USP 43 Dietary Supplements / Black Cohosh 4817

Table 2 round scarsof the earlierstalks; laterally, it is clearly curled,

Relative and the lowersurfaceis covered with thin, longitudinally
Reten- grooved, dark brown, easily breakableroots. Thefracture is
Compound tionTime horny and fibrous. The transversecut shows a thin outer
Cimicifugoside H-l 0.61 bark surrounding a ring of numerous pale, narrow wedges
of vascular tissuealternating with dark medullary rays and a
Cimiracemoside A 0.78 large central pith. Black Cohosh roots are dark brown,
(26R)-Actein 0.94 between 1 and 3 mm in diameter, brittle, nearlycylindrical
or obtuselyquadrangular, and longitudinally wrinkled. The
26-Deoxycimicifugoside 0.96 fracture isshort. The transversecut shows a distinct
(26S)·Actein 0.98 cambium lineseparating a wide outer bark from a central
region composed of three to sixwedges of lignified xylem
23-epi·26-Deoxyactein 1.00
tissue united by their apices and separated by broad
Acetyl-shengmanol-xyloside 1.03 nonlignified medullary rays.
1.08
Microscopic: In a surfaceview, suberous epidermal cells are
Cimigenol-arabinoside
tabular with moderatelythickened walls. The
Cimigenol-xyloside (cimicifugoside) 1.13 parenchymatous cortex isfilled with starch. Xylem wedges
26-Deoxyactein 1.22
are lignified and composed of numerous smallvessels with
bordered pits or reticulately thickened walls, thin-walled
2S-Acetyl·cimigenol-arabinoside 1.60 fibers, and xylem parenchyma.The parenchyma of the pith
(24S)-2S·Acetyl·cimigenol-xyloside 1.64
is unlignified. Medullary rays are filled with starch granules,
which are spherical or polygonal and are mostlysimpleor
2S-0.Methyl.cimigenol-arabinoside 1.90 two to three compounded but can be up to six '
2S-0.Methyl·cimigenol-xyloside 1.93
compounded. Individual starch granulesare between 3 and
15 IJm in diameter, each with a somewhat central
slit-shaped hilum.
Plotthe logarithmsof the peak areasagainst the logarithms • ARTICLES OF BOTANICAL ORIGIN, Foreign OrganicMatter
of the concentrations, in IJg/mL, of the 23-epi- (561): NMT 2.0% of foreign organic matter, and NMT
26-Deoxyactein standardsolutions, and establish the 5.0% of stem bases
calibration curve by least-squares regression. The • ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
correlation coefficient for the regression lineis NLT 0.995. Method 2 (561): NLT 8.0%, using a mixtureof alcohol and
From the plot, determine the concentration, C, in IJg/mL, water (1:1) instead of alcohol
of the relevantanalytes in the Sample solution. • Loss ON DRYING (731)
Separately calculate the percentages of the individual Sample: 1.0 g of Black Cohosh
triterpene glycosides in Table 2 as 23-epi-26-deoxyactein Analysis: Drythe Sample at 105° for 2 h.
(C37Hs601O) in the portion of Black Cohosh taken: Acceptance criteria: NMT 12.0%
• ARTICLES OF BOTANICAL ORIGIN, TotalAsh (561): NMT
Result = (V x Q/(F x W) x 100 10.0%
• ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
V =final volume of the Sample solution (mL) NMT4.0%
C = concentration of the relevantanalyte in the
Sample solution (uq/rnl) . ADDITIONAL REQUIREMENTS
F =factor to convert mg to IJg, 1000 IJg/mg • PACKAGING AND STORAGE: Preserve in a well-closed,
W = weight of Black Cohosh taken to prepare the light-resistant container. Protectfrom moisture, and store
Sample solution (mg) at room temperature.
• LABELING: The labelstates the Latin binomial and, following
Calculate the percentage of triterpene glycosides in the the official name, the parts of the plant contained in the
portion of Black Cohosh taken by adding the percentages article. Dosageforms prepared with this articleshould bear
of the individual analytes. the following statement: Discontinue use and consult a
Acceptance criteria: NLT 0.4% on the dried basis healthcare practitioner ifyou have a liver disorder or
developsymptomsof liver trouble, such as abdominal pain,
CONTAMINANTS
dark urine, or jaundice.
• ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental • USP REFERENCE STANDARDS (11)
Impurities (561): Meets the requirements USP Actein RS
• ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
USP Powdered Black Cohosh Extract RS
(561): Meets the requirements . USP 23-epi-26-Deoxyactein RS
• MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 105 cfu/g, the total
combined moldsand yeastscount does not exceed 103cfu/
g, and the bile-tolerantGram-negative bacteriacount does
not exceed 103 cfu/g. Powdered Black Cohosh
• ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meetsthe
requirements of the tests for the absence of Salmonella DEFINITION
species and Escherichia coli Powdered Black Cohosh is Black Cohosh reduced to a powder
SPECIFIC TESTS
or a veryfine powder. It contains NLT 0.4% of triterpene
• BOTANICAL CHARACTERISTICS
glycosides, calculatedas 23-epi-26-deoxyactein 1 (C37Hs601O)
Macroscopic: The Black Cohosh rhizome is dark brown, on the dried basis.
longitudinally grooved, rough, stronglyknotty, and
somewhat curled and irregular. It is 15 cm long and up to
2.5 cm thick. The upper surfaceis coveredwith numerous 1 23-epi-26-Deoxyactein is sometimes referred to as 27-deoxyactein.

