Development and Validation of HPLC Method for Simultaneous Estimation of Aceclofenac and Rabeprazole Sodium in Bulk and Capsule

Dosage Form

By Mr. DULAL MAHAVIR MOHATO

Under the guidance of Dr. P. S. WAKTE Reader

ISO 9001-2008 Certified UNIVERSITY DEPARTMENT OF CHEMICAL TECHNOLOGY, Dr. BABASAHEB AMBEDKAR MARATHWADA UNIVERSITY, AURANGABAD. 2010-2011

SCOPE OF PRESENTATION
       

Introduction Review of Literature Objective Materials and Methods Results & Discussion Conclusion Future Scope References

INTRODUCTION

Analytical Chemistry

Qualitative Analysis

Quantitative Analysis

Structural Analysis

Table 1: Classification of Analytical Instrumental Techniques

Spectrophotometry
UV-Visible Spectrophotometry Fluorescence and phosphorescence Spectrophotometry

Electrochemical
Potentiometric Voltammetry

Chromatographic
Gas chromatography (GC) High performance liquid chromatography (HPLC)

Hyphenated
LC-MS GC-MS

Atomic (emission & adsorption) Spectrophotometry Raman Spectrophotometry X-Ray Spectrophotometry Nuclear Magnetic spectroscopy Mass spectroscopy

Electrogravimetry

High performance thin layer chromatography (HPTLC) Column Chromatography Ion Chromatography Super Critical Fluid Chromatography Flash Chromatography

LC-IR

Conductometry Amperometry Colorimetry Stripping techniques

GC-IR LC-NMR HPTLC-MS HPTLC-IR

Analytical Method Development and Validation

Introduction
 Plays important roles in the discovery, development and manufacture of pharmaceuticals.  Used by Quality control laboratories to ensure the identity, Purity, Potency and performance of drug products.  Objective of validation is to demonstrate that, procedure is suitable for its intended purpose.  Guidelines from USP, WHO, ICH, FDA provides a framework for performing validation.  Achieving acceptance of products by the international agencies.  Mandatory requirement for registration of any pharmaceutical product.

Basic Criteria for New Analytical Method Development

 The drug or drug combination may not be official in any pharmacopoeias.  Analytical procedure not available in the literature due to patent regulations.  Analytical methods for the quantitation of the drug in biological fluids may not be available.  Analytical methods for a drug in combination with other drugs may not be available.  The existing analytical procedures may require expensive reagents and solvents.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Fig 1: Block Diagram of HPLC System

General Methodology for HPLC Method Development

Information on sample, define separation goal Need for special HPLC procedure, sample pretreatment, etc Choose detector and detector settings Choose LC method, preliminary run Estimate best separation conditions Optimize separation conditions Check for problems or requirement for special procedure Validation for release to routine laboratory

Table 2: Preferred Experimental Condition for the Initial HPLC Separation

Sr. No
1. 2.

Separation variable
Column Mobile phase

Preferred initial choice

15x46 mm, 5m, C8 or C18 ACN- H2O (neutral samples) or ACN-Buffer (ionic samples) (80-100%) Buffers: 25-50mM Potassium Phosphate at pH 2-3

3. 4 5.

Flow rate Temperature Sample Size

1.5-2.0 mL/ min 35-45 0C Volume: < 25L and Weight: < 100 g

Table 3: Separation Goals in HPLC Methods

Sr. No
1. 2. 3. 4. 5.

Goals
Resolution Separation time Quantitation Pressure Solvent consumption

Comment
Precise and rugged quantitative analysis requires that Resolution be greater than 1.5 < 5-10 min is desirable for routine procedure 2 % (1 SD) for assay; 5 % for less demanding analysis; 15 % for trace analysis < 150 bar is desirable for large signal/ noise ratios Minimum mobile phase use per run is desirable

Analytical Method Validation

 Incident like the thalidomide tragedy forced rethinking of the concept of product testing as a sole and sufficient indicator of product quality.   Validation has thus became a regulatory requirements in 1978. Analytical validation ensures that selected analytical method will give reproducible and reliable results adequate for intended purpose.

