A validated dilute-and-shoot LC-MS/MS urine screening for the analysis of 95 illicit drugs and medicines: insights from clinical and forensic Brazilian cases

Urine toxicological analysis is a relevant tool in both clinical and forensic scenarios, enabling the diagnosis of acute poisonings, elucidation of deaths, verification of substance use in the workplace, and identification of drug-facilitated crimes. For these analyses, the dilute-and-shoot technique associated with liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a promising alternative since it has demonstrated satisfactory results and broad applicability. This study developed and validated a comprehensive LC-MS-MS screening method to analyze 95 illicit drugs and medicines in urine samples and application to clinical and forensic Brazilian cases. The dilute-and-shoot protocol was defined through multivariate optimization studies and was set using 100 µL of sample and 300 µL of solvent. The total chromatographic run time was 7.5 minutes. The method was validated following the recommendations of the ANSI/ASB Standard 036 Guideline. The lower limits of quantification varied from 20 to 100 ng/mL. Within-run and between-run precision CVs% were lower than 20%, and bias was within ± 20%. Only four of the 95 analytes presented significant ionization suppression or enhancement (> 25%). As proof of applicability, 839 urine samples from in vivo and postmortem cases were analyzed. In total, 90.9% of the analyzed samples were positive for at least one substance, and 78 of the 95 analytes were detected. The most prevalent substances were lidocaine (40.2%), acetaminophen (38.0%), and benzoylecgonine (31.5%). The developed method proved to be an efficient and simplified alternative for analyzing 95 therapeutic and illicit drugs in urine samples. Additionally, the results obtained from sample analysis are essential to understanding the profile of Brazilian substance use, serving as a valuable database for the promotion of health and safety public policies.