An official analytical method for chlorophyll degradation compounds, including pheophorbide, in chlorella products, is described in notification Kanshoku No. 99 (May 8, 1981). However, this method has several operational issues, such as the formation of emulsion during liquid-liquid partitioning. Additionally, impurities present in the reagents (sodium sulfate decahydrate or anhydrous sodium sulfate) used to prepare saturated sodium sulfate solution can degrade pheophorbide and other related compounds, resulting in a significant decrease in analytical values. In this study, we thoroughly examined each step of the official method to enhance the operability and develop an alternative method that eliminates the need for saturated sodium sulfate solution. The developed method was evaluated for pheophorbide a and pyropheophorbide a at 100 mg%. Satisfactory analytical performance was achieved with trueness of 100% for pheophorbide a and 90% for pyropheophorbide a, and relative standard deviations of intra- and inter-day precision below 5% for both compounds. The proposed method is considered suitable for regulatory analysis of chlorophyll degradation compounds and would be useful for quality control of chlorella products.
Keywords: chlorella; chlorophyll; pheophorbide; pyropheophorbide; spectrophotometry.