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LWT VitaminC2 reviso

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EFFECT
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EXTRACTS. UHPLC-PDA VS IODOMETRIC TITRATION AS ANALYTICAL METHODS
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Vítor Spínola· Berta Mendes · José S. Câmara · Paula C. Castilho (*)
OF
TIME
AND
TEMPERATURE
ON VITAMIN
C STABILITY IN HORTICULTURAL
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Vítor Spínola · Berta Mendes · José S. Câmara · Paula C. Castilho (*)
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Centro de Química da Madeira (CQM),
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Centro de Ciências Exactas e da Engenharia da Universidade da Madeira,
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Campus Universitário da Penteada,
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9000-390 Funchal, Portugal
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E-mail address: [email protected] (Paula C. Castilho).
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Abstract
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Several fruits and vegetables from Madeira Island (Portugal) were evaluated by two
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analytical methods for their total vitamin C content (L-ascorbic acid, L-AA and
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dehydroascorbic acid, DHAA). DHAA was determined indirectly with DL-1,4-
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dithiotreitol (DTT) applied as a pre-column reductant. Ultra high performance liquid
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chromatography coupled to photodiode array (UHPLC-PDA) determinations were
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compared with L-AA content obtained by a classic iodometric titration method. The
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stability of vitamin C in horticultural extracts stored at different temperatures was also
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investigated. Red peppers represented the better source of vitamin C followed by green
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peppers and papayas. Passion fruits and cherimoyas were the analyzed foodstuffs with
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lowest vitamin C content. Both analytical methods were suitable for L-AA analysis in
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various food commodities, the UHPLC-PDA technique being preferred due to its
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advantages of selectivity, speed and accuracy. The degradation study showed that
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horticultural extracts were stable at least 24 h at 4º C and during 4 weeks when stored at
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-80 ºC.
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Keywords: Ascorbic acid · Dehydroascorbic acid· UHPLC · Iodometric Titration ·
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Fruits and Vegetables · Stability ·
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1. Introduction
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Vitamin C is the trivial name for compounds exhibiting full or partial biological activity
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of L-ascorbic acid (L-AA). It includes its isomers, synthetic forms and oxidized
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products (Johnston, Steinberg & Rucker, 2007; Eitenmiller, Ye & Landen, 2008).
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Vitamin C its one of the most important micronutrients postulated to have a beneficial
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role in health-promoting effects (antioxidant, biosynthesis of collagen, carnitine and
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hormones, immune response, iron absorption) (Davey et al., 2000; Hernández, Lobo &
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González, 2006; Valente, Albuquerque, Sanches-Silva & Costa, 2011). Due to the
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inability to synthesize vitamin C, humans have to meet their daily requirements
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throughout fresh vegetables and fruits food and/or supplements (Phillips et al., 2010;
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Fenoll, Martínez, Hellín & Flores, 2011). L-AA is by far the least stable nutrient and
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prone to loss immediately after harvest, being degraded to dehydroascorbic acid
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(DHAA) and latter to diketogulonic acid (DKG). (Johnston et al., 2007; Odriozola-
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Serrano, Hernández-Jover & Martín-Belloso, 2007). Thus, the nutritional quality of
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foodstuffs depends not only on the nutrient content when harvested but also on the
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changes occurring during postharvest handling, storage conditions, processing and
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preparation (Lee & Kader, 2000; Kalt, 2005; Rickman, Barrett & Bruhn, 2007).
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Refrigeration slows down the respiration of fruits and vegetables and extends the shelf
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life of seasonally available foodstuffs products. However, losses of ascorbic acid also
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occur under these conditions (Lee et al., 2000; Rickman et al., 2007). The best way of
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deriving benefit of L-AA is to eat fresh fruits and vegetables recently picked, and with a
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minimum of processing (refrigerating cutting, cooking) (Davey et al., 2000; Kalt, 2005).
