- Proposed BoNT/A and /B Peptide Substrates Cannot Detect Multiple Subtypes in the Endopep-MS Assay. [Journal Article]
- JAJ Anal Toxicol 2019 Jul 09
- Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A-BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulis…
Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A-BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism-a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.
- Identification and Quantification of 5-Fluoro ADB and the 5-Fluoro ADB Ester Hydrolysis Metabolite in Postmortem Blood Samples by LC-MS/MS. [Journal Article]
- JAJ Anal Toxicol 2019 Jul 04
- 5-Fluoro ADB, also known as 5-fluoro MDMB-PINACA, is a potent synthetic cannabinoid that is an agonist to the human cannabinoid CB1 and CB2 receptors. Adverse physiological and psychological effects …
5-Fluoro ADB, also known as 5-fluoro MDMB-PINACA, is a potent synthetic cannabinoid that is an agonist to the human cannabinoid CB1 and CB2 receptors. Adverse physiological and psychological effects that have resulted in hospitalization and/or death have been associated with 5-Fluoro ADB use. In addition, analytical confirmation of 5-Fluoro ADB use has been reported in both forensic human performance toxicology and postmortem cases. An analytical method for the identification and quantification of 5-fluoro ADB and the 5-fluoro ADB ester hydrolysis metabolite in human blood samples by liquid chromatography-tandem mass spectrometry was created and validated. The linear range of this assay was determined to be 0.01-10 ng/mL for 5-fluoro ADB and 10-500 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. The method met both precision and accuracy requirements. Endogenous and exogenous interferences were not observed. Ion suppression exceeding 25% was observed for 5-fluoro ADB. However, additional experiments were performed to ensure that the observed suppression did not affect other method validation parameters such as limit of detection and accuracy. Blood samples from 36 postmortem cases were analyzed utilizing this methodology. The average blood concentration of 5-fluoro ADB was 0.29 ng/mL in central blood specimens and 0.05 ng/mL in peripheral blood specimens. The average blood concentration of the 5-fluoro ADB ester hydrolysis metabolite was 49 ng/mL in central blood specimens and 21 ng/mL in peripheral blood specimens. A serum sample was also analyzed and had a serum concentration of 0.12 ng/mL for 5-fluoro ADB and 42 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. As the concentration of the 5-fluoro ADB ester hydrolysis metabolite was found at a greater concentration than that of 5-fluoro ADB, this metabolite may be a useful marker to monitor in an attempt to confirm 5-fluoro ADB use in toxicological investigations.
- Testing for Betel Nut Alkaloids in Hair of Papuans Abusers using UPLC-MS/MS and UPLC-Q-Tof-MS. [Journal Article]
- JAJ Anal Toxicol 2019 Jun 19
- Betel nut is the fruit of Areca palm, growing in Papua New Guinea. Mixed with limestone and stick mustard, arecoline and guvacoline, which are present in betel nut, are hydrolyzed into arecaidine and…
Betel nut is the fruit of Areca palm, growing in Papua New Guinea. Mixed with limestone and stick mustard, arecoline and guvacoline, which are present in betel nut, are hydrolyzed into arecaidine and guvacine, respectively. As part of the study on dietary habits of Papuans residents, our laboratory was asked to analyze the four alkaloids in hair to document long-term exposure. Hair samples were collected from 19 adult subjects (males = 11; females = 8), by some of the authors, and were sent to the laboratory for analysis. The four alkaloids have very similar chemical structures. In order to accurately identify the drugs, two methods were developed. First, the compounds were identified using an ultra-high-performance liquid chromatography system coupled to time-of-flight mass spectrometry. Then, they were quantified by an ultra-high-performance liquid chromatography system coupled to tandem mass spectrometry. After decontamination with dichloromethane, hair samples were cut into very small segments and 20 mg were incubated in methanol for 2 h 30 min in an ultrasound bath. After cooling, the methanol was evaporated to dryness in presence of 20-μL octanol to prevent volatilization. Nicotine-d4 was used as an internal standard. Linearity was observed for concentrations ranging from the limit of quantification to 20 ng/mg for arecoline, arecaidine, guvacine and guvacoline. Measured concentrations were in the range 60 pg/mg to 18 ng/mg for arecoline (n = 19), 14 pg/mg to 2.5 ng/mg for guvacoline (n = 11), 63 pg/mg to 3.8 ng/mg for arecaidine (n = 11) and 100 pg/mg to 3.2 ng/mg for guvacine (n = 6). There was no correlation between concentrations of arecoline and arecaidine (ratio from 0.01 to 0.18) and guvacoline and guvacine (ratio from 0.06 to 3.50). However, the identification of these substances in hair is a good marker of consumption of betel nut and allows us to document a local practice that remains difficult to evaluate just by questioning.
