Purpose: Purity determination of analytical standards for liquid chromatography is generally implemented by the area normalisation method with chromatography; however, this method cannot be always considered accurate, for reasons such as the presence of undetectable impurities. Here we described the application of purity determination of marker compounds for HPLC assays of crude drugs and their related extracts in the Japanese Pharmacopeia (JP) using an efficient and absolute quantification method for assuring the reliability.
Methods: Quantitative nuclear magnetic resonance (qNMR) efficiently achieves absolute purity determination that has traceability to the SI, and hence has recently attracted considerable attention in analytical chemistry. We applied this absolute quantification method to the purity determination of marker compounds for HPLC assays of crude drugs and their related extracts in JP. We adopted an internal standard method known as AQARI (Accurate QuAntitative NMR with Internal reference substance), which in principle is the most accurate among the multiple qNMR methods available. For internal reference substance (IRS), we utilized certified reference materials with SI traceability of 1,4-BTMSB-d4 or DSS-d6 (Wako Pure Chemical Industries, Ltd.) that emit specific signals close to 0 ppm. The sample weighed from 5 mg to 10 mg; 1 mg of IRS was measured out; both the weighed sample and IRS were dissolved in 1 ml of deuterated solvent to give the sample solution. An ultra-micro balance was used to determine the accurate weight of the sample and IRS. A 400MHz or higher NMR instrument was used for the measurement, to ensure sensitivity and resolution. The 13C satellite can be the cause of error when it overlaps with proximal signals, and hence, decoupling was performed. MPF8, which has good decoupling efficiency, was used for the pulse sequence. When the relaxation delay time is short, signal saturation occurs and then an accurate quantitative result cannot be obtained. Based on the aforementioned information, the relaxation delay was set to 60 s or longer. A system suitability test was introduced and the presence of impurity duplications in the target signal was checked. Validation test were conducted at multiple organisations based on these set conditions.
Results: From the results of the validation tests, it was ascertained that absolute purity determination was possible while ensuring accuracy of 2 significant digits, including errors among the instruments in use, when the compound had a molecular weight of around 300. Until JP 17th edition, we have built a supply system of seven HPLC analytical standards (magnolol, geniposide, paeonol, magnoflorine iodide, saikosaponin b2, (E)-cinnamic acid, and rosmarinic acid), of which their purities are determined by qNMR, used for assay of crude drugs and related extracts in JP. These analytical standards are currently available on the reagent market.
Conclusions: We established a supply system of HPLC analytical standards of which their purities are determined by qNMR. These standards have absolute purity values and are designated as the marker compounds used for assay of crude drugs and related extracts in JP. Our researches for adaption of qNMR to JP marker compounds contribute to the assuring of analytical values in the field of pharmaceuticals will continue after JP17.