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Quantitative NMR (qNMR) is applied to determine the absolute quantitative value of analytical standards for HPLC-based quantification. We have previously reported the optimal and reproducible sample preparation method for qNMR of hygroscopic reagents, such as saikosaponin a, which is used as an analytical standard in the assay of crude drug section of Japanese Pharmacopoeia (JP). In this study, we examined the absolute purity determination of a hygroscopic substance, indocyanine green (ICG), listed in the Japanese Pharmaceutical Codex 2002, using qNMR for standardization by focusing on the adaptation of ICG to JP. The purity of ICG, as an official non-Pharmacopoeial reference standard (non-PRS), had high variation (86.12 ± 2.70%) when preparing qNMR samples under non-controlled humidity (a conventional method). Additionally, residual ethanol (0.26 ± 0.11%) was observed in the non-PRS ICG. Next, the purity of non-PRS ICG was determined via qNMR when preparing samples under controlled humidity using a saturated sodium bromide solution. The purity was 84.19 ± 0.47% with a lower variation than that under non-controlled humidity. Moreover, ethanol signal almost disappeared. We estimated that residual ethanol in non-PRS ICG was replaced with water under controlled humidity. Subsequently, qNMR analysis was performed when preparing samples under controlled humidity in a constant temperature and humidity box. It showed excellent results with the lowest variation (82.26 ± 0.19%). As the use of a constant temperature and humidity box resulted in the lowest variability, it is recommended to use the control box if the reference ICG standard is needed for JP assays.

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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.

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