Powdered Echinacea purpurea
DEFINITION
Powdered Echinacea purpurea is Echinacea purpurea Root reduced to a powder or very fine powder.
IDENTIFICATION
•  A. Presence of Chicoric Acid and Absence of Echinacoside
Standard solution A:  20 mg/mL of USP Powdered Echinacea purpurea Extract RS in methanol
Standard solution B:  1 mg/mL of 1,3-dicaffeoylquinic acid in methanol
Sample solution:  Transfer about 1 g of Powdered Echinacea purpurea to a suitable extraction thimble. Transfer the thimble to a continuous extraction apparatus, and extract with 50 mL of chloroform for 1 h. Retain the chloroform extract for Identification test B. Continue the extraction with 50 mL of methanol, and concentrate to a small volume at 40 in vacuum. With the aid of methanol, transfer the extract to a 10-mL volumetric flask, and dilute with methanol to volume.
Adsorbent:  0.25-mm layer of chromatographic silica gel mixture (TLC plates)
Application volume:  10 µL
Developing solvent system:  Ethyl acetate, formic acid, and water (17:2:1)
Spray reagent A:  10 mg/mL of diphenylborinic acid, ethanolamine ester in methanol
Spray reagent B:  50 mg/mL of polyethylene glycol 4000 in alcohol
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms until the solvent front has moved NLT 18 cm, and dry the plate in a current of air. Spray the plate with Spray reagent A followed by Spray reagent B, and examine the plate under UV light at 365 nm.
Acceptance criteria:  The chromatogram from the Sample solution shows a yellowish-green zone at an RF value of 0.75 due to chicoric acid and another yellowish-green zone at an RF value of 0.45 due to caftaric acid, both zones corresponding in color and RF value to zones in the chromatogram from Standard solution A. The chromatogram from the Sample solution does not show or shows only traces of a zone at an RF value of 0.1 due to echinacoside (present in Echinacea angustifolia and in Echinacea pallida), and does not show a zone that corresponds in color and RF value to the spot for 1,3-dicaffeoylquinic acid (cynarin) (present in Echinacea angustifolia) in the chromatogram from Standard solution B. Other colored zones of varying intensities may be observed in the chromatogram from the Sample solution.
•  B. Presence of Isobutylalkenylamides
Standard solution A:  100 mg/mL USP Echinacea purpurea Extract RS in methanol
Standard solution B:  1 mg/mL of -sitosterol in methanol
Sample solution:  Evaporate the chloroform extract retained from preparation of the Sample solution in Identification test A to dryness at 40 in vacuum. To the residue, add 1 mL of alcohol, and pass through a nylon membrane filter having a pore size of 0.45 µm.
Adsorbent:  0.25-mm layer of chromatographic silica gel mixture (TLC plates)
Application volume:  10 µL
Developing solvent system:  Hexane and ethyl acetate (2:1)
Spray reagent:  Prepare a mixture of glacial acetic acid, sulfuric acid, and p-anisaldehyde (10:5:0.5) in an ice bath.
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms until the solvent front has moved NLT 12 cm, and dry the plate in a current of air. Examine the plate under UV light at 254 nm, then spray the plate with Spray reagent, and heat the plate at 100 for 5 min. Examine the plate under long-wavelength UV light.
Acceptance criteria 
Under UV light at 254 nm: The chromatogram from the Sample solution shows one main zone corresponding in RF value to the zone due to dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide in the chromatogram of Standard solution A, and below this zone there are several other zones due to ,,,-unsaturated isobutylamides.
After treatment with Spray reagent and heating: The zone due to dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide turns blue-black, and below this zone there are several other zones due to ,,,-unsaturated isobutylamides (not detectable in Echinacea pallida) that turn violet (unlike the corresponding zones in the chromatogram of Echinacea angustifolia that are mostly yellowish due to ,-unsaturated isobutylamides). A zone due to -sitosterol that corresponds in RF value to the principal spot in the chromatogram of Standard solution B is also observed.
•  C. The retention times for the relevant peaks of the Sample solution, mainly due to caftaric acid and chicoric acid, correspond to those of Standard solution A, as obtained in the test for Content of Total Phenols. An echinacoside peak is not detectable or is very weak.
COMPOSITION
•  Content of Total Phenols
Solution A:  Phosphoric acid (0.1 in 100) in water
Solution B:  Acetonitrile
Mobile phase:  See Table 1.
Table 1
Time
(min)
Solution A
(%)
Solution B
(%)
0 90 10
13 78 22
14 60 40
17.5 60 40
18 90 10
30 90 10
Solvent:  Alcohol and water (7:3)
Standard solution A:  5 mg/mL of USP Powdered Echinacea purpurea Extract RS in Solvent. Dissolve by shaking for 1 min. After dilution, pass through a membrane filter of 0.45-µm or finer pore size.
Standard solution B:  40 µg/mL of USP Chlorogenic Acid RS in Solvent
Sample solution:  Transfer about 125 mg of Powdered Echinacea purpurea (capable of passing through a 40-mesh sieve), accurately weighed, to a round-bottom flask equipped with a condenser. Add 25.0 mL of Solvent, and heat under reflux, while shaking by mechanical means for 15 min. Centrifuge, or pass through a membrane filter of 0.45-µm or finer pore size.
