高效液相色谱法测定癫痫患儿血清中左乙拉西坦的浓度

目的建立测定癫痫患儿血清中左乙拉西坦药物浓度的高效液相色谱(HPLC)法。方法用乙酸乙酯从血清中按液液萃取法提取左乙拉西坦和内标α-溴苯乙酮,经氮气恒流吹干富集浓缩,流动相复溶后20μL进样,0.4%磷酸-乙腈(92:8,V/V)为流动相,流速:1.0 mL·min~(-1),柱温35℃,以RP-C_(18)色谱分析柱(150 mm×3.9 mm,5μm)在210 nm波长处测定,记录左乙拉西坦与内标的峰面积比值并拟合浓度工作曲线。结果左乙拉西坦和内标峰面积比与左乙拉西坦浓度在0.234.～60.00 mg·L~(-1)的范围内线性关系良好,Y=0.065 14 X-0.004 7,r=0.999 5,最低定量限为0.234 mg·L~(-1)(S/N=4.8)。方法平均绝对回收率为(89.18±3.57)%,日间和日内RSD分别为(5.84±2.15)%和(5.48±2.61)%,冻融稳定性RSD<5%。该方法能完全分离临床可能合并所用药物的干扰(分离度R均大于1.5),采用单盲法对方法学质量控制结果RSD<4%。结论该方法经方法学验证符合血清样品的测定要求,可用于临床血药浓度测定和药动学研究。

Determination of serum concentration of levetiracetam in children with epilepsy by HPLC

AIM To establish a method for the determination of serum concentration of levetiracetam (LEV)in children with epilepsy by high performance liquid chromatography(HPLC).METHODS Theα-phenacyl bromide(α-BT)was used as internal standard(IS).LEV and IS were extracted with ethyl acetate from serum,and dried with N_2 gas,then dissolved with mobile phase and 20μL sample was injected into HPLC.The mobile phase was a mixture of 0.4%phosphate buffer and acetonitrile(92:8,V/V)at an isocratic flow rate of 1.0 mL·min~(-1).The HPLC analysis was carried out on a Xterra RP-C_(18)column(150 mm×3.9 mm, 5μm)with UV detection at 210 nm and column temperature at 35℃.LEV and IS peak area ratios were calculated,then standard curves were constructed using simple linear regression.RESULTS Calibration curves showed a linear and reproducible correlation between LEV serum concentrations and matched analyte to IS peak area ratios at the concentration range of 0.234-60.00μg·L~(-1).The equation of the regression line was Y=0.065 14 X-0.004 7 and r=0.999 5.The minimum quantitation limit was 0.234μg·L~(-1)(S/N=4.8)and the absolute recovery was(89.18±3.57)%.The relative standard deviation(RSD)of inter and intra-day precision were (5.84±2.15)%and(5.48±2.61)%respectively,and freeze-thawing stability was<5%.It could separate drugs combination and the interference from those drugs completely(resolving power R>1.5).RSD of quality control(use single blind method)was<4%.CONCLUSION This method meets the requirements for the determination of serum sample,and is suitable for the determination of LEV.