B American Society for Mass Spectrometry, 2014
J. Am. Soc. Mass Spectrom. (2015) 26:159Y165 DOI: 10.1007/s13361-014-1006-9
RESEARCH ARTICLE
Detection of “Bath Salt” Synthetic Cathinones and Metabolites in Urine via DART-MS and Solid Phase Microextraction Joseph LaPointe,1 Brian Musselman,1 Teresa O’Neill,2 Jason R. E. Shepard2 1
IonSense, Inc., Saugus, MA 01906, USA Department of Chemistry, University at Albany, State University of New York (SUNY), Albany, NY 12222, USA
2
Abstract. A rapid and sensitive method, direct analysis in real time mass spectrometry (DART-MS) was applied to the characterization and semiquantitative analysis of synthetic cathinones and their metabolites in urine. DART-MS was capable of detecting three different cathinones and three metabolites down to sub-clinical levels directly without any sample preparations. The process produced a spectrum within seconds because no extraction or derivatization was required for analysis and the high mass accuracy of the instrumentation allowed analysis without the need for lengthy chromatographic separations. The use of solid phase microextration demonstrated a relative increase in the detectability of both drugs and metabolites, improving the detection signal on average more than an order of magnitude over direct detection, while providing cleaner spectra devoid of the major peaks associated with urine that oftentimes dominate such samples. Keywords: Direct analysis in real time, Mass spectrometry, Cathinones, Urine analysis, Solid phase microextraction Received: 17 July 2014/Revised: 4 September 2014/Accepted: 8 September 2014/Published online: 15 October 2014
Introduction
A
n international problem has emerged over the past decade in the form of the manufacture, distribution, and abuse of numerous new psychoactive substances. New designer drug variants are continually being identified by enforcement agencies, but with a surprising turnover rate in the psychoactive species, in some cases having active components that rapidly appear then just as quickly disappear and/or shift in response to new restrictions [1–3]. In fact, the recent World Drug Report from the United Nations Office on Drugs and Crime stated that the occurrence of new psychoactive substances now outnumbers the total number of substances under international control [2]. Some of the more widely observed designer drug classes include synthetic cannabinoids, phenethylamines, synthetic cathinones, and tryptamines, among others. While all of these substances elicit a wide range of adverse effects in individual drug abusers, synthetic cathinones in particular have been documented as associated with increases in emergency hospital admissions and fatalities [1, 4–8].
Correspondence to: Jason R. E. Shepard; e-mail: [email protected]
Synthetic cathinones are central nervous system stimulants with psychoactive effects broadly characterized as being similar to amphetamines or ecstasy, and are considered to be a subset of the phenethylamine family, generally containing a core β-keto-phenethylamine functionality. This class of compounds is quite diverse because of the varied positions to which minor chemical modifications can be made. These modifications slightly alter the chemical structure enough to avoid restrictions while maintaining their bioactivity. Colloquially sold as “bath salts” or “research chemicals,” these products can be purchased over the internet as powders, tablets, or capsules and labeled as “not for human consumption” as a means to further complicate enforcement. Synthetic cathinones are an issue of concern not only because of their increasing use and adverse effects, but also because of the problems associated with their detection and analysis in a rapid and cost-efficient manner. The rapidly changing components make for a moving target as many of the emerging designer drugs involved exploit legal loopholes such that they are essentially not regulated. With new and unknown substances continually appearing, the active components are oftentimes not included in routine screening procedures or drug libraries, and are more complicated to identify because of a lack of standards for comparison. Although testing
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of an unknown white powder is generally a more straightforward procedure, toxicology studies or post-mortem situations, such as driving under the influence of drugs (DUID) or cases presented at hospital emergency rooms can be more complicated by the trace levels at which such drugs or metabolites are present and the complex background matrices in which they are found [4–6, 9]. For example, drugs detected in DUID instances or hospital cases involving these substances are generally associated with concentrations in the microgram to nanogram range (μg/mL–ng/mL) [1, 5, 6, 8, 9]. Additionally, most of these new psychoactive substances have not been studied clinically to fully know what metabolites might be present and to what extent the parent drug is metabolized. Further complicating such testing is that there are wide differences in the type of biological sample chosen for analysis, the varying sampling preparation methods involved, as well as the actual variety of analytical techniques employed [1, 4–6, 10]. Oftentimes, rapid screening methods are used as the first step in forensic drug analysis [4, 6, 11]. For traditional drugs, immunoassays and color tests are available, which can provide cost-effective analyses, and such methods are being developed for cathinones, with varied success [11]. However, it is becoming more common for GC-FID and GC-MS to be employed in forensic toxicology and clinical settings to screen a wider range of compounds at lower concentration levels and with fewer occurrences of false positives [5, 6, 12, 13]. However, such methods employed in a screening capacity are not ideal and add time and effort to the analyses, which contribute to the sample backlogs many labs are currently experiencing. More specifically, many new psychoactive substances, including cathinones and amphetamines, are in salt form, requiring basic extraction for analysis. In addition, these classes of compounds are relatively fragile and extensively fragment under normal GC-MS (electron impact) conditions [10, 14, 15]. Accordingly, such techniques produce similar or identical spectra, oftentimes precluding identification of the parent molecular weight, the key aspect of mass spectrometric analysis. Derivatization can be employed to rectify some of these concerns, but doing so adds additional labor to the preparation, further extends the time associated with chromatographic separations, and is a more complicated procedure to perform in biological samples. Therefore, a recent trend has been to employ more rapid, softer ionization, liquid chromatography, and/or high resolution mass spectrometry techniques as a means to provide better analytical performance for these emerging drugs [7, 12–20]. In particular, the use of higher resolution mass spectrometry screening applications have gained traction in recent years because of their potential in identification of drugs for which no reference standards were available. More recently, ambient ionization methods have also been demonstrated in forensic analysis, providing instantaneous spectra and a softer ionization that maintains the presence of the parent compound [14, 19–27]. Direct analysis in real time mass spectrometry (DART-MS) is one such ambient ionization method that has been applied extensively towards an extremely wide range of forensic drug analyses, including both the more traditional controlled
substances and new designer drugs [14, 19–21, 23, 25, 28]. The thermally desorbed analyte molecules are ionized by proton transfer reactions to produce [M+H]+ species, resulting in relatively simple mass spectra. The soft ionization process that produces this protonated parent ion and the high mass resolution associated with TOF-MS is critical in calculations for providing candidate molecular formulas. The versatility of DART-MS has been exploited to screen for drugs in a variety of complex backgrounds, and in relation to clinical and toxicologic settings, urine testing has been a particular area of focus [25, 29–32]. Testing urine and/or other biological fluids can add additional challenges because of their complex composition, with many components present at much higher concentration levels than that of a drug or metabolite. Regardless, DART-MS has been employed for analysis of urine directly to detect parent drugs and metabolites for cocaine and methadone [29, 30], methamphetamine and ecstasy [31], as well as other stimulants [32] and over-the-counter medications [25] without any sample preparations. A few groups demonstrated rapid extraction methods from biological materials such as solid phase microextraction (SPME) to increase detection limits and remove matrix interferents [30, 33, 34]. Herein, we demonstrate DART-MS was capable of detecting synthetic cathinones and cathinone metabolites from urine directly over a wide range of concentrations, down to clinically relevant levels. The presence and detection of both parent drug and metabolite reinforce the notion of drug use and analysis is enhanced with accurate mass measurements, which can aid identification of unknowns or in the search for other potential metabolites. In addition, SPME was employed to increase detection limits at the level of an order of magnitude or more, concentrating analytes and removing interferences, giving greater confidence in identification of drugs in complex biological samples for toxicology analyses.
