distribution of α-PVP and OH-α-PVP in body fluids and solid tissues in a fatal α-PVP poisoning case have not been reported; thus, the findings of our study will be useful for assessment of future cases of α-PVP poisoning.

Synthetic cannabinoids abused in South Korea: drug identifications by the National Forensic Service from 2009 to June 2013

Abstract: The rapid increase in the number of new psychoactive substances and their abuse is the most recent drug abuse issue worldwide. Although abuse of synthetic cannabinoids is highly restricted in South Korea, the rapid increase in the number of new substances is forcing the legal regulation authority to continuously improve the drug regulation act. As a result of drug screening by the National Forensic Service from 2009 to June 2013, 26 species of synthetic cannabinoids were identified in materials seized mainly by the Police Agency and the Prosecutor’s Office in South Korea. One of the most remarkable trends in synthetic cannabinoids is the increase in halogenated derivatives and new substances, including UR-144 and A-836,339 originally developed as analgesics by Abbott Laboratories. The N-pentyl fluorinated analog of UR-144 (XLR-11) has become the most frequently found synthetic cannabinoid in 2013 since its first appearance in 2012, whereas abuse of A-836,339 analogs has been little reported despite their abuse potential. Until early 2011, nicotine was the most frequently found active coingredient with synthetic cannabinoids. However, various psychoactive substances such as Δ9-tetrahydrocannabinol, α-pyrrolidinobutiophenone, α-pyrrolidinovalerothiophenone, and N,N-diallyl-5-methoxytryptamine have often been found as coingredients in herbal highs since late 2011. These coingredients should also be systematically regulated, because they can cause unexpected side effects. It is suggested that authorities in different countries share information about synthetic cannabinoids and their coingredients.

Metabolism of the newly encountered designer drug α-pyrrolidinovalerophenone in humans: identification and quantitation of urinary metabolites

Abstract: Urinary metabolites of α-pyrrolidinovalerophenone (α-PVP) in humans were investigated by analyzing urine specimens obtained from abusers Unambiguous identification and accurate quantification of major metabolites were realized using gas chromatography–mass spectrometry and liquid chromatography-tandem mass spectrometry with newly synthesized authentic standards Two major metabolic pathways were revealed: (1) the reduction of the β-keto moiety to 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol (OH-α-PVP, diastereomers) partly followed by conjugation to its glucuronide, and (2) the oxidation at the 2″-position of the pyrrolidine ring to α-(2″-oxo-pyrrolidino)valerophenone (2″-oxo-α-PVP) via the putative intermediate α-(2″-hydroxypyrrolidino)valerophenone (2″-OH-α-PVP) Of the metabolites retaining the structural characteristics of the parent drug, OH-α-PVP was most abundant in most of the specimens examined

Recently abused synthetic cathinones, α-pyrrolidinophenone derivatives: a review of their pharmacology, acute toxicity, and metabolism

Abstract: The aim of this review is to present the chemical aspects, pharmacology, acute toxicities, and metabolisms of α-pyrrolidinophenone derivatives, a new group of synthetic cathinones. Compared to other synthetic cathinones, α-pyrrolidinophenone derivatives have high lipophilicity due to the pyrrolidine ring substitution at the nitrogen atom, resulting in higher blood–brain barrier permeability. To date, some acute intoxication and fatal cases involving α-pyrrolidinophenone derivatives have been reported, and the symptoms induced by their high dosages are due to central nervous system and cardiovascular toxicities. Based on the previous metabolism studies, reduction of the β-ketone moiety to the corresponding alcohol metabolites and oxidation to the 2′′-oxo metabolites are the main metabolic pathways observed among α-pyrrolidinophenone derivatives. In addition to such pathways, specific metabolic pathways like hydroxylation followed by oxidation of the 4′-methyl group, O-demethylation of the 4′-methoxyl group, and demethylenation followed by O-methylation of the 3′,4′-methylenedioxy group can be observed for the corresponding ring-substituted compounds.

Two new synthetic cannabinoids, AM-2201 benzimidazole analog (FUBIMINA) and (4-methylpiperazin-1-yl)(1-pentyl-1H-indol-3-yl)methanone (MEPIRAPIM), and three phenethylamine derivatives, 25H-NBOMe 3,4,5-trimethoxybenzyl analog, 25B-NBOMe, and 2C-N-NBOMe, identified in illegal products

Abstract: Two new types of synthetic cannabinoids, an AM-2201 benzimidazole analog (FUBIMINA, 1) and (4-methylpiperazin-1-yl)(1-pentyl-1H-indol-3-yl)methanone (MEPIRAPIM, 2), and three newly emerged phenethylamine derivatives, 25B-NBOMe (3), 2C-N-NBOMe (4), and a 25H-NBOMe 3,4,5-trimethoxybenzyl analog (5), were detected in illegal products distributed in Japan. The identification was based on liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS), high-resolution MS, and nuclear magnetic resonance analyses. Different from the representative synthetic cannabinoids, such as JWH-018, which have a naphthoylindole moiety, compounds 1 and 2 were completely new types of synthetic cannabinoids; compound 1 had a benzimidazole group in place of an indole group, and compound 2 had a 4-methylpiperazine group in place of the naphthyl group. Compounds 3 and 4 were N-o-methoxybenzyl derivatives of 2,5-dimethoxyphenethylamines (25-NBOMe series), which had been previously detected in European countries, but have newly emerged in Japan. Compound 5 had an N-trimethoxybenzyl group in place of an N-o-methoxybenzyl group. Data on the chemistry and pharmacology of compounds 1, 2, and 5 have never been reported to our knowledge.

