LC MS-MS Research Articles

Lipid profile dataset of optogenetics induced optic nerve regeneration.

The optic nerve transfers visual information from the retina to the brain through the axons of retinal ganglion cells (RGCs). In adult mammals, optic nerve injuries and progressive degenerative diseases lead to the irreversible loss of RGCs, resulting in vision loss and blindness. Optogenetic models have proved useful in manipulating the growth of RGCs through expression and stimulation of channelrhodopsins (Chr2) in RGCs using the RGC-specific thy-1 promoter. Using transgenic Chr2 mouse (Thy1-ChR2-EYFP) as a model of regeneration, we profile the lipid changes which occur after traumatic optic nerve crush, light stimulation and forced RGC axonal growth. Thy1-ChR2-EYFP and control (C57BL/6) mice were divided in four groups each – 1) no crush and no stimulation, 2) no crush with stimulation, 3) crush and without stimulation, and 4) crush with stimulation. After euthanasia, the optic nerves were collected for lipidomic analysis. The Bligh and Dyer method was used for lipid extraction, followed by mass spectrometry lipid profiling with a Q-Exactive Orbitrap Liquid Chromatography-Mass Spectrometer (LC MS-MS). The raw scans were analysed with LipidSearch 4.1.3 and the statistical analysis was conducted through Metaboanalyst 4.0. This data is available at Metabolomics Workbench, study ID ST001381: [https://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST001381&StudyType=MS&ResultType=5].

First-Line Toxicological Screening with Fully Automated Extraction.

In clinical toxicology, laboratories need screening methods allowing unambiguous identification of the compounds in a short turnaround time to either confirm or exclude the hypothesis of drug overdose or poisoning with a toxicant. We developed a fully automated screening procedure designed to identify and quantify in a single run 245 compounds of interest in clinical toxicology. Sample extraction was carried out by a programmable liquid handler directly coupled to a liquid chromatography-tandem mass spectrometry (LC-MS-MS) system. Data acquisition was performed in the positive and negative ionization modes with up to 15 multiple reaction monitoring (MRM) transitions per compound, each with optimized collision energy to enable both qualitative library searching and quantitation. The method was validated according to the ISO 15189 requirements and was applied to real patient samples (n=127). The 15 MRM transitions per compound provided higher confidence for the identification of all the compounds. The quantitative method was fully validated with satisfactory intra- and inter-assay imprecision and inaccuracy with CV% lower than 20%. For only nine molecules, imprecision and inaccuracy were relatively high but never exceeded 31.7%. Comparison with dedicated quantitative methods using conventional MRM monitoring performed using 127 patient samples (n=175 pairs of measured concentrations) showed excellent correlation (R2=0.96). A robustness study showed that calibration curves prepared for up to 1 month yielded uncertainty < 20%. Retention times ranged from 0.89min for metformin to 9.72min for difenacoum. The automated sample preparation required 8min and was followed by 10min chromatographic separation. This first-line screening procedure yields high confidence in compound detection and should be useful in core labs facing clinical toxicology situations where rapid and reliable results are needed.

Comprehensive Drug Screening of Whole Blood by LC-HRMS-MS in a Forensic Laboratory.

As the number of prescriptions, over-the-counter medications and drugs of abuse continue to increase, forensic laboratories are faced with the challenge of developing more comprehensive screening methods in order to detect them in whole blood samples. Another challenge faced by forensic laboratories is detecting and identifying novel synthetic compounds as they emerge and change. Traditional drug screening methods include enzyme immunoassay (EIA) and either gas or liquid chromatography paired with mass spectrometry (GC-MS or LC-MS-MS, respectively). While these methods are good, they have their disadvantages. For example, EIA requires special reagents for each drug class, GC-MS requires extensive sample preparation, and LC-MS-MS only detects drugs on a known inclusion lists of compounds of interest. Described here is the development of a robust and comprehensive screening method for drugs in whole blood samples that eliminates the aforementioned disadvantages of the traditional methods. Using a Q Exactive Focus™ liquid chromatography-high-resolution accurate mass spectrometer (LC-HRMS-MS), a method was developed that is capable of detecting ~200 drugs at a concentration of 2μg/L for most analytes. This method also employs a more automated data processing feature which reduces processing time. Finally, it has the added benefit of retroactive data analysis, which allows it to be used for unknown drug analysis as well. Used as an initial screening method, the comprehensive drug screen using LC-HRMS-MS has the potential to take on two of the most important challenges faced by forensic laboratories today.

