Determination of Saikosaponin Derivatives in Radix bupleuri and in Pharmaceuticals of the Chinese Multiherb Remedy Xiaochaihu-tang Using Liquid Chromatographic Tandem Mass Spectrometry

Abstract

Saikosaponins are bioactive oleanane saponins derived from the Chinese medicinal herb Radix bupleuri ("chaihu" in Chinese). An LC-MS/MS-based method has been developed for characterization and quantification of 15 saikosaponin derivatives (saikosaponin a, saikosaponin b(1), saikosaponin g, saikogenin A, saikogenin H, saikosaponin c, saikosaponin h, saikosaponin i, prosaikogenin C(2), prosaikogenin B(2), saikogenin C, saikogenin B, saikosaponin d, saikosaponin b(2), and saikogenin D) in one chromatographic run. Optimization of the ionization process was performed with electrospray and atmospheric pressure chemical ionization techniques in both positive and negative ion modes. Negative ion ESI was adopted for generation of the precursor deprotonated molecules to achieve the best ionization sensitivity for the analytes. In addition, the most abundant fragment ion was chosen for each analyte to give the best CID sensitivity. Because some of the saponin derivatives are isomeric, complete resolution for the whole analytes was achieved both chromatographically and mass spectroscopically. Furthermore, optimal internal standard was successfully discovered for determination of the analytes by making use of a combinatorial chemistry approach. Good linearity over the range approximately 1.65 or 4.98 to 1200 ng/mL for the analytes was observed. The intraday accuracy and precision at nominal low, intermediate, and high concentration varied between 0.8 and 11.8% and between 80 and 116%, respectively, whereas those for interday assay were between 1.1 and 15.5% and between 86 and 119%, respectively. The lower limits of quantitation for the test compounds were approximately 16.5 to 49.4 pg on-column. The new method offered higher sensitivity and greater specificity than previously reported LC methods. After the validation, the applicability of the method for determination of these chemicals present in a variety of crude chaihu roots and in different brands of the Chinese multiherb remedy Xiaochaihu-tang (or Shosaiko-to) extract granules has been demonstrated. The sensitivity and specificity of the technique will be the basis of a method for the accurate quantification of the saikosaponin derivatives in biomatrixes.

Simultaneous quantification of multiple licorice flavonoids in rat plasma

“…We found that the concentration of HCOONH 4 had only negligible effects on the chromatographic peak widths, peak asymmetry, and peak retention times. Furthermore, a mobile phase containing 0.01‰ HCOONH 4 resulted in minimal matrix effects for all of the tested flavonoids.…”

“…Electrospray ionization was used to efficiently generate precursor deprotonated molecules of all the analytes and the [M-H] Ϫ ions were used to produce characteristic product ions for MS/MS analysis. We found that inclusion of a very low concentration of HCOONH 4 (0.01‰) in the LC mobile phase dramatically improved the detection limit for the tested flavonoids and decreased the interference by matrix effects, which have been referred to as "LC-electrolyte effects." Liquid-liquid extraction with ethyl acetate was effective for isolation of all the analytes and resulted in the lowest matrix effects of several tested sample cleanup methods.…”

“…Finally, we demonstrated the applicability of this method in a pilot pharmacokinetic study of rats receiving an oral dose of Xiaochaihu-tang, an important Chinese herbal remedy for chronic hepatitis. The use of a low concentration of HCOONH 4 number of chemical constituents that may have pharmacological activities or therapeutic effects. Because pharmacokinetic information is needed for the design and interpretation of toxicity and clinical studies, monitoring the blood levels of known active constituents, representative markers, or major chemical constituents in botanical drug products is strongly encouraged by the U.S. Food and Drug Administration (FDA) [1].…”

“…As part of our ongoing studies of Xiaochaihu-tang [4,5] [6]. We report the development of an LC-MS/MS-based method that can simultaneously quantify six licorice flavonoids in rat plasma.…”

Development and Validation of Analytical Methods for Pharmaceuticals

“…HPLC with a mass spectrometer detector (LC-MS) [39,40] showed superior sensitivity and selectivity compared to HPLC-UV methods [41].…”