Simultaneous Determination of Cathine, Cathinone, Methcathinone and Ephedrine in Oral Fluid by Gas

PP 2.17 Chromatography-Mass Spectrometry

Khaled M Mohamed, Abtehaj Hadi, Alanoud M Alasiri and Mahmoud El-Said Ali Forensic Chemistry Department, College of Forensic Sciences, Naif Arab University for Security Sciences, Saudi Arabia Add

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Abstract Introduction: The stimulating herbal drug kath and its analog methcathinone are Table I: Retention Times and Ions Monitored for GC–MS Analysis of HFBA Derivatives. commonly substances of abuse in most countries. Information on their detectability in Compound RT Quantifier Ion (m/z) Qualifier Ion (m/z oral fluid is limited therefore the aim of this work is to develop and validate a GC-MS (min) method for the detection and quantification of cathine, cathinone, methcathinone and Cathine 8.08 240 117, 330 ephedrine in oral fluid samples. Cathinone 8.85 105 77, 240 Methcathinone 9.08 254 105, 210, 359 Method: The MS was run in selected ion monitor (SIM) mode. The ions m/z 117, 240 Ephedrine 8.67 254 210, 344

and 330 (cathine), m/z 77, 105 and 240 (cathinone), m/z 105, 210 and 254 Amphetamine-d5 7.60 244 337 (methcathinone), m/z 210, 254 and 344 (ephedrine), and m/z 244 and 337 (amphetamine-d ) were used as quantifier and qualifier ions. The analytes and internal 5 Table II: Accuracy and imprecision for cathine, cathinone, methcathinone and ephedrine in oral fluid. standard (amphetamine-D5) were extracted from oral fluid with ethyl acetate and derivatized with heptafluorobutyric anhydride (HFBA) at 60 °C for 30 min. Analyte Intra-assay (n = 5) Inter-assay (n = 15) RESULTS: The calibration curves were linear (r2> 0.98) in the concentration range 20– (ng/mL) Mean Accuracy Imprecision Mean Accuracy Imprecision (ng/mL) (%Bias) (% RSD) (ng/mL) (% Bias) (% RSD) 2000 ng/mL for all analytes. The intra- and inter assay imprecisions were within (1.6- Cathine 12.5%) and (1.5-9.5%), respectively for all analytes. Intra-assay accuracies were 75 70.6 -5.9 5.0 77.9 3.8 8.2 between -5.9 and 6.7% for all analytes. The method was successfully applied to detect 750 800.5 6.7 1.6 813.8 8.5 1.5 1500 1533.7 2.2 2.1 1511.6 0.8 3.8 and quantify the target analytes from oral fluid specimens collected from khat and methcathinone users. Cathinone CONCLUSIONS: The method was well validated and successfully applied to saliva 75 71.7 -4.4 6.8 75.5 0.6 5.4 specimens collected from khat and methcathinone users. 750 730.7 -2.6 9.1 747.3 -0.4 2.3 1500 1570.2 4.7 3.9 1540.5 2.7 1.7

Methcathinone Introduction 75 71.8 -4.3 10.2 77.8 3.7 6.7 750 773.9 3.2 12.5 751.5 0.2 2.8 Herbal cathinones are phenylalkylamine alkaloids naturally found in the leaves of the 1500 1552.2 3.5 3.3 1470.6 -2.0 6.3 khat plant (Catha edulis) [1]. This plant is commonly found in East Africa and Arabian Peninsula; people chew the leaves of this plant for their stimulant effect [2]. The main Ephedrine 75 75.8 1.1 9.2 77.8 3.7% 9.5% natural cathinones present in the khat are cathinone [S-(-)-α-aminopropiophenone] and 750 775.9 3.5 9.7 797.7 6.4% 7.8% cathine [S,S-(+)-norpseudoephedrine]. Cathinone is the main psychoactive constituent 1500 1549.9 3.3 9.5 1536.6 2.4% 8.4% of this plant, and it produces an effect similar to amphetamines.

