Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1′-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry

Abstract

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1'-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 μm UPLC® column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1'-hydroxymidazolam were quantified using deuterium- and (13)C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05-250 pg/mL) and 1'-hydroxymidazolam (0.25-125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1'-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1-100 μg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1'-hydroxymidazolam for at least 10 h, even after the administration of only 1 μg of midazolam.

Midazolam microdose to determine systemic and pre‐systemic metabolic CYP3A activity in humans

“…These dilution steps were carried out according to a corresponding dilution protocol by the investigator with a four-eyes-principle shortly before application which was documented appropriately. The possibility of losses due to adsorption to tubing and vials was thoroughly evaluated and confirmed in preceding experiments [14].…”

“…For a number of reasons midazolam is well suited as a probe drug in microdosing studies: (1) It can be quantified in plasma even at concentrations less than 1 pg ml −1 using MS/MS-technology not requiring radio-labelled drugs [14], (2) plasma concentrations in the observed dose range are well below KM of 3-5 μM for the CYP3A4-mediated biotransformation of midazolam [17]. Hence first order kinetics may be anticipated for CYP3A-dependent from enzyme kinetics for all investigated doses, (3) a saturated enzymatic process becoming unsaturated would disrupt …”

“…Midazolam and 1'-OH-midazolam plasma and urine (after deconjugation) concentrations were quantified by LC/MS/MS techniques as previously described [14,15] with a lower limit of quantification (LLOQ) of 0.093 pg ml −1 for midazolam and 0.281 pg ml −1 for 1'-OHmidazolam.…”

Ultrasensitive quantification of the CYP2E1 probe chlorzoxazone and its main metabolite 6-hydroxychlorzoxazone in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry after chlorzoxazone microdosing

“…The analytical setup has been described before [25]. For chromatographic separation, a Waters Acquity BEH Shield C18 column (1.7 m, 2.1 × 50 mm) with an integrated filter disc was used at 40 • C. The eluent consisted of 0.01% (vol) aqueous formic acid and 5% acetonitrile (A), and acetonitrile including 0.01% formic acid (B).…”

“…To achieve high sensitivity, speed, and reproducibility, we developed and validated an ultrasensitive assay for the quantification of chlorzoxazone and its metabolite 6-hydroxychlorzoxazone in the picomolar range, which is based on highly successful projects on microdosing of the benzodiazepine midazolam in the past [25]. In contrast to our previous work the sample preparation was carried out using liquid/liquid extraction.…”