Showing papers in "Journal of Analytical Toxicology in 2015"
TL;DR: An evaluation of the distribution of postmortem concentrations of butyr-fentanyl in a fatality attributed principally to the drug is presented.
Abstract: In this case report, we present an evaluation of the distribution of postmortem concentrations of butyr-fentanyl in a fatality attributed principally to the drug. A man who had a history of intravenous drug abuse was found unresponsive on the bathroom floor of his home. Drug paraphernalia was located on the bathroom counter. Toxicology testing, which initially screened positive for fentanyl by enzyme-linked immunosorbent assay, subsequently confirmed butyr-fentanyl, which was then quantitated by gas chromatography-mass spectrometry-specific ion monitoring (GC-MS SIM) analysis following liquid-liquid extraction. The butyr-fentanyl peripheral blood concentration was quantitated at 58 ng/mL compared with the central blood concentration of 97 ng/mL. The liver concentration was 320 ng/g, the vitreous was 40 ng/mL, the urine was 670 ng/mL and the gastric contained 170 mg. Acetyl-fentanyl was also detected in all biological specimens tested. Peripheral blood concentration was quantitated at 38 ng/mL compared with the central blood concentration of 32 ng/mL. The liver concentration was 110 ng/g, the vitreous was 38 ng/mL, the urine was 540 ng/mL and the gastric contained <70 mg. The only other drug detected was a relatively low concentration of benzoylecgonine. The cause of death was certified as acute butyr-fentanyl, acetyl-fentanyl and cocaine intoxication, and the manner of death was certified as accident.
122 citations
TL;DR: Cannabis' psychomotor, neurocognitive, subjective and physiological effects in occasional and frequent smokers are documented to investigate potential differences between these smokers and have implications for cannabis-associated impairment in driving under the influence of cannabis cases.
Abstract: Δ9-Tetrahydrocannabinol (THC), the primary psychoactive constituent in cannabis, impairs psychomotor performance, cognition and driving ability; thus, driving under the influence of cannabis is a public safety concern. We documented cannabis' psychomotor, neurocognitive, subjective and physiological effects in occasional and frequent smokers to investigate potential differences between these smokers. Fourteen frequent (≥4x/week) and 11 occasional (<2x/week) cannabis smokers entered a secure research unit ∼19 h prior to smoking one 6.8% THC cigarette. Cognitive and psychomotor performance was evaluated with the critical tracking (CTT), divided attention (DAT), n-back (working memory) and Balloon Analog Risk (BART) (risk-taking) tasks at −1.75, 1.5, 3.5, 5.5 and 22.5 h after starting smoking. GLM (General Linear Model) repeated measures ANOVA was utilized to compare scores. Occasional smokers had significantly more difficulty compensating for CTT tracking error compared with frequent smokers 1.5 h after smoking. Divided attention performance declined significantly especially in occasional smokers, with session × group effects for tracking error, hits, false alarms and reaction time. Cannabis smoking did not elicit session × group effects on the n-back or BART. Controlled cannabis smoking impaired psychomotor function, more so in occasional smokers, suggesting some tolerance to psychomotor impairment in frequent users. These data have implications for cannabis-associated impairment in driving under the influence of cannabis cases.
104 citations
TL;DR: This is the first known reported death attributed to the combined use of α-PVP and pentedrone and the first to report the distribution of pentedrones in postmortem human samples.
Abstract: We report a fatal case of combined α-pyrrolidinovalerophenone (α-PVP) and 2-(methylamino)-1-phenylpentan-1-one (pentedrone) poisoning. A 28-year-old man was taken to hospital in asystole. Despite resuscitation efforts over 30 min, he died. The forensic autopsy showed pulmonary edema and moderately advanced atherosclerotic lesions of the arteries. Microscopic observation revealed chronic changes in the heart. Confirmation of the presence of pentedrone, α-PVP, and its metabolite 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol (OH-α-PVP) in tissues and fluids were achieved using gas chromatography-mass spectrometry analysis after liquid-liquid extraction. A quantitative validated liquid chromatography-mass spectrometry method was used to determine the concentrations of the above designer drugs in postmortem samples. Pentedrone, α-PVP, and OH-α-PVP concentrations were 8,794, 901 and 185 ng/mL in whole blood, respectively; 100,044, 2,610 and 2,264 ng/g in the liver, respectively; 22,102, 462 and 294 ng/g in the kidney, respectively; 13,248, 120 and 91 ng/g in the brain, respectively and 500,534, 4,190 and 47 ng/g in the stomach contents, respectively. This is the first known reported death attributed to the combined use of α-PVP and pentedrone. Additionally, this article is the first to report the distribution of pentedrone in postmortem human samples. Language: en
95 citations
93 citations
TL;DR: A 24-year-old man whose medical history was significant for alcohol abuse and depression was found unresponsive in bed and postmortem findings were unremarkable apart from pulmonary edema and congestion, and urinary retention, and the lack of potential for mitragynine postmortem redistribution.
Abstract: A 24-year-old man whose medical history was significant for alcohol abuse and depression was found unresponsive in bed. He had several prior suicide attempts with ‘pills’ and had also been hospitalized for an accidental overdose on a previous occasion. Autopsy findings were unremarkable apart from pulmonary edema and congestion, and urinary retention. Postmortem peripheral blood initially screened positive for mitragynine ‘Kratom’ (by routine alkaline drug screen by gas chromatography –mass spectrometry, GC–MS), which was subsequently confirmed by a specific GC–MS selective ion mode analysis following solid-phase extraction. Concentrations were determined in the peripheral blood (0.23 mg/L), central blood (0.19 mg/L), liver (0.43 mg/kg), vitreous (<0.05 mg/L), urine (0.37 mg/L) and was not detected in the gastric. Therapeutic concentrations of venlafaxine, diphenhydramine and mirtazapine were also detected together with a negligible ethanol of 0.02% (w/v). The results are discussed in relation to previous cases of toxicity, and the lack of potential for mitragynine postmortem redistribution.
