The rapid determination of erythrocyte porphyrins using reversed-phase high performance liquid chromatography

The extraction, separation and quantification of free acid erythrocyte porphyrins are described. The porphyrins are extracted from whole blood using 3:1 ethyl acetate/acetic acid (v/v) and separated using the reversed-phase mode of high performance liquid chromatography (RPLC). The compounds are detected on-line spectrofluorometrically using an excitation wavelength of 404 nm and an emission cut-off filter of 550 nm. Excellent resolution of the porphyrin free acids is achieved in less than 6 min. The analytical recoveries for protoporphyrin IX and zinc protoporphyrin IX were greater than 90% with relative standard deviations for day-to-day analysis under 6%.