Showing papers in "Forensic Toxicology in 2013"

Two new-type cannabimimetic quinolinyl carboxylates, QUPIC and QUCHIC, two new cannabimimetic carboxamide derivatives, ADB-FUBINACA and ADBICA, and five synthetic cannabinoids detected with a thiophene derivative α-PVT and an opioid receptor agonist AH-7921 identified in illegal products

Abstract: We identified two new-type cannabimimetic quinolinyl carboxylates, quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC, 1) and quinolin-8-yl 1-(cyclohexylmethyl)-1H-indole-3-carboxylate (QUCHIC, 2); and two new cannabimimetic carboxamide derivatives, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA, 3) and N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentyl-1H-indole-3-carboxamide (ADBICA, 4), as designer drugs in illegal products. Compound 3 was reported to have a potent affinity for cannabinoid CB1 receptor by Pfizer in 2009, but this is the first report of its detection in illegal products. No chemical or pharmacological data for compounds 1, 2, and 4 have appeared until now, making this the first report on these compounds. We also detected synthetic cannabinoids, APICA N-(5-fluoropentyl) analog (5), APINACA N-(5-fluoropentyl) analog (6), UR-144 N-(5-chloropentyl) analog (7), JWH-122 N-(5-chloropentyl) analog (8), and AM-2201 4-methoxynaphthyl analog (4-MeO-AM-2201, 9) herein as newly distributed designer drugs in Japan. It is of interest that compounds 1 and 2 were detected with their synthetic component, 8-quinolinol (10). A stimulant thiophene analog, α-pyrrolidinovalerothiophenone (α-PVT, 11), and an opioid receptor agonist, 3,4-dichloro-N-([1-(dimethylamino)cyclohexyl]methyl)benzamide (AH-7921, 12), were also detected as new types of designer drugs coexisting with several synthetic cannabinoids and cathinone derivatives in illegal products.

New cannabimimetic indazole derivatives, N-(1-amino-3-methyl- 1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1- (4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA) identified as designer drugs in illegal products

Abstract: Two new cannabimimetic indazole derivatives, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA, 1) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA, 2), have been identified as designer drugs in illegal products. These identifications were based on liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. Because there have been neither chemical nor pharmacological data about compound 1 until now, this is the first report of this compound. Compound 2 was reported as a potent cannabinoid CB1 receptor modulator when synthesized by Pfizer in 2009; but this is the first report of its detection in illegal products.

Changes in the prevalence of synthetic cannabinoids and cathinone derivatives in Japan until early 2012

Abstract: The changes in the prevalence of designer drugs and their legal status in Japan were investigated on the basis of the analyses of 686 different products containing synthetic cannabinoids and/or cathinone derivatives obtained from 2009 to February 2012. In the early stages of distribution of herbal-type products containing synthetic cannabinoids, cyclohexylphenols and naphthoylindoles were mostly found in the products. In November 2009, however, cannabicyclohexanol, CP-47,497 and JWH-018 were controlled as “designated substances” under the Pharmaceutical Affairs Law in Japan, and the cyclohexylphenols have since disappeared from the illegal drug market and been replaced by various analogs of the naphthoylindoles, phenylacetylindoles and benzoylindoles. These compounds, which have high affinities for the cannabinoid CB1 receptor, have become very popular, and the number of emergency hospitalizations associated with their use has dramatically increased from 2011. Other synthetic compounds with different structures and pharmacological effects, such as cathinone derivatives, have been detected together with the synthetic cannabinoids in herbal-type products since 2011. Moreover, many new types of synthetic cannabinoids, different from the four typical structures described, have also begun to appear since 2011. In addition to the synthetic cannabinoids, liquid or powdery-type products containing cathinone derivatives have been widely distributed recently. In 2009, the most popular cathinone derivative was 4-methylmethcathinone. After this compound was controlled as a designated substance in November 2009, cathinone derivatives, which have a pyrrolidine structure at the nitrogen atom and a 3,4-methylenedioxy structure, or analogs of 4-methylmethcathinone, became popular. In the present analysis, tryptamines were also detected in 31 % of the products containing cathinone derivatives. Local anesthetics such as procaine, lidocaine, benzocaine and dimethocaine were also frequently detected. In total, we identified at least 35 synthetic cannabinoids and 22 cathinone derivatives during this survey.

A fatal case of MAM-2201 poisoning

Abstract: A 59-year-old man was found dead in his house, where three sachets containing herbal blends were found on a table. The sachet contents were analyzed by gas chromatography–mass spectrometry and found to contain [1-(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl)methanone (MAM-2201). The deceased was subjected to forensic autopsy. There were neither external injuries nor endogenous diseases judged by macroscopic and microscopic observations. Liquid chromatography–electrospray ionization–tandem mass spectrometry was used to quantitate the concentrations of MAM-2201 in postmortem samples using deuterated MAM-2201 as internal standard. The MAM-2201 concentrations were: 12.4 ng/ml in whole blood; 18.1 ng/g in the liver; 11.2 ng/g in the kidney; 4.3 ng/g in the brain; and 1,535 ng/g in the adipose tissue. We concluded that the man’s death was caused by acute intoxication with MAM-2201. In addition, we propose that the adipose tissue is the specimen of choice to detect MAM-2201 in the unchanged form. To our knowledge, this is the first report of a fatal MAM-2201 poisoning case. In addition, this report is also the first to describe the distribution of the drug in postmortem human tissues and blood.

