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


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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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International Food Research Journal 24(2): 726-733 (April 2017)
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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
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.
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
Vitamin C
Ascorbic acid
Citrus fruits
High performance liquid
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.,
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
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:
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
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
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
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:
Cst = standard working solution concentration 50
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).
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
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.
Agar, T. 1995. A Sensitive Method to Determination of
L-Ascorbic Acid, Dehydroascorbic acid and Total
Vitamin C: Microuorometric Method. Cukurova
University Journal of the Faculty of Agriculture 9 (1):
Aguado, A., Frías, J., García-Tejero, I., Romero, F., Muriel,
J. L. and Capote, N. 2012. Towards the Improvement
of Fruit-Quality Parameters in Citrus under Decit
Irrigation Strategies. ISRN Agronomy 2012: 1-9
Bungau, S., Fodor, A., i, D. M. and Szabó, I. 2011.
Studies on citrus species fruits ascorbic acid content
using kinetic, spectrophotometric and iodometric
methods. Analele Universititii din Oradea, Fascicula:
Protecia Mediului 16: 212-217.
Chebrolu, K. K., Jayaprakasha, G. K., Yoo, K. S., Jifon,
J. L. and Patil, B. S. 2012. An improved sample
preparation method for quantication of ascorbic
acid and dehydroascorbic acid by HPLC. LWT-Food
Science and Technology 47(2): 443-449.
Chinnici, F., Spinabelli, U., Riponi, C. and Amati, A.
2005. Optimization of the determination of organic
acids and sugars in fruit juices by ion-exclusion liquid
chromatography. Journal of Food Composition and
Analysis 18(2): 121-130.
Cioroi, M. 2007. Study on L-ascorbic acid contents from
exotic fruits. Cercetari Agronomice Moldova1 129:
Czech Agriculture and Food Inspection Authority.
2005. Determination of Vitamins, Caffeine and
PreservativesDownloaded from http://web.vscht.
Dong, S., Zhang, S., Cheng, X., He, P., Wang, Q. and Fang,
Y. 2007. Simultaneous determination of sugars and
ascorbic acid by capillary zone electrophoresis with
amperometric detection at a carbon paste electrode
modied with polyethylene glycol and Cu2O. Journal
of Chromatography A 1161(1): 327-333.
Eitenmiller, R. R., Ye, L. and Landen, W. O. 2008. Vitamin
Analysis for Health and Food Sciences. 2nd edn. USA:
CRC Press.
Fenoll, J. andMartinez, A. 2010. Simultaneous
determination of ascorbic and dehydroascorbic acids
in vegetables and fruits by liquid chromatography with
Tandem-Mass Spectrophotometry. Food Chemistry
127(1): 340-344.
Gazdik, Z., Zitka, O., Petrlova, J., Adam, V., Zehnalek,
J., Horna, A. and Kizek, R. 2008. Determination of
vitamin C (ascorbic acid) using high performance
liquid chromatography coupled with electrochemical
detection. Sensors 8(11): 7097-7112.
Hassan, S. A., Mijin, S., Yusoff, U. K., Ding, P. and
Wahab, P. E. M. 2012. Nitrate, ascorbic acid, mineral
Table 5. Pearson’s correlations coefcient (r) test between titration and high
performance liquid chromatography (HPLC) method in term of total vitamin C content
in fruit samples
733 Fatin Najwa, R. and Azrina, A./IFRJ 24(2): 726-733
and antioxidant activities of Cosmos caudatus in
response to organic and mineral-based fertilizer Rates.
Molecules17(7): 7843-7853.
Hernandez, Y., Lobo, M. G. and González, M. 2006.
Determination of vitamin C in tropical fruits: A
comparative evaluation of methods. Food Chemistry
96(4): 654-664.
Igwe, O. U. 2013. Quantitative Estimation of Ascorbic
Acid Levels in Citrus Fruits at Variable Temperatures
and Physicochemical Properties. International Journal
of Chemical and Biochemical Sciences. 5: 67-71
Iqbal, K., Khan, A. and Khattak, M. M. A. K. 2004.
