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Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography-high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident., (© The Author(s) 2024. Published by Oxford University Press.)

Tryptophan (TRP) catabolism via the kynurenine pathway is considered to represent a major link between inflammation and various diseases, including neurodegenerative disorders, depression, schizophrenia, multiple sclerosis, cardiovascular disease, and cancer. The kynurenine pathway and levels of TRP and its metabolites kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) are well regulated under physiological conditions but may be altered as part of the activated immune response. A simple, sensitive, and specific liquid chromatography-time of flight mass spectrometry method was developed for determining levels of the four compounds in human plasma samples. The workflow involves protein precipitation with acetonitrile, chromatographic separation on a Phenomenex Luna NH2 column by applying a linear 6 min gradient of 50-5% acetonitrile in aqueous ammonium acetate solution (5 mM, pH 9.5), and mass spectrometric detection with high-resolution tandem mass spectrometry. Charcoal-treated plasma served as surrogate matrix for external standard calibration. Stable-isotope-labeled analogues were used as internal standards. The calibration ranges were 0.5-50 μg/ml for TRP, 20-1000 ng/mL for KYN und QUIN, and 1-50 ng/mL for KYNA. Validation proved fitness of the developed workflow for the intended purpose. The established method was applied to the quantification of the four targets in 100 authentic plasma samples., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Forensic toxicology and environmental water analysis share the common interest and responsibility in ensuring comprehensive and reliable confirmation of drugs and pharmaceutical compounds in samples analyzed. Dealing with similar analytes, detection and identification techniques should be exchangeable between scientific disciplines. Herein, we demonstrate the successful adaption of a forensic toxicological screening workflow employing nontargeted LC/MS/MS under data-dependent acquisition control and subsequent database search to water analysis. The main modification involved processing of an increased sample volume with SPE (500 mL vs. 1-10 mL) to reach LODs in the low ng/L range. Tandem mass spectra acquired with a qTOF instrument were submitted to database search. The targeted data mining strategy was found to be sensitive and specific; automated search produced hardly any false results. To demonstrate the applicability of the adapted workflow to complex samples, 14 wastewater effluent samples collected on seven consecutive days at the local wastewater-treatment plant were analyzed. Of the 88,970 fragment ion mass spectra produced, 8.8% of spectra were successfully assigned to one of the 1040 reference compounds included in the database, and this enabled the identification of 51 compounds representing important illegal drugs, members of various pharmaceutical compound classes, and metabolites thereof., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an indispensable analytical technique in clinical and forensic toxicology for detection and identification of potentially toxic or harmful compounds. Particularly, non-target LC-MS/MS assays enable extensive and universal screening requested in systematic toxicological analysis. An integral part of the identification process is the generation of information-rich product ion spectra which can be searched against libraries of reference mass spectra. Usually, 'data-dependent acquisition' (DDA) strategies are applied for automated data acquisition. In this study, the 'data-independent acquisition' (DIA) method 'Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra' (SWATH) was combined with LC-MS/MS on a quadrupole-quadrupole-time-of-flight (QqTOF) instrument for acquiring informative high-resolution tandem mass spectra. SWATH performs data-independent fragmentation of all precursor ions entering the mass spectrometer in 21m/z isolation windows. The whole m/z range of interest is covered by continuous stepping of the isolation window. This allows numerous repeat analyses of each window during the elution of a single chromatographic peak and results in a complete fragment ion map of the sample. Compounds and samples typically encountered in forensic casework were used to assess performance characteristics of LC-MS/MS with SWATH. Our experiments clearly revealed that SWATH is a sensitive and specific identification technique. SWATH is capable of identifying more compounds at lower concentration levels than DDA does. The dynamic range of SWATH was estimated to be three orders of magnitude. Furthermore, the >600,000 SWATH spectra matched led to only 408 incorrect calls (false positive rate = 0.06 %). Deconvolution of generated ion maps was found to be essential for unravelling the full identification power of LC-MS/MS with SWATH. With the available software, however, only semi-automated deconvolution was enabled, which rendered data interpretation a laborious and time-consuming process.

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool., (Copyright © 2012 John Wiley & Sons, Ltd.)

The usability of a quadrupole-quadrupole-time-of-flight (QqTOF) instrument for the tandem mass spectrometric sequencing of oligodeoxynuleotides was investigated. The sample set consisted of 21 synthetic oligodeoxynucleotides ranging in length from 5 to 42 nucleotides. The sequences were randomly selected. For the majority of tested oligonucleotides, two or three different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 282 fragment ion mass spectra were acquired. Computer-aided interpretation of fragment ion mass spectra was accomplished with a recently introduced comparative sequencing algorithm (COMPAS). The applied version of COMPAS was specifically optimized for the interpretation of information-rich spectra obtained on the QqTOF. Sequences of oligodeoxynucleotides as large as 26-mers were correctly verified in >94% of cases (182 of 192 spectra acquired). Fragment ion mass spectra of larger oligonucleotides were not specific enough for sequencing. Because of the occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences, tandem mass spectra obtained from oligonucleotides consisting of 30 and more nucleotides could not be used for sequence verification neither manually nor with COMPAS. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 401-409, 2009.

Abstract: A global strategy for tandem mass spectrometric de novo sequencing of oligonucleotides is presented which is based on finding among all possible sequences the sequence, whose simulated tandem mass spectrum shows the highest degree of similarity to the measured spectrum. Global de novo sequencing can become a time-consuming task due the exponential increase of the number of possible sequences with increasing oligonucleotide length. For the reduction of sequencing effort and time, simulated annealing was introduced as stochastic optimization technique that is capable to find the optimal sequence by testing only a subset of all possible configurations. We have evaluated the performance of the de novo sequencing approach by sequencing 13 synthetic oligonucleotides ranging in length from 5 to 24 nucleotides. Mass spectrometric and tandem mass spectrometric experiments were performed on a quadrupole-quadrupole-time-of-flight instrument. Accurate molecular mass measurements were used to determine the nucleotide compositions of the oligonucleotides. For the majority of tested oligonucleotides two different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 107 fragment ion mass spectra were acquired. The nucleotide compositions together with the tandem mass spectral information represented the input for the de novo sequencing algorithm. Sequences of oligonucleotides as large as 22-mers were correctly determined in more than 90% of cases. The optimal sequence was retrieved within 1–2min. Fragment ion mass spectra of larger oligonucleotides were inappropriate for de novo sequencing. The occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences gave rise to incorrect results. [Copyright &y& Elsevier]