Two reference substances for determination of multiple components (I) : linear calibration using two reference substances for identification of chromatographic peaks

Objective: To establish a new substitute reference substance method named as "two reference substances for determination of multiple components(TRSDMC) "for multi-components assay of rhubarb and its preparations.Methods: The peak area and retention time of the five free anthraquinones in rhubarb were determined on five different HPLC instruments with 14 C18 columns of different brands and types,then the data analysis was conducted.For the quantitative analysis,aloe-emodin was used as the reference substance for determination of relative correction factors of rhein,emodin,chrysophanol and physcion.For the qualitative analysis,both aloe-emodin and physcion were served as linear calibration standards to forecast the retention time of the other three components.Results: In quantitative aspect,the relative correction factors determined were stable in a certain variation range of column temperature,flow rate and proportion of the mobile phase.In qualitative aspect,linear calibration using two reference substances was proved to be more accurate than the relative retention time method for chromatographic peak identification by validation tests with 14 columns.Conclusion: It is demonstrated that TRSDMC is practical and promising for multi-component assay of traditional Chinese medicine.