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Food Chemistry 175 (2015) 100105

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Comparison of UPLC and HPLC methods for determination of vitamin C


Inga Klimczak, Anna Gliszczynska-Swigo
University of Economics, Faculty of Commodity Science, al. Niepodlegosci 10, 61-875 Poznan
, Poland
The Poznan

a r t i c l e

i n f o

Article history:
Received 6 March 2013
Received in revised form 25 October 2014
Accepted 18 November 2014
Available online 26 November 2014
Keywords:
HPLC
UPLC
Vitamin C
Food analysis
Beverages
Pharmaceutical preparations

a b s t r a c t
Ultra performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC)
methods for determination of ascorbic acid (AA) and total AA (TAA) contents (as the sum of AA and dehydroascorbic acid (DHAA) after its reduction to AA) in fruit beverages and in pharmaceutical preparations
were compared. Both methods are rapid: total time of analysis was 15 and 6 min for HPLC and UPLC
methods, respectively. The methods were validated in terms of linearity, instrument precision, limits
of detection (LOD) and quantication (LOQ), accuracy and recovery. Intra- and inter-day instrument precisions for fruit juices, expressed as RSD, were 2.2% and 2.4% for HPLC, respectively, and 1.7% and 1.9% for
UPLC, respectively. For vitamin C tablets, inter- and intra-day precisions were 0.4% and 0.5%, respectively
(HPLC), and 0.5% and 0.3%, respectively (UPLC). Both methods were sensitive: LOD was 0.049 lg/mL for
HPLC and 0.024 lg/mL for UPLC while LOQs were 0.149 and 0.073 lg/mL for HPLC and UPLC, respectively.
These methods could be useful in the routine qualitative and quantitative analysis of AA or TAA in pharmaceutical preparations or fruit beverages. However, UPLC method is more sensitive, faster and consumes less eluent.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
There is an increasing need for fast and ultra-fast separation
methods with good efciency and resolution. Ultra performance
liquid chromatography (UPLC) has become a widely used technique, which takes full advantages of chromatographic principles
to perform separation, using short columns packed with smaller
particles (sub-2 lm). This has led to a shorter analysis time,
improved peak efciency (peak width), better resolution and
decreased use of solvents compared with conventional high-performance liquid chromatography (HPLC) (de Villiers et al., 2006;
Guillarme, Nguyen, Rudaz, & Veuthey, 2007). Moreover, the UPLC
system enables the detection of analytes at very low concentrations
because of the improved signal-to-noise ratio (Guillarme et al.,
2007). The injection volume in UPLC can be signicantly reduced
without loss of sensitivity (Guillarme, Nguyen, Rudaz, & Veuthey,
2008; Spacil, Novkov, & Solich, 2008). However, the use of short
columns (50100 mm), packed with sub-2 lm particles in conventional liquid chromatography (LC), are limited by increased column
back-pressure (>40 MPa) which is not compatible with conventional instrumentation (de Villiers et al., 2006). The necessity of
using instrumentation dedicated to UPLC is a limitation of this technique (Guillarme et al., 2007).
Corresponding author. Tel.: +48 61 8569320; fax: +48 61 8543993.
E-mail address: a.gliszczynska-swiglo@ue.poznan.pl (A. Gliszczynska-Swigo).
http://dx.doi.org/10.1016/j.foodchem.2014.11.104
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

Vitamin C is the most important water-soluble antioxidant.


