Characterization of pyrroloquinoline quinone amino acid derivatives by electrospray ionization mass spectrometry and detection in human milk

We describe a HPLC method coupled to electrospray ionization mass spectrome try (ESI/MS) for quantification and identification of pyrroloquinoline quin one (PQQ) and condensation products formed upon incubation of PQQ with amin o acids (IPQ; imidazolopyrroloquinoline and I/OPQ/R; imidazolopyrroloquinol ine with attached R-group). More importantly, using these methods we demons trate the presence of both PQQ and IPQ in human milk in nanomolar to microm olar concentrations. PQQ was incubated with amino acids and condensation pr oducts were separated by HPLC. Fractions corresponding to each product were collected and molecular masses were determined using ESI/MS. Ala, Asp, Arg , Cys, Gly, Glu, Ser, Thr, Trp, and Tyr form IPQ upon incubation with PQQ Y ields of IPQ were low (<5%) for Asp and Glu, yet high (>60%) for Thr. In ad dition to IPQ, Ala, Arg, Cys, Ser, Trp, and Tyr formed IPQ/R derivatives. H is, Ile, Leu, Glu, Leu, Lys, Met, and Phe form only IPQ/R derivatives. Prol ine did not react with PQQ Mass spectra indicate that PQQ forms stable hydr ated carbonyls and decarboxylates easily. Although mass spectra were compli cated by the oxidation state of the quinone and decarboxylation of PQQ, the se methods are invaluable for the rapid detection of the full range of PQQ adducts in biological matrices. (C) 1999 Academic Press.