Powdered Ashwagandha Root
DEFINITION
Powdered Ashwagandha Root is Ashwagandha Root reduced to a fine or very fine powder.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test 201
Standard solution:  Heat gently, for 10–15 min, about 200 mg of USP Powdered Ashwagandha Root Extract 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:  Transfer about 5.0 g of Powdered Ashwagandha Root to a 250-mL flask fitted with a reflux condenser. Add 50 mL of methanol, reflux on a water bath for 10–15 min, cool to room temperature, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, filter, concentrate under vacuum to about 40 mL, and adjust the volume with methanol to 50.0 mL. [Note—Save the remaining volume of the Sample solution for use in the test for Content of Withanolides. ]
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 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 from the Standard solution. Other less intense bands are observed for the Sample solution and Standard solution.
•  B. The Sample solution from the test for Content of Withanolides shows main peaks at retention times corresponding to those of withanolide A and withanoside IV in 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. 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:  Use the Sample solution prepared in Identification test A. 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, identify the retention times of the peaks corresponding to the different 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 of Standard solution C is similar to the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS.
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 the 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 taken:
Result = 5(rT/rS)(CS/W)
rT== sum of the peak responses for withaferin A, withastramonolide, withanolide A, withanone, and withanolide B in 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 taken to prepare the Sample solution (g)
Calculate the percentage of withanolide glycosides in the portion of Powdered Ashwagandha Root taken:
Result = 5(rT/rS)(CS/W)
rT== sum of the peak responses for withanoside IV, withanoside V, and withanoside VI in the Sample solution
rS== peak response of USP Withanoside IV RS from Standard solution B
CS== concentration of USP Withanoside IV RS in Standard solution B (mg/mL)
W== weight of Powdered Ashwagandha Root taken to prepare the Sample solution (g)
Acceptance criteria:  Add the percentages of withanolide aglycones and withanolide glycosides: NLT 0.3% is found, calculated on the dried basis. [Note—Due to inherent variations, some of the withanolides mentioned in the above test may be present in minor quantities or may be totally absent. The sample will be deemed compliant as long as the sum of the total withanolides is NLT 0.3%. ]
IMPURITIES
Inorganic Impurities 
•  Heavy Metals, Method II 231: NMT 20 ppm
Organic Impurities 
SPECIFIC TESTS
•  Botanic Characteristics
Macroscopic:  It is a dusty white or grey to light brown powder with a characteristic odor and a mucilagenous, bitter, acrid taste.
Histology 
Microscopic examination:  It shows collapsed cork cells filled with starch grains and reddish-brown content; thin-walled cortex parenchyma cells filled with starch grains and occasional microsphenoidal crystals of calcium oxalate; vessels, with pitted and scalariform thickening, and generally with end walls perforated; a few fibers with thick lignified walls and simple pits; abundant starch grains, mostly simple, sometimes compound, spherical, reniform-oval with central hilum.
•  Loss on Drying 731: Dry 1.0 g at 105 for 3 h: it loses NMT 12.0% of its weight.
•  Articles of Botanical Origin, Total Ash 561: NMT 7.0%, determined on 1.0 g of Powdered Ashwagandha Root
•  Microbial Enumeration Tests 2021: The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria 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
•  Articles of Botanical Origin, Aflatoxins 561: Meets the requirements
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at room temperature.
•  Labeling: The label states the Latin binomial and, following the official name, the part of the plant contained in the article.
•  USP Reference Standards 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 1195
Pharmacopeial Forum: Volume No. 35(4) Page 886