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Spectrophotometric, titration, enzymatic and chromatographic methods have been
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reported for the analysis of L-AA in foodstuffs (Eitenmiller et al., 2008; Nováková,
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Solich & Solichová, 2008). The AOAC (Association of Official Analytical Chemists)
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standard methodology for determination of vitamin C in juices and preparations
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employs a titration method with the indicator 2,6-di-chlorophenol-indophenol (AOAC
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Official Method 967.21, 2006) (AOAC, 2006; Hernández et al., 2006). L-AA can also
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be determined directly with iodine and iodate solution in a redox titration, using starch
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as indicator. As a good reducing agent, L-AA reacts rapidly and stoichiometric with
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iodine to give iodide ions, while it is oxidized to DHAA. Once all the L-AA has been
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oxidized, the excess iodine solution will react with the starch indicator, forming a blue-
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dark starch-iodine complex as endpoint of titration (Suntornsuk, Gritsanapun,
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Nilkamhank & Paochom, 2002; Zenebon, Pascuet & Tiglea, 2008). However, these
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traditional methods suffer from lack of specificity, which limits their use in matrices
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that contain other interfering substances that are also oxidized by the applied titrants
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(Hernández et al., 2006; Eitenmiller et al., 2008; Nováková et al., 2008). This means
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that L-AA results are normally determined by excess in vegetable extracts usually rich
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in reducing organic acids, while DHAA is not quantified.
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Liquid chromatographic (LC) methods have been more successful for L-AA
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quantification (Johnston et al., 2007; Valente et al., 2011). The ultra high performance
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liquid chromatography (UHPLC) has recently become a preferred separation technique
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in many laboratories. The development of analytical columns of very small particle size
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and specially designed instruments allow for the use of much lower flows of mobile
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phase at very high pressures, which results in increased speed of analysis, higher
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separation efficiency and resolution, higher sensitivity and much lower sample and
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solvent consumption, as compared to other analytical approaches (Nováková &
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Vlcková, 2009). Moreover, unlike classical methods, they have the potential for the
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simultaneous determination of other metabolites (Eitenmiller et al., 2008; Nováková et
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al., 2008). DHAA content tends to increase after prolonged storage, mechanical and
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thermal treatment, and depends on the type of fruit and vegetable analyzed (Davey et
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al., 2000; Lee et al., 2000). Therefore, accurate quantification of both molecules is
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important, otherwise the total content of vitamin C (sum of L-AA plus DHAA contents)
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in food commodities could be underestimated (Odriozola-Serrano et al., 2007;
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Chebrolu, Jayaprakasha, Yoo, Jifon & Patil, 2012). Due to the low UV spectra
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absorptivity, DHAA is usually determined indirectly after its conversation to L-AA by
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the so-called subtraction approach. Various reducing agents, such as DL-1,4-
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dithiotreitol (DTT), have been applied successfully (Fig. 1) (eg. Hernández et al., 2006;
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Odriozola-Serrano et al., 2007; Campos, Ribeiro, Della Lucia, Pinheiro-Sant'Ana &
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Stringheta, 2009; Fenoll et al., 2011; Chebrolu et al., 2012).
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< place Fig. 1 near here >
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Recently, we developed and validated a simple and fast UHPLC-PDA method (Spínola,
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Mendes, Câmara & Castilho, 2012) for the quantitative analysis of total vitamin C in
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several vegetables and fruits.
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In the present work, L-AA contents obtained by this method were compared with those
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obtained by iodometric titration. The stability of L-AA in extracts at different
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temperatures was also investigated.
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2. Experimental
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2.1 Chemicals and reagents
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All reagents and standards were of analytical grade. L-ascorbic acid (L-AA),
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metaphosphoric acid (MPA), formic acid and potassium iodide were purchased by
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Panreac (Madrid, Spain). Acetic acid, ethylendiaminetetraacetic acid disodium salt
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(EDTA), Tris-Buffer and starch were supplied by Merck (Darmstadt, Germany); DTT
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was obtained from Acros-Organics (Geel, Belgium) and sulfuric acid and potassium
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iodine from Riedel-de Haen (Seelze, Germany). All solutions were prepared with water
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from a Milli-Q Direct 8 system (18 M  cm at 23 ºC) (Millipore, USA).
