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.
Wu Huaxin,Huang Ting,Zhang Wei,Yao Huan,Li Xueyao,Su Ping,Yang Yi
In the biomedical and chemical fields, purity assessment of compounds is a critical step in ensuring product quality and safety. In this study, a method for purity quantification was proposed as two-signal suppression–internal standard correction–high-performance liquid chromatography–quantitative nuclear magnetic resonance (TSS-ISC-HPLC-qNMR). The two-signal suppression effectively suppresses the interference of solvent signals in the NMR spectra, and the internal standard correction eliminates the influence of many variables during sample preparation and analysis. The purity result (99.89%) of the quantification of certified reference material of acesulfame (AF) by this method matched with the certified value (99.88%), which verified the accuracy of the method. The purity result (with standard deviation) of 94.42% ± 0.10% for the quantification of the low-purity drug digitoxin by the method matched with the purity result of 94.42% ± 0.11% by qNMR with deconvolution (as a validated method), which proved that the method could be used for the quantification of low-purity compounds. The methodology achieved a substantial reduction in total analysis time, from 28 to 4 h (considering only the routine analysis time after optimization), in contrast to the ISC-HPLC-qNMR approach. Additionally, it effectively decreased bias to undetectable with a standard deviation of 0.10%, while the bias of the TSS-HPLC-qNMR method was 0.93%. The present method ensures the accuracy of the quantitative results while demonstrating a low economic burden and significant time efficiency, which shows great potential for application in the accurate quantitative analysis of low-purity organic compounds. Graphical
危险品化学品经营许可证（带存储）