A fast and simple method for the simultaneous analysis of midazolam, 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, plasma and urine

For the quantification of the sedative and anesthetic drug midazolam and its main (active) metabolites 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, human EDTA plasma, human heparin plasma and human urine a single accurate method by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed. Protein precipitation as sample preparation, without the need of a time-consuming deglucuronidation step for the quantification of 1-hydroxymidazolam glucuronide, resulted in a simple and rapid assay suitable for clinical practice with a total runtime of only 1.1 min. The four components and the isotope-labeled internal standards were separated on a C-18 column and detection was performed with a triple-stage quadrupole mass spectrometer operating in positive ionization mode. The method was validated based on the Guidance for Industry Bioanalytical Method Validation (Food and Drug Administration, FDA) and the Guideline on bioanalytical method validation of the European Medicines Agency (EMA). Linearity was proven over the ranges of 51500 mu g/L for midazolam, 1-hydroxymidazolam and 4-hydroxymidazolam and 255000 mu g/L for 1-hydroxymidazolam glucuronide, using a sample volume of 100 mu L. Matrix comparison indicated that the assay is also applicable to other human matrices like EDTA and heparin plasma and urine. Stability experiments showed good results for the stability of midazolam, 1-hydroxymidazolam and 1-hydroxymidazolam glucuronide in serum, EDTA and heparin plasma and urine stored for 7 days under different conditions. At room temperature, 4-hydroxymidazo-lam is stable for 7 days in EDTA plasma, but stable for only 3 days in serum and heparin plasma and less than 24 h in urine. All four compounds were found to be stable in serum, EDTA plasma, heparin plasma and urine for 7 days after sample preparation and for 3 freezethaw cycles. The assay has been applied in therapeutic drug monitoring of midazolam for (pediatric) intensive care patients.

Automated summary

An accurate method using UHPLC-MS/MS has been developed for the quantification of midazolam and its metabolites in human serum, EDTA plasma, heparin plasma, and urine. This method is simple, rapid, and suitable for clinical practice, with good stability and applicability to multiple human matrices.