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Recent findings by high-performance thin-layer chromatographic separation for a comprehensive analysis of Withania somnifera by densitometry and mass spectrometry: an assessment to quality and adulteration

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Abstract

Withania somnifera (L.) Dunal (WS), also known as Ashwagandha, is an Indian herb classified as Rasayana in Ayurveda owing to its rejuvenating and health-promoting activities. A sensitive and robust high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of withanoside IV (WS4), withanoside V (WS5), withaferin A (WFA), and kaempferol-based glucoside (KRG) in the roots and aerial parts of WS. The present rapid HPTLC method reports the separation and simultaneous quantification of three diverse classes of WS as withanolide (WFA), withanosides (WS4 and WS5) with an analytical marker flavonoid glycoside (KRG) for effective detection, of the aerial parts separating them from roots. In normal-phase TLC plates, these markers showed good resolution in ethyl acetate‒chloroform‒methanol‒water (8:3:4.4:1.8, V/V) solvent system. Densitometric analysis was performed for KRG (at 254 nm), WFA, WS4, and WS5 (at 540 nm after derivatization) with characterization by high-performance thin-layer chromatography‒tandem mass spectrometry (HPTLC‒MS/MS). The method was found linear (R2 > 0.99) for KRG and WFA (200‒1000 ng/band) and WS4‒WS5 (400‒2000 ng/band) with excellent recoveries (80‒100%) for all compounds. In this HPTLC based assessment, three compounds of WS could be rapidly detected for quality control distinguishing from other Withania and plant species reported as adulterants. Additionally, this method separated pigment-based compounds at 366 nm in chemical fingerprinting in WS aerial parts samples. Therefore, the presence of KRG as an analytical marker with these pigment zones can identify aerial parts, leaves, stems, fruits with calyx, and fruits in the HPTLC analysis, separating them from root samples. Hence, this HPLTC method was found robust and economical for the estimation and surety of the quality of WS samples in quantitative and qualitative analysis.

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Acknowledgements

The authors express their gratitude to Mr. Shubham Jagtap and Mr. Ganesh Saste (Pharmanza Herbal Pvt. Ltd.) for their LC–MS/MS data analysis assistance. The authors are also grateful to Anchrom Lab (Mumbai) for providing instrumental facility for the study.

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The authors did not receive support from any organization for the submitted work.

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Authors and Affiliations

  1. Pharmanza Herbal Pvt. Ltd., Anand, Gujarat, 388430, India

    Sautik Ghoshal, Chetana Ghule, Amit Mirgal, Aboli Girme & Lal Hingorani

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Correspondence to Aboli Girme.

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Ghoshal, S., Ghule, C., Mirgal, A. et al. Recent findings by high-performance thin-layer chromatographic separation for a comprehensive analysis of Withania somnifera by densitometry and mass spectrometry: an assessment to quality and adulteration. JPC-J Planar Chromat 35, 439–451 (2022). https://doi.org/10.1007/s00764-022-00187-z

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