Purity Determination Of Cyclophosphamide Hydrate By Quantitative P-31-Nmr And Method Validation

Recently, quantitative NMR (qNMR), especially H-1-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-1-qNMR. In the present study, we focused on a P-31-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-31-qNMR and H-1-qNMR results were compared under three conditions with potassium dihydrogen phosphate (KH2PO4) or O-phosphorylethanolamine (PEA) as the reference standard for P-31-qNMR and sodium 4,4-dimethyl-4-silapentanesulfonate-d(6) (DSS-d(6)) as the standard for H-1-qNMR. Condition 1: separate sample containing CP and KH2PO4 for P-31-qNMR or CP and DSS-d(6) for H-1-qNMR. Condition 2: mixed sample containing CP, DSS-d(6) , and KH2PO4 . Condition 3: mixed sample containing CP, DSS-d(6), 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-1-qNMR at 11 laboratories and P-31-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-31-qNMR are in good agreement with those determined by the established H-1-qNMR method. Since the P-31-qNMR signals are less complicated than the H-1-qNMR signals, P-31-qNMR would be useful for the absolute quantification of compounds that do not have a simple and separate H-1-qNMR signal, such as a singlet or doublet, although further investigation with other compounds is needed.