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1. PHARMACIA, vol. 60, No. 4/2013 37Comparative investigation of food supplements containing ...
COMPARATIVE INVESTIGATION OF FOOD SUPPLEMENTS
CONTAINING ASCORBIC ACID
Danka Obreshkova and Boyka Tsvetkova
Medical University – Sofia, Faculty of Pharmacy, Dept. of Pharmaceutical chemistry
Abstract. A simple, specific, precise and accurate reversed phase liquid chromatographic (RP-LC) method
has been developed for the determination of ascorbic acid in different food additives. The chromatographic
separation was achieved on a LiChrosorb C18, 250 mm x 4.6 mm, 5 μm column at a detector wavelength
of 230 nm and a flow rate of 1.5 ml/min. The mobile phase was composed of acetonitrile and water (60:40
v/v). The retention time of analyte was 3.49 min. The method was validated for the parameters like specific-
ity, linearity, precision, accuracy, limit of quantitation and limit of detection. The method was found to be
specific as no other peaks of impurities and excipients were observed. The square of correlation coefficient
(R2) was 0.9997 while relative standard deviations were found to be <2.0%. The proposed RP-LC method
can be applied for the routine analysis of commercially available food additives of ascorbic acid.
Key Words: Liquid chromatography, validation, ascorbic acid, food additives, quality control
Introduction
Vitamin C (Ascorbic acid, AA) is the most im-
portant vitamin for human nutrition that is supplied
by fruits and vegetables. It is widely distributed in
plant cells where plays many crucial roles in growth
and metabolism. As a potent antioxidant, AA has the
capacity to eliminate several different reactive oxy-
gen species, keeps the membrane-bound antioxidant
α-tocopherol in the reduced state, acts as a cofactor
maintaining the activity of a number of enzymes (by
keeping metal ions in the reduced state), appears to
be the substrate for oxalate and tartrate biosynthesis
and has a role in stress resistance [1, 2]
Several analytical methods have been reported for
the determination of vitamin C using titrimetry [3, 4],
spectrometry [5-11] and amperometry [12].
The preferred choice for AA determination are
separation techniques: capillary electrophoresis [13],
gas chromatography [14] and liquid chromatogra-
phy. Liquid chromatography avoids the problems
of non-specific interference and ion-pair [15], NH2
bonded-phase [16] and reverse phase [17-20] tech-
niques have been reported.
The aim of this study was to develop and validate
the simple, accurate and precise liquid chromato-
graphic method for routine determination of ascorbic
acid in food additives. The method was applied to
comparative investigations of several food supple-
ments containing ascorbic acid.
Materials and Methods
Chemicals and Reagents
Ascorbic acid (Northeast Pharmaceutical Group
Co., LTD, Batch № DY0261310441, Exp. Date
03.2016) was used as reference substance. The fol-
lowing oral solutions containing 100 mg/ml active
substance were obtained commercially and analyzed:
Vitamin C food suppliary SOPHARMA® (Batch
№ 40613, 200 mg/2 ml), Vitamin C food suppliary
SOPHARMA® (Batch № 30513, 500 mg/5 ml), Vi-
tamin C Vetprom AD (Batch № 0050313), Vitamin C
Biopharm (Batch № 176112012), Vitamin C Planta
Pol (Batch № F0209) and Vitamin C J.X.K. Pharma-
ceiutical Co (Batch № 121058). LC-grade acetoni-
trile was supplied from Merck (Germany). All other
chemical reagents were of analytical grade.
Instrumentation and chromatographic conditions
Chromatographic separation was performed on
modular HPLC system LC-10AShimadzu (Japan) ar-
ranged with a LC-10A pump, solvent degasser DGU-
3A, Rheodyne injector, column oven CTO-10A,
SPD-M10A diode array detector and communication
bus module CBM-10A. Separation was achieved iso-
cratically with a LiChrosorb C18, 250 mm x 4.6 mm,
5 μm column eluted with a mixture of acetonitrile
and water (60:40 v/v) as the mobile phase at flow rate
of 1.5 ml/min. Detection as carried out by absorbance
at 230 nm. The analysis was carried out at an ambient
temperature and injection volume was 20 μl.

2. 38 PHARMACIA, vol. 60, No. 4/2013 D. Obreshkova, B. Tsvetkova
Preparation of reference solutions
50.0 mg of accurately weighed reference sub-
stance ascorbic acid was dissolved and made up to
mark with water in a 50 ml volumetric flask, to get
primary stock solution of 1 mg/ml. Serial dilutions
were made to obtain 50, 100, 200, 300 and 400 μg/
ml. All solutions were filtered through 0.45 μm mem-
brane filter and immediately chromatographed.
Sample preparation
A commercially available food additives contain-
ing ascorbic acid 200 mg/2 ml and 500 mg/5 ml were
analyzed using this method. Preparation of test so-
lutions was as follows: 1.0 ml of each sample was
diluted to 100.0 ml with water. 5.0 ml of this solution
were made up to 100.0 ml with water to give 100 μg/
ml and 250 μg/ml, respectively. All solutions were
filtered through 0.45 μm membrane filter and imme-
diately suspected to analysis.
Results and discussion
The Fig. 1 showed typical chromatogram obtained
from analysis of standard solution using the proposed
method. The retention time observed –3.49 min, per-
mits a rapid determination of the analyzed substance,
which is important for routine analysis.
Validation study
The method was validated for the param-
eters like specificity, linearity, precision, accu-
racy, limit of quantitation and limit of detection.
Sample preparation
A commercially available food additives containing ascorbic acid 200 mg/2 ml and 500
mg/5 ml were analyzed using this method. Preparation of test solutions was as follows:
1.0 ml of each sample was diluted to 100.0 ml with water. 5.0 ml of this solution were
made up to 100.0 ml with water to give 100 μg/ml and 250 μg/ml, respectively. All
solutions were filtered through 0.45 μm membrane filter and immediately suspected to
analysis.
Results and discussion
The Fig. 1 showed typical chromatogram obtained from analysis of standard solution
using the proposed method. The retention time observed –3.49 min, permits a rapid
determination of the analyzed substance, which is important for routine analysis.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
mV(x100)
Detector A:230nm
3.497/11446896
Fig.1. Chromatogram obtained from analysis of standard solution
Fig. 1. Chromatogram obtained from analysis of standard solution
Specificity
The specificity of the method was determined by
checking the interference with the components from
placebo. The method was found to be specific as no
other peaks of impurities and excipients were ob-
served.
Calibration and linearity
Calibration curve was constructed in the range
of 50.00-400.0 μg/ml for ascorbic acid to encom-
pass the expected concentration in measured samples.
The corresponding linear regression equation was
y=14477.2x-1746.1 with square of correlation coeffi-
cient R2
of 0.9997. An excellent correlation existed be-
tween the peak areas and concentration of ascorbic acid.
Precision
The precision of the method was evaluated by
performing six independent determinations of the
test sample preparation and calculating RSD (%).
The RSD value measured during assessment of pre-
cision was <2.0% for ascorbic acid, confirming the
method is precise (Table 1).
Accuracy
To determine the accuracy of the method, the re-
covery was checked at three different concentration
levels – 50, 100 and 150 %. Values of analytical re-
covery experiments were listed in Table2.

3. PHARMACIA, vol. 60, No. 4/2013 39Comparative investigation of food supplements containing ...
№ Amount found, mg/2 ml Statistical data
1. 200.7
Mean 199.02. 198.2
3. 197.6
SD 0.4704. 198.1
5. 201.0
%RSD 0.746. 198.3
Table 1. Precision of the method
Table 2. Results from study of accuracy
Level (%) Theoretical concen-
tration (μg/ml)
Observed concentra-
tion (μg/ml)
Mean recovery (%) ±
SD
RSD (%)
50 98.1
97.4
99.93±0.711 0.7198.8
97.9
100 197.6
194.7
99.27±0.912 0.92198.1
195.6
150 295.7
291.7
99.48±0.750 0.75296.1
294.8
Limits of detection (LOD) and quantitation
(LOQ)
LOD, which is defined as the lowest active sub-
stance concentration that can be determined by a
method, usually cannot be calculated precisely and
accurately. On the other hand, LOQ is the concen-
tration of the sample used in analysis that can be
obtained with adequate precision and accuracy. An
estimation of the limits, which was achieved by the
determination of the signal/noise ratios of 3:1 (LOD)
and 10:1 (LOQ), were 0.5 and 2.0 μg/ml, for LOD
and LOQ, respectively.
Application of the developed method to food
additives containing ascorbic acid
Assay results for the determination of ascorbic
acid in commercial food additives were shown in Ta-
ble 3. The results were presented as average values of
analysis of nine different oral solutions (in ampules)
of each batch. RSD (%) values indicated the accu-
racy of determination of the substance in the evalu-
ated food supplements. As can be seen from the table
the content of ascorbic acid was found to be higher
in food additives manufactured by Sopharma in com-
parison to other investigated formulations.
Food additive Labeled content Actual content ± SD* RSD (%)
Vitamin C food suppliary SOPHARMA® 200 mg/2 ml 201.4±0.93 0.46
Vitamin C food suppliary SOPHARMA® 500 mg/5 ml 498.2±0.87 0.17
Vitamin C Vetprom AD 200 mg/2 ml 192.5±0.99 0.51
Vitamin C Biopharm 200 mg/2 ml 187.6±0.88 0.46
Vitamin C Planta Pol 200 mg/2 ml 188.2±0.79 0.42
Vitamin C J.X.K. Pharmaceiutical Co. 500 mg/5 ml 468.2±0.84 0.18
Table 3. Assay results for the determination of ascorbic acid in different food additives
* n=9, each content was average value from nine different determinations

4. 40 PHARMACIA, vol. 60, No. 4/2013 D. Obreshkova, B. Tsvetkova
Food additive Labeled content Actual content
Vitamin C food suppliary
SOPHARMA®
200 mg/2 ml 199.8
Vitamin C food suppliary
SOPHARMA®
500 mg/5 ml 501.2
Vitamin C Vetprom AD 200 mg/2 ml 189.7
Vitamin C Biopharm 200 mg/2 ml 191.4
Vitamin C Planta Pol 200 mg/2 ml 192.0
Vitamin C J.X.K. Pharmaceiutical Co. 500 mg/5 ml 475.2
Table 4. Assay of food additives by titrimetry
Determination of ascorbic acid in presented food
additives was also performed by titrimetric analysis
according requirements of European Pharmacopoeia
7th edition [21]. The results derived from this evalu-
ation were shown in Table 4.Our investigations in-
cluded also analysis of appearance of degradation
products, especially dihydroascorbic acid by means
of thin-layer chromatography according to the rules
of European Pharmacopoeia 7th edition. The results
confirmed that all analyzed bathes were in agreement
with the European Pharmacopoeia requirements and
the food additives produced by Sopharma were with
the highest purity – no other spots apart the princi-
pal spot of ascorbic acid were observed after removal
and visualization of the chromatographic plate.
Conclusion
The newly developed LC method is specific, pre-
cise, accurate and rapid. The analytical procedure is
suitable for quality control of food additives contain-
ing ascorbic acid.
In this work, all investigated food additives were
found to correspond to requirements. The results
from chromatographic studies were confirmed by
data from titrimetric analysis and it was found that
the maximum content of active substance was in food
supplements produced by Sopharma. On the other
hand, the thin-layer chromatographic investigations
revealed that with the highest degree of purity were
food additives manifactured by Sopharma in compar-
ison to all other analyzed formulations. This means
that these solutions will be more stable.
Samples manifactured by Biopharm and J.X.K.
Pharmaceiutical Co. were found to be difficult to
open the containers hence this will be not comfort-
able for patients.
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Corresponding author:
Boyka Tsvetkova
Faculty of Pharmacy, Medical University
2 Dunav Str. 1000 Sofia
Tel: +35929236532
Fax: +35929879874
e-mail: bojka@abv.bg
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