Development and validation of analytical methodology, using the HPLC for determination of vitamin C in fruits and its main products / Desenvolvimento e validação de metodologia analitica, utilizando a CLAE, para determinação de vitamina C em frutas e seus principais produtos

AUTOR(ES)
DATA DE PUBLICAÇÃO

1998

RESUMO

Currently, amongst commercially available vitamins, vitamin C is the most important. Due to its efficient antioxidant action, it is used in foods and widely in farmaceauticals. Ofthe substances which present this vitaminic activity, L-ascorbic acid (AA) is a well known and commercialized form, though its oxidation product, dehydroascorbic acid (DHAA) possesses the same amount of antiascorbutic action. Isoascorbic acid (IAA) which possesses about 5% of this activity, is added to various foods, thus requiring efficient methods for its control. To better evaluate the vitaminic potential of the natural sources of vitamin C, i.e. fruits, legumes and their products, the need to develop methods which could simultaneously detemine these active forms imposes a great methodological challenge. Thus, a methodology for the analysis of the various active forms of vitamin C (AA, DHAA, IAA) was developed, using a High Performance Liquid Chromatography (HPLC) technique, and using a Spherisorb ODS-2 colul1lI1,5Jlm particle size, 15cmx4.6mmi.d. The aquous mobile phase was 2.5mmol/L of cetrimide and 25mmol/L of ammonium acetate, pH 5.60 adjusted with acetic acid and a flow rate of 0.8mL/min, using a diode array detector (DAD) operating at 263nrn. Quantitative data were obtained using calibration curvesconstructed by plotting peak area versus concentration. Detection and quantification limits were 0.5 and 1.0Jlg/rnL for AA, D~ and IAA, respectively. The recovery was 97.8% for AA, 98.8% for DHAA and 117.1% for IAA. Reapeated injections showed a relative standard deviation lower than 6.0%. Concomitantly, another method was also developed, with the same objectives, with a PLRP-S column, 5Jlm partide size, 100A pore size, 25cmx4.6mm i.d., mobile phase of 250mmol/L NaH2P04, pH 3.30 adjusted with HCI, flow rate of 0.50rnL/min and DAD (À=263nm).Detection and quantification limits were 0.35Jlg/rnL and 1.0Jlg/rnLrespectively. The relative standard deviation was lower than 10.0%. For the detection of DHAA, in both methods, the reaction with DL-homocistein was used and quantification by difference, using two inj.ections. A reaction of DHAA with 0- phenylenediamine (o-PDA) was also studied, for detection with single injection, but with lower sensitivity in the UV detection and a reaction which consumed much time. For application in the samples we opted for the methodology developed using the CI8 colul1lI1,mainly for presenting a better resolution and being less expensive. The determination of total vitamin C (AA and DHAA) was carried out in cashew fi-uit, which varried trom 207.0 to 300.8mgll00g in mature fi-uitsand 148.5 to 243.0mgll00g in commercial juices. Commercial ITozen cashew pulp presented 118.0mgll00g and artesanal pulp 229.O to 318.5mgll OOg,while a nectar presented 170.7mgll OOg.The West Indian cherry fi-uitspresented a variation trom 1229.2mg/100g in mature fi-uitsto 3498.1mgllOOgin green fi-uits.Commercial juices varried trom 710.0 to 881.2mgll00g; ITozen commercial pulp trom 1141.1 to 1375.2mgll00g and nectar 282.3mgllOOg. Vitamin C was determined in other samples: grape, grape juice, prune, red guava,figoda- Índia and beeroot. DHAA was only detected in prune and niagara grapes and at higher concentrations in cashew fi-uits(45.7%) and mature West Indian cherry fi-uits (20.7%). The results were compared with the official method of titration with 2,6- dichloroindophenol. The study of the validation of the method developed with the Cls column, presented satisfatory results thus being recommended for the determination of vitamin C in fi-uitsand their products, beside offering the advantages in relation to the method of AOAC in matrixes which present interference with the color during titration, and it also offers the possiblity of determining of DHAA, which is necessary in some cases

ASSUNTO(S)

high performance liquid chromatography vitamin c vitamina c fruit frutas cromatografia liquida de alta eficiencia

ACESSO AO ARTIGO

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