Analysis of Vitamin C in Fruits and Beverages

ABSTRACT

Vitamin C is one of the most important vitamins and antioxidants found in fruits and beverages. Hence, reliable information about its concentration in these foods should be available for the awareness of all concerned sectors ranging from the food processors, quality control agencies and authorities, up to the consuming population.  However, the complexity of food matrices and the instability of vitamin C during analysis pose a great challenge for accurate quantification and analysis. This review presents the available analytical methods (extraction procedures, tirimetric, enzymatic, spectrophotometric, liquid chromatographic, and capillary electrophoresis techniques) from recent publications focused on vitamin C analysis of fruits and beverages.

INTRODUCTION

Food analysis constitutes a significant area of research in chemistry nowadays. With the increasing variation of food products in the market, availability of accurate methods of analyses becomes an important commitment of the scientific community. Food composition data of new and improved commodities is a continuous need of the industry and consumers for quality control purposes and informed food choices, respectively. Some of the major trends of novel formulation in foods deal with vitamins and antioxidants as beneficial components.

For the past decade, the nutritional quality of food had been one of the major concerns of consumers and food manufacturers. Vitamin C became one of the most investigated vitamins due to its oxidation and loss during processing and storage (Serrano et al., 2007). Its labile nature made it an important quality indicator of fruits and beverages, which are important and common sources of vitamin C in the diet.

Vitamin C is the generic descriptor for all compounds exhibiting equivalent biological activity of L-ascorbic acid (L-AA), including its oxidation products, esters, and synthetic forms (Spinola et al., 2012). L-AA is the biologically active isomer of vitamin C and is rapidly and reversibly oxidized to dehyroascorbic acid (DHAA) due to the presence of two hydroxyl groups in its structure (Novakova et al., 2008). Further oxidation originates diketogulonic acid, which has no biological function, and proceeds an irreversible reaction.

The content of vitamin C in foodstuffs is reported as the sum of the contents of L-AA and DHAA (Mazurek and Jamroz, 2015). Being used as health-related quality index of fruits and beverages and considering its sensitivity to degradation along with the heterogeneity of food matrices, efforts to simultaneously quantify L-AA and DHAA had emerged as a sustained interest in food analysis. In addressing this analytical challenge, emphasis should be paid to the development of accurate analytical methods for vitamin C quantification.

This review focuses on the current and existing analytical methods for the analysis of vitamin C in fruits and beverages. The data for this review were gathered from scientific works recently published employing tirimetry, enzymatic, spectrophotometry, liquid chromatography, and capillary electrophoresis as techniques in the analysis of vitamin C in fruits and beverages. Also, several extraction techniques were discussed since this procedure can contribute as possible source of errors in vitamin C analysis.

Extraction procedures

In this section, we summarize the different extraction methods in preparation for Vitamin C analysis. Extraction is the pre-treatment process before it reaches analysis. According to the book Bioavailability and Analysis of Vitamins in Food, the appropriate extraction method depends upon the following criteria: (1) the analytical information required; (2) the nature of the food matrix; (3) the form in which the vitamin occurs naturally or is added (this is particularly because some vitamins are bound with other nutrients or food constituents such as carbohydrates and proteins); (4) the nature and relative amounts of potentially interfering substances; (5) the stability of the vitamin towards heat and extremes of pH; (6) the labile nature of the vitamins. These criteria should be considered so that the analyte can be separated from the food without loss or degradation in its quality and yield

