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Comparison of Vitamin C Content in Citrus Fruits by Titration and High Performance Liquid Chromatography (HPLC) Methods

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Comparison of Vitamin C Content in Citrus Fruits by Titration and High Performance Liquid Chromatography (HPLC) Methods International Food Research Journal 24(2): 726-733 (April 2017) Journal homepage: http://www.ifrj.upm.edu.my Comparison of vitamin C content in citrus fruits by titration and high performance liquid chromatography (HPLC) methods 1Fatin Najwa, R. and 1,2*Azrina, A. 1Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, 43400 UPM Serdang, Selangor, Malaysia 2Research Centre of Excellence, Nutrition and Non-communicable Diseases, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Article history Abstract Received: 29 June 2015 Vitamin C is one of the essential vitamins for human and animal. Many methods were Received in revised form: developed for the determination of vitamin C such as spectrophotometry, electrophoresis, 23 March 2016 titration, and high performance liquid chromatography (HPLC). This study aims to compare Accepted: 4 April 2016 vitamin C content of citrus fruits (orange, grapefruit, lemon, lime, kaffir lime and musk lime) using indophenol titration and HPLC-PDA methods. In the titration method, orange has the highest vitamin C content (58.30 mg/100g) followed by grapefruit (49.15 mg/100g), lemon Keywords (43.96 mg/100g), kaffir lime (37.24 mg/100g), lime (27.78 mg/100g) and musk lime (18.62 Vitamin C mg/100g). While, in the HPLC method orange also leads with the highest vitamin C content Ascorbic acid (43.61 mg/100g) followed by lemon (31.33 mg/100g), grapefruit (26.40 mg/100g), lime (22.36 Citrus fruits mg/100g), kaffir lime (21.58 mg/100g) and musk lime (16.78 mg/100g). Orange is the best High performance liquid source of vitamin C while musk and kaffir lime have lower content. Significant differences chromatography were observed in vitamin C of samples by both methods. Both methods are suitable for the Titration determination of vitamin C, however HPLC method is more accurate, precise and specific. © All Rights Reserved Introduction and metal ion (Wantz et al., 2005). Human is unable to synthesize their own vitamin Vitamin C, also is known as ascorbic acid is C supply as human cells cannot perform the crucial one of the most important vitamins and essential last step in vitamin C biosynthesis,the conversion for human and animal life. This water soluble of L-gulono-g-lactone into ascorbic acid which vitamin contributes to many health benefits such is catalyzed by gulonolactone oxidaseenzyme as prevention of scurvy and cancer, relief from (LinsterandVan Schaftingen, 2007). Therefore, they common cold, stimulate collagen synthesis and require vitamin C for maintaining the physiological play a significant role in wound healing process functions. To meet the requirement, vitamin C must (Iqbal et al., 2004). According to Teucher et al. be consumed from diet. The recommended nutrient (2004), vitamin C helps to enhance availability and intake of vitamin C for Malaysian adult is 70 mg per absorption of iron from non-heme sources. Vitamin day. An intake of 45 mg/day will ensure measurable C also has antioxidant properties since it can easily amount of ascorbate be present in the plasma of most lose the electron to neutralize and inhibit free radicals people and available to supply tissue requirements for from being oxidized in preventing cell damage. It is metabolism or repair at sites of depletion or damage also commonly used as food additive which acts as (MOH, 2005). antioxidant (Whitney and Rolfes, 2008). Many methods can be used for determination Vitamin C is an organic compound consists of of vitamin C such as spectrophotometry, carbon, hydrogen and oxygen (Chinnici et al., 2005). electrophoresis, titration, and high performance The terms vitamin C is not only used for ascorbic acids, liquid chromatography (HPLC) (Tang and Wu, but it includes all compounds exhibiting biological 2005; Dong et al., 2007; Spinola et al., 2012). The activity such as oxidized, ester and synthetic form. most commonly used method is oxidation-reduction The main biological form of vitamin C is L-ascorbic titration method where ascorbic acids are oxidized acid, and it can reversibly change to oxidized form to dehydroascorbic acid and the indophenol dye is called dehydroascorbic acid (Fenolland Martinez, reduced to a colorless compound. The end point of 2010). Many factors can cause oxidation of vitamin the titration can be easily detected when an excess of C such as pH, light, temperature, presence of oxygen the unreduced dye give a rose pink color in an acid *Corresponding author. Email: [email protected] 727 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733 solution (Tee et al., 1996). It is a simple and easy Preparation of standard solution for titration method to determine vitamin C in fruits and fruit method juices. However, the method is not suitable for fruits Vitamin C standard solution (0.2 mg/ml) was that have