Simultaneous determination and method validation of ranitidine hydrochloride and itopride hydrochloride by UV-spectrophotometery

An UV spectrophotometric method using simultaneous equation was developed for the simultaneous determination of ranitidine and itopride in a binary mixture. In the proposed method, the signals were measured at 315.0 and 258.0 nm corresponding to absorbance maxima of ranitidine and itopride hydrochloric acid (HCl) in double distilled water, respectively. Linearity range was observed in the concentration range of 2 to 20 μg/ml for both drugs. Concentration of each drug was obtained by using the absorptivity values calculated for both drugs at two wavelengths, 315.0 and 258.0 nm and solving the simultaneous equation. Developed method was applied to laboratory mixture. The method was validated statistically and recovery study was performed to confirm the accuracy of the method. The method was found to be rapid, simple, accurate and precise.


INTRODUCTION
Ranitidine hydrochloride (RanHCl) is a H 2 -receptor antagonist which is used as anti-ulcer drug that reduces acid secretion by blocking the histamine receptor type.Ranitidine is a H 2 -receptor antagonist, same as cimetidine.The only difference is that it contains furan ring in place of imidazole ring of cimetidine.This one is amino alkyl furan derivative.It is more potent in inhibiting the gastric acid secretion (Klaus, 2005).Itopride hydrochloride (ItoHCl) (Gupta et al., 2004) is a new gastro-prokinetic agent which is used in reflux oesophagaitis (Pillai and Singhvi, 2008).These two drugs are natural candidates for combination therapy in the treatment of reflux oesophagitis, dyspepsia, hyperacidity etc.Although no marketed product has yet been launched in India, however, those two drugs are logical candidates for combination formulation in hyperacidicty and reflux-oesophagitis.Therefore, characterization of formulations like tablets, capsules or microcapsules having this combination of drugs will require simultaneous determination of RanHCl and ItoHCl in aqueous buffer media.Because of this reason for determining the concentration of the drugs simultaneously in the acidic dissolution medium, a sensitive, validated, less time consuming and low cost analytical method by UV-spectrophotometry is required to be developed.The analytical method has been validated by different statistical parameter as calculated (Attimarad et al., 2012;Mohite et al., 2009).Therefore, in this project  (Pathak and Rajput, 2008).

MATERIALS AND METHODS
Ranitidine hydrochloride was a kind gift from Strides Acrolab, Bangalore and Itopride hydrochloride was a kind gift from Themis Pharma Pvt Ltd, Thane, Mumbai.Distilled water was obtained from Barnstead type steel distillation apparatus.Double beam UV-Vis spectrophotometer, Pharmaspec 1700, Shimadzu was used for measuring the absorbance of the solutions.Volumetric flask 100 ml, graduated pipette 10 ml, beaker 100 ml, beaker 50 ml were procured from Borisil®.Single pan analytical balance (Dwijo, 200 G), wax paper, tissue paper were used.

Preparation of stock solutions of RanHCl and ItoHCl
RanHCl and ItoHCl were dried overnight in desiccator.RanHCl, 100 mg was dissolved in 100 ml double distilled water to prepare a stock solution of concentration 1000 µg/ml.Working standard of RanHCl was prepared by diluting the stock solution 100 times to obtain a standard concentration of 100 µg/ml.Similarly, working standard of ItoHCl of 100 µg/ml was also prepared.

Preparation of standard solutions
Standard RanHCl and standard ItoHCl solutions of both the drugs were prepared by using the working standard solutions of the two drugs according to Tables 1 and 2.

Preparation of standard solutions containing both RanHCl and ItOHCl
This is seen in Table 2.

Selection of two analytical wavelengths
UV spectra of pure drugs solutions and mixture solution of two drugs were constructed by taking the solutions in a 1 cm cuvette and scanned from 200 to 400 nm in a double beam UV-Spectrophotometer (Shimadzu Pharmaspec 1700).One wavelength was selected where one molecule will produce peak absorbance and the other some lower absorbance.Similarly, at the other wavelength the second molecule will produce maximum and the first molecule some lower absorbance value.

