International Food Research Journal 24(2): 726-733 (April 2017)

Journal homepage: http://www.ifrj.upm.edu.my

Comparison of vitamin C content in citrus fruits by titration and high

performance liquid chromatography (HPLC) methods
1
Fatin Najwa, R. and 1,2*Azrina, A.
1
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, 43400 UPM
Serdang, Selangor, Malaysia
2
Research Centre of Excellence, Nutrition and Non-communicable Diseases, Faculty of Medicine
and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Article history Abstract

Received: 29 June 2015 Vitamin C is one of the essential vitamins for human and animal. Many methods were
Received in revised form: developed for the determination of vitamin C such as spectrophotometry, electrophoresis,
23 March 2016 titration, and high performance liquid chromatography (HPLC). This study aims to compare
Accepted: 4 April 2016
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
Keywords
(43.96 mg/100g), kaffir lime (37.24 mg/100g), lime (27.78 mg/100g) and musk lime (18.62
Vitamin C mg/100g). While, in the HPLC method orange also leads with the highest vitamin C content
Ascorbic acid (43.61 mg/100g) followed by lemon (31.33 mg/100g), grapefruit (26.40 mg/100g), lime (22.36
Citrus fruits mg/100g), kaffir lime (21.58 mg/100g) and musk lime (16.78 mg/100g). Orange is the best
High performance liquid source of vitamin C while musk and kaffir lime have lower content. Significant differences
chromatography were observed in vitamin C of samples by both methods. Both methods are suitable for the
Titration determination of vitamin C, however HPLC method is more accurate, precise and specific.
© All Rights Reserved

Introduction and metal ion (Wantz et al., 2005).

Human is unable to synthesize their own vitamin
Vitamin C, also is known as ascorbic acid is C supply as human cells cannot perform the crucial
one of the most important vitamins and essential last step in vitamin C biosynthesis,the conversion
for human and animal life. This water soluble of L-gulono-g-lactone into ascorbic acid which
vitamin contributes to many health benefits such is catalyzed by gulonolactone oxidaseenzyme
as prevention of scurvy and cancer, relief from (LinsterandVan Schaftingen, 2007). Therefore, they
common cold, stimulate collagen synthesis and require vitamin C for maintaining the physiological
play a significant role in wound healing process functions. To meet the requirement, vitamin C must
(Iqbal et al., 2004). According to Teucher et al. be consumed from diet. The recommended nutrient
(2004), vitamin C helps to enhance availability and intake of vitamin C for Malaysian adult is 70 mg per
absorption of iron from non-heme sources. Vitamin day. An intake of 45 mg/day will ensure measurable
C also has antioxidant properties since it can easily amount of ascorbate be present in the plasma of most
lose the electron to neutralize and inhibit free radicals people and available to supply tissue requirements for
from being oxidized in preventing cell damage. It is metabolism or repair at sites of depletion or damage
also commonly used as food additive which acts as (MOH, 2005).
antioxidant (Whitney and Rolfes, 2008). Many methods can be used for determination
Vitamin C is an organic compound consists of of vitamin C such as spectrophotometry,
carbon, hydrogen and oxygen (Chinnici et al., 2005). electrophoresis, titration, and high performance
The terms vitamin C is not only used for ascorbic acids, liquid chromatography (HPLC) (Tang and Wu,
but it includes all compounds exhibiting biological 2005; Dong et al., 2007; Spinola et al., 2012). The
activity such as oxidized, ester and synthetic form. most commonly used method is oxidation-reduction
The main biological form of vitamin C is L-ascorbic titration method where ascorbic acids are oxidized
acid, and it can reversibly change to oxidized form to dehydroascorbic acid and the indophenol dye is
called dehydroascorbic acid (Fenolland Martinez, reduced to a colorless compound. The end point of
2010). Many factors can cause oxidation of vitamin the titration can be easily detected when an excess of
C such as pH, light, temperature, presence of oxygen the unreduced dye give a rose pink color in an acid