Experimental Reagents All synthetic cathinones and cathinone metabolites were purchased from Cayman Chemical (Ann Arbor, MI, USA). Urine (Liquicheck Urinaysis Control, Liquichek Urine Tox Negative Control, and qUantify Control) were purchased from Bio-Rad Laboratories (Hercules, CA, USA).
Sample Preparation Cathinones stock solutions and serial dilutions were prepared by addition of the drug and/or metabolite to urine. Cathinone/ urine solutions (3 μL) were spotted onto QuickStrip Sample Cards (IonSense, Inc., Saugus, MA, USA) in triplicate, with up to 12 sample spots per card, aligned perpendicular to the gap between the ion source and the mass spectrometer inlet. All of the QuickStrips were run with the gas temperature at 250°C and a Linear Rail presentation speed of 0.5 mm/s.
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Figure 1. The structure and monoisotopic formula weights of the cathinones and metabolites tested, including (a) 3,4dimethylmethcathinone (3,4-DMMC) and (b) its β-hydroxy metabolite; (c) 4-ethylmethcathinone; (d) the β-hydroxy metabolite of pentedrone; (e) 2-methylmethcathinone; (f) the β-hydroxy metabolite of mephedrone
DART-MS Parameters An EXACTIVE +LC/MS (ThermoFisher, Waltham, MA, USA) equipped with DART-SVP (IonSense, Inc., Saugus, MA, USA) was utilized for DART-MS measurements. The Exactive Plus resolution was set to 70,000 for acquisition of positive ion mass spectra. The atmospheric pressure interface was typically operated at the following potentials: capillary inlet 250°C. The DART ion source was operated with helium gas (Airgas, Cambridge, MA, USA) at 250°C, at a flow rate of 2.5 L min-1, and a grid voltage of 350 V. The EXACTIVE+ Xcalibur software was utilized for data processing.
SPME Parameters Three custom-made coated metal solid phase microextraction (SPME) fibers, one coated with 200 μm of C18, the second coated with a strong cation exchange resin, and the third coated with 200 μm of PDMS/divinyl-benzene (Sigma Aldrich,
Bellefonte, PA, USA) were utilized for rapid extraction of drug and metabolites. The SPME fibers were immersed in a preconditioning solvent of 1:1 methanol:water for 30 min, after conditioning fibers were immersed in sample for 1 h with the sample plate holder being shaken with a Scientific Industries SI-0400 Microplate Genie (Cole Parmer, Vernon Hills, IL, USA) to effect good mixing. Post extraction, the fibers were rinsed in clean water for 10 sec with agitation. A custom fabricated SPME holder was used to introduce the fibers laterally to the optimal desorption/ionization position between the DART source and the mass spectrometer inlet. SPME fibers were analyzed by using DART gas heated to 250°C.
Results and Discussion The detection of new psychoactive substances from body fluids is evolving to a stage where mass spectrometry methods are predominately performed because of the detection limit
Table 1. The Signal Intensity of 3,4-Dimethylmethcathinone (3,4-DMMC) and Its β-Hydroxy Metabolite from 200 μg/mL to 2.0 ng/Ml. The Drug and Metabolites Were Successfully Detected Via DART-MS, as Well as Via the Use of Two Solid Phase Extraction Sorbent (C18 and PDMS/DVB). The Signal Enhancement Was Calculated Based on the Ratio of the Two Measurements, Providing a Signal Increase of Over an Order of Magnitude (Ranging from 10-Fold to 27-Fold) Signal intensity
Signal intensity
Concentration
3,4-DMMC
C18 SPME
C18/direct detection ratio
β-OH 3,4-DMMC
PDMS SPME
PDMS/direct detection ratio
2.0 ng/mL 20 ng/mL 200 ng/mL 2.0 μg/mL 20 μg/mL 200 μg/mL
1.12E+06 8.66E+05 2.23E+06 2.69E+06 1.60E+07 5.44E+07
1.13E+07 1.99E+07 -
10.1 23.0 -
3.70E+06 3.02E+06 4.82E+06 2.28E+06 3.55E+06 8.75E+06
9.84E+07 7.72E+07 -
26.6 25.6 -
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requirements for clinical samples, the ability to quantitate over a wide range, and the ubiquity in what compounds can be detected in a single assay. Urinalysis of drugs can target the detection of parent drug and/or metabolites based on what is known from in vitro or in silico metabolite studies that employ microsomes, metabolite prediction software, and the knowledge of common metabolic pathways [10, 16, 18, 35]. For example, the parent cathinone 3,4-dimethylmethcathinone (3,4-DMMC) has been identified as a component of drugs found on the illicit market, and its metabolites were identified as those based on the common metabolic pathways of demethylation, reduction, hydroxylation, and oxidation normally observed in urine samples [8, 18, 35]. Furthermore, each of these 100
metabolic products was detected in the urine of a drug abuser as phase I metabolites. However, certain variables are commonly unknown in toxicology casework which complicates analysis of urine for indications of drug use, such as the wide range of purity of drugs found on the illicit market, varied amounts of drug ingested, unknown times since ingestion, and the identity of specific metabolic products associated with each drug. These unknown variables have led to wide analytically relevant concentration ranges reported in urine [1, 8, 16, 36]. Most important, a study by Shima and coworkers reported a known amount of drug ingested and the time since ingestion (~30 mg ingested, with samples collected ~6 h post-intake) [35]. In this study, the
194.1546
192.1389
80 60
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40 20 0 100
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40 20
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60 40
200 pg/mL 193.1595
