ABOUT AUTHOR:
Hiren D. Antala
Department of Quality Assurance,
Noble Pharmacy College, Junagadh,
Gujarat, India
hirenantala21@gmail.com
ABSTRACT
The present manuscript describes simple, sensitive, rapid, accurate, precise and economical dual wavelength UV Spectrophotometric method for the simultaneous determination of Lafutidine and Rabeprazole Sodium in combined dosage form. The principle for dual wavelength method is “The absorbance difference between two points on the mixture spectra is directly proportional to the concentration of the component of interest”. The wavelengths selected for determination of Lafutidine were 281 nm and 287.8 nm, whereas, the wavelengths selected for determination of Rabeprazole Sodium were 269.2 nm and 276.4 nm. Methanol was taken as a solvent. Regression analysis of Beer’s plots showed good correlation in concentration range of 10-45 μg/ml for Lafutidine and 6-22 μg/ml for Rabeprazole Sodium. The mean recovery was found to be100.35% and 99.33%for Lafutidine and Rabeprazole Sodium, respectively. The precision (intraday, interday and repeatability) of method was found to be within the limits as per ICH Guideline Q2(R1). The Proposed method was successfully applied for the simultaneous estimation of both the drugs in commercial Pharmaceutical dosage form.
REFERENCE ID: PHARMATUTOR-ART-1869
INTRODUCTION:[1, 11]
Lafutidine is chemically 2-(furan-2-ylmethylsulfinyl)-N-[4-[4-(piperidin-1-ylmethyl) pyridin-2-yl]oxybut-2-enyl]acetamide. Lafutidine is not official in any pharmacopoeias. Lafutidine is the new generation H2-receptor antagonist. It blocks the production of acid by acid producing cells in the stomach and blocks histamine H2-receptors in the stomach and prevents histamine mediated gastric acid secretion. It is indicated in hyperacidity, NSAID induced gastritis, gastric and duodenal ulcers and also used as preanesthetic medication. Apart from H2-receptor blockade activity, it has additional gastro protective action. Therefore not only inhibit acid secretion but also provide gastric mucosal protection.
Rabeprazole Sodium is chemically2-[[[4-(3-Methoxypropoxy)-3-Methyl-2-Pyridinyl]-Methyl]Sulfinyl]-1H-Benzimidazole Sodium salt. Rabeprazole sodium (RBP) is a Potent Proton Pump inhibitor that suppress gastric acid secretion by specific inhibition of the gastric H+/K+-ATPase enzyme system at the secretory surface of the gastric parietal cell and is used in the treatment of Gastroesophageal reflux disease (GERD) and duodenal ulcers. It has a faster onset of action and lower potential for drug interaction compared to Omeprazole.
Figure. 1Chemical Structure of Lafutidine
Figure. 2Chemical Structure of Rabeprazole Sodium
Literature survey[4,5,6,7,8,9,10] revealed that a number of analytical methods have been reported for the estimation of Lafutidine(LAF) and Rabeprazole Sodium(RAB) in individual and combination with other drugs are spectrophotometry, HPLC, RP-HPLC, HPTLC,but not even single method was reported for the simultaneous estimation of LAF and RAB in their combined dosage form.
MATERIALS AND METHODS
Instrument:
Instrument used was an UV-Visible double beam spectrophotometer, SHIMADZU (model UV-1800) with a pair of 1 cm matched quartz cells. All weighing was done on Shimadzu analytical balance (Model AU-220).
Reagents and chemicals:
Pure drug samples of LAF and RAB were provided as a gift sample fromAlkem Laboratories Ltd, Mumbai andCadila Pharmaceuticals Ltd, Ahmedabad respectively. Methanol LR was used as solvent. Calibrated glass wares were used throughout the work.
Marketed formulation:
The commercial formulation LAFUMACPLUS (Macleods Pharmaceuticals Ltd.,Mumbai) was purchased from Local pharmacy. Each Capsule contains 10mg Lafutidine and 20mg Rabeprazole Sodium.
Preparation of standard stock solution:
Accurately weighed quantity of LAF (100 mg) and RAB (100 mg) was transferred to two separate 100 ml volumetric flasks, dissolved in Methanol and diluted to the mark with same solvent. (Stock solutions: 1000µg/ml of LAFand 1000µg/ml of RAB).