Both, ascorbic acid (AA) and its oxidation product, dehydroascorbic
acid (DHAA), have vitamin C activity. Because humans cannot synthesise AA, its main sources in the diet are fruits, vegetables and
their products as well as dietary supplements containing AA. There
are signicant variations in vitamin C content in food. For example,
in fruits it may range from 210 mg/100 g in plums and apples up
to 10001300 mg/100 g in rosehip and acerola, whereas in fruit
juices, it may range from 030 mg/L in apple juice to 280
860 mg/L in orange juice (Belitz, Grosch, & Schieberle, 2009;
Davey et al., 2000). Supplements containing vitamins or dietary
minerals are included as a category of food in the Codex Alimentarius. Currently, AA is the most widely used vitamin/antioxidant supplement worldwide. It is available as a single compound or in
multi-compound preparations. It is also used in cosmetic and food
industry as an antioxidant. Vitamin C in food can be lost, particularly during thermal processing or storage. Its loss (1060%) is
due to both oxidation and leaching (Combs, 2008). Thus, addition
of AA to fortify foods or restore vitamin C losses during processing
or storage is common.
Several analytical methods have been proposed for the determination of vitamin C, including titrimetric (Suntornsuk, Gritsanapun,
Nilkamhank, & Paochom, 2002), enzymatic (Shekhovtsova,
Muginova, Luchinina, & Galimova, 2006), chemiluminometric
(Pires, Marconi, Meneses, & Zagatto, 2006), spectrophotometric
(Llamas, Di Nezio, & Fernndez Band, 2011), uorometric (AOAC,

ska-Swigo / Food Chemistry 175 (2015) 100105


I. Klimczak, A. Gliszczyn

2007), and amperometric (Wawrzyniak, Ryniecki, & Zembrzuski,


2005) but separation techniques such as capillary electrophoresis
(Dong et al., 2007), gas chromatography (Silva, 2005) and liquid
chromatography (LC) (Novkov, Solich, & Solichov, 2008;
Spnola, Mendes, Camara, & Castilho, 2012; Tarrago-Trani,
Phillips, & Cotty, 2012), are regarded as more accurate. Among
chromatography techniques, HPLC methods are much more frequently used than gas chromatography.
Many chromatographic methods refer to the determination of
AA. Some of them are dedicated to the determination of AA and
TAA content as the sum of AA and DHAA after reduction of DHAA
_
to AA (Kauzewicz
et al., 2012; Spnola et al., 2012; Tarrago-Trani
et al., 2012). This is essential for fruits and vegetables because
many harvesting and post-harvest handling procedures promote
oxidation or degradation of AA (Hernandez, Lobo, & Gonzlez,
_
2006; Kauzewicz
et al., 2012; Lee & Kader, 2000). In many plant
crops, DHAA may represent up to 10% of total vitamin C and it
tends to increase during storage (Lee & Kader, 2000). However, in
the case of some fruit juices (e.g. stored short-term orange juices),
losses due to oxidation can be insignicant (Fernndez-Garca,
Butz, Bognr, & Tauscher, 2001; Snchez-Moreno, Plaza, de
Ancos, & Cano, 2003). For other types of juices, literature data in
this respect are limited (Brenes, Del Pozo-Insfran, & Talcott,
2005; Gonzlez-Molina, Moreno, & Garca-Viguera, 2009). Therefore, it is important to determine whether DHAA analysis is necessary for all type of juices.
Enhanced sensitivity and separation power of UPLC in comparison to conventional HPLC decreases the time and cost of analysis,
and has become increasingly important in liquid chromatography
applications. These include analysis of food and plant compounds,
e.g. vitamins (de Brouwer et al., 2010; Hampel, York, & Allen, 2012;
Spnola et al., 2012), phenolics (Gruz, Novak, & Strnad, 2008;
Herrero et al., 2011) and alkaloids (Ortega et al., 2010; Yi et al.,
2012). In the elds of food safety, UPLC is used for determination
of pesticides residues and their metabolites (Leandro, Hancock,
Fussell, & Keely, 2006; Li et al., 2013), and heterocyclic aromatic
amines (Barcel-Barrachina et al., 2006). Major applications of
UPLC in pharmaceutical analyses include quality control and stability monitoring of products, drug discovery and development
(Novkov & Vlckov, 2009; Wren & Tchelitcheff, 2006). There
are few methods dedicated solely to vitamin C determination in
foods (Spnola et al., 2012).
Due to the widespread application of vitamin C in food industry,
and its losses during processing or storage, it is necessary to monitor levels of this compound in food and pharmaceutical preparations using low-cost, fast and reliable analytical tools. Sample
preparation before analysis is crucial to obtain accurate results.
According to literature data, 110% meta-phosphoric acid can be
used to prepare samples for AA determination in beverages, fruits
and biological samples (nal concentration from 0.2% to 5%)
(Karlsen, Blomhoff, & Gundersen, 2005; Odriozola-Serrano,
Hernndez-Jover, & Martn-Belloso, 2007; Tiwari, ODonnell,
Muthukumarappan, & Cullen, 2009; Valdramidis, Cullen, Tiwari, &
ODonnell, 2010). The weakness of these methods is incomplete
data on stability of AA during sample preparation and analysis.
Therefore, the rst aim of the present study was to determine the
stability of AA in meta-phosphoric acid used for sample
preparation.
The next aim of this study was to validate and compare UPLC
and HPLC methods for AA and TAA determination. The validated
HPLC and UPLC methods were applied to different fruit juices
and vitamin supplements. Moreover, the concentration of AA and
TAA was determined in different fruit juices stored for 24 and
48 h after opening to verify the necessity of determination of both
AA and DHAA to assess the concentration of vitamin C in this kind
of product.