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Figure 2. DART-MS spectra of five serially dilutions of 3,4-dimethylmethcathinone (3,4-DMMC) and its β-OH metabolite in urine, ranging from 2 μg/mL to 200 pg/mL. The high mass accuracy of the mass spectrometer enabled direct detection of the peaks of interest for each concentration, down to the lowest of clinically relevant levels (nanograms/mL). At the lowest concentrations (2.0 ng/ mL and 200 pg/mL), the peaks of interest were still present, but background peaks became more prominent compared with the drug and metabolite, and the limits of the semiquantitative nature of the assay were reached
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100 5.5
6.5
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-3
75
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parent drug 3,4-DMMC was quantified at 4.4 μg/mL and the βOH-DMMC metabolite was found at a similar concentration, amongst six other metabolites identified. Other clinical reports of cathinones quantitated from urine, with unknown amounts ingested, indeterminate times post-ingestion, and involving various cathinone compounds have been reported in the range of 30 ng/mL to 7.6 μg/mL [8, 37–39]. DART-MS analyses were demonstrated against three cathinones and three known metabolites (conversion of the βketo group to β-hydroxy moiety) in drug-free urine. The six drug and metabolite structures, their molecular formulas, and the calculated [M+H]+ values are shown in Figure 1. Serial dilutions of 3,4-DMMC and its metabolite together in urine were prepared down to the lower range of clinically-relevant concentrations associated with cathinones, and beyond to mimic the most challenging preliminary screening situation. DART-MS analysis of the dilute cathinone solutions in urine without any processing or preparation was able to detect the cathinone and its metabolite down to nanogram/mL concentrations (Table 1, Figure 2). The spectra in Figure 2 show that the high mass accuracy [M+H]+ values for both the drug and metabolite were identified at each concentration. As the concentrations of drug and metabolite decreased, the signal intensity also decreases (Table 1), a gradual but apparent increase in the presence on background peaks was observed (Figure 2) and the quantitative nature of the measurement is reduced. At the lowest concentration level probed (~2.0 ng/mL), although the peaks for both the drug and metabolite are visible in the spectrum, the semiquantitative analysis of the assay is no longer viable. At such concentrations, ion suppression would be more likely to negatively affect the measurements and the signal intensity substantially deviates from linearity. Indeed, the relative standard deviation of triplicate measurements at this concentration was measured at 27% and 23% for the drug and its metabolite, respectively. Accordingly, the DART-MS data of the serially diluted standards in drug-free urine was evaluated for its linearity in response to 3,4-DMMC across five clinically relevant concentrations, from 200 μg/mL to 20 ng/ mL (Figure 3). The ability of this rapid screening method to probe the linear range of a sample in a semiquantitative fashion was applied without internal standards or corrections. Data were obtained in a single measurement file with curves constructed by plotting the peak area against the standard concentrations, resulting in a log–log plot with a linear regression correlation coefficient of R90.98 (Figure 4). In general, the doses of this class of drug ingested by users are usually large enough to result in levels of the parent drug on the high end of the clinical range (μg/mL) [7]. However, drug exposure oftentimes includes situations where trace levels of analysis are of interest, and such situations often probe the limit of detection where the potential for ion suppression exists. To this effect, the same solutions were examined by solid phase microextraction (SPME) with two different sorbent coated samplers (C-18 and PDMS/DVB). Both sorbents demonstrated enhanced detection and preferentially extracted the drugs and metabolites away from the high concentration of background
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-5
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Log (abundance)
-4
2.0 20 ng/ml ng/ml
200 µg/ml
200 2.0 ng/ml µg/ml
0 1.0
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Figure 3. A partial sampling chronogram showing the automated sampling profile of 3,4-dimethylmethcathinone across various concentrations and the appearance of signal over background. The raw data produced enabled direct semiquantitative analysis across five orders of clinically relevant dilutions without the use of internal standards or corrections (insert). The limit of quantitation for this assay was 2.0 ng/mL
components (Tables 1 and 2). Without the use of SPME, we have previously demonstrated that urine spectra were dominated by the metabolic components of urine, specifically urea and creatinine [32]. Spectra generated from the use of SPME resulted in cleaner spectra with the more important ions better represented (data not shown). For 3,4-DMMC and the C18 SPME sorbent, detection limit increases of 10-fold to 23-fold were demonstrated for the 2.0 ng/mL and 20 ng/mL concentrations, respectively (Table 1). More specifically, the 3,4DMMC abundance at 10 ng/mL with direct detection was 8.66E+5, whereas the abundance from analyzing the same solution with the C18 sorbent was 1.99E+7. The ratio of these
spectrometer inlet
SPME fiber ion source
Figure 4. The experimental setup for the use of SPME fibers and DART-MS, with the fiber held in an automated holder to introduce the fiber laterally into the optimal sampling position between the ionization source and the mass spectrometer inlet
