Soy Isoflavones Tablets
DEFINITION
Soy Isoflavones Tablets contain Powdered Soy Isoflavones Extract. Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of Extract, represented by the sum of the content of the isoflavones daidzin, glycitin, genistin and one or more of the following isoflavones: malonyl daidzin, malonyl glycitin, malonyl genistin, acetyl daidzin, acetyl glycitin, acetyl genistin, daidzein, glycitein, and genistein.
IDENTIFICATION
•  The retention times of the daidzin, glycitin, and genistin peaks in the chromatogram of the Sample solution correspond to those of Standard solutions A–E, as obtained in the test for Content of Isoflavones.
STRENGTH
•  Content of Isoflavones
Diluent:  Acetonitrile and water (2:3)
Internal standard solution:  2.0 mg/mL of USP Apigenin RS in dimethyl sulfoxide. [Note—This solution is stable for 6 months when stored in a tightly closed, light-resistant glass container at room temperature. ]
Malonyl/Acetyl isoflavones retention times check solution 1:  Heat 1 g of USP Defatted Powdered Soy RS in a shallow porcelain dish at 120 for 120 min, and transfer to a centrifuge tube, fitted with PTFE or polyethylene-lined screw cap. Add the following in exact volumes: 0.5 mL of Internal standard solution, 10 mL of acetonitrile (swirl to disperse), and 6.0 mL of water. Cap, shake on an orbital or wrist-action shaker for 60 min, add 8.5 mL of water, mix, and centrifuge. Pass a portion of the supernatant through a hydrophilic propylene or PVDF membrane of 0.45-µm or finer pore size, discarding the first 5 mL of filtrate.
Malonyl/Acetyl isoflavones retention times check solution 2:  Transfer 1 g of USP Defatted Powdered Soy RS to a centrifuge tube, fitted with PTFE or polyethylene-lined screw cap. Add the following in exact volumes: 0.5 mL of Internal standard solution, 10 mL of acetonitrile (swirl to disperse), and 6.0 mL of water. Cap, shake on an orbital or wrist-action shaker for 60 min, add 8.5 mL of water, mix, and centrifuge. Pass a portion of the supernatant through a hydrophilic propylene or PVDF membrane of 0.45-µm or finer pore size, discarding the first 5 mL of filtrate.
[Note—All Standard solutions and Standard stock solutions are stable for 2 months when stored in a tightly closed, light-resistant glass container at room temperature. ]
Standard stock solution:  Contains the following in dimethyl sulfoxide: 2.0 mg/mL of USP Daidzin RS, 0.5 mg/mL of USP Glycitin RS, 2.0 mg/mL of USP Genistin RS, 0.2 mg/mL of USP Daidzein RS, 0.2 mg/mL of USP Glycitein RS, and 0.2 mg/mL of USP Genistein RS
Standard solution A:  Add 0.5 mL of Standard stock solution and 0.5 mL of Internal standard solution to a 25-mL volumetric flask, and dilute with Diluent to volume.
Standard solution B:  Add 1.0 mL of Standard stock solution and 0.5 mL of Internal standard solution to a 25-mL volumetric flask, and dilute with Diluent to volume.
Standard solution C:  Add 1.5 mL of Standard stock solution and 0.5 mL of Internal standard solution to a 25-mL volumetric flask, and dilute with Diluent to volume.
Standard solution D:  Add 2.0 mL of Standard stock solution and 0.5 mL of Internal standard solution to a 25-mL volumetric flask, and dilute with Diluent to volume.
Standard solution E:  Add 2.5 mL of Standard stock solution and 0.5 mL of Internal standard solution to a 25-mL volumetric flask, and dilute with Diluent to volume.
Sample solution:  Accurately weigh and finely powder NLT 20 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to NMT 5 mg of isoflavones, to a 30-mL glass centrifuge tube, fitted with a PTFE or polyethylene-lined screw cap. Add the following in exact volumes: 0.5 mL of Internal standard solution, 10 mL of acetonitrile (swirl to disperse), and 6.0 mL of water. Cap, shake on an orbital or wrist-action shaker for 60 min, add 8.5 mL of water, mix, and centrifuge. Pass a portion of the supernatant through a hydrophilic propylene or PVDF membrane of 0.45-µm or finer pore size, discarding the first 5 mL of filtrate. [Note—Do not use nylon filters. Analyze samples containing significant amounts of acetyl and/or malonyl isoflavones within 4 h of preparation. ]
Solution A:  0.05% phosphoric acid in water
Solution B:  Acetonitrile
Mobile phase:  See the gradient table below.
Time
(min)
Solution A
(%)
Solution B
(%)
0 90 10
60 70 30
60.5 10 90
63.5 10 90
64 90 10
74 90 10
Chromatographic system 
Mode:  LC
Detector:  UV 260 nm
Column:  3.0-mm × 25-cm; 5-µm packing L1
Column temperature:  40
Flow rate:  0.65 mL/min
Injection size:  5 µL
System suitability 
Samples:  Standard solution C, Malonyl/Acetyl isoflavones retention times check solution 1, and Malonyl/Acetyl isoflavones retention times check solution 2
Suitability requirements 
Chromatogram similarity:  The chromatograms obtained from Standard solution C, Malonyl/Acetyl isoflavones retention times check solution 1, and Malonyl/Acetyl isoflavones retention times check solution 2 are similar to the Reference Chromatograms provided with the USP Defatted Powdered Soy RS.