Identification and quantitation of a new cathinone designer drug PV9 in an “aroma liquid” product, antemortem whole blood and urine specimens, and a postmortem whole blood specimen in a fatal poisoning case

Abstract: A couple bought “aroma liquid” and “bath salt” type drugs at a dubious drug shop. Both of them orally took the liquid type drug; although the male subject showed no symptoms, the female subject suffered shivering, convulsions, and low levels of consciousness. The woman was taken to an emergency hospital to receive intensive medical treatment, but died about 20 h after admission. The aroma liquid solution, and the antemortem blood and urine collected during medical treatment at the hospital were brought to our laboratory by the police for analysis of the causative drug(s). In addition, a sample of postmortem femoral vein blood was collected from the cadaver. After some screening tests, we finally identified PV9 (α-POP) in all specimens by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS–MS). The concentration of PV9 was 18.3 mg/ml in the aroma liquid solution, 45.7 ng/ml in the antemortem blood, 20.3 ng/ml in the antemortem urine, and 180 ng/ml in the postmortem femoral vein blood. The concentrations in antemortem blood and urine and in postmortem blood were greatly lowered by dilution during the intensive medical treatment, including intravenous drip infusion of a large volume of solution. The probable coexistence of a β-hydroxyl metabolite was also investigated by mass chromatography and analysis of fragment ions of the product ion spectrum obtained by LC–MS–MS. To our knowledge, this is the first reported identification and quantitation of PV9 in human specimens in a fatal PV9 poisoning case.

Identification of two new-type designer drugs, piperazine derivative MT-45 (I-C6) and synthetic peptide Noopept (GVS-111), with synthetic cannabinoid A-834735, cathinone derivative 4-methoxy-α-PVP, and phenethylamine derivative 4-methylbuphedrine from illegal products

Abstract: We identified two new-type designer drugs, piperazine derivative MT-45 [1-cyclohexyl-4-(1,2-diphenylethyl)piperazine, synonym: I-C6, 1] and synthetic peptide Noopept [ethyl 2-(1-(2-phenylacetyl)pyrrolidine-2-carboxamido)acetate, synonym: GVS-111, 2], in chemical and herbal products. MT-45 (1) was previously reported as an opiate-like analgesic substance, and Noopept (2) was reported to have nootropic (cognitive enhancer) activity. We also detected two synthetic cannabinoids, A-834735 (3) and QUPIC N-(5-fluoropentyl) analog (synonym: 5-fluoro-PB-22, 4), in the illegal products. A-834735 (3) was previously reported to act as an agonist at both cannabinoid CB1 and CB2 receptors. In addition, cathinone derivative 4-methoxy-α-pyrrolidinovalerophenone (4-methoxy-α-PVP, 5) and phenethylamine derivative 4-methylbuphedrine (6) were newly detected with known cathinone derivative 4-methylbuphedrone (7) in the products.

Application of modified QuEChERS method to liver samples for forensic toxicological analysis

Abstract: In forensic toxicological analysis, liver is commonly used as an alternative biological specimen in cases in which blood and urine cannot be obtained. Liver samples are generally purified by a solid-phase extraction (SPE) technique after homogenization. The homogenizer probe cleaning process is laborious and has a risk of cross-contamination, and the SPE technique itself is tedious and time consuming. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method is widely acknowledged in some fields, such as food analysis, as a simple, fast, and reliable method. We previously developed a modified QuEChERS method for forensic toxicological analysis in human whole blood and urine. In this study, we applied this method to liver samples from forensic cases and successfully detected not only targeted drugs (i.e., benzodiazepines, zopiclone, and zolpidem) but also various types of drugs. This method has no risk of cross-contamination because homogenization of the liver and extraction of the drugs are simultaneously performed in a disposable plastic tube. In addition, the total process time is approximately 5 min. We recommend the modified QuEChERS method for extraction of drugs from both fluid and solid samples, such as liver, in forensic cases.