A fatal case of 3,4-methylenedioxypyrovalerone poisoning: coexistence of α-pyrrolidinobutiophenone and α-pyrrolidinovalerophenone in blood and/or hair

Abstract: We report a fatal case of 3,4-methylenedioxypyrovalerone (MDPV) poisoning. A 35-year-old woman was taken to a nearby hospital after she was found unconscious. MDPV (1,200 ng/ml) and α-pyrrolidinobutiophenone (α-PBP; 200 ng/ml) were detected in her cardiac blood. Hair segments from the victim were also analyzed to confirm chronic drug use. MDPV and α-pyrrolidinovalerophenone (α-PVP) were found in many of the segments tested with the highest MDPV concentration at 22 ng/10-mm hair. We concluded that the woman died of acute MDPV poisoning, and that she had abused MDPV and α-PVP for a long time. To our knowledge, this is the first report dealing with MDPV in human hair segments, and with α-PBP in human specimens.

SPME–GC–MS analysis of α-pyrrolidinovaleorophenone in blood in a fatal poisoning case

Abstract: We provided toxicological analytical support for a fatal case of abuse of α-pyrrolidinovaleorophenone (α-PVP). Solid-phase microextraction (SPME) and capillary gas chromatography coupled to mass spectrometry (GC–MS) was employed to quantify the drug in whole blood. The whole blood concentration of the drug in the heart was 486 ng/ml. This is the first report of α-PVP intoxication as ascertained by mass spectrometric identification of α-PVP in whole blood.

Characterization of the designer benzodiazepine pyrazolam and its detectability in human serum and urine

Abstract: In 2012, online shops selling so-called research chemicals started offering pyrazolam, a new benzodiaze- pine that differs from phenazepam and etizolam, which have also recently appeared on the ''gray market'', in that it is not marketed by pharmaceutical companies anywhere in the world. This article describes the characterization of pyrazolam (8-bromo-1-methyl-6-pyridin-2-yl-4H-(1,2,4) triazolo(4,3-a)(1, 4)benzodiazepine) using gas chroma- tography-mass spectrometry, liquid chromatography- tandem mass spectrometry (LC-MS-MS), liquid chroma- tography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS), and nuclear magnetic resonance spec- troscopy. In addition, a study was carried out in which one of the authors ingested two 0.5-mg pyrazolam tablets. Serum and urine samples were then obtained to investigate the metabolism of pyrazolam and to obtain preliminary results for the elimination half-life and the detectability of a 1-mg dose in serum and urine using a highly sensitive LC-MS-MS method and immunoassays. The results showed an elimination half-life of about 17 h and no detectable metabolism. The parent compound was detected with the described LC-MS-MS method in serum for more than 50 h and in urine for approximately 6 days. Immu- noassays showed cross-reactivity, but poor detection in the study samples demonstrated that consumption or adminis- tration of this presumably potent drug could go undetected unless instrumental analytical techniques are also used.

Symptoms, toxicities, and analytical results for a patient after smoking herbs containing the novel synthetic cannabinoid MAM-2201

Abstract: We report a case of intoxication by the synthetic cannabinoid MAM-2201 ([1-(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl)-methanone). A 31-year-old man smoked about 300 mg of a herbal blend. He experienced an acute transient psychotic state with agitation, aggression, anxiety, and vomiting associated with a sympathomimetic syndrome. MAM-2201 was detected and quantified in a plasma sample using liquid chromatography-tandem mass spectrometry (LC–MS–MS). The level was 49 ng/ml 1 h after smoking. The use of other drugs was analytically excluded. The presence of MAM-2201 was confirmed in the herbal blend using gas chromatography–mass spectrometry (GC–MS) and LC–high resolution MS. This is the first description of an analytically confirmed intoxication and of the determination of MAM-2201 in human blood plasma.

Urinary excretion and metabolism of the newly encountered designer drug 3,4-dimethylmethcathinone in humans

Abstract: Cathinone-derived designer drugs have recently grown to be popular as drugs of abuse. 3,4-Dimethylmethcathinone (DMMC) has recently been abused as one of the alternatives to controlled cathinones. In the present study, DMMC and its major metabolites, 3,4-dimethylcathinone (DMC), 1-(3,4-dimethylphenyl)-2-methylaminopropan-1-ol (β-OH-DMMC, diastereomers), and 2-amino-1-(3,4-dimethylphenyl)propan-1-ol (β-OH-DMC, diastereomers), have been identified and quantified in a DMMC user’s urine by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry using newly synthesized authentic standards. Other putative metabolites including oxidative metabolites of the xylyl group and conjugated metabolites have also been detected in urine. The identified and putative phase I metabolites indicated that the metabolic pathways of DMMC include its reduction of the ketone group to the corresponding alcohols, N-demethylation to the primary amine, oxidation of the xylyl group to the corresponding alcohol and carboxylate forms, and combination of these steps. Concentrations of the identified metabolites were found to increase slightly after enzymatic hydrolysis, suggesting that these compounds are partially metabolized to the respective conjugates.

