An improved extraction and analysis protocol for the determination of heme b (iron-protoporphyrin IX) in microalgae

Abstract Microalgae could serve as an improved source of bioavailable heme b for the treatment of chronic anemia in humans and animals and/or be used as an additive for plant-based protein products to provide a ‘meaty’ taste. Unfortunately, ‘standard’ spectrophotometric assays developed for heme b -rich samples, particularly meat and blood samples, are not viable for microalgae due to lower heme b levels and spectral interference from pigments such as chlorophyll. Removing interferents in heme b extracts from photosynthetic organisms is time-consuming and risks loss of product. Analysis of 6 different microalgal strains using a spectrophotometer and literature best-practice HPLC method revealed that HPLC was consistently more sensitive and had a 1000-fold lower LoD compared to the spectrophotometric method (0.1 pmol cf 230 pmol). The HPLC method also offered the advantage of not having to remove pigment interferences in the extraction phase. The instrumental method comparison highlighted the inefficiencies in the traditional sequential acetone extraction process, prompting trials of several alternate extraction protocols. It was found that a single-step acidic N,N -dimethylformamide (DMF: HCl, 98:2 v/v) extraction improved heme b yield by ~ 50%, reduced extraction time and solvent use by ~ 90%, and extracted heme b remained stable for two weeks at -25 °C, enabling batch analysis. Coupling this extraction procedure with HPLC analysis provides a robust analytical tool for advancing the evaluation of microalgae-derived heme b in medicinal and functional food settings.