ABSTRACT
Pharmacopeias are essential for ensuring the quality of botanical raw materials, yet their utility is limited by slow adaptation to rapid innovation in the dietary supplement industry. Challenges arise from differing standards (e.g., the British Pharmacopoeia (BP) and the United States Pharmacopoeia (USP)), as well as a critical lack of comparative data for globally sourced ingredients. The post‐COVID surge in adaptogens, such as ashwagandha, underscores the urgency of these issues. The commercial use of aerial parts—despite traditional root‐only use and phytochemical variability—raises concerns about quality and safety. The situation necessitates rigorous source verification and re‐evaluation of current pharmacopeial methods. To address these deficiencies, we developed and compared an HPLC‐PDA method utilizing BP and USP standards to quantify three steroidal lactones (withaferin A, withanolide A, and withanoside IV) in ashwagandha samples and commercial products. Our analysis revealed substantial variations in steroidal lactone content. Alarmingly, over 44% of products failed to meet BP standards, and 60% failed to meet USP standards. The high failure rate was attributed to the elevated presence of co‐eluted dihydrowithaferin sulfate, primarily found in leaf samples, which led to an underestimation of withanolide A. Only 10 of 25 supplement products met both standards; only two were confirmed as root‐derived, while the remainder contained varying proportions of (un)disclosed aerial parts. These findings highlight critical plant‐part‐specific chemical variations and the need for enhanced source verification and improved quality control. Developing robust analytical methods and revisiting existing pharmacopeial guidelines are crucial for assessing the quality and safety of ashwagandha products, including novel formulations.
Keywords: ashwagandha, dietary supplements, HPLC‐PDA, pharmacopeial methods, quality
1. Introduction
Pharmacopeias play a crucial role in ensuring the quality and safety of botanical raw materials by providing authoritative standards and analytical methodologies. Their core functions include maintaining consistent product quality, safeguarding public health, facilitating international trade, and guiding industry and regulators (Coates et al. 2024; Wiggins and Albanese 2019; Zöllner and Schwarz 2013). Conversely, many pharmacopeias struggle to keep pace with the rapid innovation and the diversity of novel botanical dietary supplement formulations. Monographs for emerging ingredients or advanced processing techniques are often absent, creating a critical gap that necessitates continuous updates and global harmonization. Further complicating standardization are the inherent differences between leading pharmacopeias, such as the United States Pharmacopeia (USP) and the British Pharmacopoeia (BP). Both champion public health, but their legal authorities, scopes, and technical specifications vary considerably. The USP is legally binding under US federal law, whereas the BP holds equivalent legal weight in the United Kingdom and Commonwealth nations. These distinct regulatory philosophies yield unique monographs, preferred analytical methodologies, and divergent specifications. A notable gap exists in the direct comparison and assessment of the applicability of these pharmacopeial standards to a single‐source raw material, a crucial aspect that remains uninvestigated. As the dietary supplement industry globalizes and botanical complexity increases, aligning pharmacopoeial standards becomes increasingly imperative. Harmonization is essential to ensure consistent product quality, resolve regulatory discrepancies, and satisfy consumer expectations for safe and efficacious natural health products (Coates et al. 2024; Wiggins and Albanese 2019; Zöllner and Schwarz 2013).
The post‐COVID era has witnessed a notable surge in adaptogenic botanicals, moving them into mainstream wellness due to increased global focus on holistic health and the pervasive impact of pandemic‐related stress and fatigue. This surge highlights the necessity and applicability of pharmacopeial standards. Among these, ashwagandha ( Withania somnifera (L.) Dunal) is a prominent candidate (Mallinson et al. 2025). Traditionally used in Ayurvedic medicine for its immune‐strengthening and anti‐inflammatory effects, its relevance is further underscored by recent trials exploring its efficacy in mitigating symptoms such as fatigue, muscle weakness, cognitive dysfunction, and poor mental health (Mikulska et al. 2023; Wiciński et al. 2024). The herb's unique steroid‐derived molecules, withanolides (WLs), are recognized for their ability to modulate inflammatory pathways and serve as key quality markers (Bashir et al. 2023; Kapoor 2001; Paul et al. 2021). While classical texts and pharmacopeias (e.g., USP, BP, Indian Pharmacopeia, and the Ayurvedic Pharmacopeia of India) primarily designate the root as medicinally important, standardizing extracts to WLs content along with rising global demand has led to the commercial introduction of extracts from the aerial parts. Despite similar qualitative WLs fingerprints, aerial extracts exhibit significant quantitative and qualitative differences in other phytochemicals when compared to roots, impacting overall quality, efficacy, and safety (Chatterjee et al. 2010; Kaul et al. 2016). Such disparities have resulted in “standardized” products with potentially misleading plant part declarations, even with pharmacopoeial standards in place (e.g., USP requiring not less than (NLT) 0.3% and 2.5%) combined withanolide A (WL A) and withanoside IV (WS IV) for root powder and root extract on a dry basis, respectively (United States Pharmacopeia, 2024); BP specifying NLT 0.01% withaferin A and WL A for root powder on a dry basis (British Pharmacopoeia, 2024). The ongoing debate over plant‐part specific chemical disparities, quality assurance, and safety requires rigorous verification of the plant source (leaf, root, or both) and the development of specific analytical methods to detect aerial parts in extracts.
