Riboflavin-Sensitized Photodynamic UV Spectrophotometry for Ascorbic Acid Assay in Beverages

M.Y. JUNG, SK. KIM, and S.Y. KIM

analysis. Alkaline treatment is a rapid and simple method for determination of ascorbic acid (Fung and Luk, 1985a,b). HowRiboflavin-sensitized photodynamic ultraviolet spectrophotometric assay of various commercial beverages was carried out. The maximum con- ever, this method is less sensitive because it is based on abcentration of ascorbic acid for obtaining a background correction (15 sorbance at 243 nm instead of 265 nm for quantitation of min illumination and pH 7.5) was 2 1 l.rg/mL ascorbic acid in the testing ascorbic acid. In addition, the calibration curve was not straight solution. The upper limit of the measurementrange for a straight line in at low concentrations of ascorbic acid. the calibration graph of standard ascorbic acid was 10 pg/mL, (working Ascorbic acid reportedly oxidized extremely fast in the presrange O-10 pg/mL). The results in repeatability of the method for as- ence of photosensitizers and light (Sattar et al., 1977; Bodannes corbic acid contents in commercial fruit juices and soft-drinks showed and Chan, 1979; Chou and Khan, 1983; Rooney, 1983; Jung et a maximum of 4% relative standard deviation. Recovery tests with al., 1994). Our previous results showed that 1.2 X 10-4M asknown amounts of added ascorbic acid in different fruit juices and sports corbic acid oxidized completely after a 12 min storage in the drink showed the recovery of added ascorbic acid was 97.5-102.3%. Indophenol, iodine and/or HPLC methods were used in parallel to as- presence of 6.0 pg/mL riboflavin under 3300 lux fluorescent certain the reliability of the proposed method. This type assay could be light because of the formation of singlet oxygen which is very successfully applied to many commercial beverages for determination of reactive toward ascorbic acid (Jung et al., 1994). The bimolecular rate constant for reaction of ascorbic acid with singlet oxascorbic acid with good accuracy, precision and reliability. ygen at pH 7.5 was 6.63 X lo* M-‘ set-r (Jung et al., 1994). Key Word: Ascorbic acid, photodynamic assay, riboflavin sensitized, Thus, we expected that a rapid background correction for direct fruit juices, sports drink UV measurement of ascorbic acid could be achieved by photodynamic singlet oxygen formation. Thus, our objective was to establish a rapid riboflavin-sensitized photodynamic UV specINTRODUCTION trophotometric assay for ascorbic acid in beverages. ASCORBIC ACID occurs naturally in fruits and vegetables and is often added to fruit juices and sports drinks. Since it is easily MATERIALS & METHODS oxidized during processing, handling and storage, the ascorbic Materials acid content may be used as an index of quality. Commonly used methods for determining ascorbic acid in foods are indoL-ascorbic acid and riboflavin were purchased from Sigma Chemical phenol method, iodine method and HPLC method. The indo- Co. (St. Louis, MO). Serum bottles were obtained from Supelco Inc., phenol method, based on titrimetry using the reducing power of (Bellefonte, PA). HPLC grade acetonitrile was obtained from Fisher Sciascorbic acid, can not be used for samples containing reductants entific Co. (Fair Lawn, NJ). Commercial beverageswere purchased from or for colored samples. Iodine method has the disadvantage of local groceries. redox reagent interference. High performance liquid chromatography (HPLC) has been used for ascorbic acid assay (Doner and Comparison of photochemical methods for making background Hicks, 1981; Rose and Nahrwold, 1981; Bradbury and Singh, corrections 1986; Vanderslice and Higgs, 1993). However, the analytical To compare photochemical methods of riboflavin and methylene blue, conditions such as pre-treatment of samples, packing of columns and chemical H,O,-NaOCl treatment method for making background and mobile phase differ with samples, so that the optimum con- corrections,21 ug/mL ascorbicacid solutionscontaining6 ug/rnL of ABSTRACT
ditions must be carefully tested for each assay.