Nojavan et al. (2008) investigated two different sample preparation steps, i. e. freezing and mild-temperature drying before the determination of ascorbic acid content. In freezing procedure, about 100 g of each fully ripe, half-ripe, and unripe dog rose and orange samples were separately frozen into liquid nitrogen and mixed with 5mL of the extractant solution containing metaphosphoric acid (MPA). In this method the weight of frozen pulverized sample were optimized and the best amounts were obtained based on extraction recovery. They have studied the weight of different samples over the 0.1–3g and the suitable amount for each sample was obtained. Different concentrations of metaphosphoric acid were evaluated and a concentration of 5% was chosen. After homogenization, the time for centrifuge was studied between 1 and 20 min. According to the results, from the point of extraction recovery, the best time was 10 min. In mild-temperature drying, a modified procedure on the method by Nojavan et al. (2008), about 100g of dog rose samples in each maturity stage were separately weighed and dried at about 15–20 °C and ground to fine powder before extraction. The obtained powder was extracted with 25 mL of extractant solution at 10 °C, containing metaphosphoric acid. In this method, concentration of MPA in extractant solution and time of extraction were optimized. The concentration chosen for metaphosporic acid was also 5% for standardization. The extraction time was studied over the 0.5–10 h and the 4 h was found optimum. These parameters were selected from the point of extraction recovery. In conclusion, it was found that freezing sample preparation method is more effective than mild-temparature drying procedure when it comes to preparation for extraction because it prevents oxidation and degradation of ascorbic acid and affords enough sensitivity and selectivity in extraction of ascorbic acid in dog rose (at three maturity stages) and orange sample. Although there are different extraction procedures, in general, they adhere to the same basic principles that is to prevent vitamin degradation and loss (Acar et al., 2003). Acids are the most commonly used extraction reagents because it protects the vitamin C from oxidation and hydrolysis and precipitate proteins. One of the most common reducing agents are metaphosphoric and oxalic acid. These reducing agents can be used alone or in combination with other acids or short-chain alcohols, such as methanol or ethanol. Of the commonly used acids, metaphosphoric acid has been reported to be the most effective for plant products in general because it prevents oxidation compared to other acids (Majidi et al., 2016). However, it may cause serious analytical interactions with silica-based column materials such as RP-C₁₈ or NH₂ bonded-phases which can result in drifts in the retention time and baseline. On the other hand, oxalic acid has also been reported as an effective reducing agent and it is said that, it is less toxic than metaphosphoric acid and it is cheaper. However, sometimes it does not utilize all the ascorbic acid present in the sample and the extracts are less stable compared with metaphosphoric acid. A metal chelator such as EDTA is also usually required (Hernandez et al., 2006).

Titrimetric techniques

Vitamin C analysis must be performed at the presence of a chelating agent because it can be easily oxidized, especially at high pH conditions so it is necessary to conduct the analysis at alkaline conditions. One of the most common methods used for vitamin c analysis is titrimetry. Direct titration method using iodine as a titrant is inexpensive, simple, reliable and rugged compared with other methods. In iodine titration method, analysis of vitamin C can be possibly performed without extraction (Suntornsuk et al., 2001). However, some titration methods like 2,6-dichloroindophenol (DCIP) titration of ascorbic acid with in acidic solution is not applicable in some of the food matrices due to naturally occurring components in fruits such as tannins, sulfhydril compounds, betanins, Cu(II), Fe(II), Mn(II) and Co(II) as these are oxidized by the dye but, it can be used for fresh juices and multivitamins that do not contain excessive amounts of copper or iron. For ready to eat or processed foods known to be containing copper, iron, or tin, other methods capable of measuring dehydroascorbic acid in addition to l-ascorbic acid should be used to quantitate total vitamin C. Furthermore, the method is applicable only when the concentration of DHA is low (Hernandez et al., 2006)

According to the study of Shrestra et al. (2015 Two types of titration methods were performed: Iodine Titration Method, and Indophenol (DCPIP) Titration method. Six fruit samples were analyzed: lemon, bitter orange, pomelo, sweet orange, grapefruit, and citron. Hence, the average concentration of ascorbic acid calculated from different titration methods are found to be maximum in pomelo whereas minimum in citron among six citrus fruits. The results of iodine titration and dye titration were found to be close to each other. The concentration of ascorbic acid really depends on the variety of the fruit and also depends from one method to another method of analysis. Table 1 presents the recent studies on the use of titrimetry in the determination of vitamin C.

Table 1. Titrimetric analysis of Vitamin C in various samples

Analyte	Sample	Sample Preparation	Reference
AA	Fruit juices	Add 10 ml of 1M H2SO4 was added; Titrated with a standard iodine solution	Shrestra et al., 2015
AA	Fruit juices	Add 3%MPA solution with filtration; 2ml HPO3-HOAC titrated with indophenol dye	Shrestra et al., 2015
AA	Fruits and vegetables	Add 10% KI with 1ml of 0.3 M H2SO4 and 10ml of 0.01M KIO3; Titrated against 0.01M Na2S2O3	Majidi et al., 2016
AA	Dietary supplements	Add 1g of KI, 10ml of 0.2 M H2SO4 and 0.1g NaHCO3; Titrate excess I3 with Na2S2O3 solution	Spring, 2016
AA	Tropical fruits	2ml of 3%MPA with 8% acetic acid; Titrate  with indophenol solution	Hernandez et al., 2006