2. Materials and Methods
2.1. Chemicals and Standards
Chemical structures and UV spectra of three reference compounds, are presented in Figure 1. Kaempferol‐3‐O‐robinoside‐7‐O‐glucoside was purchased from the USP (Frederick, MD 21701, USA), and quercetin‐3‐O‐robinobioside‐7‐O‐glucoside was purchased from Advanced CHEMBLOCK (Hayward, CA 94545, USA). WS IV (1), withaferin A (2), and WL A (3) were obtained from Cayman Chemical (Ann Arbor, MI, USA). The purity of all reference compounds exceeded 97% except withaferin A, which was 92% pure.
FIGURE 1.
Chemical structures and UV spectra of three reference standards used in the Withania somnifera quantification study.
Acetonitrile, methanol, ethanol, and formic acid used for extraction were high‐performance liquid chromatography (HPLC)‐grade reagents purchased from Fisher Scientific (Suwanee, GA, USA). Potassium dihydrogen phosphate and orthophosphoric acid were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Water for the mobile phase was purified using a Milli‐Q water purification system (Millipore).
2.2. Plant Materials and Dietary Supplements
Five morphologically characterized root samples and three leaf samples (accession numbers #5218, 6087, 26125, 26128, 26131 for roots; #26129, 26132, 26142 for leaves) were included. Additionally, we included seven W. somnifera extracts from various sources: three root extracts (#4343, 4652, 26148), one leaf extract (#4341), one aerial extract (#4651), one root and leaf blend extract (#4300), and two whole plant extracts (#4342, 26149). Twenty‐five dietary supplements claiming to contain only W. somnifera were purchased from Amazon.com (USA), priced between $14 and $40 per bottle of 60 capsules or tablets. All sample specimens were deposited at the National Center for Natural Products Research repository at the University of Mississippi, University, MS, USA.
2.3. Sample Preparation
All W. somnifera samples, whether raw materials or dietary supplements (capsules and tablets), were ground to a fine powder. For capsules, five were weighed, opened, their contents pooled, and triturated. An appropriate amount of powdered capsule contents or ground tablets (equivalent to the average dosage form weight), or approximately 500 mg of plant powder, was weighed. Each sample was then sonicated in 2.5 mL of methanol for 30 min, followed by centrifugation at 10,000 rpm for 15 min. The supernatant was transferred to a 10 mL volumetric flask. This extraction procedure was repeated three times, and the combined supernatants were brought to a final volume of 10 mL with methanol. Approximately 2 mL of each solution was filtered through a 0.45 μm PTFE membrane filter into an LC sample vial.
To aid in the assessment of the overall quality of ashwagandha supplements in commerce, we prepared a series of blends with controlled ratios (0%–100% leaf) of root (#26128) and leaf (#26129) material from a single‐sourced plant.
2.4. Instrumentation and Analytical Conditions
2.4.1. HPLC‐Photodiode Array Analysis (PDA)
The HPLC system used was Waters Alliance e2695, equipped with a 2998 photodiode array detector (Waters Corp., Milford, MA), and a computerized data station running Waters Empower 3 software.
2.4.1.1. USP Method
Separation according to the USP method was performed on a Luna 5 μm C18(2) column (100 Å, 250 × 4.6 mm, Phenomenex Inc., Torrance, CA) maintained at 27°C and equipped with a 2 cm C18 guard column (Phenomenex Inc.). The mobile phase consisted of potassium dihydrogen phosphate with phosphoric acid in water (A) and acetonitrile (B) applied in the following gradient elution: 0 min, 95% A: 5% B in the next 18 min to 55% A:45% B, in the next 7 min to 20% A:80% B (isocratic for 3 min), and finally to 100% B (held for 2 min). Following each run, a 3‐min wash with 100% acetonitrile and a 13‐min equilibration period were performed. The flow rate was 1.5 mL/min. A 20 μL sample was injected, with peak identification based on retention time comparison with standard compounds and UV–Vis spectral comparison with reference compounds at 227 nm.
2.4.2. BP Method
Chromatographic separation was performed according to the BP method, utilizing a Synergi Max‐RP 4 μm column (80 Å, 150 × 4.6 mm, Phenomenex Inc., Torrance, CA) at 50°C, with a 2 cm C18 guard column (Phenomenex Inc.) preceding the analytical column. The mobile phase was comprised of water (A) and ethanol/methanol (1:1) (B), and applied via the following gradient elution: 0 min, 65% A: 35% B (isocratic for 5 min), in the next 25 min to 55% A:45% B, in the next 1 min to 100% B (held for 2 min). Each run included a 3‐min wash with 100% acetonitrile and a 13‐min equilibration period. The flow rate was 1.0 mL/min. A 20 μL sample was injected, and peak identification relied on retention time comparison with standards WLs and UV–Vis spectral comparison with reference compounds at 230 nm.
3. Results and Discussion
An HPLC‐PDA method was developed to identify and quantify three marker compounds—WS IV (1), withaferin A (2), and WL A (3)—in eight dry plant samples (five root and three leaf), eight extracts (three root, one aerial, one leaf, two whole plant, and one root–leaf blend), and 25 commercial supplements, following USP and BP methodologies. Compound identification was confirmed by comparing retention times and UV spectra with authenticated reference standards. Quantification was performed using external calibration curves, with detection at 227 nm (USP) and 230 nm (BP). The quantitative results for all samples and products are presented in Table 1.
TABLE 1.
