Studenberg S D, Long J D, Woolf J H, Bruner C J, Wilson D, Woolley J L
Division of Pharmacokinetics and Drug Metabolism, Wellcome Research Laboratories, Research Triangle Park, NC 27709, USA.
J Pharm Biomed Anal. 1995 Oct;13(11):1383-93. doi: 10.1016/0731-7085(95)01563-z.
A precise and specific robotics-based liquid chromatographic (LC) method for measuring atovaquone concentrations in plasma was developed and validated, and the method was compared with an existing manual LC method. The compound was isolated from plasma by liquid-liquid extraction, separated by reversed-phase LC, and quantitated against an internal standard with UV detection. Least-squares linear regression with 1/concentration2 weighting was used as the calibration model. The range of the calibration curve for the assay under routine conditions was 0.25-50 micrograms ml-1. No endogenous interferences with the compound or the internal standard were noted in either untreated human plasma or in plasma from patients enrolled in Phase III clinical trials of atovaquone. The accuracy of the assay (determined as the percent bias) ranged from -4.8% to -9.4% in the validation runs. The intra- and interassay precisions (determined as the relative standard deviation) were less than 6.8% and 6.4%, respectively. The contribution of an internal standard on assay accuracy and precision also was examined. Interassay variability was marginally improved by the incorporation of an internal standard to the assay; accuracy and intra-assay precision were essentially unchanged. A paired t-test between estimates of atovaquone concentrations in healthy volunteer and HIV + patient human plasma samples assayed by the automated and manual methods demonstrated no significant difference (p = 0.31) between the values determined by each method.
开发并验证了一种基于机器人的精确且特异的液相色谱（LC）方法，用于测定血浆中阿托伐醌的浓度，并将该方法与现有的手动LC方法进行了比较。该化合物通过液-液萃取从血浆中分离出来，通过反相LC进行分离，并用紫外检测法以内标物进行定量。校准模型采用1/浓度²加权的最小二乘线性回归。常规条件下该测定法校准曲线的范围为0.25 - 50微克/毫升。在未经处理的人血浆或参与阿托伐醌III期临床试验患者的血浆中，均未发现该化合物或内标物存在内源性干扰。验证运行中该测定法的准确度（以偏差百分比确定）范围为-4.8%至-9.4%。批内和批间精密度（以相对标准偏差确定）分别小于6.8%和6.4%。还研究了内标物对测定准确度和精密度的影响。在测定中加入内标物使批间变异性略有改善；准确度和批内精密度基本未变。对健康志愿者和HIV +患者人血浆样本中阿托伐醌浓度的自动和手动测定估计值进行配对t检验，结果表明两种方法测定的值之间无显著差异（p = 0.31）。
