UPLC-FLD法测定全血中原卟啉和锌原卟啉

Determination of protoporphyrin and Zinc protoporphyrin in whole blood by UPLC-FLD

目的建立全血中原卟啉和锌原卟啉的超高效液相色谱-荧光测定方法。方法全血加入间卟啉Ⅸ-二盐酸盐内标,经纯水稀释后,采用二甲基亚砜-丙酮液液萃取进行净化,经C_(18)色谱柱分离,双通道荧光检测器检测,内标法定量。结果原卟啉和锌原卟啉的线性范围均为0.1~5.0μmol/L,相关系数均为0.9995;方法的检出限分别为0.02μmol/L和0.03μmol/L,定量限分别为0.06μmol/L和0.09μmol/L;原卟啉和锌原卟啉在低、中、高三个浓度加标时,方法的加标回收率分别为85.2%~94.7%和84.8%~92.8%;批内精密度(RSD)分别为2.8%~5.3%和2.7%~6.1%,批间精密度(RSD)分别为3.4%~6.3%和3.2%~7.1%;对30份血样中原卟啉与锌原卟啉检测结果分别为:0.1~0.5μmol/L与0.4~1.2μmol/L。结论本方法适用于全血中的原卟啉和锌原卟啉的同时测定。

Objective To establish an ultra-performance liquid chromatography-fluorescence detection(UPLC-FLD)method for the determination of protoporphyrin and zinc protoporphyrin in whole blood.Methods Mesoporphyrin IX dihydrochloride was added as an internal standard to the whole blood samples,which were then diluted with pure water and purified using dimethyl sulfoxide-acetone liquid-liquid extraction.Separation was achieved on a C_(18) column with dual-channel fluorescence detection.Results The linear ranges for both protoporphyrin and zinc protoporphyrin were 0.1~5.0μmol/L,and the correlation coefficients were both 0.9995.Detection limits were 0.02μmol/L and 0.03μmol/L,with quantification limits at 0.06μmol/L and 0.09μmol/L,respectively.Recoveries ranged from 85.2%to 94.7%and 84.8%to 92.8%at low,medium and high spiked concentrations,respectively.The intra-batch precision(RSD)was 2.8%to 5.3%and 2.7%~6.1%,respectively.The inter-batch precision(RSD)was 3.4%to 6.3%and 3.2%~7.1%,respectively.In an analysis of 30 blood samples,the concentrations of porphyrins and zinc porphyrins were found to be in the ranges of 0.1 to 0.5μmol/L and 0.4 to 1.2μmol/L,respectively.Conclusion This method is effectively applied for the simultaneous determination of protoporphyrin and zinc protoporphyrin in whole blood.