Table 1. Titrimetric analysis of Vitamin C in various samples (continued)

Analyte	Sample	Sample Preparation	Reference
DHA	Sauerkraut, blackcurrant and orange juice	Add 10ml MPA; Titrate with indophenol dye solution	Danielczuk et al., 2004

Enzymatic methods

L-ascorbic acid enzyme conversion to l-dehydroascorbic acid coupled to a determinative step such as direct spectrophotometric assay following decrease of l-ascorbic acid, optical path difference (OPD) other derivatization reactions, and electrochemical determination of oxygen uptake during the reaction have been used to assay l-ascorbic acid in biological samples.Equations 1 and equations 2 shows the ascorbate oxidase and ascorbate peroxidase activity in converting the l-ascorbic acid to dehydro form

l-Ascorbic acid + 1 /2 O₂ → l-Dehydroascorbic acid + H₂O             Equation 1

l-Ascorbic acid + H₂O₂ → l-Dehydroascorbic acid + 2H₂O            Equation 2

Several sources of enzymes have been used for the enzymatic conversion. An ascorbate oxidase spatula (Boehringer–Mannheim) to convert l-ascorbic acid and isoascorbic acid to the dehydro forms before OPD derivatization and LC quantitation of the quinoxaline derivatives. It has been extensively used for quantitation of total vitamin C in foods. Amounting levels as low as 0.2 μg g−1 where vitamin C total and isoascorbic acid is quantitated wherein l-Dehydroascorbic acid can be quantified by omitting the enzymatic oxidation. Ihara et al., (2004) used ascorbate oxidase and OPD derivatization to develop a rapid automated method for the routine assay of l-ascorbic acid in serum and plasma. The assay had a sample throughput of 100 h⁻¹. The application of ascorbate peroxidase oxidation of l-ascorbic acid to dehydroascorbic acid has been used to spectrophotometric assay of total vitamin C in foods. But, quiacol peroxidases that are commercially available in Sigma Chemical Co. from horseradish have been used in some recent methods and catalyze the oxidation of l-ascorbic acid as well as quiacol. Tsumura et al., (1993) developed direct spectrophotometric assay was tested on a various number of foods and no interferences were apparent. It is said to be that this method was sharply exact as compared to assays using DCIP and DNPH (Eitenmiller et al., 2008).

Spectrophotometric techniques

Spectrophotometry is the product of modernization technique in determining various contents within the product. Wang et al., (2015) pointed out that among other technologies, spectroscopic technique have drawn great attention for their prominent advantages: (1) they are nondestructive methods which enable the acquisition of fruits’ internal quality parameters without damaging their surfaces; (2) the measurement processes are simple and rapid, as no complex pretreatments or chemical reactions on fruits samples are needed; (3) they enable the detection of several fruit internal attributes simultaneously. As a disadvantage, however, the small point-source measurements which are commonly used in spectral assessment cannot provide spatial information, which is important in many fruit evaluation instances.

Measurement of Vitamin C in some consumer goods is very important for substantiating nutritional claims and this can be conveniently accomplish by spectrophotometric measurement of L-AA. Khan et al., (2006) stated that all the parts of a fruit and all fruits have not equal amounts of edible part, in the comparative study of the vitamin C contents in various fruits, the percent of edible parts of those must also be considered. Fruits such as orange and lemon  contain high amount of vitamin C (Lidija et al., 2003; Alam. 1996). Fruit juices may be analysed directly without any preparation. However, it is recommended that samples containing suspended matter are centrifuge or filtered first, to avoid pipetting difficulties.

According to the study of Adebayo (2015), high amount of ascorbic acid were obtained from spectrophotometric methods in all the selected fruits when compared with the titrimetric method. This shows that this method is more sensitive for the determination of ascorbic acid contents of fruits. The disparity could be attributed to poor detection of end point or the presence of substance that may interfere with the reagent in the titrimetric method as suggested by Okei et al.,(2009).

