High-performance liquid chromatography of uroporphyrinogen and coproporphyrinogen isomers with amperometric detection

Abstract

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.

Separation and characterization of pentacarboxylic porphyrinogen isomers by high-performance liquid chromatography with electrochemical detection

“…The resolution of pentacarboxylic porphyrinogen isomers was superior to that of the porphyrins by reversed-phase chromatography. This is similar to previously obtained results on the separation of coproporphyrinogen isomers (Lim et al, 1986). The separation of porphyrinogens is therefore preferred for the analysis of isomers important in biochemical studies.…”

Porphyrinogen fragmentation profiles by ultra‐high‐performance liquid chromatography/electrospray ionisation tandem mass spectrometry

“…HPLC is very well established for the separation of porphyrin and porphyrinogen isomers . Uroporphyrinogen I and III were resolved when run isocratically using 2% (v/v) acetonitrile in 1 mol/L aqueous ammonium acetate (pH 5.16) (Fig.…”

Liquid chromatography and mass spectrometry of haem biosynthetic intermediates: a review

“…Porphyrinogens do not fluoresce and show a weak UV absorption at around 220-240 nm (Lim et al, 1986a). They have therefore been most commonly analysed by electrochemical detection owing to the ease of oxidation (Li et al, 1987a).…”

“…They have therefore been most commonly analysed by electrochemical detection owing to the ease of oxidation (Li et al ., ). HPLC methods have been described for the isomer separation of porphyrinogens and all systems used reversed‐phase chromatography, which required a mobile phase system of 1 mol/L ammonium acetate (pH 5.16), 0.27 mmol/L ethylenediaminetetraacetic acid (EDTA) and the following portions of acetonitrile and/or methanol: 4% acetonitrile for uroporphyrinogens (Lim et al ., ), 7% acetonitrile and 3% methanol for heptacarboxylic acid‐ (Lim et al ., ), 8% acetonitrile and 12% methanol for hexacarboxylic acid‐ (Li et al ., ), 40% methanol for pentacarboxylic acid‐ porphyrinogens (Li et al ., ), and 25% acetonitrile for coproporphyrinogens (Lim et al ., ). Isomer resolution was superior to that of porphyrins, particularly the type I and III isomers which are of interest clinically.…”