IN THE LAB - Lab Notebook | Analytical Methods Validation: Design and Execution

By Clifford Nilsen, CSSBB The mechanics of analytical methods validation

Editor’s Note: This is the third in a series of articles on analytical methods validation (the second part appeared on p. 34 of our June/July issue). The final article will continue to review the mechanics of analytical method validation, with an application of statistical methods that best support the validation effort.
This third installment of the series on analytical method validation, the process of demonstrating through laboratory studies that an analytical method is suitable for its intended use, will focus on the mechanics of analytical method validation.

Stability Indication (Forced Degradation Studies)

There are two kinds of forced degradation studies: chemical and physical. Chemical degradation is usually accomplished by acid hydrolysis, base hydrolysis, and oxidation.
Acid hydrolysis: Expose the sample to a mineral acid such as hydrochloric or sulfuric acid, starting with 100% working amount plus 10 mL of 0.5 N acid. Reflux the acidified sample for 30 minutes, cool to room temperature, neutralize it with 10 mL of 0.5N NaOH, dilute to a final volume, and assay the resulting sample using high performance liquid chromatography (HPLC) with a photodiode array detector to determine the amount of degradation, if any, and the peak purity of the principal analyte. If possible, strive to achieve between 15% and 30% degradation. If the initial experiment results in a degree of degradation far outside the 15% to 30% range, then adjust the acid strength and/or reflux time accordingly and repeat the experiment. Measuring the concentration of principal analyte in the degraded sample versus an unadulterated sample and comparing that value to the starting amount of sample can determine percent degradation.

There are two kinds of forced degradation studies: chemical and physical. Chemical degradation is usually accomplished by acid hydrolysis, base hydrolysis, and oxidation.

Base hydrolysis: Expose the sample to a base such as sodium hydroxide or potassium hydroxide, starting with 100% working amount plus 10 mL of 0.5 N base. Reflux the alkaline sample for 30 minutes, cool to room temperature, neutralize it with 10 mL of 0.5N mineral acid, dilute to a final volume, and assay the resulting sample using HPLC with a photodiode array detector to determine the amount of degradation, if any, and the peak purity of the principal analyte. If possible, strive to achieve between 15% and 30% degradation. If the initial experiment results in a degree of degradation far outside the 15% to 30% range, then adjust the base strength and or reflux time accordingly and repeat the experiment. Measuring the concentration of principal analyte in the degraded sample vs. an unadulterated sample and comparing that value to the starting amount of sample can determine percent degradation.
Oxidation: Expose the sample to 10% hydrogen peroxide starting with 100% working amount plus 10 mL of 10% hydrogen peroxide. Let the sample stand for 30 minutes at room temperature with occasional swirling, dilute to a final volume, and assay the resulting sample using HPLC with a photodiode array detector to determine the amount of degradation, if any, and the peak purity of the principal analyte. If possible, strive to achieve between 15% and 30% degradation. If the initial experiment results in a degree of degradation far outside the 15% to 30% range, then adjust the hydrogen peroxide strength and/or standing time accordingly and repeat the experiment. Measuring the concentration of principal analyte in the degraded sample versus an unadulterated sample and comparing that value to the starting amount of sample can determine percent degradation.
Physical degradation is achieved by exposure to heat and light.
Heat: Expose a portion of the sample to 60°C dry heat for five days. Allow the sample to cool down, then assay the resulting heat-exposed sample using HPLC with a photodiode array detector to determine the amount of degradation, if any, and the peak purity of the principal analyte. If possible, strive to achieve between 15% and 30% degradation. If the initial experiment results in a degree of degradation far outside the 15% to 30% range, then adjust the heating time accordingly and repeat the experiment. Measuring the concentration of principal analyte in the degraded sample versus an unadulterated sample and comparing that value to the starting amount of sample can determine percent degradation.

For each forced degradation, there should be no interfering degradants under the principal analyte peak as determined by peak purity analysis. Peak purity can be evaluated using purity parameters (factors), peak ratios, spectral overlays, or ratiograms. If this criterion is met, then the method is deemed to be stability indicating.

Light (photostability): Expose a portion of sample to 1.2 million lux-hours of cool white light and 200 watt-hours/m² 360nm ultraviolet A light, using a suitable photostability system (contact the author for more information). After light exposure, assay the resulting sample using HPLC with a photodiode array detector to determine the amount of degradation, if any, and the peak purity of the principal analyte. Report the percent degradation, if any, that is observed. Measuring the concentration of principal analyte in the degraded sample versus an unadulterated sample and comparing that value to the starting amount of sample can determine percent degradation.
For each forced degradation, there should be no interfering degradants under the principal analyte peak as determined by peak purity analysis. Peak purity can be evaluated using purity parameters (factors), peak ratios, spectral overlays, or ratiograms. If this criterion is met, then the method is deemed to be stability indicating.

Selectivity

Process a placebo (product without the active ingredient) as a sample at 10 times the working concentration. Assay the placebo as a sample to make sure that no interferences are present that will prevent detection of the active ingredient(s).
Typical interfering agents are preservatives and dyes that are often very ultraviolet active. For HPLC methods, there should be no peaks greater than noise at the retention time of the principal analyte.

Linearity and Range

Prepare a series of five (five standards that span a range of 50% to 150% of the analyte working range). For example, if one were performing a linearity on acetaminophen (APAP), and the working concentration was 0.1 mg/mL, then one might proceed as follows:

• Make 200 mL of a solution of APAP in alcohol with a concentration of 1 mg/mL (10 times the working concentration). This is the APAP stock solution.
• Prepare the five working standards according to Table 1 (see below, left).

Table 1. Acetaminophen (APAP) Linearity Standards

Using the HPLC method for the product, inject each standard six times. For each standard—50%, 75%, 100%, 125%, and 150% of the working standard, respectively—calculate the area unit’s percent relative standard deviation to determine injection precision at each level. Plot the mean counts for each standard level versus concentration, performing a linear regression on the resulting curve.
In most cases, it is desirable to have an injection precision, in terms of area unit relative standard deviation, of less than 2% for each standard level, i.e., 50% to 150% of the working concentration. The linear correlation coefficient significance (r²) for the standard curve should typically be > 99.9%. The Pearson linearity coefficient (r) should be > 0.9995. Contrary to common belief, r² is not the linear correlation coefficient, but, rather, r = correlation coefficient and r² = significance of the correlation—in this case, the percentage of area counts that can be explained by concentration.