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.

近来，定量核磁共振（qNMR），尤其是¹ H-qNMR，已被广泛用于测定有机分子的绝对定量值。我们之前报道了¹ H-qNMR的最佳且可重复的样品制备方法。在本研究中，我们专注于³¹ P-qNMR 绝对测定方法。的有机磷化合物，环磷酰胺水合物（CP），在日本药典第17版中所列被选择为目标化合物，和³¹ P-qNMR和¹ H-qNMR结果三个条件用磷酸二氢钾下进行了比较（KH ₂ PO ₄）或Ô-磷酰乙醇胺(PEA) 作为³¹ P-qNMR的参考标准，4,4-二甲基-4-硅戊烷磺酸钠-d ₆ (DSS- d ₆ ) 作为¹ H-qNMR的标准。条件1：对于³¹ P-qNMR，包含CP和KH ₂ PO _{4 的}单独样品或对于¹ H-qNMR包含CP和DSS- d _{6 的}样品。条件 2：包含 CP、DSS- d ₆和 KH ₂ PO _{4 的}混合样品。条件 3：含有 CP、DSS- d _{6 的}混合样品，和豌豆。由于条件 1 和 3 提供了良好的结果，因此进一步在多个实验室进行了验证研究。11个实验室的¹ H-qNMR和10个实验室的³¹ P-qNMR在条件1下测定的CP纯度分别为99.76±0.43和99.75±0.53%，5个实验室在条件3下测定的纯度分别为99.66±0.08和99.75±0.53%。分别为 99.61 ± 0.53%。这些数据表明³¹ P-qNMR确定的 CP 纯度与已建立的¹ H-qNMR 方法确定的那些一致。由于³¹ P-qNMR 信号不如¹ H-qNMR 信号复杂，因此³¹P-qNMR 可用于对没有简单且独立的¹ H-qNMR 信号（例如单峰或双峰）的化合物进行绝对定量，但需要对其他化合物进行进一步研究。