[Reader Insight] Are You Using The Internal Standard Method In A Right Way?

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.

Due to the complexity and labor-intensive nature, the standard addition method is seldom applied in practical quantitative analyses. On the other hand, the internal standard method, which corrects for all experimental errors by using an internal standard, is considered the most accurate approach for quantification.

Why? Assuming that the chemical properties of the external standard and the internal standard are identical, adding the internal standard during sample preparation eliminates the need for an external standard. This approach simultaneously corrects for recovery rates and matrix effects, and allows quantification across different sample matrices by preparing the calibration curve using pure solvents without considering the influence of background values on the calibration curve. Moreover, it ensures that results are not affected by environmental contaminants introduced during analysis.

Typically, isotopically labeled substances are used as internal standards. However, the high cost of such compounds makes the use of structurally similar compounds as internal standards a common alternative in many studies. This practice, however, remains controversial.

For instance, if analytes A and B are isomers, and B has an isotopic internal standard (B'), can B' also serve as the internal standard for A?

To achieve accurate quantification, the answer is no. Even if A and B are isomers, their chemical properties differ (except for enantiomers), which contradicts the fundamental premise of the internal standard method. If even isomers differ chemically, how can structurally similar compounds be identical?

Even if their chemical properties are similar, the retention times of A and B will likely differ. Compounds eluting later are less affected by matrix effects, making such quantification inherently inaccurate.

For compounds with high natural isotope abundance (such as those containing chlorine), some external standard ions may overlap in mass-to-charge ratio (m/z) with those of the internal standard. In such cases, the internal standard method is clearly unsuitable.

Readers may wonder: does the use of isotopic internal standards for compounds with low natural isotope abundance guarantee the reliability of the internal standard method?

The answer may still be disappointing. While this is a most-common scenario, it is also often overlooked.

Consider this: the internal standard method is predominantly used in mass spectrometry (MS) analyses, and during ionization and fragmentation in the MS, the changes a compound undergoes are primarily physical rather than chemical. Thus, even if the external and internal standards share identical chemical properties and experience similar losses during sample preparation, they may still be influenced differently by matrix effects in the ion source.

The author conducted a comparative experiment by preparing calibration curves using both pure solvent and matrix solutions containing isotopic internal standards. The results revealed that the slope of the curve prepared with the pure solvent was over three times lower than that prepared with the matrix solution.

The observed discrepancy of such magnitude challenges the common belief that, since both external and internal standards are equally affected by matrix effects or losses, the internal standard should theoretically correct for these influences, maintaining a consistent slope. The finding indicates that, although both external and internal standards were affected by matrix effects, the internal standard was influenced three times more significantly than the external standard.

The table below shows the mass spectral parameters of perchlorate (ClO^4- ) and chlorate (ClO^3-) derived from standard solutions, unaffected by matrix effects.

The isotopic mass of oxygen is 18. Therefore, the molecular ion of isotopic perchlorate ( ¹⁸O-ClO^4- ) is 8 m/z units heavier than perchlorate, while isotopic chlorate ( ¹⁸O-ClO^3- ) is 6 m/z units heavier than chlorate. However, the fragment ions reveal a difference of 6 m/z units between isotopic perchlorate and perchlorate, indicating the loss of one oxygen atom during fragmentation. The m/z difference of 4 between isotopic chlorate and chlorate suggests a similar fragmentation mechanism.

Notably, among the compounds studied, only perchlorate yielded two prominent fragment ions, whereas the other three compounds, including isotopic perchlorate, produced only one fragment ion. For isotopic perchlorate, no additional fragments other than m/z 89.1 were detected, likely due to insufficient signal intensity. This indicates that the mass spectrometric behavior of perchlorate and its isotope, as well as chlorate, exhibit certain discrepancies.

In conclusion, to ensure precise quantification using the internal standard method, the following are recommended:

• Prepare internal standard calibration curves using matrix-matched solutions.
• Avoid using isomers or structurally similar compounds as internal standards.
• Account for the influence of natural isotope abundance.

Therefore, a comprehensive understanding of the matrix, detector, experimental principles, and analyte structure is indispensable when selecting a quantitative method.