Chemical and Pharmaceutical Bulletin
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Purity Determination of Cyclophosphamide Hydrate by Quantitative 31P-NMR and Method Validation
Nahoko UchiyamaJunko HosoeNaoki SugimotoKyoko IshizukiTatsuo KoideMika MurabayashiNaoto MiyashitaKengo KobayashiYoshinori FujimineToshiyuki YokoseKatsuya OfujiHitoshi ShimizuTakashi HasebeYumi AsaiEri EnaJunko KikuchiKohei KiyotaKazuhiro FujitaYoshinobu MakinoNaoko YasobuYoshiaki IwamotoToru MiuraKoji MizuiKatsuo AsakuraTakako SuematsuHitomi MutoAi KohamaTakashi GotoMasu YasudaTomohiko UedaYukihiro Goda
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2021 Volume 69 Issue 7 Pages 630-638


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.

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