Thermal degradation of a new synthetic cannabinoid QUPIC during analysis by gas chromatography–mass spectrometry

Abstract: Quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC) is a newly introduced synthetic cannabinoid in the drug market. This drug was found to undergo thermal decomposition during gas chromatography–mass spectrometry (GC–MS), probably because of the presence of an ester bond in its structure. Most notably, when QUPIC dissolved in methanol or ethanol was analyzed by GC–MS, most of the QUPIC decomposed to give thermal degradation products. We identified the products as methyl 1-pentyl-(1H-indole)-3-carboxylate, ethyl 1-pentyl-(1H-indole)-3-carboxylate, and methyl indole-3-carboxylate by comparison of their mass spectra with those of reference standards synthesized in our laboratory. Nonalcoholic solvents such as acetone and chloroform gave a major peak and a minor peak for unchanged QUPIC and the degradation product 8-quinolinol, respectively. Furthermore, we studied the effects of various parameters, such as injection methods (splitless or split, and split ratio), injector temperatures, and injector liners on the thermal degradation of QUPIC. Split injection was effective in avoiding degradation. When performing splitless injection, an injector temperature of 250 °C and a surface deactivated injector liner without glass wool minimized the degradation and enhanced the sensitivity. These results indicate that special attention is required for GC–MS analysis of QUPIC, and the information presented in this study will be very useful for forensic toxicologists using GC–MS.

Rapid and simultaneous extraction of acidic and basic drugs from human whole blood for reliable semi-quantitative NAGINATA drug screening by GC–MS

Abstract: We present a rapid procedure for simultaneous extraction of a wide range of acidic and basic drugs from whole blood samples for reliable semi-quantitative NAGINATA drug screening by gas chromatography–mass spectrometry (GC–MS). To extract a wide range of drugs, the partition/extraction procedure used for the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method was employed as the initial step. Various procedures were tested as the second step for the removal of whole blood impurities, including the use of primary secondary amine and C18 for the dispersive solid-phase extraction of the QuEChERS method, four kinds of silica-based C18 columns, alumina columns, and protein–lipid removal filter cartridges (Captiva ND Lipids). Subsequent GC–MS screening used the NAGINATA software with a constructed calibration-locking database for detection of acidic and basic drugs; drug detection ability, accuracy of tentative concentrations, and drug recoveries were examined and compared. We also examined the applicability of our established method in an actual forensic case. Our results showed that the number of drugs detectable at low concentrations was greatly increased by the use of the partition/extraction procedure of the QuEChERS method as the initial step and the protein–lipid removal filter cartridge as the second step. These combined steps provided notably clean extracts. Recoveries carefully measured with each reference standard were largely more than 60 %, and tentative concentrations obtained by the established screening method without reference standards were in the range of 48–310 % of the expected values for 65 acidic and basic drugs. Therefore, relatively reliable semi-quantitative values were obtained at the screening step without the need for each reference standard. We also experienced significant time savings for the extraction and in obtaining tentative concentrations at the screening step in an actual forensic case, indicating that the method is useful for rapid diagnosis of drug intoxication. Our proposed method should prove useful for semi-quantitative screening of a wide range of drugs and poisons in whole blood samples in clinical and forensic cases.

A large amount of new designer drug diphenidine coexisting with a synthetic cannabinoid 5-fluoro-AB-PINACA found in a dubious herbal product

Abstract: Traffic police brought five dubious herbal product packages to our laboratory for toxicological analysis. These products usually contain one or multiple kinds of synthetic cannabinoids. In one of the packages, we identified the coexistence of a new type of dubious drug (diphenidine) with the synthetic cannabinoid 5-fluoro-AB-PINACA. Conclusive identification was performed by comparison of the mass spectra of the test herb extracts with those of the reference standards of diphenidine and 5-fluoro-AB-PINACA by both gas chromatography–mass spectrometry (GC–MS) and electrospray ionization–tandem mass spectrometry. Both mass spectra of the test extracts coincided with those of the reference standards for each MS method. Diphenidine and 5-fluoro-AB-PINACA were quantitated in the herbal product by GC–MS using selected ion monitoring and the standard addition method. The content of diphenidine in the herbal product was as high as 289 ± 23.2 mg/g (n = 5); that of 5-fluoro-AB-PINACA was 55.5 ± 5.8 mg/g (n = 3). Diphenidine is known as an N-methyl-d-aspartate receptor channel blocker. Although its human toxicity has not been studied, it is likely to have severe psychotropic action in humans. The very high content of diphenidine in the present herbal package should prompt law enforcement agencies to be aware of the potential harmful effects of diphenidine itself and also when consumed in combination with other drugs of abuse.

Determination of antidepressants in whole blood using hollow-fiber liquid-phase microextraction and gas chromatography–mass spectrometry

Abstract: A hollow-fiber liquid-phase microextraction (HF-LPME), used in three-phase mode, and combined with gas chromatography–mass spectrometry (GC–MS), was developed to quantify antidepressants and their major metabolites (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, desmethylclomipramine, fluoxetine, and norfluoxetine) in whole blood samples, using their deuterated analogs as internal standards. The HF-LPME system comprised a disposable 8-cm polypropylene porous hollow fiber, 4.0 ml of sample solution (0.5 ml of blood added to 3.5 ml of 0.1 M NaOH: donor phase), dodecane (organic phase), and 0.1 M formic acid (acceptor phase) for extraction. After stirring the system, the acceptor phase was evaporated under a nitrogen stream and resuspended in 30 μl of methanol. Derivatization was not required. A 2.0-μl aliquot of this solution was injected into a GC–MS system. The method was validated after the optimization of several parameters that may influence the extraction efficiency. The limits of quantification for all antidepressants were below the therapeutic levels (20.0 ng/ml). The average intraday and interday precisions were within 9.7 and 9.8 %, respectively, for all analytes. The calibration curves were linear in the concentration range of 20–1,200 ng/ml. The developed method was applied to seven actual postmortem samples. Tricyclic antidepressants were detected in all of the analyzed cases. To our knowledge, this is the first demonstration of usefulness of HF-LPME for analysis of antidepressants in postmortem forensic cases.

