LOD and LOQ

Summary

This video by Ramalinga Prasad Kupa from Pharma World explains the concepts of Limit of Detection (LOD) and Limit of Quantitation (LOQ) in analytical chemistry. It covers how to detect and measure impurities using high-sensitivity chromatograms, signal-to-noise ratios, and linear regression methods. The video provides step-by-step instructions for calculating LOD and LOQ using Excel, emphasizing their importance in determining impurities at low levels, particularly for method validation in pharmaceuticals. Viewers are encouraged to practice these techniques for better understanding and expertise in chromatography.

Takeaways

• 🔍 Limit of Detection (LOD) is the minimum amount of impurity that can be detected above the baseline noise in chromatographic methods like HPLC or GC.
• 📉 The baseline noise refers to small disturbances recorded when no impurity is present, appearing as a zigzag pattern at high sensitivity.
• 👁️‍🗨️ LOD is commonly determined visually by comparing the signal of an impurity to the noise level of the baseline.
• 📊 The Signal-to-Noise Ratio (S/N) is a key metric for LOD calculation, which is calculated by dividing the signal height by the noise height.
• 🔢 The USP and EP guidelines differ slightly in their calculations for LOD, with some methods yielding double the result.
• 📈 Linear regression methods provide a more scientific approach for LOD determination by analyzing responses at different concentrations.
• 📝 LOD is calculated as 3.3 times the ratio of the standard error to the slope from a linear regression model.
• 📏 Limit of Quantitation (LOQ) is the lowest concentration of a substance that can be accurately quantified with acceptable precision and uncertainty.
• 🔄 LOQ is typically calculated as 10 times the ratio of the standard error to the slope, and is roughly three times the LOD value.
• ⚗️ Establishing both LOD and LOQ is essential for validating methods used in estimating impurities, especially for trace contaminants in cleaning validation processes.

Q & A

What is the limit of detection (LOD)?

The limit of detection (LOD) is the minimum amount of an impurity that can be detected above the noise level of a baseline. It helps identify whether any impurity or organic entity is present in the sample, even at very low levels.

What is the baseline noise in chromatography?

Baseline noise refers to small disturbances recorded by the detector when no impurity is passing through it. At low sensitivity, the baseline appears as a straight line, but at high sensitivity, it shows a zigzag pattern due to residual noise generated by the detector.

How is the signal-to-noise ratio calculated?

The signal-to-noise ratio is calculated as the ratio of the height of the signal to the height of the noise. It helps in determining whether a signal can be considered as an impurity.

What is the basic method to find the LOD?

The basic method to determine the LOD is through visual inspection of a zoomed-in baseline. Any signal that appears above the average noise level is considered as a potential impurity.

How is the LOD calculated using linear regression?

The LOD is calculated using linear regression by preparing a set of six linear concentrations of the impurity. The signal responses are plotted in a scatter graph, and the slope and standard error are used in the formula: LOD = 3.3 * (standard error / slope).

What does a higher LOD value indicate?

A higher LOD value indicates that the method is less sensitive, meaning it can only detect impurities at higher concentrations. If the LOD is too high, the method may not be suitable for detecting very low levels of impurities.

What is the limit of quantitation (LOQ)?

The limit of quantitation (LOQ) is the smallest amount of an impurity that can be quantified with accuracy, precision, and certainty. It is determined using the same parameters as LOD, but with a different formula.

How is LOQ calculated?

LOQ is calculated using the formula: LOQ = 10 * (standard error / slope). It represents the lowest concentration of an impurity that can be measured with precision.

What is the significance of LOD and LOQ in cleaning validation?

In cleaning validation, LOD and LOQ are crucial for ensuring that any residual contaminants in the equipment wash water are detected and quantified at very low levels, making sure the cleaning process is effective.

Are LOD and LOQ necessary for all types of tests?

No, LOD and LOQ are not necessary for tests that measure higher levels of active pharmaceutical ingredients (API), such as assays where API content is between 99-100%. They are more relevant for tests detecting trace contaminants or very low levels of components.