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
Muchohi, S.N., 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; Ward, S.A., 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; Preston, L., Molecular and Biochemical Parasitology Research Group, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA Liverpool, United Kingdom; Newton, C.R.J.C., Kenya Medical Research Institute (KEMRI), Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast, P.O. Box 230, 80108-Kilifi, Kenya, Neurosciences Unit, Institute for Child Health, University of London, London, United Kingdom; Edwards, G., 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; Kokwaro, G.O., 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, 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.