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Table 2. The Signal Intensity Enhancement Provided by the Use of Solid Phase Microextration Against the Cathinones 4-Ethylmethcathinone (4-EMC) and 2-Methylmethcathinone (2-MMC), and the β-Hydroxy Metabolites of Pentadrone and Mephedrone at the Lower Limit of Clinically Relevant Concentrations (1.0 to 10 ng/mL). The Drug and Metabolites Were Successfully Detected Via DART-MS, as Well as Via the Use of Two Solid Phase Extraction Sorbent (C18 and PDMS/DVB). The Signal Enhancement was Calculated Based on the Ratio of the Two Measurements, Providing a Signal Increase Ranging from 5-Fold to 60-Fold)
two numbers (1.99E+7/8066E+5) shows a 23-fold increase in signal intensity. Similarly, the PDMS SPME enhanced detection for the 3,4-DMMC β-OH metabolite, demonstrating detection limit increases on the order of 925× compared with direct detection (Table 1). With the 3,4-DMMC metabolite and the PDMS sorbent, the abundance at 10 ng/mL with direct detection was 3.02E+6, whereas the PDMS abundance was 7.72E+7. The ratio of these two numbers (7.72E+7/3.02E+6) is a 25-fold increase in signal intensity. For comparison, a second panel of cathinones and metabolites were serially diluted in urine, including the two cathinones 4-ethylmethcathinone (4-EMC) and 2-methylmethcathinone (2-MMC), and two presumed β-OH metabolites of mephedrone and pentedrone. All studies were performed at similar concentrations as used with 3,4-DMMC, and enhanced levels of detection with SPME fibers were observed for the other two cathinones (range of ~5-fold to 33-fold) and two metabolites (range of 33-fold to 60fold) tested. Data are presented for the two different SPME fiber sorbents simultaneously, as the PDMS/DVB routinely demonstrated better specificity for the metabolites than the C18 sorbent, and vice-versa (data not shown). Analysis of two SPME fibers added negligible time to the assay. Both of these single SPME assays (testing the drug and metabolite on with a single sorbent) generated nearly equivalent results with a significant increase the detectability of both drug related compounds. This is not the case for all SPME fibers tested as a strong cation exchange sorbent (SCX) fiber did not appear to have relevant affinity to either the cathinones or the metabolites and, thus, did not provide any signal enhancement. Certainly, urine samples testing positive for multiple related compounds such as the drug and its metabolite, strengthen the narrative of identified drug use. Because drug users cannot be certain of the actual contents or purity of the drugs they ingest, and street drug formulations of “bath salts” are constantly changing, the actual exposure appears to be highly variable. In particular, mass spectrometry methods employing higher
resolution for screening applications have gained traction in recent years because of their potential to characterize unknown drugs or those for which no reference standards were available. Measurements providing higher mass accuracy allow elemental formula determinations for unknowns that could enable database searches or other calculations as to the identity of drugs and metabolites. Herein, the need for high mass accuracy measurements is advantageous at lower concentrations, where background matrix peaks are observed that could complicate analyses with lower resolution instrumentation, resulting in false positives or false negatives. SPME also was demonstrated as a means to reduce background components and preferentially concentrate drugs and metabolites to advance trace detection capabilities. Finally, phase I metabolites, such as the compounds tested herein, are further metabolized to phase II glucuronides. It has been shown that the DART-MS ionization process has enough energy to break labile bonds such as glucuronides [40], so that direct analysis of urine for detection of phase I metabolites is possible, with or without an enzymatic hydrolysis step, potentially further reducing the amount of sample preparation required for DART urine analysis.
Conclusions DART-MS has the potential to serve as a rapid, informative technique that provides data that are complementary to traditional methods of toxicological analyses, particularly as a viable alternative method for rapid screening. The high mass accuracy has the potential to also signal the presence of unknown drugs and/or metabolites simultaneously. DART-MS demonstrated the detection of drugs and metabolites at clinically relevant (trace) levels in urine. The use of SPME proved beneficial to increase the signals of both drugs and metabolites, in most cases more than an order of magnitude, compared with direct analysis. The analysis of four additional cathinones and metabolite compounds supports the data acquired for 3,4DMMC and its metabolite, in terms of limits of detection, dynamic range, and semiquantitative potential.
Acknowledgments The authors gratefully acknowledge funding of this work in part by DHS under BAA 13-007 Chemical Attribute Signature Studies.
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23. Grange, A.H., Sovocool, G.W.: Detection of illicit drugs on surfaces using direct analysis in real time (DART) time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 25, 1271–1281 (2011) 24. Leuthold, L.A., Mandscheff, J.