Original Research Article

Matrix-specific method validation for quantitative analysis of vitamin C indiverse foods

Maria Teresa Tarrago-Trani, Katherine M. Phillips *, Marlyn Cotty

Department of Biochemistry (0308), 304 Engel Hall, Virginia Tech, Blacksburg, VA 24061, USA

1. Introduction

Vitamin C (L-ascorbic acid, 2,3-enediol-L-gulonic acid-g-lac-tone) is an essential water-soluble vitamin that is a cofactor innumerous physiological reactions in central biochemical process-es, including post-translational hydroxylation of proline and lysinein collagen and other connective tissue proteins; collagen geneexpression; synthesis of norepinephrine and adrenal hormones;

activation of many peptide hormones; and synthesis of carnitine(Davies et al., 1991; Arrigoni and De Tullio, 2000; Johnston et al.,2007; Smirnoff, 2000). Besides these key functions, ascorbic acid(AA) also acts as a cellular antioxidant, and facilitates intestinalabsorption of iron and maintenance of plasma iron in reduced form(Bender, 2003; Smirnoff, 2000). The current Recommended DailyAllowance (RDA) for vitamin C is 25 mg/day for children 4–8 yearsold, 75 mg/day for adult females, and 90 mg/day for adult men(Food and Nutrition Board, Institute of Medicine, 2000; Levineet al., 1995). However, intakes substantially higher than the RDAhave been investigated for a variety of health benefits (Christenet al., 2010; Hemila, 2011; Honarbakhsh and Schachter, 2009;

Journal of Food Composition and Analysis 26 (2012) 12–25

A R T I C L E I N F O

Article history:Received 23 January 2012Received in revised form 14 March 2012Accepted 15 March 2012

Keywords:Vitamin CTotal ascorbic acidDehydroascorbic acidFruits, VegetablesSpicesCerealMethod validationQuality controlHigh performance liquid chromatography(HPLC)PotatoesOrange juiceOrangesCollardsGreensParsleyOreganoCauliflowerZucchini squashCantaloupeCornGreen onionsGreen beansBroccoliGreen pepperFood analysisFood composition

A B S T R A C T

Vitamin C, assayed as total ascorbic acid (AA), was extracted from foods using HPLC with ultravioletspectrophotometric detection, including treatment of the extract tris(2-carboxyethyl) phosphine (TCEP) toreduce any dehydroascorbic acid to AA. The method was validated for a variety of matrices including fruitsand vegetables, fruit juice, dried spices, and high-starch and high-fat foods, using spike recovery, sequentialextractions, analysis of available certified reference materials, and verification of AA peak purity. The limitsof detection and quantitation were 0.06–0.09 mg and 0.2 mg AA per 100 g food, respectively. The averagerecovery of added AA from all matrices was 97–103%.The inter-day relative standard deviation (RSD) formatrices including orange juice, fortified cereal, a fruit and vegetable composite, and freeze-driedvegetables was 1.1–2.0% and 4.8%, and HORRAT values (RSD/predicted RSD) for a wide range of foods were<0.1–0.6. Results for certified reference materials, BCR1431 (freeze-dried Brussels sprouts), BCR1421 (milkpowder) and VMA399 (dry breakfast cereal) (465 ! 4.6, 74.0 ! 1.1 and 70.5 ! 1.5 mg/100 g, respectively)were within the certified ranges. Without matrix-specific method adjustments to the method validated fororange juice, extraction problems and interferences in the AA peak for particular matrices lead to over- orunderestimation of vitamin C in many foods (0.3–70 mg/100 g; 5.5–64%).

! 2012 Elsevier Inc.. All rights reserved.

* Corresponding author. Tel.: +1 540 231 9960; fax: +1 540 231 9070.E-mail addresses: kmpvpi@vt.edu, kp.jfca@yahoo.com (K.M. Phillips).

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Jacob and Sotoudeh, 2002; Levine et al., 2009; Li and Schellhorn,2007; Lykkesfeldt and Poulsen, 2010; Willcox et al., 2008). Theprimary natural food sources of vitamin C are vegetables and fruits,particularly citrus fruit, green leafy vegetables, broccoli, cauliflow-er, Brussels sprouts, tomatoes, peppers, and potatoes (Eitenmilleret al., 2008).

Epidemiological studies, clinical feeding trials, and dietaryrecommendations rely on food composition databases such as theUSDA National Nutrient Database for Standard Reference (SR)(USDA, 2011) to estimate the vitamin C content of foods consumed.Therefore, the validity of any dietary intake estimates obviouslydepends on the accuracy of those food composition data. Whilenumerous publications and databases report analytical values forthe vitamin C content of foods, including variability among foods orsamples of a particular food, many of these studies lack matrix-specific method validation and/or quality control to prove theaccuracy and precision of quantitation.

For nutritional purposes, vitamin C is the sum of AA anddehydroascorbic acid (DHAA) (‘‘total ascorbic acid’’); DHAAabsorbed from the intestinal tract undergoes intracellular reduc-tion (via NADPH- and glutathione-dependent reductases), render-ing it biologically active (Bender, 2003; Wilson, 2005). Methodsreported for the determination of vitamin C in foods are typicallydesigned for analysis of a particular food type or matrix (e.g. fruitsand vegetables or fortified products) and validated for a specificfood or tested on a limited number of products (see for example,Barros et al., 2010; Behrens and Madere, 1987; Chebrolu et al.,2012; Fediuk et al., 2002; Vanderslice and Higgs, 1990, 1993), andvalidated methods for the analysis of vitamin C in a broad range offood matrices are lacking. Widely used existing standard methodsare titrimetric and fluorimetric techniques, such as the Associationof Official Analytical Chemists (AOAC) methods 967.21, 967.22,984.26 (AOAC, 2005a,b,c) that were developed for specificmatrices. The titrimetric method (AOAC 967.21) applies to vitaminpreparations and juices. The fluorimetric method was developedfor vitamin preparations (AOAC 967.22) and selected foods(vitamin fortified breakfast cereal, fruit juices and infant formula)(AOAC 984.26) (DeVries, 1983). In all of these cases, most of the AAis not endogenous, but added to the product. High performanceliquid chromatographic (HPLC) methods have been widely appliedin the analysis of vitamin C in various foods (e.g. Barros et al., 2010;Brause et al., 2003; Davey et al., 2003; Drivelos et al., 2010;Garrido-Frenich et al., 2005; Gokmen et al., 2000; Hernadez et al.,2006; Laur and Tian, 2011; Lee and Coates, 1999; Lykkesfeldt,2000; Nishiyama et al., 2004; Novakova et al., 2008a; Odriozola-Serrano et al., 2007; Romeu-Nadal et al., 2006; Spınola et al., 2012;Vanderslice et al., 1990; Wechtersbach and Cigic, 2007), butcurrently there is no standard method for the analysis of vitamin Cin different food matrices.

Several common deficiencies exist in methods used todetermine vitamin C in foods: lack of specificity, incompleteextraction or stabilization of AA during analysis, and failure toconfirm complete separation of AA from food-specific interfer-ences in chromatographic analyses. Titrimetric and fluorimetricmethods are simple and therefore popular (Hernadez et al., 2006)but are not chemically specific for AA. The titrimetric methodAOAC 967.21 (AOAC, 2005a) relies on reduction of the blue dye 2,6-dichloroindophenol by AA to a colorless solution. However, otherreducing compounds can react with the dye, leading to overesti-mation of AA, and intensely colored extracts resulting from somefruits and vegetables interfere with detection of the titrationendpoint (Eitenmiller et al., 2008). The fluorimetric method (AOAC967.22 and AOAC 984.26) (AOAC, 2005b,c) is based on oxidation ofAA to DHAA followed by reaction with o-phenylenediamine toproduce a fluorescent quinoxaline derivative. Substances in theextract that either quench the fluorescence or yield fluorescent

products may therefore interfere with the measurement of AA(Deutsch and Weeks, 1965; Eitenmiller et al., 2008), and starch inthe extract can physically interfere with the fluorescencemeasurement (Remmers et al., 1968). Also, because existingstandard methods were developed for AA fortified products, theextraction procedure may not be rigorous enough to recover AAcontained within the cells of intact plant foods.

AA is susceptible to oxidation in aqueous solution; therefore,temperature, light, pH, the presence of oxygen, metal catalysts(iron, copper) and enzymes such as ascorbate oxidase affect therate of AA degradation, at a variable rate in different foods. At lowpH AA is fully protonated and relatively stable, with maximalstability at 4–6; at pH > 6, AA is reversibly oxidized to DHAA, andDHAA may be further irreversibly oxidized to 2,3-diketo-L-gulonicacid, followed by degradation to other by-products includingoxalic acid, L-threonic acid, CO2, L-xylonic acid, L-lyxonic acid, andL-xylose (Johnston et al., 2007). Therefore, to measure vitamin C(AA + DHAA), the concentrations in the food must be preservedduring analysis, and either both compounds must be measured orany DHAA must be chemically converted to AA. Reduction of DHAAhas been accomplished by reducing agents including dithiothrei-tol, mercaptoethanol, glutathione, tris(2-carboxyethyl) phosphine(TCEP) (Davey et al., 2003; Gokmen et al., 2000; Lykkesfeldt, 2000;Nishiyama et al., 2004; Odriozola-Serrano et al., 2007; Wechters-bach and Cigic, 2007). Finally, although HPLC is specific to thedetection of AA, other components can interfere with detection ifnot completely resolved or removed. Detection is most commonlyby ultraviolet (UV) or fluorescence, and complex matrices containmany compounds that absorb or emit at the same wavelength usedfor detection of AA. The inadequacy of published methodstherefore demands specific validation when AA is analyzed in aparticular food for which a given procedure was not developed.

As part of the United States Department of Agriculture’s (USDA)National Food and Nutrient Analysis Program (NFNAP) (Haytowitzet al., 2008) to update data in the National Nutrient Database forStandard Reference (USDA, 2011), a broad range of commonlyconsumed fruits and vegetables, dried spices, and other foods werecollected according to statistically representative sampling plansand needed to be analyzed for vitamin C. Validation of samplepreparation and handling prior to analysis of vitamin C werepreviously reported (Phillips et al., 2010). A protocol wasdeveloped for chemical analysis of AA that was validated andapplicable to diverse foods, by adapting existing publishedmethods for extraction and HPLC and implementing matrix-specific validation and analytical quality control. The purpose ofthis communication is to describe the validated methodology, andto describe the matrix-specific modifications, validation andquality control that were essential for accurate and precisequantitation of AA in a wide variety of foods.

