Purity determination of cyclophosphamide hydrate by quantitative 31 P-NMR and method validation

• Published in: Chemical & pharmaceutical bulletin
• Main Authors: Uchiyama, Nahoko, Hosoe, Junko, Sugimoto, Naoki, Ishizuki, Kyoko, Koide, Tatsuo, Murabayashi, Mika, Miyashita, Naoto, Kobayashi, Kengo, Fujimine, Yoshinori, Yokose, Toshiyuki, Ofuji, Katsuya, Shimizu, Hitoshi, Hasebe, Takashi, Asai, Yumi, Ena, Eri, Kikuchi, Junko, Kiyota, Kohei, Fujita, Kazuhiro, Makino, Yoshinobu, Yasobu, Naoko, Iwamoto, Yoshiaki, Miura, Toru, Mizui, Koji, Asakura, Katsuo, Suematsu, Takako, Muto, Hitomi, Kohama, Ai, Goto, Takashi, Yasuda, Masu, Ueda, Tomohiko, Goda, Yukihiro
• Format: Journal Article
• Language: English
• Published: Japan 15.04.2021
• Subjects: absolute purity; certified reference material; cyclophosphamide hydrate; quantitative 31P-NMR
• ISSN: 1347-5223

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 (KH PO ) or O-phosphorylethanolamine (PEA) as the reference standard for P-qNMR and DSS-d as the standard for H-qNMR. Condition 1: separate sample containing CP and KH PO for P-qNMR or CP and DSS-d for H-qNMR. Condition 2: mixed sample containing CP, DSS-d , and KH PO . 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.