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2.2 Raw material
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Nine horticultural products, which are commonly cultivated and consumed in Madeira
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Island (Portugal), were chosen for this study. The edible portion of fruits, cherimoyas
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(Annona cherimola Mill.), purple passion fruits (Passiflora edulis Sims), papayas
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(Carica papaya L.), strawberries (Fragaria), lemons (Citrus limon (L.) Burm. F.), and
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vegetables, broccoli (Brassica oleraceae L. var. italica Plenk), green and red peppers
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(Capsicum annuum L.) and watercress (Rorippa nasturtium-aquaticum L.) was
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analyzed. Food commodities were supplied (at least 3 kg of samples) by a national food
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distributor (Sonae MC) with connections to local registered producers, from February to
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May 2011. Local products were delivered by Sonae to our laboratory (Madeira
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Chemical Center - CQM) within one or two days after harvest. All foodstuffs were
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immediately stored in a common refrigerator at 4 ºC before extraction and kept under
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these conditions for 5 consecutive days in order to assess the rate of degradation of L-
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AA during storage.
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2.3 Sample preparation
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From major size foods such as cherimoyas, papayas, green and red peppers and lemons,
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small portions were taken from multiple specimens to form a composite sample. In case
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of passion fruits, strawberries, broccoli and watercress several whole specimens from
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the same cultivar were used for analysis. Sample preparation followed the procedure
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indicated in our previous work (Spínola et al., 2012). Briefly, approximately 200 g of
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each product was homogenized in a blender and the pH was determined directly in the
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pulp (Metrohm 7444 pH meter), using the buffers 4 and 7. Then 10 mL of extraction
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solution (30 g/L MPA – 80 mL/L acetic acid – 1 mmol/L EDTA) was added to 3 mL of
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pulp and centrifuged (10 000 rpm, 10 min, 2 – 4 ºC). The resulting extracts were stored
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at -80 ºC, immediately after extraction until UHPLC-PDA analysis (within 1 week). For
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iodometric titration, samples were analyzed on the same day of extraction.
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2.4 Chromatographic conditions
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The analysis was carried out according to a previously optimized and validated
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UHPLC-PDA method, described in Spínola et al. (2012). The chromatographic system
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Acquity UPLC (Waters) was equipped with a Acquity HSS T3 analytical column (100
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mm × 2.1 mm, 1.8 µm particle size) using a isocratic mobile phase composed of
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aqueous 1 mL/L formic acid at a flow rate of 250 µL/min and the injection volume was
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2 µL. The detection wavelength for the photo-diode detector was set at 245 nm and the
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analytical column was kept at room temperature. The chromatographic analysis were
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performed in triplicate (n = 3) and the results were expressed as mg of L-AA/ 100 g of
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edible portion.
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2.5 Iodometric titration
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Undiluted extracts were used for iodometric titration, performed according to Analytical
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Standards of Adolfo Lutz Institute (Zenebon et al., 2008) which follows the general
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norm for vitamin C evaluation used in Brazil and most Latin-American countries.
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Briefly, 1 mL of 10 g/L starch solution and 1 mL of 100 g/L potassium iodide solution
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were added to accurately weighted amounts of fruit/vegetable extracts. Then the
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samples (n = 3) were titrated with 0.002 mol/L potassium iodate solution, previously
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standardized, until the mixture becomes dark blue and the color persisted for more than
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60 seconds. All solutions were prepared and standardized daily with L-AA standard
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solution (50 µg/mL). Each mL of 0.002 mol/L potassium iodate solution is equivalent to
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0.8806 mg of L-AA. The limit of detection (LOD) was determined by successive
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dilutions of standard L-AA solution until it was no longer possible to determine the
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content with accuracy. The lowest amount of standard L-AA, which could be measured
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by a 25 ± 0.05 mL burette, was considered to be the LOD. The limit of quantification
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(LOQ) was calculated by multiplying the LOD by a factor of 3.3, as suggested by
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Suntornsuk et al. (2002).
