Importance of Purity Evaluation and the Potential of Quantitative ¹H NMR as a Purity Assay

Abstract

In any biomedical and chemical context, a truthful description of chemical constitution requires coverage of both structure and purity. This qualification affects all drug molecules, regardless of development stage (early discovery to approved drug) and source (natural product or synthetic). Purity assessment is particularly critical in discovery programs and whenever chemistry is linked with biological and/or therapeutic outcome. Compared with chromatography and elemental analysis, quantitative NMR (qNMR) uses nearly universal detection and provides a versatile and orthogonal means of purity evaluation. Absolute qNMR with flexible calibration captures analytes that frequently escape detection (water, sorbents). Widely accepted structural NMR workflows require minimal or no adjustments to become practical 1H qNMR (qHNMR) procedures with simultaneous qualitative and (absolute) quantitative capability. This study reviews underlying concepts, provides a framework for standard qHNMR purity assays, and shows how adequate accuracy and precision are achieved for the intended use of the material.

Phenylbutazone purity challenge

“…Due to the importance of this topic, several methodologies for purity determination are reported in the scientific literature. In the last 15 years, however, quantitative NMR (qNMR) has become a reference technique for the characterization of organic standards containing NMR-active nuclei [1,2].…”

“…Although a number of calibration techniques have been proposed for qNMR [1], the most accurate one for quantitative results is the internal standard method [2]. In such an experiment, a solution of the candidate standard material (A) is spiked with a known amount of a primary standard of known purity (B) and the NMR spectrum of the mixture is recorded.…”

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

“…In this context, quantitative NMR (qNMR) is recommended by the metrological community as universal and powerful method to be used for purity determination of organic compounds [56,57]. The validity and broader acceptance of qNMR is supported by the use of this methodology by the pharmaceutical and chemical industries in their GMP/GLP settings [58]. Furthermore, the use of ICP-MS could be beneficial for compound-independent calibration based on sulfur or the copper complex [59], and the determination of metal traces [60], particularly copper, potentially introduced from process chemicals and nickel from stainless steel equipment.…”

Self-Managed Belief as Part of the “Scientific Method”: Part II—Examples From Published Scientific Work

“…As for structure elucidation, experimental data that is ambiguous or misleading in a way that goes unnoticed, but is believed to be correct by the spectroscopist, can lead to structural misassignments or (often hidden) structural uncertainties in both small molecules and biomolecules [2,30]. Similarly, ensuring the proper data quality brings up extremely subtle issues having a major impact on the deduced results in quantitative NMR applications, which are increasingly employed in many areas such as in the determination of the purity of the marketed drug substances [31][32][33][34][35].…”