Tailing factor:  NLT 0.8 and NMT 1.2 for the daidzin peak, Standard solution C
Relative standard deviation:  NMT 2.0%, for the genistin peak, in repeated injections, Standard solution C
Resolution:  NLT 1.0 between acetyl glycitin and malonyl genistin, and NLT 2.0 between any other consecutive pair of isoflavone peaks, Malonyl/Acetyl isoflavones retention times check solution 2
Analysis 
Samples:  Standard solution A, Standard solution B, Standard solution C, Standard solution D, Standard solution E, Malonyl/Acetyl isoflavones retention times check solution 1, Malonyl/Acetyl isoflavones retention times check solution 2, and Sample solution
Identify the peaks of daidzin, glycitin, genistin, daidzein, glycitein, and genistein in the chromatograms of Standard solutions A–E by comparison with the Reference Standard Chromatogram provided with the appropriate USP Reference Standard used. Measure the peak areas of the analytes and the internal standard peaks. Determine the ratio of the peak areas of each analyte to the internal standard peak areas. Plot the ratios of the relevant peak responses versus the concentrations, in mg/mL, of each analyte obtained from Standard solutions A–E, and determine the regression line by least-squares analysis. The correlation coefficient for each of the regression lines is NLT 0.999. From the graphs obtained, determine the concentration, C, in mg/mL, of daidzin, glycitin, and genistin, in addition to daidzein, glycitein, and genistein, if present, in the Sample solution. Identify the peaks of malonyl daidzin, malonyl glycitin, acetyl daidzin, acetyl glycitin, malonyl genistin, and acetyl genistin in the chromatograms of the Malonyl/Acetyl isoflavones retention times check solutions by comparison with the Reference Chromatograms provided with the USP Defatted Powdered Soy RS. From the graphs obtained for daidzin, glycitin, and genistin, determine the concentration, C, in mg/mL, of the corresponding malonyl and acetyl derivatives, if present, in the Sample solution:
Result = Co × F
Co== concentration obtained from the relevant graph (mg/mL)
F== conversion factor for each analyte (1.207 for malonyl daidzin, 1.101 for acetyl daidzin, 1.193 for malonyl glycitin, 1.094 for acetyl glycitin, 1.199 for malonyl genistin, and 1.097 for acetyl genistin)
Calculate the quantity, T, in mg, of total isoflavones in the portion of Tablets taken:
Result = V × CT
V== final volume of the Sample solution (mL)
CT== sum of concentrations (C) (mg/mL) of all relevant isoflavones
Calculate the percentage of Powdered Soy Isoflavones Extract with respect to the label claim:
Result = T × (AWT/W) × (100/LE) × (100/L)
T== content of total isoflavones in the portion of Tablets taken (mg)
AWT== average weight of Tablets (mg/Tablet)
W== weight of the portion of Tablets taken (mg)
LE== content of total isoflavones, mg, in 100 mg of the Extract used to prepare the Tablets
L== amount of Extract per Tablet according to label claim (mg/Tablet)
Acceptance criteria:  NLT 90.0% and NMT 110.0% of the labeled amount of Extract, represented by the sum of the content of the isoflavones daidzin, glycitin, genistin and one or more of the following isoflavones: malonyl daidzin, malonyl glycitin, malonyl genistin, acetyl daidzin, acetyl glycitin, acetyl genistin, daidzein, glycitein, and genistein
PERFORMANCE TESTS
•  Disintegration and Dissolution of Dietary Supplements 2040: Meet the requirements for Disintegration
•  Weight Variation of Dietary Supplements 2091: Meet the requirements
CONTAMINANTS
•  Microbial Enumeration Tests 2021: The total aerobic microbial count is NMT 104 cfu/g; and the total combined molds and yeasts count is NMT 103 cfu/g.
•  Absence of Specified Microorganisms 2022: Tablets meet the requirements of the tests for absence of Salmonella species and Escherichia coli.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers, and store at room temperature.
•  Labeling: The label states the Latin binomial and, following the official name, the article from which the Tablets were prepared. Label it to indicate the amount of Extract, in mg, per Tablet. Label it to indicate the content, in percentage, of isoflavones in the Extract used to prepare the Tablets.
•  USP Reference Standards 11
USP Apigenin RS
USP Daidzein RS
USP Daidzin RS
USP Defatted Powdered Soy RS
USP Genistein RS
USP Genistin RS
USP Glycitein RS Click to View Structure
USP Glycitin RS Click to View Structure
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Maged H. Sharaf, Ph.D.
Principal Scientific Liaison
1-301-816-8318
(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 1449
Pharmacopeial Forum: Volume No. 36(1) Page 167