Development and validation of a sensitive assay for analysis of midazolam, free and conjugated 1-hydroxymidazolam and 4-hydroxymidazolam in pediatric plasma: Application to Pediatric Pharmacokinetic Study

Abstract

Pharmacokinetic, pharmacodynamic and pharmacogenomic studies of midazolam are currently being performed in critically ill children to find suitable dose regimens. Sensitive assays using small volumes of plasma are necessary to determine the concentrations of midazolam and its respective metabolites in pediatric studies. Midazolam is metabolized to hydroxylated midazolam isomers, which are present as free as well as the corresponding glucuronide conjugates. A high-performance liquid chromatographic method with tandem mass spectrometry has been developed and validated for the quantification of midazolam, and free and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites in small volumes of plasma. Cleanup consisted of 96-well µ-elution solid phase extraction (SPE). The analytes were separated by gradient elution using a C18 analytical column with a total run time of 5 min. Multiple reaction monitoring was employed using precursor to product ion transitions of m/z 326.2 →291.3 for midazolam, m/z 342.1 →203.0 for 1-hydroxymidazolam, m/z 342.1 →325.1 for 4-hydroxymidazolam and m/z 330.2 →295.3 for 2H4-midazolam (internal standard). Since authentic hydroxymidazolamglucuronide standards are not available, samples were hydrolyzed with β-glucuronidase under optimized conditions. Assay conditions were modified and optimized to provide appropriate recovery and stability because 4-hydroxymidazolam was very acid sensitive. Standard curves were linear from 0.5 to 1,000 ng/mL for all three analytes. Intra- and inter day accuracy and precision for quality control samples (2, 20, 200 and 800 ng/mL) were within 85–115% and 15% (coefficient of variation), respectively. Stability in plasma and extracts were sufficient under assay conditions. Plasma samples were processed and analyzed for midazolam, and free 1-hydroxymidazolam and 4-hydroxymidazolam metabolites. Plasma samples that were hydrolyzed with β-glucuronidase were processed and analyzed for midazolam, and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites under the same assay conditions. The difference in concentration between the total and free hydroxymidazolam metabolites provided an estimate of conjugated hydroxymidazolam metabolites. The combination of 96-well µ-elution SPE and LC-MS/MS allows reliable quantification of midazolam and its metabolites in small volumes of plasma for pediatric patients. This assay is currently being successfully utilized for analysis of samples from ongoing clinical trials.

Midazolam Dose Optimization in Critically Ill Pediatric Patients With Acute Respiratory Failure: A Population Pharmacokinetic-Pharmacogenomic Study

“…Blood (1 mL for each sample) was centrifuged, plasma separated and stored at −80 degrees until shipped on dry ice. PK samples were analyzed to quantify morphine, M3G, and M6G using a validated high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) in the Bioanalytical Core within the Center for Clinical Pharmacology at CHOP [19,20]. More detailed information about the analytical assay can be found in the supplemental document.…”

Vitamin D3 Is Transformed into 1,25(OH)2D3 by Triggering CYP3A11(CYP3A4) Activity and Hydrolyzing Midazolam

“…MDZ is usually used as a specific biomarker for CYP3A11 (CYP3A4)-mediated metabolism in several cell lines [21]. MDZ is always metabolized by the CYP3A enzymes to 4-hydroxymidazolam and 1-hydroxymidazolam, both of which participate in calcium metabolism [22]. In our study, concentrations of serum MDZ at all sampling points were remarkably lower in the rats treated with vitamin D3 than in Control rats, suggesting that vitamin D3 regulates levels of CYP3A11 (CYP3A4) and calcium metabolism by hydrolyzing MDZ to 4-hydroxymidazolam and 1-hydroxymidazolam.…”

Prolonged Anesthetic Recovery after Continuous Infusion of Midazolam in 2 Domestic Cats (Felis catus)

“…33 UDP-glucuronosyltransferase is the enzyme involved in phase II of metabolism, in which 1-hydroxymidazolam undergoes glucuronidation. 30 1-Hydroxymidazolam is an active metabolite, 3,25,27,45 and although much less potent than midazolam, its accumulation in humans (for example, due to renal disease, chronic high-dose administration of the parent drug, or genetic differences in enzyme activities) has been reported to cause prolonged sedation. 7,30 In another report, elevated plasma concentrations of 1-hydroxymidazolam were detected in critically ill human patients who recovered slowly from midazolam sedation.…”

“…30 1-Hydroxymidazolam is an active metabolite, 3,25,27,45 and although much less potent than midazolam, its accumulation in humans (for example, due to renal disease, chronic high-dose administration of the parent drug, or genetic differences in enzyme activities) has been reported to cause prolonged sedation. 7,30 In another report, elevated plasma concentrations of 1-hydroxymidazolam were detected in critically ill human patients who recovered slowly from midazolam sedation. 29 Plasma samples collected from human patients still sedated 36 h or more after discontinuation of midazolam infusion indicated high plasma 1-hydroxymidazolam concentrations (3121 to 11,525 ng/mL), whereas plasma midazolam concentrations were in the subtherapeutic range (less than 100 ng/mL).…”