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.
Article Details
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Wessels AMA, Bolhuis MS, Bult W, Nijsten MWN, Kneyber MCJ, Touw DJ
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.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 1;1162:122476. doi: 10.1016/j.jchromb.2020.122476. Epub 2020 Dec 3.
- PubMed ID
- 33385770 [ View in PubMed]
- Abstract
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 5-1500 mug/L for midazolam, 1-hydroxymidazolam and 4-hydroxymidazolam and 25-5000 mug/L for 1-hydroxymidazolam glucuronide, using a sample volume of 100 muL. 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 freeze-thaw cycles. The assay has been applied in therapeutic drug monitoring of midazolam for (pediatric) intensive care patients.
DrugBank Data that Cites this Article
- Drug Enzymes
Drug Enzyme Kind Organism Pharmacological Action Actions Midazolam UDP-glucuronosyltransferase 2B4 Protein Humans NoSubstrateDetails Midazolam UDP-glucuronosyltransferase 2B7 Protein Humans NoSubstrateDetails - Drug Reactions
Reaction Details Details Details