Thus, a rapid, simple and reliable method is needed for determination of ascorbic acid. Direct UV spectrophotometry can provide such a fast and simple method (Fung and Luk, 1985a). However, the absorption of UV light by the sample matrix is the major problem with this method. Various background correction techniques such as thermal degradation, UV decomposition, enzymatic or metal catalytic oxidation, and alkaline treatment have been proposed to solve this problem. The thermal, UV, and metal catalytic decomposition of ascorbic acid was too slow to be used practically (Fung and Luk, 1985a). In the enzymatic technique, enzymes used were ascorbate oxidase (Tono and Fujita, 1981, 1982; Esaka et al., 1985), ascorbate peroxidase (Kelly and Latzko, 1980) and guaiacol peroxidase (Casella et al., 1989; Tsumura, et al., 1993). Although the enzymatic methods are simple, rapid and highly specific for ascorbic acid, the enzymes are relatively costly for routine
Author Jung is with the Department of Food Science and Technology, Jeonju Woosuk University, Samrea-Up, Wanju-Kun, Jeonbuk Province, 566400, Republic of Korea. Authors S.K. Kim and S.Y. Kim are with the Dept. of Food Science & Technology, Chungnam National Univ., Daejeon, Republic of Korea.

riboflavin or methylene blue or containing 0.26M H,O, and 0.072M NaOCl were prepared. The photochemical methods of riboflavin and methylene blue and chemical H,O,-NaOCl treatment method were well established ways to generate singlet oxygen. Samples (20 mL) treated with riboflavin and methylene blue were transferred into serum bottles and, then, placed, at room temperature (-23°C) in a light storage box, described in detail previously (Fakourelis et al., 1987; Jung and Min, 1991; Jung et al., 1991). The light intensity at the sample level was 5500 lux. The sample prepared with H,O,-NaOCl was placed under dark. The content of ascorbic acid was determined by HPLC (Bradbury and Singh, 1986). Photodynamic background correction Riboflavin was chosen to achieve the background correction (sample blank) for a new direct UV analysis because of effectiveness in ascorbic acid destruction. To study the destruction of various concentrations of ascorbic acid, 9, 21, 35, 53 or 105 pg/mL ascorbic acid solutions containing 6 @mL riboflavin (PH 7.5) were prepared by addition of calculated amounts of ascorbic acid and riboflavin into O.OlM potassium buffer (pH 7.5). Prepared sample (20 mL) was transferred into serum bottles. The bottles were placed in the light storage box as described. The content of ascorbic acid during illumination was monitored by measuring absorbance at 265 nm using a spectrophotometer (Bodannes and Chan, 1979).

