Riboflavin-Sensitized Photodynamic UV Spectrophotometry

for Ascorbic Acid Assay in Beverages

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

ABSTRACT analysis. Alkaline treatment is a rapid and simple method for

Riboflavin-sensitized photodynamic ultraviolet spectrophotometric assay determination of ascorbic acid (Fung and Luk, 1985a,b). How-
of various commercial beverages was carried out. The maximum con- ever, this method is less sensitive because it is based on ab-
centration 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 pres-
range 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 as-
known amounts of added ascorbic acid in different fruit juices and sports
drink showed the recovery of added ascorbic acid was 97.5-102.3%. corbic acid oxidized completely after a 12 min storage in the
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 bimolec-
ascorbic acid with good accuracy, precision and reliability. ular rate constant for reaction of ascorbic acid with singlet ox-
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 pho-
todynamic singlet oxygen formation. Thus, our objective was to
INTRODUCTION establish a rapid riboflavin-sensitized photodynamic UV spec-
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
acid content may be used as an index of quality. Commonly Materials
used methods for determining ascorbic acid in foods are indo- L-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 Sci-
ascorbic 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 chromatog-
raphy (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
ditions must be carefully tested for each assay. riboflavin or methylene blue or containing 0.26M H,O, and 0.072M
Thus, a rapid, simple and reliable method is needed for de- NaOCl were prepared. The photochemical methods of riboflavin and
termination of ascorbic acid. Direct UV spectrophotometry can methylene blue and chemical H,O,-NaOCl treatment method were well
provide such a fast and simple method (Fung and Luk, 1985a). established ways to generate singlet oxygen. Samples (20 mL) treated
However, the absorption of UV light by the sample matrix is with riboflavin and methylene blue were transferred into serum bottles
the major problem with this method. Various background cor- and, then, placed, at room temperature (-23°C) in a light storage box,
described in detail previously (Fakourelis et al., 1987; Jung and Min,
rection techniques such as thermal degradation, UV decompo- 1991; Jung et al., 1991). The light intensity at the sample level was 5500
sition, enzymatic or metal catalytic oxidation, and alkaline lux. The sample prepared with H,O,-NaOCl was placed under dark. The
treatment have been proposed to solve this problem. The ther- content of ascorbic acid was determined by HPLC (Bradbury and Singh,
mal, UV, and metal catalytic decomposition of ascorbic acid was 1986).
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 Photodynamic background correction
peroxidase (Kelly and Latzko, 1980) and guaiacol peroxidase Riboflavin was chosen to achieve the background correction (sample
(Casella et al., 1989; Tsumura, et al., 1993). Although the en- blank) for a new direct UV analysis because of effectiveness in ascorbic
zymatic methods are simple, rapid and highly specific for as- acid destruction. To study the destruction of various concentrations of
corbic acid, the enzymes are relatively costly for routine ascorbic acid, 9, 21, 35, 53 or 105 pg/mL ascorbic acid solutions con-
taining 6 @mL riboflavin (PH 7.5) were prepared by addition of cal-
culated amounts of ascorbic acid and riboflavin into O.OlM potassium
Author Jung is with the Department of Food Science and Tech- buffer (pH 7.5). Prepared sample (20 mL) was transferred into serum
nology, Jeonju Woosuk University, Samrea-Up, Wanju-Kun, bottles. The bottles were placed in the light storage box as described.
Jeonbuk Province, 566400, Republic of Korea. Authors S.K. Kim The content of ascorbic acid during illumination was monitored by meas-
and S.Y. Kim are with the Dept. of Food Science & Technology, uring absorbance at 265 nm using a spectrophotometer (Bodannes and
Chungnam National Univ., Daejeon, Republic of Korea. Chan, 1979).