Biological signicance of ascorbic acid (vitamin C) in
human health—a review. Pakistan Journal of Nutrition
3(1): 5-13.
Linster, C. L. and Van Schaftingen, E. 2007. Vitamin C.
Febs Journal, 274(1): 1-22.
Ministry of Health Malaysia. 2005. Recommended Nutrient
Intakes (RNI) for Malaysia. Kuala LumpurNational
Coordinating Committee on Food and Nutrition.
Quiroz A. R. B. and Fernandez-Arias M. 2009. A Screening
Method for the Determination of Ascorbic Acid in
Fruit Juices and Soft Drinks. Food Chemistry 116(2):
Sanusi, R. A., Ogunro, Y. and Nwozoh, S. 2008. Effect
of Storage Time on Ascorbic Acid Content of Some
Selected “Made in Nigeria” Fruit Preserves. Pakistan
Journal of Nutrition 7(6): 730-732.
Scherer, R., Rybka, A. C. P., Ballus, C. A., Meinhart, A. D.
and Godoy, H. T. 2012. Validation of a HPLC method
for simultaneous determination of main organic acids
in fruits and juices. Food Chemistry 135(1): 150-154.
Spinola, V., Mendes, B., Câmara, J. S. and Castilho,
P. C. 2012. An improved and fast UHPLC-PDA
methodology for determination of L-ascorbic and
dehydroascorbic acids in fruits and vegetables.
Evaluation of degradation rate during storage.
Analytical and Bioanalytical Chemistry 403(4): 1049-
Spinola, V., Mendes, B., Câmara, J. S., and Castilho, P.
C. 2013. Effect of time and temperature on vitamin
C stability in horticultural extracts. UHPLC-PDA vs
iodometric titration as analytical methods. LWT-Food
Science and Technology50: 489-495.
Stumpf, W., Conn, P. M. andPreiss, J. 1988. The
Biochemistry of Plants: Carbohydrates. California:
Academic Press.
Tang, Y. and Wu, M. 2005. A quick method for the
simultaneous determination of ascorbic acid and sorbic
acid in fruit juices by capillary zone electrophoresis.
Talanta 65(3): 794-798.
Tee E.S., Noor M.I., Azudin M. N. and Idris K. 1997.
Nutrient Composition of Malaysian Foods. 4th edn.
Kuala Lumpur: Institute for Medical Research.
Tee, E. S., Young, S. I., Ho, S. K. and Shahid, S.
M. 1988. Determination of vitamin C in fresh
fruits and vegetables using the dye-titration and
microuorometric methods. Pertanika 11(1): 39-44.
Tee, E.S., Rajam, K., Young, S. I., Khor, S. C. and Zakiyah,
H. O. 1996 Laboratory Procedures in Nutrient
Analysis of Foods. Kuala Lumpur, Malaysia: Division
of Human Nutrition, Institute for Medical Research.
Teucher, B., Manuel, O. and Héctor, C. 2004 Enhancers
of iron absorption: ascorbic acid and other organic
acids. International Journal for Vitamin and Nutrition
Research 74(6): 403-419.
Ullah, S., Hussain, A., Ali, J., Ullah, K. and Ullah, A. 2012.
A simple and rapid HPLC method for analysis of
vitamin-C in local packed juices of Pakistan.Middle-
East Journal of Scientic Research12(8): 1085-1091.
Wantz, F., Banks, C. E. and Compton, R. G. 2005.
Direct oxidation of ascorbic acid at an edge plane
pyrolytic graphite electrode: a comparison of
the electroanalytical response with other carbon
electrodes. Electroanalysis 17(17): 1529-1533.
Whitney, E. and Rolfes, S.R. 2008. Understanding
Nutrition. 11thedn. USA: Thomas Wadsworth.
... The pear is a genuinely amazing fruit that is hardy and commonly cultivated in temperate regions of the world. Its size, shape, texture, and flavor are all different [24]. Pears are a juicy, mildly sweet fruit with a buttery texture. ...