While blood and urine routinely used for drugs of abuse testing, oral fluid has become a Table III: Concentration levels of cathine, cathinone, methcathinone and ephedrine (ng/mL) in oral fluid cases. popular choice as an alternative matrix for drug testing. In contrast to blood or urine Cases Methcathinone Ephedrine Cases Cathine Cathinone samples, oral fluid is easy to collect, difficulty of adulteration and noninvasive [3-5]. 1 138.0 112.6 1 3625.9 539.1 2 1628.7 156.8 2 2447.3 709.3 To our knowledge, information on natural cathinone and methcathinone and related 3 468.3 91.1 3 1764.8 189.8 4 352.0 81.8 4 25.5 - ephedrine detectability in oral fluid using GC-MS is not reported. Here, a GC-MS 5 185.0 91.5 5 73.7 14.4 method was developed and validated to detect and quantify cathine, cathinone, 6 277.5 17.1 methcathinone and ephedrine in oral fluid samples. Human subject samples were analyzed using the developed method to demonstrate the applicability of the method.

Methods

GC-MS condition A Varian 450-GC gas chromatograph with a Varian 240-MS ion trap mass spectrometer was used for the detection of the analytes. Separation of analytes was performed with VF-5ms (30 m × 0.25 mm) column (Varian, Inc., Palo Alto, USA)..

Sample preparation

To 0.5 mL of oral fluid specimens were added 50 μL of amphetamine-d5 (IS), 100 μL of saturated sodium bicarbonate adjusted to pH 9.0 with I.0 N of sodium carbonate and 4.0 mL of ethyl acetate in tubes. The tubes were vortex mixed for 5 min and centrifuged (3000 rpm) for 3 min. The ethyl acetate was transferred to a 5-mL glass tubes and evaporated to dryness using a stream of nitrogen at room temperature.

Derivatization The extracted samples were dissolved in 50 μL of ethyl acetate, and then 50 μL of HFBA was added. Samples were capped mixed and incubated for 30 min at 65–70°C in heat block. Samples were allowed to cool at room temperature, and then evaporated to dryness under a stream of nitrogen. Samples were then reconstituted with 50 μL of ethyl acetate and 2 μL was injected in GC–MS system. Figure 2. The total ion chromatograms of HFBA derivatives of cathine (2), cathinone (4), methcathinone (5), ephedrine (4) and IS (1) for the analysis of spiked oral fluid at concentration of 2000 ng/mL (A), authentic samples received from khat (B) and methcathinone (C) users. Results Conclusions A GC-MS method was successfully developed and validated to quantify cathine, cathinone, methcathinone and ephedrine in oral fluids. The method was successfully applied to detect and quantify all analytes in human subject samples, which can be employed by clinical and forensic laboratories. Oral fluids is acceptable as alternative biological samples to use to detect and quantify the target cathinones and related ephedrines. References [1] Hassan, N.A., Gunaid, A.A., Murray-Lyon, I.M.; Khat (Catha edulis): health 515 aspects of khat chewing, Eastern Mediterranean Health Journal, (2007); 13: 706–718. [2] Coppola, M., Mondola, R.; Synthetic Cathinones: Chemistry, pharmacology and toxicology of a new class of designer drugs of abuse marketed as “bath salts” or “plant food”; Toxicology Letters, (2012); 211: 144-149. [3] De Giovanni, N., Fucci, N.; The state of the art on the use of oral fluid as alternative specimen in forensic toxicology; Current Pharmaceutical Analysis, (2008); 4: 258–273. [4] Drummer, O.; Drug testing in oral fluid; Clinical Biochemist Reviews, (2006); 27: 147–159. [5] Bosker, W.M., Huestis, M.A.; Oral fluid testing for drugs of abuse; Clinical Chemistry, (2009); 55: 1910–1931.

Figure 1. Mass spectra of HFBA derivatives of cathine, cathinone, methcathinone and amphtamine-d5.

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The International Arab Forensic Science and Forensic Medicine Conference / 8-10 November 2015