92 citations
TL;DR: Three different types of commercially available blotter papers reported to contain NBOMe derivatives were obtained and they were screened using Direct Analysis in Real Time AccuTOF(TM) mass spectrometry followed by confirmation and quantification by high-performance liquid chromatography triple quadrapole mass Spectrometry.
Abstract: In recent years, N-methoxybenzyl-methoxyphenylethylamine (NBOMe) derivatives, a class of designer hallucinogenic drugs, have become popular drugs of abuse. These drugs have been the cause of severe intoxications and even deaths. They act as 5-HT2A receptors agonists and have been reported to produce serotonin-like syndrome with bizarre behavior, severe agitation and seizures persisting for as long as 3 days. The most commonly reported derivatives are 25I-NBOMe, 25B-NBOMe and 25C-NBOMe, respectively 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl) methyl]ethanamine, N-(2-methoxybenzyl)-2,5-dimethoxy-4-bromophenethylamine and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine. Like many low dose hallucinogenic drugs these compounds are often sold on blotter paper. Three different types of commercially available blotter papers reported to contain NBOMe derivatives were obtained. These blotter papers were screened using Direct Analysis in Real Time AccuTOF(TM) mass spectrometry followed by confirmation and quantification by high-performance liquid chromatography triple quadrapole mass spectrometry. The major drug present on each of the three blotter products was different, 25I-NBOMe, 25C-NBOMe or 25B-NBOMe. The blotter papers were also found to have minute amounts of two or three NBOMe derivative impurities of 25H-NBOMe, 25I-NBOMe, 25C-NBOMe, 25B-NBOMe and/or 25D-NBOMe.
85 citations
TL;DR: The deaths of two teenage male subjects that were related to 25B- NBOMe and 25I-NBOMe in Indiana during 2014 are reported and relevant published casework is reviewed.
Abstract: Over the last few years, NBOMe substances have been used either as a legal alternative to lysergic acid diethylamide (LSD) or sold surreptitiously as LSD to unknown users. These NBOMe substances have been detected in blotter papers, powders, capsules and liquids. We report the deaths of two teenage male subjects that were related to 25B-NBOMe and 25I-NBOMe in Indiana during 2014. Samples were extracted via a solvent protein precipitation with acetonitrile and analyzed via ultra-performance liquid chromatography with tandem mass spectrometry. For these two cases, we describe the NBOMe instrumental analysis, toxicological results for postmortem heart blood and urine specimens and the relevant case history and pathological findings at autopsy. In the first case, 25B-NBOMe was detected in postmortem heart blood at 1.59 ng/mL; in the second case, 25I-NBOMe was detected in postmortem heart blood at 19.8 ng/mL. We also review relevant published casework from clinical toxicology and postmortem toxicology in which analytically confirmed 25B-NBOMe and 25I-NBOMe were determined to be causative agents in intoxications or deaths.
68 citations
TL;DR: This study offers the advantage of complete analytical methods for rapid and simultaneous multicomponent identification of nicotine, nitrosamines and PAHs in e-liquid samples obtained from the Greek market.
Abstract: The electronic cigarette (e-cig) is an invention of the past few years and its popularity is rapidly growing all over the world. A rapid multicomponent analytical protocol for the analysis of the replacement liquids (e-liquids) of e-cig was developed using gas (GC) and liquid chromatography (LC) – mass spectrometry (MS). GC – MS-based methods were developed for the determination of the main humectants and polycyclic aromatic hydrocarbons (PAHs). For the determination and quantification of nicotine (NIC) and nitrosamines, appropriate LC– MS-based methods were developed. The approbated methods were applied for the analysis of 263 e-liquid samples obtained from the Greek market. The instruments response was linear; the limits of quantification ranged from 0.003 mg/mL for three PAHs to 1.187 mg/mL for glycerol. The precision was <16% for all analytes, while the mean accuracy ranged from 99.1% for NIC to 106.6% for the flavor 2,5-dimethylpyrazine. The measured concentrations of NIC were correlated with the theoretical concentrations as reported by the manufacturers. An analog relation between the concentration of the glycerol and of propylene glycol was noticed. The frequency of detection of flavors ranged from 30.4% for the methyl cyclopentenolone to 5.3% for 3.4-dimethoxybenzaldehyde. Nitrosamines and PAHs were not detected in any sample. Because a similar analytical protocol was not available from the existing literature so far, our study offers the advantage of complete analytical methods for rapid and simultaneous multicomponent identification.
59 citations
TL;DR: It is demonstrated that extreme cannabis smoke exposure can produce positive urine tests at commonly utilized cutoff concentrations, however, positive tests are likely to be rare, limited to the hours immediately post-exposure, and occur only under environmental circumstances where exposure is obvious.
Abstract: Increased cannabis potency has renewed concerns that secondhand exposure to cannabis smoke can produce positive drug tests. A systematic study was conducted of smoke exposure on drug-free participants. Six experienced cannabis users smoked cannabis cigarettes (5.3% THC in Session 1 and 11.3% THC in Sessions 2 and 3) in a sealed chamber. Six non-smokers were seated with smokers in an alternating manner. Sessions 1 and 2 were conducted with no ventilation and ventilation was employed in Session 3. Non-smoking participant specimens (collected 0‐34 h) were analyzed with four immunoassays at different cutoff concentrations (20, 50, 75 and 100 ng/mL) and by GC-MS (LOQ 5 0.75 ng/mL). No presumptive positives occurred for non-smokers at 100 and 75 ng/mL; a single positive occurred at 50 ng/mL; and multiple positives occurred at 20 ng/ mL. Maximum THCCOOH concentrations by GC-MS for non-smokers ranged from 1.3 to 57.5 ng/mL. THCCOOH concentrations generally increased with THC potency, but room ventilation substantially reduced exposure levels. These results demonstrate that extreme cannabis smoke exposure can produce positive urine tests at commonly utilized cutoff concentrations. However, positive tests are likely to be rare, limited to the hours immediately post-exposure, and occur only under environmental circumstances where exposure is obvious.
