| Summary: | A reference extractive, containing multiple active known compounds, has been considered to be an alternative to individual reference standards. However, in the Chinese Pharmacopoeia (ChP) the great majority of reference extractives have been primarily used for qualitative identification by thin-layer chromatography (TLC) and few studies on the applicability of reference extractives for quantitative analysis have been presented. Using Salvia miltiorrhiza Bunge as an example in this paper, we first present a preliminary discussion on the feasibility and applicability of reference extractives for the quantitative analysis of TCMs. The reference extractive of S. miltiorrhiza Bunge, comprised of three pharmacological marker compounds, namely cryptotanshinone, tanshinone I and tanshinone IIA, was prepared from purchased Salvia miltiorrhiza Bunge by extraction with acetone under reflux, followed by silica gel column chromatography with stepwise elution with petroleum ether-ethyl acetate (25:1, v/v, 4.5 BV) to remove the non-target components and chloroform-methanol (10:1, v/v; 3 BV) to yield a crude reference extractive solution. After concentration, the solution was further purified by preparative reversed-phase HPLC on a C18 column with isocratic elution with 77% methanol aqueous solution to yield the total reference extractive of S. miltiorrhiza Bunge. Thereafter, the reference extractive was applied to the quality assessment of S. miltiorrhiza Bunge using high-performance liquid chromatography (HPLC) coupled with diode array detection (DAD). The validation of the method, including linearity, sensitivity, repeatability, stability and recovery testing, indicated that this method was valid, reliable and sensitive, with good reproducibility. The developed method was successfully applied to quantify seven batches of samples collected from different regions in China and the results were also similar to those obtained using reference standards, with relative standard deviation (RSD) <3%. Preparation of a reference extractive of S. miltiorrhiza Bunge was significantly less expensive and time consuming than preparation of a corresponding reference standard. Quantitative analysis using a reference extractive was shown to be simple, low-cost, time-saving and practical, with high sensitivity and good stability; and is, therefore, a strong alternative to the use of reference standards.
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