Singh Sonu S, Sharma Kuldeep, Barot Deepak, Mohan P Ram, Lohray Vidya B
Zydus Research Centre, Bioanalytical and DMPK Department, Sarkhej-Bavla N.H. No. 8A, Moraiya, Ahmedabad 382213, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Jul 25;821(2):173-80. doi: 10.1016/j.jchromb.2005.05.013.
A new HPLC method was developed for the estimation of carboxylic acid metabolite of clopidogrel bisulfate in rat plasma using atorvastatin as internal standard. Plasma samples were extracted with a mixture of ethyl acetate and di-chloro methane (80:20, v/v) followed by subsequent reconstitution in a mixture of water:methanol:acetonitrile (40:40:20, v/v). The chromatographic separation was achieved with gradient elution on Kromasil ODS, 250 mm x 4.6 mm i.d., 5 microm analytical column maintained at 30 degrees C. Carboxylic acid metabolite of clopidogrel as well as the internal standard were detected at a wavelength of 220 nm. The method was validated as per USFDA guidelines. Calibration curves were linear in the concentration range of 125.0-32,000 ng/ml and the correlation coefficient was better than 0.999. The extraction efficiency for the carboxylic acid metabolite of clopidogrel was more than 85.76%. The intra-day accuracy ranged from 98.9% to 101.5% with a precision of 1.30% to 6.06%. Similarly, the inter-day accuracy was between 96.2% and 101.1% with a precision of 3.47% to 4.30%. The drug containing plasma samples were stable at -70 degrees C for 48 days and at ambient temperature for 24h. In the auto-sampler maintained at 15 degrees C, the processed and reconstituted samples were stable for 35 h. The drug containing frozen plasma samples were stable enough to with stand three freeze thaw cycles. The method was successfully applied to the pharmacokinetic study of the two different polymorphs of clopidogrel bisulfate in Wistar rat.
建立了一种新的高效液相色谱法，以阿托伐他汀为内标物，用于测定大鼠血浆中硫酸氢氯吡格雷的羧酸代谢物。血浆样品用乙酸乙酯和二氯甲烷的混合物（80:20，v/v）萃取，随后在水：甲醇：乙腈的混合物（40:40:20，v/v）中复溶。在Kromasil ODS 250 mm×4.6 mm内径、5μm分析柱上进行梯度洗脱，实现色谱分离，柱温保持在30℃。在220 nm波长下检测氯吡格雷的羧酸代谢物和内标物。该方法按照美国食品药品监督管理局（USFDA）的指导原则进行了验证。校准曲线在125.0 - 32,000 ng/ml浓度范围内呈线性，相关系数优于0.999。氯吡格雷羧酸代谢物的萃取效率超过85.76%。日内准确度在98.9%至101.5%之间，精密度为1.30%至6.06%。同样，日间准确度在96.2%至101.1%之间，精密度为3.47%至4.30%。含药血浆样品在-70℃下可稳定保存48天，在室温下可稳定保存24小时。在保持在15℃的自动进样器中，处理和复溶后的样品可稳定保存35小时。含药冷冻血浆样品足够稳定，可经受三个冻融循环。该方法成功应用于Wistar大鼠体内硫酸氢氯吡格雷两种不同晶型的药代动力学研究。