58 citations
TL;DR: In situ thermal degradation products for 18 cathinones were identified during gas chromatography-mass spectrometry (GC-MS) analysis and presented and discussed within the context of forensic toxicological analysis, selection of appropriate instrumental methods and implications for the interpretation of results.
Abstract: The synthetic cathinones represent an important class of designer drugs. The widespread attention and publicity associated with these psychostimulants have resulted in numerous legislative actions at state and federal levels throughout the USA. These amphetamine-like compounds are characterized by a β-keto functional group. Although the synthetic cathinones share many properties of their phenethylamine counterparts, the presence of the ketone moiety is responsible for a number of unique and distinct differences in terms of their chemical characteristics and properties. Thermal degradation of methcathinone was first reported several decades ago but has received limited attention. In this study, we identified in situ thermal degradation products for 18 cathinones during gas chromatography-mass spectrometry (GC-MS) analysis. Oxidative degradation arises from the loss of two hydrogens, yielding a characteristic 2 Da mass shift. Degradation products were characterized by prominent iminium base peaks with mass-to-charge ratios 2 Da lower than the parent drug, and in the case of the pyrrolidine-containing cathinones, prominent molecular ions arising from the 2,3-enamine. Chromatographic and mass spectroscopic data are described for 4-ethylmethcathinone, 4-methylethcathinone, buphedrone, butylone, ethcathinone, ethylone, flephedrone, 3,4-methylenedioxy-α-pyrrolidinobutiophenone, 3,4-methylenedioxypyrovalerone, mephedrone, methcathinone, methedrone, methylone, 4-methyl-α-pyrrolidinobutiophenone, naphyrone, pentedrone, pentylone and pyrovalerone. Degradation was minimized by lowering injection temperatures, residence time in the inlet and eliminating active sites during chromatographic analysis. Chromatographic and mass spectral data for the cathinone degradation products are presented and discussed within the context of forensic toxicological analysis, selection of appropriate instrumental methods and implications for the interpretation of results.
57 citations
TL;DR: An analytical method was developed and validated for the purpose of detecting and quantifying 37 new designer drugs including cathinones, hallucinogenic phenethylamines and piperazines using only 100 µL whole blood.
Abstract: An analytical method was developed and validated for the purpose of detecting and quantifying 37 new designer drugs including cathinones, hallucinogenic phenethylamines and piperazines. Using only 100 µL whole blood, a salting-out-assisted liquid-liquid extraction with acetonitrile was performed to isolate target compounds followed by chromatographic separation using a Waters ACQUITY ultra performance liquid chromatograph coupled to a Waters XEVO quadrupole time-of-flight mass spectrometer. Mephedrone-d3 was used as an internal standard. A gradient elution was used in combination with a Waters ACQUITY HSS C18 column (2.1 × 150 mm, 1.8 µm). Samples were analyzed using the detector in positive electrospray ionization mode with MS(E) acquisition. All compounds of interest were resolved in a 15 min run time and positively identified based on accurate mass of the molecular ion, two product ions and retention time. All analyte calibration curves were linear over the range of 0.05-2 mg/L with most correlation coefficient (r(2)) values >0.98. The limits of detection were within the range of 0.007-0.07 mg/L and limits of quantification within 0.05-0.1 mg/L. All analytes were stable 48 h after extraction and most were stable in blood after 1 week stored in a refrigerator and 3 freeze-thaw cycles. No carryover was observed up to 10 mg/L and no interferences from common therapeutic drugs or endogenous compounds. Recoveries ranged from 71 to 100% and matrix effects were assessed for blank, post-mortem and decomposed blood. All bias and % coefficient of variation values were within the acceptable values of ±15 and ≤15%, respectively (±20 and ≤20% at lower limit of quantification). The method was applied to several forensic cases where the subject exhibited behavior characteristic of designer drug intoxication and where routine screening for a panel of drugs was negative.
TL;DR: Subjective effect measures and amounts of THC absorbed by nonsmokers (relative to smokers) indicated that extreme secondhand cannabis smoke exposure mimicked, though to a lesser extent, active cannabis smoking.
Abstract: The increasing use of highly potent strains of cannabis prompted this new evaluation of human toxicology and subjective effects following passive exposure to cannabis smoke. The study was designed to produce extreme cannabis smoke exposure conditions tolerable to drug-free nonsmokers. Six experienced cannabis users smoked cannabis cigarettes [5.3% Δ(9)-tetrahydrocannabinol (THC) in Session 1 and 11.3% THC in Sessions 2 and 3] in a closed chamber. Six nonsmokers were seated alternately with smokers during exposure sessions of 1 h duration. Sessions 1 and 2 were conducted with no ventilation and ventilation was employed in Session 3. Oral fluid, whole blood and subjective effect measures were obtained before and at multiple time points after each session. Oral fluid was analyzed by ELISA (4 ng/mL cutoff concentration) and by LC-MS-MS (limit of quantitation) for THC (1 ng/mL) and total THCCOOH (0.02 ng/mL). Blood was analyzed by LC-MS-MS (0.5 ng/mL) for THC, 11-OH-THC and free THCCOOH. Positive tests for THC in oral fluid and blood were obtained for nonsmokers up to 3 h following exposure. Ratings of subjective effects correlated with the degree of exposure. Subjective effect measures and amounts of THC absorbed by nonsmokers (relative to smokers) indicated that extreme secondhand cannabis smoke exposure mimicked, though to a lesser extent, active cannabis smoking.