Determination of 30 Synthetic Cathinones in Postmortem Blood Using LC-MS-MS.

Synthetic cathinones, commonly referred to as "bath salts," are powerful amphetamine-like psychostimulants, and new derivatives are constantly appearing in the illicit market to evade judicial consequences. To keep up with these new stimulant drugs, a low-sample-size liquid chromatography-tandem mass spectrometry method was validated to quantify 30 synthetic cathinones in postmortem blood including N-ethylpentylone and eutylone. Mixed mode cation exchange solid-phase extraction using 0.25mL postmortem blood was performed followed by detection using a triple quadrupole mass spectrometer operating electrospray ionization in positive mode. The reversed-phase chromatographic separation was achieved in 16min, resolving all isobaric compounds. The linear range of the calibration curve was 1-500ng/mL (R 2>0.99) for all compounds. Limit of quantification (LOQ) and limit of detection were determined to be at 1ng/mL. Both imprecision and bias were evaluated and had metall allowed criteria (CV and bias <20%). No matrix effect was observed with values ranging from -5.1 to 13.3% (CV 11.4-17.5%, n=10). Extraction efficiency (84.9-91.5%) and process efficiency (86.1-102.6%) were satisfactory, except for 4-chloroethcathinone which was 63 and 64.9%, respectively. No carryover after the upper LOQ was detected. Neither endogenous nor exogenous interferences were observed. Both dilution integrity and stability (24h) yielded acceptable results. This method was applied to 18 postmortem cases received between 2015 and 2019. Eight different synthetic cathinones were detected in selected postmortem cases within the past 5 years, showing a wide range of concentrations from 1.4 to >500ng/mL. While ethylone and methylone were detected in 2015, cases between 2016 and 2017 were predominantly butylone, dibutylone, pentylone and N-ethylpentylone which had also exhibited a significant increase in 2018. To our knowledge, this method is the most comprehensive methodology for the determination of up-to-date synthetic cathinones currently available in whole blood.

Determination of the Cross-Reactivity of the Biological Metabolite (-)-trans-Δ9-Tetrahydrocannabinol-Carboxylic Acid-Glucuronide (THC-COOH-Gluc) for Cannabinoid Immunoassays.

The highest concentrated metabolite of (-)-trans-Δ9-tetrahydrocannabinol (THC) in urine, the main psychoactive constituent of Cannabis sativa, is 11-nor-9-carboxy-(-)-trans-Δ9-tetrahydrocannabinol-β-D-glucuronide [(-)-trans-THC-COOH-Gluc]. Even though reference standards for THC, 11-hydroxy-THC (11-OH-THC) and THC-COOH are commercially available as the biological (-)-trans-stereoisomers, the reference standard of THC-COOH-Gluc is only available as the racemic 11-nor-9-carboxy-(±)-cis-Δ9-tetrahydrocannabinol-β-D-glucuronide. This poses the problem for immunoassays, because different stereoisomers may have different cross-reactivity (CR). The aim of the current study was to extract the biological stereoisomer (-)-trans-THC-COOH-Gluc from a urine sample of two marihuana consumers by solid-phase extraction with a Chromabond® C18 cartridge. The cannabinoids in the obtained extract were quantified by Liquid-chromatography coupled to tandem mass spectrometry (LC-MS-MS) and used after dilution for further testing of the CR of (-)-trans-THC-COOH-Gluc with a homogenous enzyme immunoassay assay (hEIA) (Urine HEIA® Cannabinoids (THC), Immunalysis™, Pomona, CA, USA). The CR was determined as the measured HEIA® signal (ng/mL) per THC-COOH-Gluc concentration (ng/mL) in percentage. Results showed that the CR (determined in concentration ratios) is concentration dependent and is 72-87% in the calibration range (20-50ng/mL). At the cut-off of the hEIA (40ng/mL), the CR was determined to be 75%. With a molecular weight quotient of 1.51 (MWTHC-COOH-Gluc/MWTHC-COOH=520.568g/mol/344.451g/mol), this means that CR (in molar ratios) is 106-131%. This finding is important, since the major metabolite of THC in urine is (-)-trans-THC-COOH-Gluc and not (-)-trans-THC-COOH, which is used for calibration and no hydrolysis is performed during the determination by hEIA.

