Pertanika 11(1), 39-44 (1988)

Determination of Vitamin C in Fresh Fruits and VegetablesUsing the Dye-titration and Microfluorometric Methods

E.S. TEE, S.I YOUNG, SJC. HO* and S. SITI MIZURADivision of Human Nutrition,

Institute for Medical Research,Kuala Lumpur, Malaysia

Key words: Vitamin C; ascorbic acid; dehydroascorbic acid; dye-titration; microfluorometry; fruits;vegetables

ABSTRAK

Kandungan vitamin C dalam 19 jenis buah-buahan segar dan 24 sayur-sayuran telah dikaji dengankaedah-kaedah rasmi AOAC, iaitu titratan pewarna dan mikrofluorometrik. Seperti dijangkakan, hasilyang telah diperoleh daripada kaedah kedua, yang menentukan asid askorbik dan asid dehidroaskorbikfDHAA), 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 diantarakedua-dua kaedah tidak bererti (p < 0.05). Jika hanya kadar asid askorbik diperlukan, kaedah titratandapat memberi hasil vang memuaskan, dan ia dapat dijalankan dengan menggunakan peralatan makmalyang mudah. Sebaliknya, jika alat fluorometer diperoleh, kadar jumlah vitamin C boleh ditentukan, daniniadalah lebih berguna dari segi pemakanan.

ABSTRACT

The vitamin C content of 19 types of fresh fruits and 24 vegetables was determined by the officialAOAC methods of dye-titration and microfluorometry. As expected, values obtained by the latter me-thod, 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 of DHAA in the foods. Largerdifferences were obtained for the vegetables. The mean recovery value obtained by the dye-titrationmethod was significantly higher than that given by the fluorometric method (p < 0.01). Reproducibilitystudies showed that the two methods did not give significantly different variances (p < 0.05). If onlyascorbic acid values were required, the titrimetric procedure would give good results, and it may be carriedout rapidly using simple laboratory equipment. If a fluorometer was available, total vitamin C values,which would be more useful from the nutritional point of view, could be determined.

INTRODUCTION factor. Chemically, it is a six carbon sugar, with aVitamin C or ascorbic acid is a water-soluble diol grouping at carbons 2 and 3 which is readilyvitamin, well recognized as an anti-scorbutic food oxidised to a diketo group to form dehydroas-

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

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

corbie acid (DHAA) (Bender, 1982). Its readyoxidation to DHAA is the most prominent che-mical 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, butsmall 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 al. (1982) and Pachlaet al. (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 (Sauberlich et al.,1982). 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 essen-tially on the chemical reactions of ascorbic acidand DHAA (Cooke and Moxon, 1981).

The Association of Official Analytical Che-mists (AOAC) has designated two official methodsfor the determination of vitamin C: the dye-titra-tion method and the microfluorometric method(AOAC, 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 dehydroa-scorbic acid to form a fluorescent quinoxalinecompound which is measured in a fluorometer.

This laboratory has been employing thedye-titration method using DCIP for the deter-mination 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 study concentrated on the analysis ofthese foods.

MATERIALS AND METHODS

Samples of fresh fruits and vegetables were pur-chased 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 re-ducing capacity of the extracts was then measuredby titrating with 2, 6-dichlorophenolindophenol(DCIP). 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 titration was de-tected when excess of the unreduced dye gave arose pink colour in acid solution. For sampleswhich yielded highly intense coloured extracts,repeated ether extraction was carried out to facili-tate detection of the end-point (details in TeeetaL, 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 de-ny droascorbic acid with Norit (active carbon).Aliquots were next reacted with a-phenylenedia-mine 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 me-thod, recovery studies were carried out by adding10 mg of ascorbic acid to the food being analysed,and treating in the same manner as the food sam-ple. Mean recovery values for the two methodswere compared using t-test. Reproducibility stu-dies were also studied using 4 types each of fruitsand vegetables, and carrying out six analyses on

40 PERTANIKA VOL. 11 NO. 1, 1988

DETERMINATION OF VITAMIN C IN FRESH FRUITS AND VEGETABLES

TABLE 1Vitamin C content of fresh fruits (mg per 100 g edible portion)

Pear, yellow (Pyrus sinensis)Apple red (Pyrus malusjGrapes, green (Vitis vinifera)Coconut, young (Cocos nuciferajApple, green (Pyrus malus)Watermelon, red (Citrullus lanatus)Water apple (Eugenia aquea)Banana (Pisang nangkaj (Musa paradisiaca)Orange, local (Citrus suhuiensisjPineapple (Ananas comosus)Sapodilla (Achras zapota)Starfruit (Averrhoa carambola)Lime (Limau nipis) (Citrus aurantifolia)Orange (Citrus nobilis)Lemon (Citrus limonjMango (Mangifera indica)Papaya (Carica papaya)Rambutan (Nephelium lappaceum)Guava, big (Psidium guajava)

