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DETERMINATION OF VITAMIN C CONTENT IN COMMERCIAL FRUIT JUICES BY

VOLUMETRIC AND SPECTROPHOTOMETRIC METHODS

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 Journal of Hygienic Engineering and Design

Original scientific paper

UDC 664.81:577.164.2

DETERMINATION OF VITAMIN C CONTENT IN COMMERCIAL FRUIT JUICES BY

VOLUMETRIC AND SPECTROPHOTOMETRIC METHODS
Ariola Devolli1*, Merita Stafasani1, Edlira Shahinasi1, Frederik Dara2, Hikmete Hamiti3
1
Department of Chemistry, Faculty of Biotechnology and Food, Agricultural University of Tirana,
Pajsi Vodica, Koder-Kamez, 1029 Tirana, Albania
2
Department of Applied Mathematics, Faculty of Natural Sciences, University of Tirana,
Boulevard Zogu I nn, 1001 Tirana, Albania
3
National Food Authority, Kukës-Prizren Street nn, 8501 Kukes, Albania

*
e-mail: [email protected]

Abstract 1. Introduction
Ascorbic acid known as Vitamin C is the main Fruit juices are consumed throughout the world
constituents of fruit juice and it is an essential due to the health benefits, the pleasant taste, their
antioxidant. Presence of this organic acid plays an characteristic aroma and flavor. Natural fruit juices
important role in human health and on the shelf life are good source of vitamins, minerals, soluble and
of the product. The purpose of this study was the insoluble fibers and major source of valuable bioactive
determination of vitamin C content in some imported compounds essential for human health [1, 2].
commercial fruit juices traded in Albania.
Due to their potential nutritional and biological fruit
Samples were chosen randomly in Tirana’s juices are foods with multiple implications for body
hypermarkets. Content of ascorbic acid was balance [3]. Consumption of fruit juices as a part of a
determined by redox titration with iodine solution and normal diet to prevent or treat cardiovascular diseases
UV-spectrophotometric methods using potassium is becoming more fashionable [4].
permanganate as a chromogenic reagent. Results
Fruit juice is unfermented but fermentable liquid or
were analyzed by two-sample t-test equal means.
juice intended for direct consumption, obtained from
Content of ascorbic acid in commercial fruit juices the edible portion of sound, appropriately mature
determined by volumetric methods varied from 5.28 and fresh fruit by mechanical extraction process and
mg/100mL to 39.63 mg/100mL. The level of ascorbic preserve, exclusive by chemical and physical means
acid according to the spectrophotometric method [5]. Fruit juice as well is a natural fluid that can be
varied from 8.7 mg/100mL to 49.33 mg/100mL. Based obtained by crushing or squeezing of fresh fruits or
on two-sample t-test for equal means of vitamin C reconstituted concentrates [6, 7].
for the content in fruit juices revealed that there is no
The most common ingredients in juices are fruits or
significant difference between two methods.
reconstituted juice, water, preservatives, sugar, acid
However, it was noticed that the level of vitamin and color [8]. Fruit juices may be produced either by
C content was about three times higher than extracting the natural fruits with water resulting in
one declared on the labels of analyzed samples. water extractable juices. Those juices have all essential
Approximately 40 - 50% of the amount of ascorbic acid physical, chemical, organoleptic and nutritional
is lost after opening the package of commercial fruit properties of the fruits they are produced from [9].
juices and seven-day storage at the temperature of 4
In terms of chemical composition fruits consist of water
Celsius degree.
and dry matter. The dry matter contains considerable
Key words: Vitamin C, Commercial fruit juices, amounts of vitamins (vitamin E and C), acids, sugars,
Volumetric method, UV-spectrophotometric method, polysaccharides, pectin, cellulose, polyphenols and
Determination. minerals.

