Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers.

Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS2. Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

The differentiation of N-butyl pentylone isomers using GC-EI-MS and NMR.

Forensic laboratories are faced with an ever-expanding seized drug landscape including the increasing prevalence of novel psychoactive substances (NPS), such as synthetic cathinones, that have varying potencies and scheduling. This study demonstrates a combined gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) and nuclear magnetic resonance (NMR) spectroscopy approach for the differentiation of N-butyl pentylone isomers based on distinct retention times, characteristic EI mass spectra, and NMR characterization. Retention time reproducibility was assessed from 60 replicate measurements for each isomer over the course of a month. In addition, the effect of the mass spectrometer tune and the stability of an identified characteristic ion ratio using spectral data from ± 1 scan on either side of the peak apex were also statistically assessed using Welch's ANOVA testing. The presence of diastereomers for N-sec-butyl pentylone was identified using the developed GC-EI-MS method, which was confirmed using one-dimensional and two-dimensional NMR spectroscopy. The retention time reproducibility of the chromatographic method was ± 0.076% or less over the course of a month. An identified characteristic ion ratio between the abundance of the fragment ion at m/z 128 and the fragment ion at m/z 72 enabled the differentiation of the four N-butyl pentylone isomers, even when accounting for the effect of the mass spectrometer tune and mass spectral scans used to calculate the characteristic ion ratio. The 95% confidence interval mean abundance ratio of the fragment ions at m/z 128 and m/z 72 was 17.14 ± 0.14 for N-butyl pentylone, 6.44 ± 0.05 for N-isobutyl pentylone, 3.38 ± 0.02 for N-sec-butyl pentylone, and 0.75 ± 0.01 for N-tert-butyl pentylone. These results highlight the capabilities of a combined GC-EI-MS and NMR approach for the differentiation and characterization of synthetic cathinone isomers.

In vitro metabolism of cathinone positional isomers: does sex matter?

Synthetic cathinones, one of the most prevalent categories of new psychoactive substances, have been posing a serious threat to public health. Methylmethcathinones (MMCs), notably 3-MMC, have seen an alarming increase in their use in the last decade. The metabolism and toxicology of a large majority of synthetic cathinones, including 3-MMC and 2-MMC, remain unknown. Traditionally, male-derived liver materials have been used as in vitro metabolic incubations to investigate the metabolism of xenobiotics, including MMCs. Therefore, little is known about the metabolism in female-derived in vitro models and the potential sex-specific differences in biotransformation. In this study, the metabolism of 2-MMC, 3-MMC, and 4-MMC was investigated using female rat and human liver microsomal incubations, as well as male rat and human liver microsomal incubations. A total of 25 phase I metabolites of MMCs were detected and tentatively identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven sex-specific metabolites were detected exclusively using pooled male rat liver microsomal incubations. In addition, the metabolites generated from the sex-dependent in vitro metabolic incubations that were present in both male and female rat liver microsomal incubations showed differences in relative abundance. Yet, neither sex-specific metabolites nor significant differences in relative abundance were observed from pooled human liver microsomal incubations. This is the first study to report the phase I metabolic pathways of MMCs using in vitro metabolic incubations for both male and female liver microsomes, and the relative abundance of the metabolites observed from each sex.

Expert Algorithm for Substance Identification Using Mass Spectrometry: Application to the Identification of Cocaine on Different Instruments Using Binary Classification Models.

This is the second of two manuscripts describing how general linear modeling (GLM) of a selection of the most abundant normalized fragment ion abundances of replicate mass spectra from one laboratory can be used in conjunction with binary classifiers to enable specific and selective identifications with reportable error rates of spectra from other laboratories. Here, the proof-of-concept uses a training set of 128 replicate cocaine spectra from one crime laboratory as the basis of GLM modeling. GLM models for the 20 most abundant fragments of cocaine were then applied to 175 additional test/validation cocaine spectra collected in more than a dozen crime laboratories and 716 known negative spectra, which included 10 spectra of three diastereomers of cocaine. Spectral similarity and dissimilarity between the measured and predicted abundances were assessed using a variety of conventional measures, including the mean absolute residual and NIST's spectral similarity score. For each spectral measure, GLM predictions were compared to the traditional exemplar approach, which used the average of the cocaine training set as the consensus spectrum for comparisons. In unsupervised models, EASI provided better than a 95% true positive rate for cocaine with a 0% false positive rate. A supervised binary logistic regression model provided 100% accuracy and no errors using EASI-predicted abundances of only four peaks at m/z 152, 198, 272, and 303. Regardless of the measure of spectral similarity, error rates for identifications using EASI were superior to the traditional exemplar/consensus approach. As a supervised binary classifier, EASI was more reliable than using Mahalanobis distances.

