Quantitation of red cell porphyrins by fluorescence scanning after thin-layer chromatography

This paper reports a new method for the quantitative determination of erythrocyte and plasma porphyrins by fluoroscanning of the methyl esters separated by thin-layer chromatography (TLC). After esterification overnight in the dark the polycarboxylated porphyrin methyl esters were extracted into chloroform and aliquots applied to the TLC plates within the range of quantities shown in a preliminary study to be directly proportional to fluorescence intensity. The accuracy and reliability of the technique was tested by comparison of RBC protoporphyrin values obtained by TLC with an established quantitative porphyrin solvent extraction method and with a rapid method presently in common use. Good correlation was demonstrated between the solvent extraction and the TLC techniques. The high sensitivity and adaptability of the TLC technique and its ability to clearly separate all the porphyrins present in the samples are discussed, along with the fluorescence mechanisms involved and the effects of instrumentation.