124
Journal of Hygienic Engineering and Design

Minerals and vitamins could be added to enrich fruit be lost from foods during storage, thermal processes,
juices [3]. preparation and cooking [24].
According to Leahu et al., [3] consumption of fruit Ascorbic acid (AA) is used commonly in the beverage
juices with pulp is recommended, being used in the industry, especially in fruit juices. The addition of
treatment and prevention of cardiovascular diseases, vitamin C in fruit juices is done for some technical
liver disease, etc., natural juices are common sources of reasons such as: to restore the vitamin C lost value
vitamins (mainly vitamin C) and minerals in daily dose. during processing and to contribute to the product’s
appearance and shelf life. AA may be added in some
Some manufacturers produce fruit juices from raw
different stages of juices production process to prevent
materials other than the fruit, or use concentrates
enzymatic browning and flavor deterioration [26, 27].
in quantities below the legal requirements [10].
Preservatives such as sodium benzoate, sulphur For all reasons mentioned above the vitamin C content
dioxide, sorbic acid and ascorbic acid may be used to in fruit juices represents an indicator of quality, which
extend fruit juice shelf-life. These preservatives works has to be carefully monitored, regarding its variation
effectively at pH usually below 4.3, therefore amount during manufacturing and storage conditions [15].
of acid added has to maintain the proper pH value. The Due to its importance and facility of degradation, it is
commonly used acid is citric acid which should not of great importance to quantify correctly the levels of
exceed range 0.39 - 1.1 % in fruit juices [11]. vitamin C in fruit juices [27].
The anti-oxidant components of fruit juice have The development of rapid, simple, and inexpensive
beneficial long term health effects, such as decreasing analytical methods is the areas of growing interest,
the risk of cancer and heart disease [12]. Vitamin C especially since quick decisions are needed in
does not provide energy to the body but it is essential environmental and industrial fields [28, 28]. Many
and necessary nutrients for the growth of the body analytical methods can be used for the quantitative
and also associated with the enzyme function in the determination of vitamin C content in fruit juices, such
body. It plays an important role not only in collagen as: titrimetry, spectrophotometry, biological methods,
biosynthesis, but also in iron absorption and immune electrochemical methods (voltametry, fluorometry,
response activation [13, 14, and 15]. and potentiometry) and chromatographic method [30,
31, and 28].
The main sources of vitamin C are fruits and
vegetables such as oranges, lemons, limes, citrus Vitamin C is usually analyzed by classical techniques
fruits, strawberries, melons, peppers, tomatoes, leafy such as volumetric methods - titration with an oxidant
vegetables, and potatoes spinach and etc. [16]. solution such as dichlorophenol indophenol (DCPIP)
[32], potassium iodates [33], or bromate [34]. Not all
Even a healthy diet contains sufficient quantities of
analytical methods are suitable for the determination
ascorbic acid; the body requires more of the vitamin
of both forms (ascorbic acid and oxidized form,
after serious injury, major surgery, burns and when
dehydroascorbic acid) of ascorbic acid because two
exposed to extremes temperature [14]. According to
forms of the vitamin C, ascorbic acid and its oxidized
some previous studies minimum daily requirement of
form dehydroascorbic acid possess the different
vitamin C for adults is 60 mg. The recommended daily
chemical, optical and electrochemical properties [30].
doses are 25 to 30 mg/1000 kcal [17, 18, and 19]. In
some cases, excessive quantities of ascorbic acid may Spectrophotometry is one of the most frequently used
result in the inhibition of natural processes occurring simple methods, because vitamin C is able to absorb
in food and can contribute to taste deterioration [20]. UV ray [35]. Direct ultraviolet (UV) spectrophotometry
can provide a fast, simple and reliable method for the
Vitamin C or L-ascorbic acid (AA) is a water soluble
determination of L-ascorbic acid. Absorption of UV
essential nutrient which is not synthesized by the
light by the sample matrix is a major problem due to
human organism; it is easily degraded by enzymes and
the interference appeared by absorption of other rays
possibly could be decomposed during the storage.
[36, 37].
The chemical formula of ascorbic acid is C6H8O6 with
a white or slightly yellow crystal or powder [21, 22, and This paper aims to determine the content of ascorbic
20]. acid in some commercial fruit juices by both titrimetric
and spectrophotometric methods. Obtained results
Vitamin C is instability because of its strong reducing
were compared in order to define if there was a
agent and it is degraded by oxidizing agents such as
significant difference between two methods and to
atmospheric oxygen, heating, light, etc. Also, vitamin C
evaluate the simplest, accurate and reliable analytical
can be easily lost by boiling due to its water-solubility
method for determination of vitamin C content in
[15, 23]. Ascorbic acid has limited stability and may
commercial fruit juices.