101

2. Materials and methods


2.1. Materials
Cartons or bottles of commercial fruit beverages (juices and
drinks) were purchased from local markets. Vitamin C pharmaceutical preparations were bought in local pharmacy. Bottles of fruit
juices (three for each type of juice originating from two different
batches; n = 6) were stored after opening for 24 and 48 h in the
cold (46 C) to assess the effect of time on the concentration of
DHAA. Meta-phosphoric acid and AA (min 99.7%) were purchased
from Merck (Darmstadt, Germany), DL-dithiothreitol (DTT; min
99%) was from SigmaAldrich (Steinheim, Germany). Deionizeddoubly distilled water was ltered through a 0.45 lm lter for
HPLC (Millipore, Bedford, MA, USA). Methanol (Chempur, Piekary
Slaskie, Polska) was HPLC grade. All other chemicals were reagent
grade.
2.2. Sample preparation
The vitamin C extraction method was adapted from Ross (1994).
Beverage sample (0.5 mL) and 10% meta-phosphoric acid (0.5 mL)
were mixed using a vortex (5 min) (nal concentration of metaphosphoric acid was 5%), centrifuged at 8500g for 10 min, and
injected onto the HPLC or UPLC column to determine AA content
(Gliszczynska-Swigo & Tyrakowska, 2003). Before injection onto
UPLC column, the samples of fruit juices or drinks were diluted
1:50 or 1:10, respectively, using 10% meta-phosphoric acid.
The sum of AA and DHAA (vitamin C content; TAA) was determined after reduction of DHAA to AA using DTT. The juice sample
(0.2 mL) and 5% DTT (0.2 mL) were mixed and diluted to 2 mL with
10% meta-phosphoric acid and injected onto HPLC or UPLC column
to determine vitamin C (Gliszczynska-Swigo et al., 2006).
Vitamin C tablets were dissolved in 25 mL 10% meta-phosphoric
acid and treated as fruit beverages samples. Before injection onto
HPLC or UPLC column, the samples of tablets were diluted 1:50
or 1:150, respectively, using 10% meta-phosphoric acid. Three
independent extractions were carried out for all samples. The only
difference between sample preparation for HPLC and UPLC was the
magnitude of sample dilution before injection.
2.3. Chromatographic determination of AA
The vitamin C was determined using Waters 600 high-performance liquid chromatograph (Waters Corp., Milford, MA, USA)
equipped with LiChrospher C18 (250  4.0 mm, 5 lm, Merck KGaA,
Germany) tted with the same guard column. A gradient of mobile
phase composed of methanol (solvent A) and 5 mmol/L KH2PO4, pH
2.65 (solvent B) was used according to the following program: linear increment starting with 522%A in 6 min and the return to the
initial conditions within the next 9 min with the ow rate of
0.8 mL/min. The eluate was detected using a Waters 996 photodiode array detector set at 245 nm (Gliszczynska-Swigo et al.,
2006). The injection volume was 20 lL.
UPLC determination of vitamin C was done using Acquity
ultra high performance liquid chromatograph equipped with
ACQUITY UPLC BEH C18 (100  2.1 mm; 1.7 lm; Waters, Milford,
MA, USA) tted with guard column. A gradient of mobile phase
composed of methanol (solvent A) and 5 mmol/L KH2PO4, pH
2.65 (solvent B) was used according to the following program: linear increment starting with 515%A in 1 min, from 15% to 35% for
the next 1 min and the return to the initial conditions within the
next 4 min with the ow rate of 0.2 mL/min. The eluate was
detected using a Waters Acquity photodiode array detector set
at 245 nm. The injection volume was 5 lL.

