Journal of analytical toxicology最新文献

Abacavir (ABC), Dolutegravir (DTG), and Lamivudine (3TC) are part of a fixed-dose combination medication for treatment of HIV. The three drugs offer different but complementary mechanisms of action by inhibiting reverse transcriptase and integrase, and ultimately inhibiting HIV replication. Due to lack of information regarding long-term safety following in utero exposure, we are evaluating potential toxicity to offspring following in utero exposure to this combination therapy in Hsd:Sprague Dawley®SD® (HSD) rats, including cardiovascular toxicity and neurotoxicity. Generating internal exposure data are integral to putting toxicological findings into context. The objective of this work was to develop and validate a method to simultaneously quantitate ABC, DTG, and 3TC in rat matrices following exposure to this combination. The method used protein precipitation of plasma, fetal, placental, brain, or heart homogenate followed by ultra-performance liquid chromatography-tandem mass spectrometry. In adult Sprague Dawley rat plasma, the method was linear (r≥0.99) over the range 10/15/5 to 10,000/15,000/5000 ng/mL for ABC/DTG/3TC and recovery was ≥92% for all three analytes at all concentration levels. The limits of detection were 2.22, 3.69, and 0.978 ng/mL for ABC, DTG, and 3TC, respectively. Intra- and inter-day precision was ≤8.7% relative standard deviation (RSD), and RE ≤±12.0% for standards prepared at 20/30/10, 400/600/200, and 5000/7500/2500 ng/mL. Matrix standards as high as 40/60/20 µg/mL could be diluted into the calibration range (RE≤±3.5% and RSD ≤2.4%). The method was evaluated for HSD rat maternal plasma and fetal, placental, brain, and heart homogenates (mean RE ≤±15.0% and RSD ≤8.6%). Analyte stability was demonstrated in extracted plasma for two days at different temperatures, and in various matrices stored at -80°C for at least 32 days (80-113% of Day 0 concentrations). These data demonstrate that this simple and efficient method is suitable for quantitation of ABC, DTG, and 3TC in rat matrices generated from toxicology studies. The method can easily be adapted to other biological matrices and species (e.g., human).

A sensitive liquid chromatography tandem mass spectrometry method for the detection of (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol and (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabinol in hair with a cutoff of 1 pg/10 mg of hair and a limit of quantitation of 0.2 pg/10 mg of hair for both analytes was developed and is herein described. A subset of samples collected from a school-age population between December 2022 and February 2023 was analyzed by this method after having screened presumptive positive by enzyme immunoassay out of a total pool of approximately 5300 samples. Of these presumptive positive samples, 66% showed the presence of one or both analytes at a concentration ≥1 pg/10 mg of hair. Of the 213 positive samples, 57% contained more (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabinol than (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol, and 6% contained more than 50-fold higher Δ8 isomer than Δ9 isomer. Of the 197 samples that were reportable for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol ≥1 pg/10 mg cutoff, 53% of them contained more (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabinol than (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol. Of the 197 samples that were reportable for (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabinol ≥1 pg/10 mg cutoff, 15.7% exceeded the upper limit of linearity of the method (200 pg/10 mg). These results suggest a high level of (-)-Δ8-tetrahydrocannabinol usage in this population relative to (-)-Δ9-tetrahydrocannabinol usage. They further suggest the possibility that the (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol reported for some of these samples may only have been present due to (-)-Δ9-tetrahydrocannabinol contamination of (-)-Δ8-tetrahydrocannabinol products being consumed in large quantities. Thus, reported results for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol alone may give a false picture of the extent of the cannabis product being consumed by a test subject.

