Extraction of Porphyrins from Biological Materials for HPLC Analysis

Sample preparation is an important and integrated part of the successful application of HPLC to the analysis of porphyrins in biological materials. A good sample preparation procedure minimizes quantitative errors and places less demand on the chromatogra-phy, allowing faster and better analysis.

Capillary Voltage Esi
Figure 1. CE-ESIMS of ALA and PBG. Capillary, 70 cm x 75 pm i.d.; running buffer, 50 mM ammonium acetate, pH 5.2:ace-tonitrile (90:10, vol/vol); running voltage, 20 kV; ESI voltage, 3.5 kV.

It is recommended that, whenever possible, porphyrins should be extracted and analyzed as the free acids. The separation of porphyrin free acids is superior to that of the corresponding methyl esters (7). Methyl esterification of porphyrins may also cause structural modification of the parent compounds. The deconjugation and transmethylation of protoporphyrin glycoconjugates following esterification and extraction of porphyrins from rat Harderian gland is a typical example. The procedure led to the incorrect identification of protoporphyrin glycoconjugates as the unconjugated protoporphyrin (9).

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