An overview on HPLC method
development
Arnab Dey
M. Pharm Pharmaceutical Analysis
1st Year, 2nd Semester
Roll – 18920724014
Date - 13/05/25
Principle of HPLC
The separation principle in both normal-phase and reverse-phase HPLC modes
is based on adsorption.
When a mixture is injected into the HPLC column, the components move at
different rates depending on their relative affinities for the stationary phase.
Components with higher affinity for the adsorbent move more slowly, while those
with lower affinity travel faster.
Fig : HPLC
HPLC Method Development
Developing and validating analytical methods are essential steps in the discovery,
development, and production of pharmaceuticals.
These methods are employed to confirm the identity, purity, strength, and effectiveness of
pharmaceutical products.
Most of the analytical development work focuses on validating an HPLC method to indicate
stability.
The HPLC method aims to separate and quantify the primary active drug, any reaction
impurities, all accessible synthetic intermediates, and any degradants.
Need of HPLC Method Development
The factors driving the development of new drug analysis methods are:
When a drug or drug combination is not available in the pharmacopoeias.
When there are no analytical methods available for the drug formulation due to
interference from the formulation excipients.
Analytical methods for quantifying the analyte in biological fluids are
unavailable.
Steps of HPLC method development
Steps in HPLC method development include:
Identifying the physicochemical properties of drug molecules.
Selecting chromatographic conditions.
Developing an analytical approach.
Preparing sample .
Optimizing the method.
Validating the method.
1. Identifying the physicochemical properties of drug molecule:
• The physicochemical properties of drug molecule play a crucial role in method
development.
• To design an effective method, it is essential to first examine the drug’s physical
characteristics, including solubility, polarity, pka, and pH. Polarity is a physical property of
a compound that helps an analyst determine the appropriate solvent and mobile phase
composition.
• Selecting an appropriate pH for ionizable analytes often produces sharp and
symmetrical peaks in HPLC.
2. Selecting Chromatographic Conditions:
Selection of column
Selection of chromatographic mode
Buffer selection
Effect of pH
Effect of organic modifier
Selection of detector and wavelength
3. Developing an analytical approach:
The initial step in developing an analytical method for RP-HPLC involves selecting key
chromatographic parameters, including the mobile phase, column, mobile phase flow rate,
and the pH of the mobile phase.
These parameters are determined through a series of trials and are subsequently evaluated
against system suitability criteria.
Common system suitability parameters include a retention time exceeding 5 minutes, a
theoretical plate count greater than 2000, a tailing factor below2, a resolution above 5, and
a percent R.S.D. of analyte peak areas in standard chromatograms not exceeding 2.0%.
4. Preparing Sample:
• Sample preparation plays a crucial role in HPLC analysis, ensuring the solution is
consistent and uniform for injection onto the column.
• The primary objective is to produce a sample aliquot that is free of significant
interferences, does not harm the column, and is compatible with the chosen HPLC
method.
5. Optimizing the method:
• Identify the method’s limitations and refine it using experimental design.
• Evaluate its performance under varying conditions, with different instrument
configurations, and across diverse sample types.
6. Method Validation:
The ICH recommend the following standard parameters for method development:
1. Range
2. Linearity
3. Specificity
4. Accuracy
5. Precision
6. Solution stability
7. Limit of detection (LOD)
8. Limit of quantification (LOQ)
9. Robustness
10. System suitability
Reference
Vidushi Y, Meenakshi B, Bharkatiya M. A review on HPLC method development
and validation. Res J Life Sci, Bioinform, Pharm Chem Sci. 2017;2(6):178.
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