1. INTRODUCTION:

Rabeprazole sodium(RS) is chemically known as 2-({[4-(3-methoxy propoxy)-3-methyl-2-pyridinyl]-methyl}sulfinyl)-1H-benzimidazole sodium salt (Figure 1)¹. It is a latest proton pump inhibitor, which suppresses gastric acid secretion by inhibiting the gastric H+ K+ ATPase at the secretary surface of the gastric parietal cell². Literature survey revealed that various analytical methods such as estimation of rabeprazole sodium in tablet dosage form³, simultaneous determination of rabeprazole and domperidone in pharmaceutical dosage form⁴, development and validation of a dissolution test for rabeprazole sodium⁵ have been reported.

Itopride hydrochloride (IH) is chemically known as N–[4-[2-(dimethylamino)ethoxy]- benzyl]-3,4-dimethoxybenzamidehydrochloride (Figure 2)⁶. Itoride hydrochloride (IH) is a gastroprokinetic agent, which increases the release of acetylcholine (Ach) through Dopamine D2 receptor antagonistic action and inhibits decomposing of released Ach through its cetylcholinesterase inhibitory action, resulting in enhancement of gastrointestinal motility.

Few methods were reported for the estimation of itopride hydrochloride such as itopride hydrochloride and its metabolites in human serum and urine⁷ have been reported. Few simultaneous estimation methods of both the drugs in formulations^8,9,10,11 were also reported but they were found to show longer retention times.

Figure 1:- Structure of Rabeprazole Sodium

Figure 2:- Structure of Itopride Hydrochloride

2. EXPERIMENTAL:

2.1. Reagents:

Rabeprazole Sodium and Itopride Hydrochloride were obtained from Sigma Aldrich, USA. HPLC Grade Methanol was obtained from S.D. Fine Chemicals Ltd., India. HPLC Grade Sodium Dihydrogen Orthophosphate Dihydrate was purchased from Qualigens Fine Chemicals Ltd., Mumbai. The capsule dosage form (Rabee ISR,) was procured from the local market (Label claim: 20 mg of rabeprazole sodium and 150 mg of itopride hydrochloride) marketed by RPG Life Sciences Ltd., India.

Stock standard solutions of Rabeprazole Sodium and Itopride Hydrochloride were prepared by dissolving appropriate amounts of compounds in a known volume of methanol and phosphate buffer mixture.

2.2. Equipments:

Shimadzu HPLC (LC-10 AT VP) system; LC system used consist of pump (Model SHIMADZU; LC- 10 AT VP) with universal loop injector(Rheodyne 7725 i) of injection capacity 20 µL. Detector consists of UV-Visible SPD-10 AVP detector, SHIMADZU; the reverse phase column used was Phenomenex Luna C18 (5µM, 25 cm×4.6 mm I.D), USA.

2.2.1. Preparation and selection of mobile phase:

The preliminary isocratic studies on reverse phase C8 and C18 columns with different mobile phase combinations like acetonitrile and water, methanol and water, methanol and phosphate buffer were studied for simultaneous separation of both the drugs. The optimal composition of mobile phase was determined to be Methanol:Buffer (65:35 v/v) and the pH was found to be 4.3±0.1 and was filtered through 0.45 micron membrane filter.

2.3. Preparation of standard and sample solutions:

RS and IH (10 mg each) were weighed accurately and transferred to separate 100 mL volumetric flasks. Both drugs were dissolved in 100 mL mobile phase to prepare standard stock solution of 100 µg/mL. for analysis of capsule dosage form (Label claim: 20 mg of rabeprazole sodium and 150 mg of itopride hydrochloride); twenty capsules were weighed, with and without shell, their average weight was determined and the content was finely powdered and powder equivalent to weight of one third of the capsule was transferred to a 100 mL volumetric flask and dissolved in 50 mL mobile phase. The solution was shaken vigorously for 15 min and filtered through 0.45 µ filter paper.

2.4. Preparation of calibration curves:

Solutions of both drugs having different concentrations were prepared by dilution of the standard solutions. These solutions were chromatographed and peak areas were measured. Peak areas were then plotted against the respective concentrations for both RS and IH. From the plots it was found that the linear range of RS was between 2.5 and 15 µg/mL whereas that for IH was between 20 and 120 µg/mL. Unknown assay samples were quantified by reference to these calibration plots.

