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.

The goal of the qNMR Summit is to take stock of the status quo and the recent developments in qNMR research and applications in a timely and accurate manner. It provides a platform for both advanced and novice qNMR practitioners to receive a well-rounded update and discuss potential qNMR-related applications and collaborations. For over a decade, scientists from academia, industry, nonprofit institutions, and governmental bodies have focused on the standardization of qNMR methodology, as well as its metrological and pharmacopeial utility. This paper reviews key content of qNMR Summits 1.0 to 4.0 and puts into perspective the outcomes and available transcripts of the October 2019 Summit 5.0, with attendees from the United States, Canada, Japan, Korea, and several European countries. Summit presentations focused on qNMR methodology in the pharmaceutical industry, advanced quantitation algorithms, and promising developments.

NMR spectroscopy has recently been utilized to determine the absolute amounts of organic molecules with metrological traceability since signal intensity is directly proportional to the number of each nucleus in a molecule. The NMR methodology that uses hydrogen nucleus ( 1 H) to quantify chemicals is called quantitative 1 H-NMR ( 1 H qNMR). The quantitative method using 1 H qNMR for determining the purity or content of chemicals has been adopted into some compendial guidelines and official standards. However, there are still few reports in the literature regarding validation of 1 H qNMR methodology. Here, we coordinated an international collaborative study to validate a 1 H qNMR based on the use of an internal calibration methodology. Thirteen laboratories participated in this study, and the purities of three samples were individually measured using 1 H qNMR method. The three samples were all certified via conventional primary methods of measurement, such as butyl p-hydroxybenzoate Japanese Pharmacopeia (JP) reference standard certified by mass balance; benzoic acid certified reference material (CRM) certified by coulometric titration; fludioxonil CRM certified by a combination of freezing point depression method and 1 H qNMR. For each sample, 1 H qNMR experiments were optimized before quantitative analysis. The results showed that the measured values of each sample were equivalent to the corresponding reference labeled value. Furthermore, assessment of these 1 H qNMR data using the normalized error, E n -value, concluded that statistically 1 H qNMR has the competence to obtain the same quantification performance and accuracy as the conventional primary methods of measurement.

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The``qNMR test'' has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019,ˆfteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the``qNMR test'' in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b 2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76％ relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.

Quantitative 1 H-NMR ( 1 H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the 31 P nucleus because of the simplicity of its signals and previously reported 31 P-qNMR in D2O. Here we report 31 P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d4 were used as reference standards for 31 P-qNMR and 1 H-qNMR, respectively, in methanol-d4. The purity of SOF determined by 31 P-qNMR was 100.63 ± 0.95%, whereas that determined by 1 H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the 31 P signal of PAA and SOF were 3.38 ± 2.39 Hz and 2.22 ± 0.19 Hz, respectively, suggesting that the T2 relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, DMSO-d6, was used as the dissolving solvent with PAA and DSS-d6 as reference standards for 31 P-qNMR and 1 H-qNMR, respectively. SOF purities determined by 31 P-qNMR and 1 H-qNMR were 99.10 ± 0.30% and 99.44 ± 0.29%, respectively. SOF purities determined by 31 P-qNMR agreed with the established 1 H-qNMR values, suggesting that an aprotic solvent is preferable for 31 P-qNMR because it is unnecessary to consider the effect of deuterium exchange.