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4818 Black Cohosh / Dietary Supplements
IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHY
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: Transfer 5 g of Powdered BlackCohosh
to a screw-cap centrifuge tube, add 10 mL of a mixture of
alcohol and water (7:3), and heat on a steam bath for
10 min. Centrifuge, and use the clear supernatant.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 J..Im (TLC plates)
Application volume: 10 J..IL
Developing solvent system: Use the upper phase of a
mixture of butyl alcohol, glacial acetic acid, and water
(5:1 :4).
Derivatization reagent: Methanol, glacial acetic acid,
sulfuric acid, and p-anisaldehyde (85: 10: 5: 0.5).
[Nort--Store in a refrigerator. The reagent is colorless;
discard if color appears.]
Analysis
Samples: StandardsolutionA, Standardsolution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about 15 em, and dry the plate in a stream of air.
Examine the plate under UV light at 365 nm. Treat the
plate with Derivatization reagent, heat at 100° for 5 min,
and examine in white light.
Acceptance criteria: Under UV light at 365 nm, the
chromatogram of the Sample solution exhibits main zones
similar in position and color to the main zones of Standard
solutionA. In the upper third of the plate, the Sample
solution exhibits a blue fluorescent zone at the level of the
zone due to isoferulic acid of Standardsolution B. Under
white light, the Sample solution exhibits main zones similar
in position and color to the main zones of Standardsolution
A. StandardsolutionB 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 Standardsolution B.
• B. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.5 mL of StandardsolutionA
prepared in Identification test A, diluted with methanol to
2 mL
Standard solution B: 1.0 mLof StandardsolutionB prepared
in Identification test A, diluted with methanol to 5 mL
Sample solution: Transfer 0.5 g of Powdered BlackCohosh
to a screw-cap tube, add 5 mL of methanol, 'sonicatefor
10 min, and filter into a 1O-mL volumetric flask. Wash the
residue on the filter paper four times, using 1 mL of
methanol for each washing; add the washings to the
volumetric flask; and dilute with methanol to volume.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 5 J..Im (HPTLC plates)
Application volume: 2 J..IL as 8-mm bands
Developing solvent system: Toluene, ethyl formate, and
formic acid (5:3:2)
, Derivatization reagent: Proceed as directed for
Identification test A.
Analysis
Samples: StandardsolutionA, Standardsolution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about two-thirds of the length of the plate, and
dry the plate with in a current of alr, Treat the plate with
Derivatization reagent, heat at 100° for 5 min, and
examine in white light.
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USP 43
Acceptance criteria: The Sample solution exhibits main
zones similar in' position and color to the main zones of
Standardsolution A. Standardsolution 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 Standardsolution, as obtained in the
test for Contentof 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
BlackCohosh 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 filter of 0.45-J..Im or finer 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 concentrations of 500, 100, 50, 25,
and 12.5 J..Ig/mL. Pass through a membrane filter of
0.45-J..Im or finer 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: Accurately weigh about 750 mg of
Powdered BlackCohosh, and place in a 20-mL PTFE-capped
centrifuge tube. Add 15 mL of methanol, sonicate for
30 min, centrifuge, and retain the supernatant. Repeat the
extraction twice. Evaporate the combined extracts under
vacuum at 45°-50°. Dissolve the residue in methanol, and
quantitatively transfer to a 1O-mL volumetric flask. Dilute
with methanol to volume, and pass through a membrane
filter of 0.45-J..Im or finer pore size.
Solution A: 0.05% Trifluoroacetic acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Water Solution A Solution B
(min) (%) (%) (%)
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
(NOTE-The Detectoris set up according to the
manufacturer's instructions in order to achieve a
signal-to-noise ratio of NLT 10 for the 12.5-J..Ig/mL
23-epi-26-Deoxyactein standardsolution.]
Column: 4.6-mm x 25-cm; 5-J..Im packing L1
Column temperature: 35°
Flow rate: 1.6 mL/min
USP 43 Dietary Supplements / Black Cohosh 4819

