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. 2025 Sep;417(21):4895-4906.
doi: 10.1007/s00216-025-06006-8. Epub 2025 Jul 22.

A multi-detector analytical approach for characterizing complex botanical extracts: a case study on ashwagandha

Vincent P Sica  1 , Constance A Mitchell  2 , Julie Krzykwa  3 , Timothy R Baker  1 , Suramya Waidyanatha  4
Affiliations

A multi-detector analytical approach for characterizing complex botanical extracts: a case study on ashwagandha

Vincent P Sica et al. Anal Bioanal Chem. 2025 Sep.

Abstract

A comprehensive analytical characterization of botanical extracts can be difficult due to their complexity, dynamic range, and limited availability of constituent standards. By using ashwagandha root extract as a case study, this work showcases how to characterize a botanical extract utilizing an analytical system with multiple detectors to support the development of toxicological tools for evaluating botanicals. The platform incorporated ultra-high-performance liquid chromatography (UHPLC) coupled with photodiode array (PDA) detection, charged aerosol detection (CAD), and high-resolution mass spectrometry (HRMS) detection to generate a detailed chemical profile. This multi-detector platform enabled both semi-quantification and the identification of the majority of constituents, ensuring accurate chemical analysis while compensating for potential detector biases. The approach provided a thorough fingerprint of the ashwagandha extract, enabling authentication of the material. The generation of a comprehensive fingerprint supports in silico modeling and bioassay-based toxicological evaluations of botanicals, particularly in the context of dietary supplement safety. This study demonstrated the utility of detailed chemical analysis supporting the authenticity of ashwagandha root extract and the advancement of tools needed for robust safety assessments of botanical products by providing the semi-quantification and identification of over 60 constituents in ashwagandha extracts.

Keywords: Ashwagandha root extract; Complex mixtures; Multi-detector platform; Ultra-high-performance liquid chromatography.

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Conflict of interest statement

Declarations. Ethics approval: N/A. Statement on animal welfare: N/A. Source of biological material: N/A. Conflict of interest: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The following authors are employed in the personal care product industry (at the time of article submission): V.S. and T.B. The opinions presented here are those of the authors. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Overall strategy to select, source, and support evidence for the authenticity and chemical characterization of ashwagandha root extract. Figure adapted from Waidyanatha et al. 2024
Fig. 2
Fig. 2
The chromatograms generated from a single injection across the multi-detector platform
Fig. 3
Fig. 3
CAD chromatogram labeling the 46 quantified peaks that were above the AET (6.75 µg/mL) for ashwagandha
Fig. 4
Fig. 4
a Correlation of m/z signals to begin determining the number of constituents contributing to the quantified CAD peak 26. b Using extracted ion chromatograms to confirm alignment of the MS signals with the CAD peak and to determine if any adducts or in-source fragments were missed during the process
Fig. 5
Fig. 5
(Top) Comparison of the retention time (37.2 min) between the withanoside IV standard and peak 26. (Bottom) Comparison of the fragmentation of withanoside IV and the ashwagandha constituent 26a
See this image and copyright information in PMC

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