YAZAWA HPLC COLUMN SCHOOL

— A Perspective from ICH Q2(R2) and Molecular Interactions —

The first step in analytical method validation is confirming reproducibility through repeated analyses.
In the pharmaceutical field, ICH Q2(R2) serves as the international guideline for analytical method validation.

Once a method has been established, several repeated injections should be performed to confirm reproducibility.
The following parameters should be carefully evaluated:

• Retention time

• Peak area

• Peak height

• Peak shape (tailing or fronting)

• Column pressure

If variations are observed in these parameters, the method cannot be considered robust.

When results are not reproducible, many people tend to suspect the column. However, in most cases, the actual causes lie in:

• Sample preparation

• Mobile phase composition (particularly ionic strength)

• pH design

• Sample solvent

• Instrument condition

HPLC separation is the cumulative result of molecular interactions between individual analyte molecules and stationary phase molecules.
If a compound forms aggregates in solution, the column may effectively “recognize” the aggregate rather than individual molecules. Therefore, the analyte in the injected solution must be completely dispersed at the molecular level (fully dissolved).

(Reference)
Is it correct to dissolve the sample in the mobile phase?

ICH Q2(R2) and Method Validation

ICH Q2(R2) requires the evaluation of the following parameters in analytical method validation:

• Specificity

• Linearity

• Range

• Accuracy

• Precision

• Limit of Detection (LOD)

• Limit of Quantitation (LOQ)

• Robustness

These are regulatory requirements, but their fundamental meaning is confirmation of reproducibility at the molecular level.

For example:

Precision
→ Are molecular interactions stable under identical conditions?

Robustness
→ How much do molecular interactions change in response to slight variations in pH or organic solvent ratio?

Specificity
→ Is the difference in interaction between the target analyte and impurities sufficiently designed?

ICH evaluates the results.
However, to establish a truly reliable HPLC method, it is essential to understand the underlying causes.

Sample Stability and Validation

Sample stability can also become a critical issue.

If the following phenomena occur during repeated analysis, the problem may not lie in the method but in changes to the sample itself:

• Gradual change in peak height

• Appearance of new impurity peaks

• Progressive shift in retention time

In such cases, sample stability must be confirmed before evaluating specificity or precision under ICH criteria.

Possible countermeasures include:

• Protection from light

• Low-temperature storage

• Use of stabilizing agents

• Fresh preparation immediately before analysis

There Is No Universal ODS Solution

Some analysts wish to analyze all compounds using a single ODS column. This is impossible.

The following interactions operate between analytes and stationary phases:

• Hydrophobic interactions

• Electrostatic interactions

• Coordination interactions

• Ionic interactions

In particular, the ionic character of the solute strongly governs separation behavior.

Is the compound:

• Cationic?

• Anionic?

• Zwitterionic?

Ignoring these differences and proceeding with validation will not lead to a robust method.

What Analytical Method Validation Truly Means

Analytical method validation is not merely filling out an ICH checklist.

It means understanding molecular structure, designing molecular interactions, and confirming their reproducibility.

That is the essential work required to establish a robust analytical method.

(Reference)
How to Find an HPLC Analytical Method