Preparation of working standard solution:
100µg/ml of LAF solution was prepared by diluting 10.0 ml of stock solution with methanol in 100 ml volumetric flask up to the mark. 100µg/ml of RAB solution was prepared by diluting 10.0 ml of stock solution with Methanol in 100 ml volumetric flask up to the mark.
Dual wavelength method:
The utility of dual wavelength data processing program is tocalculate the unknown concentration of a component ofinterest present in a mixture containing both thecomponents of interest and an unwanted interferingcomponent by the mechanism of the absorbance differencebetween two points on the mixture spectra. This is directlyproportional to the concentration of the component ofinterest, independent of the interfering components. Thepre?requisite for dual wavelength method is the selection oftwo such wavelengths where the interfering componentshows same absorbance whereas the component of interestshows significant difference in absorbance withconcentration.
Study of overlain spectra and selection of wavelength:
By appropriate dilutions from the working standardsolutions of 100μg/ml of LAF and 100 μg/ml of RAB, thesolutions of LAF (10 μg/ml) and RAB (20 μg/ml) wereprepared respectively and scanned over the range of 200?400 nm and the overlain spectra were observed fordevelopment of suitable method for analysis. The overlainspectra of LAF and RAB are shown in Figure 3. From the overlayspectra two wavelengths 281 nm and 287.8 nm wereselected as λ1 and λ2for the estimation of LAF. RAB shows thesame absorbance at these wavelengths. Similarly,wavelengths 269.2 nm and 276.4 nm were selected as λ3 and λ4 for estimation of RAB.LAF shows thesame absorbance at these wavelengths.
Analysis of marketed formulation:
Twenty Capsules were weighed and content crushed to obtain a fine powder. An accurately weighed powder equivalent to about 10mg of LAF and 20mg of RAB was transferred to 100 ml volumetric flask and the volume was made up to the mark using Methanol as solvent. The solution was sonicated for 20minutes. The solution was filtered through whatman Filter Paper No.42. First few ml of filtrate were discarded. 10 ml of the solution from above filtrate was diluted to 100 ml with Methanol.
The quantitative determination of LAF was carried out by measuring the absorbance difference value at between 281 nm and 287.8 nm, where RAB have same absorbance at both wavelengths. The absorbance difference between 281 nm and 287.8 nm is directly proportional to concentration of LAF.The quantitative determination of RAB was carried out by measuring the absorbance difference value at between 269.2 nm and 276.4 nm, where LAF have same absorbance at both wavelengths. The absorbance difference between 269.2 nm and 276.4 nm is directly proportional to concentration of RAB. (Table. 7)
METHOD VALIDATION[3]:
Linearity and range:
Aliquots of standard stock solutions of LAF and RAB were taken in volumetric flasks and diluted with Methanol to get final concentrations in range of 10-45μg/ml for LAF and 6-22 μg/ml for RAB. This calibration range was prepared six times and absorbancedifferences were measured at respective wavelengths for each drug separately.A calibration curve was obtained by plotting absorbance differences against respective concentrations(Table.1)(figure 3,4,5)
Precision:
Precision of the method was determined by performing interday variation, intraday variation and method repeatability studies. In interday precision, the absorbance of standard solutions of LAF (15, 30 and 45μg/ml) and RAB (6, 12 and 18μg/ml) were measured on Three consecutive days. In intraday variation the absorbance were measured Three times in a day. Repeatability study, one concentration of both the drugs was measured Six times.(Table.2, 3, 4)
Accuracy (Recovery studies):
To study the accuracy of the proposed method, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). A known amount of drug was added to preanalyzedCapsule powder and percentage recoveries were calculated.(Table.5, 6)
Limit of Detection (LOD):
Calibration curve was repeated for six times and the standard deviation (SD) of the intercepts was calculatedthan LOD was calculated as follow from the formula
LOD= (3.3×SD)/Slope
Where, SD = the standard deviation of Y- intercept of six calibration curves.
Slope = the mean slope of the six calibration curves.