2. Materials and methods

2.1. Overview

The protocol and method modifications began with publishedmethodology for extraction of AA using a metaphosphoric acid-based buffer containing EDTA as chelating agent and TCEP toreduce DHAA to AA (Lykkesfeldt, 2000; Musulin and King, 1936;Nishiyama et al., 2004) and analysis of AA by reversed-phase HPLCwith UV detection (Novakova et al., 2008b). Method validationfocused first on establishing adequate precision and recovery of AAfor orange juice. Subsequently the method was tested on a varietyof foods and commercially available certified reference materials(RM) having matrix characteristics that could affect extraction,stability, or HPLC separation of AA relative to juice, such as higherfiber, starch, fat, and/or lower moisture content, presence of active

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enzymes in raw foods, or occurrence of the AA within the cellmatrix. No attempt was made to systematically analyze anyinteraction among the effects of different matrix characteristicsand assay parameters. Rather, foods having the characteristicsnoted in parentheses were chosen, as follows: pineapple andbroccoli (high fiber raw fruit/vegetable); potato, yucca chips,plantain chips, and dry breakfast cereal (high starch, vitaminfortified); French fried potatoes, fried plantains, yucca chips, andwhole milk powder (high fat); dried parsley, dried oregano andfreeze-dried Brussels sprouts (dried fruit/vegetable); and pasteur-ized orange juice (product with added vitamin C); canned fruit/vegetable, and pasteurized orange juice (heat-processed foodsinactivated endogenous enzymes by heat-treatment).

Method validation included spike recovery studies, sequentialexhaustive extraction, verification of AA peak purity on HPLC,evaluation of the precision of replicate analyses, and comparison ofresults for RMs to certified values. When problems were found inthe analysis of a particular matrix using the initial method,modifications were introduced and the validation was repeateduntil satisfactory performance was achieved. Adjustments intro-duced included an increased number of extractions, dilutions orsample size adjustments, preliminary fat extraction, and/orchanges to the HPLC method.

Two orange juice control materials were prepared and usedthroughout the study to provide a consistent reference. Additionalcontrol composites (CC) (mixed fruit and vegetable composite,French-fry composite) were later developed to monitor performancefor other matrices. Quality control (QC) parameters for the CCs wereestablished for routine analysis of different matrices by the validatedmethodology. Finally a variety of foods were analyzed implementingthe matrix-specific validated methodologies and QC measures, todemonstrate precision for a range of foods.

2.2. Reagents and standards

ACS grade ascorbic acid (AA) (>99% pure), metaphosphoric acid(MPA), ethylenediaminetetraacetate disodium salt (EDTA), andformic acid were purchased from Sigma Aldrich (Saint Louis, MO).HPLC grade ortho-phosphoric acid, methanol, hexane and waterwere purchased from Fisher Scientific (Pittsburgh, PA). Tris(2-carboxyethyl) phosphine (TCEP) was obtained from ThermoScientific (Rockford, IL).

AA stock (5 mg/mL) and working solutions (0.1 mg/mL) wereprepared and diluted to make HPLC AA calibration standards in therange of 0.2–50 mg/mL. All AA solutions were prepared in AAextraction buffer (5% MPA, 1 mM EDTA, 5 mM TCEP), blanketedwith argon, and stored in the freezer at "60 8C for up to 6 weeks.

2.3. Food samples

All foods were prepared in a room with UV filtered light. Roomtemperature was #24 8C. In some cases more than one compositeof the same food were prepared. Different composites of the samefood are denoted with Roman numerals (I, II, III, IV, etc.).

2.3.1. JuicesRefrigerated juice in the original container was shaken

vigorously and, in the case that multiple containers were to becomposited, poured into a stainless steel bowl and mixedthoroughly by stirring with a stainless steel spatula. Then, workingquickly and with intermittent stirring, subsamples of approxi-mately 40–45 mL each were dispensed into polypropylene screw-cap bottles, capped under argon, and stored at "60 8C untilanalyzed. The temperature of the juice ranged from 4 to 15 8C fromstart to end of the procedure. Juices not composited were poureddirectly into containers after the initial mixing.

2.3.2. Fruits and vegetablesRaw cauliflower (Brassica oleracea, botrytis), collard greens

(Brassica oleracea var. viridis), russet potatoes (Solanum tuber-osum), green onions (Allium cepa), corn (Zea mays), zucchinisquash (Cucurbita spp.), green beans (Phaseolus vulgaris), broccoli(Brassicaoleracea var. italica), sweet green peppers (Capsicumannuum), clementines (Citrus clementina hort. ex Tanaka), pineap-ple (Ananas comosus), tomatoes (Lycopersicon esculentum), babyspinach (Spinacia oleracea), carrots (Daucus carota), celery (Apiumgraveolens), green and red leaf lettuce (Lactuca sativa var. crispa),romaine lettuce (Lactuca sativa var. logifolia), iceberg lettuce(Lactuca sativa var. capitata), banana (Musa acuminata Colla),blackberries (Rubus fruticosus), peaches (Prunus persica), nectar-ines (Prunus persica var. nucipersica), plums (Prunus spp.),cantaloupe (Cucumis melo L.), honeydew melon (Cucumis melo),watermelon (Citrullus lanatus), mango (Mangifera indica), papaya(Carica papaya), red and green grapes (Vitis vinifera), Granny Smithand red delicious apples (Malus domestica) were purchased at alocal grocery store (Kroger, Blacksburg, VA) for method validation.Yellow corn, green onions, squash, green beans, green leaf andromaine lettuce, mango, and papaya were collected as part of theNFNAP and were composites of samples from a statistical U.S.nationwide sampling plan (Haytowitz et al., 2008; Trainer et al.,2010). Homogenized composites of each vegetable/fruit wereprepared as follows. Approximately 1.0–1.5 kg of produce waswashed with distilled deionized water for about 2 min, drainedand blotted dry with a clean, lint-free cloth. Inedible portions wereremoved including bruised or damaged areas, and specifically theleaves and stems from cauliflower and broccoli; stems fromcollards and tomatoes; peels from clementines, bananas, water-melon and pineapple; peel, seeds and pulp from cantaloupes andpapaya; top of stems and roots from green onions; stalk, husks,corn silk, and dried/inedible end kernels from corn; ends fromzucchini squash, carrots and celery; end tips and strings fromgreen beans; stem and seeds from sweet green peppers andapples; stems and pits from nectarines, peaches and plums.Grapes were individually separated from stems. The stem fromiceberg lettuce, green leaf, red leaf, and romaine lettuce wasremoved first and then the leaves were separated, washed anddried as described above. Potatoes were not peeled. Composites oflarge fruits such as cantaloupe and watermelon were preparedfrom at least 3 fruits that were each subsampled to achieve a totalweight of roughly 1.5 kg. Washed and cleaned produce was thencut into pieces (#1.5 cm), then immediately frozen and homoge-nized with liquid nitrogen in a 6 L industrial food processor (RobotCoupe USA, Jackson, MS). Subsamples were distributed while stillfrozen among 60 mL glass jars with TeflonTM-lined caps, andstored at "60 8C.

Cooked vegetables were processed as follows. Cobs of corn wereboiled for 5–7 min, drained, allowed to cool to room temperature,and then corn kernels removed. Zucchini squash was cut into1.5 cm pieces and steamed for 2–3 min or until tender, drained andallowed to cool to room temperature. Potatoes halves werecovered and microwaved for 6 min at 100% power (1200 W),allowed to cool to room temperature, and cut into 1.5 cm pieces.Cooked, drained and cut produce was homogenized with liquidnitrogen in the 6 L food processor and distributed into subsamplesas described for the fresh produce.

2.3.3. Dry foods (<10% moisture)

2.3.3.1. Dry spices. Commercial brand dry parsley (Petroselinumcrispum) and oregano flakes (Origanum vulgare) were dispensedinto 30 mL glass jars (#2 g/jar) with Teflon-lined caps, blanketedwith nitrogen, and stored at room temperature. Oregano andparsley flakes were ground in a small food processor (GIRMI

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Chopper, TR30 EuroCuisine, Los Angeles, CA) before analysis toensure complete homogenization in the extraction buffer. Briefly,about 2 g of spice flakes were processed at a time in 7–12 pulses of30 s each, until flakes were ground to a fine powder. The initialtemperature of the spice was recorded (#24 8C or roomtemperature), and then monitored after every 3 blends and atthe end of blending, making sure that the final temperature didnot rise more than one degree above initial temperature (#25 8C).Ground spices were then transferred to 30 mL glass jars withTeflonTM-lined cap, blanketed with argon, stored at roomtemperature and assayed within 24 h, or frozen at "60 8C if notassayed within 24 h.

2.3.3.2. Breakfast cereal. Ready-to-eat dry breakfast cereal washomogenized in an industrial food processor (Robot Coupe USA,Jackson, MS) at room temperature. Subsamples were distributedamong 60 mL glass jars with TeflonTM-lined caps, blanketed withnitrogen and stored at "60 8C.

2.3.4. High-fat foods (>5% fat)Composites of fried yellow plantains (Musa X paradisiaca)

from restaurants in 3 American cities (#1.5 kg) and commercialbrand plantain chips (#1 kg); and commercial brand yucca chips(Manihot esculenta) (#1 kg) sampled as part of the NFNAP(Haytowitz et al., 2008; Trainer et al., 2010) were preparedindividually by homogenizing each food in liquid nitrogen in a6 L industrial food processor (Robot Coupe USA, Jackson, MS). Amixed snack food composite was prepared by mixing roughlyequal proportions (#1.4 kg each) of commercial brand cheesepuffs, potato chips, corn chips, tortilla chips, pretzels, andcrackers, and homogenizing in liquid nitrogen in a 30-quartindustrial food processor (Robot Coupe USA, Jackson, MS).Subsamples of each type of composite were dispensedinto 60 mL glass jars with TeflonTM-lined caps and storedat "60 8C.

2.3.5. Control compositesControl composites (CC) were prepared for different food

matrices to monitor assay performance. For each CC 80–200subsamples of the composite were dispensed and stored at "60 8C.Samples of orange juice CCs used to validate the method for orangejuice were included in all assays during subsequent methoddevelopment for other matrices, to monitor assay precisionindependent of matrix.

2.3.5.1. Orange juice. Two orange juice CC were prepared (orangejuice I, orange juice II). Commercial brand orange juice (refrigerat-ed, pasteurized, pulp-free, not from concentrate, two to four !gallon cartons) was purchased locally (Kroger, Blacksburg, VA) andcomposited as described in Section 2.3.1.

2.3.5.2. Mixed canned fruit/vegetable composite. A composite(#2.75 kg total) consisting of canned tomato puree (#0.55 kg),canned mandarin oranges (#0.55 kg), and canned spinach(#1.65 kg) was prepared by homogenizing the ingredients asdescribed in Section 2.3.2, except without freezing in liquidnitrogen.

