6/2/2020 USP-NF

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© 2020 USPC

Powdered Echinacea purpurea Extract

DEFINITION
Powdered Echinacea purpurea Extract is prepared from dried Echinacea purpurea Root, Echinacea purpurea Aerial Parts, or a mixture of
them, by extraction with hydroalcoholic mixtures or other suitable solvents. The ratio of the starting crude plant material to Powdered
Extract is between 2:1 and 8:1. It contains NLT 4.0% of total phenols, calculated as the sum of caftaric acid (C13H12O9), chicoric acid
(C22H18O12), and chlorogenic acid (C16H18O9), on the dried basis. It contains NLT 0.025% of dodecatetraenoic acid isobutylamides
(C16H25NO), calculated on the dried basis.

IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHY
Presence of chicoric acid and absence of echinacoside
Standard solution A: 0.2 mg/mL of USP Echinacoside RS in methanol
Standard solution B: 0.2 mg/mL of USP Caftaric Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and 0.2 mg/mL of USP Chicoric
Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered Echinacea purpurea Extract RS in methanol. Shake to disperse, sonicate for 5 min,
and centrifuge. Use the supernatant.

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Sample solution: 20 mg/mL of Powdered Echinacea purpurea Extract in methanol. Shake to disperse, sonicate for 5 min, and
centrifuge. Use the supernatant.
Chromatographic system
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(See Chromatography 〈621〉, Thin-Layer Chromatography.)
Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Application volume: 5 µL Standard solution C and Sample solution, and 2 µL Standard solution A and Standard solution B as 8-mm
bands
Relative humidity: Condition the plate to a relative humidity of about 33% using a suitable device.
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Developing solvent system: A mixture of ethyl acetate, methylethyl ketone, water, and formic acid (5:3:1:1)
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl diphenylborinate in ethyl acetate
Analysis
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Samples: Standard solution A, Standard solution B, Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer chromatographic plate, and dry in air. Develop the chromatograms in a
saturated chamber. Remove the plate from the chamber, heat at 100° for 5 min, derivatize the plate while still warm with
Derivatization reagent, dry in air, and examine under UV light at 366 nm.
System suitability: Standard solution A shows one major blue band in the lower third section of the chromatogram due to
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echinacoside. Standard solution B shows two blue bands at about the middle of the chromatogram due to caftaric acid (higher RF)
and chlorogenic acid (lower RF) that are clearly separated, and a blue band for chicoric acid in the upper third section of the
chromatogram.
Acceptance criteria: The most prominent band in the Sample solution chromatogram is a blue band in the upper third section of the
chromatogram at an RF corresponding to the chicoric acid band in the chromatograms of Standard solution B and Standard
solution C (less prominent in Echinacea pallida and absent or almost absent in Echinacea angustifolia). The second most
prominent band in the Sample solution chromatogram is a blue band at about the middle of the chromatogram due to caftaric
acid, corresponding to a band in the chromatograms of Standard solution B and Standard solution C (absent in Echinacea
angustifolia and a minor band in Echinacea pallida). The Sample solution chromatogram does not exhibit a band at the RF of
echinacoside in Standard solution A (difference from Echinacea pallida and Echinacea angustifolia). The Sample solution
chromatogram may exhibit minor blue bands corresponding to similar bands in the chromatogram of Standard solution C. One of
these is due to chlorogenic acid at an RF corresponding to chlorogenic acid in Standard solution B.
• B. THIN-LAYER CHROMATOGRAPHY
Presence of alkylamides

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Standard solution A: 0.2 mg/mL of USP β-Sitosterol RS in methanol