Injection volume: 20 IJL C = concentration of the relevant analyte in the

System suitability Sample solution (lJg/mL)
Samples: System suitability solution, Standard solution, and F =factor to convert mg to IJg, 1000 IJg/mg
1OO-lJg/mL 23-epi-26-Deoxyactein standard solution W = weight of Powdered Black Cohosh taken to
Suitability requirements prepare the Sample solution (mg)
Chromatogram similarity: The chromatogram of the
Standard solution is similarto the reference Calculatethe percentage of triterpene glycosides in the
chromatogram provided with the lot of USP Powdered portion of Powdered Black Cohosh taken by adding the
Black Cohosh ExtractRS being used. percentages of the individual analytes.
Resolution: NLT 1.0 between the (26S)-actein and the Acceptance criteria: NLT 0.4% on the dried basis
23-epi-26-deoxyactein peaks, System suitability solution'
Tailing factor: NMT 2.0 for the 23-epi-26-deoxyactein CONTAMINANTS
peak, 1OO-lJg/mL 23-epi-26-Deoxyactein standard • ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental
solution Impurities (561): Meets the requirements
Relative standard deviation: NMT 2.0% of the • ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
logarithm of the area responses for replicate injections, (561): Meets the requirements
1OO-lJg/mL 23-epi-26-Deoxyactein standard solution • MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Analysis microbial count does not exceed lOs cfu/g; the total
Samples: System suitability solution, Standard solution, combined moldsand yeasts count does not exceed 103cfu/
23-epi-26-Deoxyactein standard solutions, and Sample g; and the bile-tolerant Gram-negative bacteria count does
solution . not exceed 103 cfu/g.
Using the chromatogram of the Standard solution and the • ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meetsthe
reference chromatogram provided with the lot of USP requirements of the tests for the absence of Salmonella
Powdered Black Cohosh ExtractRS being used, identify species and Escherichia coli
the retention times of the peaks corresponding to the SPECIFIC TESTS
triterpene glycosides. The approximate relative retention • BOTANICAL CHARACTERISTICS
times of the triterpene glycosides are provided in Table Macroscopic: The material is a light to dark brown powder,
2. is odorless or has a slight odor, and has an acrid or bitter
taste.
Table 2 Microscopic: It shows numerous starch granules with
Relative concentric striations, simple or compound. The individual
Retention granules are spherical or more or less polygonal and are
Name Time
between 3 and 15 IJm in diameter, each with a somewhat
Cimicifugoside H-1 0.61 central slit-shaped hilum. Vessels with bordered pits occur,
as do lignified fibers. Reddish to brown fragments of
Cimiracemoside A 0.78
suberized epidermis with more or lesstabular cells occur.
(26R)-Actein 0.94 • ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
26-Deoxycimicifugoside 0.96
Method 2 (561): NLT 8.0%, using a mixture of alcohol and
water (1 :1) instead of alcohol
(26S)-Actein 0.98 • Loss ON DRYING (731)
23-epi-26-Deoxyactein 1.00
Sample: 1 g of Powdered Black Cohosh
Analysis: Drythe Sample at 105° for 2 h.
Acetyl-shengmanol-xyloside 1.03 Acceptance criteria: NMT 12.0%
Cimigenol-arabinoside 1.08
• ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
10.0%
Cimigenol-xyloside (cimicifugoside) 1.13 • ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
26-Deoxyactein 1.22
NMT4.0%
2S-Acetyl-cimigenol-arabinoside 1.60 ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed,
(24S)-2S-Acetyl-cimigenol-xyloside 1.64 light-resistantcontainers, and. protect from moisture.
2S-0-Methyl-cimigenol-arabinoside 1.90 • LABELING: The labelstates the Latin binomialand, following
the official name, the parts of the plant from which the
2S-0-Methyl-cimigenol-xyloside 1.93
articlewas derived. Dosage forms prepared with this article
should bear the following statement: Discontinue use and
Plotthe logarithms of the peak areas against the logarithms consult a healthcare practitioner ifyou have a liver disorder
of the concentrations, in IJg/mL, of the 23-epi- or develop symptoms of livertrouble, such as abdominal
26-Deoxyactein standard solution, and establish the pain, dark urine, or jaundice.
calibration curve by least-squares regression. The • USP REFERENCE STANDARDS (11)
correlation coefficientfor the regression lineis NLT 0.995. USP Actein RS .
From the plot, determine the concentration, C, in IJg/mL, USP Powdered Black Cohosh ExtractRS
of the relevant analytes in the Sample solution. USP 23-epi-26-Deoxyactein RS
Separately calculate the percentages of the individual
triterpene glycosides in Table 2 as 23-epi-26-deoxyactein
(C37Hs6010) in the portion of Powdered Black Cohosh
taken:
Result =(V x C)/(F x W) x 100
V = volume of the Sample solution (mL)

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4820 Black Cohosh / Dietary Supplements USP 43

Powdered Black Cohosh Extract Analysis

Samples: StandardsolutionA, Standardsolution B, and
DEFINITION Sample solution
Powdered Black Cohosh Extractisprepared from Black Cohosh Developthe chromatograms until the solventfront has
by extraction with hydroalcoholic mixtures or other suitable moved about two-thirds of the length of the plate, and
solvents. It contains NLT 90.0% and NMT 110.0% of the dry the plate with the aid of a current of air. Spraythe
0