Peripheral blood is considered the gold standard for postmortem toxicological analysis. However, vitreous fluid has been described as more resistant to postmortem redistribution and may act as an isolated matrix, preserving postmortem drug concentrations. To determine the utility of vitreous fluid analysis compared to traditional blood analysis for 6-acetylmorphine, morphine, cocaine, benzoylecgonine, fentanyl, and norfentanyl, paired peripheral blood and vitreous fluid were collected and analyzed in 122 postmortem cases from Western Pennsylvania. In this study, we tested the frequency of detection and performed analyses to correlate drug concentrations in both matrices. We demonstrate that vitreous fluid provides a viable matrix for the postmortem analysis of several drugs of abuse and their metabolites. However, when both matrices are available for analysis, vitreous fluid, when compared to whole blood, is not optimal for all analytes. These differences are likely due to differences in physiochemical properties and other pharmacokinetic parameters.

Synthetic cannabinoids remain one of the most important groups of new psychoactive substances and are responsible for many cases of poisoning in Europe. Deaths from acute 4F-MDMB-BICA poisoning have recently been reported. Severe poisonings may be underreported because 4F-MDMB-BICA is not routinely screened for in most forensic and toxicology laboratories. We report the case of a young man in France who presented with poisoning after orally consuming a powdered substance sold online as an opioid. The coma required intensive care unit management with emergent chest tube insertion and mechanical ventilation. The outcome was favorable with no sequelae due to early medical care. In the absence of remaining product and preserved urine samples, qualitative toxicological screening was performed on plasma, cerebrospinal fluid, and a hair strand. Using ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry and a molecular network data processing strategy, 4F-MDMB-BICA and two of its metabolites were identified only in plasma and cerebrospinal samples. These results were consistent with a single exposure. The identification of the substance consumed was crucial because of discrepancy between the symptoms observed and those expected after presumed exposure. Identification of 4F-MDMB-BICA and two of its metabolites was achieved in early plasma and cerebrospinal fluid samples. This documented case is helping to improve knowledge of 4F-MDMB-BICA poisoning, which could be an emerging public health issue.

Concentrations of amitriptyline and nortriptyline in postmortem blood samples may not accurately reflect the concentrations at the time of death due to postmortem redistribution or degradation. The brain is suggested as an alternative matrix since it is less subjected to postmortem redistribution and more protected against trauma and putrefaction, but reference concentrations in brain tissue are scarce. In this study, we aimed to provide concentrations in brain tissue and brain-blood ratios in 53 postmortem cases, where amitriptyline and/or nortriptyline were detected. To establish reference levels, each case was assigned to one of three classes according to the cause of death: (A) lethal intoxication by the sum of amitriptyline and nortriptyline or nortriptyline alone, (B) lethal intoxication by the drugs in combination with other drugs and (C) the cause of death was not influenced by amitriptyline and/or nortriptyline. A positive correlation between blood and brain concentrations was found with a Spearman coefficient of 0.98. In 42 cases, where both drugs were detected, the 10-90 percentiles in brain tissue ranged from 0.17-9.1 mg/kg (median: 0.78 mg/kg) for amitriptyline and 0.22-5.0 mg/kg (median: 1.43 mg/kg) for nortriptyline across all classes. In 11 cases where only nortriptyline was detected, the percentiles ranged from 0.32-7.2 mg/kg (median: 0.28 mg/kg) in brain tissue. A median brain-blood ratio of 3.4 was found for amitriptyline, 8.5 for nortriptyline as a metabolite of amitriptyline and 9.7 for nortriptyline as an individual ingested drug. No significant difference was found between the different classes. The obtained brain concentrations and brain-blood ratio can contribute to the alternative or complementary use of brain tissue for future toxicological investigations.