Analysis of the prevalence and coexistence of synthetic cannabinoids in “herbal high” products in Poland

Abstract: “Herbal highs” are a group of products marketed in recent years as legal substitutes for marijuana. This article presents the results of examinations performed on samples seized in “head shops” and from individuals during a 3.5-year period, between mid-2008 and the end of 2011 in Poland. Of over 2000 samples delivered for analysis, 420 preparations were selected for this study. Gas chromatography–mass spectrometry and liquid chromatography–quadrupole-time-of-flight–mass spectrometry were used for identification of psychoactive components, and high-performance liquid chromatography was used for their quantitation. The most common ingredients of herbal highs were: JWH-081 (144 products), JWH-018 (103), RCS-4 (92), JWH-073 (89), JWH-250 (75), JWH-122 (69), cannabicyclohexanol (55), and JWH-210 (38). Over 50 % of the products contained two or more active ingredients; 136 products (32.4 %) contained two; 56 products (13.3 %) contained three; and 22 (5.2 %) contained more than three. Common combinations of ingredients were investigated by the graph method; substances coexisted mainly with those introduced into the drug market in a similar period of time. The most common dual combinations were JWH-081 + RCS-4 (18 products), JWH-073 + JWH-250 (16), and JWH-081 + JWH-250 (12). JWH-081 was blended with almost all detected synthetic cannabinoids. The main risks of the use of these substances were due to ignorance of great variation in the content and composition of synthetic cannabinoids even if the products had identical labels. This inconsistency could cause serious health damage to users, while ignorance of the fact that more than one third of the products being sold at head shops contain illicit compound(s) could result in unexpected arrest.

Differentiation of regioisomeric fluoroamphetamine analogs by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

Abstract: In recent years, a large number of clandestinely produced controlled-substance analogs (designer drugs) of amphetamine with high structural variety have been detected in forensic samples Analytical differentiation of regioisomers is a significant issue in forensic drug analysis because, in most cases, legal controls are placed only on one or two of the three isomers In this study, we used gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the differentiation of regioisomers of fluoroamphetamine analogs (fluoroamphetamines and fluoromethamphetamines), which were synthesized in our laboratories Free bases and their acylated and silylated derivatives were subjected to GC–MS analysis using DB-1ms, DB-5ms, and DB-17ms capillary columns The separation of free bases was incomplete on all columns Trifluoroacetyl derivatives of 3- and 4-positional isomers showed slight separation on DB-1ms and DB-5ms On the other hand, trimethylsilyl derivatization enabled baseline separation of six fluoroamphetamine analogs on DB-1ms and DB-5ms columns, which was sufficient for unequivocal identification For LC–MS/MS, a pentafluorophenyl column was able to separate six regioisomeric fluoroamphetamine analogs but a conventional C18 column could not achieve separation between 3- and 4-positional isomers These results show that a suitable choice of derivatization and analytical columns allows the differentiation of regioisomeric fluoroamphetamine analogs

Identification of (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone and its 5-pentyl fluorinated analog in herbal incense seized for drug trafficking

Abstract: Four herbal incense products were seized from suspected drug abusers in Korea. The major ingredients in the herbal incense samples were purified, and their structures were elucidated using gas chromatography–electron ionization–mass spectrometry (GC–EI–MS), liquid chromatography–time-of-flight–mass spectrometry (LC–TOF–MS), and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. As a result, ingredients in the herbal incense were identified as (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone and its 5-pentyl fluorinated analog [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone. The former is being sold via the Internet as a "research chemical" named UR-144, and the latter is sold as 5F-UR-144. UR-144 is a selective full agonist of CB2 cannabinoid receptor, and was first developed by Abbott Laboratories as an analgesic. It exhibits analgesic activity against both neuropathic and inflammatory pain associated mainly with the CB2 receptor, but shows less psychotropic effects associated with the CB1 receptor. Fluorination of the N-pentyl side chain of cannabimimetic compounds increases their cannabinoid receptor affinity such as with AM-2201, which shows both increased analgesic and psychotropic effects simultaneously. UR-144 and 5F-UR-144 can be classified as research chemicals based on their analgesic effects, but in practice are abused as psychotropic agents and can cause unexpected toxic effects. Thus, the trade and diversion of these chemicals should be monitored carefully for possible abuse. To our knowledge, this is the first report disclosing cyclopropylcarbonylindoles in herbal products.

Sensitive determination of ethylene glycol, propylene glycol and diethylene glycol in human whole blood by isotope dilution gas chromatography-mass spectrometry, and the presence of appreciable amounts of the glycols in blood of healthy subjects