Occurrence and seasonal variations of pharmaceuticals and personal care products in drinking water and wastewater treatment plants in Samsun, Turkey

In this study, the occurrence and removal of five pharmaceuticals and personal care products (PPCPs) in the influent and effluent from both a drinking water treatment plant (DWTP) and a wastewater treatment plant (WWTP) in Samsun, Turkey was investigated. The concentrations of five pharmaceuticals, namely carbamazepine, diclofenac, ibuprofen, paracetamol and triclosan, were reported in different seasons from autumn 2016 to summer 2017. The targeted PPCPs were analysed using liquid chromatography tandem mass spectrometry (LCMS–MS). Results of the analysis of samples from influent of the DWTP show that the average concentrations of carbamazepine, ibuprofen and triclosan in different seasons were 0.385 μg/L, 0.038 μg/L and 0.342 μg/L, respectively. The maximum concentration of carbamazepine was recorded in spring (0.84 μg/L), while in summer, triclosan and ibuprofen with values of 0.155 μg/L and 0.735 μg/L were highest. However, the concentrations of other selected drugs were below the limit of detection (LOD) or were not detected at all. In the effluent of the DWTP, only triclosan was detected with a concentration 0.565 μg/L in summer. Similarly, the average concentrations of carbamazepine, ibuprofen, paracetamol, and triclosan in the influent of the WWTP in different seasons were 78.76 μg/L, 2.77 μg/L, 2.31 μg/L and 0.58 μg/L, respectively. Concentrations in the influent of the WWTP were also not negligible, where the maximum concentration of carbamazepine was observed in autumn, ibuprofen and triclosan in winter, paracetamol in spring, and diclofenac in summer. However, in the effluent of the WWTP, only carbamazepine was detected with an average concentration of 2.30 μg/L. Seasonal variations in the presence/detection of PPCPs in both treatment plants were observed. Statistical tools were applied to verify the obtained results.

High Throughput Detection of 327 Drugs in Blood by LC-MS-MS with Automated Data Processing.

The described procedure provides a rapid technique for the detection and semi-quantitation of a large number of drugs in blood. This procedure uses a minimal sample volume and employs a one-step liquid extraction and automated data processing to yield rapid turnaround times. A total of 327 of the most commonly used medicinal and illicit drugs in Australia were selected including various amphetamines, anesthetics, antidepressants, antipsychotics, anticonvulsants, benzodiazepines, beta blockers, opioid and nonopioid analgesics, stimulants, THC and a large number of synthetic cannabinoids and other novel psychoactive substances. The extracts were subject to 5-minute chromatography using a Kinetex C18 50×4.6mm 2.6μm solid-core analytical column and analyzed using a Sciex 3200 Q-TRAP MS-MS (+ ESI, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Matrix effects and extraction efficiencies were acceptable with most analytes showing > 80% response and low variation (within 25%RSD). Cannabinoids were most affected by the matrix and yielded poorest recovery values but were still detectable. Precision, accuracy, repeatability and multipoint linearity were assessed for all analytes. The method has been used in routine practice in the forensic toxicology service at the Victorian Institute of Forensic Medicine in over 6000 coronial investigations using both postmortem and clinical blood specimens. This technique has greatly increased throughput, reduced turnaround times and allowed for rapid same-day analysis of results when needed. The method is routinely used in routine overnight testing with results reported to pathologists within 4h of data acquisition. This rapid toxicological technique is used in conjunction with other investigative processes such as full-body CT imaging, review of case circumstances and medical histories to provide an efficient death investigation process.

Oxycodone Concentrations and Metabolic Ratios in Femoral Blood from Fatal Intoxications and Other Causes of Death using LC-MS-MS.