Content (mg/g, w/w) of compounds 1–3 from extracts, plant materials, and dietary supplements of Withania somnifera using LC‐UV at 227 nm for USP method and 230 nm for BP method.
| # | NCNPR # | 1 (withanoside IV) | 2 (withaferin A) | 3 (withanolide A) | |||
|---|---|---|---|---|---|---|---|
| USP method | BP method | USP method | BP method | USP method | BP method | ||
| Extracts (mg/g) | |||||||
| 1 | 4652‐Root | 8.5 ± 0.2 | 8.2 ± 0.1 | 6.1 ± 0.4 | 6.4 ± 0.9 | 3.2 ± 0.7 | 3.4 ± 1.2 |
| 2 | 4651‐Aerial | 7.9 ± 0.1 | 8.1 ± 0.1 | 20.7 ± 1.5 | 20.9 ± 1.1 | 0.6 ± 1.1 | NA |
| 3 | 4300‐L + R | 29.5 ± 0.8 | 30.0 ± 0.5 | 49.3 ± 0.6 | 49.9 ± 0.4 | 0.1 ± 0.6 | NA |
| 4 | 4341‐Leaf | 11.7 ± 1.1 | 12.1 ± 1.1 | 20.1 ± 0.2 | 20.4 ± 0.8 | 2.4 ± 0.7 | NA |
| 5 | 4342‐Whole | 11.5 ± 0.6 | 11.7 ± 1.5 | 26.9 ± 0.5 | 27.5 ± 0.2 | 2.4 ± 1.6 | NA |
| 6 | 4343‐Root | 5.2 ± 1.4 | 5.4 ± 1.3 | 7.2 ± 0.9 | 7.5 ± 0.9 | 6.4 ± 1.6 | 6.5 ± 1.4 |
| 7 | 26148‐Root | 1.5 ± 0.3 | 1.5 ± 0.1 | 1.5 ± 1.9 | 1.3 ± 1.5 | 7.1 ± 0.9 | 7.6 ± 1.4 |
| 8 | 26149‐Whole | 3.9 ± 1.6 | 4.1 ± 1.5 | 3.6 ± 1.1 | 3.9 ± 1.6 | 1.6 ± 1.8 | NA |
| Plant materials (mg/g) | |||||||
| 9 | 5218‐R | 0.4 ± 1.7 | 0.4 ± 0.3 | 0.33 ± 0.2 | 0.37 ± 0.7 | 0.33 ± 0.9 | 0.34 ± 1.0 |
| 10 | 26131‐R | 0.003 ± 1.2 | 0.003 ± 0.5 | 0.002 ± 0.6 | 0.003 ± 0.5 | 0.1 ± 1.2 | 0.1 ± 0.7 |
| 11 | 26128‐R | 0.01 ± 0.5 | 0.01 ± 0.4 | 0.002 ± 0.3 | 0.002 ± 0.9 | 0.06 ± 3.0 | 0.06 ± 0.2 |
| 12 | 26125‐R | 0.31 ± 1.3 | 0.33 ± 1.4 | 0.23 ± 0.6 | 0.23 ± 1.9 | 0.3 ± 0.2 | 0.27 ± 0.6 |
| 13 | 6087‐R | 0.32 ± 0.8 | 0.34 ± 0.2 | 0.13 ± 0.8 | 0.14 ± 1.4 | 0.2 ± 2.6 | 0.2 ± 1.1 |
| 14 | 26132‐L | 0.86 ± 0.1 | 0.84 ± 0.2 | 1.05 ± 0.6 | 1.02 ± 0.5 | 0.02 ± 0.6 | 0.01 ± 0.4 |
| 15 | 26142‐L | 0.58 ± 0.4 | 0.56 ± 0.5 | 1.2 ± 0.4 | 1.2 ± 0.1 | 0.02 ± 0.7 | 0.02 ± 0.1 |
| 16 | 26129‐L | 0.78 ± 0.7 | 0.75 ± 1.8 | 1.2 ± 0.3 | 1.1 ± 0.1 | 0.04 ± 0.7 | 0.05 ± 1.2 |
| Dietary supplements (mg/dose) | |||||||
| 17 | 4301PR‐R | 0.75 ± 0.5 | 0.77 ± 0.8 | 0.7 ± 0.6 | 0.67 ± 0.4 | 4.6 ± 0.2 | 4.55 ± 0.1 |
| 18 | 4290PR‐R | 2.3 ± 0.3 | 2.2 ± 0.2 | 1.2 ± 0.1 | 1.0 ± 0.5 | 4.5 ± 0.1 | 4.4 ± 0.1 |
| 19 | 4294PR | 7.1 ± 0.1 | 7.2 ± 0.3 | 10.1 ± 0.4 | 10.2 ± 0.1 | 0.03 ± 0.3 | NA |
| 20 | 4293PR‐R + L | 3.9 ± 0.1 | 3.9 ± 0.3 | 6.3 ± 0.6 | 6.2 ± 0.3 | 0.06 ± 0.3 | NA |
| 21 | 4292PR‐R + L | 3.9 ± 0.2 | 3.8 ± 0.3 | 6.3 ± 0.1 | 6.6 ± 0.1 | 0.07 ± 0.2 | NA |
| 22 | 4279PR‐R + L | 10.0 ± 0.1 | 9.6 ± 0.1 | 11.7 ± 0.1 | 11.8 ± 0.1 | 0.02 ± 0.3 | NA |
| 23 | 4287PR‐R | 1.47 ± 0.2 | 1.45 ± 0.7 | 0.3 ± 0.6 | 0.3 ± 0.5 | 1.3 ± 0.5 | 1.28 ± 1.3 |
| 24 | 4283PR‐R | 1.33 ± 0.1 | 1.32 ± 0.2 | 1.3 ± 0.3 | 1.3 ± 1.0 | 1.4 ± 0.6 | 1.3 ± 0.8 |