Abstract: Ethylene glycol (EG), propylene glycol (PG), and diethylene glycol (DEG) are widely used as components of antifreeze liquids for automobiles and in many other products. They occasionally cause severe poisonings when they are ingested in high doses. In this study, we established a detailed procedure for highly sensitive and simultaneous determination of EG, PG, and DEG in human whole blood by isotope dilution gas chromatography–mass spectrometry (GC–MS). A 0.25-ml aliquot of whole blood containing the glycols was mixed with 100 ng each of deuterated EG, PG, and DEG as internal standards. After centrifugation, the supernatant fraction was evaporated to dryness, derivatized with heptafluorobutyric anhydride, and the derivative was extracted with n-hexane; 1 μl of the n-hexane layer was subjected to GC–MS analysis. Contrary to our expectation, appreciable amounts of EG, PG, and DEG were found, even in human whole blood samples obtained from nonoccupational healthy subjects. Because such results have never been reported, in the early stages of this study, we suspected that our results were caused by carry-over of the glycols during the GC–MS procedure; thus, we cleaned the injection port, installed a new GC column, and washed the ion-source chamber. Despite these efforts, the clear peaks were detected at the same retention times as those of the authentic glycols for the extracts of healthy subjects, which led us to confirm the presence of the glycols in the blood of healthy subjects. Because the blank whole blood samples from healthy subjects already contained glycols, we constructed a standard addition calibration curve for each sample; the horizontal intercept point, where the straight calibration line intersected the horizontal concentration axis, showed the background concentration of the glycol. The calibration curves showed linearity in the range of 0.4–400 ng/ml for EG and DEG, and in the range of 4–2,000 ng/ml for PG, with correlation coefficients larger than 0.99. The limits of detection and limits of quantitation could not be exactly determined, because of the unavailability of blank human whole blood without each glycol. However, the values were estimated to be around or lower than 1 ng/ml from the signal-to-noise ratios of peaks for each glycol at the lowest concentrations obtained by selected ion monitoring. To validate the method, intraday and interday repeatability was tested; the percent relative standard deviations were 2.5–12.2 % and 0.8–8.5 %, respectively. Recoveries of the three glycols using whole blood samples of two subjects obtained by a unique method were 61.6–80.9 %. The concentration ranges (mean) of EG, PG, and DEG in whole blood obtained from ten subjects were 39.1–97.0 (64.0), 49.1–689 (181), and 8.08–22.9 (11.1) ng/ml, respectively. To test the effect of oral intake of PG, two volunteers ingested 100 ml of a commercially available energy drink containing 33.7 mg of PG, the safety of which was accredited by the Japanese Government. The PG levels in the blood of the two subjects increased by 74.0 and 158 % at 1 and 0.5 h, respectively, suggesting that the glycols present in human blood are largely derived from food. To our knowledge, the analytical method for the glycols presented in this article is the most sensitive among those so far reported. In addition, this is the first description of the presence of the glycols in healthy human subjects, which should be useful for setting cutoff levels of the glycols in poisoning cases.

In vitro and in vivo metabolisms of 1-pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122)

Abstract: 1-Pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122) is an agonist of the cannabinoid receptors CB1 and CB2. In this study, the phase I and phase II metabolisms of JWH-122 were investigated using two models. In vitro studies using incubations of JWH-122 with human liver microsomes were performed to obtain metabolites of the drug at the initial step; 11 classes of metabolites were found and analyzed by liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). Hydroxylation(s) on the naphthalene moiety and/or the indole moiety of the molecule took place as such or in combination with dehydrogenation or cleavage of the N-pentyl side chain. Furthermore, dihydrodiol metabolites were formed probably via epoxide formation on the naphthalene moiety, irrespective of the combination with hydroxylation(s). A metabolite carrying a carboxyl group on the N-pentyl side chain was also detected. As the second step of the study, in vivo experiments using chimeric mice were performed; the mice were orally administered JWH-122, and their urine samples were collected, subjected to enzymatic hydrolysis, and analyzed by LC–MS and LC–MS–MS. The urine samples without hydrolysis were also analyzed for their molecular formulae in the conjugated forms by LC–high resolution MS. The in vivo model using chimeric mice confirmed most metabolite classes and clarified the phase II metabolism of JWH-122. It was concluded that all metabolites formed in vivo were excreted conjugated as glucuronide or sulfate, with conjugation rates above 50 %.

Identification of a new synthetic cannabinoid in a herbal mixture: 1-butyl-3-(2-methoxybenzoyl)indole

Abstract: Two unknown cannabimimetic compounds were detected in a seized herbal mixture after gas chromatography–mass spectrometry (GC–MS) screening. To elucidate the chemical structures, 0.3 g of the dried plant material was extracted with methanol and concentrated under reduced pressure. The extract was purified by silica gel column chromatography with methylene chloride and methanol. Pure compounds were isolated by preparative high-performance liquid chromatography (HPLC) and then analyzed by electrospray ionization (ESI) mass spectrometry (MS) with direct flow injection, high-resolution ESI-time-of-flight (TOF)–MS and one-dimensional and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. GC–MS spectra showed that the base ion at m/z 321 for compound 1 was the same as that of 1-pentyl-3-(4-methoxybenzoyl)indole (RCS-4), and the fragment ions were almost the same as those of RCS-4. The GC–MS spectrum of compound 2 was very similar to that of compound 1 except that the mass numbers of the fragment ions at m/z 290, 200, 186, and 173 of compound 2 were equally smaller than those of compound 1 by 14 amu. From these GC–MS results, compound 1 was assumed to be the 2- or 3-methoxy isomer of RCS-4, and compound 2 was assumed to be a 1-butylindole homologue of compound 1. The ESI mass spectra showed a single peak at m/z 322.33 for compound 1 and a single peak at m/z 308.25 for compound 2, which showed the masses of the protonated ions. High-resolution TOF–MS spectra showed the accurate mass numbers of protonated molecular ions at m/z 322.180512 for compound 1 and at m/z 308.164895 for compound 2, suggesting the molecular formulas of C21H23NO2 and C20H21NO2, respectively. The 1H NMR spectra showed signals that suggested 23 and 21 protons for compounds 1 and 2, respectively, while the respective 13C NMR spectra showed 21 and 20 carbon signals. All protons and carbons were assigned by their couplings and correlations observed in 1H–1H correlation spectroscopy (COSY), 1H–13C heteronuclear multiple bond correlation (HMBC), and 1H–13C heteronuclear single quantum coherence (HSQC) spectra. On the basis of the spectral data, compound 1 was identified as the 2-methoxy isomer of RCS-4; compound 2 was identified for the first time as 1-butyl-3-(2-methoxybenzoyl)indole. Phenazepam and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) were also identified as coexisting drugs in the herbal mixture. The contents of compounds 1 and 2 in the mixture were calculated to be 22.4 and 3.45 mg/g, respectively.

