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Analytical Method Development And Validation For Simultaneous Estimation Of Meclizine And Caffeine In Their Tablet Dosage Form By Rp-Hplc


BHANU PRATAP SINGH *, R.P.S.RATHORE
Department of Quality Assurance , Bhupal Nobles’ College Of Pharmacy Udaipur  India

 

Abstract

Today, HPLC continues to evolve rapidly toward higher speed, efficiency, and sensitivity, driven by needs of life sciences and pharmaceutical applications.  Pharmaceutical products formulated with more than one drug, typically referred to as combination products the development and validation of analytical methods. There are different modes of separation in HPLC Normal phase mod, Reversed phase mode, Ion exchange chromatography, Reverse phase ion pair chromatography, Affinity chromatography and Size exclusion chromatography. Spectrophotometric, High performance liquid chromatography (HPLC) & High performance thin layer chromatography (HPTLC)] for drug products containing more than one active ingredient. This method of analysis is gaining importance due to simple, rapid, precise, highly accurate and less time consuming The spectrophotometric analysis of drugs rarely involves the measurement of absorbance of sample containing only one absorbing component. Liquid chromatography (LC) is a physical separation technique conducted in the liquid phase. High-performance liquid chromatography (HPLC), sometimes called high-pressure liquid chromatography. Normal phase mode of separation is therefore, not generally used for pharmaceutical applications because most of the drug molecules are polar in nature and hence take longer time to elute.

Key word:  HPLC, Drug, analytical, mode of searation, chromatography, spectrophotometric.

INTRODUCTION

Historical Aspect

Classical Liquid Chromatography, the term chromatography meaning “color writing,” was first discovered by Mikhail Tswett, a Russian botanist who separated plant pigments on chalk (CaCO3) packed in glass columns in 1903. Since the 1930s, chemists used gravity fed silica columns to purify organic materials and ion-exchange resin columns to separate ionic compounds and radio nuclides.In the late 1960s, Liquid Chromatography turned “high performance” with the use of small-particle columns that required high pressure pumps.

The first generation of HPLC was developed by researchers in the 1960s, including Horvath Kirkland, and Huber. Commercial development of in-line detectors and reliable injectors allowed HPLC to become a sensitive and quantitative technique leading to an explosive growth of applications. In the 1980s, the versatility and precision of HPLC rendered it virtually indispensable in pharmaceuticals as well as other diverse industries.

the emerging Today, HPLC continues to evolve rapidly toward higher speed, efficiency, and sensitivity, driven by needs of life sciences and pharmaceutical applications[1].

Figure 1 depicts the classical technique of Liquid Chromatography with a glass column that is packed with coarse adsorbents and gravity fed with solvents.Fractions of the eluent containing separated components are collected manually. This is contrasted with the latest computer-controlled HPLC operated at high pressure and capable of very high efficiency.

Fig.1: The traditional technique of low-pressure liquid chromatography using a glass    column and gravity-fed solvent with manual fraction collection, A modern automated HPLC instrument capable of very high efficiency and pressure1.

Analytical Method Development

Pharmaceutical products formulated with more than one drug, typically referred to as combination products. These combination products can present daunting challenges to the analytical chemist responsible for the development and validation of analytical methods. Development and validation of analytical methods [Spectrophotometric, High performance liquid chromatography (HPLC) & High performance thin layer chromatography (HPTLC)] for drug products containing more than one active ingredient. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products.

The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing ones. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs [1.2.3.]

MATERIALS & METHOD

Equipments

Method development and validation for estimation of Meclizine and Caffeine as active pharmaceutical ingredients and ointment dosage forms following materials are use

Table 1: Detail of instruments used

SR. No. Instruments Specification and Manufaturer
1 Double Beam UV-Visible Spectrophotometer VU-Visible 1700,

Shimadzu Limited

2 HPLC SPD-20AT, Shimadzu Limited
3 Analytical balance AX-200, Precisa Limited
4 pH Meter Chemiline, India
5 Ultra Sonic Cleaner Toshcon, Toshniwal Process Instrument Pvt. Ltd., Ajmer

Table 2: System Specification of HPLC (SPD-20AT)