| 25 | 4280PR‐R + L | 0.06 ± 0.1 | 0.05 ± 0.1 | 0.02 ± 0.9 | 0.02 ± 0.3 | 0.03 ± 0.2 | NA |
| 26 | 4291PR‐R + L | 0.08 ± 0.2 | 0.08 ± 0.7 | 0.14 ± 0.3 | 0.15 ± 1.5 | 0.04 ± 0.1 | NA |
| 27 | 4284PR‐R + L | 0.05 ± 1.8 | 0.05 ± 0.6 | 0.07 ± 0.5 | 0.06 ± 0.2 | 0.02 ± 0.4 | NA |
| 28 | 4288PR‐R + L | 0.17 ± 0.6 | 0.16 ± 0.2 | 0.2 ± 0.6 | 0.2 ± 1.3 | 0.02 ± 0.7 | NA |
| 29 | 4289PR‐R + L | 0.09 ± 0.1 | 0.09 ± 0.5 | 0.01 ± 0.1 | 0.01 ± 0.5 | 0.02 ± 0.8 | NA |
| 30 | 4286PR‐R | 0.06 ± 0.1 | 0.07 ± 1.2 | 0.18 ± 0.8 | 0.18 ± 0.7 | 0.2 ± 0.3 | 0.2 ± 1.4 |
| 31 | 4285PR‐R | 0.08 ± 1.1 | 0.08 ± 0.1 | 0.11 ± 0.9 | 0.12 ± 0.7 | 0.1 ± 0.5 | 0.1 ± 1.4 |
| 32 | 4282PR‐R | 0.04 ± 0.6 | 0.04 ± 1.7 | 0.17 ± 0.9 | 0.17 ± 0.8 | 0.2 ± 0.1 | 0.19 ± 0.4 |
| 33 | 2619PR‐R | 0.05 ± 0.6 | 0.06 ± 1.5 | 0.07 ± 2.1 | 0.07 ± 0.5 | 0.16 ± 1.1 | 0.16 ± 0.9 |
| 34 | 4281PR‐R + L | 3.3 ± 0.1 | 3.4 ± 0.2 | 2.73 ± 0.1 | 2.65 ± 0.1 | 0.02 ± 0.3 | NA |
| 35 | 4628‐Whole | 0.3 ± 0.4 a | 0.3 ± 0.6 a | 1.3 ± 0.5 a | 1.3 ± 1.0 a | 0.08 ± 0.4 a | NA |
| 36 | 4629‐R | ND | ND | ND | ND | ND | ND |
| 37 | 4630‐R | 2.1 ± 1.3 | 1.96 ± 1.3 | 3.92 ± 2.0 | 3.96 ± 0.1 | 1.18 ± 0.7 | 1.11 ± 0.1 |
| 38 | 4631‐R | 3.6 ± 1.1 | 3.8 ± 0.1 | 4.3 ± 2.1 | 4.4 ± 0.1 | 2.55 ± 0.3 | 2.36 ± 0.2 |
| 39 | 4632‐R + L | 0.08 ± 0.9 | 0.08 ± 0.1 | 0.04 ± 1.7 | 0.03 ± 1.8 | 0.02 ± 3.4 | NA |
| 40 | 4633‐R | 0.42 ± 0.2 | 0.44 ± 1.7 | 0.3 ± 0.3 | 0.29 ± 0.3 | 0.47 ± 0.6 | 0.46 ± 0.5 |
| 41 | 4634‐R | 0.2 ± 0.7 | 0.2 ± 0.3 | 0.14 ± 0.9 | 0.13 ± 0.8 | 0.40 ± 0.2 | 0.39 ± 0.2 |
Note: 1–3 compounds: withanoside IV [1], withaferin A [2], withanolide A [3]; ND = Not detected.
mg/100 mg of extract. Compounds used in the USP Method: withanoside IV [1], withanolide A [3]. Compounds used in the BP Method: withaferin A [2], withanolide A [3]. NA = Not available (provided) due to the co‐elution of dihydrowithaferin A sulfate (a significant leaf marker) with withanolide A.
3.1. Comparison and Gaps Between British Pharmacopoeial and US Pharmacopeial Monographs
The USP monograph for W. somnifera establishes distinct calibration standards for quantifying key bioactive constituents. WL A (3) is designated as the primary standard for the analysis of total WLs, including withaferin A, 12‐deoxywithastramonolide, withanone, and withanolide B. Similarly, WS IV (1) serves as the standard for quantifying withanolide glycosides (WSs), such as withanoside V and withanoside VI. Kaempferol 3‐O‐robinobioside‐7‐O‐glucoside is used to determine total flavonol glycosides, including quercetin 3‐O‐robinobioside‐7‐O‐glucoside and quercetin 3‐O‐rutinoside‐7‐O‐glucoside.