reddish-purplish color. The titration method prepared by dissolving 100 mg vitamin C in 3% of also is time-consuming and lack of specificity due to metaphosphoric acid (HPO3) solution and diluted to interference of reducing substances in the food such 500 ml with the same solvent. Then, 5 ml aliquot of the as ferrous iron, stannous tin, cuprous copper, sulphur vitamin C standard solution was diluted (containing dioxide, sulphite orthiosulphate (Eitenmiller et al., 1 mg vitamin C) with 5 ml 3% metaphosphoric acid 2008). (HPO3). Next the diluted vitamin C standard solution Several other methods to determine vitamin was titrated with dye solution 2, 6-dichlorophenol- C content like spectrometric, spectrofluorimetric, indophenol to a faint pink color. Triplicate titration and electrophorhesis, still some of them are not also was conducted for the vitamin C standard. practical and need re-evaluation due to insufficient of sensitivity and selectivity (Agar, 1995). Quiroz Quantification of Vitamin C in the extracted solution and Fernandez (2009) stated that the most preferred of titration method method of determining vitamin C content in foods is Firstly, weight of the sample in 10 ml filtrate was a chromatographic method using HPLC due to the calculated by the following equation: rapid, high accuracy and consistency. Previously, there were many studies done on citrus fruits mainly focusing on determination of vitamin C using HPLC by modifying the stationary Where, phase, mobile phase, type of detector and sample a = Weight of sample will be used preparation (Gazdik et al., 2008; Spinola et al., 2012) b = Final volume of homogenous slurry [sample but there was not many studies done comparing two + 6% metaphosphoric acids (HPO3)] or more different methods in determining vitamin c= Aliquot of homogenous slurry used for dilution C in citrus fruits (Ullah et al., 2012; Spinola et al., to 100 ml with 3% metaphosphoric acid (HPO3) 2013). Moreover, most studies are just focusing on single type of lime and do not focus on another type Then total vitamin C content (mg per 100 g of limes such as kaffir lime and musk lime.Thus, the sample) of each sample was obtained by the following purpose of this study was to provide the comparison equation: between titration and HPLC method in determination of vitamin C content in citrus fruits. Vitamin C content (mg per 100 g sample) = Materials and Methods X = volume of dye used for titration of aliquot of diluted sample (ml) Sample preparation of titration method Y = vitamin C equivalent of dye solution, mg per Sample and standard preparation and ml dye solution quantification of vitamin C in titration method was W = weight of sample in aliquot of filtrate of performed using the method described by Tee et al. diluted sample used for titration (g) (1996). In brief, fruit samples (200 g-300 g) was blended with 6% metaphosphoric acid (HPO3) with Sample preparation of HPLC method an equal volume to homogenous slurry and made it Sample preparation, chromatography conditions, up until 500 ml of volume. The homogenous mixture identificantion and quantification of vitamin C in was measured around 10 to 30 ml and diluted into HPLC method was perfomed using the method 100 ml volumetric flask with 3% of metaphosphoric described by Czech Agriculture and Food Inspection acid (HPO3). The diluted sample was then filtered to Security (2005). The homogenous solid sample remove away suspension using vacuum pump before was measured around 10 to 30g and mixed it with 10 ml aliquote of the filtrate was pipetted into a 60 to 80 ml 3% of metaphosphoric acid (HPO3) small Erlenmayer flask. The filtrate was immediately for one minute. The obtained extracted was filtered titrated with a dye solution 2, 6-dichlorophenol- through filtration paper and washed it for few times indophenol to a faint pink end point. Triplicate by using vacuum pump filtration. Next, the filtrate titration was conducted for all samples. quantitatively transferred into a 100 ml volumetric flask and 3% of metaphosphoric acid (HPO3) was added up to 100 ml volumetric mark. All the sample Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733 728 solutions was filtered again through 0.45 µm syringe Where, filter. After that, the samples were run in the HPLC Cst = standard working solution concentration 50 system. µg/ml Ast = corresponding peak area HPLC system or chromatography conditions m = weight of samples The total of vitamin C content in 6 types of fruits were determined by HPLC system. The reversed– Statistical analysis phase liquid chromatographic method was used for IBM SPSS version 21.0 software was used for determination of vitamin C consisted of an isocratic statistical analysis. The total vitamin C concentration elution procedure with photodiode assay detection at in food samples was expressed as mean ± standard 254 nm. The separations were carried out on Carbon deviation. One-way ANOVA was used to determine 18 (C18) column of 250mm x 4.6 mm (LiChro CART, significance difference of mean value of vitamin C Darmstadt, Germany).
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