determination of vitamin c in fresh fruits and vegetables

8/12/2019 Determination of Vitamin C in Fresh Fruits and Vegetables

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Pertanika 11 1 ,39-44 1988

Determination of Vitamin in Fresh Fruits n VegetablesUsing the Dye titration and Microfluorometric MethodsE.S. TEE, S.I YOUNG, S.K. HO* and SITI MIZURA

Division ofHuman Nutrition,Institute for Medical Research,

Kuala Lumpur, MalaysiaKey words: Vitamin C; ascorbic acid; dehydroascorbic acid; dye-titration;microfluorometry; fruits;

vegetablesABSTRAK

Kandungan vitamin C dalam 19 jenis buah-buahan seg r dan 24 sayur-sayuran telah dikaji aengan.kaedah-kaedah rasmi AOAC, iaitu titratan pewarna dan mikrofluorometrik. Seperti dijangkakan, hasilyang telah diperoleh daripada kaedah kedua, yang menentukan asid askorbik dan asid dehidroaskorbikDHAA , adalah lebih tinggi daripada yang didapati daripada kaedah titratan, yang hanya menentukanasid askorbik. Terdapat perbezaan yang agak besar pada hasil-hasil yang diperoleh daripada kedua-duakaedah, bergantung kepada kandungan DHAA dalam makanan. Perbezaan yang lebih besar telah didapatipada sayur-sayuran. Min hasil bilas daripada kaedah titratan adalah lebih tinggi daripada yang diberi olehkaedah fluorometrik p 0.01 . Kajian kebolehulangan menunjukkan bahawa perbezaan varians di antarakedua-dua kaedah tidak bererti p 0.05 . Jika hanya kadar asid askorbik diperlukan, kaedah titratandapat memberi hasil yang memuaskan, dan i dapat dijalankan dengan menggunakan peralatan makmalyang mudah. Sebaliknya, jika alat fluorometer diperoleh, i d r jumlah vitamin C boleh ditentukan, daniniadalah lebih berguna dari segi pemakanan.

ABSTRACTThe vitamin C content of19 types of fresh fruits and 24 vegetables w s determined by the official

O Cmethods of dye-titration and microfluorometry. s expected, values obtained by the latter method, which estimated ascorbic acid plus dehydroascorbic acid DHAA , were clearly higher than thosegiven by the titration method, which determined only ascorbic acid. There were considerable differencesin the values obtained by the two methods, depending on the concentration ofDHAA in the foods. Largerdifferences were obtained for the vegetables. The mean recovery value obtained by the dye-titrationmethod w s significantly higher than that given by the fluorometric method p0.01 . Reproducibilitystudies showed that the two methods did not give significantly different variances p 0.05 . I f onlyascorbic acid values were required, the titrimetric procedure would give good results, and it may be ca iedout rapidly using simple laboratory equipment. I f a fluorometer w s available, total vitamin values,which would be more useful from the nutritional point of view, could be determined.

INTRODUCTIONVitamin C or ascorbic acid is a water-solublevitamin, well recognized as an anti-scorbutic food

factor. Chemically, i t is a six carbon sugar, with adiol grouping at carbons 2 and 3 which is readilyoxidised to a diketo group to form dehydroas-

School of Arts and Science, Tunku Abdul Rahman College, Kuala Lumpur.


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E.S. TEE, S.I YOUNG, S .K . HO AND S. SIT MIZURAcorbic acid DHAA) Bender, 1982). Its readyoxidation to DHAA is the most prominent chemical property of the vitamin.

