[Quantitative Determination Method of Aconitum Monoester Alkaloids Using Relative Molar Sensitivity (RMS) for the Assay in the Japanese Pharmacopoeia].

Recently, a novel quantitative method using relative molar sensitivity (RMS) was applied to quantify the ingredients of drugs and foods. An important development in this regard can be observed in the Japanese Pharmacopoeia (JP) 18, where the quantification of perillaldehyde, an unstable compound, in crude drug "Perilla Herb," was revised to incorporate the RMS method. In this study, the primary objective was to improve the tester safety and reduce the amount of reagents used in the JP test. To achieve this, the quantification of three toxic Aconitum monoester alkaloids (AMAs) was explored using the RMS method, employing a single reference compound for all three targets. These AMAs, namely benzoylmesaconine hydrochloride, benzoylhypaconine hydrochloride, and 14-anisoylaconine hydrochloride, which are the quantitative compounds of Kampo extracts containing Aconite Root (AR), were quantified using the reference compound benzoic acid (BA). Reliable RMS values were obtained using both 1H-quantitative NMR and HPLC/UV. Using the RMS of three AMAs relative to the BA, the AMA content (%) in commercial AMAs quantitative reagents were determined without analytical standards. Moreover, the quantitative values of AMAs using the RMS method and the calibration curve method using the three analytical standards were similar. Additionally, similar values were achieved for the three AMAs in the Kampo extracts containing AR using the RMS and the modified JP18 calibration curve methods. These results suggest that the RMS method is suitable for quantitative assays of the Kampo extracts containing AR and can serve as an alternative to the current method specified in the JP18.

Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 2).

As a new absolute quantitation method for low-molecular compounds, quantitative NMR (qNMR) has emerged. In the Japanese Pharmacopoeia (JP), 15 compounds evaluated by qNMR are listed as reagents used as the HPLC reference standards in the assay of crude drug section of the JP. In a previous study, we revealed that humidity affects purity values of hygroscopic reagents and that (i) humidity control before and during weighing is important for a reproducible preparation and (ii) indication of the absolute amount (not purity value), which is not affected by water content, is important for hygroscopic products determined by qNMR. In this study, typical and optimal conditions that affect the determination of the purity of ginsenoside Rb1 (GRB1), saikosaponin a (SSA), and barbaloin (BB) (i.e., hygroscopic reagents) by qNMR were examined. First, the effect of humidity before and during weighing on the purity of commercial GRB1, with a purity value determined by qNMR, was examined. The results showed the importance afore-mentioned. The results of SSA, which is relatively unstable in the dissolved state, suggested that the standardization of humidity control before and during weighing for a specific time provides a practical approach for hygroscopic products. In regard to BB, its humidity control for a specific time, only before weighing, is enough for a reproducible purity determination.

[Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 1)].

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, fifteen 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 b2 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.

Collaborative Study to Validate Purity Determination by 1H Quantitative NMR Spectroscopy by Using Internal Calibration Methodology.

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 (1H) to quantify chemicals is called quantitative 1H-NMR (1H qNMR). The quantitative method using 1H 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 1H qNMR methodology. Here, we coordinated an international collaborative study to validate a 1H 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 1H 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 1H qNMR. For each sample, 1H 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 1H qNMR data using the normalized error, En-value, concluded that statistically 1H qNMR has the competence to obtain the same quantification performance and accuracy as the conventional primary methods of measurement.

Convallasaponin A, a new 5beta-spirostanol triglycoside from the rhizomes of Convallaria majalis.

The rhizomes of Convallaria majalis have been analyzed for their steroidal glycoside constituents, resulting in the isolation of a new 5beta-spirostanol triglycoside, named convallasaponin A, along with two known cardenolide glycosides and a known cholestane glycoside. The structure of convallasaponin A was determined on the basis of extensive spectroscopic analysis, including 2D NMR data, and the results of hydrolytic cleavage. The cardenolide glycosides showed tumor specific cytotoxic activity.