Preparation and separation of hydroxy derivatives of uroporphyrinogen I by high-performance liquid chromatography with electrochemical detection.
The preparation and high-performance liquid chromatography (HPLC) separation of meso-hydroxyuroporphyrinogen I, hydroxyacetic acid uroporphyrinogen I and beta-hydroxypropionic acid uroporphyrinogen I is described. meso-Hydroxyuroporphyrin I, hydroxyacetic acid uroporphyrin I and beta-hydroxypropionic acid uroporphyrin I were isolated from the urine of a patient with congenital erythropoietic porphyria. The porphyrins were reduced to the corresponding porphyrinogens with 3% (w/w) Na/Hg amalgam. The hydroxy porphyrinogens were separated on a Hypersil ODS column with 4% (v/v) acetonitrile in 1 M ammonium acetate buffer, pH 5.16, containing EDTA (0.27 mM) as the mobile phase, and detected electrochemically. Reduction of meso-hydroxyuroporphyrin I and hydroxyacetic acid uroporphyrin I, followed by HPLC analysis, showed that, in addition to the expected formation of meso-hydroxyuroporphyrinogen I and hydroxyacetic uroporphyrinogen I, respectively, uroporphyrinogen I was also produced. Reduction of beta-hydroxypropionic acid uroporphyrin I, however, gave beta-hydroxypropionic acid uroporphyrinogen I, acrylic acid uroporphyrinogen I and uroporphyrinogen I as the products. The peaks were identified by conversion into the porphyrin methyl esters and analysed by liquid secondary-ion mass spectrometry.
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MRC Toxicology Unit, University of Leicester, UK.
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D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D006900:Hydroxylation; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014578:Uroporphyrins / Q000138:chemical synthesis* / Q000302:isolation & purification
D007202:Indicators and Reagents; D014578:Uroporphyrins; C034103:uroporphyrin I