Analysis of benzodiazepines and their metabolites using DBS cards and LC–MS/MS

• A fast and reliable sample preparation method using DBS card and LC–MS/MS was developed.
• Eighteen benzodiazepines, seven metabolites and zolpidem were the target analytes.
• Among the FTA DMPK card A, B and C, the card C showed superior results.
• All the method validation parameters were satisfactory.

Dried Blood Spot (DBS) has been used a blood extraction method for inherited metabolic disorder screening since 1960s. With introduction of LC–MS/MS, not only DBS could be used to analysis drugs in small blood volume, but in various fields, such as toxicology, drug therapeutic monitoring, drug diagnostic screening, and illicit drugs. In toxicology field, many drugs (e.g. benzodiazepines, acetaminophen, small molecule drugs) have been tested with DBS. Compared with earlier blood extraction methods (SPE and LLE), DBS has lots of advantages; lower blood volume (less than 50 μL), shorter analysis time caused by a more concise analysis procedure and lower cost. We optimized the DBS procedure and LC–MS/MS conditions for 18 benzodiazepines, seven benzodiazepine metabolites, and one z-drug (zolpidem) analysis in blood. 30 μL of whole blood was spotted on FTA DMPK card C and dried for 2 h in a desiccator. A 6-mm disk was punched and vortexed for 1 min in a centrifuge tube with 300 μL methanol/acetonitrile mixture (1:1, v/v). After evaporation, redissolved in 100 μL mobile phase of LC–MS/MS and 5 μL was injected. In the analysis for 26 target compounds in blood, all of the method validation parameters – LLOD, LLOQ, accuracy (intra- and inter-assay), and precision (intra- and inter-assay) – were satisfied with method validation criteria, within 15%. The results of matrix effect, recovery, and process efficiency were good. We developed a fast and reliable sample preparation method using DBS for 26 benzodiazepines, benzodiazepine metabolites, and z-drug (zolpidem).