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Copyrights@2013
Received: 17th July-2013
Revised: 24th July-2013
ISSN: 2278-0246
Accepted: 25th Aug-2013
Coden: IJAPBS
INTRODUCTION
Ascorbic acid is a naturally occurring organic compound (fig. 1.). It is widely distributed in nature. Naturally this is
obtained from fruits and vegetables. It is in pure form a white crystalline powder. Its impure form appears yellow
colour. It dissolves in water to give mildly acidic solutions [1]. Ascorbic acid is one form "vitamer" of vitamin C.
Chemically, it is exists in D-ascorbic acid form which does not occur in nature. It may be synthesized artificially [2].
It is an essential part of human life. The IUPAC name of ascorbic acid is 2,3-dihydro-Lthreohexo-1,4-lactone.
Commonly ascorbic acid is known as vitamin C. The ascorbic acid in the form of vitamin C have various major
biologically activities such as maintenance of the organism, prevention of vitamin C deficiency (scurvy), promotion
of collagen biosynthesis, inhibition of melanogenesis and antioxidation [3-8] etc. Commercially ascorbic acid in
various formulations is available such as tablets, injections, syrups and capsules etc. Assay is an important method
for checking the commercially formulated products. There are various reported methods have been validated for the
simultaneous estimation of vitamin C by high-performance liquid chromatography using an UV detector [9-16]. The
main aim of this study is to develop and validate a new assay method for checking the quality and quantity of
ascorbic acid from formulated tablets.
HO
HO
H
HO
OH
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IJAPBS
ISSN: 2278-0246
EXPERIMENTAL
Chemicals and reagents
The 99.7% pure drug of ascorbic acid, HPLC grade methanol, distilled water and 0.45 nylon filter membrane were
purchased from Merck India Ltd. Mumbai.
Instrumentation
A binary pump CYBERLABTM HPLC chromatograph and a double beam UV-Spectrophotometry were used for
analysis. The separation was on C18-column. The analyte were monitored with UV detector at 260 nm. The HPLC
was operated at isocratic eluation mode with 50:50 (v/v) methanol-water mobile phase. The flow rate of eluation was
1.0 min./mL. An ultrasonic sonicator was used for the sonication of mobile phase, standard solution and sample
solution.
Method validation
The various method validation parameters were performed according to international conference harmonization
guide lines [17-19] such as system suitability, Linearity, limit of detection (LOD) and limit of quantification (LOQ)
and accuracy.
Linearity
The linearity ranges in both methods were 0.5-10 g/mL. The linearity ranges refers to the highest and lowest
quantity of the analyte that the method can detect with an appropriate amount of accuracy and linearity [20]. The
linearity was calculated by regression analysis.
Accuracy
Performance of the validated method is conformed by performing inter-day and intra-day recovery study at three
different concentration levels 1.5, 2.5 and 5.0g/mL. The three different concentration diluted from the stock
solution were added to an extract with a known content of ascorbic acid and the percentage recovery of the
respective constituents was calculated as R %=peak area of the drug in sample/peak area of the drug in standard x
100.
RESULTS
The mobile phase was select under reversed phase partition chromatographic condition. The mobile phase
developed was studied in order to achieve suitable system stability. The different ratios of (10:90, 20:80, 30:70,
40:60, 50:50) mobile phases compositions were tested at ambient temperature 25 0C [21]. The mobile phase
methanol/water in the ratio of 50:50 (v/v) was given suitable retention time and better resolution. There is no
interference with excipients (Fig.2).
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IJAPBS
ASCORBIC ACID
3.55
94.00
[mAU]
ISSN: 2278-0246
71.20
48.40
-20.00
0.00
0.50
1.00
1.50
2.00
2.50
3.00
4.61
4.65
4.68
2.93
2.97
3.09
3.11
3.13
3.15
2.72
2.56
2.33
2.35
2.00
2.04
2.06
2.08
1.57
1.67
1.70
1.05
1.09
1.13
2.80
0.09
0.18
4.15
25.60
3.50
4.00
4.50
5.00
[min]
Mean
69964.26
3.56
1.544
2785.42
1.646
SD
1.6562
0.036007
0.027019
0.0983362
0.027016
CV%
0.002367
1.003818
1.749904
0.035304
1.641465
SE
() 0.74
() 0.01
() 0.01
() 0.43
() 0.01
HPLC Method
0.5-10
Y=2726.x+475.2
0.997
0.031
0.093
UV Method
0.5-10
Y=0.033.x+0.079
0.983
0.3
0.9
The limit of detection (LOD) and limit of quantification (LOQ) for reversed phase-high performance liquid
chromatography (RP-HPLC) were reported 0.031 and0.093 g/mL and for UV-Spectrophotometry were reported 0.3
and 0.9 g/mL (Table 1.), respectively. The mean recoveries in inter-day and intra-day with RP-HPLC were
calculated 99.98 % and 99.77 % (Table 3.) and with UV-Spectrophotometry method were calculated 99.08 % and
99.37 % (Table 4.).
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ISSN: 2278-0246
RP-HPLC Method
RP-HPLC method
DISCUSSION
The system suitability study is indicates that the applied method was suitable for the analysis. Wave length
selection is the primary need for the chromatographic analysis. To selection the wave length for ascorbic acid were
investigated in order to determine a suitable wavelength for the assay evaluation. The suitable wave length was found
260 nm. The selection of mobile phase is an important secondary basic need for chromatographic analysis. The
mobile phase was select under reversed phase-partition chromatographic conditions. The recovery results were
showed good accuracy with less than one coefficient variation percentage in both methods. Hence, the interday and
intraday results were significant.
CONCLUSION
In this study, an isocratic RP-HPLC and UV-Spectrophotometry method were developed and validated for the
simultaneous estimation of ascorbic acid from formulated ascorbic acid products. The experimental conditions in
both methods were optimized to provide high resolution and reproducible absorbance and peak area. The results
were statistically significant. In both methods coefficient variation and standard deviation were found less than one
from interday and intraday recovery study. From the obtained results this was observed the reverse phased- high
performance liquid chromatography (RP-HPLC) is an advance technique while UV-Spectrophotometry has few
limitations. Hence, both methods are suitable for the quantitative estimation. Any one method can be used for
quantitative estimation of ascorbic acid formulated products in pharma industries.
ACKNOWLEDGEMENT
Present study was supported by Dolphin Institute of Biomedical and Natural Sciences, Dehradun, Uttarakhand,
INDIA.
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International Journal of Analytical, Pharmaceutical and Biomedical Sciences
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