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OF FOOD SCIENCE-Volume

60, No. 2, 1995

Jung et al. Waters Associates. The pH of the diluted samples was checked. To study the destruction rates of ascorbic acid at different pH’ 21 s.OlM thiosulfate solution. and the sample was then transferred into a 50 mL or a 100 mL volumetric flask.Methylerie . OF FOOD SCIENCE-361 Volume SO. 2.5 with dilute NaOH solution. Waters Associates.0 mL of juice or drink was transferred into a 250 mL beaker and . A volume of filtrate equivalent to about 2 mg of ascorbic acid was then titrated with indophenol solution using the procedure described including titration of the blank.01 phosphate buffers of pH 7. No. Preparation of calibration graph To prepare the calibration graph of standard ascorbic acid.5) was added to the mark. if the samples contained high concentrations of suspendedsolids interfering with the absorptivity at 265 nm we recommend centrifuging or filtering before UV analysis.5) were prepared by addition of calculated amounts of ascorbic acid and riboflavin into 0.5 mL or 1 mL of riboflavin stock solution (0. 0.5.tg/mL ascorbic acid to be completely destroyed during a 15 min-illumination period. Differences in absorbance of samples before and after 15 min-illumination were used for calculation of ascorbic acid. Thus. . MA) was eouiDDedwith a UV detector (Waters Associate. 5. we used photochemical methods of riboflavin and methylene blue and chemical H. 21. 1986). 63 pgl mL and 105 pg/mL ascorbic acid after 15 min-illumination were 98. Indophenol method 60 20 t 01 I \ -A0 \ \ .-NaOCI treatment method to produce singlet oxygen. For the sample titration. respectively. The mobile phase (flow rate 1.OOSM) was standardized in the usual way with O. 63 and 105 pg/mL) during light storage (Fig. 35. The proposed direct UV method measures ascorbic acid but not dehydroascorbic acid because dehydroascorbic acid does not absorb light at 265 nm. For sample analysis.-HOAC solution plus a given amount of water equivalent to the volume of indophenol solution used in the previous standardization titration. The time required to destroy 9 pg/mL ascorbic acid was 12 min. Consumption of the blank was determined by titrating indophenol solution with 7 mL of HPO. MA). 83 and 74%. Six ug/mL riboflavin in O.-HOAc solution before filtering. The ascorbic acid destruction in the systems of 35 pg/mL. Rooney. l-Comparison of the destruction rates of ascorbic acid by photochemical methods of riboflavin and methylene blue.OlM phosphate buffer into the volumetric flask. Chou and Khan. 1983. The destruction rate by riboflavin sensitized method increased rapidly with decreasing concentrations of ascorbic acid.060g riboflavin in 100 mL of O.5.Riboflavin I I blue I The indophenol solution.OlM nhosuhate buffer (nH 7. We found that ascorbic acid in the prepared sample was completely destroyed after 15 min of light storage. 1986) for ascorbic acid determination.OlM phosphate buffer. pg/mL ascorbic acid solutions containing 6 pg/mL riboflavin @ H 7. 2). Thus it was selected for more detailed study for making the sample blank (background correction). Ten pL of sample was injected. The destruction of ascorbic acid by riboflavin sensitized photochemical method was fastest among the tested methods.5 using a spectrophotometer.40 mL or 90 mL of O. 0. it was adjusted to pH 7.l . of sample was mixed with an equal volume of HPO.0 mL of standard ascorbic acid solution and 5 mL of 3% metaphosphoric acid-8% acetic acid solution (HPO. 1983. The column used was jr-Bondapak C-18 (30 cm X 3. The ascorbic acid was quantitated at 265 nm by the UV detector.-NaOCI treatment method were compared (Fig. 50 rnt. Milford. If the pH was not 7. pH 7. 1995-JOLJRNAL . 12 commercial beverage samples by comparison with established methods.5 and 5. The content of ascorbic acid was determined by measuring absorbanceof solutions at 265 nm for samples at pH 7. and that to destroy 21 pg/mL ascorbic acid was 15 min under these conditions.5) was‘ added to the volumetric flask and then O.PO. Then. None of the commercial beveragesneided to be pretreated for removal of solid materials.O. Absorbances of samples before and after light storage were measured at 265 nm using a spectrophotometer. Three methods compared were the indophenol (AOAC. and chemical H. respectively.5. v/v). 1).80 Method reliability was tested on.5) were prepared by addition of calculated amounts of ascorbic acid and riboflavin into O.6 with dilute HCl and acetonitrile (30:70. 1979.5) was added to get the approximate ascorbic acid concentration of <lo ug/mL.5). The beaker was washed with O. 5 mL of sample. was standardized by titration with 2. HPLC method A high performance liquid chromatograph (Model 45..OlM phosphate buffer (PH 7.25 g/L of 2. 1994). Iodine method 0 5 10 Time 15 (min) 20 25 30 The iodine solution (O. After 15 min of illumination.3 mL/min) was aqueous 0. RESULTS & DISCUSSION Background correction (sample blank) Ascorbic acid is reportedly easily oxidized by the reaction with singlet oxygen (Bodannes and Chart.2-1.5. 1980).5 and 3.n H202-NaOCI . However. 1 1* Fig.OlM phosphate buffer (PH 7. and chemical H202-NaOCI treatment method. This result suggested the samples should have <21 J. O-16 pg/ mL of ascorbic acid solutions containing 6 pg/mL riboflavin (pH 7. The relative rates of decomposition of ascorbic acid by photochemical methods of riboflavin and methylene blue. Differences in absorbances of standard solutions and reagent blanks measured at 265 nm were plotted vs concentrations of ascorbic acids. As decomposition rate is dependent on initial concentration less time was required to destroy smaller amounts of ascorbic acid.5 because of addition of sample.5 and 3. or at 245 nm for samples at pH 3. The calibration graph prepared previously was used to determine the actual concentrations of ascorbic acid in the sample. 20 mL of water and 2 mL of 1% starch solution were titrated with standardized iodine solution.O. Prepared sample (20 mL) was transferred into a 30 mL serum bottle and then placed in the light storage box for 15 min as described. Effects of initial ascorbic acid concentration and pH Riboflavin sensitized photodestruction of ascorbic acid was compared on different initial concentrations (9.9 mm. 5. adjusted to pH 4. 100% ascorbic acid was destroyed by riboflavin sensitized photooxidation. Milford.OlM phosphate buffer (pH 7. 1985b) and HPLC method (Bradbury and Singh.HOAc) to the end point (a persistent rosy pink color). Milford.005M KH. 0.5) was prepared and used as reagent blank. the iodine titration (Fung and Luk.6 dichloroindophenol. MA) was used (Bradbury and Singh. Determination of ascorbic acid contents To prepare a sample for analysis.