Standard solution B: 100 mg/mL of USP Powdered Echinacea purpurea Extract RS in dichloromethane. Shake to disperse, sonicate
for 5 min, and centrifuge. Use the supernatant.
Sample solution: 100 mg/mL of Powdered Echinacea purpurea Extract in dichloromethane. Shake to disperse, sonicate for 5 min,
and centrifuge. Use the supernatant.
Chromatographic system
(See Chromatography 〈621〉, Thin-Layer Chromatography.)
Adsorbent: Chromatographic silica gel with an average particle size of 5 µm (HPTLC plates)
Application volume: 5 µL Standard solution B and Sample solution, and 2 µL Standard solution A, as 8-mm bands
Relative humidity: Condition the plate to a relative humidity of about 33% using a suitable device.
Developing solvent system: A mixture of toluene, ethyl acetate, cyclohexane, and formic acid (8: 2: 1: 0.3)
Developing distance: 6 cm
Derivatization reagent: Place 85 mL of methanol in a 100-mL glass bottle, and cool it down in a water–ice cubes–salt bath or in a
freezer. To the ice-cold methanol, slowly and carefully add 10 mL of acetic acid and 5 mL of sulfuric acid, and mix well. Allow the
mixture to cool to room temperature, then add 0.5 mL of p-anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and Sample solution
Apply the Samples as bands to a suitable thin-layer chromatographic plate, and dry in air. Develop the chromatograms in a
saturated chamber. Remove the plate from the chamber, dry in air, derivatize with Derivatization reagent, heat at 100° for 3–5
min, set aside to cool, and examine under visible light.
System suitability: The chromatogram of Standard solution B exhibits the most prominent band as a pinkish violet band at about the
middle of the chromatogram, and just below this pinkish band, a violet band at a lower RF similar in position and color to the β-
sitosterol band in the chromatograms of Standard solution A. These two bands are clearly separated from each other. The
chromatogram of Standard solution B also shows a broad pink violet band close to the solvent front. The β-sitosterol band of the

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Standard solution B chromatogram and the pinkish violet band underneath are clearly separated from one another.
Acceptance criteria: The most prominent band of the Sample solution chromatogram is a pinkish violet band at about the middle of
the chromatogram similar in position and color to a band in the Standard solution B chromatogram (much less prominent in
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Echinacea angustifolia and Echinacea pallida), a violet band corresponding to β-sitosterol band in the chromatograms of Standard
solution A and Standard solution B, and a broad pink violet band close to the solvent front similar in position and color to the band
in the chromatogram of Standard solution B. The Sample solution chromatogram does not exhibit a yellow band below the β-
sitosterol band (difference from Echinacea angustifolia) or a prominent violet band at about two-thirds of the chromatogram
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(difference from Echinacea pallida).

• C. The retention time of the major peak in the Sample solution corresponds to that of the chicoric acid peak in Standard solution A,
and the second most prominent peak corresponds to that of the caftaric acid peak in Standard solution B. The Sample solution
chromatogram shows no or a very minor peak at the retention time corresponding to the echinacoside peak in the Standard solution D
chromatogram, all peaks as obtained in the test for Content of Total Phenols.
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COMPOSITION
• CONTENT OF TOTAL PHENOLS
Solvent: Alcohol and water (7:3)
Standard solution A: 30 µg/mL of USP Chicoric Acid RS in Solvent
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Standard solution B: 20 µg/mL of USP Caftaric Acid RS in Solvent

Standard solution C: 20 µg/mL of USP Chlorogenic Acid RS in Solvent
Standard solution D: 20 µg/mL of USP Echinacoside RS in Solvent
Sample solution: Transfer 60 mg of Powdered Echinacea purpurea Extract to a round-bottom ask equipped with a condenser. Add 25
mL of Solvent, and heat under re ux while shaking by mechanical means for 15 min. Centrifuge, or pass through a membrane lter
of 0.45-µm or ner pore size.
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

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Time (min) Solution A (%) Solution B (%)

13 78 22

14 60 40

17 60 40

17.5 90 10

22 90 10

Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
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 volume: 5 µL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Relative standard deviation: NMT 2% for chicoric acid peak in repeated injections, Standard solution A

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Analysis
Samples: Standard solution A, Standard solution B, Standard solution C, Standard solution D, and Sample solution
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Separately calculate the percentage of caftaric acid (C13H12O9), chicoric acid (C22H18O12), and chlorogenic acid (C16H18O9) in the
portion of Powdered Echinacea purpurea Extract taken:

Result = (rU/rS) × (CS/CU) × 100

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rU = peak response for the relevant analyte from the Sample solution

rS = peak response of the relevant analyte from the corresponding Standard solution

