Determination of midazolam in human plasma by liquid chromatography with mass-spectrometric detection

Erin R. Lepper, J. Kevin Hicks, Jaap Verweij, Suoping Zhai, William D. Figg*, Alex Sparreboom

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


A liquid chromatographic assay with mass-spectrometric detection was developed for the quantitative determination of the cytochrome P450 3A phenotyping probe midazolam in human plasma. Sample pretreatment involved a one-step extraction of 600 μl aliquots with ethyl acetate. Midazolam and the internal standard, lorazepam, were separated on a column (150 mm×4.6 mm, i.d.) packed with 5 μm Zorbax Eclipse XDB-C8 material, using a mobile phase composed of methanol and 10 mM aqueous ammonium acetate (60:40, v/v). Column effluents were analyzed using mass-spectrometry with an atmospheric pressure chemical ionization source. Calibration curves were linear in the concentration range of 1.00-200 ng/ml. The accuracy and precision ranged from 92.8 to 112% and 0.056 to 13.4%, respectively, for four different concentrations of quality control samples analyzed in triplicate on eight separate occasions. The developed method was subsequently applied to study the pharmacokinetics of midazolam in a group of 35 human subjects at a single dose of 25 μg/kg.

Original languageEnglish
Pages (from-to)305-310
Number of pages6
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Issue number2
StatePublished - 5 Jul 2004
Externally publishedYes


  • Midazolam


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