*Corresponding author.
Email: azrinaaz@upm.edu.my
727 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733

solution (Tee et al., 1996). It is a simple and easy Preparation of standard solution for titration
method to determine vitamin C in fruits and fruit method
juices. However, the method is not suitable for fruits Vitamin C standard solution (0.2 mg/ml) was
that have reddish-purplish color. The titration method prepared by dissolving 100 mg vitamin C in 3% of
also is time-consuming and lack of specificity due to metaphosphoric acid (HPO3) solution and diluted to
interference of reducing substances in the food such 500 ml with the same solvent. Then, 5 ml aliquot of the
as ferrous iron, stannous tin, cuprous copper, sulphur vitamin C standard solution was diluted (containing
dioxide, sulphite orthiosulphate (Eitenmiller et al., 1 mg vitamin C) with 5 ml 3% metaphosphoric acid
2008). (HPO3). Next the diluted vitamin C standard solution
Several other methods to determine vitamin was titrated with dye solution 2, 6-dichlorophenol-
C content like spectrometric, spectrofluorimetric, indophenol to a faint pink color. Triplicate titration
and electrophorhesis, still some of them are not also was conducted for the vitamin C standard.
practical and need re-evaluation due to insufficient
of sensitivity and selectivity (Agar, 1995). Quiroz Quantification of Vitamin C in the extracted solution
and Fernandez (2009) stated that the most preferred of titration method
method of determining vitamin C content in foods is Firstly, weight of the sample in 10 ml filtrate was
a chromatographic method using HPLC due to the calculated by the following equation:
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 Where,
phase, mobile phase, type of detector and sample a = Weight of sample will be used
preparation (Gazdik et al., 2008; Spinola et al., 2012) b = Final volume of homogenous slurry [sample
but there was not many studies done comparing two + 6% metaphosphoric acids (HPO3)]
or more different methods in determining vitamin c= Aliquot of homogenous slurry used for dilution
C in citrus fruits (Ullah et al., 2012; Spinola et al., to 100 ml with 3% metaphosphoric acid (HPO3)
2013). Moreover, most studies are just focusing on
single type of lime and do not focus on another type Then total vitamin C content (mg per 100 g
of limes such as kaffir lime and musk lime.Thus, the sample) of each sample was obtained by the following
purpose of this study was to provide the comparison equation:
between titration and HPLC method in determination
of vitamin C content in citrus fruits. Vitamin C content (mg per 100 g sample) =

Materials and Methods X = volume of dye used for titration of aliquot of

diluted sample (ml)
Sample preparation of titration method Y = vitamin C equivalent of dye solution, mg per
Sample and standard preparation and ml dye solution
quantification of vitamin C in titration method was W = weight of sample in aliquot of filtrate of
performed using the method described by Tee et al. diluted sample used for titration (g)
(1996). In brief, fruit samples (200 g-300 g) was
blended with 6% metaphosphoric acid (HPO3) with Sample preparation of HPLC method
an equal volume to homogenous slurry and made it Sample preparation, chromatography conditions,
up until 500 ml of volume. The homogenous mixture identificantion and quantification of vitamin C in
was measured around 10 to 30 ml and diluted into HPLC method was perfomed using the method
100 ml volumetric flask with 3% of metaphosphoric described by Czech Agriculture and Food Inspection
acid (HPO3). The diluted sample was then filtered to Security (2005). The homogenous solid sample
remove away suspension using vacuum pump before was measured around 10 to 30g and mixed it with
10 ml aliquote of the filtrate was pipetted into a 60 to 80 ml 3% of metaphosphoric acid (HPO3)
small Erlenmayer flask. The filtrate was immediately for one minute. The obtained extracted was filtered
titrated with a dye solution 2, 6-dichlorophenol- through filtration paper and washed it for few times
indophenol to a faint pink end point. Triplicate by using vacuum pump filtration. Next, the filtrate
titration was conducted for all samples. quantitatively transferred into a 100 ml volumetric
flask 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 filtered again through 0.45 µm syringe Where,

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

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 significantly different at
p<0.05 (ANOVA). Values in same row with different superscript symbol are significantly
different at p<0.05 (T-test).

