重组羧肽酶G2对甲氨蝶呤在Wistar大鼠体内代谢特征的影响  

Effect of CPG2 on the pharmacokinetic characters of MTX in Wistar rats

作　　者：朱鲲鹏[1] 张岭[1] 张曼[1] 孟志云[1] 甘慧[1] 朱晓霞[1] 顾若兰[1] 吴卓娜[1] 李俭[1] 孙文种[1] 窦桂芳[1] 

机构地区：[1]军事医学科学院野战输血研究所药物代谢重点实验室,北京100850

出　　处：《药物分析杂志》2013年第5期763-769,共7页Chinese Journal of Pharmaceutical Analysis

基　　金：临床前药代动力学技术平台;课题编号:2012ZX09301003-001-007

摘　　要：目的:建立灵敏稳定的高效液相色谱-二级质谱联用法(HPLC-MS/MS),用于同时测定Wistar大鼠血清中甲氨蝶呤(MTX)及其代谢产物2,4-二氨基-N10-甲基蝶酸(DAMPA)含量,并研究重组羧肽酶G2(CPG2)在健康大鼠体内对MTX的代谢特征的影响。方法:采用乙腈沉淀蛋白方法进行样品处理。采用HPLC-MS/MS进行分析。使用Thermo Hypersil GOLDC18(100 mm×2.1 mm,5#m)色谱柱,流动相A为水(含5 mmol.L-1乙酸铵,pH 3.5),流动相B为甲醇(含0.1%的甲酸),梯度洗脱(0～1.5 min,10%B;1.5～2 min,10%B→90%B;2～5 min,90%B;5～7 min,10%B),流速0.2 mL.min-1,柱温20℃;采用ESI+源,MRM离子检测模式,毛细管温度350℃,喷雾电压4.5 kV,鞘气压力172.4 kPa,辅气压力68.95 kPa,CollisionGAS(CAD)68.95 kPa,Curtain GAS(CUR)68.95 kPa,检测离子反应为MTX(m/z 455.2→308.1),DAMPA(m/z 326.1→175.1)。实验动物分为2组,尾静脉给药,单独给药组(MTX 350 mg.kg-1)和联合给药组(MTX 350 mg.kg-1给药5 min后给予CPG2 0.858 mg.kg-1),测定大鼠血清中MTX和DAMPA的浓度,并计算药代动力学参数。结果:血清中MTX和DAM-PA的线性范围均为5～1000 ng.mL-1(r>0.99),定量下限均为5 ng.mL-1。MTX的批内与批间精密度(RSD)为2.23%～14.29%,准确度(RE)为-2.83%～6.61%;DAMPA的批内与批间精密度(RSD)为1.78%～13.88%,准确度(RE)为-1.15%～7.00%。MTX和DAMPA的提取回收率范围分别为87.82%～95.09%和89.70%～96.21%,生物基质不干扰样品的测定,样品稀释后检测无稀释效应。本实验涉及条件下样品稳定。结论:建立的HPLC-MS/MS方法可用于测定大鼠血清样品中MTX及其代谢产物DAMPA的浓度,证实了CPG2在大鼠体内对MTX的酶解作用。Objective： To develop an LC - MS/MS method for the quantitive determination of methotrexate （MTX） and its metabolite 2,4 - diamino - N10 - methylpteroic acid （DAMPA） in serum, and evaluate the effect of car-boxypeptidase G2 （ CPG2 ） on MTX pharmacokinetics in healthy Wistar rats. Methods： After protein was precipitated with acetonitrile, the samples were analyzed by HPLC - MS/MS with a triple quadrupole tandem mass spectrometerin multiple reactions monitoring（MRM） mode using positive electrospray ionization（ESI）. To employ a Thermo Hypersil GOLD Cls column（100 mm×2.1 mm,5μm）and a mobile phase where the mobile phase solvent B was meth-anol（ containing 0.1% formic acid）and the mobile phase solvent A was water（ containing 5 mmol · L^-1 ammonium acetate, pH 3.5 ）. The initial mobile phase composition of 90% solvent A and 10% solvent B was maintained for 1.5 min;between 1.5 and 2 min,the percentage of solvent B was changed to 90% and maintained for 3 min;between 5 and 7 min,the percentage of solvent B was changed to 10%. The flow rate was 0.2 mL · min^-1. The settings ofthe mass spectrometer were as follows ： spray voltage 4.5 kV and capillary temperature 350 ℃. The sheath gas and auxiliary gas pressures were 172.4 kPa and 68.95 kPa, respectively. The collision gas pressure was 68.95 kPa andcurtain gas pressure was 68.95 kPa. The ion combination of m/z 455.2→308.1 ,m/z 326.1→175.1 was used to qualify MTX and DAMPA respectively. The experimental animals were divided into 2 groups, after a single dose of350 mg · kg^-1 MTX or combination doses of 350 mg · kg^-1 MTX and 0.858 mg · kg^-1 CPG2 through caudal veins to rats, the concentration of MTX and DAMPA in rat serum was detected and the pharmacokinetic parameters werecalculated. Results：The calibration curves for both MTX and DAMPA were linear in the range of 5 - 1000 ng · mL^- 1 （ r 〉 0.99 ） with the lower limit of quantitation of 5 ng · mL^- 1. The intra - and inter - batch precisions （ RS-Ds） for MTX an

关 键 词：甲氨蝶呤 2 4-二氨基-N10-甲基蝶酸 重组羧肽酶G2 高效液相色谱-二级质谱联用法 药代动力学参数 体内代谢 酶解 

分 类 号：R917[医药卫生—药物分析学]