Nanolevel of detection of ascorbic acid using horse-radish peroxidase inhibition assay

• Published in: Heliyon 2024-05, Vol.10 (10), p.e30715-e30715, Article e30715
• Main Authors: Sil, Bijon Kumar, Jamiruddin, Mohd Raeed, Haq, Md Ahsanul, Aekwattanaphol, Nattanit, K, Prem Ananth, Salendra, Limbadri, Paliwal, Himanshu, Paul, Pijush Kumar, Buatong, Wilaiporn, Srichana, Teerapol
• Format: Article
• Language: English
• Subjects: Ascorbic acid; Enzyme inhibition assay; Horse radish peroxidase; Limit of detection; Nanomolar; Percent inhibition
• ISSN: 2405-8440; 2405-8440
• DOI: 10.1016/j.heliyon.2024.e30715

Ascorbic acid plays a significant role in regulation of various bodily functions with high concentrations in immune cells and being involved in connective tissue maintenance. Commonly it is detected through various colorimetric methods. In this study, we propose a one-step simple method based on the inhibitory activity of ascorbic acid on horseradish peroxidase and hydrogen peroxide. The detection is observed by colorimetric changes to TMB (3,3′,5,5′ tetramethylbenzidine). The enzyme inhibition unit was optimized with a high level of linearity (r2 = 0.9999) and the level of detection and level of quantification were found to be 1.35 nM and 4.08 nM, respectively with higher sensitive compared to the HPLC method (11 μM). Both intra and inter-assays showed high correlations at different AA concentrations. (r2 > 0.9999). Similar results were also observed for vitamin C tablets, ascorbate salts, fruits, and market products (r2 = 0.999). There was negligible effect of interference by citric acid, lactic acid, tartaric acids, and glucose with high recoveries (>98%) at 1 mg/mL to 0.0078 mg/mL concentration ranges. The recovery error (RE%) was found to be less than 10%. Our detection method is distinguished by its simplicity, nano-level of detection, reproducibility, and potential application and adaptability as a point-of-use test. [Display omitted] •The detection of ascorbic acid is one step using horse radish peroxidase inhibition.•The method is very sensitive and reproducible with limit of quantification at 4 nM.•The methodology does not need sample purification.