Dye-titrationmethod(1)

1.6 ± 0.02.2 ± 0.1 a ' b

2.9 ± 0.05.0 ± 0.05.1 ± 0.25.2 ± 0.06.4 ± 0.0a

10.2 ± 0.412.2 ± 0.214.3 ± 1.0D

18.9 ± 1.125.4 ± 1.8b

41.7 ± 1.242.7 ± 1.546.8 ± 0.056.5 ± 1.967.9 ± 0.669.8 ± 5.298.4 ± 10.9b

Microfluorometricmethod(2)

3.0 ± 0.13.6 ± 0.4*1.2 ± 0.14.9 ± 0.12.4 ± 0.05.4 ± 0.36.7 ± 0.1

28.0 ± 0.017.9+ 0.318.0+ l . l b

26.6 ± 2.439.4 ± 2.5b

41.3 ± 0.151.7 ± 2.260.2 ± 1.577.1 ± 0.564.3 ± 1.386.7 ± 2.8

101.4 ± 8.4b

Difference( 2 ) - ( l )

1.41.4

- 1 . 7- 0 . 1- 2 . 7

0.20.3

17.85.73.77.7

14.0- 0 . 5

9.013.420.6

- 3 . 616.9

3.0

end point of titration difficult to determine due to coloured extracts

mean of 6 determinations;all others, mean of duplicate analyses

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

RESULTS AND DISCUSSION

A total of 43 types of fresh fruits and vegetableswere studied. Vitamin C content of 19 types offruits is tabulated in Table 1, whilst those for the24 fresh vegetables are given in Table 2. Resultsin both tables are tabulated in ascending order ofthe vitamin content as obtained by the dye-titra-tion method.

It is clear from results presented in the tablesthat there was a definite difference between vita-min 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 ex-pected, since in the latter 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 ob-tained by the two methods (column 3 in bothtables) may then be taken as the 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, the difference inresults obtained by the two methods was foundto be higher. Only in 3 vegetables was the dif-ference 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 signi-ficantly higher than that for the fluorometricmethod (p < 0.01). Standard deviations and coef-

PERTANIKA VOL. 11 NO. I, 1988 41

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

TABLE 2Vitamin C content of fresh vegetables (mg per 100 g edible portion)

Dye-titrationmethod

(1)

0.7 ± 0.20.9 ± 0.01.4 ± 0.0a

1.4 ± 0.1.1.5 ± 0.2b

2.4 ± 0.1a

4.2 ± 0.15.4 ± 0.6*6.2 ± 0.9b

7.7 ± 0.19.6 ± 0.8b

102± 0.412.7 ± 0.713.0 ± 1.014.0 ± 0.517.2 ± 0.217.8 ± 0.7a

20.8+ 2.525.3 ± 1.927.5 ± 1.5a

46.6 ± 3.9b

53.2 ± 6.254.5 ± 5.3b

77.2 ± 6.1a

Microfluorometricmethod

(2)

11.9 ± 0.24.2 ± 0.15.1 ± 0.2

16.4 ± 0.713.2 ± 1.14.5 ± 0.29.2 ± 0.56.2 ± 0.1

15.1 ± l . l b

13.2 ± 0.310.2 ± 1.5b

16.7 ± 0.824.9 ± 1.015.4 ± 0.930.1 ± 3.221.7 ± 1.119.0 ± 1.528.6 ± 5.638.8 ± 1.761.1 ± 4.7

64.2 ± 5.6b

50.8 ± 9.683.3 ± 5.8b

1100.0+14.9

Difference(2)-(l)

11.23.33.7

15.011.72.15.00.88.95.50.66.5

12.22.4

16.14.61.37.8

13.633.6

17.6-2.428.822.8

Four-angled bean (Psophocarpus tetragonolobus)Lettuce (Letuca sativa)Brinjal (Solarium melongena)Cucumber (Cucumis sativus)Plantain flower (Musa sapientum)Carrot (Daucas carota)Angled Loofah (Luffa acutangula)Small onion (Allium fistulosum)Winter gourd (Benincasa hispida)Green bean sprout (Phaseolus aureus)Yam bean (Pachyrrhizus erosus)French bean (Phaseolus vulgaris)Potato (Solarium tuberosum)Chives, Chinese (Allium odorrumjLadies 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)

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

Number ofdeterminations

Mean ± SD

Coefficient ofvariation

Range

TABLE 3Recovery Values

Dye-titrationmethod

26

96.5 ±4.7%

4.8

84.6 - 108%

Microfluorometricmethod

25

91.4 ±8.2%

9.0

71.4 -106%

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

observed F-ratio was calculated to be 1.07. Thetwo methods did not give significantly differenivariances (p < 0.05).