Construction of simultaneous equations
To determine the absorptivity of the pure component a standard curve was constructed by plotting absorbance (A) against standard Absorbance Wavelength (nm) concentrations (C).The equation of the straight line was obtained from linear regression.The path-length of the light was 1 cm (1 cm cuvette), therefore the slope of the straight line was the absorptivity of the component.Four absorptivities were calculated for two wave lengths and two components.Two simultaneous equations were constructed as described.The equations were solved by using Cramer's rule (Beckett and Stenlake, 2002).For large number of calculations MS Excel and VB application were used.If the two simultaneous equations are as follows: Then the solution for CX and CY were solved by the following equations:

Determination of analytical wavelengths from UVspectra
Three spetra were obtained from (i) standard solution of pure RanHCl (10 µg/ml), (ii) standard solution of pure ItoHCl (10 µg/ml) and (iii) standard solution containing 10 µg/ml of RanHCl and 10 µg/ml of ItoHCl.The spectra were provided as follows (Figures 1 to 4).RanHCl showed two peaks at 228.5 and 315 nm while ItoHCl showed a single maximum at 258 nm.Since there were two components in the mixture solution hence, two analytical wavelengths were selected at 258 and 315 nm.

Construction of simultaneous equations
Four standard straight lines were constructed by plotting the absorbances (Y-axis) of standard solutions of pure drugs, at two analytical wavelengths chosen in Equation (3) against the concentration (X-axis).The absorptivity values were calculated from the slopes of Figures 5 and 6  carried out with data of C Y also.The equations thus obtained from Figure 7 were applied for calculating the final values.

Specificity
Since this procedure determines the two drugs in a solution therefore this method alone was not sufficient to    prove the specificity.For proving the specificity another method like high performance liquid chromatography (HPLC) is required in tandem with this procedure.However, no HPLC method is reported yet for separating

Linearity
Linearity calculation has been done as shown in Table 5.
As per the acceptance criteria the correlation coefficients are higher than 0.9990.Therefore the method showed linearity within 2 to 20 µg/ml of each drug.

Limit of detection (LOD) and limit of quantitation (LOQ)
Determination of LOD and LOQ of RanHCl at 315 and 258 nm as shown in Table 6.Since it is a mixture solution therefore the LOD and LOQ will be the higher values that is 0.266 and 0.805 µg/ml, respectively for RanHCl (where ItoHCl is the background matrix).Determination of LOD and LOQ of ItoHCl at 315 and 258 nm has been done as mentioned in Table 7.Since it is a mixture solution therefore the LOD and LOQ will be the higher values i.e. 1.497 and 4.536 µg/ml, respectively for ItoHCl (where RanHCl is the background matrix).

Result of precision
As per Tables 8 and 9, the %RSD values are within 2.0 therefore, the method was found to be precise with respect to repeatability.

Accuracy
Acceptance criteria: Recovery should be within 98% to 102%.The recovery results indicating that the test method has an acceptable level of accuracy for the assay

Range
From the linearity, precision and accuracy experiments the range of the analytical method was found to be between 5 to 20 µg/ml concentration for both RanHCl and ItoHCl.

Conclusion
In this context an easy and economical UVspectrophotometeric simultaneous assay method was developed where a sample of solution containing a mixture of two drugs were subjected to UV-spectroscopy at two analytical wavelength of 315 and 258 nm.Two simultaneous equations were developed.The individual concentrations of two drugs were calculated from those equations.The method was validated and various

Figure 6 .
Figure 6.Standard straight line of ItoHCl at two 315 and 258 nm.

Figure 7 .
Figure 7. Plot of recovered values (in X-axis) and actual values (in Y-axis).

Table 1 .
Preparation of standard solutions of pure drugs.

Table 2 .
Preparation of standard solutions containing both the drugs.

Table 3 .
Absorbances of pure drug solutions at 315 and 258 nm.

Table 4 .
Recovery of CX and CY.

Table 6 .
Determination of LOD and LOQ of RanHCl at 315 and 258 nm.

Table 7 .
Determination of LOD and LOQ of ItoHCl at 315 and 258 nm.