2.3.5.3. French fries. French fried potatoes (#1.4 kg) were pur-chased from a local fast-food restaurant) and homogenized asdescribed in Section 2.3.2.

2.3.5.4. Breakfast cereal. Packaged ready-to-eat vitamin fortifiedmultigrain breakfast cereal ($8 kg total) was purchased at a localsupermarket and homogenized as described in Section 2.3.3, usinga 30-quart industrial food processor (Robot Coupe).

2.4. Analysis of vitamin C

An overview of the method is illustrated in Fig. 1. Details ofmethodology and modifications ultimately adopted for differentmatrices are given below. All work was performed in a room withUV filtered light.

2.4.1. Sample handling before analysisFrozen homogenized samples prepared as described in

Section 2.3 were handled as follows immediately prior toweighing aliquots for analysis. All samples were mixedthoroughly before and during weighing aliquots for analysis,to ensure representative subsampling. Pasteurized or cannedjuice (Section 2.3.1) and canned fruits/vegetables (Section 2.3.5)were thawed in a 30 8C water bath for 20 min, and kept on icebefore and during subsampling. Homogenized samples of fruitsand vegetables (Section 2.3.2) were weighed while still frozenand kept on ice to minimize thawing during subsampling. Dryfoods (Section 2.3.3) were allowed to equilibrate to roomtemperature for 30 min. High-fat foods (Section 2.3.4) werethawed in a 30 8C water bath for 20 min, and then kept on icebefore and during subsampling. Milk powder which is a high-fat‘‘dry’’ food was allowed to equilibrate to room temperature for30 min before weighing.

2.4.2. Original methodThis section describes the method developed and validated for

orange juice, followed by modifications validated for othermatrices (Section 2.4.3).

2.4.2.1. Extraction and sample clean-up. Samples were handledbefore weighing as described in Section 2.4.1. After thoroughlymixing the sample, 2 g were weighed into a 50 mL TeflonTM

centrifuge tube (Fisher Scientific, Pittsburgh, PA), then 8 mL ofextraction buffer (5% MPA/1 mM EDTA/5 mM TCEP; pH 1.8)were added. Tubes containing the weighed sample were kept onice before and after addition of extraction buffer when multiplesamples were being prepared for analysis. The samples in bufferwere homogenized for 2 min using an OmniTM mixer fitted witha saw tooth generator blade, 10 mm % 195 mm (Omni Interna-tional, Marietta, GA), with the centrifuge tube submerged in icewater with a flow of argon on top to minimize AA oxidation.Samples were then centrifuged at 10 8C and 91.7 Hz (7280 % g)for 30 min. The supernatant was decanted into a 50 mLdisposable polypropylene tube, capped under argon and kepton ice. The pellet was thoroughly resuspended in 4 mL ofextraction buffer, capped under argon, sonicated for 5 min, thencentrifuged for 30 min at 10 8C and 91.7 Hz (7280 % g). Thesecond supernatant was combined with the first supernatant(2X-extraction, see Fig. 1). Combined supernatants were filteredin 25 mL Maxi-SpinTM centrifuge filter tubes with 0.45 mmpolyvinylidene fluoride (PVDF) membrane (AllTech GraceTechnologies, Deerfield, IL), or by vacuum filtration using aBuchner funnel and Whatman #41 paper. The filtered extractwas quantitatively transferred to a 25 mL, 50 mL, or 100 mLvolumetric flask (depending on the dilution needed), taken tovolume with extraction buffer, capped under argon, andthoroughly mixed. The extract was transferred to a 40 mLamber glass flask with TeflonTM-lined lid, capped under argon,and stored at "60 8C. Extracts were analyzed by HPLC eitherundiluted or diluted with extraction buffer 1/5 to 1/20 to yield afinal concentration within the range of the calibration standards,depending on the estimated AA concentration in the sample, andthen aliquots (1 mL) were transferred to 2 mL amber HPLC/GCvials, capped under argon and analyzed by HPLC, or stored at"60 8C and analyzed by HPLC within 1 week.

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2.4.2.2. HPLC analysis. The HPLC column was a C18 reversed phasewith polar end-capping [Phenomenex SynergiTM 4 mm Hydro-RP(250 mm % 4.6 mm), Phenomenex, Torrance, CA]. The mobilephase was 0.05% (w/v) aqueous formic acid or, for most matrices,0.02% aqueous ortho-phosphoric acid (w/v). The HPLC system(Perkin ElmerTM, Waltham, MA) consisted of a LC binary pump 250,and a diode array detector (model 235C) operated with Turbo-chromTM 4 software. Alternatively, an Agilent 1200 HPLC system(Santa Clara, CA) equipped with a LC quaternary pump (modelG1311A) and diode array detector (model G1315D), operated withChemStationTM, version B.03.02, was used. Twenty microliters ofsample, previously filtered through a MiniUniprep syringeless0.45 mm PDVF filter (Waters Corporation, Milford, MA), wereinjected into the HPLC and eluted under isocratic conditions at1 mL/min (HPLC – method A). Under these conditions the AA peakeluted at approximately 5.2–5.5 min. When the mobile phase was0.02% aqueous ortho-phosphoric acid (w/v), the flow rate used waseither 0.7 mL/min (HPLC – method B) with the AA peak eluting atabout 7.6–7.8 min; or the flow rate used was 0.4 mL/min (HPLC –method C), and the AA peak eluted at around to 13.7 min. AA wasdetected at 254 nm (Novakova et al., 2008b). AA standards in therange of 0.2–50 mg/mL AA in extraction buffer were run induplicate with every assay and injected multiple times throughoutthe HPLC run. A calibration curve was prepared based on linearregression of all data points and used to quantify AA in the samples.

2.4.3. Method modifications for specific matricesThe methodology described in Section 2.4.2 for orange juice was

modified as follows for other matrices.

2.4.3.1. Fruits and vegetables. A third extraction of the pellet wasperformed, with 12 mL of extraction buffer being added and the

third supernatant after homogenization and centrifugation wascombined with first and second supernatants (=3X-extraction,Fig. 1). The combined supernatants were transferred to avolumetric flask for further dilutions for HPLC, that were carriedout as described in Section 2.4.2.1.

2.4.3.2. Dry foods (<10% moisture). The sample weight and volumeof AA extraction buffer were adjusted so that the ratio of extractionbuffer and dry mass were equivalent to that of wet samples. Anapproximate water content of 90% for orange juice, fruits andvegetables was used to calculate an equivalent dry mass of 0.2 g in2 g of wet sample. Considering that a sample size >0.2 g of anymaterial was required to ensure representative subsampling, aswell as limitations on the practical volume of buffer that can behandled during the extraction, 0.3 g of dry composites wereweighed. Frozen homogenized dry food samples were allowed toequilibrate to room temperature for 30 min (Section 2.4.1) andthen weighed (0.3 g) into 50 mL TeflonTM centrifuge tubes, then12 mL of extraction buffer were added, vortexed gently tothoroughly hydrate composite, and then extraction proceeded asdescribed in Section 2.4.2.1. Dry foods were extracted a total of 3times, with 12 mL of extraction buffer per each extraction (3X-extraction).

2.4.3.3. High-fat foods (>5% fat). High-fat foods were washed withhexane prior to reduce the fat content prior to AA extraction. Thethawed, homogenized food (see Section 2.4.1) (about 0.3 g for dryfoods or about 2.0 g for undried foods) was weighed into a 50 mLTeflonTM centrifuge tube and 20 mL of hexane were added. Thetube was capped under argon, mixed well in a Vortex mixer andthen shaken in an orbital platform shaker (New BrunswickScientific Co., Edison, NJ) for 10 min at 350 rpm (5.83 Hz) and

Fig. 1. Overview of the general method. All work was performed under UV filtered light. See Table 1 for method modifications for specific matrices.

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room temperature. Samples were centrifuged at 10 8C and 91.7 Hz7280 % g for 20 min, then the hexane layer (top) was carefullydecanted and discarded. Samples were then defatted a second timewith 20 mL hexane following the same procedure. Excess hexanewas evaporated from the defatted pellets using a stream of argon atroom temperature. Extraction buffer (12 mL of 5% MPA/1 mMEDTA/5 mM TCEP; pH 1.8) was added to samples, followed byhomogenization for 2 min using an OmniTM mixer as described inSection 2.4.2.1. Samples were then centrifuged at 10 8C and 43.3 Hz(RCF 1627) for 5 min and stored in freezer at "60 8C overnight (notover 24 h) to continue assay the following day. The next day, frozensamples were thawed for 1 h in ice/water, then they were mixedthoroughly and sonicated for 5 min before centrifuging at 10 8Cand 91.7 Hz (7280 % g) for 30 min. The supernatant was decantedinto a 50 mL disposable polypropylene tube, capped under argonand kept on ice. After this step, the procedure continued asdescribed in Section 2.4.2.1, with a total of 3 extractions, asdescribed in Section 2.4.3.1.

Note that the assay of high fat foods was split into two days forpractical purposes, because defatting the sample and performingthe complete AA extraction in one day would require approxi-mately 10 h.

2.5. Method validation

2.5.1. Extraction efficiencySelected matrices were subjected to sequential extractions.

Broccoli, pineapple and potatoes were chosen based on initialchallenges encountered during extraction due to high fiber contentand greater difficulty to disrupt the cells. Potatoes are also high instarch, which affected filtration. Breakfast cereal was tested as foodfortified with AA, as well as being a dry matrix with high starchcontent. Dry parsley was included as another dry matrix, and onewith high endogenous vitamin C content. Fried plantainsrepresented a food high in fat, fiber, and starch. Extraction wasperformed as described in Section 2.4, except two additional 12 mLextractions were carried out, for a maximum of five 12 mLextractions (5X-extraction). Three aliquots of each material wereanalyzed in the same assay batch for each of 2X–3X-extractions,and 5X-extractions.

2.5.2. Spike recovery experimentsAA standard was added to samples of representative matrices

(orange juice, raw cauliflower, raw broccoli, raw pineapple, driedparsley, and fried plantains) at 20–100% of the endogenous AAlevel prior to extraction (prior to defatting for fried plantains,Section 2.4.3.3). AA was added as a precisely measured volume ofstandard solution (0.5–1.0 mg/mL, 0.1–2 mL) to all samples,followed by capping of the tube under argon and gentle swirlingto ensure mixing.

2.5.3. Authenticity of AA peak on HPLCPurity and authenticity of the AA peak in the samples was

assessed first by confirming co-migration of the AA peak in thesamples with authentic AA standard, and then by comparing theUV absorption spectrum at three points in the peak, start,maximum, and end, using the program TurboscanTM (PerkinElmerTM, Waltham, MA) or the Spectral Analysis module of AgilentChemStationTM, version B.03.02.