102

ska-Swigo / Food Chemistry 175 (2015) 100105


I. Klimczak, A. Gliszczyn

AA was identied by comparing its retention time and ultraviolet spectrum with that of the AA standard. UV-spectrum was also
used to conrm the purity of AA separated from other possible
compounds present in extract. Quantication of AA or vitamin C
(as the sum of AA and DHAA) was done using the external standard
method with AA as a standard. For each extract, at least two injections were made.
The content of DHAA was determined indirectly by subtraction
of AA from TAA.

concentration of meta-phosphoric acid was 5% or 10% for juices


and tablets, respectively).
3.2. Analytical characteristics

3. Results and discussion

In this paper, HPLC and UPLC methods for vitamin C determination were compared. AA and DTT were eluted from an HPLC system
in 4.01 and 13.06 min, respectively. Using UPLC system, retention
times of AA and DTT were 1.56 and 3.30 min, respectively
(Fig. 1). Using UPLC method, the total time of analysis was 2.5
times shorter than with HPLC and solvent consumption decreased
from 12 to 1.2 mL/min.
Analytical characteristics of HPLC and UPLC methods included:
linearity, precision and sensitivity (Table 1). Quantication of AA
was performed using an external standard method. The curve for
AA in HPLC method was linear to atleast 300 lg/mL. In UPLC
method, lower concentrations must be used: a maximum concentration cannot be higher than 50 lg/mL. The RSD values of the
standard curve slopes obtained with HPLC and UPLC systems were
2.78%, 1.82%, respectively. Linear correlation coefcients (r) for
both standard curves were 0.999.
Instrument precision was checked from six consecutive injections of AA extracts from fruit juice and pharmaceutical preparation. In the case of fruit juice, the intra-day (the daily) and interday (day-to-day) RSDs obtained using HPLC were 2.2% and 2.4%,
respectively, whereas with UPLC 1.7% and 1.9%, respectively.
For vitamin C tablets, the intra- and inter-day RSDs obtained using
HPLC were 0.4% and 0.5%, respectively; with UPLC system 0.1%
and 0.3%, respectively (Table 1). Precision of AA determination in
pharmaceutical preparation using both methods was higher than
obtained for fruit juice. It is probably the effect of less complex
matrix of tablet than fruit juice.
The LOD and the LOQ for AA under HPLC conditions were
0.049 lg/mL and 0.149 lg/mL, respectively. These results are much
lower than those reported by Snchez-Mata, Camara-Hurtado,
Diez-Marqus, and Torija-Isasa (2000), Hernandez et al. (2006)
and Vidovic et al. (2008) but slightly higher than those reported
by Valente, Albuquerque, Sanches-Silva, and Costa (2011). In UPLC
method, LOD and LOQ were 0.024 and 0.073 lg/mL, respectively
(Table 1). These results are very similar to those obtained by
Spnola et al. (2012) but better than those reported by Zhang,
Chen, Liao, and Ren (2009) as well as by Talekar, Vora, Gawade,
and Gopala (2013). Moreover, the extraction solvent used in the
present study was simpler than that described by Spnola et al.
(2012) and guaranteed stability of sample within at least 24 h.
The proposed UPLC method can be an alternative for existing
methods for vitamin C determinations in juices and pharmaceutical preparations.
The efciency of the extraction was determined based on the
recovery of AA from spiked juice and tablet samples. It was found
that the recovery of AA was not lower than 98.9% (Table 2).

