A rapid and accurate UPLC/MS/MS method for the determination of benzodiazepines in human urine

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2011-02, Vol.879 (5), p.421-428
• Main Authors: Ming, Dong Sheng, Heathcote, John
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Benzodiazepines; Benzodiazepines - chemistry; Benzodiazepines - urine; Biological and medical sciences; Chromatography; Chromatography, High Pressure Liquid - methods; Detectors; drug residues; Drug Stability; Formic acid; Fundamental and applied biological sciences. Psychology; General pharmacology; Human; Humans; Ionization; Medical sciences; methanol; Methanol - chemistry; Methyl alcohol; monitoring; Pharmacology. Drug treatments; Regression Analysis; Reproducibility of Results; screening; Sensitivity and Specificity; tandem mass spectrometry; Tandem Mass Spectrometry - methods; Temperature; ultra-performance liquid chromatography; UPLC/MS/MS; Urine; Urine analysis
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2010.12.029

A rapid, sensitive, and specific ultra-performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) assay method for simultaneous determination of 13 benzodiazepine compounds in human urine was developed and validated. Aliquots of 0.5 mL of urine specimens were used for the analysis and the benzodiazepines were extracted by single step methanol (containing 0.2% formic acid) precipitation and then separated on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column with the temperature maintained at 45 °C. The mobile phases consisted of methanol and water (both containing 0.2% formic acid) and the flow rate was 0.4 mL/min. The TQ detector, equipped with an electrospray ionization ion source, was set up with a positive mode. The acquisitions were performed in multiple-reaction monitoring (MRM) and the limit of quantification was 20 ng/mL for all of the 13 compounds. The low limits of detections (LODs) of the benzodiazepines in this method were between 0.5 and 2 ng/mL. The chromatographic separation time was 4 min and calibration curves in human urine were generated over the range of 20–2000 ng/mL. The method validation parameters such as accuracy, precision, carryover, recovery, stability, and specificity for all of the 13 compounds were within the acceptable range. This method is suitable for the high throughput screening of benzodiazepines in clinical laboratories.