Comparison of vitamin C content in citrus fruits by titration and high performance liquid chromatography (HPLC) methods

Abstract

Vitamin C is one of the essential vitamins for human and animal. Many methods were developed for the determination of vitamin C such as spectrophotometry, electrophoresis, titration, and high performance liquid chromatography (HPLC). This study aims to compare vitamin C content of citrus fruits (orange, grapefruit, lemon, lime, kaffir lime and musk lime) using indophenol titration and HPLC-PDA methods. In the titration method, orange has the highest vitamin C content (58.30 mg/100g) followed by grapefruit (49.15 mg/100g), lemon (43.96 mg/100g), kaffir lime (37.24 mg/100g), lime (27.78 mg/100g) and musk lime (18.62 mg/100g). While, in the HPLC method orange also leads with the highest vitamin C content (43.61 mg/100g) followed by lemon (31.33 mg/100g), grapefruit (26.40 mg/100g), lime (22.36 mg/100g), kaffir lime (21.58 mg/100g) and musk lime (16.78 mg/100g). Orange is the best source of vitamin C while musk and kaffir lime have lower content. Significant differences were observed in vitamin C of samples by both methods. Both methods are suitable for the determination of vitamin C, however HPLC method is more accurate, precise and specific.

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*Corresponding author.
Email: azrinaaz@upm.edu.my
International Food Research Journal 24(2): 726-733 (April 2017)
Journal homepage: http://www.ifrj.upm.edu.my
1Fatin Najwa, R. and 1,2*Azrina, A.
1Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, 43400 UPM
Serdang, Selangor, Malaysia
2Research Centre of Excellence, Nutrition and Non-communicable Diseases, Faculty of Medicine
and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Comparison of vitamin C content in citrus fruits by titration and high
performance liquid chromatography (HPLC) methods
Abstract
Vitamin C is one of theessential vitamins for human and animal. Many methods were
developed for the determination of vitamin C such as spectrophotometry, electrophoresis,
titration, and high performance liquid chromatography (HPLC). This study aims to compare
vitamin C content of citrus fruits (orange, grapefruit, lemon, lime, kafr lime and musk lime)
using indophenol titration and HPLC-PDA methods. In the titration method, orange has the
highest vitamin C content (58.30 mg/100g) followed by grapefruit (49.15 mg/100g), lemon
(43.96 mg/100g), kafr lime (37.24 mg/100g), lime (27.78 mg/100g) and musk lime (18.62
mg/100g). While, in the HPLC method orange also leads with the highest vitamin C content
(43.61 mg/100g) followed by lemon (31.33 mg/100g), grapefruit (26.40 mg/100g), lime (22.36
mg/100g), kafr lime (21.58 mg/100g) and musk lime (16.78 mg/100g). Orange is the best
source of vitamin C while musk and kafr lime have lowercontent. Signicant differences
were observed in vitamin C of samples by both methods. Both methods are suitable for the
determination of vitamin C, however HPLC method is more accurate, precise and specic.
Introduction
Vitamin C, also is known as ascorbic acid is
one of the most important vitamins and essential
for human and animal life. This water soluble
vitamin contributes to many health benets such
as prevention of scurvy and cancer, relief from
common cold, stimulate collagen synthesis and
play a signicant role in wound healing process
(Iqbal et al., 2004). According to Teucher et al.
(2004), vitamin Chelps to enhance availability and
absorption of iron from non-heme sources. Vitamin
C also has antioxidant properties since it can easily
lose the electron to neutralize and inhibit free radicals
from being oxidized in preventing cell damage. It is
also commonly used as food additive which acts as
antioxidant (Whitney andRolfes, 2008).
Vitamin C is an organic compound consists of
carbon, hydrogen and oxygen (Chinnici et al., 2005).
The terms vitamin C is not only used for ascorbic acids,
but it includes all compounds exhibiting biological
activity such as oxidized, ester and synthetic form.
The main biological form of vitamin C is L-ascorbic
acid, and it can reversibly change to oxidized form
called dehydroascorbic acid (Fenolland Martinez,
2010). Many factors can cause oxidation of vitamin
C such as pH, light, temperature, presence of oxygen
and metal ion (Wantz et al., 2005).
Human is unable to synthesize their own vitamin
C supply as human cells cannot perform the crucial
last step in vitamin C biosynthesis,the conversion
of L-gulono-g-lactone into ascorbic acid which
is catalyzed by gulonolactone oxidaseenzyme
(LinsterandVan Schaftingen, 2007). Therefore, they
require vitamin C for maintaining the physiological
functions. To meet the requirement, vitamin C must
be consumed from diet. The recommended nutrient
intake of vitamin C for Malaysian adult is 70 mg per
day. An intake of 45 mg/day will ensure measurable
amount of ascorbate be present in the plasma of most
people and available to supply tissue requirements for
metabolism or repair at sites of depletion or damage
(MOH, 2005).
Many methods can be used for determination
of vitamin C such as spectrophotometry,
electrophoresis, titration, and high performance
liquid chromatography (HPLC) (Tang and Wu,
2005; Dong et al., 2007; Spinola et al., 2012). The
most commonly used method is oxidation-reduction
titration method where ascorbic acids are oxidized
to dehydroascorbic acid and the indophenol dye is
reduced to a colorless compound. The end point of
the titration can be easily detected when an excess
of the unreduced dye give a rose pink color in an
Keywords
Vitamin C
Ascorbic acid
Citrus fruits
High performance liquid
chromatography
Titration
Article history
Received: 29 June 2015
Received in revised form:
23 March 2016
Accepted: 4 April 2016

