Mass spectrometric characterisation of acondensation product betweenporphobilinogen and indolyl-3-acryloylglycinein urine of patients with acuteintermittent porphyria

Abstract

We document the presence of a previously unknown species in the urine of patients with acute intermittent porphyria (AIP). Thecompound was fully characterised by liquid chromatography tandem mass spectrometry. Interpretation of both full spectrum ac-quisition and product ion spectra acquired in positive and negative ionisation modes by quadrupole time of flight MS allowed forthe identification of a condensation product arising from porphobilinogen (PBG, increased in the urine of AIP patients) andindolyl-3-acryloylglycine (IAG, derived from indolylacrylic acid and present in human urine). The structure was unequivocally con-firmed through comparison between the selected reaction monitoring chromatograms obtained from the urinary species and thecondensation product qualitatively synthesised in the laboratory. Owing to the large amounts of both PBG and IAG in urine of AIPpatients, the possible ex vivo formation of PBG-IAG in urine samples was evaluated. The product was spontaneously formed atroom temperature, at 4 °C and even during storage at 20 °C when spiking a control sample with PBG. A positive correlationwas found between PBG and PBG-IAG in samples collected from AIP patients. However, no correlation was found between PBG-IAG and IAG. Purified PBG-IAG did not form the characteristic chromogen after application of p-dimethylaminobenzaldehyde inHCl, thus suggesting that the current techniques used to measure PBG in urine of AIP patients based on Ehlrich’sreactiondonot detect this newly characterised PBG-IAG fraction.