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 Journal of Hygienic Engineering and Design

2. Materials and Methods IO3- + 3 C6H8O6  3 C6H6O6 + 3 H2O + I- (1)

2.1 Sample collections
Recently the trend of fruit juice production and The amount of vitamin C in fruit juices was calculated
consumption has been increased significantly in by using following relationship:
Albania as well as other countries. Different imported
brands and types of packed fruit juices are traded in
markets.
Subject of this study were most preferred and
consumed brands of commercial fruit juices. 3 to 5
different types of 5 selected brands were purchased
from Tirana’s hypermarkets during April to June (2018), Where: M = molar concentration, V = volume , and n = moles number.
which were chosen randomly. Fruit juices samples
were stored in 4 0 C prior analyzing.
The analyses were carried out within 24 hours of 2.4 Evaluation of total acid ascorbic content in fruit
sample collection in the Laboratories of Chemistry juices by spectrophotometric method
Department and Food Center, Faculty of Biotechnology The determination of vitamin C in commercial fruit
and Food, Agricultural University of Tirana. juices was also conducted by spectrophotometric
method using potassium permanganate as a
chromogenic reagent. The reduction of absorbance
2.2 Reagents and equipment used for determination was measured when a potassium permanganate
of vitamin C content in fruit juices solution reacted with the solution of ascorbic acid in
All reagents were chemically pure and of analytical acid medium, as shown in equation (2):
grade (Merck and Fisher), volumetric glassware (flask,
burettes, pipettes, cylinders, etc.) and other equipment
2KMnO4 + 5C6H8O6 + 3H2SO4 = 2MnSO4 +
were calibrated. (2)
5C6H6O6 +K2SO4 + 8H2O
Determination of vitamin C content through
Spectrophotometric method was performed by using
an UV-vis spectrophotometer type SPECORD 40, where vitamin C consumes KMnO4 (violet color)
Analytic Jena AG Germany. causing a decrease in the absorbance at 525-530 nm.
This method is based also on previous studies [22, 38].

2.3 Evaluation of total acid ascorbic content in fruit

juices by titration method 2.5 Preparation of standard solutions and
calibration curve of ascorbic acid
A standard solution of potassium iodate (KIO3) was used
as an oxidizing agent for volumetric determination of Standard solutions of ascorbic acid were prepared
vitamin C. Continuous standardization of potassium within the samples range (1 to 100 mg/L). Ascorbic
iodate solution was carried out by using a standard acid (0.01 g) was dissolved in small amount of 0.5 %
solution of ascorbic acid. oxalic acid solution and completed with distilled water
to obtain a concentration of 100 mg/L.
10 mL samples of each selected fruit juices samples
was pipetted into a conical flask where was added 5 The solution of chromogenic agent with 100 mg/L
mL potassium iodide (KI 0.2 M), 2.5 mL hydrochloric concentration was prepared by dissolving previously
acid (HCl 1 M) and a few drops of starch solution. This 0.01 g of KMnO4 in 5 molar solution of sulfuric acid in
solution was titrated against standard solution of KIO3 a 100 mL volumetric flask and completed to the mark
(0.01 M) until the dark blue color was appeared. with distilled water. 1 mL of chromogenic agent was
added in a series of 10 mL standard solution with
Analysis of vitamin C content by titration was
different concentrations of ascorbic acid and after 5
conducted with three replications. Calculation of
min was performed the absorbance measurement of
vitamin C content in fruit juice samples was carried
each solution at 530 nm against blank [22, 38].
out by using the relationship of oxidation – reduction
reaction of potassium iodate with acid ascorbic (1 mol Two calibration curves with different concentration
IO3- is equal to 3 mol of C6H8O6) and based on equation range of ascorbic acid (0.1 - 4 mg/L and 10 - 100 mg/L
according to other similar studies [38 - 41]. respectively) were prepared (Figures 1 and 2).

126
Journal of Hygienic Engineering and Design

frequently consumed brands were selected. A total of

18 commercial fruit juices were analyzed in our study.
All the collected samples were labeled with specific
code ranging from L1 to L18.
The pH was recorded on pH meter Hanna, model HI
2210. There was observed no differences in pH values
of fruit juices samples as shown in Figure 3.