Analysis of cannabinoids in oral fluid by liquid chromatography–tandem mass spectrometry

Abstract: A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography–tandem mass spectrometry (LC–MS/MS). These cannabinoids include ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), ∆9-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-∆9-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and ∆9-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal™ device. The advantages of performing a liquid–liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex™ column packed with 2.6-μm core–shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000™ triple quadrupole LC–MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method’s LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection.

Mass spectrometric differentiation of the isomers of mono-methoxyethylamphetamines and mono-methoxydimethylamphetamines by GC–EI–MS–MS

Abstract: Mass spectrometric differentiation of the six isomers of mono-methoxyethylamphetamines (MeO-EAs) and mono-methoxydimethylamphetamines (MeO-DMAs) by gas chromatography–electron ionization–tandem mass spectrometry (GC–EI–MS–MS) was investigated. Based on their EI-mass spectra, the fragment ions at m/z 121 and 72 were selected as precursor ions for their regioisomeric and structurally isomeric differentiation, respectively. Collision-induced dissociation provides intensity differences in product ions among the isomers, enabling mass spectrometric differentiation of the isomers. Furthermore, high reproducibility of the product ion spectra at the optimized collision energy was confirmed, demonstrating the reliability of the method. To our knowledge, this is the first report on mass spectrometric differentiation of the six isomers of MeO-EAs and MeO-DMAs by GC–EI–MS–MS. Isomeric differentiation by GC–EI–MS–MS has a high potential to discriminate isomers of newly encountered designer drugs, making GC–MS–MS a powerful tool in the forensic toxicology field.

Simultaneous determination of new-generation antidepressants in plasma by gas chromatography-mass spectrometry

Abstract: We have developed a gas chromatography–mass spectrometry (GC–MS) method for plasma for the determination of new-generation antidepressants, including olanzapine (antipsychotic used in bipolar disorder), and antidepressant selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine and its metabolite norfluoxetine, paroxetine, sertraline, venlafaxine, and mirtazapine. Sample preparation was performed by liquid–liquid extraction with tert-butyl methyl ether. Fluoxetine, norfluoxetine, sertraline, and paroxetine required subsequent derivatization with 1-(heptafluorobutyryl) imidazole (HFBI). The GC separation lasts a total of 23.76 min. Qualitative and quantitative analysis were performed using an electron-impact ionization gas chromatograph interfaced to a mass-selective detector in selected-ion monitoring mode to increase the sensitivity of the method. Method validation was performed taking into account linearity, sensitivity, selectivity, accuracy, precision, and recovery, achieving good results for all the parameters studied. Calibration curves were prepared in the range of 0.005–2 μg/ml (according to the therapeutic and toxic concentrations of each individual compound), with all correlation coefficients R2 > 0.99. The limit of quantification was between 0.005 and 0.1 μg/ml, depending on the compound, whereas the limit of detection ranged from 0.0025 to 0.05 μg/ml. The method is fast and simple, allowing the identification and quantification of some of the most widely used antidepressants at therapeutic or toxic concentrations, and may be useful in routine clinical and forensic toxicology analysis.

Simple and rapid determination of myristicin in human serum

Abstract: Myristicin (5-allyl-1-methoxy-2,3-methylenodioxybenzene) is the main component of nutmeg (Myristica fragrans Houtt.) essential oil. The increasing use of myristicin as a cheap hallucinogenic intoxicant, frequently causing fatal cases of myristicin poisoning, requires new methods for determination of this compound in blood. This report describes the rapid, simple, and useful procedure for myristicin analysis in human serum, involving myristicin–protein complex degradation before chromatographic analysis. The developed method is characterized by a high recovery (above 99 %), a low detection limit (6.0 ng/g) and good repeatability (average RDS of 2.01 %).

