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Rituraj Singh Chundawat*, Yuvraj Singh Sarangdevot, Bhupendra Vyas, Gajendra Singh Rathore, Udaibhan Singh Rathore, Pankaj Sharma
Department of Quality Assurance, Bhupal Nobles’ College of Pharmacy,
Udaipur- 313002, Rajasthan, India

Pharmaceutical analysis may be defined as a process or a sequence of processes to identify and/or quantify a substance or drug, the components of a pharmaceutical solution or mixture or the determination of the structures of chemical compounds used in the formulation of pharmaceutical product. HPLC is the most rapid and reliable analytical technique for analysis of drugs. Its simplicity, high specificity and wide range of sensitivity makes it ideal for analysis of many drugs in both dosage forms and biological fluids. Montelukast is a CysLT1 antagonist which blocks the action of leukotriene D4 (and secondary ligands LTC4 and LTE4) on the cysteinyl leukotriene receptor CysLT1 in the lungs and used in asthma. Fexofenadine competes with free histamine for binding at H1-receptors in the GI tract, large blood vessels, and bronchial smooth muscle. It blocks the action of endogenous histamine and relieves from allergy. The analytical method development of Montelukast and Fexofinadine combination in pharmaceutical dosage form is done by using HPLC method.


1.1 Introduction to Drug Analysis

Pharmaceutical analysis is a branch of practical chemistry that involves a series of process for identification, determination, quantification and purification of a substance, separation of the components of `a solution or mixture, or determination of structure of chemical compounds. The substance may be a single compound or a mixture of compounds and it may be in any of the dosage form. The substance used as pharmaceuticals are animals, plants, micro organisms, minerals and various synthetic products.  Analytical determination is based on the measurement of some physical, Chemical or structural properties which are related directly or indirectly to the amount of desired constituent present in the sample. Drugs or pharmaceuticals are chemicals which are of organic, inorganic or other origin. Whatever may be the origin, some property of the medicinal agent is used to measure them qualitatively and quantitatively.e.g. Gravimetric analysis, Acidimetry and Alkalimetry Methods.

1.2 Introduction to selection of Analytical Method
A vital first step in any quantitative analysis is the selection of method. This will require    careful consideration of following criteria.

  • The type of analysis required.
  • Problems arising from the nature of material to be investigated.
  • Possible interference from components of the material other than those of interest.
  • The concentration range, which needs to be investigated.
  • The accuracy required.
  • The facilities available.
  • The time required to complete the analysis.

Finally, the choice of method is always governed by complexity of sample as well as number of components in the samples.

Analytical methods are generally classified into two categories

  • Instrumental
  • Non-instrumental

1.3 Analytical Method Development
1.3.1 Introduction

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 one. 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.

1.3.2 Basic criteria for new method development of drug analysis:

  • The drug or drug combination may not be official in any pharmacopoeias,
  • A proper analytical procedure for the drug may not be available in the literature due to patent regulations,
  • Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients,
  • 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. It may also involve cumbersome extraction and separation procedures and these may not be reliable.

1.4 Introduction to HPLC
1.4.1 Introduction

HPLC is fastest growing analytical technique for analysis of drugs. Its simplicity, high specificity and wide range of sensitivity makes it ideal for analysis of many drugs in both dosage forms and biological fluids.

HPLC is presently used in pharmaceutical research and development in following areas:
1) To purify synthetic or natural products.
2) To characterize metabolites.
3) To assay active ingredients, impurities, degradation products and in dissolution studies.
4) In pharmacokinetic and pharmacodynamic studies.

Most of the drugs in multi-component dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC method eliminates tedious extraction and isolation procedures. Some of the advantages are:

  • Speed (analysis can be accomplished in 20 minutes or less),
  • Greater sensitivity (various detectors can be employed),
  • Improved resolution (wide variety of stationary phases),
  • Reusable columns (expensive columns but can be used for many analysis),
  • Ideal for the substances of low volatility,
  • Easy sample recovery, handling and maintenance,
  • Instrumentation tends itself to automation and quantitation (less time and less labour),
  • Precise and reproducible,
  • Calculations are done by integrator itself.

1.4.2 Mode of Separation
There are different modes of separation in HPLC. They are normal phase mode, reversed phase mode, reverse phase ion pair chromatography, affinity chromatography. Normal phase mode, the stationary phase is polar and the mobile phase is nonpolar in nature. In this technique, nonpolar compounds travel faster and are eluted first. Reversed phase mode is the most popular mode for analytical and preparative separations of compound of interest in chemical, biological, pharmaceutical, food and biomedical sciences. The polar compound gets eluted first in this mode and nonpolar compounds are retained for longer time. As most of the drugs and pharmaceuticals are polar in nature, they are not retained for longer times and hence elute faster.

1.5 Method Development in HPLC
A typical method development scheme has the following steps:
1. Choose the mode
2. Choose the column and column packing dimensions
3. Choose the stationary phase chemistry
4. Choose the mobile phase solvents
5. If the mode requires it, adjust the mobile phase pH
6. Run some initial isocratic or gradient experiments to define boundary conditions
7. Optimize the experimental conditions

1.6 Method Optimization
The aim of this step is to achieve adequate selectivity (peak spacing). The mobile phase and stationary phase compositions need to be taken into account. To minimize the number of trial chromatograms involved, only the parameters that are likely to have a significant effect on selectivity in the optimization must be examined. For the final method optimization following parameters should be considered.

  • Shorter analysis time
  • Better Resolution
  • Better selectivity
  • Better Sensitivity

After method optimization, system parameter optimization also considered. This is used to find the desired balance between resolution and analysis time after satisfactory selectivity has been achieved. The parameters involved include column dimensions, column-packing particle size and flow rate. These parameters may be changed without affecting capacity factors or selectivity.

To develop an HPLC method for simultaneous estimation of Montelukast and Fexofenadine by HPLC.

  • Literature assessment showed that various analytical methods has been reported for the estimation of Montelukast and Fexofenadine individually and with combination also but combination does not fulfill regulatory requirement and with other drugs combination by HPLC in pharmaceutical dosage form.


Method Development:

· Study of physicochemical parameters.

-  Solubility

-  Maximum Absorbance

-  Molecular Weight

· Selection of Suitable Stationary Phase.

-  Normal Phase

-  Reversed Phase

·  Selection of Suitable Mobile Phase Depending Upon Polarity

·  Selection of Appropriate Columns.

·  Selection of Proper Temperature.

·  Selection of Proper Flow Rate.

·  Selection of Detection Wavelength.

·  Selection of Sampling Preparation.



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