3.1. Stability of AA in meta-phosphoric acid

3.3. Quantication of AA in fruit beverages and vitamin C tablets

The results of our study show that the stability of AA in juice


samples using 2.5% meta-phosphoric acid (nal concentration of
meta-phosphoric acid was 1.25%) was signicantly less than with
10% meta-phosphoric acid (meta-phosphoric acid nal concentration of 5%). The concentration of AA in juice samples (n = 6)
decreased from 252.7 to 232.2 mg/L (8%) in 1 h. In the same time,
the concentration of AA in tablet samples dissolved in 2.5% metaphosphoric acid (n = 6) decreased from 62.5 to 46.5 mg/tablet
(26%). Juice or tablet samples dissolved in 10% meta-phosphoric
acid were more stable. However, after 24 h, at least 97.3% of AA
was still present in juice or tablet samples. Based on these results,
10% meta-phosphoric acid was used for sample preparations (nal

The validated HPLC and UPLC methods were applied to different


fruit juices and vitamin supplements. The content of vitamin C in
tested products is reported in Table 3. DHAA was not present in
any sample (the results obtained for samples prepared with and
without DTT were not statistically different). The results obtained
using HPLC and UPLC methods were very similar although, for
some products, the results obtained using UPLC were slightly
higher than those obtained by HPLC. It may be due to the higher
sensitivity of UPLC than HPLC.
The changes in AA, TAA and DHAA concentrations in different
opened fruit juices stored for 24 and 48 h under refrigeration conditions are presented in Table 4. DHAA was not observed in any

2.4. Method validation


Validation of HPLC and UPLC methods included linearity, instrument precision and sensitivity. Quantication of AA was performed
using an external standard method. The eight-point (25300 lg/mL
for HPLC and 550 lg/mL for UPLC) calibration curves (n = 4; generated during a 6-month period) were prepared with the standard
solutions of AA in 10% meta-phosphoric acid at concentrations
spanning those present in samples.
Instrument precision was checked from six consecutive injections of fruit juice and vitamin C tablet extracts prepared as
described in Section 2.2.
The LOD and LOQ were calculated from the calibration curve at
concentrations from 0.1 to 3 lg/mL (HPLC) or 0.053 lg/mL
(UPLC). The following equation was used:
r
LOD 3:3
b
r;
LOQ 10
b

where r is the standard deviation of y-intercepts and b is the slope


of the calibration curve.
The efciency of the extraction method was determined by
spiking samples at two concentration levels of AA (50% and 100%
of the AA level in the product). Spiked and unspiked samples were
treated in the same way throughout the whole procedure. Three
independent extractions were carried out for each product and
AA level. Each sample was injected onto the HPLC or UPLC columns
twice.
2.5. Statistical analysis
Statistical analyses were performed using the Statistica 9.0
(StatSoft, Inc., 2000) program. Results on the effect of storage time
on the AA and TAA concentrations in juice samples were assessed
using ANOVA. Differences between fresh and stored samples were
evaluated by the Dunnetts test. The differences between HPLC and
UPLC results as well as between AA and TAA concentrations in
stored fruit beverages were evaluated by the Students t test. The
differences at 5% level were considered as signicant.

ska-Swigo / Food Chemistry 175 (2015) 100105


I. Klimczak, A. Gliszczyn

A245 [a.u.]

AA

AA

A245 [j.u.]

Juice sample
Juice sample with DTT

103

DTT

DTT

10

11

12

13

14

15

Retention time [min]

Retention time [min]

Fig. 1. Typical HPLC (A) and UPLC (B) chromatograms of ascorbic acid separated from fruit juice and pharmaceutical preparation.

