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

Abstract

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

Quantitative ³¹P-NMR for Purity Determination of Organophosphorus Compounds (Pharmaceuticals)

“…核を選択し，新たに 31 P-qNMR による 絶対定量法の検討を行った． 1 H-qNMR は，既 に 測 定 条 件 等 が 確 立 さ れ て おり，日局においてその条件等が記載されてい る． 8-10) 一 方 で， 31 P-qNMR は 論 文 等 の 報 告 も 少 な く，確立された測定方法がない．多くの医薬品の 場 合，分 子 内 の 31 P は 1-2 個 程 度 で あ る た め， 31 P-qNMR の主となるシグナルは単純な形状となり，定 量シグナルの選択は，容易と考えられる．そこで 筆者らは，わが国の薬価収載医薬品である有機リ ン化合物の中から水溶性の cyclophosphamide hydrate (CP，日 局 収 載 ) ，非 水 溶 性 の sofosbuvir (SOF)及 び brigatinib(BR)を選び(Fig. 1) ， 31 P-qNMR による 最適な測定条件の設定及び複数機関でのバリデー ション研究を行い，既に絶対定量法が確立してい る 1 H-qNMR による定量値と比較し，その有効性を 確認した．なお，BR は日本薬学会第 143 年会シン 国立医薬品食品衛生研究所(〒210-9501 川崎市川崎区 殿町 3-25-26) e-mail: nuchiyama@nihs.go.jp 本総説は， 日本薬学会第 143 年会シンポジウム S34 で 発表した内容を中心に記述したものである． ポジウム発表当時，論文未投稿であったため，本報 では，既報である CP 及び SOF の結果を中心に述べ る．11,12) …”

“…Conditions of 1 H-and 31 P-qNMR Measurements and Purities (%) of CP and SOF Determined Using 1 H-and 31 P-qNMR11,12) …”