Powdered Ginkgo Extract
Powdered Ginkgo Extract is prepared from dried and comminuted leaves of Ginkgo extracted with an acetonewater mixture or other suitable solvents. The ratio of the crude plant material to Powdered Extract is between 35:1 and 67:1. It contains NLT 22.0% and NMT 27.0% of flavonoids, calculated as flavonol glycosides, with a mean molecular mass of 756.7, on the dried basis. It contains NLT 5.4% and NMT 12.0% of terpene lactones, consisting of between 2.6% and 5.8% of bilobalide (C15H18O8) and between 2.8% and 6.2% of the sum of ginkgolide A (C20H24O9), ginkgolide B (C20H24O10), and ginkgolide C (C20H24O11), on the dried basis.
Change to read:• A. Thin-Layer Chromatographic Identification Test 201
Test for flavanoids
Standard solution: A solution of 0.6 mg/mL of USP Rutin RS, and 0.2 mg/mL each of USP Chlorogenic Acid RS and USP Quercetin RS in methanol
Sample solution: 5 mg/mL of Powdered Extract in a mixture of methanol and water (4:1)
Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Application volume: 5 µL
Developing solvent system: Ethyl acetate, water, anhydrous formic acid, and glacial acetic acid (100:26:11:11)
Spray reagent 1: 5 mg/mL of 2-aminoethyl diphenylborinate in methanol
Spray reagent 2: 50 mg/mL of polyethylene glycol 400 in alcohol
Samples: Standard solution and Sample solution
Before development of the chromatograms, saturate the chamber for 20 min with Developing solvent system. Record temperature and humidity in the laboratory. If the relative humidity exceeds 50%, condition the plate to about 35% relative humidity using a suitable device. Apply the samples separately as bands to a suitable thin-layer chromatographic plate (see Chromatography 621), and allow the bands to dry. Develop the plate over a path of 6 cm, remove the plate from the chromatographic chamber, and dry in a circulating air oven at 105 for 5 min. Immediately spray the hot plate with Spray reagent 1, then with Spray reagent 2, dry, and examine under long-wavelength UV light.
Acceptance criteria: The Standard solution shows in its lower part with increasing RF values a yellowish-brown fluorescent zone due to rutin (RF 0.28), a light blue fluorescent zone due to chlorogenic acid (RF 0.36), and a yellow fluorescent zone due to quercetin (RF 0.92). The Sample solution shows a yellowish-brown fluorescent zone, a light blue fluorescent zone, and a yellowish-brown fluorescent zone at RF similar to those of rutin, chlorogenic acid, and quercetin, respectively, in the Standard solution. Additional yellowish to yellowish-green zones due to flavonoids detected in the Sample solution chromatogram include one zone below the rutin zone, two zones between the rutin and chlorogenic acid zones, and two zones above the chlorogenic acid zone. Other zones may be seen in the Sample solution chromatogram.USP35
Change to read:• B. HPLC: In the test for Content of Flavonol Glycosides, the retention times of the peaks for quercetin, isorhamnetin, and kaempferol of the Sample solution correspond to those of the Standard solution. In the chromatogram of the Sample solution, the ratio of the kaempferol peak to the quercetin peak is NLT 0.7, and the peak for isorhamnetin is NLT 0.1 times the size of the quercetin peak.USP35
Change to read:• Content of Flavonol Glycosides
Extraction solvent: Alcohol, hydrochloric acid, and water (25:4:10)
Mobile phase: Methanol, water, and phosphoric acid (100:100:1)
Standard solution A: 0.125 mg/mL of USP Quercetin RS in methanol
Standard solution B: 0.125 mg/mL of USP Kaempferol RS in methanol
Standard solution C: 0.03 mg/mL of USP Isorhamnetin RS in methanol
Sample solution: Transfer about 0.3 g of Powdered Extract, accurately weighed, to a 250-mL flask fitted with a reflux condenser. Add 78 mL of Extraction solvent, and reflux in a hot water bath for 135 min. [NoteThe solution will turn deep red. The color of the solution is not a definitive indication of reaction completeness. ] Allow to cool at room temperature. Transfer to a 100-mL volumetric flask, dilute with water to volume, and mix.
Detector: UV 370 nm
Column: 4.6-mm × 25-cm; packing L1
Flow rate: 1.5 mL/min
Injection size: 20 µL
Samples: Standard solution A, Standard solution B, and Standard solution C
[NoteThe relative retention times for quercetin, kaempferol, and isorhamnetin are about 1.0, 1.8, and 2.0, respectively; Standard solution A, Standard solution B, and Standard solution C. ]
Relative standard deviation: NMT 2.0% determined from the quercetin peak in repeated injections, Standard solution A
Samples: Standard solution A, Standard solution B, Standard solution C, and Sample solution
Calculate the percentage of each flavonol glycoside in the portion of Powdered Extract taken:
Result = (rU/rS) × (CS/W) × F × 10
Calculate the total percentage of flavonol glycosides by adding the individual percentages calculated.
Acceptance criteria: 22.0%27.0% of flavonoids, calculated as flavonol glycosides with a mean molecular mass of 756.7, on the dried basisUSP35
• Content of Terpene Lactones
Solvent: Methanol and water (9:1)
Buffer solution: Dissolve 1.19 g of dibasic sodium phosphate and 8.25 g of monobasic potassium phosphate in 1000 mL of water, and adjust to a pH of 5.8.
Diluent: Methanol and water (1:1)
Solution A: Water
Solution B: Methanol
Mobile phase: See Table 1.
