Purity Determination of Cyclophosphamide Hydrate by Quantitative ³¹P-NMR and Method Validation

Recently, quantitative NMR (qNMR), especially ¹H-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 ¹H-qNMR. In the present study, we focused on a ³¹P-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 ³¹P-qNMR and ¹H-qNMR results were compared under three conditions with potassium dihydrogen phosphate (KH₂PO₄) or O-phosphorylethanolamine (PEA) as the reference standard for ³¹P-qNMR and sodium 4,4-dimethyl-4-silapentanesulfonate-d₆ (DSS-d₆) as the standard for ¹H-qNMR. Condition 1: separate sample containing CP and KH₂PO₄ for ³¹P-qNMR or CP and DSS-d₆ for ¹H-qNMR. Condition 2: mixed sample containing CP, DSS-d₆, and KH₂PO₄. Condition 3: mixed sample containing CP, DSS-d₆, 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 ¹H-qNMR at 11 laboratories and ³¹P-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 ³¹P-qNMR are in good agreement with those determined by the established ¹H-qNMR method. Since the ³¹P-qNMR signals are less complicated than the ¹H-qNMR signals, ³¹P-qNMR would be useful for the absolute quantification of compounds that do not have a simple and separate ¹H-qNMR signal, such as a singlet or doublet, although further investigation with other compounds is needed.