Expert Algorithm for Substance Identification Using Mass Spectrometry: Statistical Foundations in Unimolecular Reaction Rate Theory.

This study aims to resolve one of the longest-standing problems in mass spectrometry, which is how to accurately identify an organic substance from its mass spectrum when a spectrum of the suspected substance has not been analyzed contemporaneously on the same instrument. Part one of this two-part report describes how Rice-Ramsperger-Kassel-Marcus (RRKM) theory predicts that many branching ratios in replicate electron-ionization mass spectra will provide approximately linear correlations when analysis conditions change within or between instruments. Here, proof-of-concept general linear modeling is based on the 20 most abundant fragments in a database of 128 training spectra of cocaine collected over 6 months in an operational crime laboratory. The statistical validity of the approach is confirmed through both analysis of variance (ANOVA) of the regression models and assessment of the distributions of the residuals of the models. General linear modeling models typically explain more than 90% of the variance in normalized abundances. When the linear models from the training set are applied to 175 additional known positive cocaine spectra from more than 20 different laboratories, the linear models enabled ion abundances to be predicted with an accuracy of <2% relative to the base peak, even though the measured abundances vary by more than 30%. The same models were also applied to 716 known negative spectra, including the diastereomers of cocaine: allococaine, pseudococaine, and pseudoallococaine, and the residual errors were larger for the known negatives than for known positives. The second part of the manuscript describes how general linear regression modeling can serve as the basis for binary classification and reliable identification of cocaine from its diastereomers and all other known negatives.

Comparison of in-source collision-induced dissociation and beam-type collision-induced dissociation of emerging synthetic drugs using a high-resolution quadrupole time-of-flight mass spectrometer.

In-source collision-induced dissociation (CID) is commonly used with single-stage high-resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in-source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in-source CID and beam-type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in-source CID. Electrospray ionization-quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to generate product ion spectra from in-source CID and beam-type CID for a series of well-characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in-source CID and a range of collision energies for beam-type CID. The results indicate that large fragmentor potentials for in-source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam-type CID. Although conditions can be found in which in-source CID and beam-type CID provide similar overall spectra, the in-source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam-type CID.

The influence of chemical modifications on the fragmentation behavior of fentanyl and fentanyl-related compounds in electrospray ionization tandem mass spectrometry.

Fentanyl is a synthetic opioid that has been approved by the FDA as a general anesthetic because of its rapid onset and high potency. However, since 2013 an opioid epidemic involving fentanyl or fentanyl-related compounds (FRCs) has swept the United States and caused numerous deaths in every state. The identification of novel FRCs is complicated by the rapid turnover of modifications to the core fentanyl structure. In this study, a series of 16 FRCs were analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS) to gain a deeper understanding of the conserved and unique fragmentation behaviors associated with substitution to the core fentanyl structure. This work provides an approach, based on the product ions from ESI-MS/MS, to identify the modification site(s) on the core fentanyl structure for FRCs. Five common locations of substitution to the core fentanyl structure were used to assess the effect of substitution on the fragmentation behavior of FRCs. The proposed fragmentation pathways are supported through the combination of isotopic labeling, multi-stage mass spectrometry (MSn ), and accurate mass measurements with high-resolution mass spectrometry (HRMS). The identification of primary product ions specific to regions of substitution provides an additional tool for the identification of the location of substitution to the core fentanyl structure, which ultimately will assist toxicologists and seized drug analysts in the identification of emerging FRCs.

The characterization of isobaric product ions of fentanyl using multi-stage mass spectrometry, high-resolution mass spectrometry and isotopic labeling.

This study uses a combination of multi-stage mass spectrometry (MSn ), accurate mass measurements - with high-resolution mass spectrometry (HRMS) - and isotopic labeling to characterize the fragmentation behavior of fentanyl and 4-ANPP. By understanding the fragmentation behavior of fentanyl and its analogs in more detail, toxicologists and seized drug analysts will be better poised to identify new and emerging fentalogs, which are increasingly common and deadly adulterants in the growing opioid crisis. Throughout the literature the product ion at m/z 188 is often the most abundant fragment in the mass spectrometric analysis of fentanyl and fentanyl analogs, and this fragment is used for both qualitative and quantitative determinations. Our work shows there are at least three different structures for the isobaric fentanyl product ions at m/z 188, and they each form and fragment via different pathways. The development of fragmentation mechanisms to explain the observed fragmentation pathways of fentanyl and its main precursor 4-ANPP helps contribute to the advancement of knowledge about fentanyl fragmentation and could provide important information for the identification of future fentanyl analogs.