The study of Mussa et al., (2014) employed determination of total Vitamin C content of fruit juice samples using 2,4-DNPH as the derivatizing agent followed by spectrophotometric detection. This is a simplified and established method as stated by Qasi Mohammed et al., (2009). Results revealed that the declared vitamin C content is not significantly different with that obtained from direct analysis.

Revanasiddappa et al., (2007) also added that this method was simple selective and offer the advantage of sensitivity without the need for extraction or heating. The assay methods do not involve any stringent reaction conditions, and noninterference from associated substances in the dosage forms and real samples. The reliability of this method is justified by the calculation of the % of standard deviations and it was found to be varied within the range from 0.29 to 1.98% and also confirmed from the consideration of the following expected interference as stated by Mohammed et al., (2009).

Comparing the values of vitamin C content with that labeled on the packed fruit juices that are locally available that are natural source of vitamin C by Mussa et al., (2014) agreed with the international sample; and good alternative to some reported costly instrumental method and its its advantages were mainly due to its cheaper cost. Easier availability and higher stability of colour of auramine O dye (Janghel et al., 2012). Recent studies on the use of Spectrophotometric determination of Vitamin C in fruits and beverages are summarized in Table 2.

Table 2. Spectrophotometric Determination of Vitamin C in Fruits and Beverages

Analyte	Sample	Instrument Condition and Reagents	References
AA	Fruits, Biological Sample and Pharmaceuticals	Systronic UV-Vis Spectrophotometer 108 with 2 cm matched silica cells; Stock solution: Loba Chemie; Working Standard Soution: Potassium Iodide;LMG: 0.05% solution;pH 4.5	Tiwari, 2010
AA	Fruits and Vegeatable	Shimadzu Spectrophotometer (model UV-1601) with a pair 1 cm quarts cell; 5%Metaphosphoric acid-10% acetic acid; 10% Thiourea solutio;2-4,denitrophynel-hydrazine solution; 85% sulphuric acid	Khan et al., 2006
AA	Fruits, Beverages and Pharmaceuticals	Systronic VIS-spectrophotometer type-106 matched with a 1-cm quartz cell;dissolving a known amount of CuSO4 in 0.01 M H2SO4;0.2%, w/v N-phenylbenzimidoylthiourea (PBITU);pH 8.0	Shrivas et al., 2005
AA	Fruits and Vegeatable	Thermo Electronic Cooperation spectrophotometer (Model GESEYS 10uv) with 1 cm cell;0.05g standard crystalline ascorbic acid;- 10% Acetic acid,10% ThioUrea,2,4- Dinitrophenyl Hydrazine,85% Sulphuric acid,Bromine water	Mohammed et al., 2009
AA	Fruit Juices, Foods and Tablets	PerkinElmer (Lambda25) spectrophotometer and a 1.0 cm glass cell;stock solution of AgNO3 (0.01 mol L−1);Polyvinylpyrrolidone (PVP) (0.4 g L−1) solution;Robinson buffer solution (0.04 mol L−1)	Zarei et al., 2015

Table 2. Spectrophotometric Determination of Vitamin C in Fruits and Beverages (continued)

Analyte	Sample	Instrument Condition and Reagents	References
AA	Fruit Juices	Double beam UV-Visible spectrophotometer (Model GESEYS 10uv) with 1 cm cell;5% Starch Solution, Sodium Carbonate, (0.04 N) Standard Potassium Iodate Solution, (0.03 N) Sodium Thiosulfate Solution, 10% KI Solution, 0.2 M Sulphuric acid solution, Sodium Carbonate, 3% Acetic acid,10% Thiourea, 2,4- Dinitrophenyl Hydrazine, 85% Sulphuric acid and Bromine water	Mussa et al., 2014

Chromatographic techniques

Despite its limitations, classical methods are still common in food analysis. AOAC Official titration method (967.21/2006) is routinely applied for the analysis of fruits and juices due to its simplicity and low cost (Serrano et al., 2007). However, more accurate analytical techniques for vitamin C determination were developed and validated to simultaneously quantify and differentiate L-AA, DHAA, and other L-AA-related compounds. Separation techniques, including high-pressure liquid chromatographic (HPLC) methods, had demonstrated several advantages in terms of specificity, sensitivity, separation capability, and short-analysis time (Spinola et al., 2014). Table 2 presents different validated HPLC methods for vitamin C determination in fruits and beverages, which were published between 2006 and 2014.

