Gas chromatography with dual cold electron ionization mass spectrometry and vacuum ultraviolet detection for the analysis of phenylethylamine analogues
Graphical abstract
Introduction
Emerging drugs are compounds synthesized to have similar effects as other psychoactive substances and to circumnavigate drug laws [1]. Prevention efforts have been legislated such as the Controlled Substances Act (CSA [2]), which controls certain drugs based on their potential for abuse and accepted medical use. However, efforts to control the distribution and use of synthetic drugs [3] often leads to synthesis of new drugs. In addition, online availability results in easy access to many drugs [4]. There are several classes of synthetic emerging drugs that have entered the market, such as cathinones, cannabinoids, and piperazines. Phenylethylamines (PEAs) are psychoactive drugs that have become more prevalent during the past several years. Certain PEAs were popularized by Alexander Shulgin in his book PiHKAL [5]. Fig. 1 provides the general structure of PEAs, which are composed of a base phenylethylamine structure and may have a wide variety of substituents that can result in various physiological and pharmacological effects. There have been several deaths attributed to PEAs in both the US and Europe and several case studies have shown the adverse effects of PEAs in humans [6], [7]. For example, 25C-NBOMe was found to be drastically more potent than methamphetamine [8].
A wide array of techniques are available for the analysis of PEAs that are applicable to seized drug samples or toxicological samples. These include Fourier transform-infrared spectroscopy (FT-IR) [9], [10], ultra high performance liquid chromatography-ultraviolet detection (UHPLC-UV) [11], LC-mass spectrometry (LC-MS) [9], [12], UHPLC-UV-MS [13], direct analysis in real time-MS (DART-MS) [12], capillary electrophoresis-UV (CE-UV) [11], CE-laser induced fluorescence (CE-LIF) [14], and nuclear magnetic resonance (NMR) [9], [10].
The most popular method of separating drug mixtures in forensic labs is gas chromatography (GC). Coupling GC to flame ionization detection (FID) has been used for forensic analysis of illicit drugs, including PEAs [15], [16]. While robust, cost effective, providing excellent repeatability, and good dynamic range with relatively low limits of detection, compound identification with FID lacks specificity. Since no structural information is obtained for compound(s) under the peak, solutes with similar retention times may not be easily discriminated, and quantitation is unreliable due to possible co-elution. For more confident identification, most forensic laboratories perform GC analysis with MS detection. This allows for compounds to be identified by mass to charge ratios and ion fragment patterns, which provide possible insight into the mass and structure of detected molecules. The acquired electron ionization (EI) mass spectra can be compared to a library database of recorded compounds, which is useful for forensic drug chemists in performing accurate analysis.
Traditionally, forensic labs utilize GC–MS with a 70 eV EI source. However, the analysis of certain compounds may present challenges for classical EI because of excessive fragmentation of the molecular ion. Decreased ionization energies (15 eV) can aid in molecular ion preservation, as well as ring isomer differentiation [17]. Cold electron ionization (Cold EI) aids in the analysis of labile compounds by generating supersonic molecular beams (SMBs), where the vibrational energy of analytes decreases prior to ionization [18], [19]. Molecules ionized after vibrational cooling produce mass spectra with increased relative intensity for the molecular ion. Cold-EI-MS has been used for the analysis of synthetic cathinones [20], cannabinoids [21] and fentanyls [22].
MS can provide enough information to discriminate compounds based on their mass and fragmentation pattern, but may not be able to differentiate positional isomers of certain substances. This is especially true in cases where the isomers differ by the position of a substituent on the benzene ring. With promising potential for casework, statistical analysis has been used to differentiate between positional isomers of certain cathinones [17], [23] and PEAs [24]. Little research has been extended to chemometric analysis of ring isomers that vary with two substituents, such as dimethoxyamphetamine, for which 7 potential isomers exist. Orthogonal techniques such as NMR and FT-IR [9], [10] must be used for increased specificity and accurate compound identification.