Time-course profile of urinary excretion of intravenously administered α -pyrrolidinovalerophenone and α -pyrrolidinobutiophenone in a human

Abstract: We report the time-course profile of urinary excretion of α-pyrrolidinovalerophenone (α-PVP) and α-pyrrolidinobutiophenone (α-PBP) after intravenous injection in a human. The subject was a man aged in his forties, who intravenously injected unknown amounts of so-called unregulated drugs with the intention to commit suicide. The urinary concentrations of the drugs were analyzed by MonoSpin® extraction and gas chromatography–mass spectrometry. α-PVP, α-PBP, and their 2′-oxo metabolites of these compounds were identified in the urine. The highest concentration of α-PVP and α-PBP in urine was 1.2 and 1.6 μg/ml, respectively. The excretion half-lives of α-PVP and α-PBP were calculated to be 22 and 11 h, respectively. To our knowledge, this is the first report on the urinary excretion profiles of α-PVP and α-PBP in a human.

MALDI-TOF mass spectrometric determination of four pyrrolidino cathinones in human blood

Abstract: A rapid and sensitive detection method using matrix-assisted laser desorption ionization (MALDI)–time-of-flight (TOF)–mass spectrometry (MS) was developed for the analysis of four pyrrolidino cathinones: α-pyrrolidinopropiophenone (PPP), 4′-methyl-α-pyrrolidinopropiophenone (MPPP), α-pyrrolidinobutiophenone (PBP), and α-pyrrolidinovalerophenone (PVP). In this method, α-cyano-4-hydroxycinnamic acid was used as the matrix to assist ionization of the cathinones. Each MALDI–TOF–MS spectrum of the cathinones showed not only protonated molecular ion [M + H]+ but also several fragment ions having comparable intensities to that of [M + H]+. Hence, MPPP and PBP could be clearly discriminated by the mass spectra alone, although these compounds have almost the same mass numbers in their protonated molecular ions. The quantification of MPPP, PBP, or PVP was performed using PPP as internal standard, and that of PPP was performed using PBP as internal standard. The limit of detection was 1 ng/ml, and the quantification range was 2–200 ng/ml for the four cathinones using 20 μl of blood. In a fatal poisoning case in which PVP was abused, the PVP levels in whole blood samples obtained from the right heart, left heart, and femoral vein were 0.597, 0.635, and 0.580 μg/ml, respectively. We recommend the MALDI–TOF–MS method without any chromatography for both identification and quantification of the pyrrolidino cathinones in various matrices in forensic toxicological analysis, because of the simplicity, rapidness, and reliability of the method.

A simple method for the simultaneous determination of mushroom toxins by liquid chromatography–time-of-flight mass spectrometry

Abstract: A simple and rapid method for the determination of nine mushroom toxins, ibotenic acid, propargylglycine, choline, muscimol, muscarine, α-amanitin, β-amanitin, phalloidin, and phallacidin, in mushroom samples has been developed. Mushroom toxins were extracted with 0.5 % formic acid in methanol/water, purified with Oasis HLB cartridges, and analyzed by liquid chromatography–time-of-flight mass spectrometry. The separation was performed on a pentafluorophenylpropyl column, and the mass spectrometer was operated in positive ion mode with electrospray ionization. Calibration curves were linear over the range of 0.01–1 μg/ml (R 2 = 0.999). The detection limits for the toxins in mushroom samples were 0.0098–4.9 μg/g, which were low enough for the investigation of food poisoning cases. The intraday and interday recoveries for blank mushroom extracts fortified with mushroom toxins were 72.5–107 % and 75.9–108 %, and relative standard deviations were <7.9 and 8.2 %, respectively.

Analysis of purity and cutting agents in street mephedrone samples from South Wales

Abstract: Mephedrone (4-methylmethcathinone) was banned in the UK under the Misuse of Drugs Act in April 2010 and by the EU in December 2010. Banning drugs is intended to reduce harm by limiting access and causing a reduction in purity, although this may cause overdoses when users cannot predict the correct dose. In this study, 119 mephedrone samples from South Wales were collected between November 2011 and March 2013. Mephedrone purity was determined by gas chromatography–mass spectrometry, and cutting agents were identified using Fourier transform infrared spectroscopy (FTIR). Mean mephedrone purity was 68.2 %, with a standard deviation of 24.9 %. A clear time trend was observed, with mephedrone purity declining from the start of our collection period. The mean purity decreased from 80 % in the first 10 samples collected to 50 % in the last 10 samples collected. Cutting agents were identified in 48 samples by FTIR. The most common were monosodium glutamate, creatine, and sucrose. Three samples were found to contain both monosodium glutamate and sucrose and their purity was very low at 33 %, suggesting that they were cut in two phases. Surprisingly, no adulterants, such as the topical anesthetics that are used to cut cocaine, were detected. 4-Fluoromethcathinone and 4-methylethcathinone were the only other psychoactive substances detected.