The two biologically active forms of vitaminC present naturally in foods are L-ascorbic acidand L-dehydroascorbic acid DHAA). The formmost often encountered is L-ascorbic acid, bu tsmall amounts of its primary oxidation product,DHAA, may also be present Christie and Wiggins,1978).Procedures for the determination of vitaminC in foods have continued to draw the attentionof analysts. Recent reviews in this subject includethose of Christie and Wiggins 1978), Cooke andMoxon 1981), Sauberlich et ai 1982) and Pachlaet ai 1985) . The complex biological relationshipbetween the compound s) possessing vitamin Cactivity, as well as the chemical similarity of thesecompounds to others that are inactive, has madethe existence of a single, simple, and specificmethod close to impossible Sauberl ich et ai1982). Thus, there has been a proliferation ofmethods developed, resulting in hundreds andpossibly thousands of papers reported. However,they mostly describe minor variations on a fairlylimited range of possible procedures, based essentially on the chemical reactions of ascorbic acidand DHAA Cooke and Moxon, 1981).The Association of Official Analytical Chemists AOAC) has designated two official methodsfor the determination of vitamin C: the dye-titration method and the microfluorometric methodAOAC, 1984) . The former method makes use ofthe reducing power of the vitamin, and employs2, 6-dichlorophenolindophenol DCIP) as theredox indicator for the determination of ascrobicacid. In the micro-fluorometric method, ascorbicacid is oxidised with active carbon to dehydroascorbic acid to form a fluorescent quinoxalinecompound which is measured in a fluorometer.

This laboratory has been employing thedye-titration method using DCIP for the determination of ascorbic acid, values of which havebeen reported in the Food Composition Tablepublished by the Division Tee, 1985). Due toproblems encountered with the method, mainlywith foods which yielded coloured extracts e.g.fruits and vegetables), an initial study was carriedout on the microfluorometric method. The foodswere also analysed simultaneously using the dye-

titration procedure. This paper reports the resultsobtained from the study. It serves to demonstrateand quantitate the differences in vitamin C valuesobtained by the two methods for a variety oflocally available foods. Since fruits and vegetablesare the major sources of the vitamin in mostdiets, the stuqy concentrated on the analy sis ofthese foods.

MATERIALS AND METHODSSamples of fresh fruits and vegetables were purchased from the local markets for analysis. Edibleportions of each food sample were analysed byboth methods, in duplicate, on the same day ofpurchase.

The dye-titration method used was essentiallythat of the AOAC procedure AOAC, 1984).Metaphosphoric acid extracts of the foods wereprepared and pH adjusted to about 1.2. The reducing capacity of the extracts was then measuredby titrating with 2, 6-dichlorophenolindophenolDCIP). In this oxidation-reduction reaction,ascorbic acid in the extract was oxidised to DHAAand the indophenol dye reduced to a colourlesscompound. End point of the titra tion was detected when excess of the unreduced dye gave arose pink colour in acid solution. For sampleswhich yielded highly intense coloured ext racts ,repeated ether extraction was carried out to facilitate detec tion of the end-point details in Teeet ai 1987

In the microfluorometric method also asgiven in AOAC, 1984), food samples were alsofirst extracted into metaphosphoric acid solutions.Ascorbic acid in the extracts was oxidised to dehydroascorbic acid with Norit active carbon).Aliquots were next reacted with a-phenylenediamine to give a fluorescent quinoxaline derivative,which, on activation at 350 nm, fluoresces at 430nm. For each food sample analysed, a specificsample blank was carried out by the addition ofboric acid.

For each batch of analyses using either method, recovery studies were carried ou t by adding mg of ascorbic acid to the food being analysed,and treating in the same manner as the food sample. Mean recovery values for the two methodswere compared using t-test. Reproducibility studies were also studied using 4 types each of fruitsand vegetables, and carrying ou t six analyses on

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DETERMINATION OF VITAMIN C IN FRESH FRUITS AND VEGETABLESTABLE 1

Vitamin C content of fresh fruits mg per 100 g edible portion)Dye-titrationmethod1

Microfluorometricmethod2)Difference2) - 1)

Pear, yellow Pyrus sinensisApple red Pyrus mJ11usGrapes, green Vitis viniferaCoconut, young CocosnuciferaApple, green Pyrus mJ11usWatermelon, red Citru[[us lanatusWater apple Eugenia aqueaBanana PiSllng nangka MuSIl paradisiacalOrange, local Citrus suhuiensisPineapple Ananas comosusSapodilla Achras zapotaStarfruit Averrhoa carambolaLime LimJ1u nipis Citrus aurantifoliaOrange Citrus nobilisLemon Citrus limonMango Mangifera indicaPapaya Carica papayaRambutan Nephelium lappaceumGuava, big Psidium guajava