CS = concentration of the relevant analyte in the corresponding Standard solution (mg/mL)

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CU = concentration of Powdered Echinacea purpurea Extract in the Sample solution (mg/mL)

Calculate the percentage of total phenols in the portion of Powdered Echinacea purpurea Extract taken by adding the individual
percentages calculated.
Acceptance criteria: NLT 4.0% on the dried basis
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• CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES

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 lter having a 0.45-µm or ner pore size.
Standard solution B: 10 µg/mL of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in methanol
Sample solution: Transfer about 500 mg of Powdered Echinacea purpurea Extract, accurately weighed, into a 100-mL volumetric ask.
Add 80 mL of methanol, and sonicate for 30 min. Cool to room temperature, and dilute with methanol to volume. Pass through a
membrane lter of 0.45-µm or ner pore size.
Mobile phase: Acetonitrile and water (55:45)
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
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

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Injection volume: 25 µL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram 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 the 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 due to 2E,4E,8Z,10E-dodecatetraenoic acid isobutylamide and 2E,4E,8Z,10Z-dodecatetraenoic acid
isobutylamide in the chromatogram from the Sample solution by comparison with the chromatogram from Standard solution A.
Measure the areas for the relevant peaks.
Calculate the percentage of dodecatetraenoic acid isobutylamides in the portion of Powdered Echinacea purpurea Extract taken:

Result = (rU/rS) × (CS/CU) × F × 100

rU = sum of the peak responses of the relevant analytes from the Sample solution

rS = peak response from Standard solution B

CS = concentration of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in Standard solution B (mg/mL)

CU = concentration of Powdered Echinacea purpurea Extract in the Sample solution (mg/mL)

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= response factor to convert 2E,4E-hexadienoic acid isobutylamide into dodecatetraenoic acid isobutylamides, 1.353
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Acceptance criteria: NLT 0.025% on the dried basis

CONTAMINANTS
• ELEMENTAL IMPURITIES—PROCEDURES 〈233〉
Acceptance criteria
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Arsenic: NMT 1.0 µg/g

Cadmium: NMT 0.5 µg/g
Lead: NMT 5.0 µg/g
Mercury: NMT 1.0 µg/g
Change to read:
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• ▲BOTANICAL EXTRACTS 〈565〉, Preparations, General Pharmacopeial Requirements, Pesticide Residues▲ (CN 1-MAY-2020): Meets the
requirements
• MICROBIAL ENUMERATION TESTS 〈2021〉: The total bacterial count does not exceed 104 cfu/g. The total combined molds and yeasts count
does not exceed 103 cfu/g.
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• ABSENCE OF SPECIFIED MICROORGANISMS 〈2022〉: It meets the requirements of the tests for absence of Salmonella species and Escherichia
coli.
• BOTANICAL EXTRACTS, Residual Solvents〈565〉: Meets the requirements

SPECIFIC TESTS
• LOSS ON DRYING 〈731〉
Sample: 1 g
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 5.0%

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant containers, in a cool place.
• LABELING: The label states the Latin binomial and, following the o cial name, the parts of the plant from which the article was prepared.
If derived from root and aerial parts, indicate the corresponding percentages. Label it to indicate the content of total phenols and
dodecatetraenoic isobutylamides. The label bears a statement indicating that Echinacea purpurea may cause rare allergic reactions,
rashes, or aggravate asthma. It meets the requirements for Botanical Extracts 〈565〉, Labeling.

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• USP REFERENCE STANDARDS 〈11〉

USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea purpurea Extract RS
USP Echinacoside RS
USP 2E,4E-Hexadienoic Acid Isobutylamide RS
USP β-Sitosterol RS

Auxiliary Information- Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

POWDERED ECHINACEA PURPUREA Anton Bzhelyansky BDSHM2015 Botanical Dietary

EXTRACT Scienti c Liaison Supplements and Herbal Medicines 2015

Chromatographic Database Information: Chromatographic Database

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 38(6)

Page Information:

USP43-NF38 - 4958
USP42-NF37 - 4901

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USP41-NF36 - 4587

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