Time-course measurements of drugs and metabolites transferred from fingertips after drug administration: usefulness of fingerprints for drug testing

Abstract: We evaluated whether a fingerprint, which consists of secretions from the fingertips, is a suitable biological sample for drug testing. A commercially available cold medicine containing ibuprofen, dihydrocodeine, chlorpheniramine, and methylephedrine was administered to healthy subjects. The subjects washed their hands with tap water and hand soap to remove the external contaminants, and then pressed a fingertip onto wet filter paper at fixed sampling times (from 2 h to 7 days). The analytes on the filter paper were dissolved in 25 % methanol–water, and a large volume (50 μl) of the extract was analyzed by liquid chromatography–tandem mass spectrometry. The relationship between the sampling times and the concentration of analytes in fingerprints was examined. The results were compared with drug concentrations in blood samples. Most of the drugs and their metabolites were detected from fingerprints at 7 days after drug administration. The fingerprint sample preparation is rapid (ca. 3 min) and simple, and the limits of detection were 0.1 pg/fingerprint for dihydrocodeine, chlorpheniramine, and methylephedrine. We demonstrate that drugs can be detected in fingerprints at later sampling times with more rapid and simpler sample preparation than in blood. The method should be applicable to drug testing in criminal investigations.

Development of ultrasound-assisted dispersive liquid–liquid microextraction–large volume injection–gas chromatography–tandem mass spectrometry method for determination of pyrethroid metabolites in brain of cypermethrin-treated rats

Abstract: 3-Phenoxybenzoic acid (3-PBA) and 4-hydroxy-3-phenoxybenzoic acid (OH-PBA) are the two common metabolites for most pyrethroid insecticides. A rapid, sensitive, and eco-friendly method has been developed based on ultrasound-assisted dispersive liquid–liquid microextraction (DLLME) coupled to large volume injection–gas chromatography–tandem mass spectrometry for the simultaneous determination of pyrethroid metabolites in rat brain treated with cypermethrin (CYP). Brain samples were homogenized in methanol (disperser solvent) followed by derivatization with methyl chloroformate (MCF) and extraction using DLLME. Factors that influence the extraction and derivatization efficiency such as type and volume of extraction and disperser solvent, sonication time, pH, ionic strength, and volumes of MCF and pyridine were optimized. Under optimized conditions, the limits of detection were 1 and 4 ng/g for 3-PBA and OH-PBA, respectively. Mean recoveries of pyrethroid metabolites in rat brain were in the range of 83–95 %. The developed method was successfully applied for determination of 3-PBA and OH-PBA in brain samples of CYP-treated rats. The developed method can be adopted for rapid and sensitive analysis of pyrethroid metabolites in toxicological and forensic laboratories.

Analysis of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in urine samples by hollow fiber-liquid phase microextraction and gas chromatography–mass spectrometry in consideration of measurement uncertainty

Abstract: Marijuana abuse can be detected by means of toxicological analysis of the most important metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in urine samples. The aim of this study is the establishment of the detailed procedure for analysis of THC-COOH in urine by combination of hollow fiber-liquid phase microextraction (HF-LPME) and gas chromatography–mass spectrometry (GC–MS). The conditions of hydrolysis and extraction were optimized. The method was shown to be very simple and rapid, and a low amount of organic solvent was necessary for extraction. The limit of detection was 1.5 ng/ml. The calibration curves were linear over the specified range (2.0–170 ng/ml; r 2 > 0.99). The main sources of uncertainty were found to be analyte concentration, accuracy, method precision and sample volume. The effect of the analyte concentration on the overall combined uncertainty was most significant. The developed method was successfully applied to a human urine standard reference material at two levels of concentration. The obtained relative combined uncertainty was 8 %, which can be considered acceptable according to international guidelines. The present method seems very useful in clinical and forensic toxicology, because of its simplicity, rapidness and inexpensiveness.

