Christopher M. Benton1,2, Chang Kee Lim2,3, Caje Moniz2, Donald J. L. Jones1
1Cancer Studies and Molecular Medicine, RKCSB University of Leicester Leicester LE2 7LX UK
2Clinical Biochemistry King's College Hospital Denmark Hill London SE5 9RS UK
3MRC Bioanalytical Science Group, Department of Biological Sciences, Birkbeck University of London Malet Street London WC1E 7HX UK
Tóm tắt
RATIONALEHuman porphyrias, diseases caused by enzyme defects in haem biosynthesis, are characterised by the excessive production, accumulation and excretion of porphyrins and/or 5‐aminolaevulinic acid (ALA) and porphobilinogen (PBG). A method for the simultaneous separation, detection and identification of ALA, PBG and porphyrins would greatly facilitate the screening and diagnosis of porphyrias. Such a method would also be invaluable for the biochemical study of the haem, chlorophyll and corrin pathways.METHODSAn aqueous mixture containing ALA, PBG and type I isomer porphyrins was diluted with acetonitrile and infused (10 μL/min) into a Waters Synapt G2 high‐definition mass spectrometer, equipped with a Z‐Spray electrospray ionisation (ESI) source. Mass spectra were acquired in positive ionisation mode and the optimised ion mobility spectrometry (IMS) conditions were as follows: IMS wave height (V), 40; IMS wave velocity (m/s), 648; IMS gas flow (mL/min) 90.40; helium gas flow (mL/min), 182.60.RESULTSThe IMS drift‐time increased with increasing ion mass in the order of ALA, PBG, mesoporphyrin, coproporphyrin I, penta‐, hexa‐ and heptacarboxylic acid porphyrin I and uroporphyrin I. The ESI‐IMS‐MS spectra shows that PBG could form two different positively charged ions by protonation [M+H]+, m/z 227, or deprotonation [M – H]+, m/z 225. The protonated PBG (m/z 227) easily eliminated ammonia in source and the fragment ion (m/z 210) was monitored instead. Doubly charged ions of porphyrins having different drift times from the protonated singly charged molecules were observed in high abundance, providing further structural characterisation.CONCLUSIONSWe have shown, for the first time, an analytical method capable of simultaneously separating haem biosynthetic intermediates and metabolites, for a potential rapid clinical screening method for the porphyrias. IMS‐MS allowed the separation of doubly charged porphyrin ions, which will be advantageous for the analysis of natural and synthetic tetrapyrrole compounds, while reducing the misinterpretation of contaminants. Copyright © 2012 John Wiley & Sons, Ltd.