3.2. Quantitative Requirements and Sample Inclusion
The USP mandates that standardized extracts contain not less than (NLT) 2.5% total WLs and withanosides (WSs) (on a dried basis), while total flavonol glycosides must not exceed 0.04%. Furthermore, the USP applies to a variety of dosage forms, including powders, extracts, or leaf materials. In contrast, the BP monograph requires the exclusive use of dried roots and does not specify a quantitative content requirement for extracts, other plant parts, and the corresponding extracts.
Crucially, neither the USP nor the BP provides official, standardized analytical methods for evaluating blends of root and leaf materials. Although the USP includes W. somnifera leaf for flavonoid analysis, it currently lacks a standardized approach to detect or quantify adulteration involving root–leaf mixtures.
A comparative summary of the key analytical differences between the USP and BP methods is presented in the table below.
| Parameter | USP method | BP method |
|---|---|---|
| Plant parts | Finely ground dried roots, root extract (standardized for withanolides), possibly leaf for flavonoid analysis. | Only dried root material (whole or cut roots, not extracts). |
| Target compounds | Withanolide A and withanoside IV | Withaferin A and withanolide A |
| Minimum threshold | NLT 0.3% (Total WLs + WSs) for root powder and NLT 2.5% (Total WLs + WSs) on the dried basis for root extract | NLT 0.01% each for root powder |
| Focus | Total bioactive potency | Specific marker compounds for dual activity |
| Method | HPLC‐UV at 227 nm | HPLC‐UV at 230 nm |
| Flavanol glycosides | ||
| Target compound | Kaempferol‐3‐O‐robinoside‐7‐O‐glucoside | NA |
| Acceptance criteria | NMT 0.01% on the dried basis for the plant root sample and NMT 0.04% on the dried basis for the root extract | NA |
3.3. Plant Samples
The root (#5218, 6087, 26125, 26128, 26131) and leaf samples (#26129, 26132, 26142) of W. somnifera were morphologically verified to ensure their authenticity and exclude allied species, such as Weizmannia coagulans, or other closely related species. Applying USP and BP methods to morphologically verified ashwagandha samples enabled us to assess authenticity, quality, and steroidal lactone content by measuring the concentrations of WS IV, withaferin A, and WL A (Figure 2A). Analysis of five dried root samples showed high agreement between the methods, with results ranging, respectively, from 0.003 to 0.4 mg/g, 0.002 to 0.33 mg/g, and 0.06 to 0.33 mg/g (USP), and 0.003 to 0.4 mg/g, 0.002 to 0.37 mg/g, and 0.06 to 0.34 mg/g (BP). Conversely, the three leaf samples exhibited substantially higher concentration of withaferin A (USP: 1.05 to 1.2 mg/g; BP: 1.0 to 1.2 mg/g) while WL A remained low in both USP (0.02 to 0.04 mg/g) and BP (0.01 to 0.05 mg/g) measurements (Figure 2B and Table 1).
FIGURE 2.
Content (mg/g) of standard compounds—withanoside IV (1), withaferin A (2), and withanolide A (3)—in root and leaf samples of Withania somnifera were analyzed using the USP (A) and BP (B) methods.
3.3.1. Comparative Analysis of Ashwagandha Root Versus Leaf Ratios
Detection of non‐root W. somnifera material (e.g., stem, leaves), commonly used as adulterants, is feasible in crude powdered form using techniques such as microscopic analysis, high‐performance thin‐layer chromatography (Tomar et al. 2019), and liquid chromatography (LC)‐PDA with mass spectrometry (MS) (Chandra et al. 2016). However, a significant gap exists in pharmacopeial methods for quantifying such admixtures within commercial ashwagandha supplements. We hypothesize that the ratio of the aerial quality marker withaferin A to the aggregate concentration of WS IV and WL A (root quality marker) offers a plausible and rapid analytical tool for quantifying the percentage of aerial plant parts in ashwagandha root material. The absolute concentrations of the three target steroidal lactones (1, 2, and 3) were quantified in morphologically verified root and leaf materials utilizing established extraction and quantification procedures compliant with both the BP and USP methods. These quantitative data were then used to calculate the ratiometric value defined as the quantity of compound 2 divided by the sum of the quantities of compounds 1 and 3.
For the root samples, the calculated mean ratio was 0.27 ± 0.16 when using the BP method and 0.22 ± 0.18 with the USP method. Critically, the leaf samples exhibited a marked difference, showing a six‐ to seven‐fold increase in the mean ratio, resulting in values of 1.59 ± 0.36 (BP) and 1.56 ± 0.33 (USP), respectively (Figure 2A,B).
To evaluate the applicability of ratiometric analysis for identifying and quantifying the percentage of aerial plant parts present in root material, a comprehensive set of controlled binary mixtures was prepared. These blends systematically ranged from 0% to 100% leaf content, utilizing botanically verified, single‐source W. somnifera root (#26128) and leaf (#26129) material.
