Liquid Chromatography – How to Use Internal Standards

Introduction

Our last session in 2024 will cover the use of internal standard method often employed in High-Performance Liquid Chromatography (HPLC) to improve the accuracy and reliability of quantitation, particularly when dealing with potential errors associated with the absolute calibration method with challenging matrix.

Challenges Associated with the Absolute Calibration Method

In absolute calibration, the target analyte’s concentration is determined by comparing its peak area or height to a standard calibration curve. However, this method can lead to large errors, particularly when injecting very small volumes of the sample. Injection errors can arise from inaccuracies in the volume injected. Which can significantly affect the precision of quantification. Small volumes are especially problematic because precise measurement becomes difficult. Errors are almost inevitable when working with very small sample volumes.

The internal standard (I.S.) method helps mitigate these errors by adding a known volume of an internal standard to both the standard and sample solutions. Quantification is then based on the ratio of the peak sizes of the internal standard and the target compound.

How the Internal Standard Method Works

In the internal standard method, a known, stable compound (the internal standard, or I.S.) is added in equal amounts to both the standard solution and the sample solution. The key idea is that the internal standard behaves similarly to the analyte in the HPLC system, but it is chemically different. Which allows it to be distinguished from the analyte in the chromatogram. By comparing the peak areas or heights of the target analyte and the internal standard, a ratio is obtained. This ratio is used for quantification instead of relying on absolute peak areas or absolute heights.

I.S. substances must satisfy the few conditions and consequently selection can sometimes be difficult:

• Internal Standard peak must be completely separated from the peaks for other constituents contained in the sample
• It can’t be present in the sample already
• It must be eluted close to the target compound
• Chemical structure must be similar to that of the target compound
• It must be chemically stable and easy to obtain

Let us take a closer look at the internal standard method by checking an example of its practical application. In this case, we will explore the analysis of theophylline (a pharmaceutical compound) in blood serum using etofylline as the internal standard (I.S.) substance.

First, several standard solutions of theophylline with varying concentrations are prepared, such as 10, 20, 30, and 40 μg/mL. A solution of etofylline (approximately 20 μg/mL in a 1-N perchloric acid solution) is also prepared as the I.S. solution.

Next, 1 mL of each theophylline standard solution is mixed with 0.5 mL of the I.S. solution. Approximately 10 μL of each mixture is then injected into the chromatographic system. The ratio of the peak areas of theophylline and etofylline (the I.S.) is then recorded. This data allows us to construct a calibration curve, such as the one shown in Fig. 1 below. The X-axis of the calibration curve represents the concentration ratio (Cx/Cs, where Cx is the concentration of theophylline and Cs is the concentration of the I.S.), while the Y-axis represents the area ratio (Ax/As, where Ax and As are the peak areas for theophylline and the I.S., respectively).

Figure 1. Calibration curve using Internal Standard

In practice, the same I.S. solution is added to blood serum. Since the concentration of the I.S. (Cs) is constant, the X-axis in this case represents the theophylline concentration (Cx) in the graph. To analyze the blood serum, 1 mL of serum is mixed with 0.5 mL of I.S. solution. After centrifugal separation, approximately 10 μL of the supernatant is injected, and the area ratio for theophylline and the I.S. is determined from the resulting chromatogram (Fig. 2).

Figure 2. Chromatogram of injected Sample spiked with Internal Standard (sample – blood serum)

For example, if the area ratio obtained from the chromatogram is 0.75, we can use the calibration curve (Fig. 1) to deduce that the concentration of theophylline in the blood serum is 15 μg/mL. This demonstrates how the internal standard method can be effectively applied to quantify theophylline in blood serum.

In the above example, 1-N perchloric acid was used as the solvent for the internal standard (I.S.) solution, and it also serves as a protein removal agent. The primary purpose of using perchloric acid in this context is not only to compensate for inconsistencies in injection volume but also to account for variations in the pretreatment process. Specifically, it compensates for changes in liquid volume resulting from protein removal. Etofylline, a compound with a structure similar to theophylline, is selected as the internal standard substance. This choice is critical, as compounds that cause protein adsorption are unsuitable for this purpose.

It is essential to ensure that each peak remains within the range where the detector’s response maintains linearity, ensuring accurate measurements.

As previously mentioned, while the internal standard method comes with several restrictions regarding the selection of appropriate I.S. substances, it provides a quantification method that delivers a higher degree of accuracy.

Benefits of the Internal Standard Method

Compensates for Injection Variability – since both the sample and the standard solution contain the same amount of internal standard, variations in the injected sample volume can be minimized. Even if small errors occur in injection volumes, they will likely affect both the analyte and the internal standard in very same way, thereby maintaining the accuracy of the ratio.

Corrects for Instrumental Variability – it can also correct for instrumental fluctuations. For instance:

• Variations in the mobile phase flow rate – it will not affect the ratio
• Energy fluctuations in the light source of detectors.
• Solvent evaporation or changes in the sample matrix composition.

Improves Accuracy for Sample Preparation – the internal standard can also help correct for potential recovery errors during sample preparation. For instance, during extraction or other pretreatment steps, if some loss of analyte occurs, the internal standard can compensate for this loss by providing a consistent reference point.

In summary, the internal standard method reduces the potential for error by offering a relative measure, accounting for both instrumental variability and sample handling issues. This makes it a valuable tool in HPLC analysis when accuracy and precision are critical, especially when working with small volumes or complex sample preparations.

Summary

As a relative method, the internal standard approach compensates for inaccuracies in injection volumes and also accounts for variations in measurement conditions. This includes factors such as fluctuations in mobile-phase delivery, changes in lamp light intensity, sample solvent evaporation, and alterations in the mobile phase composition. Additionally, the internal standard method is effective in correcting errors (recovery-rate errors) which can occur during sample pretreatment.

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

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