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<p>Withaferin A (WFA) is a bioactive compound known for its potent anticancer, anti-inflammatory, and adaptogenic properties, predominantly derived from the medicinal plant <i>Withania somnifera</i> (L.) Dunal, commonly known as Ashwagandha. The concentration of WFA in field-cultivated plants exhibits significant variability in different parts and is also influenced by environmental parameters. The extraction protocols for WFA to date reveal significant variability, emphasizing the need for a standardized method to ensure accurate quantification. Hence, in the present study, we evaluated various extraction solvents, durations, pH levels, and purification methodologies to improve recovery rates and analytical precision. Our findings indicated that methanol served as the most efficient extraction solvent, with optimal extraction achieved through 25&#xa0;min of sonication at neutral pH. Moreover, employing C18 solid-phase extraction with a 40&#xa0;mg sorbent for 1-min reduced matrix interference. The linear range was between 2.5 and 250&#xa0;ng/g of WFA, with R<sup>2</sup> of 0.999. The LOD and LOQ were 1.2&#xa0;ng/g and 3.09&#xa0;ng/g for the optimized analytical condition. Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC–MS/MS) found WFA at 9.42&#xa0;µg/g ± 6.53% in in vitro shoot cultures of <i>Withania somnifera</i>. In contrast, field-grown shoots had significantly higher levels at 4518.55&#xa0;µg/g ± 82.07%, which may be toxic if used in formulations directly. This requires an efficient extraction protocol and estimation method. The downstream processing of field-grown shoots may be overcome using in vitro cultures grown in a controlled environment and consistent metabolite production, highlighting their potential for standardized and optimized WFA production. This method offers high-sensitivity and specificity for quantifying WFA in complex matrices, improving metabolite yield and analytical reliability for future research and standardization of plant-derived products.</p>

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Quantification of Withaferin A: An Optimization Study for Sample Pre-treatment Process Coupled with UHPLC–MS/MS Determination

  • 期刊论文

  • 发表日期：

    2025年10月29日

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  • Aparnapreethi Rajendran,
  • Karthikeyan Prakasham,
  • Vellaikumar Sampathrajan,
  • Kanimozhi Natarajan,
  • Ramya Palanisamy,
  • Senthil Natesan,
  • Kalaiselvi Senthil

摘要

Withaferin A (WFA) is a bioactive compound known for its potent anticancer, anti-inflammatory, and adaptogenic properties, predominantly derived from the medicinal plant Withania somnifera (L.) Dunal, commonly known as Ashwagandha. The concentration of WFA in field-cultivated plants exhibits significant variability in different parts and is also influenced by environmental parameters. The extraction protocols for WFA to date reveal significant variability, emphasizing the need for a standardized method to ensure accurate quantification. Hence, in the present study, we evaluated various extraction solvents, durations, pH levels, and purification methodologies to improve recovery rates and analytical precision. Our findings indicated that methanol served as the most efficient extraction solvent, with optimal extraction achieved through 25 min of sonication at neutral pH. Moreover, employing C18 solid-phase extraction with a 40 mg sorbent for 1-min reduced matrix interference. The linear range was between 2.5 and 250 ng/g of WFA, with R2 of 0.999. The LOD and LOQ were 1.2 ng/g and 3.09 ng/g for the optimized analytical condition. Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC–MS/MS) found WFA at 9.42 µg/g ± 6.53% in in vitro shoot cultures of Withania somnifera. In contrast, field-grown shoots had significantly higher levels at 4518.55 µg/g ± 82.07%, which may be toxic if used in formulations directly. This requires an efficient extraction protocol and estimation method. The downstream processing of field-grown shoots may be overcome using in vitro cultures grown in a controlled environment and consistent metabolite production, highlighting their potential for standardized and optimized WFA production. This method offers high-sensitivity and specificity for quantifying WFA in complex matrices, improving metabolite yield and analytical reliability for future research and standardization of plant-derived products.