Vacuum ultraviolet (VUV) detection for GC is a novel technique that measures the ultraviolet absorbance spectra of eluting compounds. Unlike liquid phase or supercritical fluid phase UV detection, which primarily measures a limited range of π → π* transitions, gas phase UV spectroscopy can measure a wider range of π → π* transitions as well as σ → σ* transitions. Isomers that differ by the position of substitution on the benzene ring, (which are distinguishable by liquid phase or supercritical phase UV detection [25]) are differentiable by VUV detection, but there is also emerging literature detailing differentiation of aliphatic isomers [22], [26]. When used in tandem [27] or in-parallel [22] with GC–MS, this spectroscopic method provided improved compound identification [22], [27], [28]. Simultaneous analysis of analytes provides complementary data, offering identification with higher specificity and greater confidence in confirmation. Gras et al. [29] used GC with simultaneous UV spectroscopy with a 190–350 nm wavelength range, FID, and MS for quantitative and qualitative analysis. VUV has also been used for the analysis of pesticides [30], fatty acid methyl esters [31], polychlorinated biphenyls [32], mineral oils [33] and diesel fuel [34]. Analysis of thermally labile compounds, such as explosives has recently been reported [35].
Research conducted on GC-VUV for the analysis of novel psychoactive substances have previously demonstrated the identification and resolving power of the method [36]. Drug classes studied include synthetic cathinones [36], opiates [37], and cannabanoids [38]. Roberson and Goodpaster [39] used GC-VUV to analyze a set of non-psychoactive PEA standards and street samples. They used principal component analysis (PCA) on 8 PEAs to demonstrate that GC-VUV can identify each tested compound. A comprehensive paper by Kranenburg et al. [40] described the use of GC with both classical EI ionization MS and VUV detection to analyze synthetic amphetamine and cathinone compounds. This method used sodium bicarbonate to improve peak shapes [36].
Spectral deconvolution, which is possible with accompanying VUV software, is explained and tested in the recently published paper by Reiss et al. [41]. Deconvolution is accomplished by a non-negative matrix factorization (NMF), which is a multivariate technique capable of separating a spectra of drug mixtures to identify each component. This allows for the identification of two coeluting analytes.
The combination of VUV and Cold EI-MS for emerging drug analysis was introduced in 2019 by Buchalter et al. [22]. Their research validated GC-Cold-EI-MS-VUV as a reliable tool for analysis of fentanyl analogues. This method allows drug samples to be identified by three different measurements, the combination of MS, VUV and retention data allowed for higher specificity. In this study, the GC-Cold-EI-MS-VUV combination is applied to the analysis of a wide range of PEAs, including positional isomers.
Section snippets
Materials and reagents
Compounds selected for analysis include analogues and positional isomers of substituted PEAs. Table 1 provides the compound names and substituents of the 40 compounds analyzed. Compounds marked by the same letter are positional isomers. All PEA reference standards were purchased from Cayman Chemical (Ann Arbor, MI, USA). All standards were received as 1 mg/mL solutions except for 25H-NB4OMe was received as a 10 mg/mL solution and 2-FA, 3-FA, 3,4-DMMA, 2,5-DMMA, DOM, 2C-G were received as
GC separations
Injection and oven temperature programing parameters were based on conditions used by Skultety et al. [36] to analyze synthetic cathinones. This oven program used a relatively high final temperature, which showed no effects of column degradation or bleeding over the course of this research. A GC–MS chromatogram of a mixture containing all compounds included in Table 1 is shown in Fig. 2. Three distinct elution regions were observed, the first being fluoroamphetamines, followed by species with
Conclusions
This study presents a validation of a GC instrument with dual VUV and cold EI MS detection for the analysis of a set of PEAs. Cold EI detection allowed for reduced fragmentation and more reliable identification of compounds based on increased molecular ion relative intensity. VUV provided for highly specific complementary data, including discriminating between positional isomers, allowing for increased identification confidence. No two compounds shared the same retention time, MS, or VUV data,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors greatly acknowledge Dennise Montero and Sydney Buchalter for their help with instrument operation.
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