Powdered Ashwagandha Root Extract
DEFINITION
Powdered Ashwagandha Root Extract is prepared from Ashwagandha using methanol, alcohol, water, or mixtures of these solvents. It contains NLT 2.5% of withanolides, calculated on the dried basis as the sum of withanolide aglycones and withanolide glycosides.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test 
201
201
Standard solution:
Heat gently for 10–15 min about 200 mg of USP Powdered Ashwagandha Extract Root RS in 10 mL of methanol, centrifuge, and use the supernatant. [Note—Save the remaining volume of the supernatant for use in the test for Content of Withanolides. ]
Sample solution:
Heat gently for 10–15 min about 200 mg of Powdered Ashwagandha Root Extract in 10 mL of methanol, centrifuge, and use the supernatant.
Adsorbent:
0.25-mm layer of chromatographic silica gel
Application volume:
25 µL
Developing solvent system:
A mixture of ethyl acetate, toluene, and acetic acid (45:55:3)
Spray reagent:
Mix 0.5 mL of anisaldehyde, 10 mL of glacial acetic acid, 85 mL of methanol, and 5 mL of concentrated sulfuric acid in the order mentioned.
Analysis
Samples:
Standard solution and Sample solution
Apply the Samples as bands to a suitable plate (see Chromatography 621). Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about 90% of the length of the plate. Remove the plate from the chamber, dry, spray with Spray reagent, heat for 5–10 min at 100
, and examine under visible light.
621). Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about 90% of the length of the plate. Remove the plate from the chamber, dry, spray with Spray reagent, heat for 5–10 min at 100, and examine under visible light.
Acceptance criteria:
The Sample solution exhibits five main grayish-blue bands with RF values of approximately 0.12, 0.29, 0.47, 0.67, and 0.73 that are similar in position and color to the main bands for the Standard solution. Other less intense bands are observed for the Sample solution and the Standard solution.
• B.
The Sample solution in the test for Content of Withanolides shows main peaks at retention times corresponding to those of withanolide A and withanoside IV for Standard solution A and Standard solution B, respectively. Identify other withanolide peaks in the Sample solution by comparison with Standard solution C and the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS being used. The Sample solution shows additional peaks corresponding to some of the following withanolides: physagulin D, 27-hydroxywithanone, withanoside V, withanoside VI, withaferin A, withastramonolide, withanone, and withanolide B.
COMPOSITION
• Content of Withanolides
Solution A:
Dissolve 0.14 g of potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL of phosphoric acid, dilute with water to 1000 mL, and mix.
Solution B:
Filtered and degassed acetonitrile
Standard solution A:
Dissolve, using gentle heat, a quantity of USP Withanolide A RS in methanol to obtain a solution having a known concentration of about 0.1 mg/mL.
Standard solution B:
Dissolve, using gentle heat, a quantity of USP Withanoside IV RS in methanol to obtain a solution having a known concentration of about 0.1 mg/mL.
Standard solution C:
Dilute 5 mL of the Standard solution prepared in Identification test A with methanol to 10 mL, and mix. Before injection, pass through a membrane filter of 0.45-µm or finer pore size.
Sample solution:
Transfer about 100 mg of Powdered Ashwagandha Root Extract, weighed, to a 10-mL volumetric flask, add about 7 mL of methanol, heat gently on a water bath for 15–20 min, dilute with methanol to volume, and mix. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.
Mobile phase:
See the gradient table below.
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 95 | 5 |
| 18 | 55 | 45 |
| 25 | 20 | 80 |
| 28 | 20 | 80 |
| 30 | 95 | 5 |
| 40 | 95 | 5 |
Chromatographic system
Mode:
LC
Detector:
UV 227 nm
Column:
4.6-mm × 25-cm, end-capped; packing L1
Temperature:
27 ± 1
Flow rate:
1.5 mL/min
Injection size:
20 µL
System suitability
Samples:
Standard solution A and Standard solution C
Using the chromatogram of Standard solution C and the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS being used, identify the retention times of the peaks corresponding to the various withanolide aglycones and glycosides. The approximate relative retention times of the withanolide aglycones and glycosides are provided in the following table.