Standard solutions: Dissolve accurately weighed quantities of USP Ginkgo Terpene Lactones RS in Diluent, sonicate for a few min, and dilute with Diluent to obtain solutions with known concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL. Pass through a filter of 0.45-µm or finer pore size.
Sample solution: Transfer about 120 mg of Powdered Extract, accurately weighed, to a 25-mL beaker. Add 10 mL of Buffer solution to the residue, and sonicate for 5 min. Quantitatively transfer the solution to a glass chromatographic tube filled with chromatographic siliceous earth capable of holding 20 mL of aqueous phase.1 Rinse the beaker with two 5-mL portions of Buffer solution, and transfer the washings to the column. [NoteDo not exceed 20 mL of total aqueous phase or the holding capacity of the chromatographic tube. ] Allow the Buffer solution to be absorbed into the column. After 15 min, elute the column with 100 mL of ethyl acetate, collect the ethyl acetate solution, and evaporate to dryness under vacuum in a water bath maintained at 50. Dissolve the residue in 20.0 mL of Diluent.
Detector: Evaporative light-scattering. [NoteThe parameters of the detector are adjusted to achieve the best signal-to-noise ratio, according to manufacturer recommendations. ]
Column: 4.6-mm × 25-cm; packing L1
Column temperature: 25 ± 1
Flow rate: 1 mL/min
Injection size: 15 µL
Samples: Standard solutions
Chromatogram similarity: The chromatograms obtained from the Standard solutions are similar to the reference chromatogram provided with the lot of USP Ginkgo Terpene Lactones RS being used.
Relative standard deviation: NMT 2.0% determined from the bilobalide peak in repeated injections
Correlation coefficient: NLT 0.995 for the regression line as determined in the Analysis
Samples: Standard solutions and Sample solution
Record the chromatograms, and identify the peaks of the relevant analytes in the chromatogram of the Standard solution by comparison with the reference chromatogram being used. Measure the areas of the analyte peaks. Plot the logarithms of the relevant peak responses versus the logarithms of concentrations, in mg/mL, of each analyte of the Standard solutions, and determine the regression line using a least-squares analysis.
From the graphs, determine the concentration, C, in mg/mL, of the relevant analyte in the Sample solution.
Separately calculate the percentages of bilobalide (C15H18O8), ginkgolide A (C20H24O9), ginkgolide B (C20H24O10), and ginkgolide C (C20H24O11) in the portion of Powdered Extract taken:
Result = (C/W) × 2000
Calculate the total percentage of terpene lactones in the portion of Powdered Extract taken by adding the percentages calculated for each analyte.
Total terpene lactones: 5.4%12.0%
Sum of ginkgolide A, ginkgolide B, and ginkgolide C: 2.8%6.2%
• Articles of Botanical Origin, Pesticide Residues 561: Meets the requirements
Add the following:• Heavy Metals, Method II 231: NMT 20 ppmUSP35
Change to read:• Microbial Enumeration Tests 2021: The total aerobic bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.USP35
Add the following:• Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coliUSP35
• Limit of Ginkgolic Acids
Solution A: 0.01% phosphoric acid in water
Solution B: 0.01% phosphoric acid in acetonitrile
Mobile phase: See Table 2.
Standard solution: Dissolve USP Ginkgolic Acids RS in methanol, and dilute, if necessary, with water to obtain a concentration of 0.25 µg/mL of ginkgolic acids, calculated as the sum of the congeners ginkgolic acid C 13:0, ginkgolic acid C 15:1, and ginkgolic acid C 17:1.
Sample solution: Transfer 0.5 g of Powdered Extract to a 10-mL volumetric flask. Add 8 mL of methanol to dissolve, and dilute with water to volume.
Detector: UV 210 nm
Column: 4.6-mm × 5-cm; base-deactivated packing L7
Column temperature: 35
Flow rate: 1 mL/min
Injection size: 100 µL
Sample: Standard solution
Chromatogram similarity: The chromatogram obtained is similar to the reference chromatogram provided with the lot of USP Ginkgolic Acids RS being used.
Tailing factor: NMT 2.0 for the ginkgolic acid C 15:1 peak
Relative standard deviation: NMT 5.0% for the ginkgolic acid C 15:1 peak in repeated injections
Samples: Standard solution and Sample solution
[NoteIdentify the peaks of the relevant analytes by comparison with the reference chromatogram being used. If deterioration of peak shapes is observed, wash the column using a mixture of methanol and water (9:1) for 30 min. ]
Calculate the concentration, in µg/g, of each ginkgolic acid in the portion of Powdered Extract taken:
Result = (rU/rS) × (CS/W) × P × 10
Calculate the total amount of ginkgolic acids by adding the individual contents.
Acceptance criteria: NMT 5 µg/g
• Loss on Drying 731: Dry 1.0 g of Powdered Extract at 105 for 2 h: it loses NMT 5.0% of its weight.
Change to read:• Other Requirements: Meets the requirements for Residual Solvents in Botanical Extracts 565USP35
• Packaging and Storage: Preserve in tight, light-resistant containers, protected from moisture, 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 flavonol glycosides and of terpene lactones, the extracting solvent used for preparation, and the ratio of the starting crude plant material to the Powdered Extract.
Change to read:• USP Reference Standards 11
USP Isorhamnetin RS
USP Kaempferol RSUSP35
USP Quercetin RS
1 Suitable commercially available material is Extrelut® NT 20 from E Merck Science.
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