How to Develop HPLC Method?

What is the Method of Development?

Method development measures the concentration of active pharmaceutical ingredients (API) in a dosage form. During the process of developing your drug, method development processes can also determine the multitude of constituents in your formulation.

Analytical method development is considered a critical process in pharmaceuticals.

The availability of the different types of columns, operating parameters, mobile phase composition, diluent, and pH values make it critical to develop an analytical method. A good analytical method should be simple, using column, mobile phase, and buffer should be common. It can be done easily step by step.

Following are the common HPLC method development steps.

1. Selection of HPLC Analytical Method
2. Selection of Chromatographic Conditions
3. Parameter Optimization

1. Selection of HPLC Analytical Method

• First of all, consult the literature that is available on the product. It will help you to understand the nature of the product which will help to select the different parameters.

A. Sample Preparation: 

• Select a method to prepare the sample according to its solubility, filtration requirements, extraction requirements, or other special requirements to make a clear solution of HPLC analysis.

B. Chromatography:

• Reverse-phase chromatography is used for most of the samples but when acidic or basic molecules are present in the sample then reverse-phase ion suppression (for weak acid or base) or reverse-phase ion pairing (for strong acid or base) should be used. The stationary phase should be C18 bonded. 
• Normal phase is used for low or medium polarity analyte specially when it is required to separate the product isomers.
• Choose a cyano-bonded phase for normal phase separations. Ion exchange chromatography is best used for inorganic anion or cation analysis. If the analyte has a higher molecular weight then size exclusion chromatography is the best to use.

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C. Gradient/Isotonic HPLC:

• Gradient HPLC is helpful in the analysis of complex samples having several components. It will help to get a higher resolution than isotonic HPLC has constant peak width while in isotonic HPLC's peak width increases with the retention time. Gradient HPLC has great sensitivity, especially for products having longer retention times.

D. Column Size:

• 100-150 mm columns are used for most of the samples. It reduces the method development and analysis time for the sample. Bigger columns are used for complex samples that take more time in separation. Initially, a flow rate should be kept between 1 and 1.5 ml/min and column particle size should be between 3 and 5 μm.

E. HPLC Detectors:

• If the analyte has chromophores that enable the compound to be detected by UV then it is better to use a UV detector. It is always better to use a UV detector than others. Fluorescence and electrochemical detectors should be used for trace analysis.
• Samples having high concentrations should be analyzed using refractive index detectors.

F. Wavelength:

• λmax of the sample has the greatest sensitivity to UV light. It detects the sample components that have chromophores. A wavelength above 200 nm gives greater sensitivity than the lower wavelengths. Wavelengths lower than 200 nm give more noise, therefore, it should be avoided.

2. Selection of Chromatographic Conditions:

• After the selection of the analytical method, different chromatographic conditions are selected.
• The flow of the analytes through the column depends upon the concentration of the solvent in the mobile phase. The concentration of solvent is generally used to control the retention time. Mobile phase pH and ion pairing reagents also affect the retention time of the sample. Samples having a large number of components are analyzed using the gradient to avoid the large retention time while the samples containing one or two components are analyzed on an isotonic system.

3. Parameter Optimization:

• After taking the same sample runs some parameters including column dimensions, particle size, run time and flow rate are optimized. It is done to get the best resolution and minimum run time. After proper optimization of the analysis method, it is validated to ensure the consistency of the analytical method.
• Analytical method validation is now done mandatory by all regulatory authorities.

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