Quantitative determination of porphyrins, their precursors and zinc protoporphyrin in whole blood and dried blood by high-performance liquid chromatography with fluorimetric detection.
Methods for the determination of porphyrins, delta-aminolevulinic acid (ALA), porphobilinogen (PBG) and zinc protoporphyrin of heme biosynthesis in whole blood and dried blood are described. Erythrocyte porphyrins and the precursors ALA and PBG were extracted from whole blood (50 microliter) with 0.3 ml of methanol and 1.5 M hydrochloric acid (2:1, v/v). Zinc protoporphyrin was extracted with an acetone-pyridine-Sterox solution. Other major interfering metabolites were removed by centrifugation. An aliquot of the supernatant was injected onto the reversed-phase C18 column for detection of porphyrins with excitation wavelength at 405 nm and emission wavelength at 630 nm. The mobile phase was 0.1 M phosphate-methanol-tetrahydrofuran (18:30:16, v/v/v), pH 5.38. The ALA and PBG were derivatized with o-phthalaldehyde before injection. The detection excitation wavelength occurred at 330 nm and the emission wavelength at 418 nm. The mobile phase was 0.1 M phosphate-methanol (7.5:5), pH 3.38. For the dried blood specimen of filter paper, two 0.64-cm discs punched out from the blood-impregnated filter paper were placed in a test tube containing 200 microliter of 0.9% saline for 60 min or longer at room temperature and then treated as whole blood.
Ho
J
J
Department of Pediatrics, State University of New York at Buffalo 14214.
Guthrie
R
R
Tieckelmann
H
H
D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D011166:Porphyrins / Q000097:blood*; D011524:Protoporphyrins / Q000097:blood*; D013050:Spectrometry, Fluorescence
D011166:Porphyrins; D011524:Protoporphyrins; C017803:zinc protoporphyrin