Separation of Porphyrin Mixtures from Uroporphyrin to Protoporphyrin

From uroporphyrin to protoporphyrin, the compounds differ widely in hydropho-bicity. Gradient elution is therefore essential for the simultaneous separation of these porphyrins, including their isomers.

A 15-minute linear gradient elution from 13% to 30% acetonitrile in 1 M

ammonium acetate, pH 5.16, has been described for the separation of type I and type III isomers of 8-, 7-, 6-, 5-, and 4-car-boxylated porphyrins (7). The system is applicable to analysis where the separation of dicarboxylated porphyrins is not required, e.g., urinary porphyrins. The elu-tion of dicarboxylated porphyrins requires an acetonitrile content higher than its mis-cibility with 1 M ammonium acetate. It should be emphasized that using this gradient system the acetonitrile content should not be allowed to exceed 35%, and the column must not be washed with ace-tonitrile at the end of the separation due to

Figure 4. Separation of copropor-phyrin I, II, III, and IV isomers.

(a) On Hypersil-ODS with 30% (vol/vol) acetonitrile in 1 M ammonium acetate, pH 5.16, as eluent; (b) on Hypersil-BDS C^ with 23% (vol/vol) acetonitrile in 1 M ammonium acetate, pH 5.16, as eluent. Flow-rate, 1 mL/minute.

Figure 4. Separation of copropor-phyrin I, II, III, and IV isomers.

(a) On Hypersil-ODS with 30% (vol/vol) acetonitrile in 1 M ammonium acetate, pH 5.16, as eluent; (b) on Hypersil-BDS C^ with 23% (vol/vol) acetonitrile in 1 M ammonium acetate, pH 5.16, as eluent. Flow-rate, 1 mL/minute.

the immiscibility problem. It is also important to remember that whenever acetoni-trile and 1 M ammonium acetate is used for elution, as in the separation of the individual group of porphyrin isomers by iso-cratic elution, the column should not be washed with acetonitrile before removal of ammonium acetate with a solvent in which it is completely miscible. The column may be washed with 90% methanol or acetoni-trile:methanol in water.

Porphyrin mixtures including protopor-phyrin are best separated by gradient elu-tion RP-HPLC with (1,7,17) or without ion-pairing agents (7). Columns of silica gel chemically bonded with different hydrocarbon chain lengths, from C1 to C18, have all been successfully used for the RP-HPLC separation of porphyrin mixtures in biological materials (7,8). With the increasing use of on-line HPLC-MS in analysis, including the tetrapyrroles, gradient elution solvent mixtures that are fully compatible with MS are necessary. This rules out systems that use involatile inorganic phosphate in separation.

A simple RP-HPLC system with 0.1% trifluroroacetic acid (solvent A) and ace-tonitrile (solvent B) as the gradient elution solvent mixture has been used for the separation of porphyrins. The system, fully compatible with MS, is able to resolve the type I and III isomers of 6-, 5-, and 4-car-boxylated porphyrins. Separation of uro-and heptacarboxylic acid porphyrin isomers, however, was not achieved (Figure 6). The system is best used for the separation of porphyrins with fewer numbers of carboxylic acid groups, including the dicar-boxylic acid porphyrins.

The ammonium acetate buffer system that is fully compatible with MS and provides high efficiency separation of por-phyrins is the buffer of choice. It is recommended that the following gradient mixtures are used for elution: solvent A, 10% (vol/vol) acetonitrile in 1 M ammoni um acetate buffer, pH 5.16; solvent B, 10% (vol/vol) acetonitrile in methanol. Various elution programs can be used, depending on the applications, with these two solvent mixtures for porphyrin separation (15). The pH of the buffer, 5.16, is optimal for the separation of porphyrin mixtures.

Figure 7 shows the separation of por-phyrins in the feces and urine of a patient with porphyria cutanea tarda (PCT) on a C18-bonded RP column (Hypersil-ODS). It clearly demonstrates the flexibility and applicability of the system.

Figure 5. Separation of protoporphyrin and metallopor-phyrins. Column, Hypersil-ODS; eluent, methanol: 1 M ammonium acetate, pH 5.16 (86:14, vol/vol); flow rate, 1 mL/minute. Peaks: 1 = Zn-deuteroporphyrin; 2 = heme; 3 = Zn-protoporphyrin; 4 = protoporphyrin.
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