1979. and found that no as- . considerable changes were observed in spectra around 265 nm (Fig..3 mMcm-‘ which was almost the same as previ. the calibration graph was not needed as long as the concentration of ascorbic acid in the tested sample was < 10 pg/mL. Chou and Khan.. That is. No.5. 1994).ugml-. Preparation of calibration graph The limits of the working range were determined from the calibration graph of standard ascorbic acid (Fig. 1980. 1983.105 pgml -O- 9 p9r-K: 21 pgmLl .-v- pH 7.5 increased precision of the assay. 4-Calibration I I I I I 10 acid.0 0 Fig. Since ascorbic acid destruction by riboflavin was pH-dependent. and samples at lower pH required longer. Destruction time of 15 min was selected for the optimal destruction of ascorbic acid.999. 362-JOURNAL OF FOOD SCIENCE-Volume 60.5.. 5.3 mMcm-I was used for calculation of ascorbic acid.5 was 6.5 was much greater than that at 245nm at pH 3. 5). After the riboflavin-sensitized photodynamic background correction. As previously mentioned. As pH increased. in confirmation of reported observations (Fung and Luk. Ascorbic acid destruction rate increased with increasing pH from 3.0 (Kelly and Latzko. the higher absorption at 265 nm at pH 7.5 was completely destroyed. 3 6 Time 9 (min) 12 of ascorbic 15 acid at Fig.5 required 15 min to obtain the sample blank (background correction). 2.5 and 3. 7. The molecular extinction coefficient for ascorbic acid was 14. For analysis of ascorbic acid.6 G (I) 2 0.A PHOTODYNAMIC UV METHOD FOR ASCORBIC ACID ASSAY. ously reported values at pH 7. We monitored ascorbic acid contents in the background corrected samples by the HPLC method. 3).5 to 7. :: 0 20 -0-. Jung et al. pH 7. 80 35 pgml-. ascorbic acid in solution at pH 7. The absorption at 265 nm at pH 7. 1993). 1995 1. -0. The maximum concentration of ascorbic acid was 10 pg/mL for a straight line relationship. samples adjusted to pH 7. 1983. Changes in spectra by photochemical background correction The changes in spectra of orange juice and pineapple juice samples after photochemical background correction were compared (Fig. 3-Riboflavin-sensitized photooxidation different pH during light storage. Ascorbic acid was extremely reactive to singlet oxygen (Bodamres and Chan.5) has absorption maximum at 265 nm. The correlation coefficient (r) was 0.5) on destruction of 21 pg/ mL ascorbic acid solution containing 6 pg/mL riboflavin during light storage were studied (Fig. Rooney. 5).o al F 0.4 0.5 pH 3. -A63 . 1994). the upper limit of the measurement range was 10 pg/mL ascorbic acid. the effects of pH (7. the wavelength of maximal absorbance shifted from 245 to 265 nm. Fig. The change in spectra around 265 nm by the photodynamic step seemed to be mostly due to the singlet oxygen oxidation of ascorbic acid.7J :: 0 . That is.63 X 108M-1sec-L (Jung et al.5 60 40 40 ‘ 5 :: E 60 20 i 80 0 0 3 6 Time 9 (min) acid 100 12 15 0. I 12 (pgml-‘ ) I I 2 4 6 8 14 16 Concentration curve for ascorbic ascorbic acid in aqueous solution (pH 7. 4). . 1985a).2 1 .5 pH 5. Absorption at 265 nm in samples before the photodynamic step were due to both the ascorbic acid and unknown components in the samples. The bimolecular reaction rate for reaction of ascorbic acid with singlet oxygen at pH.2 0.. Tsumura. 2-Riboflavin-sensitized photooxidation of ascorbic with different initial concentration during light storage.5.5 was used because of the higher absorption and faster destruction of ascorbic acid there. When the extinction coefficient of 14. After 15 min light storage.8 1 0. Thus.