labeled amount of triterpene glycosides, calculated as plate with Spray reagent, heat at 100 for 5 min, and
23-epi-26-deoxyactein (C37H s60 ,o) on the dried basis. examine in daylight.
Acceptance criteria: The chromatogram of the Sample
IDENTIFICATION solution exhibits main zones similar in position and color to
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST the main zones in the chromatogram of Standardsolution
Standard solution A: 100 mg/mL of U~P Powdered Black A. The chromatogram of Standardsolution B exhibits
Cohosh ExtractRS in methanol . red-violetzones due to actein and 23-epi-26-deoxyactein
Standard solution B: 1 mg/mL each of USP Actein RS, USP at R F values of about 0.5 and 0.4, respectively. The
23-epi-26-Deoxyactein RS, and isoferulic acid in methanol chromatogram of the Sample solution exhibitszones similar
Sample solution: Shake a quantity of Powdered Extract, in color and R F values to those due to actein and 23-epi-
equivalent to 25 mg of triterpene glycosides, in'10 mL of 26-deoxyactein in the chromatogram of Standard solution
methanol. Allow to stand for 15 min before use. B.
Adsorbent: Chromatographic silica gel mixture with an • C. The chromatogram of the Sample solution exhibits peaks
average particle size of 10-15 prn (TLC plates) for cimiracemosideA, 26-deoxycimicifugoside, (265)
Application volume: 10 ~L actein, 23-epi-26-deoxyactein, cimigenol-arabinoside, and
Developing solvent system: Usethe upper phase of a cimigenol-xyloside at retention times corresponding to
mixture of butyl alcohol, glacial acetic acid, and water those compounds in the chromatogram of the Standard
(5:1 :4). solution, as obtained in the test for Contentof Triterpene
Spray reagent: Methanol, glacial acetic acid, sulfuric acid, Glycosides. The ratio of the peak areas of cimigenol- .
and p-anisaldehyde (85: 10: 5: 0.5). [NOTE-Store in a . arabinoside to cimigenol-xyloside is NLT 0.4 (distinction
refrigerator. The reagent is colorless; discard ifcolor from Cimicifuga foetida).
appears.]
Analysis COMPOSITION
Samples: Standardsolution A, StandardsolutionB, and • CONTENT OF TRITERPENE GLYCOSIDES
Sample solution Standard solution: Dissolve a quantity of USP Powdered
Develop the chromatograms until the solvent front has Black Cohosh ExtractRS in methanol with shakingfor
moved about 15 em, and dry the plate with the aid of a 1 min, and dilute with methanol to obtain a solution
current of air. having a known concentration of 30 mg/mL. Pass
Acceptance criteria: Examine the plate under UV light at through a membrane filter of 0.45-~m or finer pore size.
365 nm. The chromatogram of the Sample solution exhibits 23- epi-26-Deoxyactein standard solutions: Dissolve USP
main zones similar in position and color tothe main zones 23-epi-26-Deoxyactein RS in methanol with shakingfor
in the chromatogram of StandardsolutionA. In the upper 1 min. Dilute quantitatively, and stepwise if necessary, to
third of the plate, the chromatogram of the Sample solution obtain solutions having known concentrations of 500, 100,
exhibitsa blue fluorescent zone at the level of the zone due 50, 25, and 12.5 uq/rnl., Pass through a membrane filterof
to isoferulic acid in the chromatogram of Standard solution 0.45-~m or finer pore size.
B. Spraythe plate with Spray reagent, heat at 100 for 5 min,
0 System suitability solution: 0.1 mg/mL each of USP
and examine in daylight. The chromatogram of the Sample Actein RS and USP 23-epi-26-Deoxyactein RS in methanol
solution exhibits main zones similar in position and color to Sample solution: Transfera quantity of Powdered Extract,
the main zones in the chromatogram of Standardsolution equivalent to, 7.5 mg of triterpene glycosides, to a 10-mL
A. The chromatogram of Standardsolution B exhibits volumetricflask, add 7 mLof methanol, and sonicate for
red-violetzones due to actein and 23-epi-26-deoxyactein. 30 min. Dilute with methanol to volume. Centrifuge, or
The chromatogram of the Sample solution exhibits several pass through a filter of 0.45-~m or finer pore size.
greenish-brown spots in the lower third of the plate and Solution A: 0.05% trifluoroacetic acid in water
severalviolet zones above; two of these violet zones occur Solution B: Acetonitrile
at R F values similar to those due to actein and 23-epi- Mobile phase: See Table 1.
26-deoxyactein in the chromatogram of Standardsolution
& ' Table 1
• B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Time Water Solution A Solution B
(min) (%) (%) (%)
Standard solution A: 0.5 mL of Standardsolution A
prepared in Identification test A, diluted with methanol to 0 0 80 20
2 mL
8 0 80 20
Standard solution B: 1.0 mLofStandardsolution B prepared
in Identification test A, diluted with methanol to 5 mL 15 68 0 32
Sample solution: Dilute 1 mL of the Sample solution 55 36 0 64
prepared in Identification test A with methanol to 10 mL.
Adsorbent: Chromatographic silica gel mixture with an 65 5 0 95
average particlesize of 5 urn (HPTLC plates) 70 5 0 95
Application volume: 2 ~L as an 8-mm band
Developing solvent system: Toluene, ethyl formate, and 85 0 80 20
formic acid (5:3:2)
Spray reagent: Proceed as directed for ldentliicaticn test A. Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC

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USP 43 Dietary Supplements / Black Cohosh 4821

Detector: Evaporative light-scattering 0.995. From the graphs so obtained, determine the
[NOTE-The detector isset up according to the concentration, C,in IJg/mL, ofthe relevantanalyteinthe
manufacturer's instruction in order to achievea Sample solution. Separately calculatethe percentages of
signal-to-noise ratio of NLT 10 for the 12.5-lJg/mL cimicifugoside H-l, cimiracemoside A, (26R)-actein,
23-epi-26-Deoxyactein standard solution.] 26-deoxycimicifugoside, (26S)-actein, 23-epi-
Column: 4.6-mm x 25-cm; 5-lJm packing L1 26-deoxyactein, ·acetyl-shengmanol-xyloside,
Column temperature: 35° cimigenol-arabinoside, cimigenol-xyloside
Flow rate: 1.6 mL/min (cimicifugoside), 26-deoxyactein,· 25-acetyl-cimigenol-
Injection size: 20 IJL arabinoside, (24S),.25-acetyl-cimigenol-xyloside,
System suitability 25-0-methyl-cimigenol-arabinoside, and
Samples: System sUitability solution, Standard solution, and 25-0-methyl-cimigenol-xylosideas 23-epi-
1OO-lJg/mL 23-epi-26-Deoxyactein standard solution 26-deoxyactein (C37Hs601O) in the portion of Extract
Suitability requirements taken:
Chromatogram similarity: The chromatogram of the
Standard solution is similar to the Reference Result =(V x Q/(F x W) x 100
Chromatogram providedwith the lot of USP Powdered
Black Cohosh Extract RS being used. V =volume of the Sample solution (mL)
Resolution: NLT 1.0 between the (26S)-actein and the C = concentration of the relevantanalyte in the
23-epi-26-deoxyactein peaks, System suitability solution Sample solution (lJg/mL)
Tailing factor: NMT 2.0 for the 23-epi-26-deoxyactein .F = factor to convert mg to IJg, 1000 IJg/mg
peak, 1OO-lJg/mL 23-epi-26-Deoxyactein standard W =weight of the Powdered Extract taken to
solution prepare the Sample solution (mg)
Relative standard deviation: NMT 2.0% of the
logarithm of the area response of the 23-epi- Calculate the percentage of the labeled amount of
26-deoxyactein peak in repeated injections, 100-lJg/mL triterpene glycosides in the portion of Extract taken by
23-epi-26-Deoxyactein standard solution adding all of the percentages calculatedfor individual
Analysis analytes.
Samples: System suitability solution, Standard solution, Acceptance criteria: 90.00/0-110.0% on the dried basis
23-epi-26-Deoxyactein standard solutions, and Sample CONTAMINANTS
solution • MICROBIAL ENUMERATION TESTS (2021): It meets the
Using the chromatogram of the Standard solution and the requirements of the tests for absence of Salmonella species
Reference Chromatogram provided with the lot of USP and Escherichia coli. The total bacterial count does not
Powdered Black Cohosh Extract RS, identify the exceed 104 du/g, and the total combined molds and yeasts
retention times of the peaks corresponding to the count does not exceed 103 du/g.
triterpene glycosides. The approximate relative • OTHER REQUIREMENTS: It meets the requirements under
retention times of the triterpene glycosides are Botanical Extracts (565), Pesticide Residues.
provided in Table 2.
SPECIFICTESTS
Table 2 • Loss ON DRYING (731): NMT 5.0%
Relative ADDITIONAL REQUIREMENTS
Retention
Name Time • PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store in a cool place.
Cimicifugoside H-l 0.61 • LABELING: It meets the requirements under Botanical
Cimiracemoside A 0.78 Extracts (565). Label it to indicatethe content of triterpene
glycosides, in percentage, calculated as 23-epi-
(26R)-Actein 0.94
26-deoxyactein. Dosageforms prepared with this article
26-Deoxycimicifugoside 0.96 should bear the following statement: "Discontinue use and
consult a healthcare practitionerifyou have a liver disorder
(26S)-Actein 0.98
or develop symptoms of liver trouble, such as abdominal
23-epi-26-Deoxyactein 1.00 pain, dark urine, or jaundice."
• USP REFERENC~ STANDARDS (11)
Acetyl-shengmanol-xyloside 1.03
USP Actein RS
Cimigenol-arabinoside 1.08 USP Powdered Black Cohosh Extract RS
Cimigenol-xyloside (cimicifugoside) 1.13
USP 23-epi-26-Deoxyactein RS
26-Deoxyactein 1.22