727 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733
acid solution (Tee et al., 1996). It is a simple and
easymethod to determine vitamin C in fruits and fruit
juices. However, the method is not suitable for fruits
that have reddish-purplish color. The titration method
also is time-consuming and lack of specicity due to
interference of reducing substances in the food such
as ferrous iron, stannous tin, cuprous copper, sulphur
dioxide, sulphite orthiosulphate (Eitenmiller et al.,
2008).
Several other methods to determine vitamin
C content like spectrometric, spectrouorimetric,
and electrophorhesis, still some of them are not
practical and need re-evaluation due to insufcient
of sensitivity and selectivity (Agar, 1995). Quiroz
and Fernandez (2009) stated that the most preferred
method of determining vitamin C content in foods is
a chromatographic method using HPLC due to the
rapid, high accuracy and consistency.
Previously, there were many studies done on
citrus fruits mainly focusing on determination of
vitamin C using HPLC by modifying the stationary
phase, mobile phase, type of detector and sample
preparation (Gazdik et al., 2008; Spinola et al., 2012)
but there was not many studies done comparing two
or more different methods in determining vitamin
C in citrus fruits (Ullah et al., 2012; Spinola et al.,
2013). Moreover, most studies are just focusing on
single type of lime anddo not focus on another type
of limes such as kafr lime and musk lime.Thus, the
purpose of this study was to provide the comparison
between titration and HPLC method in determination
of vitamin C content in citrus fruits.
Materials and Methods
Sample preparation of titration method
Sample and standard preparation and
quantication of vitamin C in titration method was
performed using the method described by Tee et al.
(1996). In brief, fruit samples (200 g-300 g) was
blended with 6% metaphosphoric acid (HPO3) with
an equal volume to homogenous slurry and made it
up until 500 ml of volume. The homogenous mixture
was measured around 10 to 30 ml and diluted into
100 ml volumetric ask with 3% of metaphosphoric
acid (HPO3). The diluted sample was then ltered to
remove away suspension using vacuum pump before
10 ml aliquote of the ltrate was pipetted into a
small Erlenmayer ask. The ltrate was immediately
titrated with a dye solution 2, 6-dichlorophenol-
indophenol to a faint pink end point. Triplicate
titration was conducted for all samples.
Preparation of standard solution for titration
method
Vitamin C standard solution (0.2 mg/ml) was
prepared by dissolving 100 mg vitamin C in 3% of
metaphosphoric acid (HPO3) solution and diluted to
500 ml with the same solvent. Then, 5 ml aliquot of the
vitamin C standard solution was diluted (containing
1 mg vitamin C) with 5 ml 3% metaphosphoric acid
(HPO3). Next the diluted vitamin C standard solution
was titrated with dye solution 2, 6-dichlorophenol-
indophenol to a faint pink color. Triplicate titration
also was conducted for the vitamin C standard.
Quantication of Vitamin C in the extracted solution
of titration method
Firstly, weight of the sample in 10 ml ltrate was
calculated by the following equation:
Where,
a = Weight of sample will be used
b = Final volume of homogenous slurry [sample
+ 6% metaphosphoric acids (HPO3)]
c= Aliquot of homogenous slurry used for dilution
to 100 ml with 3% metaphosphoric acid (HPO3)
Then total vitamin C content (mg per 100 g
sample) of each sample was obtained by the following
equation:
Vitamin C content (mg per 100 g sample) =
X = volume of dye used for titration of aliquot of
diluted sample (ml)
Y = vitamin C equivalent of dye solution, mg per
ml dye solution
W = weight of sample in aliquot of ltrate of
diluted sample used for titration (g)
Sample preparation of HPLC method
Sample preparation, chromatography conditions,
identicantion and quantication of vitamin C in
HPLC method was perfomed using the method
described by Czech Agriculture and Food Inspection
Security (2005). The homogenous solid sample
was measured around 10 to 30g and mixed it with
60 to 80 ml 3% of metaphosphoric acid (HPO3)
for one minute. The obtained extracted was ltered
through ltration paper and washed it for few times
by using vacuum pump ltration. Next, the ltrate
quantitatively transferred into a 100 ml volumetric
ask and 3% of metaphosphoric acid (HPO3) was
added up to 100 ml volumetric mark. All the sample

Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733 728
solutions was ltered again through 0.45 µm syringe
lter. After that, the samples were run in the HPLC
system.
HPLC system or chromatography conditions
The total of vitamin C content in 6 types of fruits
were determined by HPLC system. The reversed–
phase liquid chromatographic method was used for
determination of vitamin C consisted of an isocratic
elution procedure with photodiode assay detection at
254 nm. The separations were carried out on Carbon
18 (C18) column of 250mm x 4.6 mm (LiChro CART,
Darmstadt, Germany). The mobile phase used was
a mixture of methanol - water (5.95, v/v). The ow
rate of the mobile phase was 1.0 mL min-1 and 20
µL injection volume of samples and standard were
used in quantitative analysis. The temperature of
analytical column was kept constant at laboratory
temperature which was 25oC. The standard of the
vitamin C was an external standard which 1mg/ml
natural Vitamin C derivatives. There were different
type of concentrations of vitamin C range from 0.5
mg/ml to 200 mg/ml based on a 10 point calibration.
If the shape of the plot was deviates from the square
waveform during detection of a peak, then the peak
was considered as no longer spectrally pure. Standard
solutions were ltered through a lter paper and then
was ltered again through 0.45 µm syringe lter.
To prevent the loss of vitamin C all standards and
extracted sample solutions were protected from light
by covering it with aluminium foil.
Identication and quantication of vitamin C in
extraction solutions
Identication was performed by the comparison
of retention time of analyte in the analyzed sample
with the retention time of the calibration standard.
Quantication was carried out with the external
standard method (Vitamin C standards at various
concentrations) using the following equation formula
to calculate the Vitamin C concentration on samples.
CA (mg/ 100g) = Aa x D x RF x 100
m
Where,
Aa = Peak area of the analyte
D = Sample factor dilution
RF = Response factor (way to adjust the
proportionality of the detector response to the
concentration of vitamin C and is calculated the
following formula:
Where,
Cst = standard working solution concentration 50
µg/ml
Ast = corresponding peak area
m = weight of samples
Statistical analysis
IBM SPSS version 21.0 software was used for
statistical analysis. The total vitamin C concentration
in food samples was expressed as mean ± standard
deviation. One-way ANOVA was used to determine
signicance difference of mean value of vitamin C
content (p<0.05) among the samples for each method.
Moreover, independent t-Test was used to determine
signicance difference of mean value of vitamin C
content (p<0.05) between the titration and HPLC
method. The Pearson correlation test also was used
to test the correlation in term of vitamin C content
of the samples between oxidation- reduction titration
and HPLC method.
Results and Discussion
Weight, diameter and colourof fruit samples
Table 1 shows the weight, diameter and skin
colour of citrus fruit samples. Among the six kinds of
citrus fruit samples, grapefruit has the highest weight
of 243 g followed by orange (151 g), lemon (148 g),
kafr lime (65 g), lime (56 g) and musk lime (3.4
g). Similarly, grapefruit has the highest diameter of
8.0 cm followed by orange (6.5 cm), lemon (5.8 cm),
kafr lime (5.0 cm), lime (4.6 cm) and musk lime
(3.0 cm). Table 1 also shows skin colour of the citrus
fruits, as the orange fruit is a bright orange while
grapefruit is yellow-orange colour. The skin colour of
lemon is yellow whereas lime, musk lime and kafr
lime are the greencolour.
Total vitamin C content in fruit samples by titration
and HPLC method
Table 2 shows the total vitamin C content in
fruit samples by titration method. By comparing
the vitamin C concentration of the six citrus fruits,
orange has the highest vitamin C content of 58.30
mg/100g followed by grapefruit (49.15 mg/100g),
lemon (43.96 mg/100g), kafr lime (37.24 mg/100g)
and lime (27.78 mg/100g). Musk lime has the
least vitamin C content at only 18.62 mg/100g. To
determine the precision of the data, triplicate samples
were analysed, and standard deviation (SD) and
coefcient of variation (CV) were calculated. A
CV below 5.0% is considered precise whereby an
acceptable range for standard deviation is between
-1.00 ˂ SD ˂ 1.00. Generally, SD of the fruit samples