2.5.4. Analysis of certified reference materialsSeveral commercial RM provided with values for vitamin C

were procured and analyzed: freeze-dried Brussels sprouts (BCR1

431), milk powder (BCR1 421), and freeze-dried green beans(BCR1 383), produced by the European Commission, CommunityBureau of Reference (Geel, Belgium) and obtained from RT

Corporation (Laramie, WY); powdered adult/infant nutritionalformula (SRM1 1849) and cranberry juice cocktail (SRM1 3282)from National Institute of Standards and Technology (NIST)(Gaithersburg, MD); and fortified ready-to-eat dry breakfast cereal(VMA 399) from the American Association of Cereal Chemists (St.Paul, MN). NIST SRM1 1544 total diet and SRM1 2385 slurriedspinach which do not have certified values for vitamin C wereanalyzed to provide reference values obtained by the validatedmethodology for commercially available samples of additionalmatrices.

All RMs were stored in the freezer at "60 8C, except forcranberry juice cocktail (SRM1 3282) that was kept refrigeratedat 4–8 8C as specified in the certificate of analysis. Freeze-driedBrussels sprouts (BCR1 431), milk powder (BCR1 421), freeze-dried green beans (BCR1 383) and fortified dry breakfastcereal (VMA 399) were allowed to equilibrate to roomtemperature for 30 min (Section 2.4.1) before weighing.Cranberry juice cocktail (NIST SRM1 3282) was weighed atstorage temperature (4–8 8C).

2.5.5. Method performance parameters

2.5.5.1. Limits of detection and quantitation. The limit of detection(LOD) and limit of quantitation (LOQ) were estimated statisticallyfrom a diluted calibration curve (0.01–1.60 mg/mL of AA), as 3*SE/Sand 10*SE/S respectively (Ermer et al., 2005; Hibbert, 2007), whereSE is the standard error of the regression and S the slope of thecurve. In addition, the LOD and LOQ were estimated empirically, byprogressively measuring AA in serially diluted AA standardsolutions (0.01–0.2 mg/mL). The LOD was established as thelowest detectable concentration that yielded a visually symmetric,integratable peak, and the LOQ was defined as 3*LOD.

2.5.5.2. Repeatability and reproducibility. Repeatability was deter-mined by assaying 3–6 subsamples of each of several materials(orange juice I, orange juice II, mixed canned fruit/vegetable,freeze-dried Brussels sprouts, fortified ready-to-eat multi-grainbreakfast cereal, whole milk powder, and French fries) in a singleday. Reproducibility was assessed by assaying 10–50 subsamplesin 5–50 different days over a period of 8–42 months. Additionally, awide range of foods sampled as part of the NFNAP (Haytowitz et al.,2008) were analyzed over a period of 4 years (2007–2011) usingthe validated matrix-specific methodology. In each case thepredicted relative standard deviation (PRSD) was calculated fromthe Horwitz formula (Horwitz, 2003), PRSD = 2C"0.15, where C isthe concentration of the analyte expressed as a mass fraction (ganalyte/g sample), and the HORRAT value was calculated as RSD/PRSD (Horwitz, 2003).

2.6. Comparison of results using original and optimized methodology

The error in quantitation that would occur by use of the methodinitially developed and validated for orange juice to other foodswithout any modification was evaluated. Samples of selected foodmatrices were analyzed by the original method (Section 2.4.2) andwith the matrix-specific modifications (Section 2.4.3), with threereplicates per treatment.

2.7. Numerical calculations and statistical analysis

Numerical calculations and statistical analyses were performedwith Microsoft1 Office Excel for Mac1, version 11.1, 2004(Microsoft1 Corporation, Redmond, WA) and JMP for Mac1,version 8.0.1 (SAS Institute Inc., Cary, NC). A Student’s t-test(Hibbert, 2007) was performed to assess differences betweentreatments, with significance set at p < 0.05.

M.T. Tarrago-Trani et al. / Journal of Food Composition and Analysis 26 (2012) 12–25 17

3. Results and discussion

3.1. Problems encountered during extraction of different matrices

Table 1 summarizes the method modifications thatwere necessary for different food matrices. The followingsections describe the issues encountered when applying theunmodified method for orange juice (Section 2.4.2) to othermatrices.

3.1.1. Dry foodsIt was necessary to adjust amount of extraction buffer for dry

matrices (e.g. spices, cereal), so that the ratio of buffer to dry masswas similar to that of wet samples and to ensure completeextraction and recovery of AA. Consequently, the sample weightand volume of extraction buffer were set at 0.3 g of dry sample, anda total of 36 mL of extraction buffer (3 % 12 mL extractions (‘‘3Xextraction’’), Section 2.4.3.2). Additionally, dry flaked spices(oregano and parsley) tended to float on top of the extractionbuffer and could not be processed by the homogenizer blade. Thegrinding of these samples prior to extraction (see Section 2.3.3)was necessary to facilitate homogenization. These modificationswere incorporated in the analyses of samples for subsequentmethod validation.

3.1.2. High fat foodsThe fats/oils in samples containing more than $5% fat formed a

layer on top of the AA extract that was not possible to eliminate byfiltration. Additional problems with sample handling occurred insubsequent steps of the assay. These issues were resolved by theinitial hexane extraction (Section 2.4.3.3).

3.1.3. High-starch foodsThe extracts of raw and microwaved potatoes were very

viscous, probably due to the high starch content. The samples wereproblematic to filter with MaxiSpin centrifuge tubes and insteadvacuum filtration was applied (Section 2.4.2.1).

3.2. Method validation

3.2.1. Extraction efficiencyTable 2 shows results for vitamin C assayed in various matrices

(broccoli, potato, pineapple, and mixed canned fruit/vegetablecomposite, fried yellow plaintains, dried parsley, dry breakfastcereal, and orange juice) using multiple extractions. The extractionmethod initially designed and validated for orange juice consistedof two sequential extraction steps (8 mL and 4 mL, or 2X-extraction, see Section 2.4.2.1 and Fig. 1). Additional extractionsof orange juice did not increase the assayed AA concentration

Table 2Effect of number of extractions on vitamin C content assayed in representative food matrices.

Sample Sample weight (g) Dry weight (g) Vitamin C (mg/100 g)a

2X-extraction (8 ml, 4 ml) 3X extraction (3 % 12 ml) 5X-extraction (5 % 12 ml)

Orange juice 2.0 0.26 36.9A (0.2, 3) 36.9A (0.1, 3) 36.9A (0.1, 3)Fruit/vegetable mix, canned 2.0 0.18 11.7A (0.02, 4) 11.8A (0.2, 4) 11.7A (0.1, 2)Broccoli, raw 2.0 0.21 104.2B (1.9, 3) 110.0A (1.9, 3) 109.8A (0.4, 3)Potatoes, Russet, raw 2.0 0.43 6.6A (0.1, 3) 6.7A (0.1, 3) 6.6A (0.2, 3)Pineapple, raw 2.0 0.28 70.3A (0.8, 3) 70.7A (1.0, 3) 70.5A (0.5, 3)Parsley, dried 0.3 0.28 – 476.3A (13.0, 3) 477.3A (1.2, 3)Breakfast cereal (dry),

multigrain, ready-to-eat,vitamin fortified

0.3 0.29 – 62.0A (1.6, 3) 62.5A (1.0, 3)

Plantains, yellow, fried 1.0 0.51 – 8.6A (0.2, 3) 8.6A (0.03, 3)

a Mean (standard deviation, n). Mean values within a single row not connected by same capital letter are significantly different (p < 0.05).

Table 1Modifications to the method developed for orange juice that were necessary for other food matrices (AA = ascorbic acid).

Matrix Problems encountered when standard validatedmethod for orange juice was applied to othermatrices

Modifications Example of error in assayedvitamin C without modification

Fruits and vegetables & Incomplete extraction of AA in some fruit/vegetable matrices (e.g., broccoli) when2X-extraction was used

& 3X-extraction as standard procedure & Underestimation (for example,broccoli in Table 2, 2X-extractionyielded 5% lower AA values than3X-extraction)

& Starchy matrices (e.g. potato) difficult to filter,with long centrifugation times when filteringextract using centrifugal filtration

& Vacuum filtration & Inaccuracy and imprecisionfrom possible extract losses

& Interfering peaks eluting close to AA('10% of AA peak) on HPLC (e.g., collardgreens, potato) and

& Dilution of extract to render interferingpeaks undetectable (only applied wheninterferences '10% of AA peak)

& Inaccuracy and decreasedprecision from variabilityin integration of AA peak area,

& Deformed AA peak on HPLC, indicating lack ofresolution from interferences, with interferences upto #25–30% AA (e.g., green onions, cantaloupe, corn)

& HPLC mobile phase changed to 0.02%ortho-phosphoric acid (at 0.7 ml/min flowrate for routine analysis

& Overestimation

Dry foods: parsleyand oregano flakes

& Homogenization difficult due to large flakes floatingon top of extraction buffer and not processed by theblade; also flakes make centrifugal filtration difficult

& Parsley and oregano flakes were ground toa powder prior to AA extraction and extractsfiltered by vacuum filtration

& Underestimation andimprecision due to incompleteextraction and extract losses.

High-fat foods & Fat layer on top of AA extract that interferedwith filtration, taking extract accurately tovolume, and HPLC

& Hexane extraction of fat priorto extraction of AA

Inaccurate AA measurementsand high variability due toextract losses, inaccurate volumes,and HPLC problems.

M.T. Tarrago-Trani et al. / Journal of Food Composition and Analysis 26 (2012) 12–2518

(p = 0.9) (Table 2). Presumably AA in orange juice is readilyextracted due to the liquid matrix in which the cells are largelydisrupted during production, and because most of the AA incommercial orange juice is fortified to replace natural AA that islost during pasteurization. By comparison, in fresh fruits andvegetables AA occurs within the plant cells and must be released tobecome available for extraction.

The 3X-extraction and the exhaustive 5X-extraction yieldedvitamin C levels with no statistically significant difference(p < 0.05) for all of the foods tested. The 2X-extraction yieldedslightly lower vitamin C levels (about 0.6–1.4% lower) for themixed canned fruit/vegetable composite, pineapple, and potatoes,but these small differences were not statistically significant. On theother hand, the vitamin C concentration in broccoli assayed with

2X-extraction was 5% (5.7 mg/100 g) lower than that obtainedwith the 3X- and 5X-extractions (p = 0.004), suggesting incompleteextraction using the method validated for orange juice. Given thecompleteness of the 3X-extraction for the range of matrices tested,it was adopted as the standard method of extraction for routineanalysis of various foods.

