To develop and validate a method for analyzing cocaine and its metabolites using SPME-Tips and LC-MS/MS. Among the biological matrices used for analytical toxicology, oral fluid has advantages such as simplicity and non-invasive collection, the difficulty of adulteration, the low level of contamination risk and the possibility of recent abuse evaluation. Recently, nearly two million users have used cocaine in the US/Europe. When abused, cocaine is rapidly metabolized, mainly into methylecgonine and benzoylecgonine. Cocaethylene is formed in the concomitant use of cocaine with ethanol, and anhydroecgonine methyl ester (AEME) (in the crack abuse). Currently, in (green) analytical toxicology there is a growing interest in the use of new sample preparation techniques that are fast, efficient, automatable, and ecologically correct. Thus, methods based on microextraction processes have been developed, such as solid phase microextraction (SPME), a miniaturization of solid phase extraction. A variation of SPME technique was recently developed, consisting of a microfiber coated with extractor phase installed in a micropipette tip. In SPME-Tips, the extraction of analytes occurs in a extractor phase, allowing the direct immersion of the tip in biological fluids, causing the analytes to be adsorbed on the fiber and allowing the desorption to be carried out using small volume of more polar and less toxic solvents.

The procedure was optimized, studying parameters such as salting-out effect, extraction time, sample agitation speed, elution solvent volume, and constitution. For extraction, SPME-Tips were immersed in 150μL of oral fluid (collected with Quantisal) and kept under agitation at 500rpm for 80min. For the elution, 125μL of methanolic solution with 0.1% formic acid was used, kept under agitation at 500rpm for 30min. The elution content was taken to LC-MS/MS, in which 5μL were analyzed.

The analyses were performed in a Nexera-UHPLC chromatographic system, coupled with a LCMS8060 mass spectrometer. The chromatographic separation was performed with Atlantis-T3 column (150×3.0mm, 3.0μm), maintained at 40°C. The mobile phase consisted of ultra-pure water (A) and methanol (B), both containing formic acid (0.1%) and 2mmol/L ammonium formate. The flow rate was set to 0.4mL/min, and the elution gradient used started with 10%B for 1min, followed by a linear increase to 100%B for 2min and then maintained at 100%B for 2min and returning to the proportion start in 0.1min. This condition was maintained for another 3min, completing a total of 8min of chromatographic run. With this optimization, the method validation began, as recommended by the AAFS Standard Practices for Method Validation in Forensic Toxicology. For cocaine, benzoylecgonine, and cocaethylene, the correlation coefficient r² was greater than 0.98 (50 to 500ng/mL). Good accuracy and precision results were obtained for low, medium and high QC. In this way, low extraction efficiency was observed to AEME and methylecgonine in C18 SPME-Tips. For the other analytes, the precision and accuracy values were in accordance to the criteria of the method validation guideline. Also, to prove the applicability of the method in real situations, 10 samples of oral fluid collected from individuals who participated in university parties will be analyzed. The samples were already collected and are stored in the Laboratory.

A green analytical toxicology method was developed using SPME-Tips for the analysis of cocaine and its metabolites in oral fluid.