1.6 2.2 2.9 5.0 5.1 5.2 6.4 10.2 12.2 14.3 18.9 25.4 41.7 42.7 46.8 56.5 67.9 69.8 98.4

0.00.1a,b0.00.00.20.0O Oa0.40.21.0l.11.81.21.50.01.90.65.210.9

3.0 0 13.6 0.4b1.2 0.14.9 0.12.4 0.05.4 0.36.7 0 128.0 0.017.9 0.318.0 b26.6 2.439.4 2.5b41.3 0 151.7 2.260.2 1.577.1 0.564.3 1.386.7 2.8101.4 8,4b

1.41.4-1.7- 0 1- 2.70.20.317.85.73.77.714.0- 0.59.013,420.6- 3.616.93.0

aend point of titration difficult to determine due to coloured extractsbmean of 6 determinations; all others, mean of duplicate analyses

each food sample . Analytica l process standarddeviations of the two methods were comparedusing the F-ratios method Wernimont, 1985).

RESULTS AND DISCUSSIONA total of 43 types of fresh fruits and vegetableswere studied. Vitamin C content of 19 types offruits is tabulated in Table I, whilst those for the24 fresh vegetables are given in Table 2. Resultsin both tables are tabulated in ascending order ofth e vitamin content as obtained by the dye-titrat ion method.

is clear from results presented in th e tablesthat there was a definite difference between vitamin C values obtained by the two procedures.With a few exceptions, values obtained by thefluorometric method were higher than thosegiven by the dye-titration method. This is to be expected, since in the latte r method , only reducedascorbic acid was determined. In the fluorometricmethod, the total ascorbate estimated was derivedfrom ascorbic acid as well as preformed DHAA

present in the food. The differences in values obtained by the two methods column 3 in bothtables) may then be taken as th e content of DHAApresent in the fruits and vegetables.

In the case of the fruits analysed, thereappeared to be no major difference in the vitaminC content determined by either method for 6types of fruits difference 10 ). In another 10types , the difference was between 17 to 50 ofthe total ascorbate content. In the remaining 3fruits, the difference was greater than 60 .For the vegetables studies, th e difference inresults obtajned by the two methods was foundto be higher. Only in 3 vegetables was the difference found to be < 10 . Twelve of them gavea difference of between 13-50 , while for theremaining 9, the difference was greater than 50 .

Recovery values obtained using both methodsare tabulated in Table 3. The mean recovery valueobtained for the dye-titration method was signif icantly higher than that for th e fluOTometricmethod p < 0.0 I) . Standard deviations and coef-

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E.S. TEE, S. l YOUNG, S.K. HO AND S. nMIZURATABLE 2Vitamin C content of fresh vegetables mg pe r 100 g edible portion)

Dye-titrationmethod1

Microfluorometricmethod2)

Difference2 1

o u r ~ n g l e bean Psophocarpus tetragonolobus)Lettuce Letuca sativa)Brinjal Solanum melongena)Cucumber Cucumis sativus)Plantain flower Musa sapientuCarrot Daucas carota)Angled Loofah Lutta acutangula)Small onion Allium fistulosum)Winter gourd Benincasa hispida)Green bean sprout Phaseolus aureus)Yam bean Pachyrrhizus erosus)French bean Phaseolus vulgaris)Potato Solanum tuberosum)Chives, Chinese Allium odorrum)Ladies finger Hibiscus esculentus)String bean Vigna sinensis)Tomato Lycopersicum esculentum)Garlic leaves Allium sativum)Cabbage Brassica oleracea)Spinach, red Amaranthus gangeticus sive tricolor)Bitter gourd Momordica charantia)Mustard leaf, Chinese Brassica juncea)Cauliflower Brassica oleracea)Tapioca leaves Manihot utilissima)

0.7 0.9 1.4 1.4 1.52.4 4.2 5.4 6.27.7 9.6 10.2 12.7 13.0 14.0 17.2 17.8 20.8 25.3 27.5 46.6 53.2 54.5 77.2

