HPLC-UV色谱法检测人血浆中左亚叶酸钙浓度

Determination of L-Leucovorin in plasma by high-performance liquid chromatography using UV detection

目的建立在人血浆中测定左亚叶酸钙可靠的检测方法。方法应用高效液相紫外检测色谱法,内标为对氨基苯甲酸,流动相为乙腈:甲醇:磷酸盐缓冲液(50mM,pH4.0)=8:6:86,流速为1mL·min-1,柱温为30℃,进样量为20μL,检测波长为290nm,血浆样品采用液液萃取的方法提取。结果目标色谱峰分离效果好,无内源性杂峰干扰。提取回收率均大于90%,测定血药浓度线性范围为50.0～10000ng·mL-1(r2=0.9998),最小检出限25.0ng·mL-1。各项稳定性考察均合格。结论本法准确,灵敏度较高,可用于注射用左亚叶酸钙在人血浆中的检测和注射用左亚叶酸钙的人体内研究。

Objective To develope a method for determining L-Leucovorin in human plasma by high performance liquid chromatography.Method HPLC with UV detection excitation at 290 nm was described.Paraamino benzoic acid （PABA） was used as the internal standard.Mobile phase consisted of acetonitrile,methanol and 50 mmol·L^-1 phosphate buffer saline （adjusted to pH 4.00） with a volume ratio of 8：6：86.The flow rate was of 1.0 mL ·min^-1.The injection volume was 20μL.Plasma sample was extracted by liquid-liquid extraction.Results The target compound was eluted as separate symmetrical peak and no interfering peaks appeared.No interference from endogenous blood constituents was observed.The recovery of the method was more than 90%.The linear range was 50.0～10 000 ng·mL^-1 （r2= 0.9998） with aminimum detectable limit of 25 ng·mL^-1.The stability analysis showed that plasma sample was stable in different conditions.Conclusion The method had a good selectivity and reproducibility and can be used for the investigation of L-Leucovorin in human plasma.