Determination of cocaine, metabolites and a crack cocaine biomarker in whole blood by liquid-liquid extraction and UHPLC-MS/MS.
Cocaine is a potent stimulant drug widely abused that exists in two forms: as a hydrochloride salt and as a free base (crack). Cocaine and the inactive metabolite benzoylecgonine can be determined to reveal any kind of cocaine use, whereas the pyrolysis product anhydroecgonine methyl ester (AEME) can be determined to reveal crack smoking. There are many bioanalytical LC-MS/MS methods used for the determination of cocaine, metabolites and AEME. In these methods, chromatographic separation is usually performed by HPLC and sample preparation by solid phase extraction. For the first time, an UHPLC-MS/MS method for the simultaneous determination of cocaine, benzoylecgonine, cocaethylene and AEME in blood using a sample preparation by liquid-liquid extraction was developed and validated. Extraction recoveries were approximately 80%, 40%, 80% and 80%, respectively, obtained by using a mixture of MTBE/2-propanol (70:30, v:v). Chromatographic separation was performed on a core shell biphenyl UHPLC column (100×2.1mm ID, 1.7μm particles). Method validation showed that the method is precise, accurate, robust and sensitive for its purposes. Limit of quantification (LOQ) concentrations were 0.7-1.5ng/mL. The method was used to determine cocaine, benzoylecgonine, cocaethylene and AEME in 22 blood samples collected from victims of sudden, unexpected or violent death in Sao Paulo (Brazil). Concentrations ≥LOQ were observed in 19, 21, 10 and 10 of these samples, respectively.