Red Clover Tablets
DEFINITION
Red Clover Tablets contain Powdered Red Clover Extract. Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of Powdered Extract calculated as isoflavones.
IDENTIFICATION
•  A. HPLC Identification Test
Analysis:  Proceed as directed for the Content of Isoflavones.
Using the values obtained in the test for Content of Isoflavones, calculate the ratio of 5,7-dihydroxyisoflavones to 7-hydroxyisoflavones:
Result = (B + G)/(D + F)
B== percentage of biochanin A
G== percentage of genistein
D== percentage of daidzein
F== percentage of formononetin
Acceptance criteria:  The chromatogram of the Sample solution exhibits peaks for daidzein, genistein, formononetin, and biochanin A at retention times that correspond to those in the chromatogram of Standard solution A, and the ratio of 5,7-dihydroxyisoflavones to 7-hydroxyisoflavones is between 0.1 and 10.
STRENGTH
•  Content of Isoflavones
Solution A:  Acetonitrile and water (1:3) containing 0.05% trifluoroacetic acid
Solution B:  Acetonitrile containing 0.05% trifluoroacetic acid
Mobile phase:  See Table 1.
Table 1
Time
(min)
Solution A
(%)
Solution B
(%)
0 100 0
2 100 0
2.5 87 13
7.5 80 20
7.8 73 27
8.0 55 45
11.0 50 50
13.0 40 60
15.0 26 74
16.0 0 100
18.1 100 0
23.0 100 0
Solvent:  Alcohol and water (1:1)
Standard stock solution:  Transfer a quantity of USP Powdered Red Clover Extract RS, equivalent to 30 mg of the labeled content of isoflavones, to a 250-mL volumetric flask. Add 15 mL of dehydrated alcohol, sonicate until dissolved, and dilute with Solvent to volume.
Standard solution A:  Evaporate 50 mL of Standard stock solution to dryness under vacuum. Add 15 mL of 2 N hydrochloric acid, and heat in a water bath for 30 min. Quantitatively transfer the resulting solution, with the aid of 15 mL of alcohol, to a 50-mL volumetric flask, and dilute with Solvent to volume. Centrifuge, or filter through a membrane having a 0.45-µm or finer pore size.
Standard solution B:  0.1 mg/mL of USP Formononetin RS in a mixture of n-propanol and water (1:1). Sonicate, and filter through a membrane having a 0.45-µm or finer pore size.
Sample stock solution:  Weigh NLT 20 Tablets and pulverize. Transfer the equivalent of 40 mg of the labeled amount of isoflavones to a 250-mL volumetric flask. Add 15 mL of water, shake to disperse the powder, add 15 mL of dehydrated alcohol and 200 mL of Solvent, and sonicate for 30 min. If dark particles are present in the bottom of the flask, sonicate again for an additional 10 min or until they disappear. Cool to room temperature, dilute with Solvent to volume, and filter.
Sample solution:  Transfer 50.0 mL of the resulting solution to a round-bottom flask, and evaporate to dryness under vacuum. Add 15 mL of 2 N hydrochloric acid, and heat in a water bath for 30 min. Quantitatively transfer this solution, with the aid of 15 mL of alcohol, to a 50-mL volumetric flask, and dilute with Solvent to volume. Pass 5 mL of the solution through a filter having a 0.45-µm pore size, discarding the first 4 mL of the filtrate. Collect the remaining 1 mL of filtrate for testing.
Chromatographic system 
Mode:  LC
Detector:  UV 254 nm
Column:  4.6-mm × 25-cm; end-capped 5-µm packing L1
Column temperature:  45
Flow rate:  1 mL/min
Injection size:  10 µL
System suitability 
Sample:  Standard solution A
Suitability requirements 
Chromatogram similarity:  The chromatogram from Standard solution A is similar to the reference chromatogram provided with the lot of USP Powdered Red Clover Extract RS being used.
Tailing factor:  NMT 2.0 for the formononetin peak
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Identify the peaks corresponding to daidzein, genistein, formononetin, and biochanin A in the Sample solution chromatogram by comparison with the chromatogram obtained from Standard solution A and the reference chromatogram. Measure the areas of the analyte peaks.
Calculate the content of each isoflavone, in mg, in the portion of Tablets taken:
Result = (rU/rS) × CS × V × 1/F × D
rU== peak response for each relevant isoflavone from the Sample solution
rS== peak response for formononetin from Standard solution B
CS== concentration of USP Formononetin RS in Standard solution B (mg/mL)
V== volume of the Sample stock solution (mL)
F== conversion factor for each analyte: daidzein, 0.97; genistein, 1.13; formononetin, 1.00; biochanin A, 1.05
D== dilution factor to prepare the Sample solution from the Sample stock solution, 1
Calculate the percentage of the labeled amount of Powdered Red Clover Extract taken:
Result = CI × (AWT/W) × (100/LE) × (100/L)
CI== sum of the content of isoflavones in the portion of Tablets taken (mg)
AWT== average weight of Tablets (mg)
W== weight of the powdered Tablets taken (mg)
LE== labeled percentage of isoflavones in the Extract used to prepare the Tablets
L== label claim of Extract (mg/Tablet)
Acceptance criteria:  90.0%–110.0% as isoflavones
PERFORMANCE TESTS
•  Disintegration and Dissolution 2040: Meet the requirements for disintegration in Botanical Dosage Forms
•  Weight Variation 2091: Meet the requirements
SPECIFIC TESTS
•  Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 104 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the enterobacterial count is NLT 103 cfu/g.
•  Absence of Specified Microorganisms 2022: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers.
•  Labeling: The label states the Latin binomial and, following the official name, the article from which Tablets were prepared. The label also indicates the quantity, in mg, of Powdered Extract per Tablet. Label Tablets to indicate the content, in mg, of isoflavones per 100 mg of Powdered Extract.
•  USP Reference Standards 11
USP Formononetin RS Click to View Structure
USP Powdered Red Clover Extract RS
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Natalia Davydova
Scientific Liaison
1-301-816-8328
(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 1423
Pharmacopeial Forum: Volume No. 28(3) Page 812