25-Acetyl-cimigenol-arabinoside 1.60

(24S)-25-Acetyl-cimigenol-xyloside 1.64 Black Cohosh Fluidextract

25-0-Methyl-cimigenol-arabinoside 1.90
25-0-Methyl-cimigenol-xyloside 1.93
Plotthe logarithms of the peak area responses versus the
Io'garithms of the concentrations, in IJg/mL, of the
23-epi-26-Deoxyactein standard solutions, and determine
the reqression line using a least-squares analysis. The
correlation coefficient for the regression line is NLT
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 9 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 (C37Hs60,o).

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 4822 Black Cohosh / Dietary Supplements USP 43

IDENTIFICATION 26-deoxyactein, cimigenol-arabinoside, and cimigenol-

• A. THIN-LAVER CHROMATOGRAPHIC IDENTIFICATION TEST xyloside at retention times corresponding to those
Standard solution A: 100 mg/mL of USP Powdered Black compounds in the Standardsolution, as obtained in the
Cohosh Extract RS in methanol test for Contentof Triterpene Glycosides. The ratio of the peak
Standard solution B: 1 mg/mL each of USP Actein RS, USP areas of cimigenol-arabinoside to cimigenol-xyloside is
23-epi-26-Deoxyactein RS, and isoferulic acid in methanol NLT 0,4 (distinction from Cimicifuga foetida).
Sample solution: Fluidextract
Adsorbent: Chromatographic silica gel mixture with an COMPOSITION
average particle size of 10-15 IJm (TLC plates) • CONTENT OF TRITERPENE GLYCOSIDES
Application volume: 10 IJL Standard solution: Dissolve a quantity of USP Powdered
Developing solvent system: Use the upper phase of a BlackCohosh Extract RS in methanol with shaking for
mixture of butyl alcohol, glacial acetic acid, and water 1 min, and dilute with methanol to obtain a solution
(5:1 :4). having a known concentration of 30 mg/mL. Pass
Spray reagent: Methanol, glacial acetic acid, sulfuric acid, through a membrane filter of 0,45-lJm or finer pore size.
and p-anisaldehyde (85: 10: 5: 0.5). [NOTE-Store in a 23- epi-26-Deoxyactein standard solutions: Dissolve USP
refrigerator. The reagent is colorless; discard if color 23-epi-26-Deoxyactein RS in methanol with shaking for
appears.] 1 min. Dilute quantitatively, and stepwise if necessary, to
Analysis obtain solutions having known concentrations of 500, 100,
Samples: Standardsolution A, Standardsolution B, and 50,25, and 12.5 IJg/mL. Pass through a membrane filter of
Sample solution 0,45-lJm or finer pore size.
Develop the chromatograms until the solvent front has System suitability solution: 0.1 mg/mL each of USP
moved about 15 em, and dry the plate with the aid of a Actein RS and USP 23-epi-26-Deoxyactein RS in methanol
current of air. Sample solution: Use the Fluidextract, diluting if necessary
Acceptance criteria: Examine the plate under UV light at with methanol to obtain a concentration of about 0.75 mg/
365 nm. The Sample solution exhibits main zones similar in mL of triterpene glycosides. Centrifuge, or pass through a
position and color to the main zones of Standardsolution filter of 0,45-lJm or finer pore size.
A. In the upper third of the plate, the Sample solution Solution A: 0.05% trifluoroacetic acid in water
exhibits a blue fluorescent zone at the level of the zone due Solution B: Acetonitrile
to isoferulic acid of StandardsolutionB. Spray the plate with Mobile phase: See Table 1.
Spray reagent, heat at 100° for 5 min, and examine in .
daylight. The Sample solution exhibits main zones similar in Table 1
position and color to the main zones of Standardsolution A. Time Water Solution A Solution B
Standardsolution B exhibits red-violet zones due to actein (min) (%) (%) (%)
and 23-epi-26-deoxyactein. The Sample solution exhibits 0 0 80 20
several greenish-brown spots in the lower third of the plate
8 0 80 20
and several violet zones above; two of these violet zones
occur at R F values similar to those due to actein and 15 68 0 32
23-epi-26-deoxyactein of Standardsolution B. 55 36 0 64
• B. THIN-LAvER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 0.5 mL of StandardsolutionA 65 5 0 95
prepared in Identification test A, diluted with methanol to 70 5 0 95
2.0 mL
Standard solution B: 1.0 mLof StandardsolutionB prepared 85 0 80 20
in Identification test A, diluted with methanol to 5.0 mL
Sample solution: Use the Fluidextract, diluting if necessary Chromatographic system
with a suitable solvent to obtain a concentration of (See Chromatography (621), System SUitability.)
0.25 mg/mL of triterpene glycosides. Mode: LC
Adsorbent: Chromatographic silica gel mixture with an Detector: Evaporative light-scattering
average particle size of 5 IJm (HPTLC plates) [NOTE-The detector is set up according to the
Application volume: 2 IJL as an 8-mm band manufacturer's instruction in order to achieve a
Developing solvent system: Toluene, ethyl formate, and signal-to-noise ratio of NLT 10 for the 12.5-lJg/mL
formic acid (5:3:2) 23-epi-26-Deoxyactein standardsolution.]
Spray reagent: Proceed as directed for Identification test A. Column: 4.6-mm x 25-cm; 5-lJm packing L1
Analysis Column temperature: 35°
Samples: Standardsolution A, Standardsolution B, and Flow rate: 1.6 mL/min
Sample solution Injection size: 20 IJL
Develop until the solvent front has moved two-thirds of System suitability
the length of the plate, and dry the plate with the aid Samples: System suitability solution, Standardsolution, and
of a current of air. Spray the plate with Spray reagent, 1OO-lJg/mL 23-epi-26-Deoxyactein standardsolution
heat at 100° for 5 min, and examine in daylight. . Suitability requirements
Acceptance criteria: The Sample solution exhibits main Chromatogram similarity: The chromatogram of the
zones similar in position and color to the main zones of Standardsolution is similar to the Reference
Standardsolution A. Standardsolution B exhibits red-violet Chromatogram provided with the lot of-USP Powdered
zones due to actein and 23-epi-26-deoxyactein at R F values Black Cohosh Extract RS being used.
of about 0.5 and 0,4, respectively. The Sample solution Resolution: NLT 1.0 between the (26S)-actein and the
exhibits zones similar in color and R F values to those due to 23-epi-26-deoxyactein peaks, System suitability solution
actein and 23-epi-26-deoxyactein of Standardsolution B. Tailing factor: NMT 2.0 for the 23-epi-26-deoxyactein
• C. The Sample solution exhibits peaks for cimiracemoside A, peak, 1OO-lJg/mL 23-epi-26-Deoxyactein standard
26-deoxycimicifugoside, (26S)-actein, 23-epi- solution

