HPLC检测新生儿血清游离未结合胆红素的方法学的建立

Establishment of a method for detecting neonatal serum free bilirubin by high performance liquid chromatography

目的探索HPLC法检测新生儿血清游离未结合胆红素(Free Bilirubin,FB)。方法色谱柱ODS-C18柱(150.0mm×4.6mm×5.0μm),流动相:甲醇-醋酸铵冰乙酸溶液(96∶4),流速0.9ml/min,柱温30℃,检测波长458nm,进样量20μl。检测超滤离心血清中的FB,并考察该法的精密度、准确度、回收率、稳定性。用SPSS软件包进行统计。结果胆红素的保留时间为9.583min;定量下限为0.625μmol/L;HPLC检测血清FB的线性范围为0.625~160.000μmol/L,标准曲线:y=73403x-22975,r^2=0.9995,斜率偏差为726.6496864,截距偏差为30119.92455;不同FB浓度血清的平均日内、日间精密度RSD均<5%;准确度为95%~105%;提取回收率为68%~78%;不同时长、温度的储存稳定性平均RSD值为0.565%,反复冻融稳定性平均RSD为3.020%。用HPLC法测定13例黄疸新生儿血清FB的范围为(548.20±107.19)nmol/L。结论HPLC测定新生儿血清FB的方法操作简便,专属性强,准确度、精密度和稳定性均较高,符合生物样本分析要求。

Objective To establish the HPLC method for detecting neonatal serum FB.Methods The chromatographic column was ODS-C18 column(150.0 mm×4.6 mm×5.0μm).The mobile phase of methanolammonium acetate ice acetic acid solution was 96:4,flow rate 0.9 ml/min,column temperature 30℃,detection wavelength 458 nm,and injection volume 20 μl.The precision,accuracy,recovery rate,and stability of the method were examined.The differences between different groups were calculated by SPSS software package.Results The retention time of FB was 9.583 min.The lower limit of quantification was 0.625 μmol/L.The linear range of serum FB was 0.625-160.000 μmol/L.The standard curve:y=73403x–22975.The r^2 is 0.9995.The slope deviation was 726.6496864.The intercept deviation was 30119.92455.The average intra-day and interday precision of each gradient concentration were less than 5%.The relative recovery was 95%-105%.Different gradient concentration extraction recovery range was 68%-78%.The repeated freeze-thaw stability average RSD was 3.020%.The average RSD value of stability under different storage conditions was 0.565%.The concentration of serum FB in 13 jaundiced newborns we measured were(548.20±107.19)nmol/L.Conclusion HPLC detecting serum FB is simple in operation,and has high specificity,accuracy,precision and stability.