High Performance Liquid Chromatographic Determination of Pheophorbide a in Chlorella Tablets

A high performance liquid chromatographic (HPLC) method was established for the quantitative determination of pheophorbide a (PB-a) in chlorella tablets. PB-a was extracted from chlorella tablets with 85% acetone. The acetone aqueous extract was shaken with diethyl ether, the ether extract was washed with 5% Na2SO4 and shaken with 17% HCl. The acid extract was diluted with water and re-extracted with diethyl ether. The ether extract was evaporated to dryness, the residue of which was dissolved in methanol and applied to HPLC. PB-a was separated on Radial PAK μBondapak C18 column with methanol-0.025M ammonium acetate (88 : 12, v/v) as a mobile phase. The average recovery of PB-a added to chlorella tablets was 82.9% with coefficients of variation of 6.72%. The official method (spectrophotometric determination) was used to determine PB-a content of 19 commercial chlorella tablets in order to compare it with the proposed HPLC method. The PB-a contents obtained by the official method (8.0-111.8mg%, an average 36.4mg%) were higher than those by the proposed one (2.9-80.6mg%, an average 22.4mg%). As PB-a was separated from other chlorophyll degradative products, the proposed method was more specific and accurate than the official one.