Powdered Soy Isoflavones Extract
DEFINITION
Powdered Soy Isoflavones Extract is prepared from the seeds of Glycine max Merr. (Fam. Fabaceae) by extraction with water or hydroalcoholic mixtures. It contains NLT 90.0% and NMT 110.0% of the labeled amount of isoflavones, calculated on the dried basis as the sum of 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
• A. HPLC Identification Test
Analysis:
Proceed as directed in the test for Content of Isoflavones.
Acceptance criteria:
The retention times of the daidzin, glycitin, and genistin peaks from the Sample solution correspond to those of Standard solutions A–E.
COMPOSITION
• Content of Isoflavones
Diluent:
Acetonitrile and water (2:3)
Internal standard solution:
2.0 mg/mL 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. ]
System suitability solution 1:
Transfer 1 g of USP Defatted Powdered Soy RS to a centrifuge tube, fitted with PTFE or polyethylene-lined screw caps. 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, and centrifuge. Pass a portion of the supernatant through a hydrophilic propylene or PVDF membrane having a 0.45-µm or finer pore size, discarding the first 5 mL of filtrate.
System suitability solution 2:
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 caps. 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, and centrifuge. Pass a portion of the supernatant through a hydrophilic propylene or PVDF membrane having a 0.45-µm or finer pore size, discarding the first 5 mL of filtrate.
for 120 min, and transfer to a centrifuge tube, fitted with PTFE or polyethylene-lined screw caps. 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, and centrifuge. Pass a portion of the supernatant through a hydrophilic propylene or PVDF membrane having a 0.45-µm or finer pore size, discarding the first 5 mL of filtrate.
[Note—Standard stock solution and Standard solutions A–E 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:
Transfer a quantity of Powdered Soy Isoflavones Extract, 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, and centrifuge. Pass a portion of the supernatant through a hydrophilic propylene or PVDF membrane having a 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 Table 1.
Table 1
| 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
[Note—System suitability 1 and System suitability 2 must both be met. ]
System suitability 1
Sample:
Standard solution C
Suitability requirements
Chromatogram similarity:
The chromatogram from Standard solution C is similar to the refence chromatogram provided with the lot of USP reference standard being used for the isoflavones mentioned.
Tailing factor:
NLT 0.8 and NMT 1.2 for the daidzin peak
Relative standard deviation:
NMT 2.0% for the genistin peak
System suitability 2
Samples:
System suitability solution 1 and System suitability solution 2
Suitability requirements
Chromatogram similarity:
The chromatograms from System suitability solution 1 and System suitability solution 2 are similar to those provided with the lot of USP Defatted Powdered Soy RS being used.
Resolution:
NLT 1.0 between the acetyl glycitin and malonyl genistin peaks, and NLT 2.0 between any other consecutive pair of isoflavone peaks
Analysis
Samples:
Standard solutions A–E and Sample solution
Measure the peak areas of the analytes and the internal standard. Determine the ratio of the peak areas of each analyte to the internal standard peak area. Plot the ratios of the relevant peak responses versus the concentrations, in mg/mL, of each analyte obtained from the 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 so obtained, determine the concentration, C, in mg/mL, of the relevant analyte in the Sample solution.
Separately calculate the percentages of daidzin, glycitin, and genistin, and of daidzein, glycitein, and genistein, if present, in the portion of Powdered Soy Isoflavones Extract taken:
Result = (C/W) × V × 100
| C | = | = concentration of each isoflavone as determined above, for the Sample solution (mg/mL) |
| W | = | = weight of Powdered Soy Isoflavones Extract taken to prepare the Sample solution (mg) |
| V | = | = final solution volume of the Sample solution (mL) |
Identify the peaks of malonyl daidzin, malonyl glycitin, acetyl daidzin, acetyl glycitin, malonyl genistin, and acetyl genistin by comparison with the reference chromatograms provided with USP Defatted Powdered Soy RS and by the difference in peak abundance between System suitability solution 1 and System suitability solution 2. [Note—Malonyl derivates are converted into acetyl derivatives by heat. Malonyl derivatives are more abundant in System suitability solution 1 and acteyl derivatives are more abundant in System suitability 2. ] From the graphs obtained for daidzin, glycitin, and genistin, determine the corresponding concentration, C, in mg/mL, of the malonyl and acetyl derivatives, if present, in the Sample solution.
Separately calculate the percentages of malonyl daidzin, acetyl daidzin, malonyl glycitin, acetyl glycitin, malonyl genistin, and acetyl genistin in the portion of Powdered Soy Isoflavones Extract taken:
Result = (C/W) × V × F × 100
| C | = | = concentration of each isoflavone as determined above, for the Sample solution (mg/mL) |
| W | = | = weight of Powdered Soy Isoflavones Extract taken to prepare the Sample solution (mg) |
| V | = | = final solution volume of the Sample solution (mL) |
| F | = | = conversion factor: malonyl daidzin, 1.207; acetyl daidzin, 1.101; malonyl glycitin, 1.193; acetyl glycitin, 1.094; malonyl genistin, 1.199; and acetyl genistin, 1.097 |
Calculate the percentage of isoflavones in the portion of the Powdered Soy Isoflavones Extract taken (P) by adding the percentages calculated for all analytes present.
Calculate the percentage of the labeled amount of isoflavones in the portion of the Powdered Soy Isoflavones Extract taken:
Result = (P/L) × 100
| P | = | = sum of the individual percentages of each isoflavone in the Powdered Soy Isoflavones Extract taken, as calculated above (%) |
| L | = | = labeled amount of isoflavones in the Powdered Soy Isoflavones Extract (%) |
Acceptance criteria:
90.0%–110.0% on the dried basis
CONTAMINANTS
• Heavy Metals, Method II 
231
:
NMT 10 µg/g
231:
NMT 10 µg/g
• Articles of Botanical Origin, Test for Aflatoxins 561:
Meets the requirements
561:
Meets the requirements
• 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.
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:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
SPECIFIC TESTS
• Loss on Drying 731:
Dry 1 g at 130 for 2 h: it loses NMT 7.0% of its weight.
731:
Dry 1 g at 130 for 2 h: it loses NMT 7.0% of its weight.
• Other Requirements:
It meets the requirements for Residual Solvents and Pesticide Residues in Botanical Extracts 565.
565.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers, and store at controlled room temperature.
• Labeling:
The label states the Latin binomial and, following the official name, the part of the plant from which the article was prepared. The label also indicates the content of isoflavones. It meets other labeling requirements in Botanical Extracts 565.
565.
• USP Reference Standards 11
11
USP Apigenin RS
USP Daidzein RS
USP Daidzin RS
USP Defatted Powdered Soy RS
USP Genistein RS
USP Genistin RS
USP Glycitein RS 
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USP Glycitin RS
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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 1446
Pharmacopeial Forum: Volume No. 33(6) Page 1224