Upreti Vijay V, Mamidi Rao N V S, Katneni Kasiram, Srinivas Nuggehally R
Research Bioanalysis, Drug Metabolism and Pharmacokinetics, Discovery Research, Dr Reddy's Laboratories Ltd., Miyapur, Hyderabad 500 050, India.
Biomed Chromatogr. 2003 Sep;17(6):385-90. doi: 10.1002/bmc.253.
A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DRF-1042, a novel orally active camptothecin (CPT) analog, in human plasma. The sample preparation was a simple deproteinization with acidified methanol yielding almost 100% recovery of DRF-1042. An isocratic reverse-phase HPLC separation was developed on a Supelcosil-LC318 column (250 x 4.6 mm, 5 microm) with mobile phase consisting of 1% v/v triethylamine acetate, pH 5.5 and acetonitrile (80:20, v/v) at a fl ow rate of 1.0 mL/min. The eluate was monitored with a fluorescence detector set at excitation and emission wavelengths of 370 and 430 nm, respectively. The standard curves were linear (r(2) > 0.999) in the concentration ranges 5.0-2004 ng/mL. The lower limit of quantification (LLQ) of the assay was 5 ng/mL. The mean measured quality control (QC) concentrations (range 5 ng/mL to 40 microg/mL) deviated from the nominal concentrations in the range of -10.5-0.08 and -14.5-7.97%, inter- and intra-day, respectively. The inter- and intra-day precisions in the measurement of QC samples at four tested concentrations, were in the range 0.64-5.89% relative standard deviation (RSD) and 0.33-14.7% RSD, respectively. The method was found to be suitable for measurement of plasma concentrations above the calibration curve after serial dilutions. Stability of DRF-1042 was confirmed in a battery of studies, viz., on bench-top, in the auto-sampler, in the stock solutions, after four quick freeze-thaw cycles, up to one month at -20 degree C in human plasma and up to 2 months in the ex vivo samples. The method is simple, sensitive and reliable and has been successfully implemented to investigate the clinical pharmacokinetics of DRF-1042 in cancer patients in a phase I clinical trial.
已开发并验证了一种简单且灵敏的高效液相色谱（HPLC）方法，用于测定人血浆中新型口服活性喜树碱（CPT）类似物DRF-1042。样品制备采用酸化甲醇进行简单的脱蛋白处理，DRF-1042的回收率几乎达到100%。在Supelcosil-LC318柱（250×4.6 mm，5μm）上进行等度反相HPLC分离，流动相由1% v/v三乙胺乙酸盐（pH 5.5）和乙腈（80:20，v/v）组成，流速为1.0 mL/min。用荧光检测器监测洗脱液，激发波长和发射波长分别设置为370和430 nm。标准曲线在5.0 - 2004 ng/mL浓度范围内呈线性（r(2) > 0.999）。该测定方法的定量下限（LLQ）为5 ng/mL。平均实测质量控制（QC）浓度（范围为5 ng/mL至40μg/mL）与标称浓度的偏差，日间为 -10.5 - 0.08%，日内为 -14.5 - 7.97%。在四个测试浓度下测量QC样品的日间和日内精密度，相对标准偏差（RSD）分别在0.64 - 5.89%和0.33 - 14.7%范围内。发现该方法适用于系列稀释后高于校准曲线的血浆浓度测量。通过一系列研究证实了DRF-1042的稳定性，即台式稳定性、自动进样器稳定性、储备溶液稳定性、四次快速冻融循环后的稳定性、在人血浆中 -20℃下长达一个月的稳定性以及离体样品中长达2个月的稳定性。该方法简单、灵敏且可靠，已成功应用于一项I期临床试验中癌症患者DRF-1042临床药代动力学的研究。