... By subjecting the sample to US treatment followed by ST at room temperature, using 100% ethanol as the solvent, we extracted high amounts of ascorbic acid (3.71 mg/g). In comparison, a previous study examining aqueous extracts of lemon peels reported a significantly lower recovery of ascorbic acid, as low as 0.59 mg/g [14,51]. This striking difference of 533% highlights the pivotal role of the solvent in optimizing the extraction process and effectively isolating valuable nutrients. ...
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Citrus limon is among the species of the genus Citrus that dominates the world market. It is highly nutritious for humans as it contains twice the amount of the suggested daily intake of ascorbic acid and is also a good source of phenolic compounds, carotenoids, and other bioactive compounds. This study aimed to identify the optimal extraction procedures and parameters to obtain the maximum quantity of bioactive components from lemon peel by-products. Various extraction techniques, including stirring, ultrasound, and pulsed electric field, were evaluated, along with factors such as extraction time, temperature, and solvent composition. The results revealed that simple stirring for 150 min at 20 °C proved to be the most effective and practical method. The ideal solvent mixture consisted of 75% ethanol and 25% water, highlighting the crucial role of solvent composition in maximizing extraction efficiency. Among the extracted compounds were phenolics, ascorbic acid, and carotenoids. Under optimum extraction conditions, the extract was found to contain high total phenolic content (TPC) (51.2 mg of gallic acid equivalents, GAE/g dry weight), total flavonoid content (TFC) (7.1 mg of rutin equivalents, RtE/g dry weight), amounts of ascorbic acid (3.7 mg/g dry weight), and total carotenoids content (TCC) (64.9 μg of β-carotene equivalents, CtE/g). Notably, the extracts demonstrated potent antioxidant properties (128.9 μmol of ascorbic acid equivalents, AAE/g; and 30.3 μmol of AAE/g as evidenced by FRAP and DPPH assays, respectively), making it a promising ingredient for functional foods and cosmetics. The study’s implications lie in promoting sustainable practices by converting lemon peel into valuable resources and supporting human health and wellness through the consumption of natural antioxidants.
... Hasil ini lebih tinggi daripada penelitian-penelitian sebelumnya pada jenis jeruk lain seperti grapefruit (49.42 mg/100g), orange (58.30 mg/100g), lemon (51.78 mg/100g), Citrus aurantium (9.86 mg/piece), dan citron (7.20 mg/piece) (Najwa and Azrina 2017;Shrestha, Shrestha, and Bhattarai 2016). Variasi kandungan asam askorbat pada jeruk tergantung pada faktor alami seperti area penanaman, kondisi iklim, spesies, tingkat kematangan, dan varietas jeruk (Shrestha, Shrestha, and Bhattarai 2016). ...
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Citrus are known to be rich in antioxidant compounds such as vitamin C and phenolics, that fluctuate during ripening. This study aims to determine the content of vitamin C and sugar composition in Citrus depressa H. (CDH) fruit from Taiwan at different levels of ripeness. Analysis of vitamin C was carried out using UV spectrophotometer method, total soluble solid using a brix refractometer, and sugar composition using an HPLC ELSD. The results showed that CDH possess higher amount of vitamin C and lower sugar than other fruits in previous researches. Ripe CDH contains higher vitamin C (175.42 ± 7.43 mg/100 mL) than unripe fruit (99.17 ± 7.33 mg/100mL). The concentration of sugar component in unripe CDH (1186.09 ppm) is lower than the ripe fruit (2706.86 ppm).
... Graph 1 and Table 1 reveal that pulp and peel of grapefruit have highest contents of Vitamin C as compare to the pulp and peel of other member of citrus family [26]. The result is also comparable with a research work which shows that Grapefruit and oranges have higher contents of vitamin C as compare to lemon [27]. As the Table 1 reveals that the pulp and peel of Grapefruit has highest contents of vitamin C i.e. 114.92 ± 0.36mg/100g and 110.56 ± 0.415mg/100g respectively while orange pulp and peel showed vitamin C concentration at second position with 112.3 ± 0.45092mg/100g and 102.23 ± 0.689mg/100g respectively. ...
