Post-collection purity correction for internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance

Abstract

Quantitative nuclear magnetic resonance (qNMR) has a potential risk of inaccurate quantification of complex organic compounds with low purity due to incomplete separation of the impurity signals and the target component signals. The high performance liquid chromatography-qNMR (HPLC-qNMR) method removes impurities from the sample by HPLC and accurately determines the purity of the sample by qNMR, avoiding the laborious, time-consuming, and costly step of qualitative and quantitative determination of impurities in conventional mass balance methods. An improved method, named post-collection purity correction for internal standard correction-HPLC-qNMR (ISC-HPLC-qNMR), was developed and demonstrated on a complex compound oxytetracycline with low purity. In this method, a correction factor was introduced to compensate for the inability to achieve 100% purity through the HPLC purification procedure. The purity value with standard deviation of the oxytetracycline study material using this method was 82.00% ± 0.82%, while that obtained from the conventional qNMR with deconvolution was 81.70% ± 0.35%. The consistency of these results demonstrated that the improved method extends the applicability to the samples where HPLC is not capable of purifying complex compounds with low purity to near 100%, especially containing highly similar structural-related impurities. Furthermore, this method allows purification and quantification without the need to identify impurities in the sample, resulting in significant savings of time and cost. Additionally, it effectively compensates for the limitations of qNMR deconvolution in handling peak overlap in the sample.