-F., Fathi, M., Giroud, C., Augsburger, M., Varesio, E., Hopfgartner, G.: Desorption electrospray ionization mass spectrometry: direct toxicological screening and analysis of illicit Ecstasy tablets. Rapid Commun Mass Spectrom 20, 103–110 (2006) 25. Cody, R.B., Laramee, J.A., Durst, H.D.: Versatile new ion source for the analysis of materials in open air under ambient conditions. Anal Chem. 77, 2297–2302 (2005) 26. Cooks, R.G., Ouyang, Z., Takats, Z., Wiseman, J.M.: Ambient mass spectrometry. Science 311, 1566–1570 (2006) 27. Dalgleish, J.K., Wleklinski, M., Shelley, J.T., Mulligan, C.C., Ouyang, Z., Graham Cooks, R.: Arrays of low-temperature plasma probes for ambient ionization mass spectrometry. Rapid Commun Mass Spectrom 27, 135– 142 (2013) 28. Lesiak, A.D., Shepard, J.R.E.: Recent advances in forensic drug analysis by DART-MS. Bioanalysis 6(6), 819–842 (2014) 29. Ropero-Miller, J.D., Stour, P.R.: Forensic toxicology research and development evaluation of new and novel direct sample introduction, time of fligh mass spectrometry (AccuTOF-DART) instrument for postmortem toxicology screening, final report (2008) 30. Jagerdeo, E., Abdel-Rehim, M.: Screening of cocaine and its metabolites in human urine samples by direct analysis in real-time source coupled to timeof-flight mass spectrometry after online preconcentration utilizing microextraction by packed sorbent. J. Am. Soc. Mass Spectrom 20, 891– 899 (2009) 31. Kawamura, M., Kikura-Hanajiri, R., Goda, Y.: Simple and rapid screening for methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) and their metabolites in urine using direct analysis in real time (DART)-TOFMS. Yakugaku Zasshi 131, 827–833 (2011) 32. Lesiak, A.D., Adams, K., Domin, M.A., Henck, C., Shepard, J.R.E.: DART-MS for rapid, preliminary screening of urine for DMAA. Drug Test Anal 6, 788–796 (2014) 33. Rodriguez-Lafuente, A., Mirnaghi, F., Pawliszyn, J.: Determination of cocaine and methadone in urine samples by thin-film solid-phase microextraction and direct analysis in real time (DART) coupled with tandem mass spectrometry. Anal Bioanal Chem. 405, 9723–9727 (2013) 34. Mirnaghi, F.S., Pawliszyn, J.: Reusable solid-phase microextraction coating for direct immersion whole-blood analysis and extracted blood spot sampling coupled with liquid chromatography-tandem mass spectrometry and direct analysis in real time-tandem mass spectrometry. Anal. Chem. 84, 8301–8309 (2012) 35. Shima, N., Katagi, M., Kamata, H., Matsuta, S., Nakanishi, K., Zaitsu, K., Kamata, T., Nishioka, H., Miki, A., Tatsuno, M., Sato, T., Tsuchihashi, H., Suzuki, K.: Urinary excretion and metabolism of the newly encountered designer drug 3,4-dimethylmethcathinone in humans. Forensic Toxicol 31, 101–112 (2013) 36. Leffler, A.M., Smith, P.B.: The analytical investigation of synthetic street drugs containing cathinone analogs. Forensic Sci. Int. 234, 50–56 (2014) 37. Wright, T.H., Cline-Parhamovich, K., Lajoie, D., Parsons, L., Dunn, M., Ferslew, K.E.: Deaths involving methylenedioxypyrovalerone (MDPV) in upper east Tennessee. J. Forensic Sci. 58, 1558–1562 (2013) 38. Kesha, K., Boggs, C.L., Ripple, M.G., Allan, C.H., Levine, B., JuferPhipps, R., Doyon, S., Chi, P., Fowler, D.R.: Methylenedioxypyrovalerone (“bath salts”) related death: case report and review of the literature. J. Forensic Sci. 58, 1654–1659 (2013) 39. Ojanperä, I.A., Heikman, P.K., Rasanen, I.J.: Urine analysis of 3,4methylenedioxypyrovalerone in opioid-dependent patients by gas chromatography-mass spectrometry. Therapeut Drug Monitor. 33, 257– 263 (2011) 40. Yu, S., Crawford, E., Tice, J., Musselman, B., Wu, J.-T.: Bioanalysis without sample cleanup or chromatography: the evaluation and initial implementation of direct analysis in real time ionization mass spectrometry for the quantification of drugs in biological matrixes. Anal. Chem. 81, 193– 202 (2008)
J. Am. Soc. Mass Spectrom. (2015) 26:159Y165 DOI: 10.1007/s13361-014-1006-9
RESEARCH ARTICLE
Detection of “Bath Salt” Synthetic Cathinones and Metabolites in Urine via DART-MS and Solid Phase Microextraction Joseph LaPointe,1 Brian Musselman,1 Teresa O’Neill,2 Jason R. E. Shepard2 1
IonSense, Inc., Saugus, MA 01906, USA Department of Chemistry, University at Albany, State University of New York (SUNY), Albany, NY 12222, USA
2
Abstract. A rapid and sensitive method, direct analysis in real time mass spectrometry (DART-MS) was applied to the characterization and semiquantitative analysis of synthetic cathinones and their metabolites in urine. DART-MS was capable of detecting three different cathinones and three metabolites down to sub-clinical levels directly without any sample preparations. The process produced a spectrum within seconds because no extraction or derivatization was required for analysis and the high mass accuracy of the instrumentation allowed analysis without the need for lengthy chromatographic separations. The use of solid phase microextration demonstrated a relative increase in the detectability of both drugs and metabolites, improving the detection signal on average more than an order of magnitude over direct detection, while providing cleaner spectra devoid of the major peaks associated with urine that oftentimes dominate such samples. Keywords: Direct analysis in real time, Mass spectrometry, Cathinones, Urine analysis, Solid phase microextraction Received: 17 July 2014/Revised: 4 September 2014/Accepted: 8 September 2014/Published online: 15 October 2014
Introduction
A
n international problem has emerged over the past decade in the form of the manufacture, distribution, and abuse of numerous new psychoactive substances. New designer drug variants are continually being identified by enforcement agencies, but with a surprising turnover rate in the psychoactive species, in some cases having active components that rapidly appear then just as quickly disappear and/or shift in response to new restrictions [1–3]. In fact, the recent World Drug Report from the United Nations Office on Drugs and Crime stated that the occurrence of new psychoactive substances now outnumbers the total number of substances under international control [2]. Some of the more widely observed designer drug classes include synthetic cannabinoids, phenethylamines, synthetic cathinones, and tryptamines, among others. While all of these substances elicit a wide range of adverse effects in individual drug abusers, synthetic cathinones in particular have been documented as associated with increases in emergency hospital admissions and fatalities [1, 4–8].