... The ascorbic acid contents of lime fruits may be present at different levels depending on the lime variety. The ascorbic acid content was found in Citrus aurantifolia, Citrus histrix, and Citrus microcarpa at the level of 29.5, 21.58, and 16.78 g/100 mL, respectively [30,31]. The total phenolic content (TPC) of C. latifolia was also much higher than that of Citrus limonia which were 318.2 mgGAE/100 mL and 114.7 mgGAE/100 mL, respectively. ...
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Lime juice is rich in bioactive components and exerts a wide range of therapeutic effects, especially antioxidant activity. Freeze concentration is considered an essential method to maintain the nutritional values and bioactives of fruit juices. This study aimed to compare the ability in enriching ascorbic acid, total phenolic compounds, and major flavonoids and the antioxidant activity (DPPH) of concentrated lime juices prepared by vacuum and freeze-concentrations. The ascorbic acid in the juices was analyzed using the HPLC-PDA method. The total phenolic content and DPPH inhibition ability were measured by the colorimetric method. The polyphenol profiles of two lime varieties (C. latifolia and C. limonia) were qualitatively analyzed using LC-TOF MS/MS; then, the major juices’ flavonoids were analyzed by HPLC-PDA against/based on commercial standards. The results showed that C. latifolia was superior to C. limonia in ascorbic acid, TPC, major flavonoids, and antioxidants. C. latifolia was also more diverse than C. limonia in its polyphenol profile through the identified compounds (18 vs. 15). Freeze-concentrated lime juices were significantly higher than the vacuum-concentrated counterparts in ascorbic acid (mean difference from 9.41 to 22.01 mg, p < 0.01), and TPC (from 60.76 to 149.88 mgGAE). The quantification of major flavonoids showed that the freeze-concentrated lime juices retained high levels of hesperidin, eriocitrin, and rutin (p < 0.01) whereas the vacuum concentration preserved higher ones in diosmin and naringin (p < 0.01). The freeze-concentrated lime juice was significantly higher than vacuum-concentrated lime juices in the DPPH scavenging activity by at least 15% (p < 0.01). Overall, freeze concentration enriched bioactive compounds in lime juices almost threefold and improved antioxidants at least twofold. Thus, freeze concentration is promising for the industry in producing high-quality lime juice to preserve its thermal liable bioactive component.
... It is considered several folds richer in ascorbic acid content than many commercially cultivated fruits and vegetables including Citrus spp., Psidium guajava, Emblica officinalis, Capsicum spp., Brassica spp. etc. (Kumar and Tata 2009;Mishra et al. 2009;Choudhary et al. 2008;Fatin and Azrina 2017;Doseděl et al. 2021). Vitamin C from acerola fruits has been reported to have better absorption in human body than that from synthetic sources (Assis et al. 2008). ...
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In the era of COVID-19, fruits rich in ascorbic acid have gained considerable importance owing to their role in boosting immunity. West Indian Cherry or acerola is one of the richest sources of vitamin C among the plant sources and its plants are amenable for pot cultivation. In order to study the effect of different growing conditions on potted acerola plants, the present study was carried out. Potted plants were grown under green shade net and black shade net in comparison to open cultivation. Results revealed that shade net cultivation significantly influenced plant height, and distinct differences for leaf growth parameters were also noticed among the treatments. Black shade net promoted these parameters; however, fruit morphological parameters were not influenced by the treatments. Photosynthetic pigment accumulation was the highest (2.438 mg/g, 0.904 mg/g, 3.343 mg/g and 0.384 mg/g of chlorophyll a, b, total chlorophyll and total carotenoids, respectively) under black shade net condition. Fruits produced under open condition had more acidic pH (3.11) than those in green shade net (3.22) and black shade net (3.29). Ascorbic acid content of fruits was in the order: black shade net (2208.33 mg/100 g) > open (2046.78 mg/100 g) > green shade net condition (2000 mg/100 g). Thus, the study suggests the possibility of potted acerola cultivation even under shaded conditions.
