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

Quantitative NMR (qNMR), particularly ¹H-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 ³¹P nucleus, owing to the simplicity of its signals, and investigated the ³¹P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible ³¹P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for ³¹P-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 ³¹P-qNMR agreed well with those for the established ¹H-qNMR method in D₂O. In contrast, the SOF purity determined using ³¹P-qNMR was 1.6% higher than that for ¹H-qNMR in the protic solvent CD₃OD. Therefore, using a protic solvent, such as CD₃OD, was not suitable for ³¹P-qNMR; the deuterium exchange with the RS for ³¹P-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 ³¹P-qNMR agreed well with the established ¹H-qNMR values, indicating that the absolute quantification of SOF using both ³¹P-qNMR and ¹H-qNMR is possible in DMSO-d₆. Thus, we established an optimized and reproducible ³¹P-qNMR method in validation study across multiple laboratories.