Phosphorus-specific determination of glyphosate, glufosinate, and their hydrolysis products in biological samples by liquid chromatography–inductively coupled plasma–mass spectrometry

Abstract: Anion exchange liquid chromatography (LC) with inductively coupled plasma–mass spectrometry (ICP–MS) was used for simultaneous determination of glyphosate and glufosinate (phosphonic and amino acid group-containing herbicides; PAAHs) and their hydrolysis products, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicoacetic acid (MPPA), in biological samples. The target compounds were separated using an anion exchange resin column and gradient elution with sodium carbonate and sodium hydroxide system. The chromatographic eluates were passed through a membrane suppressor system and monitored by phosphorus-specific detection at m/z 31. PAAHs and their hydrolysis products were baseline separated from each other within 40 min of elution time, and phosphoric acid did not interfere with detection. The detection limits in serum samples were 0.1–0.7 µg/ml; those in urine samples, 0.2–1.6 µg/ml for the four compounds. The spiked recoveries for the four compounds were over 91 % in serum and urine samples. The detection of the compounds was not subject to interference from sample matrix components. The present method would be useful for toxicological analysis of PAAHs and their products in actual forensic practice. To our knowledge, this is the first trial to establish an LC–ICP–MS method for analysis of PAAHs and their products in biological samples .

Presence of appreciable amounts of ethylene glycol, propylene glycol, and diethylene glycol in human urine of healthy subjects

Abstract: In a previous study, we found that appreciable amounts of ethylene glycol (EG), propylene glycol (PG), and diethylene glycol (DEG) were present in the blood of nonoccupational healthy humans. In this study, we measured the three glycols in the urine of healthy subjects by the same isotope dilution gas chromatography–mass spectrometry method, and found the concentrations to be much higher than those in blood. The concentrations of EG, PG, and DEG in urine samples (mean ± standard deviation) obtained from 23 subjects at random were 604 ± 360, 5,450 ± 9,290, and 59.0 ± 49.3 ng/ml, respectively. These values were 9.44, 30.1, and 5.31 times higher than those in whole blood samples, respectively. The much higher concentrations of the three glycols found in urine samples of nonoccupational healthy humans suggest that the three glycols, once incorporated into the human body, are rapidly excreted into urine. To our knowledge, this is the first demonstration of the presence of relatively high concentrations of EG, PG, and DEG in urine of healthy human subjects.

Fentanyl: cause of death or incidental finding? Postmortem peripheral blood concentrations with and without documented transdermal patch use

Abstract: We reviewed postmortem fentanyl cases to compare peripheral blood (PB) concentrations between deaths caused by fentanyl and deaths in which fentanyl was incidental. Furthermore, we describe PB concentrations in fentanyl-caused deaths with and without transdermal (TD) fentanyl use. Our review produced 20 cases with PB fentanyl. Of these, 13 were determined to be fentanyl-caused deaths. Eight of the 13 involved TD fentanyl. The remaining 7 cases were decedents undergoing therapy with fentanyl (TD, n = 3; intravenous, n = 4). In the 13 fentanyl-caused deaths, the mean PB fentanyl level was 30.1 ng/ml. In the deaths involving TD fentanyl use, the mean fentanyl level was 41.7 ng/ml. Deaths without TD fentanyl use had a mean fentanyl level of 21.3 ng/ml. There were 7 other cases with incidental fentanyl. In 3 of these, therapeutic TD fentanyl was used and the mean PB concentration was 16.6 ng/ml. In the remaining 4 deaths in which therapeutic intravenous fentanyl was employed, the mean PB fentanyl level was 8.1 ng/ml. Our review suggests that a PB fentanyl concentration equal to or greater than 25 ng/ml indicates that fentanyl should be considered as being contributory to or the cause of death. However, ranges of measured PB concentrations are once again shown to overlap between subjects who overdose and those who use fentanyl as prescribed. In addition, fentanyl-caused deaths involving TD fentanyl exposure have higher PB fentanyl concentrations than those who did not use transdermal patches. Although enlightening, our study suggests that further evaluation of fentanyl concentration variability among different postmortem blood specimens is needed.

Identification of a synthetic cannabinoid A-836339 as a novel compound found in a product

Abstract: As a part of the work conducted in our laboratory, we encountered a case in which new chemical compound was contained in a certain product. This compound was found to have a molecular weight of 310 Da by liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry. Accurate mass spectrometry measurements showed that the compound had an elemental composition of C16H26N2O2S. Using these mass data together with those obtained by nuclear magnetic resonance analysis and X-ray crystallographic analysis, this compound was identified as N-[3-(2-methoxyethyl)-4,5-dimethyl-2(3H)-thiazolylidene]-2,2,3,3-tetramethylcyclopropanecarboxamide, which was reported in 2009 and named A-836339. It was described as a thiazol derivative and a selective agonist of G-protein-coupled cannabinoid receptor CB2. This is the first report to identify this compound in a dubious product.