Limit of Quantitation (LOQ):
Calibration curve was repeated for six times and the standard deviation (SD) of the intercepts was calculatedthan LOQ was calculated as follow from the formula
LOQ= (10×SD)/Slope
Where, SD = the standard deviation of Y- intercept of six calibration curves.
Slope = the mean slope of the six calibration curves.
RESULTS AND DISCUSSION:
The standard solution of LAF and RAB were prepared separately in methanol and scanned in the UV range of 200 - 400 nm. From the overlain spectra of both drugs, four specific wavelengths are selected. The absorbance at 281 nm (λ1) and 287.8 nm (λ2) wavelengths was found to be with same absorbance for RAB. These two selected wavelengths were employed to determine the concentration of LAF from the mixture of LAF and RAB. The difference in absorbance at these two wavelengths (A281– A287.8) cancels out the contribution of absorbance of RAB in mixture. Similarly, the absorbance at 269.2 nm (λ3) and 276.4 nm (λ4) wavelengths was found to be with same absorbance for LAF. These two selected wavelengths were employed to determine the concentration of RAB from the mixture of LAF and RAB. The difference in absorbance at these two wavelengths (A276.4– A269.2) cancels out the contribution of absorbance of LAF in mixture.
The proposed method was found to be simple, sensitive, rapid, accurate, precise and economic for the routine simultaneous estimation of two drugs. Linear correlation was obtained between absorbance differences and concentrations of LAF and RAB in the concentration ranges of 10-45μg/ml &6-22μg/ml(Table 1); with R2value 0.999 for LAF and 0.998 for RAB respectively.Precision was calculated as repeatability, intraday and interday variations and %RSD (Relative Standard Deviation) was found to be in between 0.27-1.73% (Table 2, 3, 4). The accuracy of method was determined at 80, 100 and 120% level. The recovery was found to be in between 98.33?101.84 %(Table5,6).The Proposed method was successfully applied for the simultaneous estimation of both the drugs in commercial Pharmaceutical dosage form.(Table 7)
Figure.3: OverlainZero order spectra of LAF and RAB in methanol.
Figure.4: Spectra for LAF and RAB for different concentration at 281 nm and 287.8 nm where, RAB has same absorbance and LAF has different absorbance
Figure.5: Spectra for LAF and RAB for different concentration at 269.2 nm and 276.4 nm where, LAF has same absorbance and RAB has different absorbance
|
Sr No. |
Concentration (μg/ml) Lafutidine |
Mean absorbance difference at 281&287.8 nm* |
Concentration (μg/ml) Rabeprazole Sodium |
Mean absorbance difference at 269.2&276.4 nm* |
|
1 |
10 |
0.097 ±0.000516 |
6 |
0.033 ±0.000816 |
|
2 |
15 |
0.143 ±0.001211 |
8 |
0.043 ±0.000816 |
|
3 |
20 |
0.184 ±0.002066 |
10 |
0.053 ±0.001414 |
|
4 |
25 |
0.236 ±0.000894 |
12 |
0.063 ±0.001472 |
|
5 |
30 |
0.279 ±0.000816 |
14 |
0.071 ±0.000816 |
|
6 |
35 |
0324 ±0.000816 |
16 |
0.083 ±0.001033 |
|
7 |
40 |
0.376 ±0.000753 |
18 |
0.094 ±0.000632 |
|
8 |
45 |
0.42 ±0.000816 |
20 |
0.106 ±0.000983 |
|
9 |
|
|
22 |
0.114 ±0.001378 |
Table 1: Linearity Study*n=6
|
LAF Concentration (μg/ml) |
Absorbance* ±S.D. |
%RSD |
RAB Concentration (μg/ml) |
