Int J Pharm Pharm Sci, Vol 6, Issue 8, 371-374Original Article
A CHEAP METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTITATIVE ESTIMATION OF ACAMPROSATE CALCIUM IN TABLETS USING UV SPECTROPHOTOMETRY
DANDU VIJAY KUMAR, PARUPUGALLA SWETHA, THOTA CHANDRA MOHAN, A. ASHOK KUMAR*
Department of Pharmaceutical analysis and Quality Assurance, Vijaya college of pharmacy, Munaganur (village), Hayathnagar (mandal), Hyderabad 501511, India.
Email: ashok576@gmail.com
Received: 25 Jun 2014 Revised and Accepted: 27 Jul 2014
ABSTRACT
Objective: To develop a cheap and a rapid UV spectrophotometric method for the quantitative estimation of Acamprosate calcium (333mg) in tablets and validate as per ICH guidelines.
Methods: The optimized method uses 100% distilled water as a solvent for the estimation of assay of Acamprosate calcium in tablets at a detection wavelength of 208 nm.
Results: The developed method resulted in Acamprosate calcium exhibiting linearity in the range 30-90μg/ml. The precision is exemplified by relative standard deviation of 0.425%. Percentage Mean recovery was found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) and limit of quantitiation (LOQ) were found to be 66µg/ml, and 200 µg/ml respectively.
Conclusion: A cheap and a rapid UV spectrophotometric method was developed and validated for the quantitative estimation of Acamprosate calcium in tablets as per ICH guidelines and hence it can be used for the routine analysis in various pharmaceutical industries.
Keywords: UV, Acamprosate calcium, Method development, Validation.
INTRODUCTION
Acamprosate calcium (Figure 1, calcium 3-acetamidopropane-1-sulfonate) is the calcium salt of acetylhomotaurine used in the treatment of alcohol dependence. It is believed to act by blocking glutaminergic N-methyl-D-aspartate receptors and activation of gamma-aminobutyric acid (GABA) type A receptors [1-3]. It is an antidipsotropic agent that was approved by the US Food and Drug Administration (FDA) in 2004 for use in alcoholic individuals to decrease alcohol hankering after alcohol detoxification [4]. Acamprosate has been commercially available since 1989, in 333 mg tablet strength [5]. Acamprosate calcium is a white, odorless or nearly odorless powder. It is freely soluble in water and practically insoluble in absolute ethanol and dichloromethane. Its chemical formula is C10H20N2O8S2Ca and molecular weight is 400.48.
Fig. 1: Structure of Acamprosate calcium
A detailed literature survey reveals capillary zone electrophoresis methods [6-7], bioanalytical methods for the analysis of Acamprosate calcium using LCMS [8-16], LC-fluorometric and electrochemical detection [8] in human plasma, dog plasma and urine and overall only one UV method has been reported for the quantitative estimation of Acamprosate calcium in tablets [17]. We here report a new, precise, accurate and linear UV method for the quantitative estimation of Acamprosate calcium in ACAMPRAL tablets.
MATERIALS AND METHODS
Materials
Instrument
A double beam UV-visible spectrophotometer (Shimadzu, model 1800) having two matched quartz cells with 1 cm light path and loaded with UV probe software (version 2.41) was used for recording of spectra and measuring absorbance. An electronic analytical weighing balance (0.1mg sensitivity, Shimadzu AY 220), digital pH meter (DELUX model 101) and a sonicator (sonica, model 2200 MH) were used in this study.
Chemicals and Reagents
Analytically pure sample of Acamprosate calcium with purities 95% was obtained as gift sample from Chandra labs, Hyderabad, India and tablet formulation [ACAMPRAL] was procured from MEDPLUS, Hyderabad, India with labelled amount 333mg of Acamprosate calcium. Triethylammonium phosphate(AR Grade) and ortho phosphoric acid (AR Grade) were obtained from SD Fine chemicals (Hyderabad, India). 0.45μm Nylon membrane filters were obtained from Spincotech Private Limited, Hyderabad, India.
Method
Solvent
100% distilled water has been used for the determination of assay of acamprosate calcium in tablets.
Selection of suitable detection wavelength
Suitable wavelength for the total experiment was determined by recording UV spectrum in the range of 200-400 nm for Acamprosate calcium and suitable wavelength selected was 208 nm (Figure 2).