Reversed-phase (RP)-HPLC, based on the available literatures, is one of the most common chromatographic techniques being used because of the non-volatile and hydrophilic nature of vitamin C (Eitenmiller et al., 2008). However, several drawbacks were noted for RP procedures including poor resolution of AA and the dead retention volume; and the low pH required by the method favoring accelerated degradation of silica-based analytical column due to the dissolution of the base silica material. To prevent degradation, the pH of the mobile phase is usually adjusted below L-AA’s pKa of 4.17. Measures to address these drawbacks were discussed in details by Novakova et al (2008). Ion exchange, ion-pair, ion-exclusion, and hydrophilic interaction liquid chromatography (HILIC) were also employed in some of the published works gathered (Table 3).

Recent studies have made use of ultra-high performance liquid chromatography (UHPLC) for vitamin C analysis in foodstuffs. Spinola and his colleagues in 2012 were able to validate a rapid, sensitive, and reproducible UHPLC-PDA-based methodology for the analysis of vitamin C in fruits and vegetables. Upon validation, the combination of shorter running time with smaller flow rate was identified as a key factor for much lower solvent consumption as compared to other chromatographic techniques.

Most HPLC methods employ UV detection of L-AA at wavelengths between 245 nm and 254 nm, although few articles had reported fluorescence detection (FD) and electrochemical detection (ECD) as well as mass spectrometry (MS) (Russel, 2000; Fenoll et al., 2011). Both FD (LOD=0.27 µg/mL) and ECD (LOD=0.02 -0.16 µg/mL) are considered more sensitive than UV (LOD=1.2•10^-3). But the wide LOD ranges of ECD render the method difficult particularly for accurate comparison while FD requires a laborious derivatization procedure compared to UV (Spinola et al., 2014).

DHAA exhibits low absorptivity in L-AA’s range of the spectrum posing a complicated analytical problem particularly in the simultaneous detection of L-AA and DHAA. To resolve this issue and increase the sensitivity of the method to DHAA, derivatization is performed before and after chromatographic separation (Spinola et al., 2012). These steps allow quantification of DHAA by obtaining the difference between the total L-AA after DHAA reduction and L-AA content of the original sample (Hernandez et al., 2006). Various thiol-containing compounds like dithiotreitol (DTT), dimercaptopropanol (BAL), and homocysteine, are popularly employed as reducing agents (Serrano et al., 2007). The efficiency, therefore, of DHAA’s reduction to L-AA is also influenced by the choice of reducing agent. Nyyssonen et al. (2000) noted that homocysteine does not affect the L-AA peak when supported with amino columns, but interferes with the separation when used with ODS columns. In addition, UV-HPLC-based determination of vitamin C in strawberries, tomatoes, and apples by Serrano et al. (2007) revealed DTT gave higher recovery rates than BAL when used with a C18 column.

Crucial to the accuracy of results of HPLC analysis is the efficiency of extraction conducted on the sample. This is very important especially when analyzing complex food matrices such as fruits, which generally contain large amounts of compounds with potentials to interfere with the identification and quantification of L-AA. Moreover, degradative enzymes have to be inactivated since these can cause destruction of AA during the extraction procedure and disruption of L-AA/DHAA redox equilibrium.

Extraction methods may also be varied among different fruits due to the differences in the matrix brought about by plant type and cultivar, maturity stage, environmental and cultural practices, and post-harvest conditions (Lee et al., 2000). For these reasons, Davey et al (2000) reported that caution should be noted in applying the methods that have been previously developed for the analysis of specific plant tissue types. Since alkaline conditions favor oxidation of L-AA, a high-ionic strength and acidic extraction solvent will be necessary for immediate precipitation of proteins and suppression of metabolic activity upon cell disruption (Hernandez et al., 2006). Most compatible extraction solvents compatible with HPLC supports and mobile phases are metaphosphoric acid, mixtures of metaphosphoric acid with glacial acetic acid, trichloroacetic acid, citric acid, mixtures of citric acid and glacial acetic acid, sulfuric acid, and phosphoric acid (Eitenmiller et al., 2008).