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2.6 L-AA degradation study
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The degradation of L-AA was evaluated in a standard L-AA solution of 50 µg/mL
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(prepared in extraction solution) and in passion fruit extracts prepared previously. The
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L-AA concentrations measured immediately after extraction was considered as the
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initially concentration. Passion fruits extracts were kept in the dark, at 4 ºC, -20 ºC and -
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80 ºC, during 8 weeks. Additionally, the stability of L-AA in passion fruits extracts kept
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at room temperature (23 ºC) was analyzed during 5 hours. The results were compared
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with those obtained for the standard solutions, maintained under the same conditions.
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All determinations were repeated three times (n = 3).
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2.7 Statistical Analysis
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Data analysis was carried out with SPSS for Windows, IBM SPSS Statistics 19 (SPSS,
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Inc., USA). Analysis of variance (ANOVA) was used to evaluate the results obtained
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from (i) the chromatographic and iodometric titration methods and (ii) L-AA
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degradation study at different temperatures. A value of p < 0.05 was considered
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statistically significant. Simple linear correlation analysis was used to measure the
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correlation between the results obtained for L-AA content from the two analytical
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methods.
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3. Results and discussion
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3.1 Analysis of vitamin C
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For all analyzed foodstuffs, results from statistical analysis showed that differences
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among the three determinations of L-AA and total vitamin C were not statistical
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significant (p < 0.05). Table 1 shows L-AA, DHAA and total vitamin C concentrations
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of nine horticultural produce obtained with the two used methods. As expected, fruits
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and vegetables exhibit different L-AA and DHAA profiles. In general, peppers (red and
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green) and papayas were the species with the highest vitamin C contents, while
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cherimoyas and passion fruits had the lowest concentration. Broccoli, watercress,
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strawberries and lemons also represented good sources of vitamin C. L-AA was always
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the predominant form of vitamin C. A simple linear correlation analysis was used to
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measure the relationship between the results obtained for L-AA by these two analytical
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methods. We obtained a relatively strong significant correlation (r2-values = 0.976).
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With the exception of peppers (red and green) and watercress, there was actually no
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statistically significant difference in L-AA content obtained by the chromatographic and
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titration methods.
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< place Table 1 near here>
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The main advantages of the iodometric titration method are its simplicity, the use of
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very elementary equipment, easily available reagents of low cost and speed of reaction
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of iodine with L-AA.
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In the present study, since the L-AA amounts of all analyzed fruit/vegetables were
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reasonably high, LOD and LOQ are not essential issues. However, in some highly
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colored extracts it is difficult to accurately determine the end point of titration. The
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LOD and the LOQ of L-AA content were 0.9 and 2.9 mg/mL, respectively. Suntornsuk
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et al. (2002) validated and applied a similar iodometric titration method to herbal juices,
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finding higher value limits. Overall, both methods showed much more restrictive limits
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than those we determined for UHPLC-PDA (22 and 67 ng/mL for LOD and LOQ,
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respectively) (Spínola et al., 2012). Besides that, iodometric titration presents the
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inconvenience of exposing samples to light and oxygen during titration which can lead
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to L-AA degradation and the method is susceptible to co-extracted interferences and
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may overestimate L-AA due to the presence of oxidizable species other than L-AA.
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Moreover, initial DHAA is never quantified in this method since L-AA is oxidized to
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DHAA by iodine.
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LC revealed to be a more specific, selective and sensitive technique for determination of
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L-AA in the different foodstuffs. Moreover, this method requires less reagents and
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material, is less time consuming than the titration method, less susceptible to systematic
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errors and allows the quantification of total vitamin C content. Since the UHPLC-PDA
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methodology requires a large financial investment in equipment, the iodometric titration
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provided satisfactory quantitative results (according to the correlation measurements)
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and can be applied in a preliminary analysis or in situations where equipment cost is an
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obstacle but availability of human resources is not.