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USP 43 Dietary Supplements / Black Cohosh 4823

Relative standard deviation: NMT 2.0% of the
logarithm of the area response of the 23-epi-
26-deoxyactein peak in repeated injections, 100-l..Ig/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
r~tention 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
Relative
Reten-
Compound tion Time
Calculate the percentage of the labeled amount of
triterpene glycosides in the portion of Fluidextract
taken:
Result =xctL x 100
LC =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%
CONTAMINANTS
• MICROBIAL ENUMERATION TESTS (2021): The total bacterial
count does not exceed 10 4 cfu/g, and the total combined
molds and yeasts count does not exceed 10 3 cfu/g.
• OTHER.REQUIREMENTS: It meets the requirements under
Botanical Extracts (565), Residual Solvents and Pesticide
Residues.

Cimicifugoside H-l 0.61

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
Cimiracemoside A 0.78 containers, and store in a cool place.
(26R)-Actein 0.94 • LABELING: It meets the requirements for Labeling under
Botanical Extracts (565). Label it to indicate the content, in
26-0eoxycimicifugoside 0.96 percentage, of triterpene glycosides, calculated as 23-epi-
(265)-Actein 0.98 26-deoxyactein. Dosage forms prepared with this article
should bear the following statement: Discontinue use and
23-epi-26-0eoxyactein 1.00 consult a healthcare practitioner if you have a liver disorder
Acetyl-shengmanol-xyloside 1.03 or develop symptoms of liver trouble, such as abdominal
pain, dark urine, or jaundice.
Cimigenol-arabinoside 1.08 • USP REFERENCE STANDARDS (11)
Cimigenol-xyloside (cimicifugoside) 1.13 USP Actein RS
USP Powdered Black Cohosh Extract RS
26-0eoxyactein 1.22 USP 23-epi-26-Deoxyactein RS
2S-Acetyl-cimigenol-arabinoside 1.60

