Coupling Supercritical Fluid Chromatography with Internal Standard Corrected Quantitative NMR for Rapid and Accurate...

Purity assessment is critical for the quality control of active pharmaceutical ingredients (APIs). However, there is a lack of rapid and accurate purity assessment methods that operate independently of target-specific certified reference materials (CRMs). Herein, we proposed a traceable quantification method, named SFC-ISC-qNMR, for purity assessment and quantitative determination of low-purity APIs, which was accomplished by tracing the test solution to the reference solution via supercritical fluid chromatography (SFC) collection with internal standard-correction (ISC), followed by an SI-traceable quantitative nuclear magnetic resonance (qNMR) analysis of the reference solution (SI = International System of Units). Digitoxin, a widely used cardiac glycoside in the treatment of heart failure and arrhythmias, was used as a model compound. By employing a mobile phase of supercritical CO₂ modified with 24%-26% methanol, digitoxin with a purity exceeding 99.87% was isolated and collected. A national CRM of ethyl para-hydroxybenzoate was used as the IS for digitoxin. The SFC-ISC reference solution can be prepared in 15 min without the need for precise weighing, simply by reconstitution of the SFC collected high-purity analyte in deuterated methanol containing IS. The feasibility and reliability of the SFC-ISC-qNMR method were exhibited through the quantitative determination of digitoxin in chemical mixtures. The purity of a commercially available digitoxin reagent was determined to be 94.17% ± 0.15%, which was consistent with high-resolution qNMR analysis. The contents of spiked digitoxin in a digoxin analytical standard were measured at 1.06%, 1.37%, 2.39%, and 4.43%, respectively, showing high consistency with the gravimetric value with a bias less than 0.13%. In contrast, direct qNMR with deconvolution and HPLC analysis failed to provide accurate measurements. These results highlight the applicability of the SFC-ISC-qNMR method for drug evaluation and research, demonstrating its significant potential for the purity assessment of APIs.