3.2.2. Spike recoveryResults from the spike recovery experiments in selected foods

assayed using matrix-specific methodology (Section 2.4) areshown in Table 3. The recovery of AA from orange juice was101.5%, 100.3% and 102.8% for spike levels corresponding to 20%,50%, and 100% of the endogenous content, respectively. For thefruit and vegetable matrices (raw broccoli, raw cauliflower, rawpineapple), dried parsley, and fried yellow plantains (defatted(Section 2.4.3.3) after spike addition) recovery ranged from 97.0 to101.4%. All recoveries were within the calculated acceptable range(Horwitz, 2003).

3.2.3. Purity of AA peak on HPLCFig. 2 shows typical chromatograms for an AA standard and

orange juice, with the AA peak symmetrical and well resolved inboth cases. Besides agreement of the retention time of the peak inthe orange juice extract with that of the AA standard, the peakpurity index (Section 2.5.3) was close to 1.0 for the AA peak in theorange juice extract, indicating that only AA was eluting. The purityof the AA peak was subsequently monitored in all samplesanalyzed during routine work. The AA peak was well resolved andsymmetric in most samples, including broccoli, cauliflower,zucchini, carrots, celery, spinach, lettuce, tomatoes, green peppers,green beans, bananas, plums, nectarines, clementines, peaches,apples, grapes, mango, papaya, pineapple, watermelon, andblackberries. On the other hand, for some foods the chromato-grams had unidentified minor shoulders on the AA peak, indicatingincomplete separation of other lower level components.

For example, in collard greens and potatoes, respectively,interferences represented approximately 2% and 10% of the AApeak in samples analyzed using the method validated for orangejuice. In these cases dilution of the extracts 1/20 placed theinterfering peaks <LOD while maintaining an adequate LOQ for AA(0.19–0.25 mg/100 g) and with an AA peak purity index compara-ble to that of the AA standard. Other foods had much largerinterferences (25–30% of the AA peak). For example, boiled corn,raw green onions (Fig. 3, panels C and D, insets), and cantaloupe(not shown) had a deformed AA peak; NIST SRM1 1849 powderedadult/infant formula (Fig. 3, panel B, inset) had a componenteluting shortly before AA that was only partially resolved. Giventhe high amount of these interferences relative to AA, dilution as

Table 3Recovery of ascorbic acid (AA) from selected foods.

Sample Endogenous AA concentration (mg/100 g)a AA spike level (%)b Percent of AA recovereda Acceptable percent recoveryc

Orange juice III 37.5 (0.6, 1.6%, 2) 20 101.5 (0.7, 0.7%, 2) 85–11050 100.3 (3.4, 3.4%, 2)

100 102.8 (1.2a, 1.2%, 2)Cauliflower, raw 59.4 (1.2, 2.0%, 2) 85 99.1 (0.6, 0.6%, 2) 85–110Broccoli, raw 113.2 (1.5, 1.3%, 3) 50 99.3 (0.4, 0.4%, 3) 90–108

100 97.7 (1.5, 1.6%, 3)Pineapple, raw 71.7 (0.1, 0.2, 3) 33 97.2 (0.8, 0.9%, 3) 85–110

66 97.0 (0.8, 0.8%, 3)Parsley, dried 500 (1.7, 0.3%, 3) 50 99.3 (1.6, 1.6%, 3) 90–108

100 99.1 (0.7, 0.7%, 3)Yellow plantains, fried 5.9 (0.03, 0.5%, 3) 80 101.4 (1.0, 0.9%, 2) 80–115

160 101.2 (0.3, 0.2%, 3)

a Mean (standard deviation, relative standard deviation, n).b Percent of endogenous AA concentration.c Acceptable recovery limits determined as described by Horwitz (2003).

Fig. 2. HPLC chromatograms of ascorbic acid (AA) standard and an orange juiceextract. HPLC was performed on a Phenomenex SynergiTM 4 mm Hydro-RP(250 mm % 4.6 mm) column with 20 mL of sample injected, and eluted with themobile phase 0.05% aqueous formic acid at a flow rate of 1 mL/min. Panel A shows achromatogram for the AA standard, 3 mg/mL; panel B, chromatogram of orangejuice extract.

M.T. Tarrago-Trani et al. / Journal of Food Composition and Analysis 26 (2012) 12–25 19

performed for potatoes and collard greens would have resulted inan unacceptably high LOD and LOQ for AA. Therefore, adjustmentsto the chromatography were made to improve resolution. Themobile phase was changed from 0.05% formic acid (HPLC methodA, Section 2.4.2.2) to 0.02% ortho-phosphoric acid and the flow ratewas decreased from 1 mL/min to 0.7 mL/min (HPLC method B,Section 2.4.2.2). Under these conditions the AA peak wassymmetric and eluted separately from the interferences in mostfoods, including corn and green onions (Fig. 3, panels C and D), andhad a peak purity index similar to that of the AA standard. A slowerflow rate (0.4 mL/min.; HPLC method C, Section 2.4.2.2) wasadditionally necessary to optimally resolve the AA peak in thepowdered formula (Fig. 3, panel B). Because the 0.02% ortho-phosphoric acid mobile phase at 0.7 mL/min (HPLC method B) wasefficient at resolving the AA peak in most foods tested, this mobilephase was later adopted for routine analysis. Nonetheless, as newfoods were analyzed it was necessary to monitor resolution of theAA peak to assure that additional dilution or a slower flow rate wasnot necessary in particular cases.

3.2.4. Results for certified reference materialsTable 4 summarizes the vitamin C content of commercially

available reference materials of different matrices determinedusing the validated methodology for each matrix. Theassayed concentration in RMs with certified values was withinthe certified range in all cases, and the HORRAT values were0.2–0.4 for all materials, indicating excellent accuracy andprecision. It is interesting to note that the certified value forBCR1 431 was generated by laboratories using both HPLC withUV detection and HPLC with fluorescence detection, and resultsfrom HPLC-UV were lower than by HPLC/fluorescence(Finglas et al., 1997), consistent with the concentrations assayedin this study by HPLC-UV being at the lower end of the certifiedrange.

Unfortunately there is a limited range of food matrix RMscurrently available with certified vitamin C values, and theuncertainty of long-term stability of vitamin C in many matriceslimits the production of such reference materials. The valuesreported for the total diet RM (NIST SRM1 1544) might provide a

Fig. 3. Representative HPLC chromatograms of matrices with interferences eluting near ascorbic acid (AA) and requiring modifications to the chromatography. Main panelsshow chromatograms using optimized HPLC separation, and insets show chromatograms before method optimization. (A) Raw potato extract run with HPLC method B(isocratic 0.02% ortho-phosphoric acid elution at 0.7 mL/min) at 1/5 dilution (inset) showing small shoulder peaks to left and right of AA, and 1/20 dilution (main panel) wherethose shoulder peaks are barely visible. (B) Powdered adult/infant formula (NIST SRM1 1849) extract with incomplete separation of AA from unknown interference elutingshortly before AA (inset) when run with HPLC method B (0.02% ortho-phosphoric acid elution at 0.7 mL/min), and resolution from interference when run with optimizedmethod (isocratic 0.02% ortho-phosphoric acid elution at 0.4 mL/min, HPLC method C). (C) Corn and (D) raw green onion extracts showing broad and asymmetric AA peak(insets) using mobile phase validated for analysis of orange juice (isocratic 0.05% formic acid elution at 1 mL/min, HPLC method A) (Fig. 1), and resolution of AA withoptimized method (isocratic 0.02% ortho-phosphoric acid elution at 0.7 mL/min, HPLC method B).

M.T. Tarrago-Trani et al. / Journal of Food Composition and Analysis 26 (2012) 12–2520

useful reference for vitamin C determination in a mixed foodmatrix using validated methodology.

3.2.5. Repeatability and reproducibilityOngoing quality control was crucial to verify routine assay

performance during method validation. Therefore, a matrixspecific control material was included in each analytical batch,after initially establishing validated tolerance limits for thematerials with the initially developed orange juice CCs assayedin parallel as a check on assay performance. Mean levels of vitaminC assayed in each of these control samples, in a single day and ondifferent days are shown in Table 5. The intra-day and inter-dayRSD for the various control materials ranged from 0.8% to 3.6% andfrom 1.1% to 4.8%, respectively, and was well below the PRSD in allcases (HORRAT values 0.1–0.8), indicating excellent precision ofthe method. Results for the two orange juice CCs are illustrated inFig. 4 and show the continuity of method performance betweenmaterials and over time. Results for analysis of vitamin C in a widerange of foods over a 4-year period using matrix-specificmethodology and quality control are shown in Table 6. TheHORRAT value of (0.6 in all cases indicates routine precision of themethod that exceeds what would be predicted and consideredacceptable based on the analyte concentration (Horwitz, 2003).

The excellent precision achieved in this study without the use ofan internal standard, and inclusion of an internal standard wouldrender the method less susceptible to variation resulting from lessthan optimal analytical technique. However, compounds consid-ered as a possible low cost and universally usable internalstandard, including isoascorbic acid (Vanderslice and Higgs,

1990, 1993), ferulic acid (Cancalon, 2001), and methyluric acid(McCoy et al., 2005), are not suitable when the analysis will beapplied to a wide range of matrices, because they are presentnaturally and artificially in some foods. Ongoing work is directed tosolve this limitation. Meanwhile, the results of this work suggestthat accurate and precise results can be obtained with careful

Table 5Precision of vitamin C assayed in control materials: intra-day and inter-day over a period of 8–42 months.

Control material Precision Time period (months) Vitamin C (mg/100 g)a N RSD (%)b HORRAT valuec

Orange juice I Intra-day 40.1 ! 0.5 6 1.3 0.2Inter-day 18 40.5 ! 0.8 50 2.0 0.3

Orange juice II Intra-day 36.8 ! 0.4 4 1.1 0.2Inter-day 8 36.6 ! 0.4 6 1.1 0.2

Mixed canned fruit/vegetable composite Intra-day 12.2 ! 0.1 4 0.8 0.1Inter-day 11 11.8 ! 0.1 22 1.2 0.2

Brussels sprouts, freeze-driedd Intra-day 463.7 ! 16.8 3 3.6 0.8Inter-day 12 463.6 ! 8.0 12 1.7 0.4

Breakfast cereal (dry), multigrain, ready-to-eat, vitamin fortified Intra-day 62.0 ! 1.6 3 2.6 0.4Inter-day 42 61.8 ! 1.3 9 2.0 0.3

French fries Intra-day 3.8 ! 0.1 3 2.3 0.3Inter-day 7 3.7 ! 0.2 14 4.8 0.5

Whole milk, powderede Intra-day 74.2 ! 0.6 3 0.8 0.1Inter-day 18 74.0 ! 0.9 7 1.2 0.2

a Mean ! standard deviation (SD).b Relative standard deviation.c HORRAT value (RSD/predicted RSD) of 0.25–1.30 considered acceptable for single laboratory validation (Horwitz, 2003).d BCR CRM1 431, Institute of Reference Materials and Methods (Geel, Belgium); AA values are shown on a dry basis.e BCR CRM1 421 certified reference material, Institute of Reference Materials and Methods (Geel, Belgium).

