[Reader Insights] The Selection of Internal Standards in the Absence of Isotopes

This article is written by an expert chromatographer under the pen name of Chromatography Mound. Welch Materials, Inc. is authorized to translate this article to English and publish it on behalf of the author.

During mass spectrometry (MS) analysis, there are often substances for which the matrix curve cannot provide accurate quantification. The main reason for this is significant deviation during sample pretreatment, resulting in unstable recovery rates. In such cases, the internal standard method is required for quantification.

However, it is not always possible to find an appropriate isotope-labelled internal standard. In this article, we will discuss how to choose an internal standard when isotopes are unavailable.

To begin, it is important to understand why isotopes are considered the best internal standards. Isotopes are compounds that have the same molecular structure as the target analyte, but differ in the number of neutrons in one or more atoms—such as deuterium (D), carbon-13 (C13), nitrogen-15 (N15), or oxygen-18 (O18). This difference in mass number allows the quadrupole of the mass spectrometer to distinguish between them.

While the physical properties of isotopes may vary slightly, their chemical properties are virtually identical to those of the target compound. This means that even when there is substantial deviation during the pretreatment process, the target analyte and its internal standard will be impacted in the same way.

For example, if substance A has a recovery rate of 80%, then the recovery of its internal standard will also be 80%. If the recovery drops to 30% during the next analysis, the internal standard will also show a recovery of 30%. This ensures that the ratio of the target analyte to its internal standard remains consistent, a key feature of the internal standard method, and is highly effective for correcting matrix effects.

Although the internal standard method is widely used, the use of isotopes as internal standards in mass spectrometry can be quite limiting, and at times even impractical.

The purchase of isotope-labeled standards is often not feasible for most laboratories, as they may be unavailable, scarce, or prohibitively expensive. Furthermore, in multi-component analyses, finding a complete set of isotope standards for all compounds is often impractical. It also complicates the data processing, and using only one isotope for internal standardization may lead to unfair results for the other components.

As the saying goes, "Shortage gives no cause for concern whilst inequality does". Given these constraints, it may be unnecessary to focus on selecting isotopes, and a compound with similar properties may suffice.

What do we mean by "similar properties"? For example, homologous compounds such as phthalates, triazines, sulfonamides, naphthalenes, and penicillins can be used as internal standards for one another.

However, it is important to remember that the reason isotopes are considered the best internal standards is because they are absent from the sample. Homologous compounds, on the other hand, may already be present in the sample.

Therefore, if this approach is adopted, it is essential to ensure that the sample does not contain the homologous compound. In fact, these types of measurements often involve numerous target compounds—sometimes a dozen or even several dozen—which makes the selection of an internal standard somewhat problematic.

When selecting an appropriate internal standard, several key rules must be followed to ensure the internal standard matches the characteristics of the target analyte:

Detector Type and Properties of the Internal Standard

It is crucial to determine the type of detector being used. For example, if a photodiode array (PDA) detector is employed, the internal standard should possess similar chromophores, such as a benzene ring or double bonds. For mass spectrometry (MS), the internal standard must share similar ionizable functional groups with the target analyte, such as amine groups or carboxyl groups.

Matching the Properties of the Internal Standard with the Target Analyte

The internal standard should exhibit similar physical and chemical properties to the target analyte, such as stability under acidic or basic conditions, heat resistance, light sensitivity, susceptibility to oxidation, and affinity for metal ions in the system.

If derivatization is involved in the pretreatment process, the reactivity of the internal standard with the derivatizing agent should also be comparable to that of the target analyte. While small discrepancies in these factors may not drastically affect the quantification accuracy, larger deviations could introduce significant bias.

For isotope-labeled internal standards, the recovery ratio of the target analyte to the internal standard should remain consistent. While slight variations in recovery (e.g., 80%:60% or 80%:50%) may occur, as long as the deviation is not too large, the results can still be reliable, especially if calibration with spiked samples is applied. This is similar to the standard addition method, where the internal standard ensures a better linear correlation coefficient.

Response Characteristics and Concentration Requirements of the Internal Standard

The internal standard should generate a signal that is sufficiently strong to match the target analyte’s response. This requirement is important for both MS and PDA detectors. For example, in mass spectrometry, different hydroxyl groups, such as phenolic versus alcoholic hydroxyl groups, have different ionization efficiencies. In PDA, the number of chromophores and the maximum absorption wavelength can significantly affect the response.

For multi-component analysis, it is sufficient to select an internal standard with properties similar to all the components being analyzed. For example, triheptadecanoin can be used as the internal standard for 1,3-dioleoyl-2-palmitoylglycerol (OPO) and its isomer OOP; heptylamine can be used as the internal standard for biogenic amines.

However, for the determination of complex mixtures such as additives (which can be classified into acids, salts, esters, etc.), it is necessary to categorize the compounds first and select an internal standard for each category.

Finally, it is worth noting that internal standards sometimes come in very small quantities, making it impossible to weigh accurately. In such cases, it is not necessary to weigh the internal standard. Instead, simply dissolve it in solvent and dilute to the desired volume. The concentration of the internal standard solution does not need to be extremely precise, as internal standards are not considered standard solutions in laboratory management.

This concludes our discussion on selecting internal standards when isotopes are not available. While the choice of internal standard requires careful consideration, ensuring that the internal standard matches the target analyte in terms of chemical and physical properties will result in more reliable and accurate quantification.