Rapid and Simultaneous Determination of Coproporphyrin and Protoporphyrin in Feces by Derivative Matrix Isopotential Synchronous Fluorescence Spectrometry

Abstract

Abstract Background: Measurement of fecal porphyrins is important in the diagnosis of porphyria, but conventional methods to measure them have drawbacks. We explored the use of derivative matrix isopotential synchronous fluorescence (MISF) spectrometry for the measurement of coproporphyrin and protoporphyrin. Methods: The MISF scanning route was selected based on information from the three-dimensional fluorescence spectrum, which was a combination of the contour line of protoporphyrin via a detection point of coproporphyrin and that of coproporphyrin via a detection point of protoporphyrin. Derivative technique eliminated the constant interfering signals. MISF was used to measure porphyrins in stools from 2 pregnant women and 20 healthy volunteers. Results: The coproporphyrin and protoporphyrin spectra were resolved with almost no mutual interference. The amplitudes of the derivative peaks were linearly related to the concentrations of coproporphyrin up to 310 nmol/L and protoporphyrin up to 590 nmol/L. The detection limits for coproporphyrin and protoporphyrin were 1.2 and 1.7 nmol/L, respectively. The within-run imprecision (CV; n = 6) was 2.2% at 175 nmol/L for coproporphyrin and 2.3% at 500 nmol/L for protoporphyrin. Bland–Altman analysis indicated no significant differences between the proposed MISF method and conventional spectrophotometry or fluorimetry. Mean (SD) recoveries of porphyrins added to fecal samples were of 98 (7)% for coproporphyrin and 102 (4)% for protoporphyrin. Conclusions: This technique provides spectral resolution of coproporphyrin and protoporphyrin, obviating the need for chromatographic separation, and measurements can be made in a single scanning. The method also appears suitable for routine testing of large numbers of samples.