0.20.0.0.OaO.l b0.20 1 a0 1 a0.6b0.9O.lb0.80.40.71.00.5020.7a2.51.9l.5a

11.9 0.24.2 0.15.1 0.216.4 0.713.2 1.14.5 0.29.2 0.56.2 0.115.1 1.1b13.2 0.310.2 1.5b16.7 0.824.9 1.015.4 0.930.1 3.221.7 1.119.0 1.528.6 5.638.8 1.761.1 4.764.2 5.6b50.8 9.683.3 5.8b

1100.0 J.4.9

11.23.33.715.011.72.15.00.88.95.50.66.512.22.416.14.61.37.813.633.617.6-2.428.822.8

ficients of variation for the former method werealso smaller than those for the latter.

observed F -i atio was calculated to be 1.07. Thetwo methods did no t give significantly differentvariances p < 0.05).

Results of reproducibility studies are tabulated in Table 4. Neither of the two methodsgave consistently higher values for coefficients ofvariation. Comparing the standard deviations, the

Number ofdeterminations 26 25Mean SO 96.5 4.7 91.4 8.2Coefficient ofvariation 4.8 9.0Range 84.6 -108 71.4 -106

TABLE 3Recovery ValuesDye-titrationmethod Microfluorometricmethod

Since the dye-titration method is based on theoxidation-reduction reaction, a number of otherreducing substances in foods besides ascorbicacid) could interfere with the determination. Manymolecules e.g., phenols, sulphydryls, and triosereductones) and ions e.g., ferrous, cuprous, orsulphite) are able to reduce the DCIP dye Pachlaet al 1985), and therefore giving rise to falselyhigh titration results. Generally, interferences canbe overcome by adjusting the pH and other reaction conditions so that most other materials reactonly very much more slowly than does ascorbateBender, 1982).

Another major practical problem associatedwith the titrirnetic method of DCIP is the difficulty .in ascertaining e n p o i n ~ when the food ex-

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,DETERMINATION OF VITAMIN C IN FRESH FRUITS AND VEGETABLESTABLE 4Variation in methods for the determination of vitamin C

Dye-titration Microfluorometricmethod methodmean SD CV mean SD V

Winter gourd 6. 2 0.9 14.4 15.1 1.1 7.4Yam bean 9.6 0.8 8.6 10.2 1.5 14.9Bitter gourd 46.6 3.9 8.3 64.2 5.6 8.7Cauliflower 54.5 5.3 9.6 83.3 5.8 6.9Apple red 2.2 0.1 4.5 3.6 0.4 10.4Pineapple 14.3 1.0 6.8 18.0 1.1 6.2Starfruit 25.4 1.8 7.2 39.4 2.5 6.4Guava big 98.4 10.9 11.1 101.4 8.4 8.3

Each value is the mean of 6 analyses1Coefficient of variation

tra cts a re c oloure d especially reddish-purplishcolours. As indicated in Tables I and 2, this problem was e nc ountered for several fruits a nd vegetables in this s tu dy . Even wi th the aid of ether todetermine the end-point unsatisfa.ctory resultshave b een e nco un te re d. Furthermore, with thea dditiona l e ther extraction steps the procedurebecame rather tedious.

The fluorometric method is a more lengthyprocedure and requires the availability of a fluorometer. t is however less susceptible to interference. The use of bori c acid i n sample blanksin order to t ak e i nt o con sid erat io n e xt ra ne ou sfluorescence under the same reaction conditions an important step in this procedure. Borateinhibits the reaction of ascorbate with the diamine but does not prevent the formation offluorophore by other compounds.

