Red Clover Tablets
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.
• 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)
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.
• 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.
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.
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
Sample: Standard solution A
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%
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
Calculate the percentage of the labeled amount of Powdered Red Clover Extract taken:
Result = CI × (AWT/W) × (100/LE) × (100/L)
Acceptance criteria: 90.0%110.0% as isoflavones
• Disintegration and Dissolution 2040: Meet the requirements for disintegration in Botanical Dosage Forms
• Weight Variation 2091: Meet the requirements
• 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.
• 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 Powdered Red Clover Extract RS
Auxiliary Information Please check for your question in the FAQs before contacting USP.
USP35NF30 Page 1423Pharmacopeial Forum: Volume No. 28(3) Page 812