Methods validation and analysis of Spirulina (<i>Arthrospira platensis</i>) microcystin contaminants by Enzyme-Linked Immunosorbent Assay (ELISA)

Sativa, Hana Aulia; Faridah, Didah Nur; Andarwulan, Nuri

doi:10.1590/1981-6723.00524

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Brazilian Journal of Food Technology

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ORIGINAL ARTICLE • Braz. J. Food Technol. 27 • 2024 • https://doi.org/10.1590/1981-6723.00524 copy

Methods validation and analysis of Spirulina (Arthrospira platensis) microcystin contaminants by Enzyme-Linked Immunosorbent Assay (ELISA)

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Spirulina (Arthrospira platensis) production dominates the global microalgae market due to its high nutritional value. Spirulina can be cultivated in ponds with quality control due to the possible growth of cyanotoxins like Microcystis sp. that produce microcystins. The Enzyme-Linked Immunosorbent Assay (ELISA) method was used and validated with linearity, precision, reproducibility, and accuracy parameters that met the criteria for microcystin analysis. The regression curve gave the linear regression, the value of the coefficient of correlation (r) 0.99 showed a very strong correlation, and coefficient determination (R²) was 0.988 (R² > 0.90). The precision analysis in repeatability (RSDanalysis < 2/3RSD Horwitz) and reproducibility (RSDanalysis < RSD Horwitz) showed an acceptable value with a significance value of 0.191 (p-value <0.05) that showed no significance difference. The accuracy from spiking analysis with a recovery percentage of 79.90±17.97% was still within the range of 40% to 120%, with a LOD value of 0.1 µg/kg. Analysis completed on spirulina powder showed that the spirulina product has a good quality with microcystin levels of 1.39 ± 0.16 µg/kg (dry basis), 719 times below the predetermined limit of 1000 µg/kg based on the Oregon Health Division Regulation for blue-green algae (BGA).

Keywords:
Cyanotoxin; ELISA validation; Microalgae cultivation; Microcystin analysis; Spirulina powder; Quality control

Highlights

Spirulina (Arthrospira platensis) dominates the global microalgae production due to its high nutritional value

Spirulina product processing ensures quality control, concerns about cyanotoxin presence such as Microcystis sp. producing Microcystin

Validated method parameters meeting the criteria for microcystin analysis

Sonication Treatment	Microcystin1⁾ (µg/kg)	RSDa2⁾ (%)	2/3 RSDH3⁾ (%)
Yes	1.19 ± 0.26^a	22.10	29.39
No	1.34 ± 0.11^a	8.13	28.88

Precision			Reproducibility
Microcystin1⁾	RSDa2⁾	2/3 RSDH3⁾	Microcystin¹⁾	RSDa²⁾	RSDH³⁾
(µg/kg)	(%)	(%)	(µg/kg)	(%)	(%)
1.46 ± 0.18	12.22	28.49	1.30 ± 0.07	5.31	43.50

Sample Source	Sample Type	Extraction Methods	Type of MCs Target	Analysis Method	Microcystin (µg/kg)	Reference
Indonesia	Powder	Methanol:Water(50:50)	MC-LR	ELISA	1.39 ± 0.16	This Research
China 1	Powder	Methanol:Water (50:50)	MC-LR	ELISA	1.64 ± 0.41	This Research
China 2	Powder	Methanol:Water (50:50)	MC-LR	ELISA	1.93 ± 0.07	This Research
Hawaii	Powder	Methanol:Water (50:50)	MC-LR	ELISA	4.67 ± 0.12	This Research
Prancis	Flakes	Methanol:Water (75:25)	MCs	ELISA	150 - 1310	(Pinchart et al., 2023Pinchart, P. E., Leruste, A., Pasqualini, V., & Mastroleo, F. (2023). Microcystins and cyanobacterial contaminants in the french small-scale productions of spirulina (Limnospira sp.). Toxins, 15(6), 354. PMid:37368655. http://doi.org/10.3390/toxins15060354 http://doi.org/10.3390/toxins15060354... )
Prancis	Flakes	Methanol:Water (80:20)	MC-LR	UHPLC-MS/MS	*ND - 15	(Pinchart et al., 2023Pinchart, P. E., Leruste, A., Pasqualini, V., & Mastroleo, F. (2023). Microcystins and cyanobacterial contaminants in the french small-scale productions of spirulina (Limnospira sp.). Toxins, 15(6), 354. PMid:37368655. http://doi.org/10.3390/toxins15060354 http://doi.org/10.3390/toxins15060354... )
Prancis	Flakes	Methanol:Water (80:20)	MCs	UHPLC-MS/MS	22 - 227
Republic of Greece	Supplement	Methanol:water (75:25), Ultrasonication (15 W, 22.5 kHz)	MCs	ELISA	35.7 – 583.5	(Papadimitriou et al., 2021Papadimitriou, T., Kormas, K., & Vardaka, E. (2021). Cyanotoxin contamination in commercial Spirulina food supplements. Journal für Verbraucherschutz und Lebensmittelsicherheit, 16(3), 227-235. http://doi.org/10.1007/s00003-021-01324-2 http://doi.org/10.1007/s00003-021-01324-... )
Pacific, Hawaii	Supplement (tablet, capsule, powder)	Lemieux oxidation	MCs (MMPB)	UHPLC	250 - 840	(Roy-Lachapelle et al., 2017Roy-Lachapelle, A., Solliec, M., Bouchard, M. F., & Sauvé, S. (2017). Detection of cyanotoxins in algae dietary supplements. Toxins, 9(3), 1-17. PMid:28245621. http://doi.org/10.3390/toxins9030076 http://doi.org/10.3390/toxins9030076... )
Belgium	Supplement	Metanol:water (80:20)	MCs	HPLC	<LOD (22.5 µg/kg)	(Van Hassel et al., 2022Van Hassel, W. H. R., Ahn, A. C., Huybrechts, B., Masquelier, J., Wilmotte, A., & Andjelkovic, M. (2022). LC-MS/MS validation and quantification of cyanotoxins in algal food supplements from the belgium market and their molecular origins. Toxins, 14(8), 513. PMid:36006175. http://doi.org/10.3390/toxins14080513 http://doi.org/10.3390/toxins14080513... )

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[1] *Corresponding Author: Didah Nur Faridah, IPB University, Faculty of Agricultural Technology, Department of Food Science and Technology, Jl. Raya Dramaga, IPB Dramaga Campuss, Bogor, West Java, 16680, Indonesia, e-mail: didah_nf@apps.ipb.ac.id