2.5. Assay of solid dosage form:

Six replicates of the required dilutions were prepared from capsule stock solution and sonicated for 15 min. These solutions were injected for quantitative analysis. The amounts of RS and IH per capsule were calculated by standard statistical procedures from the peak areas from the calibration plot. Results of analysis are reported in Table 1.

2.6. Recovery studies:

Recovery studies were carried out, by adding a known quantity of the standard drug to the sample before analysis and recovery studies were carried out at 80%, 100%, 120% level and the contents were determined from the respective chromatograms. The results were shown in table no.2. Results in table no.2 confirms that the method was accurate.

2.7. Ruggedness:

The ruggedness of the method was studied by changing the experimental condition such as,

· Changing to another column of similar type (Phenomenex C18)

· Different analyst performing the same method in the same laboratory.

Table 1:- Assay result for Replicate injections of samples

Inj .No	Area of Rabeprazole Sodium	Area of *Itopride HCl*	% of Rabeprazole Sodium recovered	% of Itopride HCl (mg)
1	237.909	1098.915	101.125	99.8177
2	236.731	1095.815	100.624	99.5361
3	232.714	1092.996	98.916	99.280
Mean	235.7847	1095.909	100.2216	99.5446
S.D	2.723723	2.417329	1.15816	0.268951
%R.S.D	1.15517	0.220577	1.15559	1.155594

Where S.D is Standard deviation and R.S.D is Relative standard deviation

Table 2:- Accuracy studies

S.I No:	Inj.Sample	Spike level	Amount Present	Amount Recovered	% Recovered
1	*Rabeprazole Sodium*	80 %	10mcg	9.9965	99.965 %
2		100 %	12.5mcg	12.5450	100.360%
3		120 %	15mcg	15.0818	100.545%
4.	*Itopride Hydrochloride*	80 %	80mcg	79.8032	99.754%
5		100 %	100mcg	99.6128	98.612%
6		120 %	120mcg	119.8875	99.906%

Table-3: Linearity Data for Rabeprazole Sodium and Itopride Hydrochloride

Concentration of *Rabeprazole Sodium* (mcg/ml)	Peak Area of *Rabeprazole Sodium*	Concentration of Itopride HCl (mcg/ml)	Peak Area of *Itopride HCl*
2.5	52.87	20	276.61
5	91.945	40	459.465
7.5	132.146	60	651.402
10	176.199	80	867.208
12.5	213.072	100	1044.58
15	260.255	120	1312.872

Table-4: System precision for Rabeprazole sodium and Itopride hydrochloride

S.N.O:	*Area of Rabeprazole Sodium* (mV)**	*Area of Itopride Hydrochloride* (mV)**
1	239.815	1094.647
2	240.759	1097.908
3	240.341	1097.488
4	239.82	1096.643
5	240.101	1096.239
Mean	240.1672	1096.585
S.D	0.396676	1.269317
%R.S.D	0.165167	0.115752

Table No: 5. Method Precision of Rabeprazole Sodium and Itopride Hydrochloride.

Sample No:	*Area of Rabeprazole Sodium* (mV)**	%Label Claim	*Area of Itopride HCl* (mV)**	%Label Claim
1	250.47	100.16	1112.696	99.6
2	250.62	100.10	1104.747	100.93
3	250.45	100.02	1097.256	100.42
4	249.147	100.05	1096.502	100.22
5	247.842	99.89	1098.266	100.8
Mean	249.7058	100.04	1101.893	100.394
S.D	1.199959	0.1011	6.141171	0.5276
%R.S.D	0.480549	0.101	0.557329	0.525

Table No: 6. System suitability Parameters

System Suitability Parameters	*Rabeprazole Sodium*	*Itopride Hydrochloride*
Resolution	10.992
Tailing Factor	1.160	1.560
Number of theoretical Plates	3013	2122
Retention time	4.587	1.660

3. RESULTS AND DISCUSSION:

The HPLC method was found to be simple, accurate, economic and rapid for routine simultaneous estimation of rabeprazole sodium and itopride hydrochloride in combined capsule dosage form at 285 nm. The slope, intercept and correlation coefficient values for Rabeprazole Sodium were found to be 16.93, 5.345 and 0.998 respectively. The slope, intercept and correlation coefficient values for Itopride Hydrochloride were found to be 10.50, 28.63 and 0.996 respectively. Results are reported in Table 3.