35.0

36.0

37.0

38.0

39.0

40.0

41.0

42.0

43.0

10/10/06

4/28/07

11/14/07

6/1/08

12/18/08

7/6/09

1/22/10

8/10/10

2/26/11

VitaminC(m

g/10

0g)

Assay DateOrange Juice I CC Ora nge Juice II CC

Fig. 4. Quality control chart for two orange juice control composites (CC) assayedover a total period of 4 years, including method development for other matrices.Heavy bold, dashed, and bold lines indicate the mean and the mean ! 2 and 3standard deviations, respectively, for each CC.

Table 4Assayed vitamin C content of commercially available reference materials.

Materiala Matrix Vitamin Cb (mg/100 g) Certification type

Assayed Certificate range

VMA 399 Fortified cereal 70.5 ! 1.5 (4) 62.7–88.8 CertifiedBCR1 431 Freeze-dried brussel sprouts 465.0c! 4.6 (4) 459–507c CertifiedBCR1 421 Whole milk powder 74.2c! 0.6 (3) 73.0–80.8c CertifiedNIST 3282 Low-calorie cranberry juice cocktail 14.4 ! 0.26 (3) 13–19 ReferenceNIST 1849 Adult/infant nutritional formula, powdered* 104.3 ! 1 (3) 103–109 ReferenceBCR1 383 Freeze-dried green beans 13.3c! 0.1 (3) 13.7–16.5c IndicativeNIST 1544 Total diet 1.7 ! 0.04 (3) N/A Not certifiedNIST 2385 Slurried spinach 0.0 (3) N/A Not certified

a NIST, National Institute of Standards and Technology (Gaithersburg, MD); BCR, Institute of Reference Materials and Methods (Geel, Belgium); VMA, American Associationof Cereal Chemists (St. Paul, MN).

b Mean ! standard deviation (SD); number of replicates given in parentheses.c Value on a dry weight basis.

M.T. Tarrago-Trani et al. / Journal of Food Composition and Analysis 26 (2012) 12–25 21

Table 6Repeatability (intra-day precision) of vitamin C content assayed in diverse foods over a period of 4 years using optimized methodology for each matrix (n/a, not applicable).

Samplea Scientific name Description Samplesize (g)

Fatextraction

Finalvolume(ml)

Vitamin Cb (mg/100 g) Relativestandarddeviation

HORRATvaluec

Fruits and vegetablesCollard greens Brassica oleracea var. viridis Raw 2 No 25 114.1 ! 1.2 (3) 1.0% 0.2Russet potatoes,

with peelSolanum tubersoum Raw 2 No 25 17.5 ! 0.4 (3) 2.4% 0.3

Microwaved 2 No 25 16.7 ! 0.3 (3) 1.6% 0.2Cauliflower Brassica oleracea

(Botrytis group)Raw 2 No 25 59.4 ! 1.2 (2) 2.0% 0.3

Broccoli Brassica oleracea var. italica Raw 2 No 25 89.7 ! 1.4 (3) 1.6% 0.3Steamed 2 No 25 83.1 ! 0.7 (3) 0.9% 0.2

Zucchini squash Cucurbita pepo Raw 2 No 25 19.7 ! 0.3 (2) 1.4% 0.2Steamed 2 No 25 10.1 ! 0.2 (2) 2.4% 0.3

Green beans Phaseolus vulgaris Raw 2 No 25 7.0 ! 0.2 (2) 2.7% 0.3Microwaved 2 No 25 6.6 ! 0.3 (2) 3.8% 0.4

Green onions Allium cepa Raw 2 No 25 12.4 ! 0.55 (2) 4.5% 0.6Sweet green peppers Capsicum annuum Raw 2 No 25 95.0 ! 2.99 (3) 3.2% 0.6Baby spinach Spinacia oleracea Raw 2 No 25 33.1 ! 0.59 (4) 1.8% 0.3Carrots Daucus carota Raw 2 No 25 3.44 ! 0.01 (2) 0.2% 0.0Celery Apium graveolens Raw 2 No 25 4.95 ! 0.15 (2) 3.0% 0.3Lettuce, green leaf Lactuca sativa var. crispa Raw 2 No 50 7.93 ! 0.16 (2) 2.1% 0.3Lettuce, red leaf Lactuca sativa var. logifolia Raw 2 No 25 13.4 ! 0.06 (2) 0.4% 0.1Lettuce, romaine Lactuca sativa var. logifolia Raw 2 No 50 6.83 ! 0.09 (2) 1.3% 0.2Lettuce, iceberg Lactuca sativa var. capitata Raw 2 No 25 2.60 ! 0.02 (2) 0.8% 0.1Tomatoes Lycopersicon esculentum Raw 2 No 25 15.2 ! 0.09 (2) 0.6% 0.1Clementines Citrus clementina hort.

ex TanakaRaw 2 No 25 42.4 ! 1.30 (3) 3.2% 0.5

Cantaloupe Cucumis melo L. Raw 2 No 25 27.3 ! 0.65 (4) 2.4% 0.3Honeydew melon Cucumis melo Raw 2 No 25 9.53 ! 0.30 (2) 3.1% 0.4Watermelon Citrullus lanatus Raw 2 No 25 10.1 ! 0.30 (4) 3.0% 0.4Bananas Musa acuminata Colla Raw 2 No 25 9.04 ! 0.26 (4) 2.8% 0.4Pineapple Ananas comosus Raw 2 No 50 71.1 ! 0.10 (3) 0.2% 0.03Mango Mangifera indica Raw 2 No 25 24.3 ! 0.51 (2) 2.1% 0.3Papaya Carica papaya Raw 2 No 25 94.3 ! 1.69 (2) 1.8% 0.3Blackberries Rubus spp. Raw 2 No 25 12.9 ! 0.05 (2) 0.4% 0.1Peaches Prunus persica Raw 2 No 25 3.40 ! 0.15 (2) 4.4% 0.5Nectarines Prunus persica var.

nucipersicaRaw 2 No 25 4.95 ! 0.06 (2) 1.1% 0.1

Red plums Prunus spp. Raw 2 No 25 3.49 ! 0.19 (2) 5.5% 0.6Apples, granny smith Malus domestica Raw 2 No 25 4.54 ! 0.04 (2) 0.9% 0.1Apples, red delicious Malus domestica Raw 2 No 25 4.50 ! 0.26 (2) 5.8% 0.7Grapes, red Vitis vinifera Raw 2 No 25 4.77 ! 0.13 (2) 2.8% 0.3Grapes, green Vitis vinifera Raw 2 No 25 2.11 ! 0.04 (2) 2.0% 0.2JuicesCranberry juice cocktail Juice 2 No 50 14.4 ! 0.26 (3) 1.8% 0.2Juice/cola mixture,

vitamin C fortifiedJuice 14.3 ! 0.57 (3) 3.9% 1.1

Dried foodsParsley flakes Petroselinum crispus 5.9% moisture 0.3 No 50 500.0 ! 1.7 (3) 0.3% 0.1Oregano flakes Origanum vulgare 9.9% moisture 0.3 No 50 1.8 ! 0.1 (2) 4.3% 0.4Breakfast cereal (dry),

multigrain, ready-to-eat,vitamin fortified

n/a 2.6% moisture 0.3 No 50 62.0 ! 1.6 (3) 2.6% 0.4

Breakfast cereal (dry),ready-to-eat,vitamin fortified

n/a 2.2% moisture 0.3 No 50 70.5 ! 1.5 (4) 2.1% 0.4

Brussels sprouts,freeze-dried

Brassica oleracea(Gemmifera Group)

7.0% moisture 0.3 No 50 465.0e! 4.6 (4) 1.0% 0.2

High fat foodsFrench fries 14.6% fat 1.0 Yes 50 3.8 ! 0.1 (3) 2.3% 0.2Yellow plantains, fried Musa X paradisiaca 7.5% fat 1.0 Yes 50 8.6 ! 0.2 (3) 1.8% 0.2Mixed snack foodsd n/a 27% fat 0.5 Yes 50 3.6 ! 0.1 (6) 3.3% 0.4Plaintain chips n/a 26% fat 0.5 Yes 50 24.1 ! 0.3 (2) 1.1% 0.2Yucca chips n/a 26% fat 0.5 Yes 50 20.2 ! 0.2 (2) 1.0% 0.1Whole milk, powdered n/a 27% fat 2.9%

moisture0.3 Yes 50 74.2e! 0.6 (3) 0.8% 0.1

Adult/infant nutritionalformula, dry

n/a 31% fat 1.6%moisture

0.3 Yes 50 104.3 ! 1.0 (4) 1.0% 0.2

a See Section 2; samples were locally procured, and intended for method testing not to generate food composition data reflecting sample-to-sample variability.b Mean ! standard deviation (SD). Number of replicates is given in parentheses.c Horrat value: relative standard deviation/predicted relative standard deviation (Horwitz, 2003)d Mixture of potato chips, pretzels, cheese puffs, corn chips, and crackers.e Results given on a dry weight basis.

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attention to method validation, good technique, and ongoingquality control without expending the additional resources toinclude an internal standard.

3.3. Errors in results determined without matrix specific methodology

Table 7 shows the vitamin C concentrations assayed in selectedfoods as they would result if the method validated for orange juicewere used, compared to the results from the optimized matrix-specific methodology in each case. The method validated fororange juice did not give accurate results in many cases. Vitamin Cwas underestimated in all dry samples (spices vegetables, cereal),by 0.32–70 mg/100 g (11.4–64.1%), and high-fat samples (Frenchfries, milk powder), by 0.37–4.6 mg/100 g (6.5–9.8%) The resultsfor fruits and vegetables could not be generalized. In some casesvitamin C determined using the optimized and non-optimizedmethodology did not differ (pineapple, potatoes). In others,vitamin C was underestimated (broccoli, 5.7 mg/100 g; 5.5%) oroverestimated (cantaloupe and green onions, 3.0 and 4.1 mg/100 g(14.3% and 31.2%), respectively). Underestimation was most likelydue to incomplete extraction. Overestimation resulted from failureto separate interferences from the AA peak in HPLC (Fig. 3). Theseresults highlight the type and magnitude of errors that can beexpected without optimization and validation of a methoddeveloped for one food to other, even similar foods. To ensureaccurate results for fruits and vegetables it is especially importantto verify the purity of the AA peak in each matrix, which was donewhen the developed method was applied in the analysis of a widerange of fruits and vegetables (Table 6).

