Purity Determination of Cyclophosphamide Hydrate by Quantitative P-NMR and Method Validation.

Recently, quantitative NMR (qNMR), especially H-qNMR, has been widely used to determine the absolute quantitative value of organic molecules. We previously reported an optimal and reproducible sample preparation method for H-qNMR. In the present study, we focused on a P-qNMR absolute determination method. An organophosphorus compound, cyclophosphamide hydrate (CP), listed in the Japanese Pharmacopeia 17th edition was selected as the target compound, and the P-qNMR and H-qNMR results were compared under three conditions with potassium dihydrogen phosphate (KHPO) or O-phosphorylethanolamine (PEA) as the reference standard for P-qNMR and sodium 4,4-dimethyl-4-silapentanesulfonate-d (DSS-d) as the standard for H-qNMR. Condition 1: separate sample containing CP and KHPO for P-qNMR or CP and DSS-d for H-qNMR. Condition 2: mixed sample containing CP, DSS-d, and KHPO. Condition 3: mixed sample containing CP, DSS-d, and PEA. As conditions 1 and 3 provided good results, validation studies at multiple laboratories were further conducted. The purities of CP determined under condition 1 by H-qNMR at 11 laboratories and P-qNMR at 10 laboratories were 99.76 ± 0.43 and 99.75 ± 0.53%, respectively, and those determined under condition 3 at five laboratories were 99.66 ± 0.08 and 99.61 ± 0.53%, respectively. These data suggested that the CP purities determined by P-qNMR are in good agreement with those determined by the established H-qNMR method. Since the P-qNMR signals are less complicated than the H-qNMR signals, P-qNMR would be useful for the absolute quantification of compounds that do not have a simple and separate H-qNMR signal, such as a singlet or doublet, although further investigation with other compounds is needed.