Development of analytical method for the determination of methylphenidate, the analog ethylphenidate and their metabolite ritalinic acid in oral fluid samples by micro-QuEChERS and liquid chromatography-tandem mass spectrometry.

The aim of this study was to develop a quantitative method for the analysis of methylphenidate, the analog ethylphenidate and their metabolite ritalinic acid in oral fluid, using micro-QuEChERS extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oral fluid samples were collected with Quantisal™ device, extracted by micro-QuEChERS technique and analyzed by LC-MS/MS. The developed method met the validation criteria of Academy Standards Board (ASB) Standard Practices for Method Validation in Forensic Toxicology (Standard 036, 2019) with limits of detection and quantification of 0.5 ng/mL and calibration curve from 0.5 to 50 ng/mL. Within-run imprecision was greater than 18.7% while between-run imprecision was greater than 17.0 % for all analytes. Bias did not vary more than 7.7 %. No evidence of carryover was found. Stability studies presented satisfactory results for 24 h on autosampler (10 °C), after 3 cycles of freeze/thaw, 7 days on freezer (-20 °C) and until 7 days on refrigerator (4 °C) for methylphenidate. The validated method was further successfully applied to the analysis of 5 authentic oral fluid samples collected from volunteers at parties and music festivals from different cities in Brazil. Four samples had positive results for methylphenidate and ritalinic acid, and only one sample was positive for methylphenidate. Ethylphenidate was not detected in the samples. The method showed acceptable analytical performance and is environmentally friendly, requiring reduced use of solvents and reagents, with potential to be applied to clinical and forensic analyses.

Quantification of amphetamine and derivatives in oral fluid by dispersive liquid-liquid microextraction and liquid chromatography-tandem mass spectrometry.

The abuse of stimulants such as amphetamine, methamphetamine, ecstasy (MDMA), and their analogues (MDEA and MDA) has been increasing considerably worldwide since 2009. In this work, an analytical method using dispersive liquid-liquid microextraction (DLLME) to determine amphetamine and derivatives in oral fluid samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Linearity was achieved between 20 to 5000 ng/mL (r>0.992, 1/x² weighted linear regression), with a limit of quantification (LOQ) of 20 ng/mL. Imprecision (%relative standard deviation) and bias (%) were not higher than 9.1 and -12.3%, respectively. The matrix effect was lower than 14.6%, with no carryover observed up to 5000 ng/mL and no interference with 10 different oral fluid matrix sources and against 14 pharmaceuticals and other common drugs of abuse. MDMA, MDA, and MDEA in processed samples were stable up to 24 h at autosampler (10°C); and amphetamine and methamphetamine up to 18 h. The developed method was successfully applied to authentic oral fluid analyses (n = 140). The proposed method is an example of the Green Analytical Toxicology, since it reduces both the amount of solvent required in samples preparation and the quantity of solvents and reagents used in analytical-instrumental stage, as well as requires a minimal sample volume, being a cheaper, quicker and more ecological alternative to conventional methods. Obtained results showed that DLLME extraction combined with LC-MS/MS is a fast and simple method to quantify amphetamine derivatives in oral fluid samples.