(orange, grapefruit, lemon, kaffir lime, lime and SD of the studied samples was within the acceptable
musk lime obtained are ± 0.53, ± 0.53, ± 0.92, ± 1.06, range except for orange while CV of all samples
± 0.529 and ± 0.53, respectively) were within the were less than 5.0%, thus indicating the precision of
range except for kaffir lime. The CV of fruit samples the data.The concentrations of vitamin C in the fruit
(orange, grapefruit, lemon, kaffir lime, lime and musk samples were also significantly different(p< 0.05).
lime obtained were 0.91%, 1.08%, 2.08%, 2.84%, Comparison of vitamin C content of fruit samples
1.90% and 2.84%, respectively) also were less than by titration method between a present and previous
5.0%, thus indicate the precision of the data. Table 2 studies is shown in Table 3. Vitamin C of orange
also shows that the concentration of vitamin C in the sample in the present study is almost similar to values
fruit samples were significantly different (p<0.05). reported by Cioroi (2006) (56.02 mg/100g), Sanusi et
Table 2 shows the total vitamin C content in fruit al. (2008) (56.00 mg/100g) and Bungau et al. (2011)
samples by high performance liquid chromatography (56.40 mg/100g) but differ in the value reported by
(HPLC) method. By comparing the vitamin C Tee et al., (1988). Vitamin C of grapefruit in this
concentration of the six citrus fruit samples, it can study is close to the value reported by Cioroi (2006)
be seen that orange remains to contain the highest (48.010 mg/100g) and Sanusi et al. (2008) (47.000
vitamin C of 43.61 mg/100g followed by lemon mg/100g) but higher than reported by Bungau et
(31.33 mg/100g), grapefruit (26.40 mg/100g), lime al. (2011) (39.40 mg/100g). Lemon in the present
(22.36 mg/100g), and kaffir lime (21.58 mg/100g). study contained almost similar amount of vitamin C
Among the fruit samples, musk lime has the lowest as published by Tee et al. (1988) (46.800 mg/100g),
vitamin C content of only 16.78 mg/100g. Similarly, Cioroi (2006) (51.780 mg/100g), Sanusi et al. (2008)
 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733 730

Table 3. Comparison of the vitamin C content in citrus fruit sample by titration

method between present and previous studies

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

Table 4: Comparison of the vitamin C content in fruit sample by HPLC method

between present and previous studies

ascorbic acids. Besides, the reduced ascorbic acids Spinola et al. (2013) also reported no significant
(dehydroascorbic acids) also are not quantified in differences in vitamin C contents by both methods.
this method. Therefore, the titration method is lack of Generally, the variations of vitamin C content
specificity, do not overcome problem with reducing in citrus fruit samples could be attributed to many
substances and might cause exposure to the air. pre-harvest and handling factors includingthe
High performance liquid chromatography (HPLC) environment. Exposure of fruits to light have strong
method is a more specific, sensitive and selective influence on chemical composition of fruits especially
technique for determining vitamin C content in vitamin C. The higher the light intensity exposed to
fruit samples. Furthermore, HPLC method requires citrus fruit trees, the higher the vitamin C content in
small amount of sample and chemicals, quite rapid the fruits as light is required for photosynthesis to
and less susceptible to systemic error due to its high produce energy in the form of glucose to produce
specificity (Quiroz and Fernandez, 2009). more ascorbic acids in the fruits (Stumpf et al., 1988).
Using simple linear correlation,the relationship Besides, Hassan et al. (2012) stated that utilization of
between titration and HPLC methodsregarding organic based fertilizer may improve the quality of
vitamin C content in citrus fruits samples was plants by reduction of nitrate content, increment of
performed. There was no correlation found between vitamin C content, antioxidant activity, nitrogen and
titration and HPLC methods in the studied samples calcium contents. Furthermore, ripening process of
(Table 5). In contrast, Hernandez et al. (2006) citrus fruits may reduce the vitamin C content. Igwe
showed that there was strong significant correlation (2013) showed that vitamin C concentration of unripe
(r2=0.980) between titration and HPLC methods. fruits was higher than the matured ripe fruits and both
Hernandez et al. (2006) also reported no significant vitamin C levels of ripe and unripe fruits decreased
differences between the titration and HPLC methods when the temperature and length of exposure of fruits
in orange, papaya, mango and pineapple samples. were increased. Storage temperature and handling
Spinola et al. (2013) also showed strong significant also are important in maintaining the vitamin C
correlation (r2=0.976) between the titration and HPLC content in fruit samples. In addition, citrus fruits
methods in samples of lemon, papayas, passion fruits grown in fully irrigated system during flowering, the
cherimoyas, strawberries and broccoli. Similarly, fruit growing stage and ripening showed increased
 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733 732

