HPLC-MS/MS测定大鼠血浆中的叶酸浓度及其药动学研究

Determination of Folic Acid in Rat Plasma by HPLC-MS/MS and Its Pharmacokinetics

目的大鼠灌胃给予叶酸溶液后,建立其在血浆中浓度的定量方法,并用于药动学研究。方法大鼠血浆采用蛋白沉淀法进行预处理,以格列本脲为内标,采用高效液相色谱-质谱联用(HPLC-MS/MS)检测大鼠血浆中叶酸的浓度。色谱分离采用Thermo Accucore C18色谱柱,流动相为乙腈和0.1%甲酸-水,梯度洗脱。MS采集选用ESI正离子及多反应监测模式。结果叶酸在10~1000 ng/ml范围内线性关系良好。方法的回收率在80.0%~95.4%范围内;批内和批间的准确度为92.7%~111.4%,批内和批间精密度为2.6%~8.3%。药动学结果显示大鼠灌胃给药后叶酸在1 h左右达到最大浓度,Cmax和暴露量随剂量的增加而增大,但和剂量并不成线性相关。结论该方法重现性好,分析时间短,能满足大鼠血浆中叶酸浓度的检测要求,并符合2015年版中国药典对生物样品分析的要求。

Objective To develop a sensitive high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)method for determination of folic acid in the plasma of rats and evaluate its pharmacokinetic characteristics.Methods Glibenclamide was selected as the internal standard(IS).The plasma samples were pretreated by protein precipitation,and the separation was achieved on a reverse phase Thermo Accucore C18 column using a gradient mobile phase system of acetonitrile-water containing 0.1%formic acid.The triple quadruple mass spectrometer was set to positive electrospray ionization mode and multiple reaction monitoring(MRM)was used for quantification.Results The results showed that folic acid at the concentration range 10-1000 ng/ml exhibited good linearity,with the correlation coefficient over 0.99.The average extraction recoveries ranged from 80.0%to 95.4%.Within-run and between-run accuracy and precisions were ranged from 92.7%-111.4%and 2.6%-8.3%,respectively The pharmacokinetic results showed that folic acid reached the maximum concentration in rats about 1 h after oral administration.Cmax and exposure increased with dose,but it was not linearly related to the dose.Conclusion The established method showed good reproducibility and short analysis time,it can be used for quantitative determination of folic acid in the plasma of rats and meet the requirements of the guidelines listed in Chinese Pharmacopoeia 2015..