A Guide to Effective HPLC Method Development in Bioanalysis

The development of sensitive, selective, and reliable HPLC methods is crucial for the quantitation of drug compounds and their metabolites. The data and analytical information obtained from this process are necessary for toxicokinetic and pharmacokinetic studies. The PK and TK studies further help in finding bioavailability, bioequivalence, and metabolism of the drug compound in human or animal trials. These parameters are necessary to prove the efficacy and safety of the drug product.

This article will explain the five stages of HPLC method development.
HPLC Method Development
Physiochemical Properties
For HPLC analysis, the scientists first analyze the physical properties of the drug molecule such as polarity, solubility, PK, and pH.

Polarity helps in deciding the solvent and mobile phase composition. In a non-polar bond, two atoms equally share the electrons. However, in a polar bond, one atom contains higher electron attraction than others.

Solubility is a concept closely related to the polarity of the relevant molecules. For instance, polar water and non-polar benzene won’t mix. Diluents are decided based on the analyte solubility.

pH defines the basic or acidic nature of the substance. Selecting the correct pH helps scientists in obtaining sharp HPLC peaks.

HPLC Conditions
Buffer Selection

Buffer is selected to resist pH fluctuations. It is nothing but a partially neutralized acid.

Generally, in HPLC method development, 10-50 mM buffer is acceptable for small molecules. The concentration of the buffer decides the organic nature of the buffer which is usually 50% or less.

In reversed HPLC, phosphoric acid and potassium salt buffers are most common. But, for organophosphate compounds, you can replace phosphonate buffers with sulfonate buffers. 

Detector Selection

Detector selection is an essential part of the HPLC analysis as the right selection of detector leads to highly sensitive, low-impurity quantitation and identification. You can select the detector based on chemical analyte nature, interference, availability, the limit of detection, and cost.

Sample Preparation

The drug compound should stay stable in the diluent solution. In the initial stages, solutions should be prepared in the amber flask until the stability of the active component is established at room temperature.

Further, you should filter the sample using 0.22 or 0.45μm pore size for filtration.

Method Optimization

To obtained required HPLC method stability, scientists are required to optimize experimental conditions. Stability, in particular, can be achieved with an assessment of parameters such as mobile phase components, pH, ratio, gradient, sample amount, flow rate, diluent solvent, etc.

Method Validation

Lastly, the HPLC method is validated to ensure it is suitable for the intended purpose. This stage evaluates several analytical parameters given below.
• Accuracy
• Precision
• Repeatability
• Linearity
• Limit of quantitation
• Limit of detection
• Specificity
• Intermediate precision
• Range
• Solution stability
• Robustness

HPLC analysis is a significant process in the pharmaceutical industry, hence, ensuring its quality through validation is necessary. The solubility, pH, pKa, and other important parameters of the method development should be accordingly assessed throughout the development. In the final stages of development, optimization should be achieved with the help gradient slope, mobile-phase modifiers, and their concentration, and changing temperature.