Loss of porphyrins from solution during analysis: Effect of sample pH and matrix on porphyrin quantification in urine by 'high-performance' liquid chromatography

Abstract

We report the effect of sample matrix and pH on quantification of porphyrins by HPLC with fluorimetric detection. For aqueous solutions of pH < 2.5, HPLC peak heights of the porphyrins increased with decreasing pH, reaching a plateau at pH < 1.0. This loss of porphyrins from solutions with pH > 1.0 appeared to be due to a combination of microprecipitation and aggregation effects. No such 'pH effect' was observed for urine samples supplemented with mixed-porphyrin standards. Addition of trace amounts of albumin to aqueous solutions also decreased these pH-related losses. These findings suggest a porphyrin-protein interaction that prevents microprecipitation and aggregation processes. We conclude that standard solutions of porphyrins for HPLC analysis should be prepared in a urine matrix. If aqueous solutions are used, then the pH must be adjusted to < 1.0. Urine samples from normal individuals require only adjustment of pH to < 2 before analysis; however, porphyric urines requiring dilution should be prepared with porphyrin-free urine diluent.