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The amounts of L-AA and DHAA naturally present in all samples have been previously
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reported in literature and those results were used to compare to ours in order to
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understand possible discrepancies (Table 2).
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< place Table 2 near here>
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Generally, vitamin C determination results are in good agreement with those reported in
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the literature using HPLC analysis. The differences could be attributed to the natural
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variation of L-AA and DHAA contents among specimens and/or cultivars (genetic
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factors) and to pre- and post-harvested factors (maturity stage, environmental and
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cultural practices, storage conditions) (Lee et al., 2000). With the exception of
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cherimoyas (17.4%), DHAA levels ranged between 1.3 and 6.5% in the extracts
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analyzed immediately after thawing from -80 ºC, generally much lower than the
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(scarce) reported data (Table 2).
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After this determination, all horticultural products (except cherimoyas) were kept at 4
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ºC and there was a sometimes sharp increase in DHAA at the expense of L-AA, as well
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as a total vitamin C decrease during storage (Spínola et al., 2012) (Fig. 2). Cold storage
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at 4 – 5 ºC, although used for most fruits, may be not suitable for cherimoya, due to the
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possibility of chilling injury (Pareek, Yahia, Pareek and Kaushik, 2011). Thus, this fruit
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is kept at room temperature after harvested. The effects of these parameters on the L-
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AA/DHAA balance are unknown.
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< place Fig. 2 near here >
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Although the degradation ratio is similar in all horticultural products, the extent of
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losses depended on the characteristic of each sample. Lemons had the lowest DHAA
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ratio found in this study, which may be justified by the low pH, 2.16, of lemon pulp
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which prevents L-AA degradation. Broccoli seem to be particularly prone to vitamin C
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loss during storage: even kept at 4 ºC in dark conditions, we observed a daily loss of
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3.93 % and 4.06% of L-AA and a loss of 1.36% and 1.43% of total vitamin C in local
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and imported broccoli, respectively (Spínola et al., 2012). Watercress was another
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highly perishable vegetable where a very high L-AA daily loss (5.59%) was observed. It
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seems that, for most of the analyzed species, the time of storage is a crucial factor for
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the formation of DHAA at expense of L-AA, the shorter the time between collection
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and use, the more L-AA remains intact (Fig. 2).
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3.2 Stability of L-ascorbic acid in extracts
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Stability is a key problem of L-AA analysis since this compound is very unstable in
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aqueous solution. Temperature has been described as one of the main factors that
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significantly influence the stability of vitamin C in solution (Iwase, 2000; Hernández et
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al., 2006; Nováková et al., 2008; Phillips et al., 2010). Thus, the effect of storage
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temperature (4, -20 and -80 ºC) on the L-AA degradation was studied in a standard
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solution (50 µg/mL in extraction mixture) and selected extracts (Fig. 3). The stability of
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samples at laboratory temperature (23 ºC) was periodically analyzed during 5 hours.
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The results obtained immediately after homogenization were therefore considered as the
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initial content of L-AA in horticultural extracts. The inclusion of standard solution in
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each analytical condition was important to establish that any changes were due to
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stability of L-AA in a particular matrix. It was found that L-AA was stable at room
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temperature after 1 hour with recoveries of 98.6% and 98.1% for standard solution and
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passion fruit extracts, respectively (data not shown). One hour later (2 hours of standing
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time) recoveries remained stable in both samples but continued to decrease thereafter.
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At 5 hours, the decrease of L-AA was 5.9% for standard solution and 6.3% for passion
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fruits extracts. Given these results, the storage of extracts at room temperature is a very
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bad option since even under these acidic conditions and protected from light, L-AA is
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stable only for 2 hours or less. This is similar with the results obtained by Iwase (2000)
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that reported that L-AA solution at laboratory temperature was stable for 1 hour.
