Direct urine analysis for the identification and quantification of selected benzodiazepines for toxicology screening

► We develop simpler and faster sample preparation method for the determination of benzodiazepines in urine. ► We overcome the problem of high cost instrumentation such as LC–MS/MS by providing similar sensitivity and specificity with other methods. ► We overcome false negative/false positive results when using immunoassay methods for benzodiazepines. ► The method is applicable in real clinical and forensic samples. A simple and rapid LC/MS method with direct injection analysis was developed and validated for the identification and quantification of ten benzodiazepines (flunitrazepam, nordiazepam, diazepam, 7-aminoflunitrazepam, flurazepam, bromazepam, midazolam, alprazolam, temazepam and oxazepam) in human urine using diazepam-d5 as internal standard (IS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as diluted urine samples were directly injected into LC/MS system. Electrospray ionization in positive mode using selected ion monitoring was chosen for the identification and quantification of the analytes. The linear range was 50–1000ng/mL for each analyte, with square correlation coefficient (r2)≥0.981. Interday and intraday errors were found to be ≤5.72%. The LC/MS method was applied at ten real samples found initially to be positive and negative, using immunoassay technique. Finally the results were confirmed with GC/MS. The method demonstrates simplicity and fast sample preparation, accuracy and specificity of the analytes which make it suitable for replacement of immunoassay screening in urine avoiding thus false negative/false positive results. Using this method, laboratories may overcome the problem of high cost instrumentation such as LC–MS/MS by providing similar sensitivity and specificity with other methods.