36O-JOlJRNAL OF FOOD SCIENCE-Volume 60, No. 2, 1995


80
60
-A- H202-NaOCI
20 \
0 - n - Methylerie blue
- l - Riboflavin
t \
\
I I I I
01
0 5 10 15 20 25
Time (min)
Fig. l-Comparison of the destruction rates of ascorbic acid by
photochemical methods of riboflavin and methylene blue, and
chemical H202-NaOCI treatment method.
To study the destruction rates of ascorbic acid at different pH’s, 21
pg/mL ascorbic acid solutions containing 6 pg/mL riboflavin @ H 7.5,
5.5 and 3.5) were prepared by addition of calculated amounts of ascorbic
acid and riboflavin into 0.01 phosphate buffers of pH 7.5, 5.5 and 3.5,
respectively. The content of ascorbic acid was determined by measuring
absorbanceof solutions at 265 nm for samples at pH 7.5 and 5.5, or at
245 nm for samples at pH 3.5 using a spectrophotometer.
Preparation of calibration graph
To prepare the calibration graph of standard ascorbic acid, O-16 pg/
mL of ascorbic acid solutions containing 6 pg/mL riboflavin (pH 7.5)
were prepared by addition of calculated amounts of ascorbic acid and
riboflavin into O.OlM phosphate buffer (PH 7.5). Six ug/mL riboflavin
in O.OlM phosphate buffer (pH 7.5) was prepared and used as reagent
blank. Differences in absorbances of standard solutions and reagent
blanks measured at 265 nm were plotted vs concentrations of ascorbic
acids.
Determination of ascorbic acid contents
To prepare a sample for analysis, 0.2-1.0 mL of juice or drink was
transferred into a 250 mL beaker and - 40 mL or 90 mL of O.OlM
phosphate buffer (PH 7.5) was added to get the approximate ascorbic
acid concentration of <lo ug/mL. The pH of the diluted samples was
checked. If the pH was not 7.5 because of addition of sample, it was
adjusted to pH 7.5 with dilute NaOH solution, and the sample was then
transferred into a 50 mL or a 100 mL volumetric flask. The beaker was
washed with O.OlM phosphate buffer into the volumetric flask. Then,
0.5 mL or 1 mL of riboflavin stock solution (0.060g riboflavin in 100
mL of O.OlM phosphate buffer, pH 7.5) was‘added to the volumetric
flask and then O.OlM nhosuhate buffer (nH 7.5) was added to the mark.
None of the commercial beveragesneided to be pretreated for removal
of solid materials. However, if the samples contained high concentrations
of suspendedsolids interfering with the absorptivity at 265 nm we rec-
ommend centrifuging or filtering before UV analysis. 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. We found that ascorbic
acid in the prepared sample was completely destroyed after 15 min of
light storage. Absorbances of samples before and after light storage were
measured at 265 nm using a spectrophotometer. Differences in absorb-
ance of samples before and after 15 min-illumination were used for cal-
culation of ascorbic acid. The calibration graph prepared previously was
used to determine the actual concentrations of ascorbic acid in the sam-
ple.
Method reliability was tested on, 12 commercial beverage samples by
comparison with established methods. Three methods compared were the
indophenol (AOAC, 1980), the iodine titration (Fung and Luk, 1985b)
and HPLC method (Bradbury and Singh, 1986). The proposed direct UV
method measures ascorbic acid but not dehydroascorbic acid because
dehydroascorbic acid does not absorb light at 265 nm.
Indophenol method
The indophenol solution, 0.25 g/L of 2,6 dichloroindophenol, was
standardized by titration with 2.0 mL of standard ascorbic acid solution
and 5 mL of 3% metaphosphoric acid-8% acetic acid solution (HPO,-
HOAc) to the end point (a persistent rosy pink color). Consumption of
the blank was determined by titrating indophenol solution with 7 mL of
HPO,-HOAC solution plus a given amount of water equivalent to the
volume of indophenol solution used in the previous standardization ti-
tration. For the sample titration, 50 rnt. of sample was mixed with an
equal volume of HPO,-HOAc solution before filtering. 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.
Iodine method
The iodine solution (O.OOSM)was standardized in the usual way with
30 O.OlM thiosulfate solution. For sample analysis, 5 mL of sample, 20 mL
of water and 2 mL of 1% starch solution were titrated with standardized
iodine solution.
HPLC method
A high performance liquid chromatograph (Model 45, Waters Asso-
ciates. Milford. MA) was eouiDDedwith a UV detector (Waters Asso-
, 1 1*
ciate, Milford, MA) was used (Bradbury and Singh, 1986) for ascorbic
acid determination. The column used was jr-Bondapak C-18 (30 cm X
3.9 mm, Waters Associates, Milford, MA). The mobile phase (flow rate
1.3 mL/min) was aqueous 0.005M KH,PO, adjusted to pH 4.6 with
dilute HCl and acetonitrile (30:70, v/v). Ten pL of sample was injected.
The ascorbic acid was quantitated at 265 nm by the UV detector.
RESULTS & DISCUSSION
Background correction (sample blank)
Ascorbic acid is reportedly easily oxidized by the reaction
with singlet oxygen (Bodannes and Chart, 1979; Chou and
Khan, 1983; Rooney, 1983; Jung et al., 1994). Thus, we used
photochemical methods of riboflavin and methylene blue and
chemical H,O,-NaOCI treatment method to produce singlet ox-
ygen. The relative rates of decomposition of ascorbic acid by
photochemical methods of riboflavin and methylene blue, and
chemical H,O,-NaOCI treatment method were compared (Fig.
1). The destruction of ascorbic acid by riboflavin sensitized pho-
tochemical method was fastest among the tested methods. After
15 min of illumination, 100% ascorbic acid was destroyed by
riboflavin sensitized photooxidation. Thus it was selected for
more detailed study for making the sample blank (background
correction).
Effects of initial ascorbic acid concentration and pH
Riboflavin sensitized photodestruction of ascorbic acid was
compared on different initial concentrations (9, 21, 35, 63 and
105 pg/mL) during light storage (Fig. 2). The destruction rate
by riboflavin sensitized method increased rapidly with decreas-
ing concentrations of ascorbic acid. As decomposition rate is
dependent on initial concentration less time was required to de-
stroy smaller amounts of ascorbic acid. The time required to
destroy 9 pg/mL ascorbic acid was 12 min, and that to destroy
21 pg/mL ascorbic acid was 15 min under these conditions. The
ascorbic acid destruction in the systems of 35 pg/mL, 63 pgl
mL and 105 pg/mL ascorbic acid after 15 min-illumination were
98, 83 and 74%, respectively. This result suggested the samples
should have <21 J.tg/mL ascorbic acid to be completely de-
stroyed during a 15 min-illumination period.
Volume SO, No. 2, 1995-JOLJRNAL OF FOOD SCIENCE-361
A PHOTODYNAMIC UV METHOD FOR ASCORBIC ACID ASSAY. ..

