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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: castilho@uma.pt (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
318
pH (≈ 2) was useful for sample preparations, ensuring sufficient stability and recovery
319
of L-AA in extracts. At these conditions, L-AA exhibits higher stability (pH < pKa) and
320
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
326
higher pH (2.41) as well as slightly higher initial vitamin C content, 57.14 mg/100g of
327
which 2.05% was DHAA and average daily losses of 2.55 % for L-AA and 2.04 % for
328
total vitamin C. These losses were remarkably low compared to other foodstuffs of
329
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
332
were obvious nor the “leafy” vegetables, broccoli and watercress. Although considered
333
“vegetables” due to their dietary use, peppers are, in fact, fruits. There is a very good
334
correlation up to pH 5, other parameters becoming more relevant for less acidic pulps.
335
< 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
338
techniques. Overall, good agreements were achieved between the two measurements
339
being both methods studied suitable for determining the content of L-AA in a wide
14
340
variety of fruits and vegetables. However, iodometric titration method lacks of
341
specificity and cannot determine DHAA concentrations. So, the titration method may be
342
applied for preliminary analysis in laboratories to identify and quantify L-AA. On the
343
other hand, UHPLC-PDA analysis affords enough sensitivity and selectivity in total
344
vitamin C determination in various horticultural products. This method is free of
345
interferences from others compounds present naturally in samples and delivers results
346
within 2 minutes after extraction. Moreover, the methodology applied here to determine
347
total vitamin C using DTT, allows the reduction of DHAA effectively and reproducibly.
348
Vegetables proved to be better source of vitamin C than the fruits analyzed in this work.
349
The results obtained show the importance of determining total vitamin C content, since
350
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
352
during long time periods.
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354
Acknowledgments
355
The authors show their gratitude to Sonae MC for supplying the fruits and vegetables
356
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).
359
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References
361
AOAC. (2006). AOAC Official Method 967.21 - Ascorbic Acid in Vitamin Preparations and
362
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Table and Figure Captions
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Table 1. pH and vitamin C contents of fruits and vegetables determined by iodometric titration
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(IT) and UHPLC-PDA methods.
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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).
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Fig. 1. Reduction of Dehydroascorbic acid (DHAA) to L-ascorbic acid (L-AA) by DTT. In
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order to determine total vitamin C content in samples, DHAA should be reduced to L-
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AA with UV-vis detection of the reduced form.
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Fig. 2. DHAA/L-AA ratio (%) during storage of fruits and vegetables at 4 ºC.
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DHAA content increased at the expense of L-AA oxidation. PF: passion fruits (--);
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PA: papayas (-▲-); S: strawberries (--); L: lemons (--); W: watercress (- -- -);
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B: broccoli (- -- -); GP: green peppers (- -- -); RP: red peppers (- -- -) (data from
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Spínola et al., 2012).
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Fig. 3. Changes in L-ascorbic acid (L-AA) content: (A) in standard solutions, (B) passion fruit
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(PF) extracts in 30 g/L MPA – 80 mL/L acetic acid - 1 mmol/L EDTA, during 8 weeks
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of storage at 4 ºC (- -- -/- -- -), -20 ºC (- -- -/- -- -) and -80 ºC (- -- -/- - - -).
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Fig. 4. Correlation between L-AA daily loss (%) and pulp pH (not including cherimoyas and
green leafy vegetables).
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