A strong linear correlation between the percentage of leaf material in the blend and the measured ratio of 2/(1 + 3) suggests the ratio could be a dependable quantitative metric for detecting aerial part adulteration (Figure 3). The linear model, y = 0.2254x + 0.001, exhibited a high coefficient of determination (r 2 = 0.94), validating the model's reliability across the entire range of adulteration. The ratio for pure root material (0% leaf) was nearly zero, contrasting sharply with the maximum ratio of approximately 1.45 for pure leaf material (100% leaf, similar to samples studied for BP and USP methods, see Figure 2A,B). Furthermore, the analysis confirmed the method's sensitivity, showing that the initial inclusion of leaf material (up to 9%) resulted in a steep, analytically distinct increase in the ratio, thus confirming the detectability of low‐level adulteration. Since the presence and relative abundance of these steroidal lactones significantly influence the biological effects of W. somnifera products (Singh et al. 2011; Wiciński et al. 2024), quantifying the percentage of aerial parts in commercial ashwagandha is crucial for resolving current safety concerns. Although this ratiometric method demonstrates strong potential for detecting undisclosed aerial part extracts used as adulterants, achieving broad applicability and statistically robust conclusions requires a critical expansion of the study, specifically through the analysis of paired root and leaf samples sourced from multiple, diverse plant origins.
FIGURE 3.
Standard (calibration) curve generated by plotting the ratio of withaferin A (2) to the sum of withanoside IV (1) and withanolide A (3) against the different percentages of leaf content in root samples.
3.4. Commercial Extracts
Using the USP and BP methods, we quantified WS IV, withaferin A, and WL A (all in mg/g) across multiple ashwagandha commercial extracts. The three dried root extracts (#4652, #4343, #26148) showed ranges of 1.5–8.5, 1.3–7.5, and 3.2–7.6, respectively, for the three analytes. In sharp contrast, the leaf extract (#4341) exhibited much higher concentrations of withaferin A (≈approximately 20) and WS IV (≈approximately 12), yet maintained a low WL A level (≈approximately 2.4). The root‐leaf blend extract (#4300) exhibited high levels of withaferin A (≈49%) and WS IV (≈30%), with virtually no WL A (≈0.1%). Finally, the two whole extract samples (#4342, 26,149) resulted in intermediate ranges of 3.9–11.7, 3.6–27.5, and 1.6–2.4 mg/g, indicating variable compositions likely containing both root and aerial parts.
3.5. Dietary Supplements
We analyzed 25 commercial W. somnifera supplements and found that most were capsules (21), with the rest being powders or tablets. Labeling varied widely regarding the plant parts used: 13 claimed root samples, 10 claimed a root‐and‐leaf blend, one claimed the whole plant, and one failed to specify the plant part. Sixteen products provided a quantitative claim for “total WLs content” (WLs), ranging from 0.44% to 35%. A major issue is the imprecision of the “total WLs” term, which often ambiguously combines steroidal lactones as WLs and their glycosides, WSs. Furthermore, the claimed “total WLs” is usually only the sum of selected, quantified compounds, not a true total of all steroidal lactones.
3.5.1. Individual Analytes and Compositional Variation
Using the USP method, we quantified WS IV, withaferin A, and WL A (in mg/dosage form) across the commercial supplement products: WS IV, withaferin A, and WL A, revealing significant variations tied to the claimed plant part. The 13 products labeled as root‐derived showed low‐to‐moderate ranges for all three analytes (0–3.6, 0–4.3, and 0–4.6, respectively). Conversely, the 10 products claiming to be root/leaf blends exhibited substantially higher levels of WS IV (0.05–10) and withaferin A (0.01–11.7), but consistently very low WL A (0.02–0.07). The presence of steroidal lactones (7, 10, and 0.03 mg/dosage form for the three analytes, respectively) in one product (#4294PR) that claimed “no plant part”, and the absence of all three analytes in another product (# 4629PR) underscores the significant inconsistencies between label claims and the actual steroidal lactone content across the marketed products.
3.5.2. Non‐Compliance With Label Claims and Pharmacopeial Standards
Analysis of total components showed wide‐ranging content for total WSs from 0% to 3.3%, total WLs from 0% to 8.7%, and total flavonoid glycosides from 0% to 0.1% (Table 2 and Figure 4). A major finding was the widespread non‐compliance with label claims for total WLs content. Of the 25 products tested, nine (36%) did not report a total WLs percentage, and several of these (#4286PR, 4285PR, 2619PR, and 4629PR) fell below the minimum 2.5% USP threshold for root extract, with concentrations as low as 0%–0.64%. Among the 16 products that did report specific percentages, nine contained concentrations 3‐ to 83‐fold lower (Table 2) than the declared (on product label) values (#4287PR, 4282PR, 4294PR, 4293PR, 4292PR, 4279PR, 4280PR, 4289PR, and 4632PR). These findings align with reports suggesting that the active, steroidal lactone levels in many ashwagandha supplements frequently fail to meet manufacturer claims of 5%, 10%, or 35% median WLs contents (Thalhamer et al. 2024), with detected concentrations often being more typical of raw herbal material than of concentrated extracts.
TABLE 2.
Comparison of label‐declared information with measured quantities (%) of total withanolides (WLs) and withanolide glycosides (WSs) in 25 dietary supplements, determined using the USP method.
