Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2010-06, Vol.878 (19), p.1629-1633
• Main Authors: Dostalek, Miroslav, Macwan, Joyce S., Chitnis, Shripad D., Ionita, Ileana A., Akhlaghi, Fatemeh
• Format: Article
• Language: English
• Subjects: 1′-hydroxymidazolam; 4-hydroxymidazolam; Acetonitrile; Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography; Chromatography, Liquid - methods; Cytochrome P450 3A; Fundamental and applied biological sciences. Psychology; General pharmacology; Human; Humans; LC–MS/MS; Least-Squares Analysis; Liquid chromatography; Medical sciences; Metabolites; Methyl alcohol; Microsomes, Liver; Midazolam; Midazolam - analogs & derivatives; Midazolam - analysis; Midazolam - blood; Midazolam - chemistry; Pharmacology. Drug treatments; Phenotyping; Phenyls; Reproducibility of Results; Run time (computers); Sensitivity and Specificity; Tandem Mass Spectrometry - methods
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2010.04.001

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D 5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.