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

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 C₁₈ column with dual-channel fluorescence detection.

Results The linear ranges for both protoporphyrin and zinc protoporphyrin were 0.1~5.0 μmol/L, and the correlation coefficients were both 0.999 5. Detection limits were 0.02 μmol/L and 0.03 μmol/L, with quantification limits at 0.06 μmol/L and 0.09 μmol/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 0.5 μmol/L and 0.4 to 1.2 μmol/L, respectively.

Conclusion This method is effectively applied for the simultaneous determination of protoporphyrin and zinc protoporphyrin in whole blood.