Table 1
Method validation parameters for determination of AA in juices and vitamin C tablets.
HPLC

UPLC

0300
0.999

050
0.999

Standard linearity

Range (lg/mL)
r

Instrument precision
(n = 6)
Juice

Intra-day
Mean (mg/L) SD
RSD (%)

248.2 5.4
2.2

255.9 4.3
1.7

Vitamin C tablet

Mean (mg/tablet)
RSD (%)

67.6 0.3
0.4

67.7 0.1
0.1

Instrument precision
(n = 6)
Juice

Inter-day
Mean (mg/L) SD
RSD (%)

245.5 5.8
2.4

253.5 4.9
1.9

Vitamin C tablet

Mean (mg/
tablet) SD
RSD (%)

65.8 0.3

66.2 0.2

0.5

0.3

LOD (lg/mL)
LOQ (lg/mL)

0.049
0.149

0.024
0.073

Table 3
Comparison of vitamin C content in analysed samples as determined by HPLC and
UPLC methods.
Sample

Vitamin C content SD (%RSD) (mg/L or mg/


tablet)
HPLC

Limits

fresh juice (the results obtained for samples prepared with and
without DTT were not statistically different). The decrease in AA
of 02.9% and 4.16.4% in comparison to fresh juice was observed
for juices stored for 24 and 48 h, respectively. The concentration of
DHAA was from 0% to 5.4% depending on storage time and a kind of
juice. It was reported that phenolic compounds protect AA from
degradation but this protection depends on the type of compound
(Miller & Rice-Evans, 1997; zkan, Krca, & Cemeroglu, 2004).
Moreover, it was found that apple juice had the lowest protective

UPLC

Fruit juices and beverages


Orange juice
White grapefruit juice 1
Red grapefruit juice 2
Blackcurrant nectar
Multifruit drink 1*
Multifruit drink 2*
Ice Tea drink*

224.3 0.9a
255.6 3.6a
467.1 1.5a
209.6 2.7a
86.6 1.3a
87.6 0.6a
100.0 0.4a

(0.4)
(1.4)
(0.3)
(1.3)
(1.5)
(0.7)
(0.4)

230.9 1.8b (0.8)


267.2 2.4b (0.9)
473.7 1.4b (0.3)
208.2 2.7a (1.3)
84.7 0.3a (0.4)
88.7 0.6a (0.7)
97.5 0.9b (0.9)

Vitamin preparations
Multivitamin preparation
Vitamin C
Vitamin C + rutin*

64.4 0.3a (0.5)


65.8 0.6a (0.9)
100.0 0.1a (0.1)

68.7 1.6b (2.3)


67.7 0.1b (0.1)
99.7 0.4a (0.4)

Values are mean SD (at least three independent extractions were carried out for
three products (n = 9) and at least two injections into UPLC or HPLC column were
done for each sample).
a,b
Means in each row are signicantly different at a = 0.05.
*
In accordance with producer declaration.

effect on AA in fortied fruit juices comparing to orange and blackcurrant juices. Apple juice phenolic acids were recognised as less
efcient protectors of AA than avonoids of orange and blackcurrant juices (Miller & Rice-Evans, 1997). These results may explain
higher losses of AA in apple juices than in multifruit juices analysed in the present study. It was observed that oxidation process
was only partly responsible for the loss of AA because the concentration of DHAA did not compensated the decrease of AA in fresh
juice. This was especially apparent for multifruit juices, in which

Table 2
Recovery of AA from selected fruit beverages and pharmaceutical preparation.
Product

a
b

AA content SD (mg/L) or (mg/tablet)

AA spiked levela (%)

Recoveryb (%)

HPLC

UPLC

HPLC

UPLC

Grapefruit juice

468.3 0.8

454.2 0.5

50
100

99.8 0.3
100.2 0.2

99.9 0.2
100.0 0.1

Blackcurrant nectar

208.1 07

209.2 0.7

50
100

99.0 0.7
98.9 0.6

99.3 0.4
99.5 0.5

Vitamin C supplement

100.1 0.1

99.8 0.2

50
100

99.9 0.3
99.8 0.4

99.9 0.2
99.8 0.3

Percent of AA content in product.