Preparation of stock and working standard solution
10mg of Acamprosate calcium was accurately weighed and taken in 100ml clean and dry volumetric flask containing 80ml of solvent and then the solution was made up to the mark using the solvent. This is considered as standard stock solution (100µg/ml). 6ml of the stock solution was pipetted out and made up to 10 ml to get a concentration 60µg/ml, treated as working standard, 100% target concentration.
Preparation of stock and working sample solution
Ten tablets were weighed separately and the average weight was determined. The average weight was weighed from the ten tablets grinded in a pestle and mortar, transferred to a 100 ml volumetric flask containing 100ml diluent and then sonicated for 3 minutes, followed by filtration through 0.45µ nylon membrane filter to get sample stock solution of 3330µg/ml. 1.8 ml of the above stock solution was pipetted out and made up to 100 ml to get working sample solution equivalent to a concentration of working standard of 60 µg/ml.
Fig. 2: UV spectrum of Acamprosate calcium
RESULTS AND DISCUSSION
Method Development
Various solvents were explored, including 1005 distilled water, potassium dihydrogen orthophosphate, triethyl ammonium phosphate and ammonium acetate buffers varying pH in the ranges of 2-7. Acamprosate calcium was found to be soluble in distilled water and hence this solvent was initiated for the determination of suitable detection wavelength and working concentration of standard. In order to test the applicability of the developed method to a commercial formulation, ACAMPRAL was studied at working concentration. Absorbance and assay for working concentration of sample at 208 nm was in acceptance limits (98-102%) with the standard working concentration during extraction of drug in the sample using the solvent. The protocol affords reproducible quantification of the drug in the sample ranging between 98 and 102%, which is the standard level in any pharmaceutical quality control. Hence the method is optimized.
Method validation
Validation of the analytical method is the process that establishes by laboratory studies in which the performance characteristics of the method meet the requirements for the intended analytical application. UV spectrophotometric method developed was validated according to International Conference on Harmonization (ICH) guidelines[18] for validation of analytical procedures. The method was validated for the parameters like linearity, accuracy, system precision, intra-day precision, inter-day precision/ intermediate precision/ ruggedness, robustness, limit of detection (LOD) and limit of quantitiation (LOQ).
Precision
System precision
Six replicate recording of absorbance at 208nm of standard solution at working concentration showed % RSD (Relative Standard Deviation) less than 2 concerning absorbance for the drug, which indicates the acceptable reproducibility and thereby the precision of the system. System precision results are tabulated in Table 1.
Method precision
Method precision was determined by performing assay of sample under the tests of (i) repeatability (Intra day precision) and (ii) Intermediate precision (Inter day precision) performed during 3 consecutive days by three different analysts, at working concentration.
Repeatability (Intra day precision)
Six consecutive recording of absorbance at 208nm of the sample from the same homogeneous mixture at working concentration showed % RSD less than 2 concerning % assay for the drug which indicate that the method developed is method precise by the test of repeatability and hence can be understood that the method gives consistently reproducible results (Table 2).
Table 1: System precision results of Acamprosate calcium.
| n | Absorbance |
| 1 | 0.332 |
| 2 | 0.335 |
| 3 | 0.331 |
| 4 | 0.329 |
| 5 | 0.332 |
| Average | 0.332 |
| SD | 0.002 |
| % RSD | 0.6 |
Table 2: Intra day precision results of Acamprosate calcium
| n | % Assay |
| 1 | 102.2 |
| 2 | 102.4 |
| 3 | 101.8 |
| 4 | 102.2 |
| 5 | 101.4 |
| Average | 101.8 |
| S.D. | 0.433 |
| % RSD | 0.425 |
Intermediate Precision (Inter day precision / Ruggedness)
Six consecutive recording of absorbance at 208nm of the sample solution from the same homogeneous mixture at working concentration on three consecutive days by three different analysts, showed % RSD less than 2 for % assay for the drug within and between days, which indicate the method developed is inter day precise / rugged (Table 3).
Table 3: Inter day precision results.
| n | Day 1 | Day 2 | Day 3 |
| 1 | 102.2 | 101.1 | 99.01 |
| 2 | 102.4 | 100. 3 | 100.06 |
| 3 | 101.8 | 99.86 | 101.00 |
| 4 | 102.2 | 100.00 | 99.06 |
| 5 | 101.4 | 99.76 | 100.25 |
| Average | 101.8 | 100.18 | 99.876 |
| SD | 0.433 | 0.62 | 0.844 |
| % RSD | 0.425 | 0.61 | 0.845 |
Linearity
Standard solutions of Acamprosate calcium at different concentrations level (50%, 75%, 100%, 125%, 150%, 175% and 200%) were prepared. Calibration curve was constructed by plotting the concentration level of drug versus corresponding absorbance at 208nm.