Metaphosphoric acid may provide efficient AA extraction by preventing oxidation compared to other acids. On the other hand, when employed with silica-based column materials, analytical interactions were noted such as baseline and retention time drifts as identified by Kall and Andersen (1999). Oxalic acid, moreover, when used in its usual concentrations is basically cheaper and less toxic than metaphosphoric acid. But it is usually associated with less recovery of total L-AA present in the sample and less stability of extracts as compared to L-AA extracts of metaphosphoric acid (Spinola et al., 2012). Results of several published works recognize the influence of the extraction solution in the selectivity of the method depending on its interaction with the mobile phase. Campos et al. (2009) was able to obtain an increasingly selective method by modifying MPA concentration (from 4.5% to 3%), changing the pH of the mobile phase (from 2.2 to 3.0), and using O-phosphoric acid instead of MPA. But the modifications led to poorer resolution of L-AA peak, which directed the team to try a new mixture of extracting solution. A mixture containing MPA, acetic acid, sulfuric acid, and EDTA was then used that resulted to the same extraction efficiency but improved peak resolution. Hernandez et al. (2006) employed the same set of extracting solution and mobile phase and satisfactory results were observed, although differences among varied matrices were identified. Hence, existing publications are of valuable help to other authors in terms of matrix-specific method validation or even minor modifications to chromatographic support and conditions.

TABLE 3. Overview of validated chromatographic methods for vitamin C determination in fruits and beverages.
Analyte	Sample	Sample Preparation	HPLC Conditions	Reference
L-AA, DHAA, TAA   (reduction with DTT)	Tropical fruits	SLE with 3% MPA – 8% acetic acid; centrifugation	Shodex RSpak KC-811   (250 x 4.6 mm, 5 µm) 0.2% o-phosphoric acid (UV 245 nm)	Hernandez et al., 2006.
L-AA, vitamin A	Banana and papaya	SLE (3% MPA – 8% CH3COOH- 1 mM EDTA); centrifugation; clean-up (C18 cartridges)	PLRP-S column   (250 x 2.1 mm, 5 µm) 0.2 M NaH2PO4 (pH 2.14)	Wall, 2006.

TABLE 3. Overview of validated chromatographic methods for vitamin C determination in fruits and beverages (continued)

Analyte	Sample	Sample Preparation	HPLC Conditions	Reference
TAA, iso-AA   (reduction with TCEP)	Fortified products; infant formula; cereals; soup; juice	Dilution with mobile phase;   Filtration	Ion-pair LiChrospher RP-18   (250 x 4.6 mm, 5 µm) CAN, sodium acetate eluent (pH 5.4), TCEP, and decylamine DAD (265 nm)	Fontannaz et al., 2006.
L-AA, DHAA, TAA   (reduction with DTT or BAL)	Fruits	SLE with 4.5% MPA;   Centrifugation and filtration	a) C18 Spherisorb ODS2   (250 x 4.6 mm, 5 µm), 0.01% H2SO4 (pH 2.6) b) NH2 Spherisorb S5 (250 x 4.6 mm, 5 µm), 10mM KH2PO4 buffer (pH 3.5); CAN (60:40) UV (245 nm)	Serrano et al., 2007.
L-AA, iso-L-AA, L-AA-2G, L-AA-2ßG	Teas and dried fruits	a) Dilution with mobile phase   b) USLE; centrifugation	HILIC Interstil Diol   (250 x 4.6 mm, 5 µm) CAN: 66.7 mM CH3COONH4 (85:15) UV (260 nm)	Tai et al., 2007.
AA, DHAA, TAA   (reduction with TCEP)	Grapevines   (berries, rachis, leaves, roots)	SLE with 3% MPA – 1 mM EDTA;   centrifugation	Synergi Fusion   (150 x 4.6 mm, 4 µm) A: 25 mM KH2PO4 – 0.1 mM EDTA (pH 2.5); B: 100% MeOH (gradient elution) UV (245 nm)	Melino et al., 2009.
L-AA, iso-AA	Fruit juices	Dilution with 6.25% MPA – 2.5mM EDTA	TSKgel Amide-80   (4.6 x 100 mm, 5 µm) ACN: 0.1% TFA (90:10) UV (244 nm)	Barros et al., 2010.
L-AA, DHAA	Fruits and vegetables	SLE with 0.05 EDTA; centrifugation; clean-up with C18 cartridges	Prontosil C18 (250x 3 mm, 3µm)   0.2% (v/v)  formic acid ESI-MS	Fenoll et al., 2011.
L-AA, DHAA, TAA, carotenoids	Fruits	SLE with 3% MPA-8% acetic acid, 0.15 M H2SO4-1 mM EDTA; filtration and centrifugation	Lichrosper 100 RP18   (250 x 4mm, 5 µm) 1 mM NaH2PO4 – 1 mM EDTA (pH 3.0) UV245 nm	Cardoso et al., 2011.

