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Whereas generic, LC-based pharmaceutical control quality procedures depend largely on the detection mode and can be particularly ‘blind’ to certain impurities, NMR is a more versatile and, thus, often more judicious detector. While adulteration presents ever-evolving challenges for the analysis of active pharmaceutical ingredients (APIs) and finished products sold in the worldwide (online) marketplace, research chemicals are usually trusted rather than being considered flawed or even adulterated.

This report shows how NMR analysis uncovered the unanticipated presence of substantial amounts of mannitol (20 and 43% w/w) as undeclared constituent in two custom synthetic peptides, DR and DRVYI, that were sourced commercially. Quantitative 1H NMR (qHNMR) readily detected the contaminant, even on a 60 MHz benchtop instrument, and quantified the highly polar and UV-transparent adulterant. Quantum-mechanical 1H iterative Full Spin Analysis (HiFSA) not only achieved unambiguous identification of both the mannitol and the peptides, but also confirmed the quantitative results.

Ensuring identity, purity, and reproducibility are equally essential during synthetic chemistry, drug discovery, and for pharmaceutical product safety. Many peptidic APIs are large molecules that require considerable effort for integrity assurance. This study builds on quantum mechanical 1H iterative Full Spin Analysis (HiFSA) to establish NMR peptide sequencing methodology that overcomes the intrinsic limitations of principal compendial methods in identifying small structural changes or minor impurities that affect effectiveness and safety. HiFSA sequencing yields definitive identity and purity information concurrently, allowing for API quality assurance and control (QA/QC). Achieving full peptide analysis via NMR building blocks, the process lends itself to both research and commercial applications as 1D 1H NMR (HNMR) is the most sensitive and basic NMR experiment. The generated HiFSA profiles are independent of instrument or software tools and work at any magnetic field strength. Pairing with absolute or 100% qHNMR enables quantification of mixtures and/or determination of peptide conformer populations. Demonstration of the methodology uses single amino acids (AAs) and peptides of increasing size, including the octapeptide, angiotensin II, and the nonapeptide, oxytocin. The feasibility of HiFSA coupled with automated NMR and qHNMR for use in QC/QA efforts is established through case-based examples and recommended procedures.