Correspondence to: Jason R. E. Shepard; e-mail: [email protected]
Synthetic cathinones are central nervous system stimulants with psychoactive effects broadly characterized as being similar to amphetamines or ecstasy, and are considered to be a subset of the phenethylamine family, generally containing a core β-keto-phenethylamine functionality. This class of compounds is quite diverse because of the varied positions to which minor chemical modifications can be made. These modifications slightly alter the chemical structure enough to avoid restrictions while maintaining their bioactivity. Colloquially sold as “bath salts” or “research chemicals,” these products can be purchased over the internet as powders, tablets, or capsules and labeled as “not for human consumption” as a means to further complicate enforcement. Synthetic cathinones are an issue of concern not only because of their increasing use and adverse effects, but also because of the problems associated with their detection and analysis in a rapid and cost-efficient manner. The rapidly changing components make for a moving target as many of the emerging designer drugs involved exploit legal loopholes such that they are essentially not regulated. With new and unknown substances continually appearing, the active components are oftentimes not included in routine screening procedures or drug libraries, and are more complicated to identify because of a lack of standards for comparison. Although testing
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of an unknown white powder is generally a more straightforward procedure, toxicology studies or post-mortem situations, such as driving under the influence of drugs (DUID) or cases presented at hospital emergency rooms can be more complicated by the trace levels at which such drugs or metabolites are present and the complex background matrices in which they are found [4–6, 9]. For example, drugs detected in DUID instances or hospital cases involving these substances are generally associated with concentrations in the microgram to nanogram range (μg/mL–ng/mL) [1, 5, 6, 8, 9]. Additionally, most of these new psychoactive substances have not been studied clinically to fully know what metabolites might be present and to what extent the parent drug is metabolized. Further complicating such testing is that there are wide differences in the type of biological sample chosen for analysis, the varying sampling preparation methods involved, as well as the actual variety of analytical techniques employed [1, 4–6, 10]. Oftentimes, rapid screening methods are used as the first step in forensic drug analysis [4, 6, 11]. For traditional drugs, immunoassays and color tests are available, which can provide cost-effective analyses, and such methods are being developed for cathinones, with varied success [11]. However, it is becoming more common for GC-FID and GC-MS to be employed in forensic toxicology and clinical settings to screen a wider range of compounds at lower concentration levels and with fewer occurrences of false positives [5, 6, 12, 13]. However, such methods employed in a screening capacity are not ideal and add time and effort to the analyses, which contribute to the sample backlogs many labs are currently experiencing. More specifically, many new psychoactive substances, including cathinones and amphetamines, are in salt form, requiring basic extraction for analysis. In addition, these classes of compounds are relatively fragile and extensively fragment under normal GC-MS (electron impact) conditions [10, 14, 15]. Accordingly, such techniques produce similar or identical spectra, oftentimes precluding identification of the parent molecular weight, the key aspect of mass spectrometric analysis. Derivatization can be employed to rectify some of these concerns, but doing so adds additional labor to the preparation, further extends the time associated with chromatographic separations, and is a more complicated procedure to perform in biological samples. Therefore, a recent trend has been to employ more rapid, softer ionization, liquid chromatography, and/or high resolution mass spectrometry techniques as a means to provide better analytical performance for these emerging drugs [7, 12–20]. In particular, the use of higher resolution mass spectrometry screening applications have gained traction in recent years because of their potential in identification of drugs for which no reference standards were available. More recently, ambient ionization methods have also been demonstrated in forensic analysis, providing instantaneous spectra and a softer ionization that maintains the presence of the parent compound [14, 19–27]. Direct analysis in real time mass spectrometry (DART-MS) is one such ambient ionization method that has been applied extensively towards an extremely wide range of forensic drug analyses, including both the more traditional controlled
substances and new designer drugs [14, 19–21, 23, 25, 28]. The thermally desorbed analyte molecules are ionized by proton transfer reactions to produce [M+H]+ species, resulting in relatively simple mass spectra. The soft ionization process that produces this protonated parent ion and the high mass resolution associated with TOF-MS is critical in calculations for providing candidate molecular formulas. The versatility of DART-MS has been exploited to screen for drugs in a variety of complex backgrounds, and in relation to clinical and toxicologic settings, urine testing has been a particular area of focus [25, 29–32]. Testing urine and/or other biological fluids can add additional challenges because of their complex composition, with many components present at much higher concentration levels than that of a drug or metabolite. Regardless, DART-MS has been employed for analysis of urine directly to detect parent drugs and metabolites for cocaine and methadone [29, 30], methamphetamine and ecstasy [31], as well as other stimulants [32] and over-the-counter medications [25] without any sample preparations. A few groups demonstrated rapid extraction methods from biological materials such as solid phase microextraction (SPME) to increase detection limits and remove matrix interferents [30, 33, 34]. Herein, we demonstrate DART-MS was capable of detecting synthetic cathinones and cathinone metabolites from urine directly over a wide range of concentrations, down to clinically relevant levels. The presence and detection of both parent drug and metabolite reinforce the notion of drug use and analysis is enhanced with accurate mass measurements, which can aid identification of unknowns or in the search for other potential metabolites. In addition, SPME was employed to increase detection limits at the level of an order of magnitude or more, concentrating analytes and removing interferences, giving greater confidence in identification of drugs in complex biological samples for toxicology analyses.
Experimental Reagents All synthetic cathinones and cathinone metabolites were purchased from Cayman Chemical (Ann Arbor, MI, USA). Urine (Liquicheck Urinaysis Control, Liquichek Urine Tox Negative Control, and qUantify Control) were purchased from Bio-Rad Laboratories (Hercules, CA, USA).
Sample Preparation Cathinones stock solutions and serial dilutions were prepared by addition of the drug and/or metabolite to urine. Cathinone/ urine solutions (3 μL) were spotted onto QuickStrip Sample Cards (IonSense, Inc., Saugus, MA, USA) in triplicate, with up to 12 sample spots per card, aligned perpendicular to the gap between the ion source and the mass spectrometer inlet. All of the QuickStrips were run with the gas temperature at 250°C and a Linear Rail presentation speed of 0.5 mm/s.
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Figure 1. The structure and monoisotopic formula weights of the cathinones and metabolites tested, including (a) 3,4dimethylmethcathinone (3,4-DMMC) and (b) its β-hydroxy metabolite; (c) 4-ethylmethcathinone; (d) the β-hydroxy metabolite of pentedrone; (e) 2-methylmethcathinone; (f) the β-hydroxy metabolite of mephedrone
DART-MS Parameters An EXACTIVE +LC/MS (ThermoFisher, Waltham, MA, USA) equipped with DART-SVP (IonSense, Inc., Saugus, MA, USA) was utilized for DART-MS measurements. The Exactive Plus resolution was set to 70,000 for acquisition of positive ion mass spectra. The atmospheric pressure interface was typically operated at the following potentials: capillary inlet 250°C. The DART ion source was operated with helium gas (Airgas, Cambridge, MA, USA) at 250°C, at a flow rate of 2.5 L min-1, and a grid voltage of 350 V. The EXACTIVE+ Xcalibur software was utilized for data processing.