The results show an excellent correlation between absorbance and concentration level of drug within the concentration range (30-90µg/ml) for the drug and the results are given in Table 4. The correlation coefficients were greater than 0.99 and hence the method is said to be linear in the range of 30-90µg/ml.
Accuracy
Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of sample at three different levels (50-150%). At each level, three determinations were performed. Percent mean recovery was calculated as shown in Table 5. The accepted limits of recovery are 98% - 102% and all observed data are within the required range which indicates good recovery values and hence the accuracy of the method developed.
Table 4: Calibration data for Acamprosate calcium
| % Level | Concentration (µg/ml) | Absorbance 1 | Absorbance 2 | Absorbance 3 |
| 50 | 30 | 0.167 | 0.170 | 0.168 |
| 75 | 45 | 0.234 | 0.269 | 0.274 |
| 100 | 60 | 0.313 | 0.327 | 0.328 |
| 125 | 75 | 0.387 | 0.405 | 0.429 |
| 150 | 90 | 0.480 | 0.492 | 0.510 |
| Regression equation | y=0.005x+0.0046 | y=0.0052x+0.206 | y=0.0055x+0.0062 | |
| Regression coefficient | 0.996 | 0.994 | 0.992 |
Table 5: Results of Accuracy studies for Acamprosate calcium
| Concentration level (%) | *%Mean recovery (% rsd) |
| 50 | 99.6(1.158) |
| 100 | 101.9(0.112) |
| 150 | 98.6(0.585) |
Robustness
The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is concluded that the method is robust as it is found that the % RSD is less than 2 for the drug concerning % assay despite deliberate variations done concerning pH ± 0.2 and detection wavelength ± 2nm (Table 6).
Table 6: Robustness results of Acamprosate calcium sample
| Variation parameter | Variation | %RSD |
Wave length (± 2nm) |
210 | 1 |
| 208 | 0.112 | |
| 206 | 0.55 |
Sensitivity
The sensitivity of measurement of Acamprosate calcium by use of the proposed method was estimated in terms of the limit of quantitation (LOQ) and limit of detection (LOD). The limit of detection (LOD) and limit of quantitiation (LOQ) were found to be 66µg/ml, and 200 µg/ml respectively. Optical characteristics and validation parameters results are summarized in Table 7.
Table 7: Optical characteristics and validation parameters of Acamprosate calcium
| Parameters | Results |
| Detection wavelength (nm) | 208 |
| Beer’s Law limits (µg/ml) | 30-90 |
| Regression equation (y = mx+c) | y=0.005x+0.1127 |
| Correlation coefficient (r2) | 0.9885 |
| Slope (m) | 0.0052 |
| Intercept (c) | 0.072 |
| % Relative Standard Deviation (% RSD) System precision | 0.6 |
| (% RSD) Intra-day precision | 0.425 |
| (% RSD) Inter-day precision | ≤2 |
| Accuracy (% Mean Recovery) | |
| 50 % Level | 99.6 % |
| 100 % Level | 101.9 % |
| 150 % Level | 98.6 % |
| LOD (µg/ml) | 66 |
| LOQ (µg/ml) | 200 |
| Sandell’s sensitivity (µg/cm2/0.001 absorbance unit) | 0.001 |
| Robustness | |
| Wavelength (± 2nm) (% RSD) | ≤2 |
CONCLUSION
A rapid UV spectrophotometric method was developed and validated for the quantitative estimation of Acamprosate calcium in tablets as per ICH guidelines. The developed method resulted in Acamprosate calcium exhibiting linearity in the range 30-90μg/ml. The precision is exemplified by relative standard deviation of 0.425%. Percentage Mean recovery was found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) and limit of quantitiation (LOQ) were found to be 66µg/ml, and 200µg/ml respectively. Accordingly it is concluded that the developed UV spectrophotometric method is accurate, precise, linear, rugged and robust and therefore the method can be used for the routine analysis of Acamprosate calcium in tablets in various pharmaceutical industries.
CONFLICT OF INTERESTS
Declared None
ACKNOWLEDGEMENT
The authors would like to thank the management of Vijaya college of pharmacy (VJYH), Hyderabad, for providing the necessary facilities to carry out of this research work. The authors are grateful to Chandra labs, Hyderabad for providing drug in form of gift sample.
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