毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究

Determination of Methotrexate Enantiomers in Human Blood Samples with Capillary Electrophoresis

建立拆分氨甲喋呤(MTX)对映体的毛细管电泳方法,讨论消旋药物MTX对映体立体选择的手性识别机理.运用聚丙酰胺涂渍毛细管的毛细管电泳方法,考察了缓冲液中添加α-CD、γ-CD、HP-β-CD和Me-β-CD对MTX的拆分.最佳拆分条件为pH7.4磷酸盐缓冲液添加0.04 mmol/LHP-β-CD,20kV分离电压,柱温25℃,280nm紫外波长检测,氨甲喋呤对映体基本上得到了基线分离.(+)MTX在18.18～636.16mmol/L浓度范围,(-)MTX在45.44～636.16mmol/L浓度范围内有良好的线性关系,两对映体的定量下限(+)MTX为18.18 mmol/L,(-)MTX为45.44mmol/L,并用该方法测定了血清中MTX对映体含量,计算了在不同CDs下两对映体的稳定常数,结果表明MTX对映体和环糊精手性匹配与配合物的键合常数和选择因子(α)有关.

A capillary electrophoresis for the chiral separation of racemic methotrexate(rac-MTX)was estabˉlished and validated.The method for recognizing the enantioselectivity of racemic drug MTX was discussed.The two enantiomers were separated with coated-silica capillary and a running buffer containing phosphate and different cyclodextrins(α-cyclodextrins(α-CD)、γ-cyclodextrins(γ-CD)、2-hydroxypropyl-β-cyclodexˉtrins(HP-β-CD)and methyl-β-cyclodextrins(Me-β-CD)).The MTX enantiomers were found to be best sepˉarated under the following conditions:pH=7.4for the phosphate solution,0.04mmol/L HP-β-CD,a20kVvoltage and an oven temperature of25℃.Under these conditions,linear calibration curves were obtained in the ranges of18.18～636.16mmol/Lfor(+)MTX,45.44～636.16mmol/Lfor(-)MTX,respectiveˉly,using UV detection.The limits of quantition were18.18mmol/L for(+)MTX and45.44mmol/L for(-)MTX(S/N=3,injection6s),respectively,at280nm.This method applied to the assay of MTX enanˉtiomers in human serum,based on which the binding constants for MTX enantiomers were calculated with difˉferent CDs.The results indicated that the coupling between MTX enantiomers and the chirality of cyclodexˉtrins were related to the equilibrium constants of binding complexes and selectivity factor(α).