Key Parameters
       Accuracy Precision Linearity and Range Limit of Detection and Limit of Quantification Selectivity and Specificity Robustness and Ruggedness Stability and System Suitability Test

REVIEW OF LITERATURE

Table 4: Drug Profiles of Rabeprazole Sodium and Aceclofenac

Drug Chemical Name Rabeprazole Sodium (RS)-2-([4-(3-methoxypropoxy)-3-methylpyridin- 2-[2-[2-[(2,62-yl]methylsulfinyl)-1H benzo[d]imidazole. dichlorophenyl)amino]phenyl]acetyl]oxy acetic acid Molecular Formula C18H20N3NaO3S C16H13Cl2NO4 Aceclofenac

Structure

Molecular weight Appearance

381.42 white to slightly off white crystalline powder

354.2 white powder or almost white crystalline

Solubility Storage Dosage Mechanism of Action Indication and usage

Water, Methanol, Ethanol 15-30 0C. Keep away from moisture. 20mg daily, tablet or capsules Proton-pump inhibitors Duodenal ulcer, Gastric ulcer

Acetone, Methanol, Ethanol Room temperature. 100 mg twice daily, tablet or capsules. Inhibitor of cyclooxygenase Rheumatoid arthritis, Osteoarthritis

Literature Survey
Rabeprazole Sodium
         Spectrophotometry Polarography Anodic voltammetry Spectrofluorometry RP-HPLC Chiral HPLC HPTLC LC-MS Super Critical Fluid Chromatography     

Aceclofenac

  

Spectrophotometry Polarography Adsorptive Stripping Voltammetry Spectrofluorometry RP-HPLC HPTLC QqLIT-MS QqTOF-MS

OBJECTIVE

Need of Present Study

 A new fixed dose combination of Aceclofenac and Rabeprazole Sodium is available in market as , ALRADAY (Ranbaxy Ltd.) and ZIX-R OD (Jenburkt Ltd.) Capsules.  The review of the literature revealed that no method yet reported for the simultaneous estimation.

Objectives
 To develop a simple, specific, accurate, precise and economic HPLC method.  To study Validation parameters on HPLC method as per ICH guidelines.  To develop HPLC method suitable for the quality control of the raw materials, formulations and dissolution studies.

MATERIALS & METHODS

Working standards & Chemicals

Chemicals
Methanol Water Aceclofenac (ACECLO) Rabeprazole (RABE) ZIX-R OD Capsule (20 mg RABE and 200 mg ACECLO)

Grade
HPLC HPLC Pharmaceutical Pharmaceutical Pharmaceutical

Supplier
Rankem, Mumbai Milli Q Wockhardt, Ltd., Aurangabad Wockhardt, Ltd., Aurangabad JENBURKT Ltd., Mumbai

Instrument
QUATERNARY HPLC SYSTEM (JASCO)  Pump (Jasco-PU-2089 plus)  Detector (Jasco-UV-2075 plus)  Connecting Module (Jasco LC NetII/ADC)  Vaccum Degasser  Rheodyne Injector with 20 L loop  Column (Grace Smart RP C18 5m, 250 mm 4.6 mm)

Method Development
 Selection of common solvent  Wavelength selection  Flow rate selection  Peak identification  Method optimization  Standard solution preparation  Construction of calibration curves  Sample solution preparation  Assay of sample

Method Validation
 Linearity and Range  Selectivity and Specificity  Limit of Detection and Limit of Quantification  Precision System repeatability Intra and Inter-day precision  Accuracy  Robustness and Ruggedness  Stability and System Suitability Test

Application of proposed method for analysis of capsule formulation

To determine the content of RABE and ACECLO simultaneously in capsules

    

Twenty capsules were uncapped and content of capsules were finely powdered in glass mortar pestle. Powder equivalent to 20 mg RABE and 200 mg ACECLO was weighed Final concentration of 3 g /ml of RABE and 30 g /ml of ACECLO was prepared Sample solution was assayed using developed HPLC method. % Assay of RABE and ACECLO was determined.

RESULTS & DISCUSSION