It is important to note that while the focus of this study was onthe analytical methodology, care in sample preparation was criticalto the accuracy and precision of results. Maintaining stability ofvitamin C in the original foods, ensuring the homogeneity ofsamples and representative subsampling for analysis wereintegral. Careful attention should be paid to these steps (Sections2.3 and 2.4.1), with specific attention to ensuring that the samplesize taken for analysis is homogenous with respect to the originalmaterial.

4. Conclusions

In this study, methodology for the extraction and HPLCquantitation of vitamin C in a variety of food matrices wasvalidated, and a reliable protocol was established for routineanalysis of a wide range of foods. Significant errors in quantitationof vitamin C resulted for many foods when the method validatedfor orange juice was applied without matrix-specific modifica-tions, even for similar foods (e.g. some fruits and vegetables).Successful implementation in a particular laboratory of publishedand validated methods, including those developed in this work,requires in-house testing to establish the precision and accuracy ofthe analysis as performed by that laboratory. Differences inanalytical technique and skill, equipment and other aspects ofmethod performance unique to a particular laboratory cansignificantly affect the results.

The method validated for orange juice (Section 2.4.2) did giveacceptable result for some foods (Table 7). Therefore if a particularstudy is limited to a defined food or set of foods for which thevalidity can be established using the approach described, certainlythe simplest procedure would be preferred (e.g. 2X vs. 3Xextraction). The formic acid mobile phase gave acceptableresolution of AA for most foods and is less corrosive to instrumentcomponents than the phosphate acid mobile phase, so the formermay be preferable if it can be validated to separate AA in the food(s)being tested. In addition, formic acid is compatible with massspectrometry detection systems, and thus provides versatility inthe detection configurations to which the method could beadapted, whereas mobile phases containing phosphates are notsuitable for mass spectrometry. On the other hand, the phosphateacid mobile phase would be a better choice if a wide range of foodswill be analyzed. The following approach is recommended forroutine analysis of vitamin C in foods using the reportedmethodology: (1) implementation of appropriate sample handlingand preparation prior to analysis, to ensure stability of vitamin Cand representative subsampling; (2) selection of the appropriateextraction method, sample size and dilution (Table 1); (3)verification of the purity of the AA peak in the specific food(s)

Table 7Vitamin C content of selected foods determined using matrix-specific optimized methodology versus method validated for orange juice (non-optimized). Statisticallysignificant differences (p < 0.1) are marked with an asterisk (*).

Matrix Vitamin C, mg/100 g (mean !standard deviation, n = 3)

Vitamin C difference Optimizationb Certified range

Non-optimizeda Optimized mg/100 g as percent of optimized

Dry samplesBrussels sprouts, freeze-driedc 404 ! 1.4 463 ! 10.8 "59* "12.7 A, B 459–507Oregano leaf, dried (sample 1) 1.99 ! 0.12 2.31 ! 0.05 "0.32* "13.9 A, B, COregano leaf, dried (sample 2) 0.92 ! 0.02 1.81 ! 0.08 "0.88* "48.8 A, B, CPaprika, ground 0.34 ! 0.06 0.95 ! 0.03 "0.61* "64.1 A, BParsley, dried 109 ! 2.6 127 ! 2.2 "18* "14.3 A, B, CBreakfast cereal 62.5 ! 0.4 70.5 ! 2.5 "8.0* "11.4 A, B, EFruits and vegetablesBroccoli, raw 104 ! 1.9 110 ! 1.9 "6* "5.5 BPineapple, raw 70.3 ! 0.8 70.7 ! 1.0 BPotatoes, raw (sample 1) 18.0 ! 0.7 17.8 ! 0.8 B, E, FPotatoes, raw (sample 1) 27.5 ! 0.2 27.5 ! 0.4 B, E, FGreen onions, raw 17.1 ! 1.9 13.1 ! 0.8 4.1* 31.2 B, GCantaloupe 24.3 ! 0.7 21.3 ! 0.7 3.0* 14.3 B, GHigh fat samplesFrench fries 3.39 ! 0.07 3.76 ! 0.07 "0.3*7 "9.8 B, DMilk powderd 67.0 ! 1.4 74.2 ! 0.6 "7.2* "9.7 A, B, D 73.0–80.8

a Method validated for orange juice.b Relative to method validated for orange juice: A, sample weight 0.3 g; B, 3 % 12 mL extraction; C, ground to powder prior to extraction; D, fat removed with hexane prior to

extraction; E, filtered with Buchner funnel; F, extract diluted for HPLC analysis to resolve AA peak; G, HPLC program modified to resolve AA peak.c BCR1 431 (Institute of Reference Materials and Methods, Geel, Belgium); results given on dry weight basis.d BCR1 421 (Institute of Reference Materials and Methods, Geel, Belgium); results given on dry weight basis.* Statistically significant (p < 0.1)

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being analyzed, with modification of HPLC conditions or sampledilution if needed to completely separate AA or depending on thenumber of different foods to be analyzed; (4) analysis of availablecertified reference materials, preferably of a similar matrix; (5)establishment of acceptable method precision by repeatedmeasurements over multiple assays of a control sample of thesame or similar matrix as samples to be analyzed, prior toanalyzing unknown samples; (6) inclusion of a food matrix-matched control sample with established tolerance limits todocument inter-assay precision and facilitate detection andcorrection of analytical problems (for example, see Phillips et al.(2006) and Taylor (1987) on the preparation and use of controlmaterials); (7) inclusion of method validation data and results forquality control samples and certified reference materials in anyliterature report of food composition data.

The reliability of results in any given laboratory dependsfundamentally upon matrix-specific method validation andprecise implementation. Published data for vitamin C in foodsshould be conditionally interpreted if not accompanied byevidence of matrix-specific method validation and quality-controldata. These data are especially critical when attempting to measureseasonal, cultivar, or storage effects on vitamin C concentrationwhere not only the samples but the day-to-day analyticalconditions vary among treatments (for example, Byers and Perry,1992; Lee and Kader, 2000). It is always advisable to include in anypublication of food composition data the results for commerciallyavailable reference material of the same or similar matrix analyzedby the same method to validate method performance and also toallow comparison of data among different literature reports.

Acknowledgements

This work was supported as part of Cooperative Agreement#59-1235-7-146 between the USDA Nutrient Data Laboratory andVirginia Polytechnic Institute and State University. The detailedwork of Amy Rasor and Nancy Conley in composite preparationand of Karen Amanna in data compilation and quality control/assurance is appreciatively acknowledged.

References

Arrigoni, O., De Tullio, M.C., 2000. The role of ascorbic acid in cell metabolism:between gene-directed functions and unpredictable chemical reactions. Journalof Plant Physiology 157, 781–788.

Association of Official Analytical Chemists, 2005a. Vitamin C in juices and vitaminpreparations. Official Method 967.21. In: AOAC Official Methods of Analysis,18th ed. Association of Official Analytical Chemists, Gaithersburg, MD, USA, p.45.1.14.

Association of Official Analytical Chemists, 2005b. Vitamin C in vitamin prepara-tions. Official Method 967.22. In: AOAC Official Methods of Analysis, 18th ed.Association of Official Analytical Chemists, Gaithersburg, MD, USA, p. 45.1.15.

Association of Official Analytical Chemists, 2005c. Vitamin C in foods. OfficialMethod 967.26. In: AOAC Official Methods of Analysis, 18th ed. Associationof Official Analytical Chemists, Gaithersburg, MD, USA, p. 45.1.16.

Barros, A.I., Silva, A.P., Goncalves, B., Nunes, F.M., 2010. A fast, simple, and reliablehydrophilic interaction liquid chromatography method for the determination ofascorbic and isoascorbic acids. Analytical and Bioanalytical Chemistry 396,1863–1875.

Behrens, W.A., Madere, R., 1987. A highly sensitive high-performance liquid chro-matography method for the estimation of ascorbic and dehydroascorbic acid intissues, biological fluids, and foods. Analytical Biochemistry 165, 102–107.

Bender, D.A., 2003. Vitamin C (ascorbic acid). In: Bender, D.A. (Ed.), NutritionalBiochemistry of the Vitamins. 2nd ed. Cambridge University Press, Cambridge,UK, pp. 357–384.

Brause, A.R., Woolllard, D.C., Indyk, H.E., 2003. Determination of total vitamin C infruit juices and related products by liquid chromatography: interlaboratorystudy. Journal of the Association of Official Analytical Chemists International 86,367–374.

Byers, T., Perry, G., 1992. Dietary carotenes, vitamin C, and vitamin E as protectiveantioxidants in human cancers. Annual Review of Nutrition 12, 139–159.

Cancalon, P.F., 2001. Routine analysis of ascorbic acid in citrus juice using capillaryelectrophoresis. Journal of the Association of Official Analytical ChemistsInternational 84, 987–991.

Chebrolu, K.K., Jayaprakasha, G.K., Yoo, K.S., Jifon, J.L., Patil, B.S., 2012. Animproved sample preparation method for quantification of ascorbic acidand dehydroascorbic acid by HPLC. LWT Food Science and Technology 47,443–449.

Christen, W.G., Glynn, R.J., Sesso, H.D., Kurth, T., MacFadyen, J., Bubes, V., Buring, J.E.,Manson, J.E., Gaziano, J.M., 2010. Age-related cataract in a randomized trial ocvitamins E and C in men. Archives of Ophthalmology 128, 1397–1405.

Davey, M.W., Dekempeneer, E., Keulemans, J., 2003. Rocket-powered high-perfor-mance liquid chromatographic analysis of plant ascorbate and glutathione.Analytical Biochemistry 316, 74–81.

Davies, M.B., Austin, J., Partridge, D.A., 1991. Vitamin C: Its Chemistry and Biochem-istry. Royal Society of Chemistry, Cambridge, UK.

DeVries, J.W., 1983. Semiautomated fluorometric method for the determination ofvitamin C in foods: collaborative study. Journal of the Association of OfficialAnalytical Chemists 66, 1371–1376.

Deutsch, M.J., Weeks, C.E., 1965. Microfluorometric assay for vitamin C. Journal ofthe Association of Official Analytical Chemists 48, 1248–1256.

Drivelos, S., Dasenaki, M.E., Thomaidis, N.S., 2010. Determination of isoascorbic acidin fish tissue by hydrophilic interaction liquid chromatography-ultravioletdetection. Analytical and Bionalytical Chemistry 397 2199-2110.

