Derivative variable-angle synchronous fluorescence (DVASF) spectrometry improves the spectral resolution and selectivity of the fluorescence method. The feasibility of DVASF spectrometry for the simultaneous determination of protoporphyrin IX (PP) and zinc protoporphyrin IX (ZnPP) was investigated. PP and ZnPP were distinguished from each other simultaneously and rapidly by the DVASF method. The spectra were resolved well, and the two components were determined in a single scan, avoiding the spectral compensation factor for PP and chromatographic separation. The linear range of the calibration curve for PP was from 0.190 to 152 nmol/L and for ZnPP was from 0.383 to 230 nmol/L. The detection limits of PP and ZnPP were 0.098 nmol/L and 0.088 nmol/L, respectively. The within-run imprecision (RSD, <i>n</i> = 5) for PP was 4.1%, and for ZnPP was 4.2%. Mean recoveries (SD) of PP and ZnPP added to a blood sample were 86.4 (7.3)% and 72.9 (6.6)%, respectively. This method should be a potential tool in the rapid routine screening of large quantities of samples.
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