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Our results at 4 ºC were in agreement to those reported by Hernández et al. (2006) and
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Davey et al. (2000). L-AA remained stable kept at 4 ºC and protected against daylight
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for at least 24 hours (Fig. 3), with recoveries of 98.2% and 97.8% for standard and
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extract solutions, respectively. However, there was a notable decrease in L-AA content
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for the assayed samples throughout the study. These conditions were not suitable to
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stabilize L-AA for longer time periods.
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< place Fig. 3 near here >
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Degradation study at -20 ºC provided more satisfactory results. These conditions were
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suitable for storage of standard solution and passion fruits extracts for 1 week or less,
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with stability of 97.2% and 96.7% from the initial content of L-AA, respectively (Fig.
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3).
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Loss of L-AA was minimal up to 4 weeks or less at -80 ºC in both samples (< 2%) but
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the degradation continued to accumulate thereafter, as can be seen in Figure 3. These
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results showed that storage at -80 ºC was the most effective condition on preventing L-
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AA degradation. Thus, storage of standard solutions and horticultural extracts at -80 ºC
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for at least one month is acceptable for this kind of analysis. This is in agreement with
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the results described by Hernández et al. (2006). These authors reported that L-AA of
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standard solution or fruit extract is stable during at least one month of storage at -80 ºC.
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Phillips et al. (2010), also found that storage of homogenized samples of clementines,
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collard greens and potatoes at -60 ºC (in darkness under nitrogen) provided excellent
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stabilization of L-AA for 4 weeks. There were no significant differences between
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standard solutions and horticultural extracts for all values measured during the
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degradation studies. However, in all described conditions of storage L-AA losses were
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always higher in the horticultural extracts. This could be related to matrix-specific
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characteristics that are known to affect L-AA stability. The preservation of the samples
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at -80 °C proved to be the most effective method of preservation, showing much lower
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losses in comparison with the other conditions experimented in this study. This is
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consistent with the fact that the decrease of temperature helps preventing L-AA
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oxidation.
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Besides lowering temperature, pH could also play a role in stabilizing L-AA during the
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period of study since this molecule is well preserved in acid solutions. Generally, acidic
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pH (≈ 2) was useful for sample preparations, ensuring sufficient stability and recovery
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of L-AA in extracts. At these conditions, L-AA exhibits higher stability (pH < pKa) and
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the formation of its oxidation products is not favored (Hernández et al., 2006; Nováková
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et al., 2008). Lemons clearly demonstrate this influence: with a pH of 2.16 and an initial
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vitamin C content of 52.07 mg/100 g of which only 1.33% was DHAA, locally
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produced lemons showed a daily loss of only 2.48% for L-AA and 1.75% for total
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vitamin C. We compared them with lemons imported from Spain for which there was
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no information as to date or place of collection (Spínola et al, 2012); these showed a
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higher pH (2.41) as well as slightly higher initial vitamin C content, 57.14 mg/100g of
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which 2.05% was DHAA and average daily losses of 2.55 % for L-AA and 2.04 % for
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total vitamin C. These losses were remarkably low compared to other foodstuffs of
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higher pH, analyzed under the same circumstances. As a tentative study, we plotted
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pulp pH values against daily decrease of L-AA (Fig. 4) occurred in produce stored at 4
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ºC (Spínola et al, 2012), not considering cherimoyas since other degradation factors
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were obvious nor the “leafy” vegetables, broccoli and watercress. Although considered
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“vegetables” due to their dietary use, peppers are, in fact, fruits. There is a very good
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correlation up to pH 5, other parameters becoming more relevant for less acidic pulps.