Eitenmiller, R.R., Ye, L., Landen Jr., W.O., 2008. Ascorbic acid: vitamin C. In:Eitenmiller, R.R., Ye, L., Landen, Jr., W.O. (Eds.), Vitamin Analysis for the Healthand Food Sciences. 2nd ed. CRC Press, Boca Raton, FL, USA, pp. 231–289.

Ermer, J., Burgess, C., Miller, M., 2005. Performance parameters, calculations andtests. In: Ermer, J., Burgess, C., Miller, M. (Eds.), Method Validation in Pharma-ceutical Analysis. Wiley-VCH, Weinheim, Germany, pp. 21–194.

Fediuk, K., Hidiroglou, N., Madere, R., Kuhnlein, H.V., 2002. Vitamin C in Inuittraditional food and women’s diets. Journal of Food Composition and Analysis15, 221–235.

Finglas, P.M., van den Berg, H., de Froidmont-Gortz, I., 1997. The Certification of theMass Fractions of Vitamins in Three Reference Materials: Margarine (CRM 122),Milk Powder (CRM 421) and Lyophilized Brussels Sprouts Powder (CRM 431).Report EUR 18039 EN. Luxembourg, Belgium: Office for Official Publications ofthe European Communities.

Food and Nutrition Board, Institute of Medicine, 2000. Dietary Reference Intakes forVitamin C, Vitamin E, Selenium, and Carotenoids. National Academies Press,Washington, DC, USA Available online: http://www.iom.edu/Activities/Nutri-tion/SummaryDRIs/$/media/Files/Activity%20Files/Nutrition/DRIs/5_Sum-mary%20Table%20Tables%201-4.pdf. Accessed 22.12.11.

Garrido-Frenich, A., Hernandez Torres, M.E., Belmonte Vega, A., Martinez Vidal, J.L.,Plaza Bolanos, P., 2005. Determination of ascorbic acid and carotenoids in foodcommodities by liquid chromatography with mass spectrometry detection.Journal of Agricultural and Food Chemistry 53, 7371–7376.

Gokmen, V., Kahraman, N., Demir, N., Acar, J., 2000. Enzymatically validated liquidchromatographic method for the determination of ascorbic and dehydroascor-bic acid in fruit and vegetables. Journal of Chromatography A 881, 309–316.

Haytowitz, D.B., Pehrsson, P.R., Holden, J.M., 2008. The National Food and AnalysisProgram: a decade of progress. Journal of Food Composition and Analysis 21(Suppl. 1), S94–S102.

Hemila, H., 2011. The effect of vitamin C on the common cold. Journal of PharmacyPractice 24, 241–242.

Hernadez, Y., Lobo, M.G., Gonzalez, M., 2006. Determination of vitamin C in tropicalfruits: a comparative evaluation of methods. Food Chemistry 96, 654–664.

Hibbert, D.B., 2007. Method validation. In: Quality Assurance for the AnalyticalChemistry Laboratory, Oxford University Press, Oxford, UK, pp. 227–261.

Honarbakhsh, S., Schachter, M., 2009. Vitamins and cardiovascular disease. BritishJournal of Nutrition 101, 1113–1131.

Horwitz, W., 2003. Validation: an invisible component of measurement. AOACInternational, Gaithersburg, MD. Retrieved January 16, 2009 from: http://www.aoac.org/dietsupp6/Dietary-Supplement-web-site/HorwitzValid.pdf.

Jacob, R.A., Sotoudeh, G., 2002. Vitamin C function and status in chronic disease.Nutrition in Clinical Care 5, 66–74.

Johnston, C.S., Steinberg, F.M., Rucker, R.B., 2007. Ascorbic acid. In: Zempleni, J., R-ucker, R.B.,McCormick, D.B.,Suttie, J.W. (Eds.),Handbook of Vitamins. 4th ed. CRCPress, Boca Raton, FL, USA, pp. 489–520.

Laur, M., Tian, L., 2011. Provitamin A and vitamin C contents in selected California-grown cantaloupe and honeydew melons and imported. Journal of Food Com-position and Analysis 24, 194–201.

Lee, H.S., Coates, G.A., 1999. Vitamin C in frozen, fresh squeezed, unpasteurized,polyethylene-bottled orange juice: a storage study. Food Chemistry 65,165–168.

Lee, S.K., Kader, A.A., 2000. Preharvest and postharvest factors influencing vitamin Ccontent of horticultural crops. Postharvest Biology and Technology 20,207–220.

Levine, M., Dhariwal, K.R., Welch, R.W., Wang, Y., Park, J.B., 1995. Determination ofoptimal vitamin C requirements in humans. American Journal of ClinicalNutrition 62, 347S–1356S.

Levine, M., Espey, M.G., Chen, Q., 2009. Losing and finding a way at C: new promisefor pharmacologic ascorbate in cancer treatment. Free Radical Biology andMedicine 47, 27–29.

Li, Y., Schellhorn, H.E., 2007. New developments and novel therapeutic perspectivesfor vitamin C. Journal of Nutrition 137, 2171–2184.

Lykkesfeldt, J., 2000. Determination of ascorbic acid and dehyroascorbic acid inbiological samples by high-performance liquid chromatography using subtrac-tion methods: reliable reduction with tris[2-carboxyethyl]phosphine hydro-chloride. Analytical Biochemistry 282, 89–93.

M.T. Tarrago-Trani et al. / Journal of Food Composition and Analysis 26 (2012) 12–2524

Lykkesfeldt, J., Poulsen, H.E., 2010. Is vitamin C supplemention benefeicial? Lessonslearned from randomized controlled trials. The British Journal of Nutrition 103,1251–1259.

McCoy, L.F., Bowen, M.B., Xu, M., Chen, H., Schleicher, R.L., 2005. Improved HPLCassay for measuring serum vitamin C with 1-methyluric acid used as anelectrochemically active internal standard. Clinical Chemistry 51, 1062–1064.

Musulin, R.R., King, C.G., 1936. Metaphosphoric acid in the extraction and titrationof vitamin C. Journal of Biological Chemistry 116, 409–413.

Nishiyama, I., Yamashita, Y., Yamanaka, M., Shimohashi, A., Fukuda, T., Oota, T.,2004. Varietal difference in vitamin C in the fruit of kiwifruit and other Actinidiaspecies. Journal of Agricultural and Food Chemistry 52, 5472–5475.

Novakova, L., Solich, P., Solichova, D., 2008a. HPLC methods for simultaneousdetermination of ascorbic and dehydroascorbic acids. TrAC Trends in AnalyticalChemistry 27, 942–958.

Novakova, L., Solichova, D., Pavlovicova, S., Solich, P., 2008b. Hydrophilic interactionliquid chromatography method for the determination of ascorbic acid. Journalof Separation Science 31, 1634–1644.

Odriozola-Serrano, I., Hernandez-Jover, T., Martin-Belloso, O., 2007. Comparativeevaluation of UV-HPLC methods and reducing agents to determine vitamin C infruits. Food Chemistry 105, 1151–1158.

Phillips, K.M., Patterson, K.Y., Rasor, A.R., Exler, J., Haytowitz, D.M., Holden, J.M.,Pehrsson, P.R., 2006. The role of quality control and reference materials in thenational food and nutrient analysis program. Analytical and BioanalyticalChemistry 384, 1341–1355.

Phillips, K.M., Tarrago-Trani, M.T., Gebhardt, S.E., Exler, J., Patterson, K.Y., Haytowitz,D.B., Pehrsson, P.R., Holden, J.M., 2010. Stability of vitamin C in frozen raw fruitand vegetable homogenates. Journal of Food Composition and Analysis 23,253–259.

Remmers, P., Booij, M.H., Bergevoet, M.E., 1968. The vitamin C level of potatoproducts heated in oil. The determination of vitamin C in the presence ofreductones. International Journal of Vitamins and Nutrition Research 38,392–408.

Romeu-Nadal, M., Morera-Pons, S., Castellote, A.I., Lopez-Sabater, M.C., 2006. Rapidhigh-performance liquid chromatographic method for vitamin C determination

in human milk versus enzymatic method. Journal of Chromatography B 830,41–46.

Smirnoff, N., 2000. Ascorbic acid metabolism and functions of a multifacetedmolecule. Current Opinion in Plant Physiology 3, 229–235.

Spınola, V., Mendes, B., Camara, J.S., Castilho, P.C., 2012. An improved and fastUHPLC-PDA methodology for determination of L-ascorbic and dehydroascorbicacids in fruits and vegetables. Evaluation of degradation rate during storage.Analytical and Bioanalytical Chemistry, doi:10.1007/s00216-011-5668-x pub-lished online Jan 6, 2012. Accessed 10.01.12.

Taylor, J.K., 1987. Quality Assurance of Chemical Measurements. Lewis Publishers,Chelsea, MI, USA.

Trainer, D., Pehrsson, P.R., Haytowitz, D.B., Holden, J.M., Phillips, K.M., Rasor, A.S.,Conley, N.A., 2010. Development of sample handling procedures for foods underUSDA’s National Food and Nutrient Analysis Program. Journal of Food Compo-sition and Analysis 23, 843–851.

U.S. Department of Agriculture, Agricultural Research Service, 2011. USDA NutrientDatabase for Standard Reference, Release 24. Retrieved January 11, 2012 fromthe Nutrient Data Laboratory Home Page: http://www.nal.usda.gov/fnic/food-comp/search/.

Vanderslice, J.T., Higgs, D.J., Block, G., 1990. Ascorbic acid and dehydroascorbic acidcontent of foods-as-eaten. Journal of Food Composition and Analysis 3, 105–118.

Vanderslice, J.T., Higgs, D.J., 1990. Separation of ascorbic acid, isoascorbic acid,dehydroascorbic acid and dehydroisoascorbic acid in food and animal tissue.Journal of Micronutrient Analysis 7, 67–70.

Vanderslice, J.T., Higgs, D.J., 1993. Quantitative determination of ascorbic, dehy-droascorbic, isoascorbic, and dehydroisoascorbic acids by HPLC in foods andother matrices. Journal of Nutritional Biochemistry 4, 184–190.

Wechtersbach, L., Cigic, B., 2007. Reduction of dehydroascorbic acid at low pH.Journal of Biochemical and Biophysical Methods 70, 767–772.

Willcox, B.J., Curb, J.D., Rodriguez, B.L., 2008. Antioxidants in cardiovascular healthand disease: key lessons from epidemiologic studies. American Journal ofCardiology 101, 75D–86D.

Wilson, J.X., 2005. Regulation of vitamin C transport. Annual Review of Nutrition 25,105–125.

M.T. Tarrago-Trani et al. / Journal of Food Composition and Analysis 26 (2012) 12–25 25