SR. No Name Specification
1 Pump LC-20AT
2 Detector SPD-20AT
3 Column BDS Hypersil C18, 250mm X 4.6 mm, 5µ(particle size), Thermo scientific
4 Injector Rheodyne injector (20µl Capacity)
5 Syringe Hamilton (25µl)
6 Chromatographic Software Spinchrom

Table 3: Detail of chemicals used

SR. NO Chemicals Grade Manufactirer
1 Acetonitrile HPLC Merck, Rankem
2 Potassium Dihydrogen Phosphate AR Merck, Rankem
3 Water HPLC HPLC Grade
4 Orthophosphoric Acid AR Merck, Rankem
5 Tri Ethyl Amine AR Merck, Rankem
6 Methanol HPLC Merck, Rankem

MARKET FORMULATION USED; PREGNITOXIN

Meclizin…………25mg

Caffeine………… 20mg

RESULT

 In the present work a simple, accurate, rapid, specific, sensitive and selective reverse phase HPLC method was developed for simultaneous estimation of Meclizine and caffeine in their combined dosage forms and Developed Method was validated.

Trail 16
Mobile phase: Buffer-Methanol (70:30) pH 3.5
Conditions:
Column: BDS Hypersil C 18 , 250 mm X 4.6 mm, 5 µ(partical size),Thermo scientific
Injection volume :  20 ul
Column temperature: 250C
Sample temperature: 250C
Flow rate : 1 ml/min
λ  max: 226 nm

Figure: 2 Pure Peaks

Buffer-Methanol (70:30) pH 3.5
Retention Time was found to be 4.227 for Meclizine and 5.413 for Caffeine.

Validation Parameters:

Assay

The results of assay were found to be 100±0.64 and 100±0.25 for Meclizine and Caffeine respectively The result of assay was found close to 100%.

Linearity

The linearity was observed in the range of 10-30 and 0.5-1.5 μg/ml with Correlation Coefficient, r = 0.999 for Caffeine. The linearity was observed in the range of 12.5-37.5 and 0.5-1.5 μg/ml with Correlation Coefficient, r = 0.999 for Mclizine.

Precision

The result of interday and intraday precision were found to be satisfactory within limit of % RSD 2 %

Recovery

The accuracy (% recovery) was found to be 99.17-100.63 % for Caffeine and 100.59-102.29% for Meclizine .

Repeatability

The result of repeatability were found to be within range of % RSD 2%

Robustness

. The result of robustness were found to be satisfactory within range of  %RSD 2% by deliberating variation in method parameters i.e. Floe rate, Mobile Phase, pH.

CONCLUSION

Analytical methods were attempted to developed and validated for Simultaneous estimation of Meclizine and Caffeine Tablet dosage form by RP-HPLC. The Trail 16 Works found to be accurate & reliable comparative to remaining trails. This trail 16 was used for Validation.A reverse phase liquid chromatography (LC) method was developed and validated for simultaneous estimation of Meclizine and Caffeine  in Tablet Formulation. The isocratic LC analysis was performed on Hypersil BDS C18 column (250 mm x 4.6 mm ) using mobile phase composed of  Buffer: Methanol pH 3.5 (70:30, v/v) at a flow rate of 1.0 ml/min. Quantitation was performed using UV detector at 226 nm. The retention times were found to be 4.2 min for Meclizine and 5.4 min for Caffeine. The analytical method was validated according to ICH guidelines The linearity was observed in the range of 10-30 and 0.5-1.5 μg/ml with Correlation Coefficient, r = 0.999 for Caffeine. The linearity was observed in the range of 12.5-37.5 and 0.5-1.5 μg/ml with Correlation Coefficient, r = 0.999 for Mclizine. The results of assay were found to be 100±0.64 and 100±0.25 for Meclizine and Caffeine respectively The result of assay was found close to 100%.  The relative standard deviation values for repeatability and intermediate precision studies were less than 2%. The method was successfully applied for market sample analysis and mean percentage assay values were 99.17-100.63 % for Caffeine and 100.59-102.29% for Meclizine. The present method is precise and accurate and can be used for the routine estimation of Meclizine and Caffeine in tablet formulation.

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