A simple liquid-liquid extraction procedure (LLE) followed by liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-QTOF-MS) analysis for drugs in oral fluid collected with the Quantisal™ device has been developed. The decision point cut-off concentrations were at or below those recommended by the National Safety Council's Alcohol, Drugs, and Impairment Division (NSC-ADID) for toxicological investigation of driving under the influence of drugs cases (DUID). Currently ANSI/ASB standard 120 does not cover the analysis of oral fluid collected in impaired driving investigations, instead guidance from the NSC-ADID was used. The supporting mass spectral based screening library was adapted from commercially available databases and included Tier 1 and Tier 2 recommended compounds. Further, the additional inclusion of novel psychoactive substances and synthetic cannabinoids was based on the Center for Forensic Science Research and Education's (CFSRE) quarterly publications of 2023. Metabolites from those publications were not included in this method since, with some exceptions, parent drugs are the dominant compounds in oral fluid. Briefly, Quantisal™ (1mL) was mixed with organic solvents, centrifuged, and decanted; followed by a second liquid-liquid process which extracted all the drugs in a single aliquot. A gradient liquid chromatography program using 0.1% formic acid in water and 0.1% formic acid in methanol was used and the runtime was 10 minutes. LC-QTOF-MS settings were optimized to promote greater sensitivity for a wide range of drug classes. The method was fully validated using ANSI/ASB 036 Standard Practices for Method Validation in Forensic Toxicology as guidance. Interference studies, limit of detection, precision at and around the decision points, ionization suppression/enhancement, and processed sample stability up to 96 hours were completed for each drug in the library database. While ion suppression or enhancement of the analytes varied greatly, the decision point was not significantly affected and internal standards that mimicked similar responses were chosen for each analyte. The method was applied to proficiency program samples, routine samples received into the laboratory, and blind samples screened against the search engine. The optimization of specific tune characteristics and instrument settings allowed the user to meet or exceed recommended screening limits for drugs in Quantisal™ collected oral fluid samples without the need for immunoassay testing.

The problem of finding a suitable biomarker to widen the detection window of γ-hydroxybutyric acid (GHB) intake remains a challenge in forensic toxicology. Based on previously published results, the present study deals with the evaluation of a fatty acid ester of GHB (4-palmitoyloxy butyrate [GHB-Pal]) in whole blood as a potential biomarker to extend the detection window of GHB use, e.g. in drug-facilitated sexual assaults. A liquid chromatography-mass spectrometry (LC-MS-MS) method for the quantification of GHB-Pal in whole blood was validated. Whole blood samples were collected from subjects involed in police roadside controls (n = 113) and from narcolepsy patients (n = 10) after the controlled administration of Xyrem® (sodium oxybate). Both sample collectives were previously tested for GHB using two different methods: ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) and gas chromatography-mass spectrometry (GC-MS). In samples from routine police casework, GHB-Pal was detected in 67 out of 113 analysed GHB-positive samples with a mean concentration of 0.8 ng/mL ± 0.5 ng/mL (standard deviation). Among samples that were tested positive for both compounds, no linear correlation was observed between GHB and GHB-Pal concentrations (r = 0.508). In contrast, GHB-Pal was not detected in any of the blood samples analysed from the patients. The absence of GHB and GHB-Pal in the patient cohort may be attributed to the time interval between dose intake and blood collection (approximately 3 and 6 h), during which GHB was eliminated from the body. Furthermore, GHB-Pal was only detectable at a GHB concentration of at least 16 µg/mL, which indicates that endogenous concentrations or low GHB doses may not be sufficient for GHB-Pal formation. Due to missing correlation between both compounds and the lack of GHB-Pal detection several hours after GHB administration, it can be assumed that GHB-Pal in blood is not a suitable biomarker to widen the detection window of GHB.

