Evaluation and optimization of high-throughput enzymatic assays for fast l-ascorbic acid quantification in fruit and vegetables |
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Authors: | Vermeir S Hertog M L A T M Schenk A Beullens K Nicolaï B M Lammertyn J |
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Affiliation: | a BIOSYST-MeBioS, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium b Flanders Centre of Postharvest Technology, Willem De Croylaan 42, B-3001 Leuven, Belgium |
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Abstract: | In this paper, we compare and evaluate the applicability of three UV-VIS absorbance based assays for high-throughput quantification of ascorbic acid in horticultural products. All the methods involve the use of a common enzyme (ascorbate oxidase) in combination with a different indicator molecule. The three methods were retrieved from literature: a direct oxidase-method, an OPDA coupled oxidase-method and a PMS-method, which is commercially available. The analysis in high-throughput context involved the analysis in microplates in combination with the use of an automated liquid handling system. We checked (i) the performance factors of the selected methods on standard solutions, (ii) the applicability of the defined methods in high-throughput context, and, (iii) the accuracy of the methods on real samples using HPLC as a reference technique. The OPDA-method was found to be the most appropriate method for the quantification of ascorbic acid in high-throughput context with a linear range between 7.0 and 950 mg L−1 and excellent correlation parameters (slopes close to 1, intercepts close to 0, R2 > 0.91) with the reference technique when real samples were analyzed. Finally, this method was optimized for assay cost and assay time. Hereto the enzymatic reaction was mathematically described using a model for enzyme kinetics, which was then used to calculate the optimal concentrations of ascorbate oxidase and OPDA. As a result of the modeling the amount of enzyme in the assay could be reduced with a factor 2.5 without affecting significantly the reaction time. In a last step the optimal concentrations were used for a successful validation with the HPLC-reference technique. |
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Keywords: | AA, ascorbic acid AAint, intermediair complex of AA AOX, ascorbate oxidase CV, coefficient of variation DHAA, dehydroascorbic acid EDTA, ethylene-diamine-tetraacetate LOD, limit of detection LOQ, limit of quantification MPA, meta-phosphoric acid OPDA, ortho-phenylenediamine PMS, 5-methyl-phenazinium methosulfate PVPP, polyvinylpolypyrroldine S.D., standard deviation seconds, s MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide |
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