Analysis of porphyrins from serum and the benefit of inert biphenyl columns for liquid chromatography separation

Reversed-phase liquid chromatography (LC) is a major separation technique for porphyrins. We coupled C18-based LC with mass spectrometry (MS) for the detection of protoporphyrin IX (PPIX) in the context of neurosurgery, where it is used as a fluorescence marker for tumor visualisation. Here, we demonstrate advantages of the replacement of the C18 with an inert biphenyl (iBP) column, which improved separation resolution and increased detection sensitivity in LC-MS of six porphyrins (uroporphyrin I, hepta-, hexa-, and pentacarboxyporphyrin I, coproporphyrins I and III) associated with the heme biosynthesis pathway. They are of potential interest as intermediates and side products in our research on 5-aminolevulinic acid-induced fluorescence-guided neurosurgery. Porphyrins consist of conjugated π-systems which interact with aromatic stationary phases such as BP by taking advantage of π-π stacking interactions. Carryover observed with PPIX on endcapped C18 stationary phase was reduced. This improvement was due to the reduction of non-specific adsorption on metallic surfaces from the LC instrument by using inert column hardware combined with the BP stationary phase. Passivation by chemical vapor deposition was shown to reduce non-specific adsorption in the LC system better than methods using mobile phase additives. PEEK tubing in conjunction with the iBP-column provided the best results and shortened run time. Furthermore, we present a Python-based software tool for automated data processing and analyte quantification. This work contributes to our ongoing developments of porphyrin LC-MS in blood and tissue samples from glioma patients.