Hollow fiber–liquid phase microextraction of barbiturates in liver samples

Abstract: Barbiturates, especially phenobarbital, are involved in intoxication cases in Brazil and other countries. Liver tissue can be a useful alternate specimen when blood is not available (decomposition, severe fire, and exsanguination cases). Because the liver is a very complex matrix, it is essential is to eliminate potential interfering substances in this specimen before instrumental analysis of target compounds. In recent years, novel strategies for sample preparation have gained acceptance in the field of analytical toxicology. The objective of this work was to develop a gas chromatography–mass spectrometry (GC–MS) method using hollow fiber–liquid phase microextraction (HF–LPME) for determination of barbiturates (phenobarbital, secobarbital, pentobarbital, and butalbital) in liver samples. An aliquot of homogenized and acidified liver in aqueous solution (equivalent to 50 mg tissue) was submitted to extraction by the use of a 9-cm hollow fiber. The fiber was filled with eucalyptus oil in its pores and its lumen was filled with a NaOH solution (pH 13; acceptor phase). After extraction in an ultrasonic bath for 5 min, the acceptor phase was withdrawn and derivatized in the injector port of the GC–MS with trimethylanilinium hydroxide for flash methylation. A totally “green chemistry” approach of the sample extraction was obtained, because relatively large amounts of organic solvent generally used for extraction could be substituted by an essential oil used as a supported liquid membrane for HF–LPME. This method was validated and successfully applied to liver samples collected from five deceased persons with previous history of exposure to barbiturates. Phenobarbital concentrations in the liver found in these actual cases ranged from 1.3 to 16.7 μg/g.

Cross-reactivities of 41 new amphetamine designer drugs to EMIT immunoassays

Abstract: Amphetamine designer drugs are central nervous system stimulants, and are widely diffused in illegal markets. Monitoring of drugs of abuse in biological fluids is successfully used for clinical and forensic applications. In particular, the urine matrix allows the verification of drug intake in the short and medium term. In a forensic toxicology laboratory, typical analysis for these drugs involves an immunoassay screening method. Here we describe the cross-reactivity profiles of 41 new amphetamine designer drugs to the urine drug tests EMIT® II Plus (Amphetamines assay and Ecstasy assay).

Determination of ibotenic acid and muscimol, the Amanita mushroom toxins, in human serum by liquid chromatography–tandem mass spectrometry

Abstract: In our previous article, we reported the analysis of ibotenic acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of ibotenic acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for ibotenic acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of ibotenic acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for ibotenic acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both ibotenic acid and muscimol with correlation coefficients not <0.999. The detection limits (signal-to-noise ratio = 3) were 1.0 and 2.5 ng/ml for ibotenic acid and muscimol, respectively. The recovery rates of the target compounds in sera at five different concentrations were 87.9–103 %. The intraday and interday accuracy and precision data were also generally satisfactory. Using the modified method, the actual concentrations of ibotenic acid and muscimol were measured for a serum sample obtained from an ill patient thought to have ingested Amanita ibotengutake; they were 95.9 and 105 ng/ml, respectively. To our knowledge, this is the first report of analysis of ibotenic acid and muscimol in human serum by an MS technique, which we believe will be very useful in forensic and clinical toxicology.

Analysis of azepane isomers of AM-2233 and AM-1220, and detection of an inhibitor of fatty acid amide hydrolase [3′-(aminocarbonyl)(1,1′-biphenyl)-3-yl]-cyclohexylcarbamate (URB597) obtained as designer drugs in the Tokyo area

Abstract: During our careful survey of unregulated drugs from November 2011 to January 2012 in the Tokyo area, we found two new compounds in commercial products. The first was identified as the benzoylindole (2-iodophenyl)[1-(1-methylazepan-3-yl)-1H-indol-3-yl]methanone (2), which is the azepane isomer of AM-2233 (1). Compound 2 was isolated by silica gel column chromatography, and was identified through a combination of liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The second compound was identified as [3′-(aminocarbonyl)(1,1′-biphenyl)-3-yl]-cyclohexylcarbamate (URB597, 5) by comparing analytical data with that of the authentic compound. For quantitation of these three compounds, each commercial product was extracted with methanol under ultrasonication to prepare the solution for analysis by liquid chromatography with ultraviolet detection. The occurrence of compounds 1 and 2, and AM-1220 (3) and its azepane isomer (4) in 29 commercial products found in the Tokyo area are also shown in this report.

Simultaneous determination of amphetamine-type stimulants in abusers’ hair: clinical usefulness of hair analysis in prehospitalization for abusers

Abstract: Simultaneous determination of amphetamine-type stimulants (ATSs), such as methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and p-methoxymethamphetamine (PMMA), has been made for hair of 32 female abusers from different ethnic backgrounds. One milligram of abuser's hair spiked with 1-methyl-3-phenylpropylamine as internal standard was used for each determination of the ATSs. After digestion with 1 M NaOH, the samples were extracted with n-heptane; the organic layer was evaporated to dryness, dissolved in 75 mM of borate buffer solution (pH 8.5), and labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The determination of the ATSs in hair was performed by the semi-micro high-performance liquid chromatography-chemiluminescence method. Thirty-two subjects who had self-report histories of ATS use for more than 1 week prior to hair sample collection participated in this study. Concentration ranges of ATSs in abusers’ hair were 0.07–6.73 ng/mg for MA (n = 24), 0.18–2.47 ng/mg for AP (n = 13), 0.05–0.86 ng/mg for MDMA (n = 12), 0.52–1.38 ng/mg for MDA (n = 3), and 0.09–1.88 ng/mg for PMMA (n = 6). Several discrepancies between detected drugs and information obtained from the interviewed abusers were found. Most of the drugs determined in this study could not be detected by the urine test performed at hospitalization. Correlation between ATS concentration and psychiatric symptoms on the basis of Brief Psychiatric Rating Scale (BPRS) was also studied. Simultaneous determination of ATSs in hair of outpatients and inpatients at psychiatric clinics and hospitals should be effective in providing proper diagnosis and treatment. This is the first report to demonstrate the presence of PMMA in hair of human subjects.