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< place Fig. 4 near here>
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4. Conclusions
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In the current study we evaluated L-AA contents obtained by two different analytical
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techniques. Overall, good agreements were achieved between the two measurements
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being both methods studied suitable for determining the content of L-AA in a wide
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variety of fruits and vegetables. However, iodometric titration method lacks of
341
specificity and cannot determine DHAA concentrations. So, the titration method may be
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applied for preliminary analysis in laboratories to identify and quantify L-AA. On the
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other hand, UHPLC-PDA analysis affords enough sensitivity and selectivity in total
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vitamin C determination in various horticultural products. This method is free of
345
interferences from others compounds present naturally in samples and delivers results
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within 2 minutes after extraction. Moreover, the methodology applied here to determine
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total vitamin C using DTT, allows the reduction of DHAA effectively and reproducibly.
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Vegetables proved to be better source of vitamin C than the fruits analyzed in this work.
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The results obtained show the importance of determining total vitamin C content, since
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the evaluation of L-AA only leads to an underestimation of nutritional value. Finally,
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storage of acid extracts at -80 ºC was the most effective procedure on stabilizing L-AA
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during long time periods.
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Acknowledgments
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The authors show their gratitude to Sonae MC for supplying the fruits and vegetables
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samples used in this study. This research was supported by Fundação para a Ciência e a
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Tecnologia (FCT) with funds from the Portuguese Government (Project PEst-
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OE/QUI/UI0674/2011).
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References
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AOAC. (2006). AOAC Official Method 967.21 - Ascorbic Acid in Vitamin Preparations and
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Juices: 2,6-Dichloroindophenol Titrimetric Method. Official Methods of Analysis of the
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Association of Official Analytical Chemists.
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Campos, F. M., Ribeiro, S. M. R., Della Lucia, C. M., Pinheiro-Sant'Ana, H. M., & Stringheta,
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P. C. (2009). Optimization of methodology to analyze ascorbic and dehydroascorbic
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Cardoso, P. C., Tomazini, A. P. B., Stringheta, P. C., Ribeiro, S. M. R., & Pinheiro-Sant'Ana, H.
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M. (2010). Vitamin C and carotenoids in organic and conventional fruits grown in
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Brazil. Food Chemistry, 126, 411-416.
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Chebrolu, K. K., Jayaprakasha, G. K., Yoo, K. S., Jifon, J. L., & Patil, B. S. (2012). An
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improved sample preparation method for quantification of ascorbic acid and
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dehydroascorbic acid by HPLC. LWT - Food Science and Technology, 47, 443-449.
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466
Table and Figure Captions
467
Table 1. pH and vitamin C contents of fruits and vegetables determined by iodometric titration
468
(IT) and UHPLC-PDA methods.
19
469
470
Table 2. Overview of L-ascorbic acid (L-AA), total vitamin C (T vitamin C) and (%) DHAA
determinations in fruits and vegetables (mg/100 g edible portion).
471
472
Fig. 1. Reduction of Dehydroascorbic acid (DHAA) to L-ascorbic acid (L-AA) by DTT. In
473
order to determine total vitamin C content in samples, DHAA should be reduced to L-
474
AA with UV-vis detection of the reduced form.
475
Fig. 2. DHAA/L-AA ratio (%) during storage of fruits and vegetables at 4 ºC.
476
DHAA content increased at the expense of L-AA oxidation. PF: passion fruits (--);
477
PA: papayas (-▲-); S: strawberries (--); L: lemons (--); W: watercress (- -- -);
478
B: broccoli (- -- -); GP: green peppers (- -- -); RP: red peppers (- -- -) (data from
479
Spínola et al., 2012).
480
Fig. 3. Changes in L-ascorbic acid (L-AA) content: (A) in standard solutions, (B) passion fruit
481
(PF) extracts in 30 g/L MPA – 80 mL/L acetic acid - 1 mmol/L EDTA, during 8 weeks
482
of storage at 4 ºC (- -- -/- -- -), -20 ºC (- -- -/- -- -) and -80 ºC (- -- -/- - - -).
483
484
Fig. 4. Correlation between L-AA daily loss (%) and pulp pH (not including cherimoyas and
green leafy vegetables).
485
486
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