-0- pH 7.5
9 p9r-K: -.- pH 5.5
21 pgmLl
80 -O- 35 pgml-, .-7J 20 -v- pH 3.5
-A- 63 ,ugml-, ::
-0- 105 pgml 0
.-
60 ; 40
::
0

40 ‘5 60
::
E
20 i 80

0 100
0 3 6 9 12 15 0. 3 6 9 12 15
Time (min) Time (min)
Fig. 2-Riboflavin-sensitized photooxidation of ascorbic acid Fig. 3-Riboflavin-sensitized photooxidation of ascorbic acid at
with different initial concentration during light storage. different pH during light storage.

Since ascorbic acid destruction by riboflavin was pH-depend- 1.2

ent, the effects of pH (7.5, 5.5 and 3.5) on destruction of 21 pg/
mL ascorbic acid solution containing 6 pg/mL riboflavin during
light storage were studied (Fig. 3). Ascorbic acid destruction
rate increased with increasing pH from 3.5 to 7.5. After 15 min 1 .o
light storage, ascorbic acid in solution at pH 7.5 was completely
destroyed. That is, samples adjusted to pH 7.5 required 15 min
to obtain the sample blank (background correction), and samples 0.8
at lower pH required longer. As pH increased, the wavelength al
of maximal absorbance shifted from 245 to 265 nm, in confir- F
mation of reported observations (Fung and Luk, 1985a). The
absorption at 265 nm at pH 7.5 was much greater than that at 1 0.6
245nm at pH 3.5. Thus, the higher absorption at 265 nm at pH G
(I)
7.5 increased precision of the assay. For analysis of ascorbic
acid, pH 7.5 was used because of the higher absorption and 2
faster destruction of ascorbic acid there. Destruction time of 15 0.4
min was selected for the optimal destruction of ascorbic acid.

