Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection
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; Walter Reed Project/KEMRI Centre for Clinical Research, New Nyanza Provincial General Hospital, P.O. Box 54, Kisumu, Kenya; Department of Pharmaceutics and Pharmacy Practice, Faculty of Pharmacy, University of Nairobi, Nairobi, 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 of Child Health, University of London, London, United Kingdom
A simple, sensitive, selective, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of lorazepam (LZP) in human plasma, using oxazepam (OZP) as internal standard. LZP and OZP were extracted from alkalinized (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with a mixture of n-hexane-dichloromethane (70:30%; v/v). Chromatographic separation was performed on a reversed-phase Synergi ® Max RP analytical column (150 mm × 4.6 mm i.d.; 4 μm particle size), using an aqueous mobile phase (10 mM KH2PO 4 buffer (pH 2.4)-acetonitrile; 65:35%, v/v) delivered at a flow-rate of 2.5 ml/min. Retention times for OZP and LZP were 10.2 and 11.9 min, respectively. Calibration curves were linear from 10 to 300 ng with correlation coefficients (r2) better than 0.99. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 10 ng/ml, respectively, using 0.5 ml samples. The mean relative recoveries at 20 and 300 ng/ml were 84.1 ± 5.5% (n = 6) and 72.4 ± 5.9% (n = 7), respectively; for OZP at 200 ng the value was 68.2 ± 6.8% (n = 14). The intra-assay relative standard deviations (R.S.D.) at 20, 150 and 270 ng/ml of LZP were 7.8%, 9.8% (n = 7 in all cases) and 6.6% (n = 8), respectively. The inter-assay R.S.D. at the above concentrations were 15.9%, 7.7% and 8.4% (n = 7 in all cases), respectively. Intra- and inter-assay accuracy data were within the acceptance interval of ±20% of the nominal values. There was no interference from other commonly co-administered anticonvulsant, antimicrobial, antipyretic, and antimalarial drugs. The method has been successfully applied to a pharmacokinetic study of LZP in children with severe malaria and convulsions following administration of a single intravenous dose (0.1 mg/kg body weight) of LZP.
Bioassay; Concentration (process); Drug products; High performance liquid chromatography; Pharmacokinetics; Plasmas; Separation; Solvent extraction; Analysis in plasma; Children; Lorazepam; Oxazepam (OZP); Ultraviolet detectors; acetonitrile; analgesic agent; anticonvulsive agent; antiinfective agent; antimalarial agent; antipyretic agent; artesunate; buffer; chloramphenicol; chloramphenicol hemisuccinate; chloroquine; cycloguanil; diazepam; dichloromethane; fosphenytoin sodium; gentamicin; hexane; lorazepam; midazolam; oxazepam; paracetamol; penicillin G; phenobarbital; phenytoin; potassium derivative; proguanil; pyrimethamine; quinine; salicylic acid; sulfadoxine; accuracy; alkalinity; article; calibration; child; controlled study; correlation coefficient; drug blood level; drug determination; drug isolation; flow rate; human; liquid liquid extraction; particle size; priority journal; reproducibility; reversed phase high performance liquid chromatography; ultraviolet radiation; Anticonvulsants; Calibration; Child; Chromatography, High Pressure Liquid; Humans; Injections, Intravenous; Lorazepam; Reproducibility of Results; Spectrophotometry, Ultraviolet; Artesunate; Cinchona pubescens