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Chemotherapeutic treatment of Plasmodium falciparum malaria has relied heavily on the quinoline-containing drugs quinine, chloroquine, mefloquine and halofantrine. In some cases, resistance to these drugs may be a result of a multi-drugresistance (MDR) mechanism involving the P-glycoprotein ATP-binding-cassette transporters. Some strains of parasite exhibit cross-resistance to drugs such as quinine, mefloquine and halofantrine, and some resistant strains exhibit amplification of the P. falciparum mdr homologue PfMDR1. The difficulty of manipulating P. falciparum experimentally makes the use of heterologous systems, for the investigation of parasite genes and their homologues, attractive. Systems based on the yeast Saccharomyces cerevisiae have the advantages of mutant strains that are readily available and ease of handling. In S. cerevisiae, MDR is referred to as pleiotropic drug resistance (PDR) and is associated with a number of P-glycoprotein homologues.

There has been burgeoning interest in psilocybin-use for the treatment of various neurological and neurodegenerative diseases. Psilocybin is mistakenly perceived as the principal pharmacologically active compound due to its high concentrations found in magic mushrooms; however, it is the prodrug of psilocin. Despite the expanding body of clinical research seeking to understand the pharmacodynamic/pharmacokinetic properties of psilocin, and its role in inducing dramatic changes to cognitive function, there has not been a corresponding increase in the development of sensitive analytical methods that can quantify psilocin in different biological fluids. Existing analytical methods have been developed using plasma, serum, and urine as the matrix of choice, but with the unknown blood-to-plasma ratio of psilocin, any pharmacokinetic conclusions drawn solely on plasma data may be misleading. Thus, the main objective of this study is to develop the first analytical method that utilizes SPE and LC-MS/MS to quantify psilocin in human whole blood. The SPE procedure yielded a high recovery efficiency (≥89%) with minimal matrix effects. The method was validated according to ANSI/ASB 036 guidelines. Linearity was between 0.7-200 ng/mL and encompassed previously reported ranges found in plasma/serum. Bias, within- and between-run precision for all quality controls met ANSI/ASB 036 acceptability criteria. Endogenous/exogenous interferences and carryover were negligible. Psilocin stability was assessed at 4°C over 48 h and was considered stable. Although a proof-of-concept study will need to be performed to characterize the method, this analytical workflow was able to detect and quantify psilocin in human whole blood at low limits of quantification., (© 2023 American Academy of Forensic Sciences.)

Liquid-chromatography mass spectrometry (LC-MS) is a powerful bioanalytical tool that is gaining widespread use in operational forensic toxicology laboratories. However, changes in ionization efficiency caused by endogenous or exogenous species must be carefully considered. While different modes of ionization can be used, electrospray ionization (ESI) can be especially prone to this phenomenon due to capacity-limited ionization. This decreased ionization efficiency can influence the accuracy and sensitivity of analytical methods. While quantitative matrix effects are evaluated routinely during method development and validation, drug-mediated ion suppression is not always assessed quantitatively, or in sufficient depth. Although stable isotope labeled internal standards (SIL-IS) can mitigate this issue, they are not always commercially available, particularly for new or emerging substances. In this study, the hypnotic drug suvorexant was used as a model compound for the investigation of such interferences. The potential for significant bias in quantitative analysis was demonstrated using this previously validated assay. In this study, quantitative biases due to ionization suppression are discussed, and techniques to overcome this challenge are presented. Decreases in specimen and injection volume were shown to significantly reduce quantitative bias due to drug-mediated suppression. This straight-forward approach can improve the robustness of analytical methodology, which is particularly important when quantitative measurements are relied upon for medicolegal and other purposes., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Suvorexant (Belsomra®) is a sedative hypnotic that was approved for use in 2015. It has a novel mechanism of action and was the first dual orexin receptor antagonist (DORA) to be approved for the treatment of sleep disorders. Sedative hypnotics often feature prominently in forensic investigations such as impaired driving and drug-facilitated sexual assault (DFSA) cases. As such, suvorexant is a drug of interest and its identification in forensic toxicology investigations is of significance. However, limited studies have been published to date and the disposition or importance of its metabolites has been largely uninvestigated. In this report, we investigate the enzymes responsible for metabolism and explore the prevalence of metabolites in blood from a series of thirteen forensic investigations. Recombinant cytochrome P450 enzymes (rCYPs) were used to generate phase I metabolites for suvorexant in vitro, and metabolites were identified using liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS). Four rCYP isoenzymes (3A4, 2C19, 2D6, and 2C9) were found to contribute to suvorexant metabolism. The only metabolite identified in blood or plasma arose from hydroxylation of the benzyl triazole moiety (M9). This metabolite was identified in seventeen blood and plasma specimens from twelve medicolegal death investigations and one impaired driving investigation. In the absence of a commercially available reference material, the metabolite was confirmed using rCYP-generated in vitro controls using high resolution mass spectrometry., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 0.5 ng/mL suvorexant were achieved in blood., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Suvorexant is a novel drug for the treatment of insomnia that is marketed under the trade name Belsomra®. Unlike other hypnotics, suvorexant is a dual orexin receptor antagonist that is believed to have a lower abuse potential compared to other therapeutics. Although sedative hypnotics feature prominently in forensic toxicology investigations, there have been limited reports that describe the analysis of suvorexant in biological samples. Following a 10-mg oral dose, peak concentrations are typically <200 ng/mL. A highly sensitive assay is required because forensic toxicology laboratories are often required to identify a drug several hours after a single dose. A new analytical procedure for the quantification of suvorexant in whole blood was developed that will aid in the identification of this new drug in forensic toxicology casework. A simple acidic/neutral liquid-liquid extraction (LLE) was used to isolate suvorexant from whole blood followed by liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry analysis using positive electrospray ionization (ESI). The extraction efficiencies of various solvents in blood were evaluated in addition to limit of detection, limit of quantitation, precision, accuracy and bias, calibration model, matrix effects, interferences, and carryover. The recovery of suvorexant was evaluated using four different extraction solvents (N-butyl chloride, ether/toluene (1:1), hexane/ethyl acetate (9:1), and methyl tert-butyl ether (MTBE). Although no significant differences in analytical recovery were observed, N-butyl chloride demonstrated improved reproducibility, efficiency and convenience. A weighted (1/x) quadratic calibration model was selected over a range of 2-200 ng/mL (R 2  = 0.995). Using only 0.5 mL whole blood, limits of detection and quantification were 0.5 ng/mL. Intra-assay (n = 5) and inter-assay (n = 15) precision (% CV) were ≤ 13% and bias ranged from -5 to 2% at concentrations of 5, 50, and 160 ng/mL. Matrix effects were 16% (9% CV) and 15% (8% CV) for 20 ng/mL and 100 ng/mL (n = 20), respectively. No qualitative interferences or carryover were observed; however, a quantitative interference with the internal standard (estazolam-D5) could be attributed to sertraline when present at a 10-fold higher concentration. In the absence of a commercially available deuterated internal standard, the potential for quantitative interferences using LC-based methods are discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)