Oxycodone (OC) is an opioid with strong analgesic effects widely used to treat acute and chronic pain. Interpretation of OC concentrations in postmortem cases is complicated due to tolerance and overlapping concentrations for fatal and non-fatal levels. In this study, our aim was to develop and validate a method for OC and its three metabolites: noroxycodone (NOC), oxymorphone (OM) and noroxymorphone (NOM) in postmortem femoral blood. Our goal was to define reference concentrations for intoxications and non-intoxications and investigate metabolic ratios in different causes of death. A rapid LC-MS-MS method using protein-precipitated postmortem blood was developed. Lower limit of quantitation was 0.005μg/g blood for all analytes; upper limit of quantitation was 1.0μg/g for OC and NOC and 0.25μg/g for OM and NOM. The method displayed high precision (3.3-7.7%) and low bias (-0.3 to 12%). In total, 192 cases were analyzed and concentrations ranged from 0.005 to 13μg/g for OC, 0.005 to 2.0μg/g for NOC, 0.005 to 0.24μg/g for OM, and 0.005 to 0.075μg/g for NOM. We found a significant difference in OC concentration between the cases where OC contributed and those where it did not. In spite of that, we do not recommend the use of a specific blood concentration to distinguish fatal intoxications. Instead, the percentiles from our data set suggest that concentrations >0.2μg/g are likely to have contributed to toxicity, but that concentrations as high as 0.3 might be tolerated without toxic effects. In addition, we also found that a low NOC/OC ratio could point toward an acute fatal intoxication. In conclusion, the OC concentration alone may not be sufficient to diagnose a fatal intoxication.

Performance of Two Fentanyl Immunoassays against a Liquid Chromatography–Tandem Mass Spectrometry Method

Rapid and automated fentanyl screening assays are in need due to the prevalence of fentanyl abuse. In the present study, we evaluated the clinical performance of two FDA-cleared automated fentanyl immunoassays, the Immunalysis SEFRIA fentanyl assay and the ARK fentanyl assay. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) was used as a gold standard. Two groups of urine specimens were tested, including 225 specimens from patients presenting to the emergency department (ED) for whom urine drugs of abuse screens were ordered and 57 specimens from patients in chronic pain management programs. The SEFRIA assay generated higher assay imprecision than ARK assay (intraday CV%, 7.15 vs. 4.7%; interday CV%, 6.6 vs. 5.3%). Clinical sensitivity and specificity for detection of fentanyl exposure were 100 and 96% for the ARK assay and 95 and 80% for the SEFRIA assay. An 'auto-repeating' issue was observed for some validation specimens flagged with high absorbance values (OD>3.0), generating false repeat results. The frequency of auto-repeating was lower in the ARK assay than SEFRIA (0.7 vs. 15.5%). Auto-repeating occurred for only previously frozen specimens in the ARK assay, but 9% of fresh specimens were also flagged and repeated in the SEFRIA assay. Positive predictive value (PPV) of the ARK assay was 73% in the ED population and 67% in the non-ED populations. The concentrations of fentanyl and norfentanyl were higher in specimens from ED patients than patients from pain management programs. High prevalence of morphine, methamphetamine, benzoylecgonine and 6-MAM was observed in specimens positive for fentanyl in both populations.

Assessment of Tobacco Exposure During Pregnancy by Meconium Analysis and Maternal Interview.

Smoking during pregnancy can have serious obstetric and fetal complications. Therefore, it is essential to identify in utero exposure to tobacco, being meconium the matrix of choice for this purpose. Meconium (n=565) was analyzed for nicotine, cotinine and hydroxycotinine by LC-MS-MS. Then, tobacco meconium results were compared with smoking habits during pregnancy and neonatal outcomes measures (birth weight, length, head circumference, gestational age and Apgar scores). Although meconium analysis increased identification of in-utero exposure to tobacco (17.7% meconium positive specimens vs 13.5% mothers admitting tobacco use during pregnancy), there was a statistically significant relationship between meconium results and interview answers (P<0.001). Birth weight was significantly lower for newborns with meconium positive results in males (P=0.023) and females (P=0.001), while for length significance was only observed in females (P=0.001); however, when excluding meconium specimens positive for other drugs, a statistically significant difference was only found for female weight (P=0.045). Meconium analysis proved to be more reliable for tobacco prenatal exposure detection than maternal interview. In addition, positive meconium results increased the probability for low birth weight, especially in females.

Quantification of Furanylfentanyl and its Metabolites in Human and Rat Plasma Using LC-MS-MS.