2 Ascorbic IND 522.scavenger of singlet acid as a oxygen.423. The recovery test results with known amounts of added as. The third HPLC method for determination Casella. Commun.98 337. The proposed method could measure ascorbic Repeatability of the method for analysis of ascorbic acid con. was used.9 120. the ascorcorbic acid contents in commercial beverages gave precise re. Res.of and Chan. High-performance liquid chromatolished methods were compared (Table 3). W. M. ascorbic acid varied greatly among the different samples.0 331.9 80.7 - acid (~gmL-lF IOD 9T.2 348. vegetables using perzymatic vitamin C assay fruits and of ascorbic acid was used. 1989.1 406.9 237.2 After illumination 456.6 411.2 6 f%) 1.bic acid contents in 9 samples were successfully determined by the proposed method.77 450. and Khan. Gullotti. pared. Biophys. and U. Ascorbic acid content in commercial beverages a UV. No. respectively.2 186. -Continued on page 368 Volume 60. IOD and HPLC represent the proposed iodine and high performance chromatographic UV spectrophotometry.).4 6 4.7 6 2.C.W. 240 260 ‘ I”. P. solutions. methods. Doner. erythorbic acid. (w-0 Fig. 115: 225-230. Fifteen minutes of further illumination after the background correction did not induce further changes in spectra.3 acid contents by proposed 529.0 102. IND.0 indophenol.. considerable differences were sults in analyzing real fruit juices and sport drink. Anal.2 80.4 Before illumination Orange juice la Orange juice 2 Pineapple juice Soorts drink a Orange juices 1 and 2 are two different brands illWninatiOn 0. p.2 HPLC 529.H.5 After * “I”. of tropical Singh.7 375.4 241.S. Among the 12 tested samples. L.1 348. Ascorbw Washington.1 of juice acid in different Beverage Ascorbic acid added lugmL-l) 402.3 426.7 256. 1979..method for M.3 90. and parallel determinations using well established methods and the proposed procedure were com. The juice. delta molecular oxygen in aqueous media. To test the reliability of the method. 280 *’ 300 No.3 Recovery I%) 97. 5-Changes in spectra for A) orange juice and B) pineapple juice samples by the riboflavin-sensitized photodynamic background correction.observed between references and the proposed method for accorbic acid in four different commercial beverages were com. and diketogluconic acid. L-ascorbic acid quenching of singlet oxygen aration of ascorbic acid was difficult in some beverages. R.6. proposed method had a distinct advantage over the AOAC inBiochem. Horwitz (Ed. samples (especially red).2 319. However. generalized antioxidant propThe results with the proposed method.:) Ascorbic hrgml-‘ ) method acid contents by indophenol Orange juice Pineapple juice sports drink 522. vegetable juice cocktail and apple juice.1” 2. 746. 115: 932-936.00 0.5 408. diketogluonic acid.Table l-Repeatability commercial beverages of methods for analysis of ascorbic acid contents in Beverages Before illumination Ascorbic 21. The dehydroerythorbic acid.erola drink.5 101.2 402. which has less interference form colored Bradbury. redox impurities can cause prob975-978.0 1. Food Sci.5 51. but with interfering constituents. Official Methods of Analysis. Association Official Analytical Chemists. A. and the three estaberty of vitamin C.5 8 c s 1. 2. Marchesini.1 249. The concentration of graphic separation of ascorbic acid.0 ii $ 0.0 ““““‘ srm 200 2. and Hicks. Ascorbic acid contents in tomato juice and grape juice results show ~4% relative standard deviation for three samples could not be measured by AOAC indophenol method because studied.3 98. observed significant deviation for any of the samples tested. sepoxidase. 54: 374-378.6 456.0 -r..4 78. The indophenol method has interference from colored Bodannes.0 450. 1 2 1 2 cocktail corbic acid remained in samples after the photodynamic background correction. L.5 424.1 463. 1983. The riboflavin sensitized photochemical method for as. P. 12 commercial beverage REFERENCES samples were collected.acid in some colored samples such as tomato juice and grape tents in selected commercial beverage was tested (Table 1). K.2 294.2 442. 1981. Biochem. FEBS Letters. for some samples. 1995JOURNAL OF FOOD SCIENCE-363 .of color interferences.42 4. 105: 195-196.U. However. dehydroascorbic 51(4): content root from the south 3. This suggested that ascorbic acid was selectively destroyed by the formation of singlet oxygen under these conditions. 13th ed.AOAC 1980..0 200 220 240 260 280 300 Wavelength Table 3-Comparison ltiical methods of results by proposed UV method with other ana- Beverage Orange juice 1 Orange juice 2 Pineapple juice Pineapple juice Grapefruit juice Grapefruit juice Acerola drink Sports drink Vegetable juice Tomato juice Grape juice Apple juice uv 529. J. Ascorbic acid pacific.4 120.B.3 406. crops1986.987. inand Petrarulo. the second J. thus. Almost 100% recovery was shown with no for these differences are uncertain. of determinations Relative standard deviation 220 Table 2--Recovery beverages with known amounts of added ascorbic Ascorbic acid recovered fugrnL-I) 392. dophenol method. dehydroascorbic acid. DC. Chou. A. Rapid enlems in that analysis.2 452. Food Sci. and acid iodine titration method.6 315.3 332. Reasons pared (Table 2).4 245.

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