Preparation of calibration graph 0.2

The limits of the working range were determined from the
calibration graph of standard ascorbic acid (Fig. 4). The corre- I I I I I I I I
lation coefficient (r) was 0.999. The maximum concentration of 0.0
ascorbic acid was 10 pg/mL for a straight line relationship. That 0 2 4 6 8 10 12 14 16
is, the upper limit of the measurement range was 10 pg/mL
ascorbic acid. The molecular extinction coefficient for ascorbic Concentration (pgml-‘)
acid was 14.3 mMcm-‘, which was almost the same as previ- Fig. 4-Calibration curve for ascorbic acid.
ously reported values at pH 7.0 (Kelly and Latzko, 1980; Tsu-
mura, 1993). When the extinction coefficient of 14.3 mMcm-I
was used for calculation of ascorbic acid, the calibration graph ascorbic acid in aqueous solution (pH 7.5) has absorption max-
was not needed as long as the concentration of ascorbic acid in imum at 265 nm. After the riboflavin-sensitized photodynamic
the tested sample was < 10 pg/mL. background correction, considerable changes were observed in
spectra around 265 nm (Fig. 5). The change in spectra around
Changes in spectra by photochemical background 265 nm by the photodynamic step seemed to be mostly due to
correction the singlet oxygen oxidation of ascorbic acid. Ascorbic acid was
extremely reactive to singlet oxygen (Bodamres and Chan, 1979;
The changes in spectra of orange juice and pineapple juice Chou and Khan, 1983; Rooney, 1983; Jung et al., 1994). The
samples after photochemical background correction were com- bimolecular reaction rate for reaction of ascorbic acid with sin-
pared (Fig. 5). Absorption at 265 nm in samples before the glet oxygen at pH. 7.5 was 6.63 X 108M-1sec-L (Jung et al.,
photodynamic step were due to both the ascorbic acid and un- 1994). We monitored ascorbic acid contents in the background
known components in the samples. As previously mentioned, corrected samples by the HPLC method, and found that no as-

362-JOURNAL OF FOOD SCIENCE-Volume 60, No. 2, 1995

 Table l-Repeatability of methods for analysis of ascorbic acid contents in
commercial beverages
Beverages
Pine-
Before illumination Orange apple sports
juice juice drink
Ascorbic acid contents
21;:) by indophenol
522.3 450.1 348.2
Ascorbic acid contents
hrgml-‘) by proposed
After illumination method 529.2 456.7 337.4
No. of determinations 6 6 6
0.0 ““““‘srm * “I”, ‘I”, *’ Relative standard deviation f%) 1.77 2.98 4.00
200 220 240 260 280 300
Table 2--Recovery with known amounts of added ascorbic acid in different
2.0 beverages
Ascorbic
Ascorbic acid re-
acid added covered Recovery
1.5 Beverage lugmL-l) fugrnL-I) I%)
8
c Orange juice la 402.2 392.0 -r- 6.3 97.5
Before illumination Orange juice 2 402.2 406.1” 2.2 101.0
sii 1.0 Pineapple juice 442.4 452.423.9 102.3
Soorts drink 241.3 237.42 4.1 98.4
a Orange juices 1 and 2 are two different brands of juice
$ 0.5
After illWninatiOn
Table 3-Comparison of results by proposed UV method with other ana-
0.0 ltiical methods
200 220 240 260 280 300 - Ascorbic acid (~gmL-lF
Beverage uv IND IOD HPLC
Wavelength (w-0 Orange juice 1 529.2 522.3 - 529.1
Fig. 5-Changes in spectra for A) orange juice and B) pineapple Orange juice 2 319.6 332.0 - -
juice samples by the riboflavin-sensitized photodynamic back- Pineapple juice 1 456.7 450.1 - 463.6
ground correction. Pineapple juice 2 256.3 249.1 - -
Grapefruit juice 1 426.6 406.2 - 411.5
Grapefruit juice 2 315.7 294.5 - -
Acerola drink 375.0 408.2 - 424.9
corbic acid remained in samples after the photodynamic back- Sports drink 331.4 348.2 - -
ground correction. Fifteen minutes of further illumination after Vegetable juice cocktail
the background correction did not induce further changes in Tomato juice 245.4
120.9 186.7
- 9T.4 120.3
-
Grape juice 80.2 - 78.5 90.0
spectra. This suggested that ascorbic acid was selectively de- Apple juice 80.2 - 51.2 -
stroyed by the formation of singlet oxygen under these condi- a UV, IND, IOD and HPLC represent the proposed UV spectrophotometry, indophenol,
tions. iodine and high performance chromatographic methods. respectively.