Fentanyl analogs (novel and traditional) continue to impact the ever-growing opioid epidemic. Furanylfentanyl (FuF) is one analog equipotent to fentanyl that has documented involvement in thousands of intoxication and fatality cases around the world. Due to its prevalence, toxicologists need to improve detection and understanding of this analog. A method for the quantification of FuF and its metabolites (4-ANPP, furanyl norfentanyl (FuNorF)) in a small volume (100μL) of human plasma by LC-MS-MS was developed and validated according to ANSI/ASB Standard. The method was cross validated in rat plasma for a future pharmacokinetic (PK)/pharmacodynamic (PD) study. In human plasma, calibration ranges were 0.025-25ng/mL (FuF and 4-ANPP) and 0.5-25ng/mL (FuNorF). Limits of detection were 0.0125ng/mL (FuF and 4-ANPP) and 0.25ng/mL (FuNorF). Lower limits of quantification coincided with lowest calibrator concentrations of 0.025ng/mL (FuF and 4-ANPP) and 0.5ng/mL (FuNorF). Precision and bias values were determined to be acceptable for all analytes. Matrix effects were acceptable for all analytes (-8.6-25.0%), except FuNorF with suppression >25%. Extraction recoveries ranged from 84.5 to 98.1%. No carryover or endogenous interferences were observed. Qualitative interferences with 4-ANPP were observed from some n-acyl substituted fentanyl analogs predicted to be low-concentration standard impurities. Analytes were stable under all conditions and dilution integrity was sustained. The method was successfully cross validated in rat plasma with acceptable bias (-7.4-8.4%), precision (within-run<19%CV and between-run<12.6%CV), matrix effects (-9.3-17.2%, except FuNorF with >25% suppression), recoveries (79.2-94.5%) and dilution integrity (1/2 and 1/10).

LC-MS-MS Method for the Determination of Antidepressants and Benzodiazepines in Meconium.

An LC-MS-MS method for the determination of 14 benzodiazepines (BZDs) (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (ADs) (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25±0.02g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed phase, with a gradient of 0.1% formic acid in 2mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of ADs and another for BZDs. Analytes were monitored by tandem mass spectrometry employing electrospray positive mode in MRM mode (2 transitions per compound). Method validation included: linearity [n=5, limit of quantification (LOQ) to 400ng/g], limits of detection (n=6, 1-20ng/g), LOQ (n=9, 5-20ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n=15, 90.6-111.5%), imprecision (n=15, 0-14.6%), matrix effect (n=10, -73 to 194.9%), extraction efficiency (n=6, 35.9-91.2%), process efficiency (n=6, 20.1-188.2%), stability 72h in the autosampler (n=3, -8.5 to 9%) and freeze/thaw stability (n=3, -1.2 to -47%). The method was applied to four meconium specimens, which were analyzed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC-MS-MS method allows a high throughput determination of the most common BZDs and ADs in meconium, which could be useful in clinical and forensic settings.

A Retrospective of Prevalence of Drugs of Abuse by Hair Analysis in Shanghai using LC-MS-MS.

This study presents a retrospective analysis of the prevalence of drug abuse in Shanghai by hair analysis. Files and toxicology analysis results of a total of 5,610 cases requesting for hair analysis of abused drugs at the Academy of Forensic Science (AFS) in Shanghai over 12months between August 2018 and July 2019 were reviewed. All cases of drug abuse identified by hair analysis were from the public security organs in Shanghai, China. Hair samples were analyzed for drugs of abuse and related metabolites, mainly including amphetamine (AMP), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), tetrahydrocannabinol (THC), ketamine (K), norketamine (NK), cocaine (COC), benzoylecgonine, morphine, 6-acetylmorphine, flunitrazepam, and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Among the 5,610 cases, 1,713 (30.5%) were positive for drugs of abuse, with amphetamine-type stimulants (ATS) (57%), including amphetamines (AMP and MA) (48%), MDMA and MDA (9%), being the most frequently detected drugs, followed by THC (14%), COC (8%), 5-MeO-DIPT (8%), and K (7%). The majority (75%) of positive hair samples were from male subjects. Overall, 77% of abusers were younger than 44years old. The proportion of female subjects (22.3%) under 24years was larger than that of male subjects (7.8%). There were 132 cases (7.7%) in which more than one type of drug was detected among 1,713 drug-positive cases. The most common combination was MDMA and K. The present study characterizes the current toxicological profile of drug abuse cases and provides a scientific basis for drug abuse prevention. Moreover, the hair concentration distributions of the commonly abused drugs in positive cases have been reported.

Method Consolidation to Improve Scope and Efficiency in Postmortem Toxicology.