| Analyte | Relative Retention Time |
|---|---|
| Withanoside IV | 0.70 |
| Physagulin D | 0.75 |
| 27-hydroxywithanone | 0.80 |
| Withanoside V | 0.89 |
| Withanoside VI | 0.89 |
| Withaferin A | 0.92 |
| Withastramonolide | 0.96 |
| Withanolide A | 1.00 |
| Withanone | 1.01 |
| Withanolide B | 1.14 |
Suitability requirements
The chromatogram for Standard solution C is similar to the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS being used.
Resolution:
NLT 1.0 for the withanolide A and withanone peaks, and NLT 3.0 between the peak corresponding to withaferin A and the peak corresponding to coeluting withanoside V and withanoside VI, Standard solution C
Tailing factor:
NMT 1.5 for the withanolide A peak, Standard solution A
Relative standard deviation:
NMT 2.0% for replicate injections, withanolide A peak, Standard solution A
Analysis
Samples:
Standard solution A, Standard solution B, Standard solution C, and Sample solution
Calculate the percentage of withanolide aglycones in the portion of Powdered Ashwagandha Root Extract taken:
Result = (rT/rS)(CS/W)
| rT | = | = sum of the peak responses for withaferin A, withastramonolide, withanolide A, withanone, and withanolide B from the Sample solution |
| rS | = | = peak response of withanolide A from Standard solution A |
| CS | = | = concentration of USP Withanolide A RS in Standard solution A (mg/mL) |
| W | = | = weight of Powdered Ashwagandha Root Extract taken to prepare the Sample solution (g) |
Calculate the percentage of withanolide glycosides in the portion of Powdered Ashwagandha Root Extract taken:
Result = (rT/rS)(CS/W)
| rT | = | = sum of the peak responses for withanoside IV, withanoside V and withanoside VI from the Sample solution |
| rS | = | = peak response of USP Withanoside IV from Standard solution B |
| CS | = | = concentration of USP Withanoside IV RS in Standard solution B (mg/mL) |
| W | = | = weight of Powdered Ashwagandha Root Extract taken to prepare the Sample solution (g) |
Acceptance criteria:
The sum of the percentages of the withanolide aglycones and withanolide glycosides is NLT 2.5%, calculated on the dried basis. [Note—Because of inherent variations, some of the withanolides mentioned in this test may be present in minor quantities or may be totally absent. The sample will be deemed compliant if the sum of the total withanolides is NLT 2.5%. ]
IMPURITIES
Inorganic Impurities
• Heavy Metals, Method II 231:
NMT 20 ppm
231:
NMT 20 ppm
Organic Impurities
• Procedure: Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
SPECIFIC TESTS
• Loss on Drying 731:
Dry 2.0 g at 105 for 3 h: it loses NMT 6.0% of its weight.
731:
Dry 2.0 g at 105 for 3 h: it loses NMT 6.0% of its weight.
• Microbial Enumeration Tests 2021:
The total aerobic bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
2021:
The total aerobic bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
• Microbiological Procedures for Absence of Specified Microorganisms 2022:
Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
2022:
Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
• Articles of Botanical Origin, Aflatoxins 561:
Meets the requirements
561:
Meets the requirements
• Botanical Extracts, Residual Solvents 565:
Meets the requirements
565:
Meets the requirements
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers, protected from light and moisture, and store at controlled room temperature.
• Labeling:
The label states the Latin binomial and, following the official name, the part of the plant from which the article was prepared. It meets other labeling requirements under Botanical Extracts 565.
565.
• USP Reference Standards 11
11
USP Powdered Ashwagandha Root Extract RS
USP Withanolide A RS
USP Withanoside IV RS
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Maged H. Sharaf, Ph.D.
Principal Scientific Liaison 1-301-816-8318 |
(DS2010) Monographs - Dietary Supplements |
| 2021 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 2022 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1197
Pharmacopeial Forum: Volume No. 35(4) Page 888