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

Ulfat Jahan Lithi; Damian W. Laird; Reza Ghassemifar; Navid R. Moheimani

doi:10.1007/s10811-025-03642-6

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

Journal article

Open access

Peer reviewed

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

Ulfat Jahan Lithi, Damian W. Laird, Reza Ghassemifar and Navid R. Moheimani

Journal of applied phycology

2025

DOI: https://doi.org/10.1007/s10811-025-03642-6

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Abstract

Anemia

Bioavailable Heme

Extraction

HPLC

Quantification

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.

Details

Title: An improved extraction and analysis protocol for the determination of heme b (iron-protoporphyrin IX) in microalgae
Authors/Creators: Ulfat Jahan Lithi - Hajee Mohammad Danesh Science and Technology University
Damian W. Laird - Murdoch University, Centre for Water, Energy and Waste
Reza Ghassemifar - Murdoch University
Navid R. Moheimani - Murdoch University, Centre for Water, Energy and Waste
Publication Details: Journal of applied phycology
Publisher: Springer Nature
Number of pages: 16
Identifiers: 991005810350107891
Murdoch Affiliation: School of Mathematics, Statistics, Chemistry and Physics; School of Environmental and Conservation Sciences; Centre for Water, Energy and Waste; Algae R&D Centre
Language: English
Resource Type: Journal article

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Collaboration types: International collaboration
Citation topics: 3 Agriculture, Environment & Ecology; 3.171 Photoproductivity; 3.171.477 Microalgae Biotechnology
Web Of Science research areas: Biotechnology & Applied Microbiology; Marine & Freshwater Biology
ESI research areas: Plant & Animal Science