Development and validation of a ‘dilute and shoot’ LC–MS/MS method in urine suitable for screening and confirmation analyses in a clinical setting

Urine toxicological analysis serves as a significant tool in both clinical and forensic contexts, facilitating the diagnosis of acute intoxications, determination of causes of death, monitoring of substance use in occupational settings, and identification of drug-facilitated crimes. In this regard, the dilute-and-shoot method, when integrated with liquid chromatography-tandem mass spectrometry (LC–MS/MS), represents a promising analytical approach due to its efficacy, reliability, and wide-ranging applicability. This study presents the development and validation of an LC–MS/MS method that simultaneously detects and quantitates 115 drugs and metabolites comprising analytes from different categories, such as drugs of abuse (DOA), new psychoactive substances (NPS), prescription and over-the-counter drugs. Sample pretreatment was studied by applying different solvents (acetonitrile, water or methanol), and the best results were obtained after adding 100 μL of sample, 200 μL of a mixture of methanol: acetonitrile (3:1, v/v). The mixture was vortexed, centrifuged and the supernatant directly injected into the instrument. The analysis took place on a C18 column with a gradient elution over 7.5 min. The method was found to be selective and sensitive, offering LOD/LOQ ranging from 0.01 to 1.5/ 0.05–5 ng/mL, respectively. Validation of the method included evaluation of recovery, carryover, matrix effect, accuracy, precision, selectivity, stability and dilution integrity. The method performed satisfactorily and was therefore applied to urine samples that collected over a 12-week period from individuals enrolled in a rehabilitation program. The proposed method was applied as a follow-up tool aiming to detect prescribed (or not) medicine, as well as other illegal substances.