ON LUS ONThe study has quantitated the differences in vitamin C values o bt ai na bl e by the dye-titration andf lu or omet ri c m et ho ds f or a number of fruits andvegetables. There is considerable variation in thesedifferences depending on the amount of DHAApresent in the food.

t is clear that the use of the dye-titrationmethod would result in underestima tion of totalvitamin C activity in a food. However if onlyascorbic acid values are r ~ q u i r e the titrimetricprocedure as described would give good results for

mo st samples. T he ex ce pt io n woul d be t ho se thatyield highly intense coloured extracts. Carefultitration using ether extraction should be carriedou t for the se foods. Attention should also be paidto ensure that no interfering substances are contributing to the titration results. I n spite of thesepotential difficulties the t itration method hasbec om e widely used due to its convenience. Rapiddeterminati ons may be carried ou t with simplelaboratory e quipme nt. In the comparative studiesreported by Wills l 1983 a nd B ra dbury andSingh 1986 , t he re was fairly g oo d agreement inascorbic acid levels estimated by the simple dyetitration method and the more sophisticated liquidchromatography procedure.

a fluorometer was available ascorbic acidplus DHAA values m ay be determined. t is however a m ore lengthy proce dure a nd grea te r carehas to be given to the various steps involved.Reproducibility studies have to be carried out,and effort made to minimize variations. Recoveryruns have to be underta ke n fre quently to monitorperformance. From the nutritional point of viewtotal vitamin C values would be more useful.

Either method may be used depending on theobjectives and resources available to the analyst.The important point is that in reporting vitamin Cvalues what the analytical data represents must beclearly stated.

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E.S. TEE, S.I YOUNG, S.K. HO AND S. SITI MIZURA KNOWLEDGEMENT

The authors thank Datuk Dr. Lim Teong Wah,Director, Institute for Medical Research for granting permission to publish this work.

REFERENCESASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS 1984 : Official Methods of Analysis. 14th

edn. S. Williams, ed. , pp. 844-846. AOAC: Virginia.

BENDER, D.A. 1982 : Vitamin C In: Vitamins inMedicine, Volume 2 4t h edn. B.M. Barker andD.A. Bender, eds. , pp. 1-68. William HeinemannMedical Books Ltd: London.

BRADBURY J.H. and U SINGH 1986 : Ascorbic acidand dehydro-ascorbic acid content of tropical rootcrops from the Sou th Pacific. Fd Sci., 51 :975978,987.

CHRISTIE, A.A. and R.A. WIGGINS 1978 : Developments in vitamin analysis. In: Developments inFood Analysis Techniques - R.D. King, ed. ,pp. 18-23, Applied Science Publishers Ltd: London.

COOKER, J .R. and R.E.D. MOXON 1981 : The detection and measurement of vitamin C In: Vitamin CAscorbic Acid J.N. Counsell and D.H. Hornig,eds. , pp. 167-198. Applied Science: London.

PACHLA, L.A., D.L. REYNOLDS and P.T. KISSINGER1985 : Review of ascorbic acid methodology. Assoc. Of Analyt Chern., 68:1 12.

SAUBERLICH, H.E., M D GREEN and S.T. OMAYE1982 : Determination of ascorbic acid and dehydroascorbic acid. In: Ascorbic Acid: Chemistry, Metabolism, and Uses P.A. Seib and B M Tolbert, eds. ,Advances in Chemistry Series 200, pp. 199-221.American Chemical Society: Washington, D.C.

TEE E.8. 1985 : Nutrient Composition of MalaysianFoods - A Preliminary Table First Update .ASEAN Sub-Committee on Protein: Kuala Lumpur.

TEE, E.8., S SITI MIZURA, R KULADEVAN, S.l.YOUNG, S.c . KHOR and S K CHIN eds. 1987 :Laboratory Procedures in Nutrient Analysis ofFoods, pp. 47-54. Division of Human Nutrition,Institute for Medical Research: Kuala Lumpur.

.WERNIMONT, G.T. 1985 : Use of Statistics to Developand Evaluate Analytical Methods. Association ofOfficial Analytical Chemists: Virginia.

WILLS, R.B.H., P WIMALASIRI and H GREENFIELD1983 : Liquid chromatography, microfluorometry,and dye-titration determination of vitamin C in freshfruit and vegetables. Assoc. Of Anal Chern66:1377-1379.

Received 29 September, 1987

44 PERTANIKA VOL. II NO.1, 1988

determination of vitamin c in fresh fruits and vegetables

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