(245)-2S-Acetyl-cimigenol-xyloside 1.64

2S-0-Methyl-cimigenol-arabinoside 1.90
2S-0-Methyl-cimigenol-xyloside 1.93
Plot the logarithms of the peak area responses versus the
logarithms of the concentrations, in I..IgJrnL, of the
23-epi-26-Deoxyactein standard solutions, and determine
the regression line using a least-squares analysis. The
correlation coefficient for the regression line is NLT
0.995. From the graphs so obtained, determine the
concentration, C, in I..Ig/mL, of the relevant analyte in the
Sample solution. Separately calculate the concentrations,
in I..Ig/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,
25-0-methyl-cimigenol-arabinoside, and
25-0-methyl-cimigenol-xyloside as 23-epi-
26-deoxyactein (C37Hs601O) in the portion of Fluidextract
taken:
Result = (0 xC/V)
o = dilution factor for the Sample solution, if
applicable: final volume of Sample solution/
. volume of aliquot of Fluidextract taken (mL/mL)
C = concentration of the relevant analyte in the
Sample solution (l..Ig/mL)
V .= volume of the Fluidextract taken to prepare the
Sample solution (mL)
Black Cohosh Tablets
DEFINITION
Black Cohosh Tablets contain Powdered Black Cohosh Extract
or Black Cohosh Fluidextract. Tablets contain NLT 90.0% and
NMT 110.0% of the labeled amount of Powdered Extract or
Fluidextract, represented by the content of triterpene
glycosides, calculated as 23-epi-26-deoxyactein (C37Hs6010)'
IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201)
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 I..Im (TLC plates)
Sampl~. sOI.ution: 10 mL of the Sample solution prepared for
tdentitication test B. Evaporate to dryness, and redissolve in
1 mL of methanol.
Standard solution A: 100 mg/mL of USP Powdered Black
Cohosh Extract RS in methanol
Standard solution B: 1 mg/mL each of USPActein RS, USP
23-epi-26-Deoxyactein RS, and isoferulic acid in methanol
Application volume: 10 I..IL
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.]

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 4824 Black Cohosh / Dietary Supplements USP 43

Analysis Time Water Solution A Solution B

Samples: StandardsolutionA, Standardsolution B, and
Sample solution
Develop untilthe solventfront has moved 15 em, and dry
the plate with the aid of a current of air. Examine the
plate under UV light at a wavelength of 365 nm. Spray
the plate with Sprayreagent, 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
Standardsolution A. Examined under UV light, the Sample
solution exhibits a blue fluorescentzone at the level of the
zone due to isoferulic acid in Standardsolution B "in the
upper third of the plate. Examined after treatme~t with
Spray reagent, the Sample solution exhibitsseveral
greenish-brown spots in the lowerthird of the plate and
several violetzones above; two of these violetzones occur
at RF values similar to those due to actein and 23-epi-
26-deoxyactein in Standardsolution B.
• B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201 )
Adsorbent: Chromatographic silica gel mixturewith an
average particle sizeof 5 urn (HPTLC plates)
Sample solution: Transfer the equivalent of the labeled
amount of Powdered Extract or Fluidextract, containing
25 mg of triterpene glycosides from a portion of powdered
Tablets, to 25 mL of water; shake to disperse; and sonlcate .
for 10 min. Add 75 mL of methanol, and sonicate for
10 min. Allow to stand for 15 min, and use the clear
supernatant.
Standard solution A: Methanol and Standardsolution A
prepared in Identification test A (3:1)
Standard solution B: Methanol and Standardsolution B
prepared in Identification test A (4:1)
Application volume: 2 fJL as an 8-mm band
Developing solvent system: Toluene, ethyl formate, and
formic acid (5:3:2) . and transfer the washings to the volumetric flask. Dilute
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 ifcolor appears.]
A~~ili . (See Chromatography(621), System Suitability.)
Samples: StandardsolutionA, Standardsolution B, and'
Sample solution '.
Develop until the solventfront has moved two-thirds of
the length of the plate, and dry the plate with the aid
of a current of air. Spraythe 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
StandardsolutionA, two of which are red-violet zones at RF
values of 0.5 and 0.4, similar in colorand RF values to those
due to actein and 23-epi-26-deoxyactein in Standard
solution B . " . .:
• C. The Sample solution exhibits peaksfor cimiracemoside A,
26-deoxycimicifugoside, (265) actein, 23-epi-
26-deoxyactein, cimigenol-arabinoside, and cimigenol-
xyloside at retention times corresponding to those
compounds in the Standardsolution, as obtained in the
test for Contentof Triterpene Glycosides. Theratioofthe peak
areas of cimigenol-arabinoside to cimigenol-xyloside is
NLT 0.4 (distinction from Cimicifuga foetida).
STRENGTH
• CONTENT OF TRITERPENE GLYCOSIDES
Solution A: Filtered and degassed 0.05% trifluoroacetic acid
in water
Solution B: Filtered and degassed acetonitrile
Mobile phase: See the gradient table below.
(min) (%) (%) (%)
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
System suitability solution: 0.1 mg/mL each of USP
Actein RS and USP 23-epi-26-Deoxyactein RS in methanol
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 filter havinga 0.45-fJm or finer
porosity.
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 solutionshaving knownconcentrationsof 500, 1DO,
50,25, and 12.5 fJg/mL. Pass through a membrane filter
having a 0.45-fJm or finer porosity.
Sample solution: Weigh NLT 20 Tablets, and finely powder.
Transfer a quantity of the powder, equivalentto 8 mg of
triterpene glycosides, to a suitable polytef-capped
centrifuge tube. Add 3 mL of water, shake to disperse, and
sonicate for 10 min at 60°. Add 3 mL of methanol, and
sonicate for 10 min. Centrifuge, and transfer the clear
supernatant to a 1O-mL volumetric flask. Wash the residue
twicewith 1.5 mL of a mixtureof methanoland water (1:1),
with a mixtureof methanol and water (1:1) to volume, and
pass through a membrane filter having a 0.45-fJm or finer
porosity.
Chromatographic system
Mode: LC
Detector: Evaporative light-scattering
[NoTE-Detectorisset up according to the
manufacturer's instruction in order to achieve a
signal-to-noise ratio of NLT 10 for the 12.5 fJg/mL
23-epi-26-Deoxyactein standardsolution.]
Column: 4.6-mm x 25-cm; 5-fJm packing L1
Column temperature: 35°
Flow rate: 1.6 mL/min
Injection size: 20 fJL
System suitability
Samples: System suitability solution and 100 fJg/mL of
23-epi-26-Deoxyactein standardsolution
Suitability requirements
Chromatographic profile: The chromatogram of the
Standardsolution is similar to the Reference
Chromatogram provided with the lot of USP Powdered
Black Cohosh Extract RS.
Resolution: NLT 1.0 between the (265)-actein and the
23-epi-26-deoxyactein peaks, System suitability solution
Tailing factor: NMT 2.0 for the 23-epi-26-deoxyactein
peak, 100 fJg/mL 23-epi-26-Deoxyactein standard
solution
Relative standard deviation: NMT 2.0% for the
logarithm of the area responses for replicate injections
100 fJg/mL 23-epi-26-Deoxyactein standardsolution '