Identification and quantitation of N , α -diethylphenethylamine in preworkout supplements sold via the Internet

Abstract: Shortly after we reported the seizure of the amphetamine derivative N,α-diethylphenethylamine (NADEP) as bulk powder, we have identified NADEP in preworkout supplements branded as “Craze” and sold via the Internet. A gas chromatography–mass spectrometry method was validated and used to quantitate NADEP in the supplements. The authentic NADEP sample of the previous study was used as the reference standard for quantitative analysis after purity assay using quantitative nuclear magnetic resonance spectroscopy. Using these methods, NADEP concentrations in two Craze supplements (Berry Lemonade Flavor and Candy Grape Flavor) were quantitated as 0.40 and 0.44 %, respectively. With the label suggesting a serving size of 5.3–5.8 g, this was equivalent to about 23 mg of NADEP. NADEP was patented in 1988 by Knoll Pharmaceuticals with claims of psychoactive effects (e.g., cognitive enhancement and pain tolerance). For unknown reasons, the compound was never developed into a medicine, and important data about its effects and risks are lacking. Nevertheless, the patent suggested an intended oral dose range of 10–150 mg with a target of 30 mg. Therefore, it could be assumed that NADEP was added to the supplements intentionally for its pharmacological effects without adequate labeling. Because NADEP is a structural analog of methamphetamine in which the two methyl groups are only replaced by ethyl groups, it is possible that the toxicity of NADEP is similar to that of methamphetamine. Thus, supplements containing NADEP should be removed from the market immediately. In countries where NADEP is not regulated as a controlled substance, it should be enforced under the Medicines Act.

Cross-reactivities of 39 new amphetamine designer drugs on three abuse drugs urinary screening tests

Abstract: The detection of illicit drugs is important both in the management of substance misuse and in the postmortem identification of drug abuse. Urinary screening assays have been available for many years, and have been successfully utilized for detecting misuse. Urine remains the biological tool of choice to verify drug-free behavior because of noninvasive sampling and high concentrations of drugs and metabolites. Herein we describe the cross-reactivities of 39 new amphetamine designer drugs on three different on-site abuse drugs urinary screening tests (Screen®7, SureStep™, InstAlert™), which are commonly available in forensic laboratories and in the clinical environment. They are chromatographic immunoassay tests for the qualitative detection of amphetamine, methamphetamine, and methylenedioxymethamphetamine at the recommended cutoff according to Italian legislation (500 ng/ml). The speed and sensitivity of these tests have made them the most widely accepted method to screen urine for drugs of abuse.

Interpretation of a highly positive ethyl glucuronide result together with negative fatty acid ethyl esters result in hair and negative blood results

Abstract: Considering the widespread nature of alcohol-related problems, the diagnosis of excessive alcohol consumption is an important task from medical and legal viewpoints. Alcohol abuse can be documented by usual blood [e.g., carbohydrate deficient transferrin (CDT)] and liver function [e.g., γ-GT or mean corpuscular volume (MCV)] tests, and, over a long-term basis, by hair analysis. Major markers of ethanol consumption in hair are ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). Detection of EtG in hair is said to be associated with excessive alcohol consumption, whereas a negative result does not unambiguously exclude alcohol abuse. Investigations on FAEEs can also be used to monitor excessive alcohol consumption. Four FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) are the most suitable markers for the detection of heavy alcohol consumption and show different concentrations in hair of children, adult teetotalers, and social drinkers in comparison with FAEEs concentrations found in the hair of alcoholics. The Society of Hair Testing has provided guidelines for hair testing for chronic excessive alcohol consumption, and states positive cutoffs for a 0–3 cm hair segment as 30 pg/mg and 0.5 ng/mg for EtG and FAEEs, respectively. This study reviews the difficulties in interpreting blood and hair results of a 41-year-old woman involved in a child custody legal dispute. Her EtG and FAEEs concentrations in a 0–3 cm segment were 203 pg/mg and 0.29 ng/mg, respectively, and blood parameters were in the normal range (0.6 %, 93 fl, 14 IU/l for CDT, MCV, and γ-GT, respectively). Although the high EtG concentration suggested excessive drinking behavior, the second hair marker, FAEEs, and the three bloods tests were inconspicuous and in accordance with the claim of abstinence from alcohol by the subject. The woman declared having dyed her hair more than 6 months prior to sampling, use of weight-loss medication, and consumption of about four energy drinks per day. A hair lotion based on ethanolic plant extracts was regularly used by the subject, and could have been a source of contamination. Therefore, we recommend that possible external sources of hair contamination always be taken into consideration, especially if contradictory biological results are obtained and if the subject denies any alcohol intake.

