Pygeum Extract is prepared from pulverized Pygeum, using suitable solvents. It contains NLT 90% and NMT 110% of the labeled amount of docosyl ferulate and NLT 90% and NMT 110% of the labeled amount of total sterols as -sitosterol, calculated on the dried basis.
• A. Thin-Layer Chromatographic Identification Test
Standard solution A: 15 mg/mL of USP Pygeum Extract RS in chloroform
Standard solution B: 2 mg/mL of USP -Sitosterol RS in chloroform
Sample solution: Dissolve 150 mg of Extract in 10 mL of chloroform.
Adsorbent: 0.25-mm layer of chromatographic silica gel
Application volume: 10 µL
Developing solvent system: Methylene chloride in a saturated chamber
Spray reagent: Sulfuric acid and water (1:1)
Samples: Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms to a length of NLT 15 cm, and dry the plate in a current of air. Spray the plate with Spray reagent, and heat the plate at 100 for 10 min. Examine the plate under white light.
Acceptance criteria: The chromatogram from the Sample solution shows one red-violet zone turning to grayish-brown near the origin that corresponds in color and RF value to that of Standard solution A, and one red-violet zone turning to grayish-brown at an RF of 0.08 corresponding in color and RF value to that in the chromatogram of Standard solution B; above these spots a grayish-brown zone may be present, corresponding in color and RF value to that of Standard solution A; and other colored zones of varying intensities may be observed in the Sample solution.
• B. HPLC Identification Test
Analysis: Proceed as directed in the test for Content of Docosyl Ferulate.
Acceptance criteria: The chromatogram of the Sample solution presents a peak for docosyl ferulate that corresponds in retention time to the principal peak in the chromatogram of the Standard solution.
• 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 100 mg of Extract into a 100-mL round-bottom flask. Add 2.0 mL of 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 L81 that has been conditioned with a 2-column volume of n-hexane . 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; G27 phase coating of 0.25-µm thickness
Column: See Table 1.
Carrier gas: Helium. [NoteThe carrier gas flow rate should be adjusted to obtain a retention time of about 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.
Separately calculate the individual percentages of campesterol, stigmasterol, and -sitosterol, as -sitosterol, respectively, in the portion of Pygeum Extract taken:
Ci = (RU/RS) × CS × (V/W) × 100
Calculate the percentage of the labeled amount of total sterols as -sitosterol:
Result = (SCi /L) × 100
Acceptance criteria: 90%110% of the labeled amount of total sterols as -sitosterol on the dried basis
• 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 with acetonitrile to obtain a concentration of 0.02 mg/mL. Pass through a filter of 0.45-µm or finer pore size.
Sample solution: To 250 mg of Extract add 5 mL of chloroform, and dilute with acetonitrile to 25 mL. Pass through a filter of 0.45-µm or finer pore size, discarding the first 4 mL of filtrate.
Detector: UV 323 nm
Column: 4-mm × 25-cm; packing L7
Column temperature: 25
Flow rate: 1 mL/min
Injection size: 20 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
Calculate the percentage of docosyl ferulate (P) in the portion of Extract taken:
P = (rU/rS) × CS × (V/W) × 100
Calculate the percentage of the labeled amount of docosyl ferulate:
Result = (P/L) × 100
Acceptance criteria: 90%110% of the labeled amount of docosyl ferulate on the dried basis
• Heavy Metals, Method II 231: 20 µg/g
• Articles of Botanical Origin, Test for Aflatoxins 561: NMT 4 µg/kg of total aflatoxins B1, B2, G1, and G2; NMT 2 µg/kg of aflatoxin B1
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirement
• Botanical Extracts, Preparations 565: Meets the requirements in General Pharmacopeial Requirements, Residual Solvents
• Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
• Absence of Specified Microorganisms 2022: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
• Loss on Drying 731: Dry 1.0 g of Extract for 3 h at 110: it loses NMT 10% of its weight.
• Articles of Botanical Origin, Total Ash 561: NMT 0.5%
• Packaging and Storage: Store in tight containers, protected from light.
• Labeling: The label states the Latin binomial and, following the official name, the part of the plant from which the article was prepared. Label the content as a percentage of total sterols as -sitosterol and the content as a percentage of docosyl ferulate. It also meets the requirements in Botanical Extracts 565, Labeling.
• USP Reference Standards 11
USP Docosyl Ferulate RS
USP Pygeum Extract RS
1 A suitable cartridge is Chromabond NH2, manufactured by Macheray Nagel, or equivalent.
Auxiliary Information Please check for your question in the FAQs before contacting USP.
USP35NF30 Page 1415Pharmacopeial Forum: Volume No. 30(3) Page 956