Volume: 2: Issue-3: July-Sept-2013 Copyrights@2013 Received: 17th July-2013 Revised: 24th July-2013
ISSN: 2278-0246 Accepted: 25th Aug-2013
Coden: IJAPBS
www.ijapbs.com Research Article
QUANTITATIVE ASSAY EVALUATION OF VITAMIN ‘C’ FROM FORMULATED TABLETS: APPLICATION ON RP-HPLC AND UV-SPECTROPHOTOMETRY Raju Chandra* Sujeet Kumar, Sanjay Singh, Keshav Dutt Sharma, Md. Naushad Alam and Daleep Verma Department of Pharmaceutical Chemistry, Dolphin PG Institute of Biomedical and Natural Sciences, Dehradun-248001, Uttarakhand, India *Author to whom correspondence should be addressed. Email:
[email protected] ABSTRACT: Assay is an important method for the accurate quantity determination of drug analyte in the formulated products. For which a rapid simple sensitive isocratic reproducible reversed phase-high performance liquid chromatographic technique has been developed for the quantitative assay evaluation of ascorbic acid. The chromatographic separation of ascorbic acid samples were performed on a C18-column by isocratic elusion at the column 250C. The mobile phase was used methanol and water in the ratio of 50:50 (v/v). The flow rate was 1.0 ml/min. The maximum peak area was resolved at 260 nm. The limit of detection and limit of quantification were 0.031 and 0.093 µg/mL by RP-HPLC and 0.3 and 0.9 µg/mL by UV-Spectrophotometry, respectively. Good results were obtained with respect to linearity R2=0.997 by RP-HPLC and R2=0.983 by UV-Spectrophotometry. The mean recoveries in inter-day and intra-day for RP-HPLC were calculated 99.98 % and 100.2 % and for UVSpectrophotometry 99.08 % and 99.37 %. The method was validated for linearity, accuracy, repeatability, limit of detection (LOD) and limit of quantification (LOQ). The method is simple, accurate and precise and was successfully applied to the determination of ascorbic acid from formulated products. Key words: Ascorbic acid, RP-HPLC, UV-Spectrophotometry, Method Validation.
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
O
O
H HO
OH
Figure. 1. Chemical structure of ascorbic acid
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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.
Preparation of mobile phase A methanol /water mobile phase was prepared. The ratio of methanol and water was inthe ratio of 50:50 (v/v). The mobile phase were filtered through a 0.45µm nylon membrane and degassed by ultrasonic sonication.
Preparation of stock solution A 100 µg/mL stock solution was prepared. In a 100 mL capacity volumetric flask 10 mg ascorbic acid was mixed with mobile phase and make up it to 100 mL volume. The stock solution was filtered with 0.45µm nylon filter membrane and degassed by sonication.
Preparation of sample solution The 20 tablets (Brand name Celin, label claim: 500 mg ascorbic acid per tablet) were weighed accurately and crushed by mortar pistol. The crushed tablets were mixed well, and then an equivalent amount of 10 mg was transferred into a small conical flask and extract with methanol/water 50:50 (v/v) mobile phase. The extract was filtered into a 100 ml volumetric flask and the volume make up to 100 mL. Achieved aliquots was covered the working concentration range 100 µg/mL.
Preparation of calibration curve A calibration curve was constructed by injecting the different concentration of serial dilutions (standard drug) 0.5, 1.0, 1.5, 2.0, 2.5 5.0, 10 µg/mL in thrice replication. The calibration curve was obtained by plotting the average peak areas against these different known concentrations.
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.
System suitability test The system suitability was checked to insure that, the system is working correctly. The system suitability parameters peak area, retention time, resolution factor, theoretical plates, and tailing factor were checked according to international conference harmonization guide line. This test was performed during development of the method. This test was performed by injecting the standard mixture in n=5 replicates.
Linearity The linearity ranges in both methods were 0.5-10 µg/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.
Limit of detection (LOD) and limit of quantification (LOQ) The Limit of detection (LOD) and limit of quantification (LOQ) were calculated using the equations, LOD =3.3σ/s and LOQ =10.σ/s. Where σ is the standard deviation of y-intercept and s is the slope of the curve.
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 5.0µg/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]
Figure. 2. Chromatogram of ascorbic acid The system suitability test is statistically significant. The coefficient of variation percentage of each parameter is less than 10 (Table 1.). Table 1. Summary of system suitability test (n=5) for RP-HPLC Parameters Peak area Retention Time Resolution Theoretical Plate Tailing Factor
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
SD=standard deviation CV%=coefficient of variation percentage SE=standard error (±) The linearity was measured by regression analysis. The linearity range of ascorbic acid was 0.5-50 µg/mL for both methods. Both methods reversed phase-high performance liquid chromatography (RP-HPLC) and UVSpectrophotometry results were showed good linearity with regression coefficient (R2) 0.997 and 0.983. The linear regression equations for ascorbic acid are described in Table 1. Table 2. Summary of linearity (n=3) and limit of detection (LOD) and limit of quantification (LOQ) Parameters Correlation range (µg/mL) Regression equation Regression coefficient LOD (µg/mL) LOQ (µg/mL)
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 µg/mL and for UV-Spectrophotometry were reported 0.3 and 0.9 µg/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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Table 3. Recovery study in inter-day UV-Spectrophotometry Method Recovery (%) Recovery Recovery (%) Recovery in µg/mL (w/w) in µg/mL (w/w) 1.49 99.33% 1.48 98.66% 2.49 99.6% 2.47 98.80% 5.05 101% 4.99 99.80% Mean=99.98% Mean=99.08% SD=0.89 SD=0.61 CV%=0.89 CV%=0.62 SD=standard deviation CV%= coefficient variation percentage
Added pure drug in µg/mL 1.5 2.5 5.0
RP-HPLC Method
Table 4. Recovery study in intra-day UV-Spectrophotometry method Recovery in Recovery (%) Recovery Recovery (%) µg/mL (w/w) µg/mL (w/w) 1.5 1.499 99.33% 1.49 99.33% 2.5 2.48 99.2% 2.48 99.2% 5.0 5.01 100.2% 4.98 99.6% Mean=99.77% Mean=99.37% SD=0.51 SD=0.20 CV%=0.51 CV%=0.20 SD=standard deviation CV%=coefficient variation percentage
Added pure drug in µg/mL
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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