Muchohi Simon N, Obiero Kenneth, Kokwaro Gilbert O, Ogutu Bernhards R, Githiga Isaiah M, Edwards Geoffrey, Newton Charles R J C
Kenya Medical Research Institute (KEMRI)/Wellcome Trust Research Programme, P.O. Box 43640, 00100 GPO, Nairobi, Kenya.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Sep 25;824(1-2):333-40. doi: 10.1016/j.jchromb.2005.07.040.
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 mmx4.6 mm i.d.; 4 microm particle size), using an aqueous mobile phase (10 mM KH2PO4 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.
建立了一种简单、灵敏、选择性好且可重现的反相高效液相色谱（HPLC）法，并采用紫外检测，以奥沙西泮（OZP）为内标物，用于测定人血浆中的劳拉西泮（LZP）。使用正己烷 - 二氯甲烷（70:30%；v/v）混合物进行单步液 - 液萃取，从碱化（pH 9.5）的加标血浆样品和临床血浆样品中提取LZP和OZP。在反相Synergi Max RP分析柱（150 mm×4.6 mm内径；4μm粒径）上进行色谱分离，使用水相流动相（10 mM KH2PO4缓冲液（pH 2.4） - 乙腈；65:35%，v/v），流速为2.5 ml/min。OZP和LZP的保留时间分别为10.2分钟和11.9分钟。校准曲线在10至300 ng范围内呈线性，相关系数（r2）优于0.99。使用0.5 ml样品时，检测限（LOD）和定量限（LOQ）分别为2.5和10 ng/ml。在20和300 ng/ml时，LZP的平均相对回收率分别为84.1±5.5%（n = 6）和72.4±5.9%（n = 7）；在200 ng时，OZP的回收率为68.2±6.8%（n = 14）。在20、150和270 ng/ml的LZP浓度下，批内相对标准偏差（R.S.D.）分别为7.8%、9.8%（所有情况下n = 7）和6.6%（n = 8）。在上述浓度下，批间R.S.D.分别为15.9%、7.7%和8.4%（所有情况下n = 7）。批内和批间准确度数据均在标称值±20%的可接受区间内。未受到其他常用的同时服用的抗惊厥药、抗菌药、退热药和抗疟药的干扰。该方法已成功应用于对患有严重疟疾和惊厥的儿童单次静脉注射剂量（0.1 mg/kg体重）的LZP进行药代动力学研究。