Recovery mean SD (n = 3 in each level).

ska-Swigo / Food Chemistry 175 (2015) 100105


I. Klimczak, A. Gliszczyn

104

Table 4
The changes in vitamin C content in fruit juices (mg/L) during short-term storage under refrigeration conditions.
Product
Multifruit juice 1

Storage (h)
0
24
48

AA1

DHAA2

TAA
a

392.3 3.9
381.1 4.8A,a
(2.9%)
376.1 1.5A,a
(4.1%)

394.2 2.7
389.2 1.5A,b
(1.3%)
386.9 2.0A,b
(1.9%)

0
8.1 3.7
(2.1%)
10.8 2.2
(2.8%)

Multifruit juice 2

0
24
48

435.8 4.0a
428.4 1.4a
414.4 2.3A,a
(4.9%)

433.1 2.1a
429.4 1.7a
426.2 2.9A,b
(1.6%)

0
0
11.8 2.9
(2.7%)

Apple juice 1*

0
24
48

254.6 6.7a
247.2 1.5a
238.3 4.9A,a
(6.4%)

251.2 2.2a
245.8 3.1a
251.9 1.1b

0
0
13.6 3.5
(5.4%)

Apple juice 2*

0
24

360.3 1.6a
354.2 2.9A,a
(1.7%)
340.8 1.5A,a
(5.4%)

357.5 2.5a
360.3 3.9a

0
0

353.0 2.2b

12.2 2.3
(3.4%)

48

Values are mean SD (three juices from two different batches were analysed; at least three independent extractions were carried out for each bottle of product (n = 18) and at
least two injections into column were done for each sample).
A
Means in the column for AA or TAA are signicantly different from appropriate fresh sample (Dunnetts test, a = 0.05).
a,b
Means in each row are signicantly different (Students t test, a = 0.05).
1
In parenthesis the loss of AA in comparison with fresh juice.
2
Calculated as the difference between TAA and AA; in parenthesis the percentage of TAA.
*
Enriched with ascorbic acid (according to the producer declaration).

probably not only oxidation but also degradation of AA occurred.


The changes in the concentration of AA in apple juices were mainly
due to the oxidation thus the decrease in TAA during storage was
not observed. Determination of vitamin C in fruit juices stored
for at least 24 h under refrigeration may require determination of
TAA because oxidation of AA may occur during storage.
4. Conclusions
Although many methods have been developed for determination of AA or TAA, both HPLC and UPLC methods proposed in this
study for determination of AA or TAA (vitamin C) in fruit beverages
and pharmaceutical preparations can be recommended as rapid,
precise and sensitive. Extraction of AA and DHAA from tested products using 10% meta-phosphoric acid guaranteed determination of
vitamin C within 24 h without its essential loss. For fresh commercial beverages as well as for pharmaceutical preparations, it is not
necessary to determine both AA and DHAA. Determination of vitamin C in stored fruit juices may require determination of TAA
because oxidation of AA may occur during storage. Both methods
can be useful in the routine qualitative and quantitative analysis
of vitamin C in beverages and pharmaceutical preparations. However, UPLC method is faster, more sensitive, consumes less eluent
and it is more eco-friendly than the conventional HPLC method.
In consequence, UPLC is cheaper than HPLC because a higher number of analyses per unit of time can be performed and consumption
of eluent is much lower.
References
AOAC. (2007). Ofcial methods of analysis. Method 967.21 vitamin C (total) in food.
Washington: Association of Ofcial Analytical Chemists, pp. 2425.
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M. A. (2006). Ultra-performance liquid chromatographytandem mass
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