FIG 4 .

Retention times for identification of coproporphyrin. HPLC retention times (400 nm) confirm identity of zinc coproporphyrin III along with UV profiles. A gradient of 20% to 50% acetonitrile over 30 min gave retention times of standards and extract between 7 and 10 min. Extract did not match either standard despite the tandem mass spectrometry data indicating identity of coproporphyrin. This observation is consistent with zinc chelation, which has been reported to shift retention times to slightly more polar elution (27, 28). When standards were individually coinjected with the extract, the appearance of a small peak shoulder with coproporphyrin III that matched extract retention time indicated that the extract was in a chelated form. UV profiles also indicated metal chelation by extract as the Soret maximum was shifted slightly higher and smaller peaks correlated well with known zinc coproporphyrin III peaks (27, 28).