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Kava, the rhizomes and roots of Piper methysticum Forst, is a popular edible medicinal herb traditionally used to prepare beverages for anxiety reduction. Since the German kava ban has been lifted by the court, the quality evaluation is particularly important for its application, especially the flavokawains which were believed to be responsible for hepatotoxicity. Now, by employing two different standard references and four different methods to calculate the relative correction factors, eight different quantitative analyses of multicomponents by single-marker methods have been developed for the simultaneous determination of eight major kavalactones and flavokawains in kava. The low standard method difference on quantitative measurement of the compounds among the external standard method and ours confirmed the reliability of the mentioned methods. A radar plot clearly illustrated that the contents of dihydrokavain and kavain were higher, whereas flavokawains A and B were lower in different kava samples. Only one of eight samples did not detect flavokawains that may be related to hepatotoxicity. In summary, by using different agents as an internal standard reference, the developed methods were believed as a powerful analytical tool not only for the qualitative and quantitative of kava constituents but also for the other multicomponents when authentic standard substances were unavailable.

Multi-component analysis is one of the key techniques for the overall quality control of traditional Chinese medicines. However, the shortage and high cost of reference substances are the greatest obstacles. The substitute method is an alternative solution. In the present study, 11 compounds of rhubarb were simultaneously determined by a method named "two reference substances for determination of multiple components", which includes a qualitative method with linear calibration using two reference substances and a quantitative method with a relative correction factor combined with ultra high performance liquid chromatography. Using aloe-emodin-8-O-β-D-glucopyranoside and chrysophanol as reference compounds, chromatographic peak identification was performed. The results demonstrated that linear calibration using two reference substances method showed higher accuracy, less deviation, and better column adaptability compared to the relative retention time method. Using chrysophanol as a reference compound, the relative correction factors were determined and showed good reproducibility and stability in different laboratories with different instruments, columns, and wavelength fluctuations. The results had no significant difference compared with the external standard method. The strategy of two reference substances for determination of multiple components coupled with ultra high performance liquid chromatography is economical, efficient, accurate, reliable, and environmentally friendly and is suitable for the quality control of traditional Chinese medicines.

Xingnaojing Injection is a traditional Chinese medicine extensively used for stroke and cerebral ischemia. For better in-process quality control of Xingnaojing Injection, a method for the analysis of its intermediate (i.e., the distillate of Fructus Gardeniae and Radix Curcumae) is needed to monitor and optimize the hydrodistillation extraction process. In this work, nine major volatile components in the intermediate were identified: isophorone, 4-methylene-isophorone, curcumenone, curcumenol, curdione, curzerenone, furanodienone, curcumol, and germacrone. A quantitative analysis of multi-component with a single-marker method based on high-performance liquid chromatography with diode array detection was developed for the simultaneous determination of the nine components. In this method, only curdione was needed as the reference substance, and the other eight components were determined using their relative correction factors to curdione. In the method validation, good linearity (r > 0.9999), sensitivity, repeatability, and accuracy (recoveries within 95.3-105.4%) were shown. The repeatability and robustness of the relative correction factors were studied with different column temperatures, flow rates, detection wavelengths, columns, and instruments. In sample analyses, consistent results between the proposed method and the external standard method were shown. The proposed method provides a comprehensive and low-cost tool for the quality assessment of the intermediate of Xingnaojing Injection.

A quantitative analysis of multiple components with a single-marker method was established for the simultaneous determination of five sesqutiterpenoids in Xingnaojing injection. This method was established with Xingnaojing injection determined by high-performance liquid chromatography coupled with diode array detection. The durability and system suitability of the established method were evaluated, and the reliable relative correction factors were obtained with curdione selected as an internal reference. The contents of the five components in all Xingnaojing injections were determined by external standard method and the contents of curcumenone, curcumenol, curzerenone, and germacrone were also calculated with the obtained relative correction factors. Then, relative error was investigated to estimate the difference of the two methods. As a result, the established new method possesses good adaptability, and there is no significant difference between the two methods, except for the content of curzerenone in eight samples. To put the established method into practice, the limits of quantitation of the established method of the five components were proposed and defined. Thus, the developed methodology can also be utilized to the quality evaluation of Xingnaojing injection, in spite of the difference found in the content of curzerenone between the external standard method and the newly established method.