Determination of midazolam and its major metabolite 1′- hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Kenya Medical Research Institute (KEMRI), Wellcome Trust Research Programme, P.O. Box 43640, 00100 GPO, Nairobi, Kenya; Kenya Medical Research Institute (KEMRI), Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast, P.O. Box 230, 80108-Kilifi, Kenya; Molecular and Biochemical Parasitology Research Group, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA Liverpool, United Kingdom; Department of Pharmacology and Therapeutics, University of Liverpool, L69 3GE Liverpool, United Kingdom; Neurosciences Unit, Institute for Child Health, University of London, London, United Kingdom; Department of Pharmaceutics and Pharmacy Practice, Faculty of Pharmacy, University of Nairobi, Nairobi, Kenya
We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1′-hydroxymidazolam (1′-OHM) in a small volume (200 μl) of human plasma. Midazolam, 1′-OHM and 1′-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY™ Elite C18 (5 μm particle size; 100 mm × 2.1 mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 μl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M + l] + of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 ≥ 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1′-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1′-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4 ± 3.1% (n = 6) and 84.2 ± 4.7% (n = 8), respectively; for 1′-OHM at 30 and 200 ng/ml the values were 89.9 ± 7.2% (n = 6) and 86.9 ± 5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1′-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1′-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.). © 2005 Elsevier B.V. All rights reserved.
Assays; Drug products; Ionization; Mass spectrometry; Metabolites; Solvent extraction; Antimalarials; Electrospray ionization (ESI); Midazolam; Protonated molecular ions; High performance liquid chromatography; 1' chlordiazepoxide; alpha hydroxymidazolam; analgesic agent; antibiotic agent; anticonvulsive agent; antimalarial agent; antipyretic agent; artesunate; ceftriaxone; chloramphenicol; chlordiazepoxide; chloroquine; cycloguanil; deethylchloroquine; midazolam; midazolam maleate; paracetamol; penicillin G; proguanil; pyrimethamine; quinine; salicylic acid; sulfadoxine; unclassified drug; accuracy; alkalinity; analytic method; anticonvulsant activity; antimalarial activity; article; blood sampling; child; controlled study; device; drug blood level; drug determination; drug isolation; electrospray mass spectrometry; high performance liquid chromatography; human; liquid liquid extraction; malaria; priority journal; reliability; seizure; Anticonvulsants; Child; Chromatography, High Pressure Liquid; Humans; Kenya; Malaria, Falciparum; Midazolam; Reproducibility of Results; Seizures; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization