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Original Article
EFFECT OF STORAGE CONDITIONS ON THE STABILITY OF ASCORBIC ACID IN SOME
FORMULATIONS
MICHAEL WORLAKO KLU*, BRIGHT SELORM ADDY, ESTHER ESHUN OPPONG, EMMANUEL SEYRAM SAKYI,
DAVID NTINAGYEI MINTAH
Department of Pharmaceutical Sciences, Central University, Miotso, Ghana
Email: michaelwkklu2009@yahoo.com
Received: 16 Jul 2016, Revised and Accepted: 07 Sep 2016
ABSTRACT
Objective: The stability of ascorbic acid is affected by temperature, pH, sunlight and the presence of metals like copper and iron.
The study seeks to investigate the effect of storage conditions on the stability of ascorbic acid in tablets (buccal tablets) and syrups sampled from
the Ghanaian market.
Methods: Ascorbic acid tablets were sampled and stored separately at room temperature and under refrigeration (in a fridge) and assayed
periodically for 35 d. Ascorbic acid syrups were also sampled and stored at room temperature, in a bowl of water and under refrigeration and also
assayed periodically for 35 d. The mode of assay was iodimetry.
Results: For both formulations, storage under refrigeration saw the least breakdown and at room temperature, the breakdown of ascorbic acid
was greatest. The syrups stored in a bowl of water were more stable than those stored at room temperature. The % breakdown of ascorbic acid
in the syrups and tablets stored at room temperature were statistically significant in comparison to that under refrigeration as determined by a
T-test. The % breakdown of ascorbic acid in the syrups stored in a bowl of water was not statistically significant in comparison to that under
refrigeration.
Conclusion: Ascorbic acid formulations should be stored under refrigeration or at low temperatures if possible. In the absence of refrigeration,
patients should be advised to store syrups of ascorbic acid in a bowl of water and the tablets at cool places in homes.
Keywords: Ascorbic acid, Room temperature, Refrigeration, Bowl of water and iodimetry
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2016v8i4.14131
INTRODUCTION
L-ascorbic acid, commonly known as vitamin C, is a very important
water-soluble vitamin. It is a low molecular weight antioxidant that
has diverse uses in both plants and animals [1].
In plants, ascorbic acid functions as a redox buffer, a cofactor in the
process of photosynthesis, biosynthesis of hormones and the
regeneration of other antioxidants. It also plays a role in the
regulation of cell division, cell growth and signal transduction [2].
In humans, ascorbic acid or vitamin C helps in the synthesis of
collagen, acts as an immune system booster by scavenging free
radicals in the body, aids in enzyme activation and oxidative stress
reduction. There is a lot of evidence that ascorbic acid offers
protection against respiratory tract disease, reduces risks for
cardiovascular diseases and some cancers [3, 4].
Scurvy is a condition that results when there is a deficiency of
ascorbic acid in the body, and its clinical manifestations are lethargy,
purpuric lesions, myalgia and bleeding of the gums [5].
Some sources of ascorbic acid are oranges, tomatoes, sweet pepper,
vegetable oils like sunflower oil and olive oil, and nuts and seeds [6].
Synthetic ascorbic acid are available as formulations of effervescent
tablets, chewable tablets, syrups, drops and injections [7].
The stability of ascorbic acid is affected by a lot of factors;
temperature, pH, sunlight and the presence of certain metals like
Iron and Copper. Temperature changes have effects on the
concentrations or amounts of ascorbic acid in formulations and
natural sources like fruits. High storage temperatures cause an
increase in the breakdown of ascorbic acid whereas low
temperatures cause a reduction in the rate of breakdown [8].
In Ghana and some parts of West Africa and the developing world,
most people do not have refrigerators in their homes so when they
are given formulations of ascorbic acid to use and administer over a
period, they have no means of storage at such low temperatures.
Since they are not being stored at recommended low temperatures
where the breakdown is minimal, the products could experience
appreciable breakdown over the course of usage. The erratic power
supply (frequent and prolonged power outages) also means that
such products cannot be stored consistently at such low
refrigeration temperatures. These issues of storage raise stability
concerns of ascorbic acid in formulations.
The study seeks to investigate and compare the effect of storage
conditions on the stability of ascorbic acid in syrups, tablets and the
pure powder stored under refrigeration (in the fridge), at room
temperature and in a bowl of water with a view to recommending the
best possible storage condition for ascorbic acid.
MATERIALS AND METHODS
Reagents and chemicals used
Potassium iodide, potassium iodate, iodine crystals, and sulphuric acid
(all from Fisher chemicals Ltd, UK), sodium thiosulphate pentahydrate
(Fizmerk chemicals Ltd, UK) and starch powder (Merck private Ltd,
India). All the chemicals used were of analytical grade.
The pure ascorbic acid powder used was of laboratory grade.
Collection of samples of formulations of ascorbic acid
The brands of ascorbic acid syrups and tablet (buccal tablets were used)
used were obtained from pharmacies at Prampram in the Greater Accra
region of Ghana. All the primary and secondary packaging of the ascorbic
acid syrups and tablets were assessed and did not show any sign of
physical or chemical degradation in their packaging.
Storage of samples
Samples of the ascorbic acid syrup in amber coloured bottles were
stored at room temperature (33 °C), under refrigeration or in a
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ISSN- 0975-7058
Vol 8, Issue 4, 2016
Klu et al.
Int J App Pharm, Vol 8, Issue 4, 2016, 26-31
27
fridge (4–6 °C) and in a bowl of water (26-27 °C) such that half the
height of the bottle was immersed in the water. The samples were
stored under these various storage conditions for 35 d.
Samples of the ascorbic acid tablets in blisters were stored at room
temperature (33 °C) and in a fridge (4–6 °C). The samples were
stored under the various storage conditions for 35 d.
Sufficient amounts of the pure ascorbic acid powder were put into
very clean and dry amber coloured bottles and tightly covered. They
were then stored at room temperature (33 °C), in a fridge (4–6 °C)
and in a bowl of water (26-27 °C) such that half the height of the
bottle was immersed in the water. The samples were stored under
the various storage conditions for 35 d.
Assay of samples
Each of the samples of ascorbic acid syrups, tablets and powder was
assayed on the same days predetermined over 35 d. The mode of the
assay was iodiometry [9]. Triplicate assays were performed for each
sample and the percentage content of each sample expressed as the
mean with standard deviation.
Reaction rate equations
Zero order rate equation
C=Co −kt
For the zero order graphs, the final concentration, C, was plotted
against time of assay, t, and the slope, k, is the zero order rate
constant and Co is the initial concentration.
First order rate equation
In C=In Co −k′t
For the first order graphs, In C, was plotted against time of assay, t,
and the slope,k′, is the first order rate constant. Co is the initial
concentration and C is the final concentration after a certain time, t.
Second order rate equation
1
C=1
Co + k′′t
For the second order graphs,
was plotted against time of assay, t, and
the slope,k′′, is the second order rate constant. Co is the initial
concentration and C is the final concentration after a certain time, t [10].
Determination of the order of breakdown of ascorbic acid
The percentage contents of the various samples of ascorbic acid for
each formulation (syrups and tablets) were converted to
concentration terms needed to plot line graphs for zero order, first
order and second order rates of breakdown. The line graphs for a
particular order of breakdown for each formulation were obtained
by plotting the correct concentration terms against the times of
assay [11].
The coefficients of correlation and the slopes were obtained for each
line graph.
The line graphs were drawn with Microsoft excel 2013.
Statistical analysis
The significance of the % breakdown of ascorbic acid in tablets and
syrups stored at room temperature and in a bowl of water in
comparison to storage under refrigeration was determined by a t-
test calculated as:
t=x− µ√
s
t is the critical value; µ true mean is the mean % content of
ascorbic acid in formulation stored in fridge after 35 d; x is the
mean % content of ascorbic acid formulation stored at room
temperature or in bowl of water; s is the standard deviation of the
three determinations that gave x and n is the number of assays per
sample [12].
The t-test was carried out at a confidence level of 95%.
RESULTS AND DISCUSSION
The assay results for all the formulations of ascorbic acid
including the pure powder under the various storage conditions
show that the concentrations of a scorbic acid in the various
products reduced with time under the storage conditions
investigated (tab les 1, 2 and 3).
Table 1: Assay results for ascorbic acid in tablets stored under the different storage conditions for 35 d
Time of
assay
(d)
Mean concentration
(
% w/w)
Tablets stored at room temperature
Tablets
stored
in Fridge
0
105.57
±
0.0572
105.57
±
0.0572
2
104.75
±
0.3581
104.91
±
0.2247
7
102.77
±
0.2738
104.83
±
0.0796
14
101.73
±
0.1392
103.91
±
0.3018
21
100.60
±
0.2002
103.41
±
0.2831
28
100.06
±
1.2522
102.92
±
0.2337
35
98.52
±
0.2133
101.87
±
0.5137
Mean concentration of ascorbic acid given as mean±SD (n=3).
Table 2: Assay results for pure ascorbic acid powder stored under the different storage conditions for 35 d
Time of
assay
(d)
Mean concentration
(
% w/w)
Pure Powder
stored
at room
temperature
Pure powder stored in a bowl of
water
Pure powder
stored
in fridge
0
104.13
±
0.6770
104.13
±
0.6770
104.13
±
0.6770
2
102.53
±
1.1877
102.95
±
0.0699
103.33
±
1.3260
7
101.12
±
2.0541
101.59
±
1.3052
103.02
±
0.2572
14
100.11
±
2.4162
100.73
±
1.8326
102.84
±
0.9806
21
99.72
±
0.8214
100.01
±
2.4957
101.22
±
0.9975
28
97.90
±
1.4671
99.31
±
0.1252
101.14
±
0.9268
35
96.31
±
0.8546
97.67
±
0.5811
100.35
±
0.5613
Mean concentration of ascorbic acid given as mean±SD (n=3).
Klu et al.
Int J App Pharm, Vol 8, Issue 4, 2016, 26-31
28
Table 3: Assay results for ascorbic acid in syrups stored under the different storage conditions for 35 d
Time of
assay
(d)
Mean concentration
(
% w/w)
Syrup
stored
at room temperature
Syrup stored in a bowl of
water
Syrup
stored
in fridge
0
115.50
±
0.0000
115.50
±
0.0000
115.50
±
0.0000
2
115.50
±
0.0000
115.50
±
0.0000
115.50
±
0.0000
7
114.30
±
0.5196
114.57
±
0.8505
115.13
±
0.4619
14
113.43
±
0.4619
113.70
±
0.0000
114.30
±
0.5196
21
110.80
±
0.5196
112.83
±
0.4619
113.40
±
0.5196
28
105.40
±
0.0000
110.23
±
0.4619
112.57
±
0.4619
35
102.87
±
0.8505
107.70
±
1.7776
110.23
±
1.3204
Mean concentration of ascorbic acid given as mean±SD (n=3)
Also from the experiment, it was observed that the concentrations of
ascorbic acid in the various formulations under different storage
conditions reduced at different rates with time. For the tablets, the
concentrations of ascorbic acid reduced at a faster rate at room
temperature than in the fridge (fig. 1).
Fig. 1: Rates of degradation of ascorbic acid (AA) in tablets
stored under the different storage conditions for 35 d
In both the pure powder and syrups, storage under refrigeration
gave the highest stability or experienced the least breakdown while
storage at room temperature experienced the most breakdown. The
stability of ascorbic acid in the powder and syrup stored in a bowl of
water was better than that at room temperature (fig. 2 and 3).
Fig. 2: Rates of degradation of ascorbic acid (AA) in pure
powder stored under the different storage conditions for 35 d
Fig. 3: Rates of degradation of ascorbic acid (AA) in syrup stored
under the different storage conditions for 35 d
Table 4: Concentration terms for the various orders of breakdown for ascorbic acid in tablets stored at room temperature
Time of assay (d)
Concentration terms
Zero order
(
% w/w)
First order
Second order
/
0
105.57
4.659
0.00947
2
104.75
4.652
0.00955
7
102.77
4.632
0.00973
14
101.73
4.622
0.00983
21
100.60
4.611
0.00994
28
100.06
4.605
0.00999
35
98.52
4.590
0.01015
The mean concentrations of ascorbic acid were used for the conversions.
Table 5: Concentration terms for the various orders of breakdown for ascorbic acid in tablets stored in fridge
Time of assay (d)
Concentration terms
Zero order
(
% w/w)
First order
Second order
/
0
105.57
4.659
0.00947
2
104.91
4.653
0.00953
7
104.83
4.652
0.00954
14
103.91
4.644
0.00962
21
103.41
4.639
0.00967
28
102.92
4.634
0.00972
35
101.87
4.624
0.00982
The mean concentrations of ascorbic acid were used for the conversions.
Klu et al.
Int J App Pharm, Vol 8, Issue 4, 2016, 26-31
29
Table 6: Concentration terms for the various orders of breakdown for ascorbic acid in syrup stored in a fridge
Time of assay (d)
Concentration terms
Zero order
(
% w/w)
First order
Second order
/
0
115.50
4.749
0.00866
2
115.50
4.749
0.00866
7
115.13
4.746
0.00869
14
114.30
4.739
0.00875
21
113.40
4.731
0.00882
28
112.57
4.724
0.00888
35
110.23
4.703
0.00907
The mean concentrations of ascorbic acid were used for the conversions.
Table 7: Concentration terms for the various orders of breakdown for ascorbic acid in syrup stored at room temperature
Time of assay (d)
Concentration terms
Zero order
(
% w/w)
First order
Second order
/
0
115.50
4.749
0.00866
2
115.50
4.749
0.00866
7
114.30
4.739
0.00875
14
113.43
4.731
0.00882
21
110.80
4.708
0.00903
28
105.40
4.658
0.00949
35
102.87
4.633
0.00972
The mean concentrations of ascorbic acid were used for the conversions.
Table 8: Concentration terms for the various orders of breakdown for ascorbic acid in syrup stored in a bowl of water
Time of assay (d)
Concentration terms
Zero order
(
% w/w)
First order
Second order
/
0
115.50
4.749
0.00866
2
115.50
4.749
0.00866
7
114.57
4.741
0.00873
14
113.70
4.734
0.00880
21
112.83
4.726
0.00886
28
110.23
4.703
0.00907
35
107.70
4.679
0.00929
The mean concentrations of ascorbic acid were used for the conversions.
To be able to quantify the amount of ascorbic acid lost or broken
down in the formulations under the various storage conditions with
time, it was necessary to determine the reaction order of
breakdown. The mean concentrations of ascorbic acid in the
formulations under the different storage conditions were converted
to concentration terms needed to plot line graphs for zero order,
first order and second order reaction rates (Tables 4-8). The graphs
were obtained by plotting the various concentration terms against
their corresponding times of assay [11]. To obtain the order of
breakdown for ascorbic acid in any of the formulations, the order
whose graph gave the most linearity was taken to be the order of
breakdown. The linearity was measured by using the coefficient of
correlation, R
2
, which shows the strength of correlation between the
terms on the y-axis and those on the x-axis. The larger the R
2
, the
greater the strength of correlation and the more linear the graph
[13]. The R
2
were obtained from the line graphs plotted with
Microsoft excel.
The breakdown of ascorbic acid in the tablets stored under the
conditions studied followed a second order kinetic. The R
2
for their
second order graphs were generally higher than the R
2
obtained for
their zero order and first order graphs (table 9). This implied that,
their second order graphs were more linear. Fig. 4, 5 and 6
respectively show zero order, first order and second order sample
line graphs for the breakdown of ascorbic acid in tablets stored at
room temperature for 35 d.
Fig. 4: A zero order graph for ascorbic acid (AA) in tablets stored at room temperature
Klu et al.
Int J App Pharm, Vol 8, Issue 4, 2016, 26-31
30
Table 9: Data from reaction order graphs for ascorbic acid tablets
Storage condition
Zero
order
First
order
Second
order
R
2
k
R
2
k
¹
R
2
k
¹¹
Room temp.
0.9539
0.1866
0.9572
0.0018
0.9589
2× 10
-5
Fridge
0.9779
0.0959
0.9801
0.0009
0.9801
9× 10
-6
Fig. 5: A first order graph for ascorbic acid (AA) in tablets stored at room temperature
Fig. 6: A second-order graph for ascorbic acid (AA) in tablets stored at room temperature
The breakdown kinetics for ascorbic acid in the syrups stored under
the three different conditions followed a zero-order. The R
2
obtained
for the zero order line graphs under the three different storage
conditions were higher than those obtained for the line graphs for
first and second orders.
The extent or degree of breakdown of ascorbic acid in the tablets
and syrups under the various conditions of storage can be
determined by comparing their rate constants, k and k’’ for zero
order and second order respectively. The higher the rate constant,
the faster the rate of breakdown and the less stable the product
whereas a low rate constant is indicative of a slow breakdown and a
more stable product [11].
For the tablets, the second order rate constant for ascorbic acid
under refrigeration was smaller than that for at room temperature
(table 9). This implies that refrigeration offered a more stable
condition of storage for ascorbic acid than at room temperature.
The breakdown of ascorbic acid in the syrups was via a second order
kinetic hence the second order rate constant, k
¹¹
, was used. The rate
constant for storage under refrigeration (in a fridge) was the smallest,
followed by that in a bowl of water and that at room temperature was
the biggest (table 10). It follows that the syrup formulations stored in a
fridge were the most stable and those stored at room temperature
were the least stable. The syrups stored in a bowl of water were,
however, more stable than those stored at room temperature.
Table 10: Data from reaction order graphs for ascorbic acid syrup
Storage condition
Zero
order
First
order
Second
order
R
2
k
R
2
k
¹
R
2
k
¹¹
Room temp.
0.9324
0.3683
0.9259
0.0034
0.9234
3× 10
-5
Fridge
0.9339
0.1397
0.9270
0.0012
0.9251
1× 10
-5
Bowl of water
0.9398
0.2125
0.9307
0.0019
0.9273
2× 10
-5
In general, storage under refrigeration stored the products more
stable as it gave the least percentage breakdown and storage at
room temperature gave the most percentage breakdown.
Storage in a bowl of water gave a breakdown percentage better
than at room temperature for the syrups and pure powder (fig.
7).
Klu et al.
Int J App Pharm, Vol 8, Issue 4, 2016, 26-31
31
Key: blue-room temperature; red-fridge; green-bowl of water
Fig. 7: Percentage breakdown of ascorbic acid after 35d in the
various formulations under the different storage conditions
The significance of the percentage breakdown of ascorbic acid in the
formulations stored at room temperature and in a bowl of water was
calculated using a T-test in comparison to the percentage
breakdown under refrigeration (table 11). The T-test was done at a
confidence level of 95%.
Table 11: Statistical significance of the effects of the storage
conditions on the stability of ascorbic acid using a T-test
Formulation
T
-
value
(n=3)
Tablets at room temperature
-
27.203
Syrup at room temperature
-
15.0
Syrup in a bowl of water
-
2.465
The T-test was carried out at a confidence level of 95%.
The critical value at a confidence level of 95% is ±4.303. From table
11, the T-values for the % breakdown of ascorbic acid in tablets and
in the syrups at room temperature lie outside the range –4.303 to
4.303. These imply that the percentage breakdown of ascorbic acid
is very significant in tablets and syrups stored at room temperature
when compared to storage in a fridge. Storage at room temperature
should therefore not be encouraged. Storage of syrups in a bowl of
water gave a % breakdown that was not significant according to the
T-test since the calculated T-value of –2.465 was within the range of
–4.303 to 4.303. This implies that storage in a bowl of water can be
done in the absence of refrigeration.
CONCLUSION
Ascorbic acid breaks down with time in tablets and syrups and even
in the pure powder during storage. Storage under refrigeration
minimizes breakdown whereas storage at room temperature
encourages significant breakdown with time in comparison to
breakdown under the other storage conditions studied.
Storage of syrups in a bowl of water should be preferred to storage
at room temperature in the absence of refrigeration because the
syrups stored in a bowl of water were found to be more stable.
In the absence of refrigeration, ascorbic acid tablets should be stored
at cool places in homes to minimize degradation.
ACKNOWLEDGEMENT
The authors are very grateful to the staff of the Chemistry laboratory
of Central University for providing the needed reagents for the
experiment.
CONFLICT OF INTERESTS
The authors declare they have no competing interest
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How to cite this article
• Michael Worlako Klu, Bright Selorm Addy, Esther Eshun
Oppong, Emmanuel Seyram Sakyi, David Ntinagyei Mintah.
Effects of storage conditions on the stability of ascorbic acid in
some formulations. Int J Appl Pharm 2016;8(4):26-31.