- Oral Fluid Drug Testing: Analytical Approaches, Issues and Interpretation of Results. [Journal Article]
- JAJ Anal Toxicol 2019 Jun 28
- With advances in analytical technology and new research informing result interpretation, oral fluid (OF) testing has gained acceptance over the past decades as an alternative biological matrix for de…
With advances in analytical technology and new research informing result interpretation, oral fluid (OF) testing has gained acceptance over the past decades as an alternative biological matrix for detecting drugs in forensic and clinical settings. OF testing offers simple, rapid, non-invasive, observed specimen collection. This article offers a review of the scientific literature covering analytical methods and interpretation published over the past two decades for amphetamines, cannabis, cocaine, opioids, and benzodiazepines. Several analytical methods have been published for individual drug classes and, increasingly, for multiple drug classes. The method of OF collection can have a significant impact on the resultant drug concentration. Drug concentrations for amphetamines, cannabis, cocaine, opioids, and benzodiazepines are reviewed in the context of the dosing condition and the collection method. Time of last detection is evaluated against several agencies' cutoffs, including the proposed Substance Abuse and Mental Health Services Administration, European Workplace Drug Testing Society and Driving Under the Influence of Drugs, Alcohol and Medicines cutoffs. A significant correlation was frequently observed between matrices (i.e., between OF and plasma or blood concentrations); however, high intra-subject and inter-subject variability precludes prediction of blood concentrations from OF concentrations. This article will assist individuals in understanding the relative merits and limitations of various methods of OF collection, analysis and interpretation.
- Characterization of an Amphetamine Interference from Gabapentin in an LC-HRMS Method. [Journal Article]
- JAJ Anal Toxicol 2019 Jun 19
- An amphetamine interference was observed during the development of an liquid chromatography-high-resolution mass spectrometry (LC-HRMS) multi-class confirmation method for the determination of 47 dru…
An amphetamine interference was observed during the development of an liquid chromatography-high-resolution mass spectrometry (LC-HRMS) multi-class confirmation method for the determination of 47 drugs and metabolites in urine. The interference passed all qualitative criteria for amphetamine leading to potential false-positive results. Upon investigation, it was found that the amphetamine interference was correlated with the presence of high levels of gabapentin. Gabapentin is routinely detected in patient urine specimens at levels in excess of 1 mg/mL as it is widely prescribed at high doses and does not undergo significant metabolism. The source of the interference was identified as a gabapentin in-source fragment isomeric with protonated amphetamine. Here we describe the characterization of this interference and how its effect was mitigated in the LC-HRMS method.
- Corrigendum to "HighResNPS.com: An Online Crowd-Sourced HR-MS Database for Suspect and Non-targeted Screening of New Psychoactive Substances". [Journal Article]
- JAJ Anal Toxicol 2019 Jun 26
- A Tool for Automatic Correction of Endogenous Concentrations: Application to BHB Analysis by LC-MS-MS and GC-MS. [Journal Article]
- JAJ Anal Toxicol 2019 May 29
- Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human in…
Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human insulin, to name only a few. The presence of significant amounts of these endogenous substances in the biological matrix used to prepare calibration standards and quality control samples (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, including using an analog matrix or analyte, relying entirely on standard addition analyses for these analytes, or simply ignoring the endogenous contribution provided that it is small enough. Although these approaches side-step the issue of endogenous analyte presence in spiked matrix-matched samples, they create serious problems with regards to the accuracy of the analyses or production capacity. We present here a solution that addresses head-on the problem of endogenous concentrations in matrices used for calibration standards and quality control purposes. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration, plus the spiked concentration are then used as the de facto analyte concentration present in the sample. These de facto concentrations are then used in data analysis software (MultiQuant, Mass Hunter, etc.) as the sample's concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This de facto correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC-MS-MS), allowing the rectification of BHB biases of up to 30 μg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding four mouse clicks to the data analysis operations.