TL;DR: This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument and finds no designer drug used in this study generated a positive result for all five immunoASSay kits.
Abstract: The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), β-keto amphetamines, substituted amphetamines, piperazines, α-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits.
TL;DR: Three deaths which involved the detection of 2-MXP in post-mortem blood and urine were encountered in forensic casework and analytical data is presented to assist analytical toxicologists with future casework.
Abstract: 2-Methoxydiphenidine, i.e. 1-[1-(2-methoxyphenyl)-2-phenylethyl]piperidine, also known as 'MXP' or '2-MeO-diphenidine' (or 2-MXP), has been available as a 'research chemical' since 2013 as a purported alternative to the 'dissociative anesthetics' methoxetamine and ketamine. Three deaths which involved the detection of 2-MXP in post-mortem blood and urine were encountered in forensic casework. The 2-, 3- and 4-methoxyphenyl positional isomers were synthesized to confirm the identity and concentration of 2-MXP. The 2-MXP femoral blood concentrations in the cases were found to be 24.0, 2.0 and 1.36 mg/L (the latter with an alternative cause of death). Some additional prescription drugs were encountered at therapeutic concentrations in all three cases. Analysis of the biofluids allowed the detection and characterization of various metabolites, including the suggested presence of hydroxy-2-MXP as the main metabolite with the hydroxyl group located on the piperidine rather than the phenyl or benzyl moiety. Additional metabolites included O-desmethyl-2-MXP and hydroxylated O-desmethyl-2-MXP. Diphenidine and hydroxy-diphenidine, also showing the presence of the hydroxyl group on the piperidine ring, were also detected. It was not possible to identify whether these arose from 2-MXP biotransformation or whether they represented the presence of diphenidine as a separate substance. These are the first published fatalities involving 2-MXP and presents analytical data to assist analytical toxicologists with future casework.
TL;DR: Investigations of authentic urine samples from forensic cases in combination with human liver microsome experiments were used for identification of metabolites, illustrating the need for a systematic approach to identify unique metabolites of AKB-48 and 5F-AKb-48.
Abstract: The occurrence of structurally related synthetic cannabinoids makes the identification of unique markers of drug intake particularly challenging. The aim of this study was to identify unique and abundant metabolites of AKB-48 and 5F-AKB-48 for toxicological screening in urine. Investigations of authentic urine samples from forensic cases in combination with human liver microsome (HLM) experiments were used for identification of metabolites. HLM incubations of AKB-48 and 5F-AKB-48 along with 35 urine samples from authentic cases were analyzed with liquid chromatography quadrupole tandem time of flight mass spectrometry. Using HLMs 41 metabolites of AKB-48 and 37 metabolites of 5F-AKB-48 were identified, principally represented by hydroxylation but also ketone formation and dealkylation. Monohydroxylated metabolites were replaced by di- and trihydroxylated metabolites within 30 min. The metabolites from the HLM incubations accounted for on average 84% (range, 67-100) and 91% (range, 71-100) of the combined area in the case samples for AKB-48 and 5F-AKB-48, respectively. While defluorinated metabolites accounted for on average 74% of the combined area after a 5F-AKB-48 intake only a few identified metabolites were shared between AKB-48 and 5F-AKB-48, illustrating the need for a systematic approach to identify unique metabolites. HLMs in combination with case samples seem suitable for this purpose.
TL;DR: In principle, HETE-CP undergoes a dynamic on-column equilibrium of cis-trans isomerism thus requiring separation at 50°C to obtain one narrow peak, and a novel longer lasting but more sensitive microbore is developed.
Abstract: Sulfur mustard (SM) is a chemical warfare agent that causes painfulblisters and chemically modifies endogenous biomacromolecules byalkylation to hydroxyethylthioethyl (HETE) adducts representing valu-able long-term markers for post-exposure analysis. The albumin ad-duct formed in human plasmain vitro (HETE bound to the sidechain of cysteine 34) was isolated and cleaved by current lots ofpronase primarily generating the internal modified dipeptide(HETE–cysteine–proline, HETE–CP) instead of the formerly reportedHETE–CPF tripeptide. The analyte was detected by liquid chromatog-raphy–electrospray ionization tandem-mass spectrometry (LC–ESI–MS-MS). In principle, HETE–CP undergoes a dynamic on-columnequilibrium of cis–trans isomerism thus requiring separation at508C to obtain one narrow peak. Accordingly, we developed both anovel longer lasting but more sensitive microbore (1 mm i.d., flow30 mL/min, cycle time 60 min, LOD 50 nM) and a faster, less sensi-tive narrowbore (2.1 mm i.d., 200 mL/min,cycletime16min,LOD100 nM, both on Atlantis T3 material at 508C) LC–ESI–MS-MS meth-od suitable for verification analysis. The corresponding tri- and tetra-peptide, Q(HETE)–CPF were monitored simultaneously. HETE–CPpeak areas were directly proportional to SM concentrations addedto plasma in vitro (0.05–100mM). Albumin adducts formed bydeuterated SM (d8-SM) served as internal standard.IntroductionSulfur mustard (SM, bis(2-chloroethyl)sulfide, CAS no. 505-60-2)is a chemical warfare agent belonging to the class of vesicants.Exposed skin areas may develop erythema and painful blistersthat are characterized by complicated and delayed woundhealing (1).Worldwide stockpiled SM is controlled by the Organisation forthe Prohibition of Chemical Weapons (OPCW, Nobel Peace Prizelaureate of2013) and scheduled for destruction accordingto theChemical Weapons Convention. Very recently, stocks of SM de-claredbytheSyrian ArabRepublicweredestroyedbyalkalinehy-drolysis on the specially equipped US container ship MV CapeRay (2). Nevertheless, SM that might be synthesized in smallerscalewithmoderateexpertisestillrepresentsathreatforthemil-itary and civilian population especially in asymmetric or terroris-tic scenarios. Therefore, bioanalytical methods are demandedallowing evidence