Development and Validation of RP-HPLC-Dad Method for Estimation of Aceclofenac and Rabeprazole Sodium in Bulk and Combined Dosage Form

R. N. Jinde*, V.D. Chipade, V. A. Shinde, M.D. Kshirsagar A.V. Chandewar

Department of Quality Assurance, P. Wadhawani College of Pharmacy, Yavatmal-445001, Maharashtra, India.

*Corresponding Author E-mail: vaibhavchipade@gmail.com

ABSTRACT:

The present work describes a validated reverse phase high performance liquid chromatographic method involved with diode array detection for simultaneous estimation of Rabeprazole sodium (RAB) and Aceclofenac (ACL) in combined dosage form. Chromatographic separation was performed on Prontosil C18(4.6 id x 250 mm 5µm particle size) Column with mobile phase consisting methanol: water pH 2.5 (75:25 v/v) .The flow rate was 1.0 ml/min and eluents were monitored at 273nm by using Photo Diode Array(PDA) detector at ambient temperature. The separation of both drug were found effectively on 3.23min of RAB and 7.78min of ACL with total run time upto10 mins. The proposed method was found to be simple as was involved with water as mobile phase component it was fast, accurate, precise, reproducible, linear, robust and rugged which have been further used for simultaneous analysis of RAB and ACL in combined pharmaceutical formulations.

KEYWORDS: Rabeprazole sodium , Aceclofenac, Reversed phase- HPLC, PDA

INTRODUCTION:

Rabeprazole is chemicaly 2-[[4-(3-methoxypropoxy)-3-methyl- pyridine yl]methylsulfinyl]-1H-Benzoimidazole Sodium salt .It is a substituted benzimidazole that inhibits gastric acid secretion and primarily used in the treatment of Ulcerative Gastroesophageal Reflux Disease (GERD). it is Rabeprazole belongs to a class of antisecretary compounds that suppress gastric acid.[2-4]

Figure 1. Chemical structure of Rabeprazole sodium

ACL is chemically [2-[(2, 6 dichlorophenyl) amino] phenyl acetoxy acetic acid a non-steroidal anti- inflammatory drug.

It has potent analgesic and anti-inflammatory properties and an improved gastrointestinal tolerance.

It is used for the relief of pain and inflammation rheumatoid arthritis.[5-7]

Figure 2. Chemical structure of Aceclofenac

Several method were reported for estimation of Aceclofenac or its derivatives with combination of several drugs by using UV-spectrophotometry[8], HPTLC[9,10], derivative spectrophotometry[11], assay using RP-HPLC[12-17].

EXPERIMENTAL:

Instrumentation:

The HPLC system consisted of Waters ^TM600E Controller system, Waters 996 PDA Detector and Water In-line Degasser AF. Data acquisition was performed by the Empower software. Analysis was carried out at 273 nm with Prontosil C18 (4.6(id) x 250mm, 5 µm partical size,) or equivalent column, using a mixture of Methanol: Water pH 2.5 (75:25 v/v) maintained by Orthophosphoric acid(OPA) and Triethylamine(TEA)( 0.02%) as the mobile phase using a low pressure gradient mode with flow rate at 1.0 ml/min .The mobile phase was degassed and filtered before pumping into HPLC system.

Reagents and Chemicals:

Methanol HPLC grade was procured from Spectrochem Pvt. Limited, Mumbai .Water of HPLC grade by LOBA CHEMIE Pvt. Ltd .Mumbai. Reference standards of RAB and ACL were provided by RANBAXY Pharmaceuticals Ltd, Nagpur. OPA was procured from RANBAXY Fine chemicals ltd. New Delhi. TEA procured from LOBA CHEMIE Pvt. Ltd .Mumbai. Handy pH meter was used to modify pH belonging to HANNA.

Chromatographic conditions:

Prontosil C18 column (250 mm x 4.6 mm (i.d.), 5 μm particle size) was used at ambient temperature. The mobile phase consisted of Methanol : Water(pH 2.5) (75:25 v/v) at a flow rate of 1.0 ml/min. The mobile phase was filtered through nylon 0.45 μm membrane filter (47mm diameter) and degassed before use. The elution was monitored at 273 nm, and the injection volume was 20 μl.

Preparation of solutions:

Preparation of mobile phase

Methanol and water in the ratio of 75:25 v/v were employed as a mobile phase .Water was sonicated after maintaining the pH by OPA and TEA upto 2.5 .

Rabeprazole sodium and Aceclofenac standard stock solution

Accurately weighed RAB (10 mg) and ACL (10 mg) were transferred to two separate 10 ml volumetric flask. 10 ml methanol was added to the flask. The drug was dissolved with sonication and the final volume was adjusted with methanol up to the mark to prepare a 1000µ g/ml stock solution of both drugs.

Rabeprazole sodium and Aceclofenac working standard solution(100µg/ml):

From the above stock solutions (1000µ g/ml) of both drugs, an accurately measured 1.0 ml volume of the stock solutions were transferred into separate 10 ml volumetric flasks and the final volume were adjusted with methanol to prepare 100 µg/ml working solutions.

Determination of wavelength of maximum absorbance:

The standard solutions of RAB and ACL were scanned in the range of 200 -400 nm against mobile phase as a blank. RAB and ACL showed maximum absorbance at 273 nm. So the wavelength selected for the determination of RAB and ACL was 273 nm.

Method Validation [1]:

Accuracy and Linearity plot:

Calibration curves were plotted over a concentration range of 50-150 μg /ml for RAB and ACL. Accurately measured standard stock solutions of RAB and ACL (0.5, 0.75, 1.0,1.25 and 1.5ml) were transferred to a series of 10 ml volumetric flasks and the volume in each flask was adjusted to 10 ml with methanol. The resulting solutions were injected into the column and the peak area obtained at flow rate of 1.0 ml/min for RAB and ACL respectively at 273 nm.