4.0

3.5

3.0

pH
Figure 1. Calibration curve for determination of vitamin
C content (concentration of AA range 0.1 to 4 mg/L)
2.5

2.0

L1
L2
L3
L4
L5
L6
L7
L8
L9

0
1
2
3
4
5
6
7
8
L1
L1
L1
L1
L1
L1
L1
L1
L1
Fruit juices samples

Figure 3. pH values of fruit juices samples

Table 1 presents statistical results of pH values of fruit

juices samples with range 2.68 to 3.66.

Table 1. Statistical results of pH data

Statistical parameters
min max x̅ std C.I C.V
pH 2.68 3.66 3.14 0.28 0.08 8.92

Figure 2. Calibration curve for determination of vitamin The results of ascorbic acid content in fruit juices
C content (concentration of AA range 10 to 100 mg/L) samples obtained from two employed methods are
shown in Figure 4. A standard solution of 10 mg/100 mL
2.6 Statistical analysis of AA was evaluated by using both analytical methods.
Based on results almost all analyzed samples presented
Measurements of vitamin C in commercial fruit juices
a higher level of vitamin C content determined by
were carried out with 3 replications and presented as
spectrophotometric method compared to volumetric
min, max, mean, standard deviation (std), confidence
one. Samples L1 to L4 of the same brand, practically
interval (C.I), and coefficient of variation (C.V). Statistical
showed no differences on amounts of vitamin C.
analysis of data was performed by using the SPSS ver.
22 software and graphs were drawn by using software
GraphPad Prism version 8.02 (2019). vitamin C (mg/100ml) spectrophotometric method
vitamin C (mg/100ml) volumetric method
50

The comparison of two methods used for determination 45

of ascorbic acid content in fruit juices samples was 40

done by paired sample t-test for equal means of 35

Vitamin C (mg/100 ml)

vitamin C content. A p value < 0.05 was considered 30

25
statistically significant [42].
20

15

10

3. Results and Discussion 5

0
The main goal of our research was the determination of
L1
L2
L3
L4
L5
L6
L7
L8
L9

d
0
1
2
3
4
5
6
7

st 8
L1
L1
L1
L1
L1
L1
L1
L1
L1
an

vitamin C content on commercial fruit juices available Fruit juices samples

in Tirana market.
Figure 4. Vitamin C content in commercial fruit juices
Different types of commercial fruit juices (apple, determined by volumetric and spectrophotometric
orange, cherry and blueberry) of 5 most preferred and methods

127
 Journal of Hygienic Engineering and Design

The highest differences on vitamin C content were method could be more consistent and reliable if we
observed on samples L9 and L16, which belongs to take into account the impact of the color interference
blueberry types of fruit juices. According to Zanini et [22, 38].
al., [22], the color and reducing sugar of industrial fruit
The content of vitamin C in commercial fruit juices
juices influence on absorbance values.
samples was measured a week after opened and
Potassium permanganate as chromogenic reagent stored at room temperature (20 - 25 0C). Determination
reacts with other reducing substances present of vitamin C was conducted according to iodometric
in the juice and not only with the vitamin C [22]. method.
Based on obtained results, as well as other studies,
As shown in Figure 5, a significant reduction of vitamin
a higher level of vitamin C content determined by
C level in all analyzed samples there was observed.
spectrophotometric method than titrimetric method
was reported.
vitamin C mg/100ml (after one week)

Statistical results of data obtained by both analytical 45 vit C mg/100ml (in the beginning)

methods employed for determination of vitamin C 40

content are shown in Table 2. The vitamin C content of 35

Vitamin C (mg/100 ml)

fruit juices samples was found in range of 5.28 mg/100 30

mL to 39.63 mg/100 mL and 15.67 mg/100 mL to 25

49.33 mg/100 mL with confidence interval 2.53 to 2.35 20

for volumetric and spechtrophotometric methods, 15

respectively. 10

5
Evaluation of analytical methods used for 0
determination of vitamin C content in fruit juices

d
L1
L2
L3
L4
L5
L6
L7
L8
L9

0
1
2
3
4
5
6
7

st 8
L1
L1
L1
L1
L1
L1
L1
L1
L1
an
samples was performed by paired samples t-test. Fruit juices samples
Due to the reasons mentioned above, results of two
Figure 5. Vitamin C content of commercial fruit juice
cases (L9 and L16) from the set of data underlying the
samples before and a week after
statistical analysis were excluded. Based on paired
samples t-test was observed no statistically significant As noted in Table 4, the paired samples t-test indicates
difference between two analytical methods (Table 3). a statistically significant difference (p ˂ 0.05) between
Following the obtained results, statistical tests and means of vitamin C content measured before and a
based on other studies, the spectrophotometric week after, which was decreased.

