A: Thin-Layer Chromatographic Identification Test 201—

Standard solution— Dissolve about 100 mg of USP Powdered Bilberry Extract RS in 25 mL of methanol, centrifuge, and use the clear supernatant.

Test solution— Proceed as directed for the Standard solution, except to use Powdered Extract.

Adsorbent— Use suitable thin-layer chromatographic plates coated with a layer of cellulose.

Application volume: 10 µL.

Developing solvent system A— Use a mixture of water, glacial acetic acid, and hydrochloric acid (82:15:3).

Developing solvent system B— Use a mixture of glacial acetic acid and water (6:4).

Procedure— Use a saturated chamber. Develop the chromatograms using Developing solvent system A until the solvent front has moved about three-fourths of the plate, and dry the plate with the aid of a current of warm air. Develop the chromatograms in the same direction using Developing solvent system B until the solvent front has moved about three-fourths of the plate, and dry the plate with the aid of a current of warm air. Examine the plate under visible light: the chromatogram of the Test solution exhibits three main red bands with RF values of approximately 0.55, 0.65, and 0.70 that are similar in position and color to the corresponding main bands in the chromatogram of the Standard solution.

B: The retention times of the anthocyanoside peaks in the chromatogram of the Test solution correspond to those in the chromatogram of Standard solution 3, as obtained in the test for Content of anthocyanosides and anthocyanidins; the peaks due to delphinidin-3-O-galactoside chloride and delphinidin-3-O-glucoside chloride are the most intense peaks in the chromatogram and are of similar intensity, and each is more intense than the peak due to cyanidin-3-O-glucoside chloride; the peaks due to cyanidin-3-O-galactoside chloride, delphinidin-3-O-arabinoside chloride, and cyanidin-3-O-glucoside chloride are of similar intensity; and each of the remaining anthocyanoside peaks in the chromtogram is of lower intensity than the peak due to cyanidin-3-O-glucoside chloride.

Solvent: a mixture of methanol and hydrochloric acid (98:2).

Diluent: a mixture of water and 85% phosphoric acid (9:1).

Solution A— Prepare a filtered and degassed mixture of water and formic acid (9:1).

Solution B— Prepare a filtered and degassed mixture of water, acetonitrile, methanol, and formic acid (40:22.5:22.5:10).

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 solution 1— Dissolve, using sonication, an accurately weighed quantity of USP Cyanidin-3-O-glucoside Chloride RS in Solvent to obtain a solution having a known concentration of about 0.4 mg per mL. Transfer 2.0 mL of this solution to a 10-mL volumetric flask, dilute with Diluent to volume, and mix. [note—This solution is stable for 48 hours at 4.]

Standard solution 2— Dissolve, using sonication, an accurately weighed quantity of USP Cyanidin Chloride RS in Solvent to obtain a solution having a known concentration of about 0.5 mg per mL. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, dilute with Diluent to volume, and mix. [note—This solution is stable for 36 hours at 4.]

Standard solution 3— Transfer about 125 mg of USP Powdered Bilberry Extract RS, accurately weighed, to a 100-mL volumetric flask, add 25 mL of Solvent, sonicate to dissolve, dilute with Diluent to volume, and mix. [note—This solution is stable for 48 hours at 4.]

Test solution— Proceed as directed for Standard solution 3, except to use Powdered Extract.

Chromatographic system (see Chromatography 621)— [note—Use deactivated silanized HPLC vials.] The liquid chromatograph is equipped with a refrigerated autosampler maintained at 4, a 535-nm detector, and a 4.6-mm × 25-cm column that contains 5-µm L1 packing. The flow rate is about 1.0 mL per minute. The column temperature is maintained at 30 ± 1. The chromatograph is programmed as follows: Chromatograph Standard solution 1, and record the peak responses as directed for Procedure: the tailing factor for the cyanidin-3-O-glucoside chloride peak is not less than 0.8 and not more than 2.0; and the relative standard deviation determined from the cyanidin-3-O-glucoside chloride peak for replicate injections is not more than 2.0%. Chromatograph Standard solution 3, and record the peak responses as directed for Procedure: the chromatogram obtained is similar to the Reference chromatogram provided with the lot of USP Powdered Bilberry Extract RS being used; the resolution, R, for the delphinidin-3-O-arabinoside, malvidin-3-O-galactoside, and petunidin-3-O-arabinose peaks is not less than 0.8; and the resolution, R, for other components is not less than 1.0.

Procedure— Separately inject equal volumes (about 10 µL) of Standard solution 1, Standard solution 2, Standard solution 3, and the Test solution into the chromatograph; record the chromatograms; and measure the areas of the analyte peaks. Using the chromatogram of Standard solution 3 and the Reference chromatogram provided with the lot of USP Powdered Bilberry Extract RS being used, identify the retention times of the peaks corresponding to the different anthocyanosides and anthocyanidins. The approximate relative retention times of the anthocyanosides and anthocyanidins are provided in the following table: Separately calculate the percentages of delphinidin-3-O-galactoside chloride, delphinidin-3-O-glucoside chloride, cyanidin-3-O-galactoside chloride, delphinidin-3-O-arabinoside chloride, cyanidin-3-O-glucoside chloride, petunidin-3-O-galactoside chloride, cyanidin-3-O-arabinoside chloride, petunidin-3-O-glucoside chloride, peonidin-3-O-galactoside chloride, petunidin-3-O-arabinoside chloride, peonidin-3-O-glucoside chloride, malvidin-3-O-galactoside chloride, peonidin-3-O-arabinoside chloride, malvidin-3-O-glucoside chloride, and malvidin-3-O-arabinoside chloride in the portion of Powdered Extract taken using the formula: in which C is the concentration, in mg per mL, of USP Cyanidin-3-O-glucoside Chloride RS in Standard solution 1; W is the weight, in mg, of Powdered Extract taken to prepare the Test solution; rU is the peak response obtained for each of the anthocyanosides in the Test solution; and rS is the peak response obtained for cyanidin-3-O-glucoside chloride in Standard solution 1. Add the percentages calculated for all the anthocyanosides: not less than 36.0% is found. Separately calculate the percentages of delphinidin chloride, cyanidin chloride, petunidin chloride, peonidin chloride, and malvidin chloride in the portion of Powdered Extract taken using the formula: in which C is the concentration, in mg per mL, of USP Cyanidin Chloride RS in Standard solution 2; W is the weight, in mg, of Powdered Extract taken to prepare the Test solution; rU is the peak response obtained for each of the anthocyanidins in the Test solution; and rS is the peak response obtained for cyanidin chloride in Standard solution 2. Add the percentages calculated for all the anthocyanidins: not more than 1.0% is found.