TABLE 3. Overview of validated chromatographic methods for vitamin C determination in fruits and beverages (continued)

Analyte	Sample	Sample Preparation	HPLC Conditions	Reference
L-AA and other organic acids	Fruit juices	Dilution with mobile phase	RP-C18   (150 x 4.6 mm, 3 µm) 0.01 M KH2PO4 buffersolution (pH 2.60) DAD (250 nm)	Scherer et al., 2012.
L-AA, DHAA, TAA   (reduction with TCEP, BME, or DTT)	Fruits and vegetables	SLE with MPA (3 g/100 mL);   centrifugation	Spherisorb C18   (150 mm x 4.6 mm, 3 µm) 0.01 M dihydrogen ammonium phosphate (pH 2.6) PDA (254 nm); ESI-MS	Chebrolu et al., 2012.
L-AA	Beverages	MEPS with methanol-water solution (10:90, v/v)	Lichrospher_100 RP-18e   (250 x 4 mm, 5 µm) A: water acidified with acetic acid (pH 2.94); B: MeOH (80:20) UV: 265 nm	Adam et al., 2012.
L-AA, DHAA, TAA   (reduction with DTT)	Fruits and vegetables	SLE with 3% MPA-8% acetic acid- 1mM EDTA; centrifugation	Acquity HSS T3   (100 x 2.1 mm, 1.8 µm) 0.1% formic acid in water (v/v) PDA (245nm)	Spinola et al., 2012.
L-AA, TAA   (reduction with DTT)	Strawberries	USLE with 3% MPA-8% acetic acid; centrifugation	Phenomenex C18   (250 x 3 mm, 5 µm) 0.03 M sodium acetate/ acetic acid buffer, 5% MeOH UV (251 nm)	Van de Velde et al., 2012.
L-AA	Grapes	USLE with 96% acetic acid; USLE with 2% MPA; centrifugation	Kromasil C18   (100 x 2.1 mm, 3.5 µm) 0.1% (v/v) acetic acid + MeOH UV (245 nm) ESI-MS	Matei, et al., 2013.
L-AA and organic acids	Citrines juices	Dilution with H2O;   filtration and centrifugation	ZirChrom-SAX   (150 x 4.6 mm; 5 µm) 50 mM ammonium phosphate (pH 5.8) PDA (254 nm)	Carballo et al., 2014.

TABLE 3. Overview of validated chromatographic methods for vitamin C determination in fruits and beverages (continued)

Analyte	Sample	Sample Preparation	HPLC Conditions	Reference
L-AA, TAA   (reduction with TCEP)	Exotic fruits, juices, and fruits’ pulp	Dilution with 10% PCA- 1% MPA + TCEP; filtration	Synergi Hydro-RP   (150 x 4.6 mm, 4 µm) 20 mM NH4H2PO4 (pH 3.5) + 0.015% (w/v) MPA PDA (246 nm)	Valente et al., 2014.

L-AA= L-ascorbic acid; DHAA= dehydroascorbic acid; ACN=acetonitrile; SLE – solid-liquid extraction; USLE=ultrasound assisted solid-liquid extraction; TFA=trifluoroacetic 4 acid; PCA=perchloric acid; TCEP=Tris-[2-carboxyethyl] phosphine hydrochloride (TCEP·HCl); CTAB= hexadecyltrimethylammonium bromide; All metaphosphoric acid (MPA) solutions are expressed in % w:v; they are all prepared in H₂O.

Capillary electrophoresis technique

Capillary electrophoresis (CE) is a separation technique which utilizes capillary tubing, high electric field strengths, and detector such as electropherogram and UV-Vis maintained at specific pH. Though relatively new as compared to the chromatographic methods, the use of CE had been attractive due to wide range of analytes that it can be applied to, small sample size requirement, high separation efficiency, rapid separation speed, and short analysis time. It is also advantageous over chromatography as the derivatization step need not be carried out (Boyce, 2007; Liu, Ding, & Tang, 2014).