Lemon and limes are considered as mystical and evergreen magical fruits which attracts the attention of scholars worldwide. Being a juicy fruit, they are widely being used in the preparation of different food products as well as in formulations of medicinal importance. Distinctive feature of lemon juice makes it an important substrate for preparation of lemon meringue pie and lemonade. Lemons and lime are good source of minerals like magnesium, calcium and potassium which are necessary for sustaining health life style. Component specific studies on lemon and lime fruits indicates that they could be used for various health benefiting features further they have no side effects. Specific nutrients and bioactive metabolites of lemon and limes makes them a good industrial substrate which could be utilized in the preparation of cosmetic products and health benefiting formulations.
Barriers to adequate hygiene are important problems throughout the world, as billions of people do not have access to sanitary conditions. In particular, unsatisfactory hand hygiene leads to the spread of bacterial infections from person to person. To address this problem, we developed the PeelTowel, an antibacterial and water-absorbing towel made of a combination of fruit peels and recycled paper waste, which has the potential to make hand-hygiene accessible, sustainable, and environmentally friendly. Kiwi, orange, and lime peels were chosen for this purpose because they contain antibacterial factors such as vitamin C and citric acid as well as water-absorbing cellulose. PeelTowels were produced by creating a paste of crushed fruit peels and paper and then drying thin films of this paste on screens. PeelTowels were tested for their ability to inhibit the growth of bacteria and absorb water. They were incubated with Escherichia coli, and bacterial survival was measured by counting colonies on agar plates. Similarly, absorption was quantified by exposing PeelTowels to varying amounts of water. The Lime PeelTowel had the highest antimicrobial activity. It eradicated 50-91% of E. coli after exposure for 1 hour and 95-98% after exposure for 18 hours. It also absorbed three times the amount of water as a commercially available paper towel. Our results suggest that Lime PeelTowels have the potential to be an environmentally friendly option for antibacterial and absorptive hand towels.
Background: Ascorbic acid is a potent natural antioxidant that protects against oxidative stress and performs various bodily functions. It is commonly found in fruits and vegetables. Objective: The manuscript has been written to provide valuable insights into ascorbic acid in managing Alzheimer's disease. Methods: The data has been gathered from web sources, including PubMed, Science Direct, Publons, Web of Science, and Scopus from 2000-2022 using AA, ascorbic acid, Alzheimer's diseases, memory, dementia, and antioxidant Keywords. Results: In the present manuscript, we have summarized the impact of ascorbic acid and its possible mechanism in Alzheimer's disease by, outlining the information currently available on the behavioral and biochemical effects of ascorbic acid in animal models of Alzheimer's disease as well as its usage as a therapeutic agent to slow down the progression of Alzheimer disease in human beings. Oxidative stress plays a significant role in the advancement of AD. AA is a wellknown antioxidant that primarily reduces oxidative stress and produces protein aggregates, which may help decrease cognitive deficits in Alzheimer's disease. The current paper analyses of ascorbic acid revealed that deficiency of ascorbic acid adversely affects the central nervous system and leads to cognitive defects. However, the results of clinical studies are conflicting, but some of the studies suggested that supplementation of ascorbic acid improved cognitive deficits and decreased disease progression. Conclusion: Based on clinical and preclinical studies, it is observed that ascorbic acid supplementation improves cognitive deficits and protects the neurons from oxidative stress injury.