Systematic toxicological approaches that employ both ideology changes and improvements in instrumentation and sample extraction allow for improved toxicology testing efficiency through lower sensitivities, higher specificity and minimized resource use. Historically, the San Francisco Office of the Chief Medical Examiner relied heavily on a gas chromatography mass spectrometry (GC-MS) testing regime, comprised of individual drug-class confirmation and quantitation assays. Traditional methods utilizing GC-MS typically require iterations of testing, exhausting sample volume, and hindering productivity and turnaround times, particularly for polypharmacy cases frequently seen in modern postmortem toxicology. The method described here consolidated the scope of seven legacy methods into a single liquid chromatography tandem mass spectrometry (LC-MS/MS) method for better sensitivity, higher throughput, minimal sample consumption for the quantitation of drugs of abuse and improved quality assurance with the incorporation of smart, automated processing. About 100 μL of blood or urine were rapidly extracted using a simple acetonitrile protein crash and subsequent in-vial filtration and injected on to an LC-MS-MS system. The developed method was fully validated to SWGTOX and international guidelines and incorporated 55 analytes along with a customized query that facilitates rapid and consistent application of acceptability criteria for data processing and review. Applicability was demonstrated with the analysis of 1,389 samples (858 blood and 531 urine) where at least 41% of positive results may have been missed due to their decreased sensitivity and 11% of results were not within the scope of the previous analytical methods estimated. On average, cases in this study would have previously required three distinct GC-MS assays, 3mL of blood, and upwards of 30h of active staff time. The described LC-MS-MS analytical approach has mitigated the need to perform multiple assays, utilized only 0.1mL of sample, significantly reduced analyst work time, incorporated 10 additional analytes and allowed for a more comprehensive testing regime to better inform cause of death determinations.

Segmental Analysis of R/S-Methamphetamine and R/S-Amphetamine in Abusers' Head Hair.

In this study, the relationships between the concentrations of R/S-methamphetamine (MA) and its metabolite R/S-amphetamine (AP), the AP/MA ratio in hair samples, and MA dependence were investigated by performing segmental hair analysis in MA users. Authentic hair samples collected from 10 chronic MA abusers were cut into 1-cm sections (a total of 120 segments). The concentrations of MA and AM enantiomers were quantitatively measured by the LC-MS-MS method. The S-MA concentrations ranged from 1.17 to 256.41ng/mg and the S-AP concentrations ranged from 0.11 to 23.31ng/mg in the 120 segments. S-MA and S-AP were the most common analytes identified in hair; no R-MA or R-AP was found. The S-AP/S-MA ratios ranged from 0.03 to 0.32, indicating that the subjects primarily consumed S-MA rather than R-MA or AP. The S-AP/S-MA ratios in the long hair of all chronic MA abusers showed some variation, but there was an overall trend of gradual increase from the distal to the proximal end. This trend was independent of the drug concentrations. Therefore, we could conclude that the AP/MA ratios increased with the duration of MA abuse, and a higher AP/MA ratio suggested high MA dependence. There was no chiral conversion of MA or AP in the hair matrix. The segmental hair analysis showed that all subjects continuously used S-MA, and some users showed an increase in drug dose or the frequency of use.

Development and Validation of Chromatographic Methods for Simultaneous Determination of Paracetamol, Orphenadrine Citrate and Caffeine in Presence of P-aminophenol; Quantification of P-aminophenol Nephrotoxic Impurity Using LC-MS/MS.

Three chromatographic methods were developed, optimized and validated for Paracetamol (PAR), Orphenadrine citrate (Or.cit) and Caffeine (CAF) determination in their mixture and in presence of PAR toxic impurity; P-aminophenol (PAP) in tablets. The first method is based on a thin layer chromatography combined with densitometry. Separation was achieved using silica gel 60F254 TLC plates and dichloromethane:methanol:acetone:glacial acetic acid (9:1:0.5:0.3, v/v/v) as a developing system at 230nm. The second method is based on high-performance liquid chromatography with diode array detection. The proposed compounds are separated on a reversed phase C18 analytical column using phosphate buffer (pH9; 0.05M) and methanol (80:20, v/v) at 1.2mL/min. Linear regressions are obtained in the range of 1-500μg/mL, 25-1000μg/mL and 1-400μg/mL for PAR, Or.cit and CAF, respectively. Quantification of the toxic PAP is carried out using LC-MS-MS by electrospray ionization in the positive mode using triple quadrupole mass spectrometry. The limit of quantification for PAP is 1ng/mL. All methods are validated according to the ICH guidelines and successfully applied to determine PAR, Or.cit, CAF and PAP in pure powder and in combined tablets dosage form without interference from excipients.