Identification of N -ethyl-α-ethylphenethylamine in crystalline powder seized for suspected drug trafficking: a research chemical or a new designer drug?

Abstract: A crystalline powder was found in an unclaimed lost article shipped from Vietnam to South Korea, and it was seized by narcotics agents as an item of suspicious trade. The chemical was suspected to be methamphetamine crystals, and was sent to the National Forensic Service for forensic identification. Elucidation of the chemical structure was carried out using gas chromatography–electron impact ionization–mass spectrometry, liquid chromatography–time-of-flight–mass spectrometry, and 1D and 2D nuclear magnetic resonance spectroscopy. The compound was identified as N-ethyl-α-ethylphenethylamine. Although the narcotic effect of this compound remains unverified, it may be classified as a phenethylamine-based designer drug on the basis of its structure. It appeared that the recipient of this article sought to abuse this chemical in the same way as amphetamines. There is a possibility that this chemical will be widely abused for recreational use in the near future.

Assessment of the presence of damiana in herbal blends of forensic interest based on comprehensive two-dimensional gas chromatography

Abstract: The popularity of 'herbal highs' as an alterna- tive to common illegal drugs is becoming an emerging concern for forensic laboratories and medical facilities. One of the herbal ingredients often claimed by the sup- pliers to be present in their products is Turnera diffusa, commonly known as 'damiana,' which has a long history as a pharmaceutical drug and an intoxicating substance due to its alleged psychoactive effects. The present project focused on the analysis of the chemical signature of damiana for its identification in different herbal blends of forensic interest. Comprehensive two-dimensional gas chromatography proved to be a powerful tool for the extensive analysis of the wide range of volatile compounds present in extracts of damiana and related herbal products. Due to the absence of unique compounds in Turnera dif- fusa, the chemical pattern has to be considered to ascertain the presence of this species in herbal blends. Differentia- tion to the closely related species Turnera ulmifolia was provided based on the chemical profile. Compound align- ment based on both retention times and mass spectral data revealed the presence of damiana in all 'herbal high' products, regardless of whether the presence was declared on the product or not. For several blends, damiana appeared to be the exclusive herbal component. Thus, it confirms the presumption that Turnera diffusa is one of the preferred herbs used as a matrix for 'herbal high' products. Utilizing principle component analysis, 31 compounds were selected, which provided discrimination of five commercial damiana batches.

[1-(Tetrahydropyran-4-ylmethyl)-1 H -indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone: a new synthetic cannabinoid identified on the drug market

Abstract: A new synthetic cannabinoid, [1-(tetrahydropyran-4-ylmethyl)-1H-indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone, was identified in several resinous samples seized by law enforcement officers in Poland. Its identification was based on liquid chromatography–electrospray ionization–quadrupole time-of-flight–mass spectrometry, gas chromatography–electron ionization–mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The reported substance was first developed by Abbott Laboratories and patented under the name “A-834,735”. It is a potent agonist of both CB1 and CB2 receptors. Although A-834,735 shows moderate selectivity to CB2 receptor, it exhibits a CB1 affinity similar to that of ∆9-tetrahydrocannabinol. The drug has recently become available in online shops. To our knowledge, this is the first report to disclose a synthetic cannabinoid containing a (tetrahydropyran-4-yl)methyl structure in products seized from the drug market.

Combination of a short middle-bore capillary column with a thicker stationary phase and a short narrow-bore separation column with a thinner stationary phase for the rapid screening of non-volatile drugs by gas chromatography–mass spectrometry

