Identification of Designer Drugs Using Gas Chromatography High-Resolution Mass Spectrometry and a Soft-Ionization Source
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Journal of Forensic Science & Criminology Volume 1 | Issue 3 ISSN: 2348-9804 Research Article Open Access Identification of Designer Drugs using Gas Chromatography High-Resolution Mass Spectrometry and A Soft-Ionization Source Lopez-Avila V* Agilent Technologies, USA *Corresponding author: Lopez-Avila V, Agilent Technologies, Santa Clara, CA 95051, USA, Fax: (408) 553- 3677, Tel: (408) 553-2709, E-mail: [email protected] Citation: Lopez-Avila V (2013) Identification of Designer Drugs using Gas Chromatography High-Resolution Mass Spectrometry and a Soft-Ionization Source. J Forensic Sci Criminol 1(3): 301. doi: 10.15744/2348- 9804.1.301 Received Date: September 27, 2013 Accepted Date: December 11, 2013 Published Date: December 16, 2013 Abstract A small set of amphetamines has been analyzed by gas chromatography (GC) high-resolution time-of-flight mass spectrometry (TOFMS) using a microplasma photoionization (MPPI) soft-ionization source. This plasma-based, wavelength selectable ionization source enables ionization of the test compounds and their corresponding derivatives at ~8-12 eV that is a softer alternative to electron ionization at 70 eV. Three plasma gases were used in this study: Xe plasma that emits photons at resonance lines of 9.57 eV and 8.44 eV; Kr plasma at 10.63 eV and 10.02 eV, and Ar plasma at 11.82 eV and 11.61 eV. Derivatization of the test compounds with trifluoroacetic anhydride and α-methoxy-α-(trifluoromethyl)-phenylacetyl pyrazole was evaluated because the MPPI mass spectra of the underivatized amphetamines yield primarily iminium ions, which make the identification of the test compounds by GC-TOFMS inconclusive. The MPPI mass spectra of the TFA-derivatized amphetamines yield abundant molecular ions, when using Xe as plasma gas, and enough fragment ions with the Ar plasma that can help in formula generation. The structure elucidation of two “known unknowns” designer drugs using this “tunable” soft-ionization source and a high-resolution TOF mass spectrometer is presented in this study. Introduction GC-MS with conventional EI using 70 eV electrons is often preferred over LC-MS, despite the fact that for certain Gas chromatography-mass spectrometry (GC-MS) with applications derivatization is needed to reduce polarity and electron ionization (EI) or chemical ionization (in positive or increase analyte volatility, because of its higher sensitivity, negative mode) plays an important role in the forensic analysis far better chromatographic resolution of the GC column as of designer drugs. Whereas targeted GC-MS with EI works compared to the LC column and the low susceptibility to matrix well whenever reference analytical standards are available effects in the case of biological samples [5]. Among the LC- for testing or the EI mass spectra of targeted compounds are MS approaches, LC-atmospheric pressure chemical ionization included in databases, the detection and characterization of (APCI) was found to work very well for a series of illicit designer unknown substances is difficult because reference standards drugs including MDA (3,4-methylenedioxy-amphetamine), for newly released designer drugs are commonly not available MDMA (3,4-methylenedioxymethamphetamine), and MDEA and the mass spectra of such designer drugs often lack the (3,4-methylenedioxyethylamphetamine) because of the soft molecular ion from which a molecular formula could be ionization achieved with the APCI source, thus yielding very derived. Alternative approaches to the conventional EI for abundant molecular ions [6]. The use of LC-high resolution GC-MS include softer ionization techniques (i.e., EI with 15 MS (i.e., TOFMS and Orbitrap systems) in clinical toxicology, eV electrons [1], chemical ionization [2], photoionization which has been discussed in detail by Wu et al. [7], offered [3]) but with the exception of the chemical ionization source, unique opportunities in clinical analysis of untargeted drugs the low-energy EI and the photoionization sources are not because ESI is a soft ionization yielding intact molecular ions, commercially available on the GC low-resolution MS systems. and high resolution facilitated the generation of molecular Liquid-chromatography (LC) with electrospray ionization formulae of such ions. A HR-MS system [i.e., quadrupole(Q) (ESI) MS has become a frequently employed tool because TOFMS], interfaced to a GC was not commercially available ESI is a soft ionization technique, which typically yields until early 2012, and a HR TOFMS is not yet available protonated molecular ions, and no derivatization is required commercially. to separate compounds by liquid chromatography. A review of the analytical techniques used in forensic science can be found elsewhere [4]. Annex Publishers | www.annexpublishers.com Volume 1 | Issue 3 Journal of Forensic Science & Criminology 2 Differentiation of positional isomers is valuable in forensic stirrer (Optichem digital hotplate stirrer, Chemglass, Life analysis