Development of the MAE/UHPLC-MS-TOF method for determination of benzodiazepines in human bio-fluids for toxicological analysis

• Microwave-assisted extraction (MAE) of benzodiazepines from human serum and blood.
• Novel approach in forensic toxicology – especially important from the point of view of green chemistry.
• Fast, simple and efficient method focused on biological material by use MS detection.

A rapid method of microwave-assisted extraction (MAE) followed by ultrahigh performance liquid chromatography with mass spectrometry with time of flight detection (UHPLC-MS-TOF) was optimized and validated for the purpose of determination of five benzodiazepines in human serum and blood samples. Extraction parameters and conditions of the UHPLC-MS-TOF method were defined. Validation of the developed method was performed at three concentration levels: 10, 100 and 250 ng/mL of each drug for both serum and blood samples. For serum and blood the limit of detection was found in the ranges 0.46–2.58 ng/mL and 0.43–1.87 ng/mL, precision (RSD): 0.3–6.7% and 0.9–8.4%, accuracy of the assay (RE): −5.3 to +2.4% and −5.7 to +7.6%, recovery: 80.5–104.3% and 79.9–106.9%, matrix effects: 95.9–110.5% and 97.5–114.2%, respectively. Moreover, the optimized and validated MAE/UHPLC-MS-TOF method was applied to analysis of blood samples.

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