LC-MS/MS法测定大鼠血浆中低聚仿刺参糖胺聚糖的浓度及初步药物动力学研究  

LC-MS/MS method for the determination of the concentration of depolymerized Apostichopus japonicus holothurian glycosaminoglycans in rat plasma and its preliminary pharmacokinetics

作　　者：贺丹丹 王远红[1] 杨爽[1,2] 吕志华 HE Dan-dan;WANG Yuan-hong;YANG Shuang;LV Zhi-hua(Key Laboratory of Marine Drugs,Ministry of Education,Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China;Laboratory for Marine Drugs and Bioproducts of Qingdao Pilot National Laboratory for Marine Science and Technology,Qingdao 266237,China)

机构地区：[1]中国海洋大学海洋药物教育部重点实验室,山东省糖科学与糖工程重点实验室,医药学院,山东青岛266003 [2]青岛海洋科学与技术试点国家实验室海洋药物与生物制品功能实验室,山东青岛266237

出　　处：《中国海洋药物》2020年第6期52-58,共7页Chinese Journal of Marine Drugs

基　　金：国家自然科学基金青年科学基金项目(31700704);山东省“蓝色药库”技术创新工程专项项目(2018SDKJ0401);中央高校基本科研业务费专项(201964019)资助。

摘　　要：目的建立大鼠血浆中低聚仿刺参糖胺聚糖(DAHG-1W)的液相色谱-质谱联用(LC-MS/MS)测定方法,研究大鼠单次静脉注射DAHG-1W后的血浆药物动力学。方法大鼠血浆样品经灰色链霉菌蛋白酶预处理、温和酸水解脱除岩藻糖支链、硫酸软骨素ABC酶降解成主链二糖并用2-氨基吖啶酮(AWAC)衍生后,进行LCMS/MS分析。色谱分离采用Cortecs UPLC T3色谱柱(2.1 mm×150 mm,1.6μm),流动相为10 mmol·L-1乙酸铵水溶液-甲醇,梯度洗脱。质谱检测采用加热电喷雾离子源(H-ESI),以负离子方式检测,AMAC标记的CS-4S6S和CS-2S4S(内标)的多反应监测(MRM)离子对均为m/z 732.0→652.4,保留时间分别为9.0和7.4 min。应用上述LC-MS/MS方法测定大鼠尾静脉注射DAHG-1W(5 mg·kg-1)后的血浆药物浓度,采用WinNonLin 6.0版软件计算药代动力学参数。结果大鼠血浆中DAHG-1W在3.91~125.00μg·mL-1浓度范围内线性关系良好;日内和日间精密度均小于15%,准确度为102.60%~111.17%;回收率为89.16%~103.35%;无明显基质效应且血浆样品稳定性好。大鼠静脉注射给药后DAHG-1W的血药峰浓度(Cmax)为(36.73±2.10)μg·mL-1,半衰期(t1/2)为(277.31±56.22)min,清除率(Cl)为(0.82±0.09)mL·min-1·kg-1,0~300 min药时曲线下面积(AUC0-t)为(3638.30±45.84)min·μg·mL-1。结论本研究建立了大鼠血浆中DAHG-1W的LC-MS/MS分析方法,可应用于DAHG-1W在大鼠体内的药物动力学研究。Objective To establish a LC-MS/MS method for the determination of depolymerized Apostichopus japonicus holothurian glycosaminoglycans(DAHG-1 W)in rat plasma and to study the plasma pharmacokinetics in rats after a single intravenous injection of DAHG-1 W.Methods Rats plasma was digested with protease by Streptomyces griseus.After fucose branching was removed by mild acid hydrolysis,chondroitin sulfate ABC enzyme was degraded to main chain disaccharides,and 2-aminoacridone derived main chain disaccharides were analyzed by liquid chromatography-mass spectrometry LC-MS/MS.Chromatographic separation was carried out using Cortecs UPLC T3 column(2.1 mm×150 mm,1.6μm).The mobile phase with gradient elution was 10 mmol·L-1 ammonium acetate in water-methanol.Mass spectrometry used a heated electrospray ion source(H-ESI)in negative ion mode.The multiple reaction monitoring(MRM)ion pair of AMAC labeled CS-4 S6 S and CS-2 S4 S(internal standard)was m/z 732.0→652.4.The retention times were 9.0 and 7.4 min,respectively.The plasma concentration of DAHG-1 W after intravenous administration at 5 mg·kg-1 in rats was measured using the above-mentioned LC-MS/MS method.WinNonLin version 6.0 software was used to calculate the pharmacokinetic parameters.Results The calibration curve was linear over the concentration range of 3.91~125.00μg·mL-1 for DAHG-1 W.The intra-day and inter-day precision were less than 15%,and the accuracy was 102.60%~111.17%.The recovery was 89.16%~103.35%.There was no obvious matrix effect and the plasma sample was stable.After intravenous injection,the blood peak concentration(Cmax)of DAHG-1 W was(36.73±2.10)μg·mL-1 in rats.The half-life(t1/2)was(277.31±56.22)min,and the clearance rate(Cl)was(0.82±0.09)mL·min-1·kg-1.The area under the curve between 0 and 300 min(AUC0-t)was(3638.30±45.84)min·μg·mL-1.Conclusion The LC-MS/MS analysis method of DAHG-1 W in rat plasma established in this study could be applied to the pharmacokinetic study of DAHG-1 W in rats.

关 键 词：仿刺参 糖胺聚糖 LC-MS/MS 药物动力学 

分 类 号：R917[医药卫生—药物分析学]