HPLC conditions were optimized to obtain an adequate separation of eluted compounds. Amongst the various mobile phases used, methanol: buffer in (65:35 v/v) was found robust with 1 mL/min. flow rate. Mobile phase and flow rate selection was based on peak parameters such as height, tailing, theoretical plates, capacity factor, run time, resolutions etc.

System precision and method precision studies were performed and the results of the studies were shown in table no 4 and 5.

Figure 3. Typical chromatogram of itopride hydrochloride and rabeprazole sodium in marketed formulation.

A typical chromatogram of rabeprazole sodium and itopride hydrochloride is shown in Figure 3. The optimum wavelength for detection was 285 nm at which detector response was obtained best. The average retention time for rabeprazole sodium and itopride hydrochloride was found to be 4.587 ±0.05 min. and 1.660 ±0.05 min. respectively. According to USP XXIV (621)12 system suitability tests are an integral part of chromatographic method. They are used to verify reproducibility of the chromatographic system. To ascertain its effectiveness system suitability tests were carried out and its results are shown in Table 6.

Hence it can be concluded that the developed RP- HPLC method is an accurate, precise and robust method and can be employed successfully for the estimation of RS and IH in formulation.

4. CONCLUSIONS:

The proposed high-performance liquid chromatographic method has been evaluated over the linearity, precision, accuracy, specificity and proved to be convenient and effective for the quality control of rabeprazole sodium and itopride hydrochloride in given application. The proposed validated procedure was found to be precise, accurate and linear over the concentration range tested (2.5–15.0 µg/mL for RS and 20-120 µg/mL) with a correlation coefficient better than 0.998. Thus, the proposed methodology is rapid, selective, requires a simple sample preparation procedure and represents a good procedure of rabeprazole sodium and itopride hydrochloride determination in pharmaceutical dosage forms.

5. REFERENCE:

1. Martindale, The Complete Drug Reference, 33rd Ed., Great Britain: The Bath Press, 2002.

2. Current Index of Medical Specialities - 77 [update -2] 2002.

3. Kulkarni V L and Mahulikar P P, Sci Ind Res., 2006, 65, 992.

4. Patel B H, Patel M M, Patel J R and Suhagia B N, J iq Chromatogr Relat Technol., 2007,30, 439.

5. Cassia V, Garcia C S, Paim M S and Elfrides E S, J pharm Biomed Anal., 2006, 41, 833-837.

6. Taisei M, Rika Douya Eiji T and Osamu N, Drug Metab Disps., 2000, 28,1231.

7. Takahara E, Fukuoka H, Takagi Nagata O and Kato H, J Chromatogr., 1992, 576,174-178.

8. Rajesh Sharma, Ganesh Prasad Mishra,Subash Chandra Chaturvedi.,

“Development and validation of RP-HPLC method for the simultaneous determination of Rabeprazole Sodium and Itopride Hydrochloride in solid dosage form” E-Journal of Chemistry, www.e-journals.net, 2010, 7(3),947-952.

9. Umamaheswari.D, M.Kumar, B.Jayakar, Rajesh Chatakonda., “Method development and validation of Itopride Hydrochloride and Rabeprazole Sodium in pharmaceutical dosage form by RP-HPLC” Journal of Chemical and Pharmaceutical Research, 2010, 2(5):399-417.

10. Pillai.S, Singhivi.I, “Quantitative estimation of Itopride Hydrochloride and Rabeprazole Sodium from capsule formulation” Indian journal of Pharmaceutical sciences,2008, vol.70, pp.658-661.

11. Gunasekaran.V, Maheshwari.S.D, “Validated simultaneous estimation of Rabeprazole Sodium and Itopride Hydrochloride in pure and pharmaceutical capsule formulation”, Analytical chemistry – An Indian Journal, Volume 5, issue 1-6, 2007.

Received on 10.04.2011 Modified on 13.05.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1077-1080