11/9/2020 USP-NF Black Cohosh Fluidextract

Printed on: Mon Nov 09 2020, 10:53:39 am

Printed by: Shruti Kharidia
O cial Status: Currently O cial on 09-Nov-2020
O cial Date: O cial as of 1-Jan-2018
Document Type: DIETARY SUPPLEMENTS
DocId: 1_GUID-F4137E6E-6DBD-40DF-A6B7-BF6231279C08_3_en-US
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© 2020 USPC

Black Cohosh Fluidextract

DEFINITION
Black Cohosh Fluidextract is prepared from Black Cohosh by extraction with hydroalcoholic mixtures or isopropanol–water mixtures.
Each mL contains the extracted constituents of 1 g of the plant material. It contains NLT 90.0% and NMT 110.0% of the labeled
amount of triterpene glycosides, calculated as 23-epi-26-deoxyactein (C37H56O10).

IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 100 mg/mL of USP Powdered Black Cohosh Extract RS in methanol
Standard solution B: 1 mg/mL each of USP Actein RS, USP 23-epi-26-Deoxyactein RS, and isoferulic acid in methanol
Sample solution: Fluidextract
Adsorbent: Chromatographic silica gel mixture with an average particle size of 10–15 µm (TLC plates)
Application volume: 10 µL
Developing solvent system: Use the upper phase of a mixture of butyl alcohol, glacial acetic acid, and water (5:1:4).
Spray reagent: Methanol, glacial acetic acid, sulfuric acid, and p-anisaldehyde (85:10:5:0.5). [NOTE—Store in a refrigerator. The reagent
is colorless; discard if color appears.]
Analysis
Samples: Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms until the solvent front has moved about 15 cm, and dry the plate with the aid of a current of air.

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Acceptance criteria: Examine the plate under UV light at 365 nm. The Sample solution exhibits main zones similar in position and
color to the main zones of Standard solution A. In the upper third of the plate, the Sample solution exhibits a blue uorescent zone at
the level of the zone due to isoferulic acid of Standard solution B. Spray the plate with Spray reagent, heat at 100° for 5 min, and
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examine in daylight. The Sample solution exhibits main zones similar in position and color to the main zones of Standard solution A.
Standard solution B exhibits red-violet zones due to actein and 23-epi-26-deoxyactein. The Sample solution exhibits several greenish-
brown spots in the lower third of the plate and several violet zones above; two of these violet zones occur at RF values similar to
those due to actein and 23-epi-26-deoxyactein of Standard solution B.
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• B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST

Standard solution A: 0.5 mL of Standard solution A prepared in Identi cation test A, diluted with methanol to 2.0 mL
Standard solution B: 1.0 mL of Standard solution B prepared in Identi cation test A, diluted with methanol to 5.0 mL
Sample solution: Use the Fluidextract, diluting if necessary with a suitable solvent to obtain a concentration of 0.25 mg/mL of
triterpene glycosides.
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Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Application volume: 2 µL as an 8-mm band
Developing solvent system: Toluene, ethyl formate, and formic acid (5:3:2)
Spray reagent: Proceed as directed for Identi cation test A.
Analysis
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Samples: Standard solution A, Standard solution B, and Sample solution

Develop until the solvent front has moved two-thirds of the length of the plate, and dry the plate with the aid of a current of air.
Spray the plate with Spray reagent, heat at 100° for 5 min, and examine in daylight.
Acceptance criteria: The Sample solution exhibits main zones similar in position and color to the main zones of Standard solution A.
Standard solution B exhibits red-violet zones due to actein and 23-epi-26-deoxyactein at RF values of about 0.5 and 0.4, respectively.
The Sample solution exhibits zones similar in color and RF values to those due to actein and 23-epi-26-deoxyactein of Standard
solution B.
• C. The Sample solution exhibits peaks for cimiracemoside A, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, cimigenol–
arabinoside, and cimigenol–xyloside at retention times corresponding to those compounds in the Standard solution, as obtained in the
test for Content of Triterpene Glycosides. The ratio of the peak areas of cimigenol–arabinoside to cimigenol–xyloside is NLT 0.4
(distinction from Cimicifuga foetida).

COMPOSITION
• CONTENT OF TRITERPENE GLYCOSIDES
Standard solution: Dissolve a quantity of USP Powdered Black Cohosh Extract RS in methanol with shaking for 1 min, and dilute with
methanol to obtain a solution having a known concentration of 30 mg/mL. Pass through a membrane lter of 0.45-µm or ner pore
size.
23-epi-26-Deoxyactein standard solutions: Dissolve USP 23-epi-26-Deoxyactein RS in methanol with shaking for 1 min. Dilute
quantitatively, and stepwise if necessary, to obtain solutions having known concentrations of 500, 100, 50, 25, and 12.5 µg/mL. Pass

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through a membrane lter of 0.45-µm or ner pore size.

System suitability solution: 0.1 mg/mL each of USP Actein RS and USP 23-epi-26-Deoxyactein RS in methanol
Sample solution: Use the Fluidextract, diluting if necessary with methanol to obtain a concentration of about 0.75 mg/mL of triterpene
glycosides. Centrifuge, or pass through a lter of 0.45-µm or ner pore size.
Solution A: 0.05% tri uoroacetic acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.

