Advancing drug testing in oral fluid: Comparison of microflow and analytical flow LC-Orbitrap analysis

• First method for sustainable microflow LC-HRMS/MS focusing on abused drugs in OF. • Validated qualitative procedure according to international guidelines. • A proof-of-concept study using pooled OF samples demonstrated the applicability. • Comparison of microflow LC-HRMS/MS and analytical flow LC-HRMS/MS. Drug abuse is still rising and being a serious issue for healthcare systems and workplace safety amongst others. Fast, sensitive, and reliable methods to confirm abuse with straightforward analytical techniques are thus required. Microflow liquid chromatography (LC)-high-resolution tandem mass spectrometry (HRMS/MS) offering sensitivity and sustainability merits further investigation. Thus, this study aimed to develop a qualitative microflow-based analytical strategy and to evaluate it in comparison to analytical flow LC-HRMS/MS for the detection of abused drugs in oral fluid (OF). The presented procedure allowed the detection of 20 abused drugs as well as nine of their metabolites. Liquid-liquid extraction proved to be suitable for all analytes except for the cannabinoids, which required solid-phase extraction. Analytical flow LC required higher injection volume (five microliter) and an increased flow rate (500 µL/min) while microflow LC allowed reduced injection volume (one microliter), reduced solvent consumption, and reduced flow rate (100 µL/min). Chromatographic separation using two Hypersil Gold C 18 columns with different inner column diameters followed by Orbitrap-based MS resulted in better peak shapes and considering the different injection volumes to an increased analyte sensitivity with limits of identification between 0.2–25 ng/mL. Microflow LC was shown to be suitable and sustainable in analytical toxicology for small molecule analysis in OF. The current procedure allowed the detection of 29 compounds while providing sufficient analytical performance also in comparison to analytical flow LC making it a valuable strategy in the clinical and forensic setting.