Identification of Suvorexant in Blood Using LC-MS-MS: Important Considerations for Matrix Effects and Quantitative Interferences in Targeted Assays.

Suvorexant (Belsomra®) is a novel dual orexin receptor antagonist used for the treatment of insomnia. The prevalence of suvorexant in forensic samples is relatively unknown, which demonstrates the need for robust analytical assays for the detection of this sedative hypnotic in forensic toxicology laboratories. In this study, suvorexant was isolated from whole blood using a simple acidic/neutral liquid-liquid extraction followed by analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Matrix effects were evaluated qualitatively and quantitatively using various extraction solvents, proprietary lipid clean-up devices and source conditions. The method was validated in terms of limit of detection, limit of quantitation, precision, bias, calibration model, carryover, matrix effects and drug interferences. Electrospray is a competitive ionization process whereby compounds in the droplet compete for a limited number of charged sites at the surface. As such, it is capacity-limited, and LC-MS-based techniques must be carefully evaluated to ensure that matrix effects or coeluting drugs do not impact quantitative assay performance. In this report, we describe efforts to ameliorate such effects in the absence of an isotopically labeled internal standard. Matrix effects are highly variable and heavily dependent on the physico-chemical properties of the substance. Although there is no universal solution to their resolution, conditions at the electrospray interface can mitigate these issues. Using this approach, the LC-MS/MS assay was fully validated and limits of detection and quantitation of 0.1 and 0.5ng/mL suvorexant were achieved in blood.

Analysis for Alpha-Pyrrolidinovalerophenone and Its 2-Oxo-PVP Metabolite in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Alpha-pyrrolidinovalerophenone (alpha-PVP), a novel psychoactive substance, has widespread recreational use. This with interest in its pharmacological effects creates a need for methods that measure alpha-PVP concentrations. We therefore developed a LC-MS/MS method that can quantitate alpha-PVP and 2-oxo-PVP in rat plasma using a 0.1-mL sample volume. Addition of internal standards (2.5ng/mL alpha-PVP-d8/2-oxo-PVP-d6) was followed by liquid-liquid extraction with 1-chlorobutane:acetonitrile (4:1), evaporation and reconstitution with 0.1% formic acid. Extracts were analyzed by LC-MS/MS using an Agilent 1100 HPLC and a Thermo Scientific TSQ Quantum Access MS/MS, with a YMC ODS-AQ, 50mm×2mm, 3μm column. The mobile phase was 0.1% formic acid:acetonitrile gradient at a 0.2-mL/minute flow rate with positive ion electrospray. SRM was used for the analysis with transitions: alpha-PVP, 232→91; alpha-PVP-d8, 240→91; 2-oxo-PVP, 246→91; 2-oxo-PVP-d6, 252→91. Alpha-PVP and 2-oxo-PVP eluted at 6.4 and 8.9min. Calibrators range from 0.25 to 500ng/mL. Accuracy and precision evaluated quality control samples prepared at 0.75, 10 and 400ng/mL. The intra-assay evaluation also included the 0.25-ng/mL LOQs prepared in six different blank plasma sources. The intra-assay accuracy ranged from 88.9 to 117.8% of the target, and the intra-assay precision ranged from 0.9 to 16.0%. The inter-assay accuracy ranged from 98.7 to 110.7% of the target, and the inter-assay precision ranged from 4.5 to 12.0%. Extraction recovery was at least 52% for alpha-PVP and 67% for 2-oxo-PVP. Ionization recoveries were at least 64% for alpha-PVP and 82% for 2-oxo-PVP. These losses did not adversely affect assay performance. Alpha-PVP and 2-oxo-PVP controls were stable at room temperature for up to 24h and frozen for at least 36days. Alpha-PVP and 2-oxo-PVP were also stable in processed samples (extracts) stored at room temperature for at least 24days. The procedure was used to analyze rat plasma samples from a pharmacokinetic study.

