title: Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children.
abstract: 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 microl) 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 microm particle size; 100 mm x 2.1mm 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 microl/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 > or = 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.).
authors.affiliation: Kenya Medical Research Institute (KEMRI)/Wellcome Trust Research Programme, PO Box 43640, 00100 GPO, Nairobi, Kenya.sndirangu@wtnairobi.mimcom.net
authors.forename: Simon N
authors.initials: SN
authors.lastname: Muchohi
authors.forename: Steve A
authors.initials: SA
authors.lastname: Ward
authors.forename: Louise
authors.initials: L
authors.lastname: Preston
authors.forename: Charles R J C
authors.initials: CR
authors.lastname: Newton
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Edwards
authors.forename: Gilbert O
authors.initials: GO
authors.lastname: Kokwaro
mesh_terms: D000927:Anticonvulsants / Q000097:blood* / Q000493:pharmacokinetics; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007630:Kenya; D016778:Malaria, Falciparum / Q000150:complications; D008874:Midazolam / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012640:Seizures / Q000188:drug therapy / Q000209:etiology; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000927:Anticonvulsants; C040081:1-hydroxymethylmidazolam; D008874:Midazolam