of exposure to SM. Due to the low stabilityand high reactivity of that poison, detection of the original com-pound or its hydrolysis products in biological specimens is amajor challenge for verification analysis. Sample drawing even afew hours after exposure prevent successful detection of SM,whereas thiodiglycol (TDG, the most prominent product afterhydrolysis) can be measured in urine for days. The most long-lived markers are covalent reaction products (adducts) to pro-teins (e.g., albumin or hemoglobin) and DNA (1). SM-adductswith diverse valine and histidine residues of hemoglobin wereidentified by Noort et al. (3) and corresponding peptide andamino acid derivatives were established as targets of sophisticat-ed analytical procedures for verification (4). Additional analyticalmethods are based on non-enzymatic hydrolytic cleavage of theSM-derived moiety from any protein thus generating TDG forsubsequent gas chromatography–mass spectrometric (GC–MS)analysis (5).SM–albumin adducts have been shown to have a half-lifein vivo of 3–4 weeks (6)thusprovidingamuchlongertimeframe for verification post-exposure. SM is known to alkylatetheonlyfree,notdisulfide-linkedcysteineresidueinhumanalbu-min producing an S-linked hydroxyethylthioethyl-derivative(HETE–albumin) (6). This chemical modification has been thetarget of diverse bioanalytical procedures (7–13) that are allbased on the fundamental work of Noort et al. (6). Accordingto this strategy albumin and its HETE adducts are extractedfrom plasma or serum by affinity chromatography, desalted onPD-10 columns and proteolytically cleaved by pronase.Pronase—a mixture of diverse endo- and exopeptidases of unde-fined composition (14)—was reported to generate an alkylatedtripeptide, HETE–CPF, suitablefor reliable analysis by liquidchromatography–electrospray ionization tandem-mass spec-trometry (LC–ESI–MS-MS) (6). In reversed-phase (RP) chroma-tography this peptide derivative elutes as a narrow peakproducing two major product ions after collision-induced-dissociation (CID) at m/z 105 and m/z 137 (6).When trying to establish this procedure in our laboratory wefailed to detect that HETE–CPF tripeptide. This unexpected re-sult presumed to be due to variable enzyme activity of the differ-ent lots of pronase used. Instead of the tripeptide we identifiedthe HETE–CP dipeptide nearly exclusively. Even though the oc-currence of this proteolysis product has been mentioned before(6), no report has focused on that marker for verification analysissofar.Thisfactmightprimarilybeduetothenon-favorablebroadelution profile of the dipeptide in RP-chromatography that ham-pers the most sensitive analysis.However, based on the fact that many if not most current lotsof commercially available pronase predominantly produce thedipeptide instead of the tripeptide, we present the developmentof a method targeting the HETE–CP molecule for verificationanalysis. Following this idea, we unraveled the scientific natureof its broad peak phenomenon, modified chromatographic
TL;DR: The developed method enabled successful analysis of the target drugs in plasma samples obtained from 51 schizophrenic patients and revealed altered plasma concentrations of the analyzed drugs resulted from pharmacokinetic interactions among the medications prescribed to treat schizophrenia.
Abstract: This work describes the development of a simple, sensitive and selective method based on high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) to determine antipsychotics (olanzapine, quetiapine, clozapine, haloperidol and chlorpromazine) along with antidepressants (mirtazapine, paroxetine, citalopram, sertraline, imipramine, clomipramine and fluoxetine), anticonvulsants (carbamazepine and lamotrigine) and anxiolytics (diazepam and clonazepam) in plasma samples obtained from schizophrenic patients. The samples were prepared by protein precipitation. The target drugs were separated on an XSelect SCH C18 column (100 mm × 2.1 mm × 2.5 µm) within 8.0 min by means of gradient elution. The drugs were then detected on a quadrupole tandem mass spectrometer equipped with an electrospray ionization source, operating in the multiple reactions monitoring mode and in the positive ionization mode. The LC-MS-MS method was linear range from subtherapeutic to toxic concentrations with lower limit of quantification values ranged from 0.2 to 5.0 ng mL(-1), precision with coefficient of variation values lower than 12%, and accuracy ranged from 90 to 108%. The developed method enabled successful analysis of the target drugs in plasma samples obtained from 51 schizophrenic patients. Therapeutic drug monitoring revealed that many of the evaluated schizophrenic patients presented altered plasma concentrations of the analyzed drugs. These altered concentrations resulted from pharmacokinetic interactions among the medications prescribed to treat schizophrenia.
TL;DR: A 15-year-old male went to a party where he ingested 25I-NBOMe and mushrooms and began seizing until he eventually passed out, and died three days later of multi-system organ failure following cardiopulmonary arrest.
Abstract: This case was submitted to the Washington State Patrol Toxicology Laboratory in September 2014. A 15-year-old male went to a party where he ingested 25I-NBOMe and mushrooms. A short time later, he started to vomit and began seizing until he eventually passed out. Resuscitation efforts were made, but were unsuccessful. He was transported to a local hospital, where he died three days later of multi-system organ failure following cardiopulmonary arrest. The hospital admission samples were negative for ethanol and basic drugs and their metabolites. The hospital serum confirmed positive for delta-9-tetrahydrocannabinol (THC) and carboxy-THC at 4.1 and 83 ng/mL, respectively. On the basis of the case history, the hospital blood and urine were sent to NMS Labs for NBOMe and psilocin confirmation. The blood was positive for 25I-NBOMe, and the urine was positive for 25C-, 25H- and 25I-NBOMe, as well as, psilocin. Antemortem and postmortem blood were also sent to AIT Laboratories for NBOMe confirmation. The antemortem blood confirmed positive for 25I-NBOMe with a concentration of 0.76 ng/mL. The manner of death was ruled an accident as a result of combined 25I-NBOMe and psilocin intoxication.
