Simultaneous analysis of 203 drugs of abuse and metabolites in urine samples using liquid chromatography–tandem mass spectrometry

The issue of drug abuse is increasingly becoming a significant concern worldwide. However, the simultaneous detection of a wide spectrum of drug of abuse (DOA), especially in biofluids, is challenging due to their diverse and varied physicochemical properties and matrix effects. Herein, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of 203 DOAs, including amphetamines, opiates, cathinones, phencyclidines, synthetic cannabinoids, cocaine, and metabolites, in 100 μL of urine. A mass analysis was performed using multiple reaction monitoring mode with an electrospray ionization source. The run time of the developed method was 16 min. Two sample preparation methods were compared for urine samples: enzymatic hydrolysis followed by either dilution or QuEChERS. Both methods demonstrated good linearity and low matrix effects; however, the dilution method showed superior accuracy and precision. Method validation was conducted to assess the efficacy of the developed method. The LC-MS/MS method with the dilution method demonstrated good linearity with the coefficients of determination (R²) above 0.99 for 200 compounds. The limit of quantification (LOQ) ranged from 0.1 to 20.0 ng/mL. The inter-day precision was below 20 % for all 203 compounds, with a bias of ±20 % for 198 compounds. This method was successfully applied to 40 real urine samples. The developed method allows the simultaneous separation and detection of a variety of DOAs with diverse physicochemical properties in a small amount of urine. Furthermore, the technique could be adapted to fit the practical requirements of forensic investigations, thereby enhancing its effectiveness and reliability.