729 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733
(orange, grapefruit, lemon, kafr lime, lime and
musk lime obtained are ± 0.53, ± 0.53, ± 0.92, ± 1.06,
± 0.529 and ± 0.53, respectively) were within the
range except for kafr lime. The CV of fruit samples
(orange, grapefruit, lemon, kafr lime, lime and musk
lime obtained were 0.91%, 1.08%, 2.08%, 2.84%,
1.90% and 2.84%, respectively) also were less than
5.0%, thus indicate the precision of the data. Table 2
also shows that the concentration of vitamin C in the
fruit samples were signicantly different (p<0.05).
Table 2 shows the total vitamin C content in fruit
samples by high performance liquid chromatography
(HPLC) method. By comparing the vitamin C
concentration of the six citrus fruit samples, it can
be seen that orange remains to contain the highest
vitamin C of 43.61 mg/100g followed by lemon
(31.33 mg/100g), grapefruit (26.40 mg/100g), lime
(22.36 mg/100g), and kafr lime (21.58 mg/100g).
Among the fruit samples, musk lime has the lowest
vitamin C content of only 16.78 mg/100g. Similarly,
SD of the studied samples was within the acceptable
range except for orange while CV of all samples
were less than 5.0%, thus indicating the precision of
the data.The concentrations of vitamin C in the fruit
samples were also signicantly different(p< 0.05).
Comparison of vitamin C content of fruit samples
by titration method between a present and previous
studies is shown in Table 3. Vitamin C of orange
sample in the present study is almost similar to values
reported by Cioroi (2006) (56.02 mg/100g), Sanusi et
al. (2008) (56.00 mg/100g) and Bungau et al. (2011)
(56.40 mg/100g) but differ in the value reported by
Tee et al., (1988). Vitamin C of grapefruit in this
study is close to the value reported by Cioroi (2006)
(48.010 mg/100g) and Sanusi et al. (2008) (47.000
mg/100g) but higher than reported by Bungau et
al. (2011) (39.40 mg/100g). Lemon in the present
study contained almost similar amount of vitamin C
as published by Tee et al. (1988) (46.800 mg/100g),
Cioroi (2006) (51.780 mg/100g), Sanusi et al. (2008)
Table 1. Weight, diameter and colour of fruit samples
Table 2. Total Vitamin C content (mg/100g) in fruits sample by different method
Values in the same column with different superscripts letters are signicantly different at
p<0.05 (ANOVA). Values in same row with different superscript symbol are signicantly
different at p<0.05 (T-test).

Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733 730
(41.000 mg/100g) and Bungau et al. (2011) (49.000
mg/100g). However, vitamin C in lime sample in this
study exhibited considerable difference compared
with Tee et al. (1988). However, there was no previous
study reported the value of vitamin C content in musk
lime and kafr lime.
Table 4 shows comparison of vitamin C content
in fruit samples by HPLC method between a present
and previous studies. Orange fruit had the almost
similar amount of vitamin C with the value reported
by Scherer et al. (2012) (43.133mg/100g). However,
the differences in vitamin C between the studied
orange with other studies (52.50 to 64.27 mg/100g)
were quite large (Tee et al., 1997; Chebrolu et al.,
2012). For grapefruit, the vitamin C content of the
present study was smaller than reported by Tee et al.
(1997) (40.000 mg/100g) and Cherbrolu et al., (2012)
(30.670 mg/100g).However, the vitamin C content
in present study was closeto the value reported by
Scherer et al. (2012). Previous studies on vitamin
C of lemon indicated a large difference of 19.40 to
55.50 mg/100g (Tee et al., 1997; Chebrolu et al.,
2012). In contrast, the vitamin C in lime sample of
the present study was lower compared with the report
by Tee et al. (1997) (36.900 mg/100g) and Cherbrolu
et al. (2012) (38.960 mg/100g). Similarly, musk lime
(41.60 mg/100g) and kafr lime (37.00 mg/100g) in
this study exhibited substantial differences in terms
of vitamin C content as compared with value by Tee
et al. (1997).
Differences of vitamin C content between titration
and high performance liquid chromatography
(HPLC) methods
Table 2 shows a comparison of mean vitamin
C content between titration and high performance
liquid chromatography (HPLC) methods. There were
signicant differences in the content of vitamin C
between both methods (p <0.05). Generally, the main
advantage of using oxidation-reduction titration
method is because of its simplicity using simple
equipment and inexpensive chemicals. Furthermore,
the reaction of indophenol dye with the ascorbic
acid is very fast. However, in some condition, the
oxidation-reduction titration may overestimate
the vitamin C content of fruit as the end point of
titration could be difcult to be detected especially
when high colored (e.g.,reddish-purplish)fruit was
used (Hernandez et al., 2006). Besides, the presence
of reducing substances (ferrous ion, copper ion,
sulphur dioxide, sulphite and thiosulphate) in the
fruit samples can react with the indophenol dye and
cause overestimation of vitamin C in fruit samples
(Spinola et al., 2013). When oxidation-reduction
titration is not rapid, the exposure of samples to
oxygen and light may cause degradation of the
Table 3. Comparison of the vitamin C content in citrus fruit sample by titration
method between present and previous studies