Table 2. Statistical results of vitamin C determined by two analytical methods

Statistical parameters
min max x̅ std C.I. C.V.
Volumetric method 5.28 39.63 22.99 9.49 2.53 41.25
Spectrophotometric
15.67 49.33 27.87 8.80 2.35 31.57
method

Table 3. Paired Samples t-test of vitamin C determined by volumetric and spectrophotometric methods
Paired Differences
95% confidence interval Sig.
Std. Std. Error of the difference t df
Mean (2-tailed)
Deviation Mean
Lower Upper
Volumetric -
Pair 1 -2.78738 6.93189 1.73297 -6.48112 .90637 -1.608 15 .129
Spectrophotometric

Table 4. Paired Samples t-Test of vitamin C content determined before and a week after
Paired Differences
95% confidence interval Sig.
Std. Std. Error of the difference t df
Mean (2-tailed)
Deviation Mean
Lower Upper
Before -
Pair 1 10.9699 5.75857 1.35731 8.10626 13.83359 8.082 17 .000
a week after

128
Journal of Hygienic Engineering and Design

Table 5 presents statistical results of vitamin C content of vitamin C than the stored one. The storage
content evaluated before and a week after opening temperature plays an important role in the decrease of
the package of commercial fruit juices samples. The total vitamin C amounts [16, 3, and 43].
concentration of vitamin C a week after was found in
It was observed that the concentrations of vitamin C
range of 1.94 mg/100 mL to 25.16 mg/100 mL. Storage
in all analyzed commercial fruit juices samples were
of commercial fruit juices in opened conditions and
higher than those indicated on labels by producers, as
at room temperature for 7 days results that total
presented in Table 6 the p value is smaller than 0.05.
amount of vitamin C losses ranged from 14.2% to 76%
Declared level of vitamin C varied from 9 mg/100 mL to
(calculated average losses was 47.7%).
12 mg/100 mL. The producer added a higher quantity
Consumption of commercial fruit juices in a short of ascorbic acid due to the fact that time and conditions
period of time (after it opened) ensures a higher of storage influence on its losses [22, 44, and 45].

Table 5.Statistical results of vitamin C content measured before and a week after
Statistical parameters
min max x̅ std C.I. C.V. Losses
Before 5.28 39.63 22.99 9.49 2.53 41.25
A week after 1.94 25.16 12.02 6.27 1.67 52.14 47.7%

Table 6. Paired Samples t-Test of vitamin C concentration analyzed and declared on the label
Paired Differences
95% confidence interval
of the difference
Std. Std. Error Sig.
Mean Deviation Mean Lower Upper t df (2-tailed)
Pair 1 Analyzed - Label 12.1046 9.60049 2.26286 7.33034 16.87877 5.349 17 .000

4. Conclusions
- Ascorbic acid content in fruit juices plays an important (juice color, reducing sugars and other reducing
role to human health and is considered as an indicator substances).
parameter of quality. Hence, a carefully quantification
- The time and storage temperature significantly affects
of the level of vitamin C in fruit juices is of great
on the amount of ascorbic acid loses after opening the
importance.
package of commercial fruit juices. Nearly 50 % of total
- This research was focused mainly on the determination amount of vitamin C in opened commercial fruit juices
of vitamin C content in commercial fruit juices by two stored for seven-day at room temperature losses.
analytical methods.
- Obtained results noticed that the concentration of
- The amount of vitamin C in commercial fruit vitamin C of analyzed commercial fruit juices samples
juices measured by volumetric methods ranged was significantly higher (nearly three times more) than
from 5.28 mg/100 mL to 39.63 mg/100 mL and by those indicated on the labels by producers.
spectrophotometric method ranged from 15.67
mg/100 mL to 49.33 mg/100 mL.
- From the paired samples t-test was observed no
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