as legal controls are often placed on one of the Sciences, Vineland, NJ ). After cooling to room temperature, positional isomers [8] and a combination of chemical the excess TFA/ethyl acetate was removed by evaporation to derivatization GC-low resolution MS and LC-MS/MS were dryness under a gentle stream of nitrogen and redisolved in 50 used to differentiate between the positional isomers of three µL of ethyl acetate. fluoroamphetamines and three fluoromethamphetamines [8]. In another recent publication, Sekula and Zuba [9] report that GC-MS with 70 eV EI and LC-QTOFMS with ESI are complementary analytical tools because the mechanisms of fragmentation under different ionization conditions lead to different mass spectra for 2-(4-iodo-2,5-dimethoxyphenyl)- N-[(2,3-methylenedioxyphenyl)-methyl]-ethanamine, a psychoactive drug discovered in 2012. This study was undertaken to explore the use of chemical derivatization and soft ionization GC-TOFMS in the identification of a few known designer drugs, and thus to establish whether such methodology could be applied to the identification of similar designer drugs. Soft ionization is achieved here with vacuum ultraviolet (VUV) light produced by a split-ring resonator microplasma device. This windowless device uses rare gases at 3-5 mL/min and delivers approximately 1 W of RF power at 2.5 GHz. The three plasma Figure 1: Chemical structures of the test compounds gases used in this study were pure Xe, 10% Kr in helium, and pure Ar. A schematic diagram of the prototype MPPI Derivatization with MTPA pyrazole soft-ionization source has been presented previously [10]. A 100 μL aliquot of the stock solution of MTPA-pyrazole [11] Although this study was performed with a research TOFMS, a in ethyl acetate at 12.3 mg/mL was added to the vial containing commercial QTOFMS with an EI source is now commercially the test compound(s) that needed to be derivatized and the available from Agilent Technologies. vial was capped, its contents were mixed, and then heated at Experimental: (Materials and Methods) 80 ºC for 10 min on a hotplate stirrer. After cooling to room temperature, the excess reagent was removed by evaporation Chemicals and reagents to dryness under a gentle stream of nitrogen and the residue was redisolved in 100 µL of ethyl acetate. Test compounds (Figure 1) were purchased from Cerilliant (Round Rock, TX). Stock solutions of the individual GC chromatographic conditions amphetamines were in methanol at concentrations of 1 mg/ mL and were stored at 4 ºC. Ten to 100 µL working standard The chromatographic separation was performed using an solutions of the individual compounds or the composite Agilent (Santa Clara, CA) 7890A GC modified to accommodate solutions were evaporated to dryness under a gentle stream a low-thermal module (LTM) column. Standards were of nitrogen and the residue was used for derivatization. introduced into the GC via a 30 m x 0.25 mm id x 0.25 µm film Trifluoroacetic anhydride ReagentPlus (> 99 %), was purchased thickness LTM HP-5MS capillary column also from Agilent from Sigma-Aldrich (St. Louis, MO). The derivatization Technologies. The column temperature was programmed reagent, (+)-α-methoxy-α-(trifluoromethyl)-phenylacetyl from 50 ºC to 245 ºC at 35 ºC and then to a final temperature of pyrazole (MTPA-pyrazole) was purchased from Wako 300 ºC at 15 ºC/min, where it was held for 3 min. Helium was Chemicals USA, Inc. (Richmond, VA) and was stored at -20 ºC used as carrier gas at a flowrate of 1.2 mL/min. The injector prior to use. Spectroscopic-grade ethyl acetate and methylene temperature was 250 ºC, the source temperature was 175 ºC chloride were purchased from J.T. Baker (Phillipsburg, NJ) and the GC-MS transfer line temperature was 300 ºC. The and EMD (Gibbstown, NJ), respectively. Research Plus grade injector fitted with a double tapered liner was set in splitless Xe and ultra-high purity 10% Kr in helium were purchased mode for 2 min after the injection (purge flow was 50 mL/ from Scott Specialty Gases (Plumsteadville, PA) and ultra- min). high purity Ar was purchased from Airgas USA, LLC (Long Beach, CA). Mass Spectrometry Derivatization with trifluoroacetic anhydride The exact mass measurements were collected with a research TOF mass spectrometer, equipped with the above mentioned A 100 µL aliquot of trifluoroacetic (TFA) anhydride/ethyl microplasma photoionization (MPPI) source, and operated acetate (5:1, v/v) was added to each vial containing either in the positive ion mode. The TOF mass spectrometer was a residue from either a single test compound or a composite modified Agilent 6200 Accurate -Mass TOF LC/MS system solution, and the vial was capped, its contents were mixed with with a flight path of 2 m that was upgraded with a 4-GHz data a vortex mixer, and then heated at 80 ºC for 10 min on a hotplate Annex Publishers | www.annexpublishers.com Volume 1 | Issue 3 3 Journal of Forensic Science & Criminology acquisition system. Spectral data were acquired at a rate of 5 amphetamines, etilefrine, and fenfluramine, which have scans/sec and the mass range for data acquisition was 42 to 600 not been tested yet.