Macroscopic: Dried whole, folded, or fragmented leaves, with or without attached petiole, varying from khaki green to greenish brown in color, often more brown at the apical edge, and darker on the adaxial surface. Lamina broadly obcuneate (fan-shaped), 2 to 12 cm in width and 2 to 9.5 cm in length from petiole to apical margin; mostly 1.5 to 2 times wider than long. Base margins entire, concave; apical margin sinuate, usually truncate or centrally cleft, and rarely multiply cleft. Surface glabrous, with wrinkled appearance due to prominent dichotomous venation appearing parallel and extending from the lamina base to the apical margin. Petiole of a similar color to leaf, channeled on the adaxial surface, 2 to 8 cm in length.

Histology—

A: Transfer 0.2 g of finely powdered Ginkgo to a test tube, add 10 mL of methanol, and heat on a water bath at 65 for 10 minutes. Shake the mixture frequently during the heating. Allow to cool to room temperature, filter, concentrate the filtrate on a hot water bath at 60 to half its volume, and cool. Apply separately, as bands, 20 µL each of the test solution and a Standard solution of USP Rutin RS and chlorogenic acid in methanol containing about 0.6 mg per mL and 0.2 mg per mL, respectively, to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel, and allow the bands to dry. Develop the chromatograms in a mixture of ethyl acetate, water, anhydrous formic acid, and glacial acetic acid (67.5:17.5:7.5:7.5) until the solvent front has moved about 10 cm from the origin. Remove the plate from the chromatographic chamber, and dry it in a circulating air oven at 100 to 105. Immediately spray the hot plate with a solution of diphenyl boryloxyethylamine in methanol containing 10 mg per mL, then spray with a solution of polyethylene glycol 400 in alcohol containing 50 mg per mL. Allow the plate to cool for 30 minutes, and examine it under long-wavelength UV light. The chromatogram of the Standard solution shows in its middle part, with increasing RF values, the yellowish brown fluorescent zone due to rutin and a light blue fluorescent zone due to chlorogenic acid. The presence of flavone glycosides in the test solution is shown by three yellowish brown to green fluorescent zones, and slightly above a blue to green fluorescent zone below the zone due to rutin, a light blue fluorescent zone appears in the same location as that due to chlorogenic acid as well as two greenish brown-yellow fluorescent zones, located above. Other, less intense zones may be seen in the chromatogram of the test solution.

B: Prepare a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.50-mm layer of chromatographic silica gel as follows. Immerse the plate for 20 seconds in a solution of sodium acetate in methanol containing 1 g per 10 mL. Allow the excess coating liquid to drip from the plate, and dry in a forced-air oven at 70 for 30 minutes. Cool in a desiccator. Transfer 0.8 g of the dried test specimen retained from the test for Loss on drying to a suitable flask fitted with a reflux condenser, add 5 mL of a mixture of methanol and water (1 in 10), and heat under reflux for 15 minutes. While still hot, filter the contents of the flask with the aid of vacuum. Rinse the flask and the test specimen with 2 mL of a mixture of methanol and water (2 in 100), and transfer the rinsings to the filter with the aid of vacuum. Return the powdered Ginkgo to the flask, add 4 mL of a mixture of methanol and water (1 in 10), and repeat the extraction. After filtration, wash the residue of powdered Ginkgo twice with 1.5 mL of a mixture of methanol and water (2 in 100). Combine the filtrates, and transfer the combined filtrates (about 12 mL) to a solid-phase extraction column containing L1 packing with a sorbent mass-to-column volume ratio of 1000 mg per 3 mL or equivalent. [NOTE—Initially pass 10 mL of methanol and then 10 mL of a mixture of methanol and water (2 in 100) through the column to condition it. Do not allow the column to dry.] Collect the eluate at the rate of 1 drop per second. Evaporate the eluate to dryness, and dissolve the residue in 2 mL of methanol. Separately apply several 10-µL spots of the test solution to the impregnated plate, and allow the spots to air-dry. Develop the plate in a mixture of ethyl acetate and methyl acetate (1:1) in a chromatographic chamber without filter paper attached to the walls until the solvent front travels about three-fourths of the length of the plate. Remove the plate from the chamber, mark the solvent front, and dry in an oven at 105 for 15 minutes. Spray the plate with acetic anhydride, and heat in an oven at 140 for 25 minutes. Cool, and examine the plate under short- and long-wavelength UV light. [NOTE—The compounds present in high concentrations may be visible in daylight as light brown spots.] The presence of terpene lactones in the test solution is shown by the following spots detected in the chromatogram at both the short and long wavelengths: bilobalide (RF about 0.75), ginkgolide A (RF about 0.68), ginkgolide B (RF about 0.52), ginkgolide J (RF about 0.39), and ginkgolide C (RF about 0.27). Other spots of varying intensities also may be seen.

Extraction solvent— Prepare a mixture of alcohol, water, and hydrochloric acid (50:20:8).

Solvent— Prepare a mixture of methanol and water (9:1).

Solution A— Use filtered and degassed water.

Solution B— Use filtered and degassed methanol.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solutions— Dissolve accurately weighed quantities of USP Ginkgo Terpene Lactones RS in methanol, sonicating for a few minutes, and dilute with methanol to obtain solutions having known concentrations of about 0.5, 1, 2, 4, and 8 mg per mL. Pass through a filter having a 0.45-µm or finer porosity.

Test solution— Transfer about 2.5 g of Ginkgo, accurately weighed, to a 30-mL glass tube with screw cap and PTFE gasket. Add 10.0 mL of Solvent, seal the tube, and mix well on a vortex mixer. Heat in a water bath at 90 for 30 minutes. Mix the hot suspension on a vortex mixer, and repeat the heating at 90 for 30 minutes. Allow to cool to room temperature, and pass through a filter having a 0.45-µm or finer porosity.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with an evaporative light-scattering detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. The column temperature is maintained at 25 ± 1. [NOTE—The parameters of the detector are adjusted to achieve the best signal-to-noise ratio.] The chromatograph is programmed as follows. Chromatograph the Standard solutions, and record the peak responses as directed for Procedure: the chromatograms obtained are similar to the Reference Chromatogram provided with USP Ginkgo Terpene Lactones RS; and the relative standard deviation determined from the bilobalide peak for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 15 µL) of each of the Standard solutions and the Test solution into the chromatograph, record the chromatograms, and identify the peaks of the relevant analytes in the chromatogram of the Standard solution by comparison with the Reference Chromatogram. Measure the areas of the analyte peaks. Plot the logarithms of the relevant peak responses versus logarithms of concentrations, in mg per mL, of each analyte obtained from the Standard solutions, and determine the regression line using a least-squares analysis. The correlation coefficient for the regression line is not less than 0.995. From the graphs so obtained, determine the concentration, C, in mg per mL, of the relevant analyte in the Test solution. Separately calculate the percentages of bilobalide (C15H18O8), ginkgolide A (C20H24O9), ginkgolide B (C20H24O10), ginkgolide C (C20H24O11), and ginkgolide J (C20H24O10) in the portion of Ginkgo taken by the formula: in which W is the weight, in mg, of Ginkgo taken to prepare the Test solution. Calculate the total percentage of terpene lactones in the portion of Ginkgo taken by adding the percentages calculated for each analyte.