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Ascorbic acid levels in matured ripe and unripe sweet oranges (Citrus sinensis), lemons (Citrus lemonum) and grapefruits (Citrus grandis) were investigated using redox titration involving oxidation-reduction conversion of ascorbic acid. The analyses were carried out under refrigeration temperature, ambient temperature and intense sunlight to ascertain the extent of the effects of temperature. Other parameters probed were ripening stage, time of exposure and specie. The results suggest that the ascorbic acid levels in the unripe fruits were higher than the ripe ones but generally decreased upon increase in temperature, ripening and time of exposure. Orange has the highest ascorbic acid content (59.00±0.3 - 50.10±0.1 and 49.50±0.3 - 43.00±0.1 mg/100 g juice) followed by lemon (50.10±0.1 - 41.30±0.2 and 41.00±0.1 - 35.20±0.2 mg/100 g juice) and finally by grapefruit (44.80±0.2 - 36.40±0.2 and 34.20±0.3 - 24.00±0.3 mg/100 g juice) both in the unripe and ripe fruits at the various temperature conditions. Since the ascorbic acid content of citrus fruits depends on a lot of factors a few of which include climatic/environmental conditions, soil chemical composition, specie, maturity state and temperature, it is pertinent to explore the ascorbic acid levels in these citrus fruits with respect to a particular geographical location so as to establish information on the value of these fruits as raw materials for neighbouring outfits using ascorbic acid especially the Fruit Juice Industries.
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Three different methods (kinetic, spectrophotometric and potentiometric method) were used to determinate the ascorbic acid content in different type of citric fruits. The results obtained have demonstrated that because of various environmental factors (such as the vegetal material sources area, the meteorological conditions, growth and harvest period, the storage conditions and the period before the analysis), the ascorbic acid contain are altering in quite large limits.
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ABSTRAK Kandungan vitamin C dalam 19 jenis buah-buahan segar dan 24 sayur-sayuran telah dikaji aengan . kaedah fluorometrik (p < 0.01). Kajian kebolehulangan menunjukkan bahawa perbezaan varians di antara kedua-dua kaedah tidak bererti (p < 0.05). Jika hanya kadar asid askorbik diperlukan, kaedah titratan dapat memberi hasil yang memuaskan, dan ia dapat dijalankan dengan menggunakan peralatan makmal yang mudah. Sebaliknya, jika alat fluorometer diperoleh, icadar jumlah vitamin C boleh ditentukan, dan ini adalah lebih berguna dari segi pemakanan. ABSTRACT The vitamin C content of19 types offresh fruits and 24 vegetables was determined by the official AOAC methods of dye-titration and microfluorometry. As expected, values obtained by the latter me-thod, which estimated ascorbic acid plus dehydroascorbic acid (DHAA), were clearly higher than those given by the titration method, which determined only ascorbic acid. There were considerable differences in the values obtained by the two methods, depending on the concentration ofDHAA in the foods. Larger differences were obtained for the vegetables. The mean recovery value obtained by the dye-titration method was significantly higher than that given by the fluorometric method (p <0.01). Reproducibility studies showed that the two methods did not give significantly different variances (p < 0.05). If only ascorbic acid values were required, the titrimetric procedure would give good results, and it may be ca"ied out rapidly using simple laboratory equipment. If a fluorometer was available, total vitamin C values, which would be more useful from the nutritional point of view, could be determined.
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Water shortage is becoming a severe problem in arid and semiarid regions worldwide, reducing the availability of agricultural land and water resources. In Spain, citrus is one of the most economically important crops, with 74,000 ha devoted to its cultivation. Since water resources are increasingly more insufficient, the efficient use of water is becoming more essential. Deficit irrigation in many agricultural crops has frequently proved to be an efficient tool for improving water-use efficiency. This paper examines the effects a deficit irrigation during the ripening period on yield and the most representative fruit quality properties. The study was conducted during two consecutive years (2009-2010) in a commercial 12-year-old orange orchard ( Citrus sinensis L. Osb. cv. Navelina) grafted onto Carrizo citrange ( Citrus sinensis L. Osb. × Poncirus Trifoliata L. Osb.). A regulated deficit irrigation (DI) was applied, which was fully irrigated during the flowering and fruit-growth stage, and during the ripening period it was subjected to a water-stress ratio of 0.75. A control treatment was established, this being irrigated at 100% of crop evapotranspiration. Along the water stress period, it was tested the temporal evolution of the main organoleptic and nutraceutical fruit properties (color index, the total soluble solids, titrable acidity, maturity index, rind weight, juice weight, the ratio of juice weight versus fruit weight, and the total C vitamin and flavonoids). It was not observed a descend in juice content, or fruit weight, or in the final yield. In terms of fruit organoleptic and nutraceutical properties, there were not detected negative effects in the studied properties. Furthermore, DI treatment showed higher values of maturity index than control treatment, which can be considered as a positive aspect in the fruit quality. Considering these results, we can affirm that a moderate water stress applied during the maturity period is a sustainable strategy for saving water, increasing the irrigation productivity and obtaining fruits with similar properties to those without deficit irrigation.