CE can be employed in different modes of operation which includes Capillary Zone Electrophoresis (CZE), Micellar Electrokinetic Chromatography (MEKC), Capillary Electrochromatography (CEC), Capillary Gel Electrophoresis (CGE), Capillary Isoelectric Focusing (CIEF), and  Capillary Isotachophoresis (CITP) (Laurer & Rozing, 2010) and its use in the determination of fat-soluble and water soluble vitamins had been extensively studied not only in pharmaceutical preparations (Hu, Zhou, Zzhang, Li, & Fang, 2001; Yin, Cao, Ding, Wang, 2008; Suntornsuk, 2007; Franco, Jasionowska, & Salvatore, 2012) but also in food samples and food preparations (Cheung, Hughes, Marriott & Small, 2009; Liu et al., 2014; Cataldi, Nardiello, Carrara, Ciriello, De Benedetto, 2003).

Considerations in the CE and separation conditions such as pH, column, voltage and detectors, are important and need to be optimized to achieve effective determination. These conditions vary from sample to sample, thus, validation of the method must be done to cover as much food matrix as possible. In a study by Schreiner, Razzazi and Luf (2003) for the determination of water-soluble vitamins (B-vitamins) in softdrinks and vitamin supplements, optimum separation conditions for all the components are achieved at 50 mM borate buffer containing 25 mM SDS at pH 8.5 with applied voltage of 30 kV and detection was carried out in 214 nm using UV detector. The relative standard deviations of the method ranged from 1. .08 to 3.68% (intra-day precision) and 1.26 to 3.35% (inter-day precision). In another study by Franco et al. (2012), optimum experimental conditions for B-complex vitamins using CE were pH 9.2 with 20 mM tetraborate buffer, 20 kV applied voltage and  detection was carried out using diode array at 214 nm for all vitamins except B₅ (190 nm) and B_2a (260 nm). Recent studies on the use of CE in the determination of Vitamin C in fruits and beverages are summarized in Table 4.

Table 4. Analysis of Vitamin C by Capillary Electrophoresis

Analyte	Sample	CE Conditions	Reference
AA	Beverages	CZE   Phosphate buffer (7.5 mM NaH2PO4 and 2.5 mM Na2HPO4), 2.5 mM TTAOH as electroosmotic flow modifier and 0.24 mM CaCl2 as selectivity modifier, pH 6.40, fused-silica capillary: 60 cm x 75 µm, -25 kV, 25 A, direct UV-absorption at 185 nm	Mato et al., 2006
L-AA, D-IAA	Fruit juice	CZE   Buffer: 50 mM tricine, pH 8.8, fused-silica capillary: 50 cm x 75 µm,  +11kV	Versari et al., 2004
AA	Citrus juice	CZE   Buffer: 35mM sodium borate containing 5% (v/v) acetonitrile, pH 9.3, fused-silica capillary: 70 cm x 50 µm, +21 kV, Detection: high speed scanning between 200 and 360 nm with electrophrogram at 270 nm	Cancalon, 2001
AA	Energy drink, sports drink and fruit nectar	MEKC   Buffer: 40 mM borate, pH 8.5, fused-silica capillary: 50 cm x 75 µm, +20 kV, diode array spectrophotometric detector at 265 nm	Navarro-Pascual-Ahuir et al., 2016
AA	Soft drinks	CZE   histidine/tartrate buffer at pH 6.5, containing 0.025% HP-beta-CD and 0.1 mM CTAB, fused-silica capillary: 60 cm x 50 m, -15kV	Law et al., 2005

AA=ascorbic acid; L-AA=L-ascorbic acid; D-IAA=D-isoascorbic acid

The variations in the separation conditions and preparations in using the capillary electrophoresis technique make it difficult to establish standardized method. Thus, a study by Spudeit et al. (2016) will aid in the development of the capillary electrophoresis method.

CONCLUSION

The complexity in the sample preparation, the amount of reagents required, and the instrumentation used in the determination of vitamin C bring about new methodologies. With the several methods presented, careful considerations in choosing the appropriate technique in the determination of vitamin C in fruits and beverages must be done. Official methods had been published and are available but the development of the new methods must be validated and verified vis a vis these official methods without sacrificing the accuracy and precision of the analyte being quantified.

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