Table 1

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

0 0 80 20

8 0 80 20

15 68 0 32

55 36 0 64

65 5 0 95

70 5 0 95

85 0 80 20

Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: Evaporative light-scattering
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[NOTE—The detector is set up according to the manufacturer's instruction in order to achieve a signal-to-noise ratio of NLT 10 for the
12.5-µg/mL 23-epi-26-Deoxyactein standard solution.]
Column: 4.6-mm × 25-cm; 5-µm packing L1
Column temperature: 35°
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Flow rate: 1.6 mL/min

Injection size: 20 µL
System suitability
Samples: System suitability solution, Standard solution, and 100-µg/mL 23-epi-26-Deoxyactein standard solution
Suitability requirements
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Chromatogram similarity: The chromatogram of the Standard solution is similar to the Reference Chromatogram provided with the
lot of USP Powdered Black Cohosh Extract RS being used.
Resolution: NLT 1.0 between the (26S)-actein and the 23-epi-26-deoxyactein peaks, System suitability solution
Tailing factor: NMT 2.0 for the 23-epi-26-deoxyactein peak, 100-µg/mL 23-epi-26-Deoxyactein standard solution
Relative standard deviation: NMT 2.0% of the logarithm of the area response of the 23-epi-26-deoxyactein peak in repeated
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injections, 100-µg/mL 23-epi-26-Deoxyactein standard solution

Analysis
Samples: System suitability solution, Standard solution, 23-epi-26-Deoxyactein standard solutions, and Sample solution
Using the chromatogram of the Standard solution and the Reference Chromatogram provided with the lot of USP Powdered Black
Cohosh Extract RS, identify the retention times of the peaks corresponding to the triterpene glycosides. The approximate
relative retention times of the triterpene glycosides are provided in Table 2.

Table 2

Compound Relative Retention Time

Cimicifugoside H-1 0.61

Cimiracemoside A 0.78

(26R)-Actein 0.94

26-Deoxycimicifugoside 0.96

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Compound Relative Retention Time

(26S)-Actein 0.98

23-epi-26-Deoxyactein 1.00

Acetyl-shengmanol–xyloside 1.03

Cimigenol–arabinoside 1.08

Cimigenol–xyloside (cimicifugoside) 1.13

26-Deoxyactein 1.22

25-Acetyl-cimigenol–arabinoside 1.60

(24S)-25-Acetyl-cimigenol–xyloside 1.64

25-O-Methyl-cimigenol–arabinoside 1.90

25-O-Methyl-cimigenol–xyloside 1.93

Plot the logarithms of the peak area responses versus the logarithms of the concentrations, in µg/mL, of the 23-epi-26-
Deoxyactein standard solutions, and determine the regression line using a least-squares analysis. The correlation coe cient for
the regression line is NLT 0.995. From the graphs so obtained, determine the concentration, C, in µg/mL, of the relevant analyte
in the Sample solution. Separately calculate the concentrations, in µg/mL, of cimicifugoside H-1, cimiracemoside A, (26R)-
actein, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, acetyl-shengmanol–xyloside, cimigenol–arabinoside,
cimigenol–xyloside (cimicifugoside), 26-deoxyactein, 25-acetyl-cimigenol–arabinoside, (24S)-25-acetyl-cimigenol– xyloside, 25-

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O-methyl-cimigenol–arabinoside, and 25-O-methyl-cimigenol–xyloside as 23-epi-26-deoxyactein (C37H56O10) in the portion of
Fluidextract taken:
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Result = (D × C/V)

D = dilution factor for the Sample solution, if applicable: nal volume of Sample solution/volume of aliquot of Fluidextract
taken (mL/mL)

C = concentration of the relevant analyte in the Sample solution (µg/mL)

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V = volume of the Fluidextract taken to prepare the Sample solution (mL)

Calculate the percentage of the labeled amount of triterpene glycosides in the portion of Fluidextract taken:
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Result = ΣC/L × 100

ΣC = sum of concentrations of the individual triterpene glycosides (mg/mL)

L = labeled concentration of triterpene glycosides of the Fluidextract (mg/mL)

Acceptance criteria: 90.0%–110.0%

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CONTAMINANTS
• MICROBIAL ENUMERATION TESTS 〈2021〉: The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts
count does not exceed 103 cfu/g.
• OTHER REQUIREMENTS: It meets the requirements under Botanical Extracts 〈565〉, Residual Solvents and Pesticide Residues.

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant containers, and store in a cool place.
• LABELING: It meets the requirements for Labeling under Botanical Extracts 〈565〉. Label it to indicate the content, in percentage, of
triterpene glycosides, calculated as 23-epi-26-deoxyactein. Dosage forms prepared with this article should bear the following statement:
Discontinue use and consult a healthcare practitioner if you have a liver disorder or develop symptoms of liver trouble, such as
abdominal pain, dark urine, or jaundice.
• USP REFERENCE STANDARDS 〈11〉
USP Actein RS
USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS

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

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11/9/2020 USP-NF Black Cohosh Fluidextract

Topic/Question Contact Expert Committee

BLACK COHOSH FLUIDEXTRACT Anton Bzhelyansky BDSHM2020 Botanical Dietary

Scienti c Liaison Supplements and Herbal Medicines

Chromatographic Database Information: Chromatographic Database

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 33(5)

Page Information:

USP43-NF38 - 4821
USP42-NF37 - 4777
USP41-NF36 - 4480

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