A practical and engaging introduction to the core principles of nutrition. A thorough introductory guide, this text will equip students with the knowledge and skills required to optimise health and well-being. With its focus on Australasia, the text incorporates current nutrition recommendations and public health nutrition issues relevant to those studying and working in nutrition in this region of the world. The text begins with core nutrition topics, such as diet planning, macronutrients, vitamins and minerals, and follows with chapters on diet and health, fitness, life span nutrition and food safety. With a consistent level and readability, careful explanations of all key topics (including energy metabolism and other complex processes), this is a book that connects with students; engaging them as it teaches them the basic concepts and applications of nutrition.
The growing number of various tetra packed juices industries requires reliable quality control to insure the actual amount of ingredient and additives reported on the labels of packed juices by the companies. Here our research related to the development and validation of a simple and rapid RP-HPLC method to estimate the actual amount of Vitamin-C present in packed juices. It was estimated by Hitachi D-2000 Elite HPLC system manager using gradient pump system, separation was carried out by using C-18 column and detection by UV-Visible Detector. The retention time was within 1.63-1.65 minutes. Standard curves were linear over the concentration range of 0.1 to 2.5mg/ml. The extraction recovery was within 94 and 101%. The proposed method was found to be rapid, accurate, repeatable and consistent. This method was also compared with oxidation-reduction methods using standardized 2,6-Dichloroindophenol (DCP), to quantifying verifying ascorbic acid levels. We observed that the amounts of ascorbic acid reported by the two methods were mostly same and identical. In the result we found that Most of our local tetra packed juices companies may underestimate or overestimate the actual content of vitamin-C.
A liquid chromatographic method for fast and simultaneous determination of tartaric, malic, ascorbic and citric acids was validated for further application to fruits and juices. Moreover, the organic acids content of commercial samples of fruits and juices were evaluated, as well as the ascorbic acid stability during the storage. Determination of organic acids was carried out using a liquid chromatograph coupled to a diode array detector, with reversed phase (C18 column) and isocratic elution with 0.01 mol L−1 KH2PO4 (pH = 2.60) mobile phase. The validation parameters showed efficiency, adequate linearity, relative standard deviation values between 0.4% and 2.3% (n = 10) for repeatability and from 1.2% to 5.0% (n = 18) for reproducibility, limits of detection (LD) were between 0.03 and 3.31 μg mL−1 and quantification (LQ) were between 0.10 and 11.03 μg mL−1, recovery rates were between 82% and 110%, for two levels. In addition, the method is fast (10 min) and generates low and non-toxic residues. The values found for vitamin C were about 10 times above the values declared at the package. Ready to drink juices have a composition similar to the fruit, concerning to organic acids, except for the powder juice, in which only ascorbic and citric acids were found, for all tastes. After opening the package, a decrease of 14.0% and 27.0% in ascorbic acid content was observed for orange powder and ready to drink juices, respectively.
A simple and rapid liquid chromatographic method based on a new stationary phase Teknokroma, Tr-010065 Mediterranea sea18 (15 cm × 0.4 cm, id 3 μm), to determine ascorbic acid in beverages is reported. With the proposed method the samples were analysed by direct injection without a previous treatment. The total analysis time does not exceed 6 min. The method showed a good repeatability (RSD < 2%: n = 6) and an excellent sensitivity (LOD = 0.01 mg/l). Seventeen samples were analysed, including fruit juices, soft drinks and isotonic beverages. Ascorbic acid contents ranged from 6.6 to 840 mg/l. The ascorbic acid stability in some beverages during their shelf-life was also evaluated. Degradation of about 54% was observed in a tea drink.