Ascorbic acid content in commercial beverages dophenol method. The proposed method could measure ascorbic
Repeatability of the method for analysis of ascorbic acid con- acid in some colored samples such as tomato juice and grape
tents in selected commercial beverage was tested (Table 1). The juice. 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. The riboflavin sensitized photochemical method for as- of color interferences. Among the 12 tested samples, the ascor-
corbic acid contents in commercial beverages gave precise re- bic acid contents in 9 samples were successfully determined by
sults in analyzing real fruit juices and sport drink. the proposed method. However, considerable differences were
The recovery test results with known amounts of added as- observed between references and the proposed method for ac-
corbic acid in four different commercial beverages were com- erola drink, vegetable juice cocktail and apple juice. Reasons
pared (Table 2). Almost 100% recovery was shown with no for these differences are uncertain.
observed significant deviation for any of the samples tested.
To test the reliability of the method, 12 commercial beverage
samples were collected, and parallel determinations using well REFERENCES
established methods and the proposed procedure were com- AOAC 1980. Official Methods of Analysis, 13th ed. W. Horwitz (Ed.), p. 746.
Association of Official Analytical Chemists,, Washington, DC.
pared. The indophenol method has interference from colored Bodannes, R.S. and Chan, P.C. 1979. Ascorbw acid as a scavenger of singlet
samples (especially red), thus, for some samples, the second oxygen. FEBS Letters. 105: 195-196.
iodine titration method, which has less interference form colored Bradbury, J.H. and Singh, U. 1986. Ascorbic acid and dehydroascorbic
content of tropical root crops from the south pacific. 3. Food Sci. 51(4):
acid
solutions, was used. However, redox impurities can cause prob- 975-978,987.
lems in that analysis. The third HPLC method for determination Casella, L., Gullotti, M., Marchesini, A., and Petrarulo, M. 1989. Rapid en-
zymatic method for vitamin C assay in fruits and vegetables using per-
of ascorbic acid was used, but with interfering constituents, sep- oxidase. J. Food Sci. 54: 374-378.
aration of ascorbic acid was difficult in some beverages. Chou, P. and Khan, A.U. 1983. L-ascorbic acid quenching of singlet oxygen
delta molecular oxygen in aqueous media; generalized antioxidant prop-
The results with the proposed method, and the three estab- erty of vitamin C. Biochem. Biophys. Res. Commun. 115: 932-936.
lished methods were compared (Table 3). The concentration of Doner, L.W. and Hicks, K.B. 1981. High-performance liquid chromato-
graphic separation of ascorbic acid, erythorbic acid, dehydroascorbic acid,
ascorbic acid varied greatly among the different samples. The dehydroerythorbic acid, diketogluonic acid, and diketogluconic acid. Anal.
proposed method had a distinct advantage over the AOAC in- Biochem. 115: 225-230. -Continued on page 368

Volume 60, No. 2, 1995JOURNAL OF FOOD SCIENCE-363