TL;DR: A novel LC-MS-MS assay that simultaneously detects and quantitates 78 drugs and metabolites was developed and validated for chronic pain management, improving on currently existing assays by including glucuronide conjugates, allowing direct detection of metabolites that might otherwise be missed by existing methods.
Abstract: A novel LC-MS-MS assay that simultaneously detects and quantitates 78 drugs and metabolites was developed and validated for chronic pain management. Urine specimen was diluted and mixed with internal standards (ISs) before injected into LC-MS-MS. Seventy-two analytes were detected with positive electrospray ionization mode and the remaining six analytes with negative mode. Two separate gradient elution chromatographic programs were established with the same mobile phases on the same bi-phenyl HPLC column. The assay was linear for all analytes with linear regression coefficient ranging 0.994-1.000. The intra-assay precision was between 1.7 and 8.8% and inter-assay precision between 1.9 and 12.2%, with bias <20% for all but six analytes. All analytes in urine specimens were stable for 7 days at 4°C, and no significant matrix effect or carryover was observed. A suboptimal recovery rate (60.0-156.8%) was observed for six analytes, potentially due to the lack of available deuterated ISs, requiring comparison to a chemically different IS. Method comparison using patient and proficiency testing samples demonstrated that this assay was sensitive and accurate. The assay improves on currently existing assays by including glucuronide conjugates, allowing direct detection of metabolites that might otherwise be missed by existing methods.
TL;DR: Postmortem blood initially screened positive for methamphetamine by ELISA, but 5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-APDB) was presumptively identified in both peripheral blood and urine and Alcohol, the only other drug identified, was confirmed at a concentration of 0.02% (w/v).
Abstract: A 20-year-old man, a college student, became unresponsive in front of his girlfriend. He was known to consume alcohol and take an unknown drug at some point while in attendance at a local music festival earlier in the day/evening. Upon arrival of emergency personnel, he was noted to be asystolic and apneic. Despite aggressive medical intervention by emergency personnel and at a local hospital emergency room, he was pronounced deceased within 1.25 h of initial medical attention. Postmortem blood initially screened positive for methamphetamine by ELISA. An alkaline drug screen detected 5-(2-aminopropyl)benzofuran (5-APB) which was subsequently confirmed and quantified by a specific GC-MS SIM analysis following solid-phase extraction. Concentrations were determined in the peripheral blood (2.5 mg/L), central blood (2.9 mg/L), liver (16 mg/kg), vitreous (1.3 mg/L), urine (23 mg/L) and gastric contents (6 mg). No other common amphetamine-like compound was detected, although 5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-APDB) was presumptively identified in both peripheral blood and urine. Alcohol, the only other drug identified, was confirmed at a concentration of 0.02% (w/v). Language: en
TL;DR: The chronic frequent cannabis smokers exhibited extended detection windows for plasma cannabinoids, reflecting a large cannabinoid body burden, and greatly expand prior research findings on cannabinoid excretion profiles in chronic frequent Cannabis smokers during ad libitum smoking.
Abstract: More Americans are dependent on cannabis than any other illicit drug. The main analytes for cannabis testing include the primary psychoactive constituent, Δ(9)-tetrahydrocannabinol (THC), equipotent 11-hydroxy-THC (11-OH-THC) and inactive 11-nor-9-carboxy-THC (THCCOOH). Eleven adult chronic frequent cannabis smokers resided on a closed research unit with unlimited access to 5.9% THC cannabis cigarettes from 12:00 to 23:00 during two ad libitum smoking phases, followed by a 5-day abstinence period in seven participants. A single cigarette was smoked under controlled topography on the last day of the smoking and abstinence phases. Plasma cannabinoids were quantified by two-dimensional gas chromatography-mass spectrometry. Median plasma maximum concentrations (Cmax) were 28.3 (THC), 3.9 (11-OH-THC) and 47.0 μg/L (THCCOOH) 0.5 h after controlled single cannabis smoking. Median Cmax 0.2-0.5 h after ad libitum smoking was higher for all analytes: 83.5 (THC), 14.2 (11-OH-THC) and 155 μg/L (THCCOOH). All 11 participants' plasma samples were THC and THCCOOH-positive, 58.3% had THC ≥5 μg/L and 79.2% were 11-OH-THC-positive 8.1-14 h after last cannabis smoking. Cannabinoid detection rates in seven participants 106-112 h (4-5 days) after last smoking were 92.9 (THC), 35.7 (11-OH-THC) and 100% (THCCOOH), with limits of quantification of 0.5 μg/L for THC and THCCOOH, and 1.0 μg/L for 11-OH-THC. These data greatly expand prior research findings on cannabinoid excretion profiles in chronic frequent cannabis smokers during ad libitum smoking. Smoking multiple cannabis cigarettes led to higher Cmax and AUC compared with smoking a single cigarette. The chronic frequent cannabis smokers exhibited extended detection windows for plasma cannabinoids, reflecting a large cannabinoid body burden.
TL;DR: A new headspace-gas chromatography-mass spectrometry method applicable to the routine determination of hydrogen sulfide (H(2)S) concentrations in biological and gaseous samples is presented and a full validation using accuracy profile based on the β-expectation tolerance interval is presented.
Abstract: The aim of our study was to present a new headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable to the routine determination of hydrogen sulfide (H2S) concentrations in biological and gaseous samples. The primary analytical drawback of the GC/MS methods for H2S measurement discussed in the literature was the absence of a specific H2S internal standard required to perform quantification. Although a deuterated hydrogen sulfide (D2S) standard is currently available, this standard is not often used because this standard is expensive and is only available in the gas phase. As an alternative approach, D2S can be generated in situ by reacting deuterated chloride with sodium sulfide; however, this technique can lead to low recovery yield and potential isotopic fractionation. Therefore, N2O was chosen for use as an internal standard. This method allows precise measurements of H2S concentrations in biological and gaseous samples. Therefore, a full validation using accuracy profile based on the b-expectation tolerance interval is presented. Finally, this method was applied to quantify H2 Si n an actual case of H2S fatal intoxication.
