Wang Xiaojing, Chen Zhihang, Che Jinjing, Meng Qingfang, Shan Chengqi, Hou Yunan, Liu Xiaolei, Chai Yifeng, Cheng Yuanguo
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China; Department of Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Nov 15;877(30):3813-21. doi: 10.1016/j.jchromb.2009.09.027. Epub 2009 Sep 25.
This study details the development and validation of a simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the quantification of combretastatin A-4 3-O-phosphate (CA4P), combretastatin A4 (CA4) and its main metabolite, combretastatin A4 glucuronide (CA4G), in beagle dog plasma. Sample pretreatment includes simple protein precipitation by adding methanol to the plasma sample containing an internal standard (colchicine). LC separation was successfully accomplished on a Waters RP8 Symmetryshield column (3.0mmx150mm, i.d., 5microm) with a gradient mobile phase of methanol (0.1% formic acid, v/v) and water (20mM ammonium acetate) at a flow rate 0.8mLmin(-1). The three analytes were detected in the positive/negative ion alternate mode, negative ion mode for CA4G and positive ion mode for CA4P and CA4. Multiple reaction monitoring (MRM) was used for determination of three analytes. Calibration curves were linear in the concentration range of 5-5000ngmL(-1) for CA4P (r>or=0.999), 5-3000ngmL(-1) for CA4 (r>or=0.999) and 5-5000ngmL(-1) for CA4G (r>or=0.999). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was reliable and has been successfully applied to a pharmacokinetic study of CA4P in beagle dogs via intravenous drop infusion at dose rates of 1, 3 and 9mgkg(-1). After daily intravenous drop infusions at 1mgkg(-1) for 7 consecutive days, the accumulation ratio was approximately 1.0, indicating no accumulation from multiple doses in beagles.
本研究详细阐述了一种简单且灵敏的液相色谱/串联质谱（LC-MS/MS）方法的开发与验证，该方法用于定量比格犬血浆中的康普瑞他汀A-4 3-O-磷酸酯（CA4P）、康普瑞他汀A4（CA4）及其主要代谢物康普瑞他汀A4葡萄糖醛酸苷（CA4G）。样品预处理包括向含有内标（秋水仙碱）的血浆样品中加入甲醇进行简单的蛋白沉淀。在Waters RP8 Symmetryshield柱（3.0mm×150mm，内径，5μm）上成功实现了LC分离，流动相为甲醇（0.1%甲酸，v/v）和水（20mM醋酸铵）的梯度洗脱，流速为0.8mL·min⁻¹。三种分析物在正/负离子交替模式下进行检测，CA4G采用负离子模式，CA4P和CA4采用正离子模式。采用多反应监测（MRM）法测定三种分析物。CA4P在5 - 5000ng·mL⁻¹浓度范围内校准曲线呈线性（r≥0.999），CA4在5 - 3000ng·mL⁻¹浓度范围内校准曲线呈线性（r≥0.999），CA4G在5 - 5000ng·mL⁻¹浓度范围内校准曲线呈线性（r≥0.999）。所有验证数据，如准确度、精密度和日间重复性，均在规定限度内。该方法可靠，已成功应用于比格犬静脉滴注1、3和9mg·kg⁻¹剂量率的CA4P的药代动力学研究。连续7天每天以1mg·kg⁻¹的剂量进行静脉滴注后，蓄积比约为1.0，表明比格犬多次给药后无蓄积现象。
