[Quantitative 31 P-NMR for Purity Determination of Organophosphorus Compounds (Pharmaceuticals)].

Quantitative NMR (qNMR), particularly 1 H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for quantification is difficult, because of the overlap and complexity of organic molecules. Therefore, we focused on the 31 P nucleus, owing to the simplicity of its signals, and investigated the 31 P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible 31 P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for 31 P-qNMR], hygroscopicity and solution stability of the analyte and RS, and qNMR measurements-such as acquisition time, relaxation delay time, and spectral width-were examined. The CP purities determined using 31 P-qNMR agreed well with those for the established 1 H-qNMR method in D2 O. In contrast, the SOF purity determined using 31 P-qNMR was 1.6% higher than that for 1 H-qNMR in the protic solvent CD3 OD. Therefore, using a protic solvent, such as CD3 OD, was not suitable for 31 P-qNMR; the deuterium exchange with the RS for 31 P-qNMR (i.e., phosphonoacetic acid) resulted in a small integrated intensity. Consequently, the aprotic solvent DMSO-d6 was employed to determine the SOF purity. The data revealed that the SOF purities determined using 31 P-qNMR agreed well with the established 1 H-qNMR values, indicating that the absolute quantification of SOF using both 31 P-qNMR and 1 H-qNMR is possible in DMSO-d6 . Thus, we established an optimized and reproducible 31 P-qNMR method in validation study across multiple laboratories.