TL;DR: A novel LC-MS-MS method for the detection of 23 commonly prescribed antihypertensive medications in urine that has the potential to significantly improve investigation and management of resistant hypertension.
Abstract: Arterial hypertension is one of the most preventable causes of premature morbidity and mortality with resistant hypertension reported to be present in 5-30% of the total hypertensive population. Despite the poor prognosis, as many as 53% of those with resistant hypertension are reported to be nonadherent to their prescribed medication. An objective test of adherence, which is easy to administer, quick, inexpensive and reliable, is therefore needed to identify patients with true resistance to antihypertensive drugs to optimize their treatment. We have developed a novel LC-MS-MS method for the detection of 23 commonly prescribed antihypertensive medications in urine. The validated method was subsequently applied to the analysis of urine from a cohort of 49 individuals who were taking at least one antihypertensive agent in the screening profile to determine their adherence. The screening method was found to be reproducible, sensitive and specific with the limit of detection ranging from 0.1 to 1.0 µg/L. Sample preparation is rapid (30 s) and simple, with a total analysis time of 11 min. The assay successfully identified the majority of drugs our cohort had admitted to taking (88%) with drugs not detected in urine, potentially indicating nonadherence to prescribed medication. The performance of this simple, robust LC-MS-MS procedure is suitable for screening urine for the presence of commonly prescribed antihypertensive medications. The assay, which can easily be implemented in other laboratories, has the potential to significantly improve investigation and management of resistant hypertension.
TL;DR: The developed DLLME-auto-IPS-GC-MS-MS method is time, labor, solvent and reagent saving, which can be routinely used for the analysis of urinary PAH metabolites, and has been successfully applied for the determination ofPAH metabolites in urine samples of exposed workers.
Abstract: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and well-known carcinogens. Hydroxy derivatives of PAH are considered as biomarkers of PAH exposure, and there is a need to measure these metabolites at low concentrations. So, a precise and eco-friendly analytical method has been developed for rapid determination of PAH metabolites. For the first time, a new analytical method based on coupling of dispersive liquid-liquid microextraction (DLLME) with auto-injector port silylation (auto-IPS) followed by gas chromatography-tandem mass spectrometry (GC-MS-MS) analysis is reported for the analysis of seven urinary PAH metabolites. Factors affecting DLLME and IPS, such as type and volume of extraction and disperser solvent, pH, ionic strength, injector port temperature, volume of N,O-bis(trimethylsilyl)trifluoroacetamide and type of solvent were investigated. Under optimized conditions, the limit of detection and limit of quantification were found to be in the range of 1-9 and 3-29 ng/mL, respectively. Satisfactory recoveries of metabolites in urine samples in the range of 87-95% were found. The developed method has been successfully applied for the determination of PAH metabolites in urine samples of exposed workers. DLLME-auto-IPS-GC-MS-MS method is time, labor, solvent and reagent saving, which can be routinely used for the analysis of urinary PAH metabolites.
TL;DR: A new analytical method was developed and applied to determine concentrations of 10 toxic metals in little cigar tobacco including uranium, which was added as an analyte in the new method because of the elimination of interfering ions at 'shifted analyte masses'.
Abstract: Smoking remains the leading cause of preventable death in the USA. Much of the focus on harmful and potentially harmful constituents (HPHCs) in tobacco products has been on cigarettes. Little cigars gained popularity over the last decade until tobacco taxes made cigarettes more expensive in the USA. Many little cigar brands are similar in size with cigarettes and may be smoked in a similar manner. Scant data are available on HPHC concentrations in little cigars, therefore we developed and applied a new analytical method to determine concentrations of 10 toxic metals in little cigar tobacco. The method utilizes 'triple quadrupole' ICP-MS. By optimizing octapole bias, energy discrimination and cell gas flow settings, we were able to accurately quantify a range of elements including those for which the cell gas reactions were endothermic. All standard modes (Single Quad No Gas, MS-MS NH3/He and MS-MS O2) were utilized for the quantitation of 10 toxic metals in little cigar tobacco, including uranium, which was added as an analyte in the new method. Because of the elimination of interfering ions at 'shifted analyte masses', detection limits were lower compared with a previous method. Tobacco selenium concentrations were below the limit of detection in the previous method, but the new technology made it possible to report all selenium concentrations.
TL;DR: Nails (fingernails and toenails) are made of keratin, and as the nail grows, substances incorporate into the keratin fibers where they can be detected 3-6 months after use.
