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. 2021 Jul;32(4):466-473.
doi: 10.1002/pca.2994. Epub 2020 Sep 14.

Quantification by LC-MS/MS of astragaloside IV and isoflavones in Astragali radix can be more accurate by using standard addition

Bijay Kafle  1 , Jan Baak  2   3 , Cato Brede  1   4
Affiliations

Quantification by LC-MS/MS of astragaloside IV and isoflavones in Astragali radix can be more accurate by using standard addition

Bijay Kafle et al. Phytochem Anal. 2021 Jul.

Abstract

Introduction: Astragali radix (AR), the root of Astragalus, is an important medical herb widely used in traditional Chinese medicine. Bioactive components include isoflavones and a unique class of triterpenoid saponins (named astragalosides).

Objectives: Accurate measurement of bioactive components, especially astragaloside IV, is necessary for confirming AR authenticity, quality control and future medical research.

Methodology: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a suitable technique but suffers from ion suppression effects due to sample matrix. This can be corrected by using isotopic labelled internal standards, but these are not available for many phytochemicals. We explored the use of standard addition to circumvent this issue.

Results: LC-MS/MS and liquid chromatography coupled with ultraviolet (LC-UV) detection provided linear calibration curves (R2 > 0.99). LC-MS/MS provided superior selectivity and detection limits below 10 ng/mL, which was 2-3 magnitudes lower than LC-UV detection. Precision and accuracy were overall improved by using LC-MS/MS with diluted sample extracts, resulting in an inter series coefficient of variation (CV) of 12% or less and mean recovery estimates in the 85-115% range. LC-MS/MS quantification by standard addition resulted in significantly higher concentrations of astragaloside IV measured in the samples. Concentrations calculated by standard addition were unaffected by large variation in signal response caused by matrix effects, independent of variation in slope of the standard addition curves.

Conclusion: Sample dilution was helpful but not sufficient for reducing effects of ion suppression. We have shown that LC-MS/MS quantification by standard addition can be a powerful approach for accurate measurement of phytochemicals in the absence of isotopic labelled internal standards.

Keywords: Astragalus; LC-MS/MS; astragaloside; isoflavone; standard addition.

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