- A Novel Liquid Chromatography Tandem Mass Spectrometry Technique using Multi-Period-Multi-Experiment of MRM-EPI-MRM3 with Library Matching for Simultaneous Determination of Amphetamine Type Stimulants Related Drugs in Whole Blood, Urine and Dried Blood Stain (DBS)-Application to Forensic Toxicology Cases in Malaysia. [Journal Article]
- JAJ Anal Toxicol 2019 May 29
- A novel mass spectrometry detection technique based on a multi-period and multi- experiment (MRM-EPI-MRM3) with library matching in a single run for fast and rapid screening and identification of amp…
A novel mass spectrometry detection technique based on a multi-period and multi- experiment (MRM-EPI-MRM3) with library matching in a single run for fast and rapid screening and identification of amphetamine type stimulants (ATS) related drugs in whole blood, urine and dried blood stain was developed and validated. The ATS-related drugs analyzed in this study include ephedrine, pseudoephedrine, amphetamine, methamphetamine, MDMA (3,4-Methylenedioxymethamphetamine), MDA (3,4-Methylenedioxyamphetamine), MDEA (3,4-Methylenedioxy-N-ethylamphetamine) and phentermine. The relative standard deviation for inter and intraday was less than 15% while recoveries ranged from 80% to 120% for all three matrices, i.e., whole blood, urine and dried blood stain. All compounds gave library matching percentage of more than 85% based on the purity. This method was proven to be simple and robust, and provide high confident results complemented with library matching confirmation.
- Simultaneous Determination of Cocaine and Metabolites in Human Plasma Using Solid Phase Micro-Extraction Fiber Tips C18 and UPLC-MS/MS. [Journal Article]
- JAJ Anal Toxicol 2019 May 16
- The determination of cocaine (COC) and its metabolites ecgonine methyl ester (EME), benzoylecgonine (BZE), norcocaine (NCOC) and cocaethylene (CE) in human plasma is relevant in clinical and forensic…
The determination of cocaine (COC) and its metabolites ecgonine methyl ester (EME), benzoylecgonine (BZE), norcocaine (NCOC) and cocaethylene (CE) in human plasma is relevant in clinical and forensic toxicology. An efficient extraction and clean-up of plasma specimens for the simultaneous determination of BZE along with COC and basic metabolites is challenging due to their widely different polarities and ionization characteristics. Recently, biocompatible SPME LC tips C18 became commercially available. We applied SPME LC tips C18 to the simultaneous extraction of COC, BZE, EME, NCOC, and CE by direct immersion of the fiber in plasma diluted with a buffer at pH 8.0. Analytes were desorbed from the fiber to methanol containing formic acid and injected into a UPLC-MS/MS system. The assay was linear from 5 to 500 ng mL-1. Precision assays presented CV% in the range of 2.22 to 10.54%, and accuracy was in the range of 93.4-108.1%. The assay requires minimal quantities of plasma and organic solvents, allowing multiple extractions in parallel. Biocompatible SPME is a promising alternative for preparing biological samples prior to drug measurement by UPLC-MS/MS.
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- Pregabalin and Its Involvement in Coronial Cases. [Journal Article]
- JAJ Anal Toxicol 2019 May 16
- Pregabalin is an anticonvulsant and analgesic designed to treat neuropathic pain and partial seizure disorders and has been available in Australia as a prescription medication since 2005. Studies hav…
Pregabalin is an anticonvulsant and analgesic designed to treat neuropathic pain and partial seizure disorders and has been available in Australia as a prescription medication since 2005. Studies have found high rates of polydrug use associated with pregabalin and it is reportedly used recreationally for its euphoric and relaxing effects as well as to self-manage opioid withdrawal symptoms. A robust analytical method for the analysis of pregabalin using protein precipitation and LC/MS/MS was developed, validated and employed in routine case work. In recent years a substantial increase in pregabalin detections in coronial case submissions had been noted. This study examines the case characteristics and outcomes of 332 coronial cases submitted to the laboratory and analyzed for pregabalin between 2015 and 2017. Pregabalin was identified in approximately 5% of all coronial cases submitted during this time. A high rate of concurrent drug use with pregabalin was evident with the predominant classes being opioids, benzodiazepines and anti-depressants. Post-mortem blood pregabalin concentrations ranged from <0.05 to 140 mg/kg (median 5.5 mg/kg); however, limited interpretation of levels could be achieved as the drug was rarely identified in the absence of other drugs. Cause of death (COD) was found to be drug related in 58% of all cases, with mixed drug toxicity specifically mentioned as related to COD in 40% of cases.