Amphetamine and derivatives are the second most commonly used group of illicit drugs worldwide. However, in the last years new psychoactive substances with stimulant effects have appeared in the illegal market, which are not detected with traditional analytical methods. The aim of this study was to develop and validate a method for the determination of amphetamine derivates (amphetamine (A), methamphetamine (M), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA)), synthetic cathinones (methylone, methedrone, mephedrone, methylenedioxypyrovalerone (MDPV), fluoromethcathinone and fluorometamphetamine), and piperazines (metachlorophenylpiperazine (mCPP) and trifluoromethylphenylpiperazine (TFMPP)) in hair using LC-MS/MS.

Thirty mg hair were incubated in 2mL methanol with 0.1% HCl for 1 hour at 60°C. After centrifugation, the solvent was evaporated until dry, reconstituted in 2mL 2% formic acid (FA) and submitted to solid phase extraction with Strata-XC cartridges. After cartridge conditioning, samples were loaded, and two washing steps were performed with 2mL 2% FA and 2mL methanol: water:FA (47.5:47.5:5). Analytes were eluted with 2mL dichloromethane: isopropanol:ammonium hydroxide (47.5:47.5:5). Eluates were evaporated and reconstituted in 75μL ammonium formate 2mM pH 3, to inject 30μL into the LC-MSMS. Chromatographic separation was performed using an Atlantis T3 (3μm; 2.1×50mm) analytical column in gradient mode with ammonium formate 2mM pH 3 and acetonitrile as mobile phase. The method was validated, including specificity (endogenous and exogenous interferences); linearity (n=4); limit of detection (LOD) and quantification (LOQ); accuracy and imprecision (between and within-day) (n=20) at low, medium and high concentrations; extraction recovery (ER) (n=6), matrix effect (ME) (n=10), and process efficiency (PE) (n=6) at low and high concentrations; and stability after 72 hours in the autosampler. The method was applied to the analysis of 7 amphetamine derivatives positive hair specimens.

All the analytes eluted within 9min, with a total run time of 13 minutes. Validation parameters were within the accepted criteria. The method was specific (no endogenous or exogenous interferences were detected) and linearity (r²>0.99) was achieved between 2–4000pg/mg for methylone, methedrone, mephedrone and MDPV; 10–4000pg/mg for fluoromethcathinone, mCPP and TFMPP; 20–4000pg/mg for A, M, MDMA and MDA; and 10–2000pg/mg for fluoromethamphetamine. Accuracy was ±9.6% of target concentration; between and within-day imprecision were <15%, except for fluoromethamphetamine at medium concentration (20.6%). ER was 47.8–92.1%, and all the analytes showed ion enhancement up to 127.2%, except mCPP, MDMA and MDA (no matrix effect observed). PE was between 54.4–165.7%. All real specimens were positive for A and MDMA, 3 for M, 2 for MDA and 1 for mCPP, with concentrations ranging between 44.9–777.1, 98.7–3654.5, 120.4–1538.9, 27.8–135.4, and 6527.9pg/mg, respectively.

A sensitive and specific method for the determination of ATS drugs in hair was developed and validated, achieving lower cut-offs than SoHT recommendations for amphetamine derivates. The method was successfully applied to the analysis of 7 real hair specimens.