Estimation of Rabeprazole sodium and Aceclofenac in capsule dosage forms:

20 Capsule granules were weighed to obtain the average Capsule granules weight. A sample of the claimed granules to contain 20 mg of RAB and 200mg of ACL active ingredients , was mixed with 20 ml of methanol. The mixture was allowed to stand with intermittent sonication to ensure complete solubility of drug. Further the resulting solution was passed through 0.45 μm membrane filter . An aliquote of this solution (1 ml) was transferred to a 10ml volumetric flask and made up to a sufficient volume with methanol to get 100µg/ml of RAB and 1000µg/ml of ACL concentration.

RESULTS AND DISCUSSIONS:

Method development:

Methanol and water in the ratio of 75:25 v/v were employed as a mobile phase .The present RP – HPLC method for the quantification of RAB and ACL in bulk and pharmaceutical dosage forms, revealed as simple, accurate and precise method with significant retention time of 3.233 min of RAB and 7.78 min of ACL . The typical chromatograms of RAB and ACL was shown in Fig.3

Fig.3.Typical Chromatogram of Rabeprazole sodium and Aceclofenac

Method Validation:

Method precision:

The intraday and inter-day variations of the method were determined using five replicate injections of three concentrations and analysed on the same day and three different days over a period of two weeks. Results were shown in Table–1.

Accuracy (% Recovery):

Accuracy of the method was assured by use of the standard addition technique, involving analysis of formulation samples. The results obtained for both drugs were compared to those expected. The recovery experiments were carried out in triplicate by spiking previously analyzed samples of the tablets (50%,100%,150%) with three different concentrations of standards (RAB 100,150,200 μg/ml and ACL 1000,1500,2000μg/ml). Relative standard deviation of all the parameters was less than 2%, which indicates that the proposed method is repeatable.Table no. 2

Table 2 : accuracy of Rabeprazole sodium and Aceclofenac

Drug	% recovery	%RSD
Rabeprazole sodium	100.43	1.039
Aceclofenac	99.83	0.67

System suitability:

To know reproducibility of the method system suitability test was employed to establish the parameters such as tailing factor, theoretical plates ,LOQ and LOD.

Table 3 : System suitability of Rabeprazole sodium and Aceclofenac

Suitability Parameters

Observation

Rabeprazole sodium

Aceclofenac

Retention time (min.)

Theoretical Plates

Tailing factor

Linearity Range (μg/ml)

Relative standard deviation (%RSD)

3.231

2187.13

1.59

10-50

1.63

7.707

2241.6

2.0

10-50

0.6385

Ruggedness:

Ruggedness of the method (intermediate precision) was estimated by preparing five dilutions of the RAB and ACL. The results were shown in Table-4.

Table 4 : Ruggedness of Rabeprazole sodium and Aceclofenac

Drugs	%Label Claim
Drugs	Rabeprazole sodium	Aceclofenac
Mean	99.89	100.49
%RSD	2.0	0.788

Robustness:

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters (+10% flow rate, +0.1 unit pH, +10% organic mobile phase) and provides an indication of its reliability during normal usage.

Specificity:

The ICH document defines specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. For the current method the blank chromatogram do not exhibited any interferences.

Analysis RAB and ACL in combined dosage forms:

Pharmaceutical formulation (Altraday capsule) of RAB and ACL was purchased from local pharmacy. The responses of formulations were measured at 273 nm for quantification of RAB and ACL by using RP-HPLC. The amounts of RAB and ACL present in sample solution were determined. Results are given in Table 6.

CONCLUSION:

The results of the study reveal that the proposed RP-HPLC method for the estimation of RAB and ACL is simple as it involves water as component of mobile phase and not involving any buffer which is considered to be disadvantageous for column life. The method was accurate, precise and linear and was successfully applied for estimation of RAB and ACL in bulk and combined dosage form.

Table 5 : Robustness of Rabeprazole sodium and Aceclofenac

Sr. No	System sutability parameter		observation
			As such	flow		Organic phase		pH		Limit
			As such	+10%	-10%	+10%	-10%	+0.1unit	-0.1unit	Limit
1.	The % RSD of peak area response for five replicate injections	RAB	1.6	0.06	0.97	1.3	1.56	0.97	0.72	NMT 2.0
1.		ACL	0.6	0.21	0.04	1.8	1.7	1.42	1.57	NMT 2.0
2.	Theoretical plates	RAB	2184	2231	2168	3206	2193	3187	3174	NLT 2000
2.	Theoretical plates	ACL	2241	2218	3177	2236	3191	2264	2245	NLT 2000
3.	Tailing factor	RAB	1.5	1.135	1.3	1.11	1.37	1.43	1.59	NMT 2.0
3.	Tailing factor	ACL	2	1.65	2	1.38	1.9	1.7	1.95	NMT 2.0

Sr. No.	Rabeprazole sodium and		Aceclofenac
Sr. No.	Assay(mg)	Assay%	Assay(mg)	Assay%
Mean	197.93	98.69	198.65	99.32
%RSD	0.086	0.089	0.257	0.259

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Received on 25.11.2012 Modified on 18.12.2012

Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 162-165

Drugs	Observed Concentration*
Drugs	System precision Area Response	%RSD	Method precision Area Response	%RSD
Rabepr azole sodium	90572	0.97318	90108	0.080
Aceclofenac	18735739	2.098061	1845607	1.187