Recommendations on the quantitative analysis of pheophorbides, photosensitizers present in algal biomass intended as food supplement

Pheophorbide a is the major photosensitizer present in algal biomass and its presence in food supplement was linked to human health issues in the past. Although several pheophorbide a quantification methods have been proposed including the United States Pharmacopeia-National Formulary protocol (USP-NFP), there are open methodological issues which make their results questionable. In the present study we have tested optimal extraction conditions for pheophorbide a from a disintegrated biomass of green algae Chlorella and Haematococcus, widely used as food supplement. Further we have compared the quantification of pheophorbide a in algal extracts using HPLC-HRMS with the spectrophotometric quantification after USP-NFP in Haematococcus biomass and oleoresin samples. We have demonstrated that USP-NFP provide considerably (1.7–2.4 times) higher pheophorbide contents compared to HPLC-HRMS quantification using a pheophorbide a analytical standard. This was mainly caused by the fact that USP-NFP integrates the concentration of all pheophorbide variants present in the samples. We have detected nine pheophorbide analogues in the original algal sample as well as in the final sample of USP-NFP, of which pheophorbide a represented only ~40%. However, this explains the values obtained by USP-NFP only partly. We also show that 60% acetone is the most suitable extraction solvent for pheophorbide a from various algal samples; however, we noted that such extracts might exhibit high activity of chlorophyllase generating pheophorbide a during the extraction process. This activity differed between studied Chlorella and Haematococcus biomass extracts. We have achieved the chlorophyllase inactivation by a short-term high-temperature treatment (90 °C, 5 min). Acceptable stability of pheophorbide a and only minor degradation of chlorophyll to pheophorbide a has been observed at these conditions.This study demonstrates the need for 1) an HPLC-DAD-based method employing multiple pheophorbide standards and 2) involvement of chlorophyllase inactivation protocol, to ensure proper quantification of pheophorbide content in algal biomass.