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),2to 12cm in width and 2to 9.5cm in length from petiole to apical margin;mostly 1.5to 2times 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,2to 8cm in length.

Histology—

A: Transfer 0.2g of finely powdered Ginkgo to a test tube,add 10mLof methanol,and heat on a water bath at 65for 10minutes.Shake the mixture frequently during the heating.Allow to cool to room temperature,filter,concentrate the filtrate on a hot water bath at 60to half its volume,and cool.Apply separately,as bands,20µLeach of the test solution and a Standard solution of USP Rutin RSand chlorogenic acid in methanol containing about 0.6mg per mLand 0.2mg per mL,respectively,to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)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 10cm from the origin.Remove the plate from the chromatographic chamber,and dry it in a circulating air oven at 100to 105.Immediately spray the hot plate with a solution of diphenyl boryloxyethylamine in methanol containing 10mg per mL,then spray with a solution of polyethylene glycol 400in alcohol containing 50mg per mL.Allow the plate to cool for 30minutes,and examine it under long-wavelength UVlight.The chromatogram of the Standard solution shows in its middle part,with increasing RFvalues,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 á621ñ)coated with a 0.50-mm layer of chromatographic silica gel as follows.Immerse the plate for 20seconds in a solution of sodium acetate in methanol containing 1g per 10mL.Allow the excess coating liquid to drip from the plate,and dry in a forced-air oven at 70for 30minutes.Cool in a desiccator.Transfer 0.8g of the dried test specimen retained from the test for Loss on dryingto a suitable flask fitted with a reflux condenser,add 5mLof a mixture of methanol and water (1in 10),and heat under reflux for 15minutes.While still hot,filter the contents of the flask with the aid of vacuum.Rinse the flask and the test specimen with 2mLof a mixture of methanol and water (2in 100),and transfer the rinsings to the filter with the aid of vacuum.Return the powdered Ginkgo to the flask,add 4mLof a mixture of methanol and water (1in 10),and repeat the extraction.After filtration,wash the residue of powdered Ginkgo twice with 1.5mLof a mixture of methanol and water (2in 100).Combine the filtrates,and transfer the combined filtrates (about 12mL)to a solid-phase extraction column containing L1packing with a sorbent mass-to-column volume ratio of 1000mg per 3mLor equivalent.[NOTE—Initially pass 10mLof methanol and then 10mLof a mixture of methanol and water (2in 100)through the column to condition it.Do not allow the column to dry.]Collect the eluate at the rate of 1drop per second.Evaporate the eluate to dryness,and dissolve the residue in 2mLof methanol.Separately apply several 10-µLspots 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 105for 15minutes.Spray the plate with acetic anhydride,and heat in an oven at 140for 25minutes.Cool,and examine the plate under short-and long-wavelength UVlight.[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 (RFabout 0.75),ginkgolide A(RFabout 0.68),ginkgolide B(RFabout 0.52),ginkgolide J(RFabout 0.39),and ginkgolide C(RFabout 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 Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

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

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

Chromatographic system (see Chromatography á621ñ)— 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 1.0mLper 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 15µL)of each of the Standard solutionsand the Test solutioninto the chromatograph,record the chromatograms,and identify the peaks of the relevant analytes in the chromatogram of the Standard solutionby 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 Wis 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.