731 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733
ascorbic acids. Besides, the reduced ascorbic acids
(dehydroascorbic acids) also are not quantied in
this method. Therefore, the titration method is lack of
specicity, do not overcome problem with reducing
substances and might cause exposure to the air.
High performance liquid chromatography (HPLC)
method is a more specic, sensitive and selective
technique for determining vitamin C content in
fruit samples. Furthermore, HPLC method requires
small amount of sample and chemicals, quite rapid
and less susceptible to systemic error due to its high
specicity (Quiroz and Fernandez, 2009).
Using simple linear correlation,the relationship
between titration and HPLC methodsregarding
vitamin C content in citrus fruits samples was
performed. There was no correlation found between
titration and HPLC methods in the studied samples
(Table 5). In contrast, Hernandez et al. (2006)
showed that there was strong signicant correlation
(r2=0.980) between titration and HPLC methods.
Hernandez et al. (2006) also reported no signicant
differences between the titration and HPLC methods
in orange, papaya, mango and pineapple samples.
Spinola et al. (2013) also showed strong signicant
correlation (r2=0.976) between the titration and HPLC
methods in samples of lemon, papayas, passion fruits
cherimoyas, strawberries and broccoli. Similarly,
Spinola et al. (2013) also reported no signicant
differences in vitamin C contents by both methods.
Generally, the variations of vitamin C content
in citrus fruit samples could be attributed to many
pre-harvest and handling factors includingthe
environment. Exposure of fruits to light have strong
inuence on chemical composition of fruits especially
vitamin C. The higher the light intensity exposed to
citrus fruit trees, the higher the vitamin C content in
the fruits as light is required for photosynthesis to
produce energy in the form of glucose to produce
more ascorbic acids in the fruits (Stumpf et al., 1988).
Besides, Hassan et al. (2012) stated that utilization of
organic based fertilizer may improve the quality of
plants by reduction of nitrate content, increment of
vitamin C content, antioxidant activity, nitrogen and
calcium contents. Furthermore, ripening process of
citrus fruits may reduce the vitamin C content. Igwe
(2013) showed thatvitamin C concentration of unripe
fruits was higher than the matured ripe fruits and both
vitamin C levels of ripe and unripe fruits decreased
when the temperature and length of exposure of fruits
were increased. Storage temperature and handling
also are important in maintaining the vitamin C
content in fruit samples. In addition, citrus fruits
grown in fully irrigated system during owering, the
fruit growing stage and ripening showed increased
Table 4: Comparison of the vitamin C content in fruit sample by HPLC method
between present and previous studies

Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733 732
concentration of vitamin C compared to control
(Aguado et al., 2012).
Conclusion
In this study, orange contained the highest vitamin
C among the citrus fruits followed by grapefruit,
lemon, kafr lime and lime by titration method.
Similarly, orange also has the highest vitamin C by
HPLC method followed by lemon, grapefruit, lime
andkafr lime.Musk lime contained the least vitamin
C by both methods.Comparison between titration
and HPLC method in terms of vitamin C content,
showed signicant differences in all fruit samples.
The vitamin C contents in fruits samples were higher
in titration method compared with HPLC method.
The signicant differences between the two methods
could be affected due to many factors such as lack
of specicity, presence of the reducing substances,
time consuming and exposure to the air. Furthermore,
the value of vitamin C content in HPLC method was
lower than titration method. This could be due to
the high sensitivity, selectivity and specicity of the
HPLC method in isolating actual amount of vitamin
C in fruit samples without any interference of other
substances.
Acknowledgement
The authors are grateful to staffs of Department
of Nutrition and Dietetics, Faculty of Medicine and
Health Sciences, UniversitiPutra Malaysia for the
assistance in analysing samples of this study.
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