Abstract: Dear Editor, An increase in requests for drug analysis has led to techniques that can analyze many samples in a short amount of time. For the purpose of enhancing the efficiency of analysis and increasing sample throughput, we looked at increasing the analysis speed of gas chromatography–mass spectroscopy (GC–MS), currently the most widely used technique for forensic drug analysis. The fast GC–MS techniques and the difficulty of their practical implementation have been reviewed in the literature [1]. Our group reported high-throughput chiral analysis of urinary amphetamines by fast GC–MS [2]. It seems very easy to establish a fast GC–MS method with a short narrow-bore capillary GC column and fast temperature programming, if using a modern GC–MS instrument that enables the rapid scanning of mass spectra and powerful pumping to maintain vacuum against the relatively large gas inflow into the ionization chamber. There are short narrow-bore capillary GC columns for fast GC–MS in the presence [3–5] and absence [2, 6, 7] of retention gaps, which are sometimes useful for obtaining narrow peak widths and symmetrical peak shapes. The retention gap is usually a short and deactivated capillary column without stationary phase. In this communication, we have tried to create a drug screening method by fast GC–MS using a short middle-bore capillary column with a thicker stationary phase in place of the inactive retention gap and a short narrow-bore capillary column. Our results showed that more than 20 drugs in whole blood could be screened within 4.1 min of GC–MS analysis. In this study, we mainly selected 30 psychotropic drugs [8, 9] as target compounds for analysis. Pure powder standards of fenfluramine-HCl, imipramine, diphenidolHCl, clomipramine-HCl, diazepam, chlorpromazine-HCl, nordiazepam, and trazodone were purchased from Sigma Aldrich (St. Louis, MO, USA); diphenhydramine-salicylate and nortriptyline-HBr from Wako (Osaka, Japan); and ethenzamide from Nacalai Tesque (Kyoto, Japan). Other drugs in the powder form were obtained from the corresponding pharmaceutical companies. Lidocaine-d10 was purchased from C/D/N Isotopes (Québec, Canada); diazepam-d5 and nordiazepam-d5 (both 1 mg/ml methanol solution) from Cerilliant (Round Rock, TX, USA). Caffeine-d3 (7-methyl-d3-theophylline) and 7-ethyltheophylline were synthesized in our laboratory. Other common reagents were of the highest purity commercially available. Blank whole blood was obtained from a healthy volunteer under permission; it was stored at -20 C until use. Methanolic stock solutions were made of the powder standards at a concentration of 1.0 mg/ml. A working stock solution of the 30 drug standards listed in Table 1 was made up in n-propyl acetate at a concentration of 20 lg/ml. Calibrators were prepared by adding enough of the standard working stock solution to 1 g of blank blood to yield the final concentrations of 0.10, 0.25, 0.50, 1.0, and 5.0 lg/ml in 16 9 125-mm screw cap tubes. A working internal standard (IS) solution containing caffeine-d3 (100 lg/ml), This article is for the special issue TIAFT2012 edited by Osamu Suzuki.

High-throughput determination of barbiturates in human plasma using on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry

Abstract: A high-throughput method was developed for determinations of eight barbiturates (barbital, allobarbital, phenobarbital, cyclobarbital, amobarbital, secobarbital, thiopental, and thiamylal) in human plasma by on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry (MS–MS). Plasma samples (100 μl) spiked with the eight barbiturates and 5-(4-methylphenyl)-5-phenylhydantoin (internal standard) were diluted with 300 μl of 13.3 mM ammonium acetate/acetonitrile (33:67, v/v). After centrifugation and filtration, the clear supernatant was injected directly onto the extraction column (Oasis HLB cartridge column). The following procedure was fully automated. The analytes retained on the extraction column were eluted by backflushing of the extraction column and introduced onto the analytical column (Phenomenex Onyx monolithic C18 column, 100 mm × 4.6 mm i.d.) by column switching. Quantification was performed by multiple reaction monitoring with negative-ion atmospheric pressure chemical ionization. Good peak separation and peak shapes of the eight drugs were achieved within an analysis time of 3 min, including the extraction time. All drugs spiked into plasma showed recoveries of 80–93 %. The regression equations for the eight drugs showed excellent linearities in the range of 10–5000 ng/ml of plasma, and the limits of detection ranged from 1.0 to 10 ng/ml. The lower and upper limits of quantitation were 10–50 ng/ml and 5000 ng/ml, respectively. Intraday and interday coefficients of variation for all the drugs were not >9.1 %. The accuracies of quantitation were 92.0–108 %. The method was successfully applied to determination of the level of amobarbital in human plasma after its oral administration to a volunteer.

Sequential extraction of amphetamines, opiates, and 11-nor-Δ 9 -tetrahydrocannabinol-9-carboxylic acid from a limited volume of urine using a monolithic silica spin column coupled with gas chromatography–mass spectrometry

Abstract: A method for sequential extraction of amphetamines (AMPs), opiates (OPIs), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from a limited volume of urine was developed. The method used two different spin columns packed with octadecyl (C18) and mixed-mode C18-strong anion exchange (SAX)-bonded monolithic silica for extraction before analysis by gas chromatography–mass spectrometry (GC–MS). The urine (0.5 ml), which was hydrolyzed with 10 M NaOH solution (0.1 ml), and 0.3 M Sorensen’s glycine buffer (pH 13, 0.1 ml) were poured into the preactivated C18-SAX spin column and then centrifuged at 10,000 rpm for 2 min to load the sample solution. AMPs and THC-COOH, which adsorbed to the column, were eluted with methanol containing 2% HCOOH. For the second extraction, the remaining solution, which was hydrolyzed by acid, and 0.2 M Sorensen’s glycine buffer (pH 12, 0.1 ml) were poured into the other preactivated C18 spin column. The adsorbed OPIs were subsequently eluted with methanol containing 2% NH4OH. The eluates were combined and the solvent evaporated under nitrogen before the target drugs in the residue were successively converted to O-trimethylsilyl-N-trifluoroacetyl derivatives by N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)-trimethylchlorosilane (TMCS) and N-methyl-bis(trifluoroacetamide) (MBTFA) followed analysis by GC–MS. Linearity from 10 to 1,000 ng/ml was observed for all the tested drugs using an internal standard method. The correlation coefficients of the calibration curves were greater than 0.990. The coefficients for intraday and interday variations at 50, 200, and 800 ng/ml in urine were between 0.7 and 11.1 %. The proposed method was applied to forensic and clinical poisoning cases and is considered suitable for routine analysis of drugs in biological materials.