SPME Parameters Three custom-made coated metal solid phase microextraction (SPME) fibers, one coated with 200 μm of C18, the second coated with a strong cation exchange resin, and the third coated with 200 μm of PDMS/divinyl-benzene (Sigma Aldrich,
Bellefonte, PA, USA) were utilized for rapid extraction of drug and metabolites. The SPME fibers were immersed in a preconditioning solvent of 1:1 methanol:water for 30 min, after conditioning fibers were immersed in sample for 1 h with the sample plate holder being shaken with a Scientific Industries SI-0400 Microplate Genie (Cole Parmer, Vernon Hills, IL, USA) to effect good mixing. Post extraction, the fibers were rinsed in clean water for 10 sec with agitation. A custom fabricated SPME holder was used to introduce the fibers laterally to the optimal desorption/ionization position between the DART source and the mass spectrometer inlet. SPME fibers were analyzed by using DART gas heated to 250°C.
Results and Discussion The detection of new psychoactive substances from body fluids is evolving to a stage where mass spectrometry methods are predominately performed because of the detection limit
Table 1. The Signal Intensity of 3,4-Dimethylmethcathinone (3,4-DMMC) and Its β-Hydroxy Metabolite from 200 μg/mL to 2.0 ng/Ml. The Drug and Metabolites Were Successfully Detected Via DART-MS, as Well as Via the Use of Two Solid Phase Extraction Sorbent (C18 and PDMS/DVB). The Signal Enhancement Was Calculated Based on the Ratio of the Two Measurements, Providing a Signal Increase of Over an Order of Magnitude (Ranging from 10-Fold to 27-Fold) Signal intensity
Signal intensity
Concentration
3,4-DMMC
C18 SPME
C18/direct detection ratio
β-OH 3,4-DMMC
PDMS SPME
PDMS/direct detection ratio
2.0 ng/mL 20 ng/mL 200 ng/mL 2.0 μg/mL 20 μg/mL 200 μg/mL
1.12E+06 8.66E+05 2.23E+06 2.69E+06 1.60E+07 5.44E+07
1.13E+07 1.99E+07 -
10.1 23.0 -
3.70E+06 3.02E+06 4.82E+06 2.28E+06 3.55E+06 8.75E+06
9.84E+07 7.72E+07 -
26.6 25.6 -
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requirements for clinical samples, the ability to quantitate over a wide range, and the ubiquity in what compounds can be detected in a single assay. Urinalysis of drugs can target the detection of parent drug and/or metabolites based on what is known from in vitro or in silico metabolite studies that employ microsomes, metabolite prediction software, and the knowledge of common metabolic pathways [10, 16, 18, 35]. For example, the parent cathinone 3,4-dimethylmethcathinone (3,4-DMMC) has been identified as a component of drugs found on the illicit market, and its metabolites were identified as those based on the common metabolic pathways of demethylation, reduction, hydroxylation, and oxidation normally observed in urine samples [8, 18, 35]. Furthermore, each of these 100
metabolic products was detected in the urine of a drug abuser as phase I metabolites. However, certain variables are commonly unknown in toxicology casework which complicates analysis of urine for indications of drug use, such as the wide range of purity of drugs found on the illicit market, varied amounts of drug ingested, unknown times since ingestion, and the identity of specific metabolic products associated with each drug. These unknown variables have led to wide analytically relevant concentration ranges reported in urine [1, 8, 16, 36]. Most important, a study by Shima and coworkers reported a known amount of drug ingested and the time since ingestion (~30 mg ingested, with samples collected ~6 h post-intake) [35]. In this study, the
194.1546
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Figure 2. DART-MS spectra of five serially dilutions of 3,4-dimethylmethcathinone (3,4-DMMC) and its β-OH metabolite in urine, ranging from 2 μg/mL to 200 pg/mL. The high mass accuracy of the mass spectrometer enabled direct detection of the peaks of interest for each concentration, down to the lowest of clinically relevant levels (nanograms/mL). At the lowest concentrations (2.0 ng/ mL and 200 pg/mL), the peaks of interest were still present, but background peaks became more prominent compared with the drug and metabolite, and the limits of the semiquantitative nature of the assay were reached
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100 5.5
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Relative abundance
parent drug 3,4-DMMC was quantified at 4.4 μg/mL and the βOH-DMMC metabolite was found at a similar concentration, amongst six other metabolites identified. Other clinical reports of cathinones quantitated from urine, with unknown amounts ingested, indeterminate times post-ingestion, and involving various cathinone compounds have been reported in the range of 30 ng/mL to 7.6 μg/mL [8, 37–39]. DART-MS analyses were demonstrated against three cathinones and three known metabolites (conversion of the βketo group to β-hydroxy moiety) in drug-free urine. The six drug and metabolite structures, their molecular formulas, and the calculated [M+H]+ values are shown in Figure 1. Serial dilutions of 3,4-DMMC and its metabolite together in urine were prepared down to the lower range of clinically-relevant concentrations associated with cathinones, and beyond to mimic the most challenging preliminary screening situation. DART-MS analysis of the dilute cathinone solutions in urine without any processing or preparation was able to detect the cathinone and its metabolite down to nanogram/mL concentrations (Table 1, Figure 2). The spectra in Figure 2 show that the high mass accuracy [M+H]+ values for both the drug and metabolite were identified at each concentration. As the concentrations of drug and metabolite decreased, the signal intensity also decreases (Table 1), a gradual but apparent increase in the presence on background peaks was observed (Figure 2) and the quantitative nature of the measurement is reduced. At the lowest concentration level probed (~2.0 ng/mL), although the peaks for both the drug and metabolite are visible in the spectrum, the semiquantitative analysis of the assay is no longer viable. At such concentrations, ion suppression would be more likely to negatively affect the measurements and the signal intensity substantially deviates from linearity. Indeed, the relative standard deviation of triplicate measurements at this concentration was measured at 27% and 23% for the drug and its metabolite, respectively. Accordingly, the DART-MS data of the serially diluted standards in drug-free urine was evaluated for its linearity in response to 3,4-DMMC across five clinically relevant concentrations, from 200 μg/mL to 20 ng/ mL (Figure 3). The ability of this rapid screening method to probe the linear range of a sample in a semiquantitative fashion was applied without internal standards or corrections. Data were obtained in a single measurement file with curves constructed by plotting the peak area against the standard concentrations, resulting in a log–log plot with a linear regression correlation coefficient of R90.98 (Figure 4). In general, the doses of this class of drug ingested by users are usually large enough to result in levels of the parent drug on the high end of the clinical range (μg/mL) [7]. However, drug exposure oftentimes includes situations where trace levels of analysis are of interest, and such situations often probe the limit of detection where the potential for ion suppression exists. To this effect, the same solutions were examined by solid phase microextraction (SPME) with two different sorbent coated samplers (C-18 and PDMS/DVB). Both sorbents demonstrated enhanced detection and preferentially extracted the drugs and metabolites away from the high concentration of background