| NCNPR code # | Plant part | Average weight (mg) | The label recommended serving size | Daily maximum serving size | % withanolide glycosides or withanolides per serving | Measured total withanolides (WLs) quantity (%) | Measured total withanolide glycosides quantity (WSs) (%) | %WLs + %WSs | ||
|---|---|---|---|---|---|---|---|---|---|---|
| Per serving size | Maximum recommended daily serving | Per serving size | Maximum recommended daily serving | Per serving size | ||||||
| 4301PR | Root | 344.4 | 1 capsule | 1 | NA | 6.4 | 6.4 | 0.7 | 0.7 | 7.1 |
| 4290PR | Root | 360.2 | 1 capsule | 1 | NA | 8.7 | 8.7 | 1.4 | 1.4 | 10.1 |
| 4287PR | Root | 543.9 | 1 capsule | 1 | 2.5% withanolides and 0.625 mg withaferin A | 0.1 | 0.1 | 0.04 | 0.04 | 0.14 |
| 4283PR | Root | 728.7 | 2 capsules | 1 | 0.75% withanolides | 1.8 | 1.8 | 0.6 | 0.6 | 2.4 |
| 4286PR | Root | 512.9 | 2 capsules | 1 | NA | 0.6 | 0.6 | 0.04 | 0.04 | 0.64 |
| 4285PR | Root | 898.4 | 2 tablets | 1 | NA | 0.2 | 0.2 | 0.02 | 0.02 | 0.22 |
| 4282PR | Root | 691.1 | 1 capsule | 1 | Minimum 5% withanolides and < 0.1% withaferin A | 0.3 | 0.3 | 0.01 | 0.01 | 0.3 |
| 2619PR | Root | 648 | 1 capsule | 1–2 | NA | 0.2 | 0.4 | 0.01 | 0.02 | 0.2 |
| 4629PR | Root | NA a | ¼ tsp. = 600 mg | 1–3 | NA | 0 | 0 | 0 | 0 | 0 |
| 4630PR | Root | 608.5 | 1 capsule | 1–3 | 2.5% withanolides | 3.4 | 10.2 | 0.6 | 1.8 | 4.0 |
| 4631PR | Root | 567.8 | 1 capsule | 1–3 | 3.5% withanolides | 4.8 | 14.4 | 1.2 | 3.6 | 6.0 |
| 4633PR | Root | 582 | 1 capsule | 1–2 | 2.5 mg withanolides |
0.6 (2.1 mg) |
1.2 |
0.1 (0.35 mg) |
0.2 |
0.7 (2.45 mg) |
| 4634PR | Root | 670.2 | 1 caplet | 1–2 | 0.44% withanolides | 0.4 | 0.8 | 0.05 | 0.1 | 0.45 |
| 4628PR | Whole | NA a | NA | NA | 1.5% withanolides | 4.7 b | — | 0.6 b | — | 5.3 b |
| 4294PR | NA | 438.6 | 1 capsule | 1 | 35% withanolides | 4.6 | 4.6 | 1.7 | 1.7 | 6.3 |
| 4293PR | Root, leaf | 190.0 | 1 capsule | 2 | 35% withanolides | 1.7 | 3.4 | 2.3 | 4.6 | 4.0 |
| 4292PR | Root, leaf | 217.8 | 1 capsule | 2 | 35% withanolides | 8.4 | 16.8 | 2.6 | 5.2 | 11.0 |
| 4279PR | Root, leaf | 328.2 | 1 capsule | 2 | 35% withanolide glycosides | 0.1 | 0.2 | 3.3 | 6.6 | 3.4 |
| 4280PR | Root, leaf | 660 | 2 capsules | 1 | 10% withanolide glycosides | 0.1 | 0.1 | 0.02 | 0.02 | 0.12 |
| 4291PR | Root, leaf | 224.1 | 1 capsule | 1 | NA | 0.2 | 0.2 | 0.06 | 0.06 | 0.26 |
| 4284PR | Root, leaf | 1098.6 | 2 tablets | 1 | NA | 1.0 | 1.0 | 0.6 | 0.6 | 1.6 |
| 4288PR | Root, leaf | 287.5 | 1 capsule | 1 | NA | 0.2 | 0.2 | 0.1 | 0.1 | 0.3 |
| 4289PR | Root, leaf | 298.2 | 2 capsules | 1 | 5% withanolide glycosides | 0.1 | 0.1 | 0.1 | 0.1 | 0.2 |
| 4281PR | Root, leaf | 576.5 | 1 capsule | 2–3 | 2.5% total withanolides | 2.3 | 6.9 | 1.1 | 3.3 | 3.4 |
| 4632PR | Root, leaf | 335.4 | 1 capsule | 1–2 | 10% withanolide glycosides | 0.1 | 0.2 | 0.03 | 0.06 | 0.13 |
Note: Reported values represent the sum of WSs and WLs quantified in products labeled as containing ashwagandha root extracts, expressed as a percentage of the label claim on a per‐serving basis. NA = label claim not available.
For powders, the average weight is not applicable, as this requirement applies only to solid dosage preparations like tablets or capsules.
Values are expressed as a percentage (%); calculation per serving size was not possible due to the unavailability of serving size information.
FIGURE 4.
Bar charts showing the content of combined withanolides (withaferin A, 12‐deoxywithastramonolide, withanolide A, withanone, and withanolide B, withanoside IV, V, VI) and combined three flavonoids (quercetin 3‐O‐robinobioside‐7‐O‐glucoside, quercetin 3‐O‐rutinoside‐7‐O‐glucoside, kaempferol 3‐O‐robinobioside‐7‐O‐glucoside) in different extracts (mg/g) (A), plant root and leaf samples (mg/g) (B), and dietary supplements (mg/dosage form) (C) based on the USP method.
