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.

本研究的目的是开发一种使用 micro- QuEChERS萃取和液相色谱-串联质谱 (LC-MS/MS) 分析口腔液中哌醋甲酯、哌醋甲酯类似物及其代谢物利他林酸的定量方法。使用 Quantisal™ 设备收集口腔液样品，通过 micro- QuEChERS提取技术并通过 LC-MS/MS 进行分析。开发的方法符合美国科学院标准委员会 (ASB) 法医毒理学方法验证标准实践（标准 036, 2019）的验证标准，检测和定量限为 0.5 ng/mL，校准曲线为 0.5 至 50 ng/mL。所有分析物的批内不精密度大于 18.7%，而批间不精密度大于 17.0%。偏差的变化不超过 7.7%。没有发现残留的证据。对于哌醋甲酯，稳定性研究在自动进样器 (10 °C) 上 24 小时、在 3 个冷冻/解冻周期后、在冰箱 (-20 °C) 上 7 天和在冰箱 (4 °C) 上 7 天后呈现令人满意的结果。经验证的方法进一步成功地应用于分析从巴西不同城市的派对和音乐节的志愿者身上采集的 5 份真实口腔液样本。4份样本的哌甲酯和利他林酸呈阳性，只有1份样本的哌甲酯呈阳性。样品中未检测到哌醋甲酯。该方法显示出可接受的分析性能并且对环境友好，需要减少溶剂和试剂的使用，具有应用于临床和法医分析的潜力。