Distribution of synthetic opioids in postmortem blood, vitreous humor and brain

In the US, the use of synthetic opioids (e.g. fentanyl and derivatives) has become an increasing health issue with thousands of overdose deaths being observed since 2013. With the high mortality rate associated with these substances, postmortem analyses and interpretation of synthetic opioids has become extremely important. However, due to the novelty of these compounds, the available data are limited and provides challenges to toxicologists. The objectives of this study were (1) to develop and validate analytical methods for the determination of synthetic opioids in vitreous humor and brain, and (2) to investigate the postmortem distribution of new synthetic opioids in blood, vitreous humor, and brain tissue. Vitreous humor (0.5mL) and brain tissue (5g) homogenized in water (diluted 1:3, w/w) were extracted by mixed mode cation exchange-reversed phase solid phase extraction. Extracts were analyzed by liquid chromatography tandem mass spectrometry (LC–MSMS). The chromatographic separation was performed by reversed-phase with 0.1% formic acid in water and in acetonitrile as mobile phases in gradient mode, with a total run time of 21min. Data were acquired with ESI+ in dynamic multiple reaction mode (dMRM), monitoring 2 transitions per compound. The methods were succesfully validated following SWGTOX guidelines, with limits of quantification of 0.1ng/mL in vitreous humor and 0.1ng/g in brain. Fifty-eight authentic case samples from the New York City Office of the Chief Medical Examiner (NYC-OCME) were analyzed to assess the distribution and detectability of synthetic opioids in these postmortem samples. Of the fifteen synthetic opioids included in the method, six synthetic opioids and metabolites (4-ANPP, acetylfentanyl, fentanyl, furanylfentanyl, norfentanyl, U-47700) were detected in the authentic cases. Concentrations for most analytes were within the 0.1 to 100ng/mL or ng/g calibration range across all three matrices, with only concentrations from acetylfentanyl and U-47700 exceeding 100ng/mL or ng/g. The highest concentrations were observed in brain (except norfentanyl), followed by blood and vitreous humor. Most analytes were detected in all three matrices in a given case. This was followed by detection of an analyte in combinations of brain and another matrix or brain only. Through the case analyses, vitreous humor and brain demonstrated to be viable alternatives to blood when performing postmortem analyses of synthetic opioids. Brain exhibited a higher detectability for most analytes when compared to blood and vitreous humor.

Identification of synthetic cannabinoids that were seized, consumed, or associated with deaths in Kuwait in 2018 using GC–MS and LC–MS-MS analysis

Synthetic cannabinoids are gaining much popularity worldwide. Although the death rate associated with their use is rising, these drugs are the largest and fastest growing class of novel psychoactive substances. Despite increased concerns regarding adverse effects stemming from the use of synthetic cannabinoids, there is no published data on the subject for the Gulf region or Kuwait, specifically. The current study investigates the diversity of synthetic cannabinoids in Kuwait in 2018. In total, 434 cases from the Narcotics and Psychotropic Laboratory, 70 cases from the Toxicology Laboratory, and six cases from the Forensic Medicine Department were reviewed and analyzed. Numerous synthetic cannabinoid types were identified using GC–MS and LC–MS-MS. The majority of synthetic cannabinoids were members of the indazole-3-carboxamide or indole-3-carboxamide families. Members from the indazole-3-carboxamide family identified in Kuwait were 5F-ADB, FUB-AMB, ADB-FUBINACA, AB-FUBINACA, 5F-ADB-PINACA, 5F-AKB-48, 5Cl-AKB-48, MDMB-FUBINACA, 5F-AB-PINACA, APINACA, and AB-PINACA whereas MDMB−CHMICA, 5F-MDMB-PICA, ADB-BICA, and MMB−CHMICA belonged to the indole-3-carboxamide family. In addition, members of other families were identified, including CBL2201 and UR-144, which belonged to indole-3-carboxylate and cyclopropylindole families, respectively. The most common synthetic cannabinoids were 5F-ADB, FUB-AMB, and 5Cl-AKB-48. Various mixes of two, three, or four types of synthetic cannabinoids were identified, and mixtures of synthetic cannabinoids with other illicit drugs were also present. Our findings show that in Kuwait, the most common mix of synthetic cannabinoids is FUB-AMB with 5F-ADB. These two types were mixed, either together or individually, with methamphetamine, tramadol, heroin, Δ9THC, and ketamine. Most importantly, our results reveal the synthetic cannabinoid types that were associated with six reported deaths.