Table 5. Pearson’s correlations coefficient (r) test between titration and high
performance liquid chromatography (HPLC) method in term of total vitamin C content
in fruit samples

*
p<0.05

concentration of vitamin C compared to control J. L. and Capote, N. 2012. Towards the Improvement
(Aguado et al., 2012). of Fruit-Quality Parameters in Citrus under Deficit
Irrigation Strategies. ISRN Agronomy 2012: 1-9
Conclusion Bungau, S., Fodor, A., Ţiţ, D. M. and Szabó, I. 2011.
Studies on citrus species fruits ascorbic acid content
using kinetic, spectrophotometric and iodometric
In this study, orange contained the highest vitamin methods. Analele Universitităţii din Oradea, Fascicula:
C among the citrus fruits followed by grapefruit, Protecţia Mediului 16: 212-217.
lemon, kaffir lime and lime by titration method. Chebrolu, K. K., Jayaprakasha, G. K., Yoo, K. S., Jifon,
Similarly, orange also has the highest vitamin C by J. L. and Patil, B. S. 2012. An improved sample
HPLC method followed by lemon, grapefruit, lime preparation method for quantification of ascorbic
andkaffir lime.Musk lime contained the least vitamin acid and dehydroascorbic acid by HPLC. LWT-Food
C by both methods.Comparison between titration Science and Technology 47(2): 443-449.
and HPLC method in terms of vitamin C content, Chinnici, F., Spinabelli, U., Riponi, C. and Amati, A.
showed significant differences in all fruit samples. 2005. Optimization of the determination of organic
acids and sugars in fruit juices by ion-exclusion liquid
The vitamin C contents in fruits samples were higher
chromatography. Journal of Food Composition and
in titration method compared with HPLC method. Analysis 18(2): 121-130.
The significant differences between the two methods Cioroi, M. 2007. Study on L-ascorbic acid contents from
could be affected due to many factors such as lack exotic fruits. Cercetari Agronomice Moldova1 129:
of specificity, presence of the reducing substances, 23-27.
time consuming and exposure to the air. Furthermore, Czech Agriculture and Food Inspection Authority.
the value of vitamin C content in HPLC method was 2005. Determination of Vitamins, Caffeine and
lower than titration method. This could be due to PreservativesDownloaded from http://web.vscht.
the high sensitivity, selectivity and specificity of the cz/~kohoutkj/ENG/LAPP_ANGL_HPLC_UV.pdf
HPLC method in isolating actual amount of vitamin Dong, S., Zhang, S., Cheng, X., He, P., Wang, Q. and Fang,
Y. 2007. Simultaneous determination of sugars and
C in fruit samples without any interference of other
ascorbic acid by capillary zone electrophoresis with
substances. amperometric detection at a carbon paste electrode
modified with polyethylene glycol and Cu2O. Journal
Acknowledgement of Chromatography A 1161(1): 327-333.
Eitenmiller, R. R., Ye, L. and Landen, W. O. 2008. Vitamin
The authors are grateful to staffs of Department Analysis for Health and Food Sciences. 2nd edn. USA:
of Nutrition and Dietetics, Faculty of Medicine and CRC Press.
Health Sciences, UniversitiPutra Malaysia for the Fenoll, J. andMartinez, A. 2010. Simultaneous
assistance in analysing samples of this study. determination of ascorbic and dehydroascorbic acids
in vegetables and fruits by liquid chromatography with
Tandem-Mass Spectrophotometry. Food Chemistry
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