Linearity and Range in Analytical Method Validation by HPLC
Linearity
earity is one of the key parameters assessed during method validation. It refers to the ability of an analytical method to produce results that are directly proportional to the concentration or amount of the analyte within a specific range
When a method is linear, it means that the response of the measurement instrument changes consistently as the concentration of the analyte changes. This property is essential for quantitative analysis, where the goal is to determine the exact concentration of a substance in a sample.
Without linearity, the relationship between the instrument’s response and the analyte’s concentration becomes unpredictable, leading to inaccurate or biased results.
Preparation of Standards
A set of standard solutions containing the API at various concentrations e.g., 10%, 25%, 50%, 75%, 100% and 120 % of the target concentration is prepared.
Instrumental Analysis
The prepared standard solutions are analyzed using a HPLC instrument, which separates and quantifies the API in each solution.
Calibration Curve
The peak areas (instrument responses) corresponding to each standard concentration are recorded. A calibration curve is constructed by plotting the API concentration (x-axis) against the instrument response (y-axis).
Linear Regression
A linear regression analysis is performed to fit a straight line through the data points on the calibration curve. The slope and intercept of the line are determined.
R-squared Calculation
The R-squared value is calculated. A high R-squared value (close to 1) indicates good linearity between the API concentration and the instrument response.
Results
In this case, let’s say the obtained an R-squared value of 0.998 for the calibration curve. This high R-squared value suggests that the API’s concentration and the instrument response exhibit strong linearity within the specified concentration range.
If linearity fails during the validation of an analytical method, it indicates that the relationship between the concentration of the analyte and the instrument’s response is not linear within the specified concentration range.
To address linearity failure, it’s important to investigate the specific cause and take appropriate corrective actions. These actions may involve recalibrating the instrument, reevaluating the sample preparation process, adjusting the concentration range, optimizing the analytical conditions, or exploring alternative methods for calibration and quantification.
Range
The term “range” refers to the interval or span of values within which the analytical procedure’s results are considered accurate, reliable, and suitable for its intended purpose.
The range of an analytical method typically includes the lowest and highest levels of the target analyte that the method is expected to measure accurately.
Read also:
- Why Precision is an Important Parameter in Analytical Method Validation?
- Validation of Compendial Methods
- The Lifecycle of a Pharmaceutical Test Method
