MALDI-TOF mass spectrometric determination of eight benzodiazepines with two of their metabolites in blood

• Benzodiazepines and their metabolites were determined by MALDI-TOF-MS.
• The limits of detection of compounds ranged from 0.07 to 2 ng/ml in blood.
• MALDI-MS(-MS) spectra of zopiclone were different from those of ESI-MS(-MS).
• Present method provides a high throughput method using only 20 μl of blood.
• This method was applied to biological fluids obtained from two victims.

A rapid and sensitive method was developed for the determination of benzodiazepines and benzodiazepine-like substances (BZDs) by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of BZDs. Determination of 8 BZDs (with two of their metabolites) belonging to top 12 medical drugs detected in poisonous cases in Japan, was performed using diazepam-d5 as the internal standard. The limit of detection of zolpidem was 0.07 ng/ml with its quantification range of 0.2–20 ng/ml in blood, in the best case, and the limit of detection of flunitrazepam was 2 ng/ml with its quantification range of 6–200 ng/ml in blood, in the worst case. The spectra of zopiclone in MALDI-MS and MS/MS were different from those in electrospray ionization MS and MS/MS. Present method provides a simple and high throughput method for the screening of these BZDs using only 20 μl of blood. The developed method was successfully used for the determination of BZDs in biological fluids obtained from two victims.