Ensuring Accuracy and Long-Term Stability in qNMR Methods with External Calibration

Several recent reviews have highlighted the broad effectiveness and potential of quantitative nuclear magnetic resonance spectrometry (qNMR) techniques.^1,2

This approach satisfies the highest metrological standards, its results are SI-traceable, and the Commité Consultatif pour la Quantité de Matière credits it as a primary ratio analytical method,³ suggesting that qNMR does not demand analyte-specific reference compounds.

For other prevalent analytical approaches, including high-performance liquid chromatography, infrared spectroscopy, or mass spectrometry, the analyte must be accessible as a certified reference material (CRM) to standardize the tools.

For NMR, any CRM can be employed as a calibrant in theory.

qNMR methods can be theoretically split into three distinct methodologies:

1. Ratio measurements (relative [100 %] qNMR)
2. Potency determination (IC-qNMR)
3. Content measurements (EC-qNMR) (Figure 1).

All techniques agree that the ratio of two signal integrals is calculated alongside additional data like sample weight, stoichiometry, molar mass, etc., to verify quantitative results.

Figure 1. Concept of relative (100 %) qNMR without reference, IC-qNMR with internal reference and EC-qNMR with external reference. Image Credit: Bruker BioSpin - NMR, EPR and Imaging

This article explores content measurements from the external calibration (EC)-qNMR method. Unlike other qNMR methods, this approach analyzes separate samples, one with the reference compound in a solution of known concentration and the others with the analytes.

The EC-qNMR technique can be additionally divided as follows:

1. The reference solution is held in a coaxial insert and measured with the analyte solution.
2. The reference signal is electronically produced.⁴
3. Direct measurement of concentrations derives from the PULCON principle.^5,6

The third approach does not require special hardware or software for electronic referencing, and a coaxial insert does not reduce the measurement’s sensitivity.

The PULCON EC-qNMR is as easy to implement as IC qNMR, making them both outstanding candidates for routine quantitative analyses.

Although both techniques are relatively similar, the IC-qNMR approach is deemed the gold standard for quantification as it eradicates selected error sources (variation of sample volume or flip angle, long-term instrument stability, etc.) by measuring analyte and reference standard simultaneously in the same sample.

EC-qNMR avoids standard disadvantages of internal referencing, including signal overlap and chemical reactions between analyte and standard. As the reference standard and the analyte are prepared separately, the EC-qNMR approach provides more adaptability concerning the choice of NMR solvent. This approach is also more economical as smaller amounts of CRMs are required.

This article investigates the capability of PULCON EC-qNMR, demonstrating how to reduce potential bias, and highlighting the convenience of the process organized with the TopSpin component ERETIC2.

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References

1. Pauli, G.F. et al. (2014). Importance of purity evaluation and the potential of Quantitative ¹H NMR as a purity assay, Journal of Medicinal Chemistry, 57(22), pp. 9220–9231. https://doi.org/10.1021/jm500734a.
2. Giraudeau, P. (2016). Challenges and perspectives in quantitative NMR, Magnetic Resonance in Chemistry, 55(1), pp. 61–69. https://doi.org/10.1002/mrc.4475.
3. Jancke, H., Malz, F. and Haesselbarth, W. (2005). Structure analytical methods for quantitative reference applications, Accreditation and Quality Assurance, 10(8), pp. 421–429. https://doi.org/10.1007/s00769-005-0004-9.
4. Akoka, S., Barantin, L. and Trierweiler, M. (1999). Concentration measurement by proton NMR using the ERETIC method, Analytical Chemistry, 71(13), pp. 2554–2557. https://doi.org/10.1021/ac981422i.
5. Wider, G. and Dreier, L. (2006). Measuring protein concentrations by NMR spectroscopy, Journal of the American Chemical Society, 128(8), pp. 2571–2576. https://doi.org/10.1021/ja055336t.
6. Nishizaki, Y. et al. (2021). Accurate and precise external calibration enhances the versatility of quantitative NMR (QNMR), Analytical Chemistry, 93(5), pp. 2733–2741. https://doi.org/10.1021/acs.analchem.0c02967.

This information has been sourced, reviewed and adapted from materials provided by Bruker BioSpin - NMR, EPR and Imaging.

For more information on this source, please visit Bruker BioSpin - NMR, EPR and Imaging.