This study is part of a larger project designed to investigate the prevalence of psychoactive drug (PAD) use among Brazilian drivers. In this paper we describe the development and validation of an analytical method to analyze 32 prescription and illicit PADs (amphetamines, benzodiazepines, cocaine, cannabis, opioids, ketamine and m-CPP) and metabolites in oral fluid samples collected with a Quantisal™ device. Samples were extracted with ethyl acetate:hexane and analyzed by LC-MS/MS. Instrumental LOD ranged from 0.26 to 0.65 ng/mL. Mean procedural recoveries at 1.3 ng/mL (LLOQ) ranged from 50% to 120% for 24 compounds. Recoveries were concentration independent, with the exception of femproporex, heroin and ecgonine methyl-ester (EME) for which the recovery decreased significantly at higher levels (13 and 52 ng/mL). RSD was <20% for all compounds at all spiking levels. Ion suppression due to the matrix was <20% for most compounds, and higher than 60% for EME and diethylpropion. Analysis was performed against a in-matrix standard curve. About 10% of the 2235 oral fluid samples collected from drivers on Brazilian Federal highways were positive (≥LOD) for at least one analyte investigated. Alone or in combination with other drugs, cocaine/metabolites were the analytes most detected in the samples (129; 5.8%), followed by amphetamines/metabolite (69; 3.1%), benzodiazepines (28; 1.2%), cannabinoids (23; 1.1%) and opioids (8; 0.4%). Detection of at least two PADs from different classes accounted for 9.3% of the 236 positive samples. Cocaine was found at higher levels in the samples (up to 1165 ng/mL). Preventive measures aimed at reducing the use of PADs by drivers in Brazil will certainly contribute to decrease the country's highway death rates.
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