Effective HPLC method development(Part I)

Effective HPLC method development(Part I)

3. Define Method Objectives

There is no absolute end to the method development process.  The question is what is the “acceptable method performance”?  The acceptable method performance is determined by the objectives set in this step.  This is one of the most important considerations often overlooked by scientists.  In this section, the different end points (i.e., expectations) will be discussed in descending order of significance.

3.1 Analytes:

For a related substance method, determining the “significant and relevant” related substances is very critical. With limited experience with the drug product, a good way to determine the significant related substances is to look at the degradation products observed during stress testing. Significant degradation products observed during stress testing should be investigated in the method development.

Based on the current ICH guidelines on specifications, the related substances method for active pharmaceutical ingredients (API) should focus on both the API degradation products and synthetic impurities, while the same method for drug products should focus only on the degradation products.  In general practice, unless there are any special toxicology concerns, related substances below the limit of  quantitation (LOQ) should not be reported and therefore should not be investigated.

In this stage, relevant related substances should be separated into 2 groups:

3.1.1. Significant related substances: Linearity, accuracy and response factors should be established for the significant related substances during the method validation.  To limit the workload during method development, usually 3 or less significant related substances should be selected in a method.

3.1.2 Other related substances: These are potential degradation products that are not significant in amount.  The developed HPLC conditions only need to provide good resolution for these related substances to show that they do not exist in significant levels.

3.2 Resolution (Rs)

A stability indicating method must resolve all significant degradation products from each other. Typically the minimum requirement for baseline resolution is 1.5.  This limit is valid only for 2 Gaussian-shape peaks of equal size.  In actual method development, Rs = 2.0 should be used as a minimum to account for day to day variability, non-ideal peak shapes and differences in peak sizes.

3.3 Limit of Quantitation (LOQ)

The desired method LOQ is related to the ICH reporting limits.  If the corresponding ICH reporting limit is 0.1%, the method LOQ should be 0.05% or less to ensure the results are accurate up to one decimal place.  However, it is of little value to develop a method with an LOQ much below this level in standard practice because when the method is too sensitive, method precision and accuracy are compromised.

3.4 Precision, Accuracy

Expectations for precision and accuracy should be determined on a case by case basis.  For a typical related substance method, the RSD of 6 replicates should be less than 10%.  Accuracy should be within 70 % to 130% of theory at the LOQ level.

 3.5 Analysis time

A run time of about 5-10 minutes per injection is sufficient in most routine related substance analyses.  Unless the method is intended to support a high-volume assay, shortening the run time further is not recommended as it may compromise the method performance in other aspects (e.g., specificity, precision and accuracy.)

3.6 Adaptability for Automation

For methods that are likely to be used in a high sample volume application, it is very important for the method to be “automatable”. The manual sample preparation procedure should be easy to perform.  This will ensure the sample preparation can be automated in common sample preparation workstations.

4. Understand the Chemistry

Similar to any other research project, a comprehensive literature search of the chemical and physical properties of the  analytes (and other structurally related compounds) is essential to ensure the success of the project.

4.1 Chemical Properties

Most sample preparations involve the use of organic-aqueous and acid-base extraction

techniques.  Therefore it is very helpful to understand the solubility and pKa of the analytes. Solubility in different organic or aqueous solvents determines the best composition of the sample solvent. pKa determines the pH in which the analyte will exist as a neutral or ionic species.  This information will facilitate an efficient sample extraction scheme and determine the optimum pH in mobile phase to achieve good separations.

4.2 Potential Degradation Products

Subjecting the API or drug product to common stress conditions provides insight into the stability of the analytes under different conditions.  The common stress conditions include acidic pH, basic pH, neutral pH, different temperature and humidity conditions, oxidation, reduction and photo-degradation. These studies help to determine the significant related substances to be used in method development, and to determine the sample solvent that gives the best sample solution stability.

In addition, the structures of the analytes will indicate the potential active sites for degradation. Knowledge from basic organic chemistry will help to predict the reactivity of the functional groups. For example, some excipients are known to contain trace level of peroxide impurities. If the analyte is susceptible to oxidation, these peroxide impurities could possibly produce significant degradation products.

4.3 Sample Matrix

Physical (e.g., solubility) and chemical (e.g., UV activity, stability, pH effect) properties of the sample matrix will help to design an appropriate sample preparation scheme.  For example, Hydroxypropyl Methylcellulose (HPMC) is known to absorb water to form a very viscous solution, therefore it is essential to use mostly organic solvents in sample preparation.

Continue ....... Part II