Absorbance* ±S.D. |
%RSD |
|
15 |
0.14767± 0.00057735 |
0.39 |
6 |
0.03467± 0.00057735 |
1.66 |
|
30 |
0.291± 0.001 |
0.34 |
12 |
0.06133± 0.00057735 |
0.94 |
|
45 |
0.42767± 0.001154701 |
0.27 |
18 |
0.091± 0.001 |
1.09 |
S.D. = Standard Deviation*n=3
RSD= Relative Standard Deviation
Table 2: Intra-Day Precision Study
|
LAF Concentration (μg/ml) |
Absorbance* ±S.D. |
%RSD |
RAB Concentration (μg/ml) |
Absorbance* ±S.D. |
%RSD |
|
15 |
0.14833 ±0.001527525 |
1.03 |
6 |
0.03333±0.00057735 |
1.73 |
|
30 |
0.29233 ±0.002081666 |
0.71 |
12 |
0.061±0.001 |
1.64 |
|
45 |
0.43033 ±0.004163332 |
0.97 |
18 |
0.09233±0.001527525 |
1.65 |
*n=3
Table 3: Inter-Day Precision Study
|
LAF Concentration (μg/ml) |
Absorbance* ±S.D. |
%RSD |
RAB Concentration (μg/ml) |
Absorbance* ±S.D. |
%RSD |
|
10 |
0.10167±0.0015055 |
1.48 |
20 |
0.106±0.001264 |
1.19 |
*n=3
Table 4: Repeatability Study
|
Lafutidine |
Level of Recovery |
Amt. of Drug taken(μg/ml) |
Amt. of Std. drug taken(spiked amt.)(μg/ml) |
% Recovery* ± S.D. |
%RSD |
|
80% |
10 |
8 |
101.84± 0.79 |
0.78 |
|
|
100% |
10 |
10 |
98.9± 1.1 |
1.11 |
|
|
120% |
10 |
12 |
100.31± 1.06 |
1.06 |
*n=3
Table 5: Recovery Study of Lafutidine
|
Rabeprazole Sodium |
Level of Recovery |
Amt. of Drug taken(μg/ml) |
Amt. of Std. drug taken(spiked amt.)(μg/ml) |
% Recovery* ± S.D. |
%RSD |
|
80% |
10 |
8 |
98.33± 1.44 |
1.46 |
|
|
100% |
10 |
10 |
101.33± 1.15 |
1.13 |
|
|
120% |
10 |
12 |
98.33± 1.67 |
1.69 |
*n=3
Table 6: Recovery Study of Rabeprazole Sodium
|
BRAND NAME: LAFUMAC PLUS |
Drugs |
Label Claim (mg) |
Amount Found (mg) |
% Label Claim* |
S.D. |
%RSD |
|
LAF |
10 |
10.183 |
101.83% |
1.502886112 |
1.48 |
|
|
RAB |
20 |
19.5 |
97.5 % |
1.048808848 |
1.08 |
*n=6
Table 7: Results of simultaneous estimation of LAF and RAB in Marketed Formulation.
|
SR No. |
PARAMETERS |
LAFUTIDINE |
RABEPRAZOLE SODIUM |
|
1 |
Wavelength
|
281 &287.8 nm |
269.2&276.4 nm |
|
2 |
Linearity Range |
10-45μg/ml |
6-22μg/ml |
|
3 |
Correlation Coefficient |
R2= 0.999 |
R2= 0.998 |
|
4 |
Precision a)Intraday (n=3) b)Interday (n=3) c)Repeatability (n=6) |
%RSD 0.27-0.39 0.71-1.03 1.48 |
%RSD 0.94-1.66 1.64-1.73 1.19 |
|
5 |
Accuracy (n=3) |
100.35% |
99.33% |
|
6 |
Limit of detection (LOD) |
0.52μg/ml |
0.27μg/ml |
|
7 |
Limit ofQuantification(LOQ) |
1.57μg/ml |
0.82μg/ml |
|
8 |
Assay (n=6) |
101.83% |
97.5 % |
Table 8:Results of Validation Parameters
CONCLUSION:
The proposed dual wavelength method gives accurate and precise results for determination of Lafutidine and Rabeprazole Sodium in marketed formulation (Capsule) without prior separation and is easily applied for routine analysis. The most striking feature of the dual wavelength method is its simplicity and rapidity. Method validation has been demonstrated by variety of tests for linearity, accuracy, precision and ruggedness. The proposed method was successfully applied to determination of these drugs in commercial Pharmaceutical dosage form.
ACKNOWLEDGEMENT:
It is great pleasure for me to acknowledge my parents and all of my friends,who have contributed towards the conception, origin and nurturing and successful completion of this research work.
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