高效液相色谱法测定癫痫患儿血清中左乙拉西坦的浓度

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

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。

AIM To establish （LEV） in children with epilepsy by a method for the determination of serum concentration of levetiracetam performance liquid chromatography （HPLC）. METHODS The a- phenacyl bromide （a-BT） was used as internal standard （IS）. LEV and IS were extracted with ethyl acetate from serum, and dried with N2 gas, then dissolved with mobile phase and 20 μL 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-C18 column （150 mm ×3.9 mm, 5 μm） 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 0.234 μg.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.