Abstract: Nails (fingernails and toenails) are made of keratin. As the nail grows, substances incorporate into the keratin fibers where they can be detected 3-6 months after use. Samples are collected by clipping of 2-3 mm of nail from all fingers (100 mg). We present drug testing results from 10,349 nail samples collected from high-risk cases during a 3-year period of time. Samples were analyzed by validated analytical methods. The initial testing was performed mostly using enzyme-linked immunosorbent assay, but by liquid chromatography-tandem mass spectrometry (LC-MS-MS) as well. Presumptive positive samples were subjected to confirmatory testing with sample preparation procedures including washing, pulverizing, digestion and extraction optimized for each drug class. The total of 7,799 samples was analyzed for amphetamines. The concentrations ranged from 40 to 572,865 pg/mg (median, 100-3,687) for all amphetamine analytes. Amphetamine and methamphetamine were present in 14% of the samples, 22 samples were positive for 3,4-methylenedioxymethamphetamine (0.3%), 7 for methylenedioxyamphetamine (0.09%) and 4 for 3,4-methylenedioxy-N-ethylamphetamine (0.05%). Cocaine and related analytes were found in 5% samples (7,787 total), and the concentration range was 20-265,063 pg/mg (median 84-1,768). Opioids overall ranged from 40 to 118,229 pg/mg (median 123-830). The most prevalent opioid was oxycodone (15.1%) and hydrocodone (11.4%) compared with 1.0-3.6% for the others, including morphine, codeine, hydromorphone, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and oxymorphone. Carboxy-Δ-9-tetrahydrocannabinol positivity rate was 18.1% (0.04-262 pg/mg, median 6.41). Out of 3,039 samples, 756 were positive (24.9%) for ethyl glucuronide (20-3,754 pg/mg, median 88). Other drugs found in nails included barbiturates, benzodiazepines, ketamine, meperidine, tramadol, zolpidem, propoxyphene, naltrexone and buprenorphine. Nail analyses have become a reliable way of determining the long-term use and abuse of drugs. Extraction techniques are simple and produce accurate and precise results. Sensitive analytical instrumentation, mainly LC-MS-MS, allows for detection of femtogram (10(-15) g) quantities of substances in nails. Samples were from a high-risk population, therefore the extraordinary positivity rate was observed.
TL;DR: The present report documents postmortem blood concentrations of ethylone, a novel synthetic cathinone, along with other concurrently identified substances, providing valuable information for developing analytical assays and evaluating a toxic concentration range of Ethylone.
Abstract: Synthetic cathinones are an emerging class of designer drugs, frequently with deceptive labels and a multitude of analogs to circumvent drug control regulations. Research regarding the pharmacological effects and toxicity of these amphetamine derivatives is scarce, heightening the risk to the public health and safety. The composition of synthetic cathinone products continually changes and laboratories began to notice ethylone-positive products in late 2011. This report presents nine postmortem cases in whom ethylone was identified. Ethylone was isolated using solid-phase extraction and detected by gas chromatography-mass spectrometry. Seven of the cases had measurable concentrations of ethylone in blood, ranging from 38 to 2,572 ng/mL; ethylone was detected in the blood sample of one case with a concentration below the assay limit of quantification (25 ng/mL), and one case did not have detectable ethylone in blood. Besides ethylone, all but one case were also positive for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; seven cases had other drugs quantified in blood, including ethanol, alprazolam, benzoylecgonine, diphenhydramine, morphine and tramadol. In five cases where ethylone was present at blood concentrations >400 ng/mL, no other drugs excluding ethanol, cannabis metabolite and doxylamine (one case) were found. The assay also tested for mephedrone, methylone and three dimethoxyamphetamine analogs; no case was positive for these analytes. The present report documents postmortem blood concentrations of ethylone, a novel synthetic cathinone, along with other concurrently identified substances. The findings provide valuable information for developing analytical assays and evaluating a toxic concentration range of ethylone.
TL;DR: A fast, 'dilute-and-shoot' method that removes the unnecessary, costly and time-consuming extraction steps found in traditional methods and still surpasses all analytical requirements is highlighted.
Abstract: Opioid testing represents a dominant share of the market in pain management clinical testing facilities. Testing of this drug class in oral fluid (OF) has begun to rise in popularity. OF analysis has traditionally required extensive clean-up protocols and sample concentration, which can be avoided. This work highlights the use of a fast, 'dilute-and-shoot' method that performs no considerable sample manipulation. A quantitative method for the determination of eight common opioids and associated metabolites (codeine, morphine, hydrocodone, hydromorphone, norhydrocodone, oxycodone, noroxycodone and oxymorphone) in OF is described herein. OF sample is diluted 10-fold in methanol/water and then analyzed using an Agilent chromatographic stack coupled with an AB SCIEX 4500. The method has a 2.2-min LC gradient and a cycle time of 2.9 min. In contrast to most published methods of this particular type, this method uses no sample clean-up or concentration and has a considerably faster LC gradient, making it ideal for very high-throughput laboratories. Importantly, the method requires only 100 μL of sample and is diluted 10-fold prior to injection to help with instrument viability. Baseline separation of all isobaric opioids listed above was achieved on a phenyl-hexyl column. The validated calibration range for this method is 2.5-1,000 ng/mL. This 'dilute-and-shoot' method removes the unnecessary, costly and time-consuming extraction steps found in traditional methods and still surpasses all analytical requirements.
TL;DR: Ten phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified and one human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations.
Abstract: 'NBOMe' (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing.
TL;DR: The stability of fentanyl was assessed when stored at three different temperatures in synthetic urine and the excellent comparability between DLLME and LLE is demonstrated and the use of this methodology in the analysis of forensically relevant samples is discussed.
Abstract: Fentanyl is a synthetic narcotic anesthetic ∼80-100 times more potent than morphine. Owing to the potential for its abuse, the drug may be included in a forensic toxicology work-up, which requires fast, precise and accurate measurements. Here, the stability of fentanyl was assessed when stored at three different temperatures (-20, 4 and 25°C) in synthetic urine. Stability at those three temperatures was demonstrated over 12 weeks upon analysis by gas chromatography-mass spectrometry with a deuterated internal standard (fentanyl-D5) utilizing three different extraction techniques: liquid-liquid extraction (LLE), solid-phase extraction and dispersed liquid-liquid microextraction (DLLME). The DLLME method was then optimized before use in the analysis of fentanyl in urine samples obtained from autopsy cases at the El Paso County Coroner's Office. Accuracy of the DLLME method was assessed by completing spike and recovery studies at three different fortification levels (10, 100 and 250 ng/mL) with excellent recovery (89.9-102.6%). The excellent comparability between DLLME and LLE is demonstrated (Bland-Altman difference plot with a mean difference of 4.9 ng/mL) and the use of this methodology in the analysis of forensically relevant samples is discussed.