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USP43 Dietary Supplements / Black Pepper 4825

Analysis PERFORMANCE TESTS

Samples: System suitability solution, Standard solution, • DISINTEGRATION AND DISSOLUTION OF DIETARY
23-epi-26-Deoxyactein standard solutions, and Sample SUPPLEMENTS (2040): Meet the requirements for
solution Disintegration
Using the chromatogram of the Standard solution and the • WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091):
Reference Chromatogram provided with the lot of USP Meet the requirements
Powdered Black Cohosh Extract RS, identifythe
retention times of the peaks corresponding to the CONTAMINANTS
triterpene glycosides. The approximate relative • MICROBIAL ENUMERATION TESTS-NuTRITIONAL AND
retention times of the triterpene glycosides are provided DIETARY SUPPLEMENTS (2021): The total bacterial count
in the following table. does not exceed 104 du/g, and the total combined molds
and yeasts count does not exceed 103 du/g.
• MICROBIAL PROCEDURES FOR ABSENCE OF SPECIFIED
Relative MICROORGANISMS-NuTRITIONAL AND DIETARY
Retention
Name Time SUPPLEMENTS (2022): Tablets meet the requirements of
the tests for absence of Salmonella species and
Cimicifugoside H-l 0.61 Escherichia coli.
Cimiracemoside A 0.78 ADDITIONAL REQUIREMENTS
(26R)-Actein 0.94 • PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at room temperature.
26-Deoxycimicifugoside 0.96
• LABELING: The labelstates the Latin binomialand, following
(26S)-Actein 0.98 the official name, the article from which the Tablets were
prepared. The label also indicates the amount, in mgl
23-epi-26-Deoxyactein 1.00
Tablet, of Powdered Extractor Fluidextract; the solvents
Acetyl-shengmanol-xyloside 1.03 used to prepare the Powdered Extractor Fluidextract; and
1.08
the ratio of starting crude plant material to Powdered
Cimigenol-arabinoside
. Extractor Fluidextract. Label it to indicate the content, in
Cimigenol-xyloside (cimicifugoside) 1.13 percentage, of triterpene glycosides as 23-epi- .
26-Deoxyactein 1.22
26-deoxyactein in the Powdered Extractor Fluidextract
used to prepare the Tablets.
2S-Acetyl-cimigenol-arabinoside 1.60 The label bears the following statement: Discontinueuse and
(24S)-2S-Acetyl-cimigenol-xyloside 1.64
consult a healthcare practitioner ifyou have a liver
disorder or develop symptoms of liver trouble, such as
2S-0-Methyl-cimigenol-arabinoside 1.90 abdominal pain, dark urine, or jaundice.
2S-0-Methyl-cimigenol-xyloside 1.93 • USP REFERENCE STANDARDS (11)
USP Actein RS
USP Powdered Black Cohosh ExtractRS
Plot the logarithms of the peak area responses versus the USP 23-epi-26-Deoxyactein RS
logarithms of the concentrations, in IJg/mL, of the
23-epi-26-Deoxyactein standard solutions, and determine
the regression line using a least-squares analysis. The
correlation coefficientfor the regression line is NLT
0.995. From the graphs so obtained, determine the Black Pepper
concentration, C, in IJg/mL, of the relevantanalyte in the
Sample solution. DEFINITION
Calculate the quantity, in mg, of triterpene glycosides in Black Pepper consists of the dried fully developed unripe
the portion of Tablets taken: fruits of Piper nigrum L. (Fam. Piperaceae). It contains NLT
2.5% of piperine, calculated on the dried basis.
Result = CT/l 00
IDENTIFICATION
CT = sum of the concentrations C, in IJg/mL, of allthe • A. Black Pepper meets the requirements under Specific Tests,
relevant triterpene glycosides, calculated as Botanical Characteristics.
23-epi- 26-deoxyactein • B. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.9 mg/mL of USP Piperine RS in
Calculatethe percentage of the labeled amount of Extract methanol '
in the portion of Tablets taken: Standard solution B: 2.0 mg/mL of borneol in methanol
Standard solution C: 5 mg/mL of USP Powdered Black
Result =(Awr/W) x (100/LE) x (100/L) x Cn Pepper Extract RS in methanol. Sonicate for about 10 min,
centrifuge, and use the supernatant.
Awr = average weight of Tablets Sample solution: Sonicate for 10 min about 0.5 g of Black
W = weight of sample Pepper, finely powdered, in 5 mLof methanol, centrifuge,
LE = labeled content, as percentage, of triterpenes in and use the supernatant.
the Extractused to prepare the Tablets Chromatographic system
L = labeled amount of Extract per Tablet Adsorbent: Chromatographic silica gel mixture with an
Cn = content,in mg, of triterpenes in the sample average particle size of 5 IJm (HPTLC plates)
Application volume: 15 IJL of Standard solution C, 7 IJL
Acceptance criteria: 90.0%-110.0% of the labeled amount of the Sample solution, and 3 IJL of the Standard solution A
of Powdered Extractor Fluidextract, represented by the and Standard solution 8, as bands
content of triterpene glycosides Developing solvent system: Hexanes and ethyl acetate
(5:3)

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