3.5.3. Flavonoid Glycosides as Markers of Leaf/Aerial Parts Adulteration
The presence of combined flavonoid glycosides strongly suggests aerial parts adulteration, as they were absent in authentic root samples but present at approximately 0.3% in leaf samples (Mundkinajeddu et al. 2014). Of the 13 root‐derived dietary supplements, seven contained no combined flavonoid glycosides, while the remaining six (#4301PR, 4290PR, 4287PR, 2619PR, 4630PR, 4633PR) contained 0.02–0.6 mg/dose. One product (#4294PR) with no plant part specified contained 0.98 mg/g these flavonoid glycosides, suggesting both root and leaf/aerial components. Among the 10 supplements labeled as root/leaf blends, eight products (#4293PR, 4292PR, 4279PR, 4291PR, 4288PR, 4289PR, 4281PR, 4632PR) contained 0.03–1.02 mg/g (Figure 4C).
3.5.4. Analytical Method Discrepancies and Ratiometric Confirmation
Methodological challenges were noted, as dihydrowithaferin A sulfate, a compound abundant in leaf/aerial parts, was identified to co‐elute with and interfere with the quantification of WL A, a key primary root quality marker. However, in some leaf or leaf–root blend samples, minimal interference was observed, likely due to the low abundance of this sulfate compound. The BP method proved more susceptible to this interference than the USP method, although overall steroidal lactone profiles were generally comparable (Table 1). This analytical issue likely accounts for the 16% of products that passed the BP criteria but failed the USP criteria. When evaluated against pharmacopeial limits, 44% of products failed both BP and USP criteria, while 40% passed both. The remaining 16% passed only the BP method (Figure 5).
FIGURE 5.
Pie chart showing the percentage of passed versus failed results among 25 dietary supplements based on USP and BP methods.
Using the proposed ratiometric analysis [withaferin A/(WS IV + WL A)], which circumvents some co‐elution issues and is less susceptible to absolute concentration variability, 40% of 25 products met both BP and USP criteria. This ratio confirmed distinct compositional patterns independent of label claims—two products (#4290, #4301) were exclusively root‐derived; two (#4281, #4631) were predominantly root‐based; four (#4279, #4293, #4294, #4630) primarily contained leaf or aerial parts; and two (#4292, #4628) were entirely leaf‐derived, despite being labeled as mixtures.
Although the USP method and ratiometric analysis reveal a wide spectrum of adulteration in commercial ashwagandha supplements, the observed variability in WLs and flavonoid concentrations may not be solely attributable to adulteration of aerial parts. Factors such as cultivation practices, storage, edaphic conditions (Gafner et al. 2023), and chemotypes (Kaul et al. 2009; Kushwaha et al. 2012), along with variations in extraction methodology and stability (Sangwan et al. 2004; Thalhamer et al. 2024), can severely impact the absolute concentrations of these key analytes. Collectively, these manifold factors explain the wide range of active ingredient levels observed across commercial supplements and underscore the critical need for standardized sourcing, careful selection of plant parts, and rigorous analytical verification. Such inherent variability may directly impact product efficacy, as supplements with inconsistent or low levels of bioactive compounds are less likely to deliver the expected therapeutic benefits.
4. Conclusions
The findings reveal a critical disconnect between current pharmacopeial standards and the complex reality of the global ashwagandha market. While not mandated, the cross‐comparison of pharmacopoeias is invaluable for ensuring the quality and authenticity of botanical raw materials and is crucial for global harmonization efforts regarding ingredient safety. By utilizing morphologically verified samples, we established a robust metric using the ratio of withaferin A to the sum of WS IV and WL A. Although it is derived from a single plant sample, the ratio tool enabled clear differentiation of ashwagandha root extracts from aerial part extracts, independent of the products' label claims. We identified that only 40% of commercial ashwagandha products met both BP and USP standards. Strikingly, out of 10 products that met both pharamcopieal standards, only two products were exclusively root‐derived; the remainder contained an undisclosed amount of aerial and/or leaf parts. Since multiple currently marketed products were identified with these blends, the existing USP or BP methods—designed only for root powder and/or root extract analysis—are inadequate. These methods must be improved, and robust analytical tools must be developed to ensure the overall quality and accurately determine the plant parts used in current ashwagandha supplements. Such efforts are not merely about quality and authenticity, but they are critical for addressing ongoing global safety concerns on ashwagandha.
Author Contributions
Bharathi Avula: methodology, investigation, data curation, formal analysis, writing – original draft, writing – review and editing. Kumar Katragunta: formal analysis, data curation, writing – review and editing. Kiran Kumar Tatapudi: formal analysis, data curation. Yan‐Hong Wang: writing – review and editing. Ikhlas A. Khan and Amar G. Chittiboyina: conceptualization, review and editing, resources, funding acquisition, and project administration.
Funding
This work was supported by the U.S. Food and Drug Administration, 5U01FD004246 and U.S. Department of Agriculture, 58‐6060‐6‐015.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
The authors acknowledge Dr. Thomas Brendler for his technical expertise and advice on comparing pharmacopoeias for quality assurance on Ashwagandha supplements. This research is supported in part by “Science Based Authentication of Botanical Ingredients” funded by the Center for Food Safety and Applied Nutrition, US Food and Drug Administration, grant number 5U01FD004246, and “Discovery & Development of Natural Products for Pharmaceutical & Agricultural Applications” funded by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58‐6060‐6‐015. The authors would like to thank Jeff Solomon for his support and for proofreading the current manuscript.
Contributor Information
Bharathi Avula, Email: bavula@olemiss.edu.
Ikhlas A. Khan, Email: ikhan@olemiss.edu.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.