Is there something more about synthetic cannabinoids

Abstract: Many healthcare providers anticipate clinical effects of synthetic cannabinoids from what is known about cannabis (marijuana) and the endogenous cannabinoid system. However, clinical experience suggests otherwise. The acute clinical effects that follow smoking marijuana have been well documented [1, 2]. These effects include anxiety, panic reactions, depressive feelings, euphoria, relaxation, perceptual alterations, time distortion, intensified sensory experiences, infectious laughter, talkativeness, impaired attention, short-term memory and psychomotor function, and sinus tachycardia. After discovery of the CB1 cannabinoid receptor and anandamide, pharmacology of the endogenous cannabinoid system has been characterized, and research efforts have yielded xenobiotics that interact with most of the main endogenous cannabinoid system elements [3]. The CB1 cannabinoid receptor is one of the most abundantly expressed of the neuronal receptors, and has high density within the basal ganglia. One of the most striking effects of cannabinoids is the depression of locomotor activity by inhibition of c-aminobutyric acid (GABA) uptake at striatopallidal and striatonigral terminals, by inhibition of dopamine uptake in the striatum, and by a decrease in glutamate release from the nucleus subthalamicus terminals; inhibiting the firing of the substantia nigra. Mechanisms leading to catalepsy include decreased GABA reuptake into the presynaptic terminal by cannabinoids resulting in an increase in GABA concentration in the synaptic cleft and potentiation of inhibitory GABA action on the nigrostiatal pathway [4]. In mice, anandamide produces a pharmacological profile similar to that of the classical cannabinoid receptor agonists, including analgesia, hypothermia, hypomobility, and catalepsy, which constitutes the ‘‘mouse tetrad,’’ and is classically considered a signature of cannabimimetic activity [4]. The acute clinical effects that follow smoking herbs sprayed with synthetic cannabinoids [e.g., disorientation, agitation, paranoia/fear, combativeness, hallucinations, hyperreflexic, myoclonic jerks, fasciculations, gait disturbance, collapse/syncope, seizures (status), xerostomia, sweating, hypertension, and mydriasis] are, for the most part, unlike the acute clinical effects that follow smoking marijuana. These effects are inconsistent with the effects of endocannabinoids on CB1 receptors and contradict the cannabimimetic signature observed in animals [5–11]. The contrasting clinical effects may be due to the fact that synthetic cannabinoids are not delta-9-tetrahydrocannabinol (THC), are devoid of cannabidiol, have a higher affinity for cannabinol receptors and higher potency than THC, have longer half-lives than THC, have active metabolites, have contaminants, or, is there something more...? Interestingly, while some THC effects may be attenuated (e.g., anxiety) when cannabidiol is co-administered with THC in a controlled study environment, there is little evidence to suggest cannabidiol attenuates the effects of synthetic cannabinoids [12]. We have read with interest an article published in this Journal [Uchiyama et al. (2013) Forensic Toxicol 31:223–240], in which an ongoing survey of designer drugs in the Japanese illegal market revealed structures of newly detected synthetic cannabinoids, detected but known synthetic cannabinoids, and related compounds [13]. We were intrigued by Figure 1 in the article, and noted in the L. Yip (&) R. C. Dart Rocky Mountain Poison and Drug Center, 990 Bannock Street, Denver, CO, USA e-mail: Luke.Yip@rmpdc.org

Occurrence of postmortem production of ethylene glycol and propylene glycol in human specimens

Abstract: We previously established a sensitive gas chromatography–mass spectrometry method for analysis of ethylene glycol (EG), propylene glycol (PG), and diethylene glycol (DEG), and disclosed the presence of appreciable amounts of EG, PG, and DEG in fresh whole blood and urine specimens obtained from nonoccupational healthy humans. These results led us to analyze EG and PG in specimens taken from three human cadavers. EG and PG concentrations in the postmortem blood and solid tissues were much higher than those found in fresh whole blood; their concentrations in many postmortem specimens were more than tenfold those in fresh whole blood specimens, suggesting the postmortem production of EG and PG. Therefore, we examined the time courses of EG and PG concentrations in blood specimens in the absence and presence of saprogenic blood (10 % volume) and/or glucose (3 mg/ml) in vitro, which were left at room temperature for 7 days. EG concentration in fresh blood without any addition decreased slightly during the 7 days. EG concentration in the blood with addition of glucose, and PG concentrations in the blood with and without addition of glucose did not change appreciably during the 7 days. EG and PG concentrations in the blood after addition of 10 % saprogenic blood increased 3.1-fold and 3.5-fold after 7 days, respectively; those after addition of saprogenic blood plus glucose increased 9.1-fold and 11.9-fold after 7 days, respectively. These results show that microorganisms present in the saprogenic blood caused the postmortem production of EG and PG, and the addition of glucose further enhances the EG and PG concentrations, probably acting as the substrate for glycol production by the microorganisms. To our knowledge, this is the first report to describe postmortem production of EG and PG in human specimens.

High-throughput analysis of ramelteon, agomelatine, and melatonin in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry

Abstract: A high-throughput method for analysis of ramelteon, agomelatine, and melatonin in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS) is presented. The LC system, MS–MS system, and separation column used were Waters Acquity UPLC, Acquity TQD, and Poroshell 120 EC-C18, respectively. For extraction of the target compounds, solid-phase extraction was performed with Oasis HLB cartridges. All compounds were detected with retention times of <3 min. The calibration curves for the compounds spiked into human plasma showed good linearities in the nanogram-per-milliliter range. The detection limit (signal-to-noise ratio = 3) was as low as 0.2–0.5 ng/ml. The method gave satisfactory recovery rates, accuracy, and precision for quality control samples spiked with these drugs. The present method should prove very useful in forensic and clinical toxicology and pharmacokinetic studies, because of its high sensitivity and rapidness. To our knowledge, this is the first trial to analyze ramelteon in a biological sample by LC–MS–MS.