Dexamphetamine, lisdexamphetamine, and methylphenidate are central stimulant drugs widely used to treat attention-deficit/hyperactivity disorder (ADHD), but poor adherence may lead to treatment failure, and the drugs are also subject to misuse and diversion. Drug analysis in oral fluid may thus be useful for monitoring adherence and misuse. We measured drug concentrations in oral fluid and urine after controlled dosing to investigate detection windows and evaluate the chosen cutoffs. Healthy volunteers ingested single oral doses of 10 mg dexamphetamine (n = 11), 30 mg lisdexamphetamine (n = 11), or 20 mg methylphenidate (n = 10), after which they collected parallel oral fluid and urine samples every 8 h for 4-6 days. Amphetamine (analytical cutoff, oral fluid: 1.5 ng/mL; urine: 50 ng/mL), methylphenidate (oral fluid: 0.06 ng/mL), and ritalinic acid (urine: 500 ng/mL) were analyzed using fully validated chromatographic methods. The median time from ingestion to the last detection in oral fluid was 67 ± 4.9 h (lisdexamphetamine) and 69 ± 8.8 h (dexamphetamine) for amphetamine and 36 ± 2.5 h for methylphenidate. This was comparable to urine (77 ± 5.1 h for lisdexamphetamine, 78 ± 4.5 h for dexamphetamine, and 41 ± 2.4 h for ritalinic acid). The interindividual variability in detection times was large, probably in part due to pH-dependent disposition. Using a logistic regression approach, we found similar detection rates as a function of time since intake in urine and oral fluid with the chosen cutoffs, with a high degree of probability for detection at least 24 h after intake of a low therapeutic dose. This demonstrates the usefulness of oral fluid as a test matrix to assess adherence to ADHD medications, provided that the analytical method is sensitive, requiring a cutoff as low as 0.1 ng/mL for methylphenidate. Detection windows similar to those in urine may be achieved for amphetamine and methylphenidate in oral fluid.

A comparative analysis of the metabolites and metabolic pathways of fentanyl was conducted in liver microsomes and zebrafish models utilizing ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS). A total of 21 metabolites were identified across both liver microsomes and zebrafish models. These included 9 phase I metabolites, such as N-dealkylated, N-oxidated and hydroxylated products, and 12 phase II metabolites, including glucuronidated, methylated, and sulfated products, as well as a series of products derived from conjugation with glutathione. Notably, the products derived from conjugation with glutathione are reported here for the first time. This study provides a comprehensive and in-depth comparative analysis of fentanyl metabolism in liver microsomes and zebrafish, offering a foundation for analyzing and identifying biological samples in cases of fentanyl misuse and fatalities.

The synthetic cathinone (SC) 3,4-methylenedioxy-α-pyrrolidinohexanophenone (MDPHP) is structurally correlated to the 3,4-methylenedioxypyrovalerone (MDPV). In recent years, the number of intoxication cases has increased even if little is known about the pharmacokinetics properties. The Postmortem (PM) distribution of MDPHP remains largely unexplored. In these reports, MDPHP levels were quantified in blood, gastric content, and urine. This study aimed to describe the MDPHP PM distribution in several specimens, i.e. central and peripheral blood (CB and PB), right and left vitreous humor (rVH and lVH), gastric content (GCo), urine (U), and hair. The samples were collected from a cocaine-addicted 30-year-old man with a PM interval estimated in 3-4 h. Autopsy examination revealed unspecific findings, i.e. cerebral and pulmonary edema. No injection marks were observed. Toxicological analyses were performed using a multi-analytical approach: headspace gas chromatography for blood alcohol content (BAC), gas chromatography-mass spectrometry (GC-MS) for the main drugs of abuse, liquid chromatography-tandem mass spectrometry (LC-MS-MS) for benzodiazepines, and new psychoactive substances (NPS). BAC was negative (0.02 g/L). MDPHP concentrations were as follows: 1,639.99 ng/mL, CB; 1,601.90 ng/mL, PB; 12,954.13 ng/mL, U; 3,028.54 ng/mL, GCo; 1,846.45 ng/mL, rVH; 2,568.01 ng/mL, lVH; 152.38 (0.0-1.5 cm) and 451.33 (1.5-3.0 cm) ng/mg, hair. Moreover, hair segments were also positive for 3,4-dimethylmethcathinone (DMMC < limit of quantification: 0.01 ng/mg), α-PHP (0.59 ng/mg, 0.0-1.5 cm; 3.07 ng/mg, 1.5-3.0 cm), cocaine (6.58 ng/mg, 0.0-1.5 cm; 22.82 ng/mg, 1.5-3.0 cm), and benzoylecgonine (1.13 ng/mg, 0.0-1.5 cm; 4.30 ng/mg, 1.5-3.0 cm). MDPHP concentrations were significantly higher than those reported in the literature for fatal cases. For these reasons, the cause of death was probably the consumption of a lethal amount of MDPHP. Because CB and PB were similar, PM redistribution was not relevant.