Stability of synthetic cathinones in oral fluid samples

Synthetic cathinones are new stimulant drugs derived from cathinone that have been sold as "legal highs" worldwide. These compounds can elicit powerful effects such as delusions, hallucinations as well as other potentially dangerous behavior. New analogs with varying effects and potencies are constantly introduced in the market to evade legislation, and they are not detected by routine screening and confirmation methods. Oral fluid is an alternative matrix of increasing interest in forensic toxicology. Its collection is non-invasive and easily supervised, and positive drug findings typically reflect recent drug exposure. The focus of this research was to develop a method for the determination of 10 synthetic cathinones (cathinone, methcathinone, buphedrone, mephedrone, 4-methylethcathinone, 3,4-methylenedioxypyrovalerone (MDPV), methylone, naphyrone, alpha-pyrrolidinovalerophenone (PVP) and N-ethylcathinone) in preserved oral fluid (Quantisal™), as well as evaluate their stability in preserved (Quantisal and Oral-Eze™) and neat oral fluid samples stored under different conditions, using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MSMS). Four-hundred microliters oral fluid-Quantisal buffer mixture (100μL oral fluid and 300μL buffer) were subjected to cation exchange solid phase extraction. The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1% formic acid in water and in acetonitrile in 5min. We used a Shimadzu triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The assay was linear from 1 to 250ng/mL, with the limits of detection of 0.75-1ng/mL. Imprecision (n=15) was <20.7% and accuracy (n=15) was 84-115.3%. Extraction efficiency was 87.2-116.8% (n=6), process efficiency was 30.9-103.7% (n=6), and matrix effect was -65.1 to -6.2% (CV 2.5-15.1%, n=6). The stability was performed for neat oral fluid, oral fluid in Quantisal buffer, and oral fluid in Oral-Eze buffer samples stored up to one month at room temperature, 4°C and -20°C, and after 3 freeze-thaw cycles. Losses up to -71.2 to -100% were observed in neat and preserved samples stored at room temperature up to one month. At 4°C, losses up to -88.2% occurred in neat OF and Oral-Eze samples, while Quantisal samples showed losses up to -34%. All types samples were stable if stored at -20°C and after 3 freeze-thaw cycles.