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.y 1985), and therefore giving rise to falselyhigh titration results. Generally, interferences canbe overcome by adjusting the pH and other reac-tion conditions so that most other materials reactonly very much more slowly than does ascorbate(Bender, 1982).

Another major practical problem associatedwith the titrimetic method of DCIP is the diffi-culty in ascertaining end-point when the food ex-

42 PERTANIKA VOL. 11 NO. 1, 1988

DETERMINATION OF VITAMIN C IN FRESH FRUITS AND VEGETABLES

TABLE 4Variation in methods for the determination of vitamin C

Dye-titrationmethod

mean

6.29.6

46.654.5

2.214.325.498.4

±

+

±±±±+

±±

SD

0.90.83.95.30.11.01.8

10.9

cv1

14.48.68.39.64.56.87.2

11.1

Microfluorometricmethod

mean

15.110.264.283.33.6

18.039.4

101.4

± SD

± 1.1± 1.5± 5.6± 5.8± 0.4± 1.1± 2.5± 8.4

CV

Winter gourdYam beanBitter gourdCauliflowerApple, redPineappleStarfruitGuava,big

7.414.9

8.76.9

10.46.26.48.3

Each value is the mean of 6 analysesCoefficient of variation

tracts are coloured, especially reddish-purplishcolours. As indicated in Tables 1 and 2, this pro-blem was encountered for several fruits and vege-tables in this study. Even with the aid of ether todetermine the end-point, unsatisfactory resultshave been encountered. Furthermore, with theadditional ether extraction steps, the procedurebecame rather tedious.

The fluorometric method is a more lengthyprocedure, and requires the availability of a fluoro-meter. It is, however, less susceptible to inter-ference. The use of boric acid in sample blanksin order to take into consideration extraneousfluorescence under the same reaction conditionsis an important step in this procedure. Borateinhibits the reaction of ascorbate with the dia-mine, but does not prevent the formation offluorophore by other compounds.

CONCLUSION

The study has quantitated the differences in vita-min C values obtainable by the dye-titration andfluorometric methods for a number of fruits andvegetables. There is considerable variation in thesedifferences, depending on the amount of DHAApresent in the food.

It is clear that the use of the dye-titrationmethod would result in underestimation of totalvitamin C activity in a food. However, if onlyascorbic acid values are required, the titrimetricprocedure as described would give good results for

most samples. The exception would be those thatyield highly intense coloured extracts. Carefultitration using ether extraction should be carriedout for these foods. Attention should also be paidto ensure that no interfering substances are contri-buting to the titration results. In spite of thesepotential difficulties, the titration method hasbecome widely used due to its convenience. Rapiddeterminations may be carried out with simplelaboratory equipment. In the comparative studiesreported by Wills et ah (1983) and Bradbury andSingh (1986), there was fairly good agreement inascorbic acid levels estimated by the simple dye-titration method and the more sophisticated liquidchromatography procedure.

If a fluorometer was available, ascorbic acidplus DHAA values may be determined. It is, how-ever, a more lengthy procedure, and greater carehas to be given to the various steps invowed.Reproducibility studies have to be carried out,and effort made to minimize variations. Recoveryruns have to be undertaken frequently to monitorperformance. From the nutritional point of view,total 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.

PERTANIKA VOL. 11 NO. 1, 1988 43

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

ACKNOWLEDGEMENT

The authors thank Datuk (Dr.) Lim Teong Wah,Director, Institute for Medical Research for grant-ing permission to publish this work.

REFERENCES

ASSOCIATION OF OFFICIAL ANALYTICAL CHE-MISTS (1984): Official Methods of Analysis. 14thedn. (S. Williams, ed.), pp. 844-846. AOAC: Vir-ginia.

BENDER, D.A. (1982): Vitamin C. In: Vitamins inMedicine, Volume 2. 4th 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 South Pacific. / Fd ScL, 51:975-978, 987.

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

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

PACHLA, L.A., D.L. REYNOLDS and P.T. KISSINGER!(1985): Review of ascorbic acid methodology./Assoc. Off. Analyt Chem., 68:7-72

5AUBERLICH, H.E., M.D. GREEN and S.T. OMAYE(1982): Determination of ascorbic acid and dehydro-ascorbic acid. In: Ascorbic Acid: Chemistry, Meta-bolism, and Uses (PA. Seib and B.M. Tolbert, eds.),Advances in Chemistry Series 200, pp. 199-221.American Chemical Society: Washington, D.C.

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

TEE, E.S., S. SITI MIZURA, R. KULADEVAN, S.IYOUNG, 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.j

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. GREENFIELD(1983): Liquid chromatography, microfluorometry,and dye-titration determination of vitamin C in freshfruit and vegetables. /. Assoc. Off Anal Chem.,66:1377-1379.

(Received 29 September, 1981

44 PERTANIKA VOL. 11 NO. 1, 1988