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.

应用定量 NMR（qNMR）来确定基于HPLC的定量分析标准品的绝对定量值。先前我们已经报道了用于吸湿剂（如皂苷皂苷a）的qNMR的最佳且可重现的样品制备方法，该方法在日本药典（JP）的粗药部分测定中用作分析标准品。

在这项研究中，我们研究了吸湿性物质吲哚菁绿（ICG）的绝对纯度测定，该物质是在日本药典2002中列出的，使用qNMR进行了标准化，重点是针对ICG对JP的适应性。当在非受控湿度下（常规方法）制备qNMR样品时，作为官方非药物参考标准（non-PRS）的ICG纯度具有较高的变化（86.12±2.70％）。此外，在非PRS ICG中观察到残留乙醇（0.26±0.11％）。接下来，当使用饱和溴化钠溶液在受控湿度下制备样品时，通过qNMR确定非PRS ICG的纯度。纯度为84.19±0.47％，其变化低于非控制湿度下的变化。而且，乙醇信号几乎消失了。我们估计，在可控湿度下，非PRS ICG中的残留乙醇被水代替。随后，当在恒温恒湿箱中在受控湿度下制备样品时，进行qNMR分析。结果显示出极好的结果，变化最小（82.26±0.19％）。由于使用恒温恒湿箱会导致最低的可变性，因此，如果JP分析需要参考ICG标准，则建议使用控制箱。