Transverse section of lamina: A thin but marked cuticle occurs over a single layer of epidermal cells on both surfaces. Stomata are present on the lower surface only, with guard cells sunken with respect to adjacent epidermal cells. Palisade elements, elongated at right angles to the surface and often irregular in appearance, occur just below the upper epidermis. Vascular bundles occur at intervals along the width of the blade, with adjacent cluster crystals of calcium oxalate. Cells of the mesophyll are smaller than the palisade cells, elongated parallel to the leaf surface and separated by large intercellular spaces.

Powdered lamina and petiole: Under the microscope, transverse fragments of the leaf display a smooth cuticle, present on both leaf surfaces and staining pinkish orange with sudan III TS. In surface view, cells of the upper epidermis are elongated and wavy-walled, with abundant yellow droplets 2 to 12 µm in diameter visible in mature and old leaves but not in young leaves. Cells of the lower epidermis are similar in shape but have straighter walls and are interrupted by anisocytic stomata. Numerous lignified elements derived from the lamina and petiole are present, including xylem vessels with annular thickening, tracheids, and vessels with bordered pits. The extent of lignification, particularly in the petiole, increases with age of leaf. Calcium oxalate crystals are numerous, present scattered or associated with vessels, ranging in size from 5 to 50 µm in young leaves to 15 to 100 µm in mature leaves. Under crossed polaroids, numerous smaller prism- or tear-shaped shiny features of indeterminate nature may be present. Very occasional, highly elongated, uniseriate, covering trichomes with no obvious cross walls and smooth or warty surfaces may be seen. Mature leaves may show the presence of very rare, polygonal to circular starch granules approximately 20 µm in diameter, with a central hilum and exhibiting a marked Maltese cross under crossed polaroids.

Test solution— 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.

Standard solution: a solution of USP Rutin RS and USP Chlorogenic Acid RS in methanol containing about 0.6 mg per mL and 0.2 mg per mL, respectively.

Developing solvent system: a mixture of ethyl acetate, water, anhydrous formic acid, and glacial acetic acid (67.5:17.5:7.5:7.5).

Spray reagent 1: a solution of 2-aminoethyl diphenylborinate in methanol containing 10 mg per mL.

Spray reagent 2: a solution of polyethylene glycol 400 in alcohol containing 50 mg per mL.

Procedure— Apply separately, as bands, 20 µL each of the Test solution and the Standard solution 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 the Developing solvent system 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 Spray reagent 1, and then spray with Spray reagent 2. 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 chromatogram of the Test solution shows a yellowish-brown fluorescent zone and a light blue fluorescent zone at RF similar to those of rutin and chlorogenic acid, respectively, in the chromatogram of the Standard solution. Additional yellowish-green zones due to flavonoids are detected in the chromatogram of the Test solution. These include one zone below the rutin zone, two zones between the rutin and chlorogenic acid zones, and four zones above the chlorogenic acid zone. Other, less intense zones may be seen in the chromatogram of the Test solution.

Adsorbent: a 0.50-mm layer of chromatographic silica gel.

Test solution— 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 a 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 a 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.

Developing solvent system: a mixture of ethyl acetate and methyl acetate (1:1).

Procedure— Prepare a suitable thin-layer chromatographic plate (see Chromatography 621) coated with Adsorbent 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. 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 the Developing solvent system 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.

Mobile phase— Prepare a mixture of methanol, water, and phosphoric acid (100:100:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solutions— Transfer accurately weighed quantities of USP Quercetin RS, kaempferol, and isorhamnetin to separate volumetric flasks, dissolve each in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain Standard solutions 1, 2, and 3 having known concentrations of 0.02, 0.02, and 0.005 mg per mL, respectively.

Test solution— Transfer about 1.0 g of Ginkgo, finely powdered and accurately weighed, to a 250-mL flask fitted with a reflux condenser. Add 78 mL of Extraction solvent, and reflux on a hot water bath for 135 minutes. [note—The solution will turn deep red. The color of the solution is not a definitive indication of reaction completeness.] Allow to cool at room temperature. Decant to a 100-mL volumetric flask. Add 20 mL of methanol to the 250-mL flask, and sonicate for 30 minutes. Filter, collect the filtrate in the 100-mL volumetric flask, wash the residue on the filter with methanol, collect the washing in the same 100-mL volumetric flask, dilute to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 270-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph Standard solution 1, and record the peak responses as directed for Procedure: the relative retention times are about 1.0 for quercetin, 1.8 for kaempferol, and 2.0 for isorhamnetin; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of each of the Standard solutions and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the percentage of each flavonol glycoside in the portion of Ginkgo taken by the formula: in which 2.51 is the mean molecular mass factor to convert each analyte into flavonol glycoside with a mean molecular mass of 756.7; C is the concentration, in mg per mL, of USP Quercetin RS in Standard solution 1; W is the weight, in g, of Ginkgo taken to prepare the Test solution; rU is the peak area for the relevant analyte obtained from the Test solution; and rS is the peak area of USP Quercetin RS in Standard solution 1. Calculate the total percentage of flavonol glycosides by adding the individual percentages calculated.

Time (minutes)	Solution A (%)	Solution B (%)	Elution
0–23	75®52	25®48	linear gradient
23–28	52	48	isocratic
28–30	52®25	48®75	linear gradient
30–35	25®10	75®90	linear gradient
35–40	10®75	90®25	linear gradient
40–50	75	25	isocratic

1000(C/W)