Pygeum Capsules contain Pygeum Extract. Capsules contain NLT 90.0% and NMT 110.0% of the labeled amount of Extract, calculated as sterols and docosyl ferulate.
• A. The retention times of the peaks for campesterol, stigmasterol, and -sitosterol, of the Sample solution, correspond to those of the Standard solution, as obtained in the test for Content of Sterols.
• B. The retention time of the peak for docosyl ferulate in the Sample solution corresponds to that in the Standard solution, as obtained in the test for Content of Docosyl Ferulate.
• Content of Sterols
Derivatizing solution: Bis(trimethylsilyl)acetamide and trimethylchlorosilane (9:1)
Internal standard solution: 2 mg/mL of 5-cholestane in chloroform
System suitability stock solution: 2 mg/mL each of campesterol, stigmasterol, and USP -Sitosterol RS
System suitability solution: Mix 2.0 mL of the System suitability stock solution and 2.0 mL of the Internal standard solution, and dilute with chloroform to 10 mL. Evaporate 500 µL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 80 µL of Derivatizing solution and 20 µL of pyridine. Allow to stand for NLT 10 min at room temperature.
Standard stock solution: 2.0 mg/mL of USP -Sitosterol RS in chloroform
Standard solution: Mix 2.0 mL of the Standard stock solution and 2.0 mL of the Internal standard solution, and dilute with chloroform to 10 mL. Evaporate 500 µL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 80 µL of Derivatizing solution and 20 µL of pyridine. Allow to stand for NLT 10 min at room temperature.
Sample solution: Transfer a quantity of Capsules, equivalent to 100 mg of the labeled amount of Extract, into a 100-mL round-bottomed flask. Add 2.0 mL of the Internal standard solution and 20 mL of diluted hydrochloric acid. Attach a condenser, and reflux in a bath at 100 for 30 min. Cool the solution to room temperature, and adjust by the addition of about 5 mL of 10 N sodium hydroxide to a pH of 8. Extract twice using 50 mL of ether each time, wash the collected organic phases with 50 mL of water, and evaporate the organic phase to dryness under vacuum. Dissolve the residue with 4 mL of chloroform, and transfer to a cartridge containing 500 mg of packing L8 that has been conditioned with a 2-column volume of n-hexane. [NoteA suitable cartridge is Chromabond NH2, manufactured by Macheray Nagel, or equivalent. ] Collect the eluate. Elute twice with a 1-column volume of a mixture of chloroform and isopropanol (2:1). Combine the eluates, and evaporate to dryness. Dissolve the residue in 10 mL of chloroform. Evaporate 500 µL of this solution to dryness under a stream of nitrogen. Dissolve the residue with 80 µL of Derivatizing solution and 20 µL of pyridine. Allow to stand for NLT 10 min at room temperature.
Detector: Flame ionization
Column: 0.32-mm × 30-m capillary column coated with a G27 phase of 0.25-µm thickness
Column: See the temperature program table below.
Carrier gas: Helium. [NoteThe carrier gas flow rate should be adjusted to obtain a retention time of 19 min for -sitosterol. ]
Makeup gas: Helium
Injection size: 2 µL
Injection type: Split injection system
Split ratio: 1:50
Sample: System suitability solution
[NoteThe relative retention times for 5-cholestane, campesterol, stigmasterol, and -sitosterol are about 0.66, 0.94, 0.96, and 1.00, respectively. ]
Resolution: NLT 2 between campesterol and stigmasterol
Column efficiency: NLT 150,000 theoretical plates for the 5-cholestane peak
Tailing factor: NMT 2.0 for each relevant peak
Samples: Standard solution and Sample solution
Identify the signals corresponding to the relevant analytes by comparison with the chromatograms obtained with the System suitability solution.
Calculate the percentages of the labeled amount of Extract as sterols in the portion of the Capsules taken:
Result = (SRU/RS) × (CS × V/W) × (AW × 100/LE) × 100/L
Acceptance criteria: 90%110% of the labeled amount of Extract, calculated as sterols
• Content of Docosyl Ferulate
Solution A: Methanol and water (95:5)
Solution B: Acetonitrile
Mobile phase: Solution A and Solution B (17:3)
Standard solution: Dissolve a quantity of USP Docosyl Ferulate RS in chloroform, and dilute stepwise if necessary, with acetonitrile to obtain a concentration of 0.01 mg/mL. Pass through a membrane filter of 0.45-µm or finer pore size.
Sample solution: Weigh the contents of NLT 20 Capsules and transfer a quantity of the material, equivalent to 0.2 mg of docosyl ferulate, to a 50-mL beaker. Add 5 mL of chloroform and dissolve in an ultrasonic bath. Transfer to a 20-mL volumetric flask with the aid of NMT 2 mL of chloroform. Dilute with acetonitrile to volume and mix. Pass through a membrane filter of 0.45-µm or finer pore size.
Detector: UV 323 nm
Column: 4-mm × 25-cm; packing L7
Column temperature: 25
Flow rate: 1 mL/min
Injection size: 320 µL
Sample: Standard solution
Column efficiency: NLT 1700 theoretical plates for the docosyl ferulate peak
Tailing factor: NMT 2.0 for docosyl ferulate
Samples: Standard solution and Sample solution
Measure the areas of the analyte peaks.
Calculate the percentage of the labeled amount of Extract as docosyl ferulate in the portion of the Capsules taken:
Result = (rU/rS) × (CS × V/W) × (AW × 100/LE) × 100/L
Acceptance criteria: 90%110% of the labeled amount of Extract, calculated as docosyl ferulate and sterols (from Content of Sterols)
• Disintegration and Dissolution of Dietary Supplements 2040: Meet the requirements for Rupture Test for Soft Shell Capsules
• Weight Variation of Dietary Supplements 2091: Meet the requirements
• Microbial Enumeration Tests 2021: The total bacterial count does not exceed 1000 cfu/g. The total combined molds and yeasts count does not exceed 1000 cfu/g.
• Microbiological Procedures for Absence of Specified Microorganisms 2022: The Capsules meet the requirements of the tests for absence of Salmonella species and Escherichia coli.
• Packaging and Storage: Preserve in tight containers, and store at controlled room temperature.
• Labeling: The label states the Latin binomial and, following the official name, the article from which the Capsules were prepared. The label also indicates the quantity of Extract, in mg/Capsule. Label the Capsules to indicate the quantity of sterols and docosyl ferulate in percentage of the Extract contained in the Capsules.
• USP Reference Standards 11
USP Docosyl Ferulate RS
USP Pygeum Extract RS
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USP35NF30 Page 1417Pharmacopeial Forum: Volume No. 36(1) Page 162