Chromatographic system 
Mode:  LC
Detector:  UV 330 nm
Column:  4.6-mm × 25-cm; 5-µm packing L1
Column temperature:  35
Flow rate:  1.5 mL/min
Injection size:  5 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
Chromatogram similarity:  The chromatogram of Standard solution A is similar to the Reference Chromatogram for total phenols provided with USP Powdered Echinacea purpurea Extract RS.
Relative standard deviation:  NMT 2% for chlorogenic acid peak in repeated injections, Standard solution B
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Identify the relevant analytes in the chromatogram obtained from the Sample solution by comparison with the chromatogram obtained from Standard solution A. Measure the areas for the relevant peaks.
Separately calculate the percentage of caftaric acid (C13H12O9), chicoric acid (C22H18O12), and chlorogenic acid (C16H18O9) in the portion of Powdered Echinacea purpurea taken:
Result = (rU/rS) × CS × (V/W) × F × 100
rU== peak area for the relevant analyte from the Sample solution
rS== peak area for chlorogenic acid from Standard solution B
CS== concentration of USP Chlorogenic Acid RS in Standard solution B (mg/mL)
V== volume of the Sample solution (mL)
W== weight of Powdered Echinacea purpurea used to prepare the Sample solution (mg)
F= = response factor: chicoric acid, 0.695; caftaric acid, 0.881; and chlorogenic acid, 1.000
Calculate the percentage of total phenols in the portion of Powdered Echinacea purpurea taken by adding the individual percentages calculated.
Acceptance criteria:  NLT 0.5% of total phenols on the dried basis
•  Content of Alkamides
Mobile phase:  Acetonitrile and water (55:45)
Standard solution A:  5 mg/mL of USP Powdered Echinacea purpurea Extract RS in methanol. Dissolve using sonication and shaking for 10 min. After dilution, pass through a membrane filter having a 0.45-µm or finer pore size.
Standard solution B:  10 µg/mL of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in methanol
Sample solution:  Transfer about 2.5 g of Powdered Echinacea purpurea (capable of passing through a 40-mesh sieve) into a round-bottom flask. Add 80 mL of methanol, and reflux for 30 min. Cool to room temperature, and filter into a 100-mL volumetric flask, using small portions of methanol to rinse the flask and the filter. Dilute with methanol to volume. Pass through a membrane filter having a 0.45-µm or finer pore size.
Chromatographic system 
Mode:  LC
Detector:  UV 254 nm
Column:  4.6-mm × 25-cm; 5-µm packing L1
Column temperature:  30
Flow rate:  1.5 mL/min
Injection size:  25 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
Chromatogram similarity:  The chromatogram obtained from Standard solution A is similar to the Reference Chromatogram for alkamides provided with USP Powdered Echinacea purpurea Extract RS.
Resolution:  NLT 1.0 between dodecatetraenoic acid isobutylamide peaks, Standard solution A
Tailing factor:  NMT 2.0 for 2E,4E-hexadienoic acid isobutylamide peak, Standard solution B
Relative standard deviation:  NMT 2.5% for the 2E,4E-hexadienoic acid isobutylamide peak in repeated injections, Standard solution B
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Identify the peaks of the 10 major alkamides in the chromatogram obtained from the Sample solution by comparison with the chromatogram obtained from Standard solution A.
Calculate the percentage of alkamides in the portion of Powdered Echinacea purpurea taken:
Result = (rU/rS) × CS × (V/W) × F × 100
rU== sum of the peak areas of the relevant analytes from the Sample solution
rS== peak area of 2E,4E-hexadienoic acid isobutylamide from Standard solution B
CS== concentration of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in Standard solution B (mg/mL)
V== volume of the Sample solution (mL)
W== weight of Powdered Echinacea purpurea used to prepare the Sample solution (mg)
F= = response factor for 2E,4E-hexadienoic acid isobutylamide, 1.353
Acceptance criteria:  NLT 0.025% on the dried basis
CONTAMINANTS
•  Heavy Metals, Method III 231: NMT 10 ppm
SPECIFIC TESTS
•  Botanic Characteristics: Under a microscope, the following characteristics are observed: vessels (80 × 30 µm) with slanted end walls and spiral or pitted secondary walls; rectangular cork cells (150 × 60 µm) with brown inclusions; rectangular parenchymatous cells (120 × 30 µm), some pitted; elongated fiber cells having a narrow lumen with funnel-shaped end (20 to 40 µm wide); polygonal sclereids; a melanogenic layer of variable thickness, interspersed between the cell walls of the parenchyma; and lignified sclereids, vessels, and fibers. Starch is present; calcium oxalate and inulin crystals are absent.
•  Loss on Drying 731: Dry a sample at 105 for 2 h: it loses NMT 10.0%.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed, light-resistant containers.
•  Labeling: The label states the Latin binomial and, following the official name, the part of the plant from which the article was derived.
•  USP Reference Standards 11
USP Chlorogenic Acid RS Click to View Structure
USP 2E,4E-Hexadienoic Acid Isobutylamide RS Click to View Structure
USP Powdered Echinacea purpurea Extract 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
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 1280
Pharmacopeial Forum: Volume No. 26(6) Page 1593