A: Ultraviolet Absorption 197U—

B: The retention time of the major peak in the chromatogram of the Test solution corresponds to that in the chromatogram of the Standard solution, as obtained in the test for Content of lutein.

Solvent: dimethylformamide or dimethyl sulfoxide.

Solvent, Mobile phase, Standard solution, Test solution, and Chromatographic system— Proceed as directed under Content of lutein.

Procedure— Inject a volume (about 10 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. Calculate the percentage of zeaxanthin relative to total carotenoids in the Preparation taken by the formula: in which ri is the individual peak response of zeaxanthin, and rs is the sum of the responses of all the peaks. Calculate the percentage of other related compounds relative to total carotenoids in the Preparation taken by the formula: in which ri is the individual peak response of any other peak in the chromatogram (excluding zeaxanthin and lutein), and rs is the sum of the responses of all the peaks: not more than 0.1% of any other impurity is found.

Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).

Mobile phase— Prepare a filtered and degassed mixture of hexane and ethyl acetate (75:25). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve a suitable quantity of USP Lutein RS in Mobile phase to obtain a solution containing about 150 µg per mL.

Test solution— Transfer 1.0 mL of Test stock solution 1, or 1.0 mL of Test stock solution 2, or 2.0 mL of Test stock solution 3 from the test for the Content of total carotenoids into a suitable vial. Evaporate the solvent to dryness under a stream of nitrogen. Add about 1.0 mL of Mobile phase, and sonicate to dissolve.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 446-nm detector and a 4.6-mm × 25-cm column that contains 3-µm packing L3. The flow rate is about 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 1.05 for zeaxanthin, and 1.0 for lutein; the resolution, R, between lutein and zeaxanthin is not less than 1.0; the tailing factor is not more than 2; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Inject a volume (about 10 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. Calculate the percentage of lutein relative to total carotenoids in the Preparation taken by the formula: in which ri is the individual peak response of lutein, and rs is the sum of the responses of all the peaks: not less than 93.5% of lutein is found.

Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).

Test stock solution 1 (for solid lutein preparations labeled as containing gelatin)— Transfer the amount of Preparation, equivalent to about 4.5 mg of lutein, to a 100-mL flask. Add about 20 mL of warm water, about 60 Units of a bacterial alkaline protease preparation, and 1 mg of bromelain. Insert the stopper into the flask, and sonicate for 30 minutes with occasional swirling. Cool to room temperature, and add 30.0 mL of methylene chloride. Shake the flask for 1 minute, and place in the dark for 30 minutes to allow separation of the layers. Transfer about 10 mL of the red lower layer to a test tube containing 2 to 3 g of anhydrous sodium sulfate. Insert the stopper into the tube, and shake gently.

Test stock solution 2 (for other solid lutein preparations)— Transfer the amount of Preparation, equivalent to about 4.5 mg of lutein, to a 100-mL flask. Add about 20 mL of warm water, insert the stopper into the flask, and sonicate for 30 minutes with occasional swirling. Cool to room temperature, and add 30.0 mL of methylene chloride. Shake the flask for 1 minute, and place in the dark for 30 minutes to allow separation of the layers. Transfer about 10 mL of the red lower layer to a test tube containing 2 to 3 g of anhydrous sodium sulfate. Insert the stopper into the tube, and shake gently.

Test stock solution 3 (for liquid lutein suspensions in oil)— Transfer an accurately weighed amount of Preparation, equivalent to about 6 mg of lutein, to a 100-mL volumetric flask, and dilute with Solvent to volume. Add a magnetic bar, and stir for 30 minutes.

Test solution— Transfer 1.0 mL of Test stock solution 1, or 1.0 mL of Test stock solution 2, or 2 mL of Test stock solution 3 into a 100-mL volumetric flask, and dilute with dehydrated alcohol to volume.

Procedure— Determine the absorbance of the Test solution at the wavelength of maximum absorbance at about 446 nm, with a suitable spectrophotometer, using dehydrated alcohol as a blank. Calculate the percentage of total carotenoids as lutein (C40H56O2) in the Preparation by the formula: in which V is the volume of organic solvent, (30.0 mL for Test stock solution 1, 30.0 mL for Test stock solution 2, and 100.0 mL for Test stock solution 3) used in preparing the Test stock solution; D is the dilution factor used in preparing the Test solution; A is the absorbance of the Test solution; W is the weight, in mg, of Preparation taken to prepare the Test stock solution; and 255 is the absorptivity of the pure lutein.

(Official January 1, 2007)