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-4
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200 µg/ml
200 2.0 ng/ml µg/ml
0 1.0
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Figure 3. A partial sampling chronogram showing the automated sampling profile of 3,4-dimethylmethcathinone across various concentrations and the appearance of signal over background. The raw data produced enabled direct semiquantitative analysis across five orders of clinically relevant dilutions without the use of internal standards or corrections (insert). The limit of quantitation for this assay was 2.0 ng/mL
components (Tables 1 and 2). Without the use of SPME, we have previously demonstrated that urine spectra were dominated by the metabolic components of urine, specifically urea and creatinine [32]. Spectra generated from the use of SPME resulted in cleaner spectra with the more important ions better represented (data not shown). For 3,4-DMMC and the C18 SPME sorbent, detection limit increases of 10-fold to 23-fold were demonstrated for the 2.0 ng/mL and 20 ng/mL concentrations, respectively (Table 1). More specifically, the 3,4DMMC abundance at 10 ng/mL with direct detection was 8.66E+5, whereas the abundance from analyzing the same solution with the C18 sorbent was 1.99E+7. The ratio of these
spectrometer inlet
SPME fiber ion source
Figure 4. The experimental setup for the use of SPME fibers and DART-MS, with the fiber held in an automated holder to introduce the fiber laterally into the optimal sampling position between the ionization source and the mass spectrometer inlet
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Table 2. The Signal Intensity Enhancement Provided by the Use of Solid Phase Microextration Against the Cathinones 4-Ethylmethcathinone (4-EMC) and 2-Methylmethcathinone (2-MMC), and the β-Hydroxy Metabolites of Pentadrone and Mephedrone at the Lower Limit of Clinically Relevant Concentrations (1.0 to 10 ng/mL). The Drug and Metabolites Were Successfully Detected Via DART-MS, as Well as Via the Use of Two Solid Phase Extraction Sorbent (C18 and PDMS/DVB). The Signal Enhancement was Calculated Based on the Ratio of the Two Measurements, Providing a Signal Increase Ranging from 5-Fold to 60-Fold)
two numbers (1.99E+7/8066E+5) shows a 23-fold increase in signal intensity. Similarly, the PDMS SPME enhanced detection for the 3,4-DMMC β-OH metabolite, demonstrating detection limit increases on the order of 925× compared with direct detection (Table 1). With the 3,4-DMMC metabolite and the PDMS sorbent, the abundance at 10 ng/mL with direct detection was 3.02E+6, whereas the PDMS abundance was 7.72E+7. The ratio of these two numbers (7.72E+7/3.02E+6) is a 25-fold increase in signal intensity. For comparison, a second panel of cathinones and metabolites were serially diluted in urine, including the two cathinones 4-ethylmethcathinone (4-EMC) and 2-methylmethcathinone (2-MMC), and two presumed β-OH metabolites of mephedrone and pentedrone. All studies were performed at similar concentrations as used with 3,4-DMMC, and enhanced levels of detection with SPME fibers were observed for the other two cathinones (range of ~5-fold to 33-fold) and two metabolites (range of 33-fold to 60fold) tested. Data are presented for the two different SPME fiber sorbents simultaneously, as the PDMS/DVB routinely demonstrated better specificity for the metabolites than the C18 sorbent, and vice-versa (data not shown). Analysis of two SPME fibers added negligible time to the assay. Both of these single SPME assays (testing the drug and metabolite on with a single sorbent) generated nearly equivalent results with a significant increase the detectability of both drug related compounds. This is not the case for all SPME fibers tested as a strong cation exchange sorbent (SCX) fiber did not appear to have relevant affinity to either the cathinones or the metabolites and, thus, did not provide any signal enhancement. Certainly, urine samples testing positive for multiple related compounds such as the drug and its metabolite, strengthen the narrative of identified drug use. Because drug users cannot be certain of the actual contents or purity of the drugs they ingest, and street drug formulations of “bath salts” are constantly changing, the actual exposure appears to be highly variable. In particular, mass spectrometry methods employing higher
resolution for screening applications have gained traction in recent years because of their potential to characterize unknown drugs or those for which no reference standards were available. Measurements providing higher mass accuracy allow elemental formula determinations for unknowns that could enable database searches or other calculations as to the identity of drugs and metabolites. Herein, the need for high mass accuracy measurements is advantageous at lower concentrations, where background matrix peaks are observed that could complicate analyses with lower resolution instrumentation, resulting in false positives or false negatives. SPME also was demonstrated as a means to reduce background components and preferentially concentrate drugs and metabolites to advance trace detection capabilities. Finally, phase I metabolites, such as the compounds tested herein, are further metabolized to phase II glucuronides. It has been shown that the DART-MS ionization process has enough energy to break labile bonds such as glucuronides [40], so that direct analysis of urine for detection of phase I metabolites is possible, with or without an enzymatic hydrolysis step, potentially further reducing the amount of sample preparation required for DART urine analysis.
Conclusions DART-MS has the potential to serve as a rapid, informative technique that provides data that are complementary to traditional methods of toxicological analyses, particularly as a viable alternative method for rapid screening. The high mass accuracy has the potential to also signal the presence of unknown drugs and/or metabolites simultaneously. DART-MS demonstrated the detection of drugs and metabolites at clinically relevant (trace) levels in urine. The use of SPME proved beneficial to increase the signals of both drugs and metabolites, in most cases more than an order of magnitude, compared with direct analysis. The analysis of four additional cathinones and metabolite compounds supports the data acquired for 3,4DMMC and its metabolite, in terms of limits of detection, dynamic range, and semiquantitative potential.
Acknowledgments The authors gratefully acknowledge funding of this work in part by DHS under BAA 13-007 Chemical Attribute Signature Studies.
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