Development and application of a multi-component LC-MS/MS method for determination of designer benzodiazepines in urine

•A multi-component method for detection of designer benzodiazepines in urine has been developed and validated.•The method includes hydrolysis of glucuronide conjugates and a simple direct injection into the LC-MS/MS system.•When applied to authentic urine samples, the method detected at least one of the analytes in 40% of the collected samples.

New psychoactive substances (NPS) have become an increasing drug problem in the past decade. For detection of NPS, new analytical methods have to be developed, and the methods also have to be updated regularly. This study aimed at developing a multi-component LC-MS/MS method for detection and quantification of 11 NPS of the benzodiazepine sub-class (“designer benzodiazepines”) in urine specimens. The method involves dilution of urine with internal standard and hydrolysis of any glucuronide conjugated forms. Separation of the compounds was achieved on a BEH Phenyl column, followed by MS/MS detection in positive electrospray mode. Method validation was performed following the EMA guideline. The method was applied to study the occurrence of designer benzodiazepines in Sweden in 2014-2015, by analysis of 390 samples retrieved from a routine drug testing laboratory. In 40% of these samples, selected based on a positive immunoassay benzodiazepine screening but a negative MS confirmation for the standard set of prescription benzodiazepines, intake of designer benzodiazepines was revealed. These results stress the importance of using and updating confirmation methods to include the increasing number of designer benzodiazepines appearing on the NPS market.