A: Ultraviolet Absorption á197Uñ—

B: The retention time for 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,Mobile phase,Standard solution,Test solution,andChromatographic system— Proceed as directed under Content of lutein.

Procedure— Inject a volume (about 10µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the peak responses.Calculate the percentage of zeaxanthin in the portion of Lutein taken by the formula: in which Tis the content,in percentage,of total carotenoids as determined in the test for Content of total carotenoids;riis the individual peak response of zeaxanthin;andrsis the sum of the responses of all the peaks:not more than 8.0%of zeaxanthin is found.Calculate the percentage of other related compounds in the portion of Lutein taken by the formula: in which riis the individual peak response of any other peak in the chromatogram (excluding zeaxanthin and lutein);and rsis the sum of the responses of all the peaks:not more than 0.1%of any single other impurity is found.

Solvent: dimethylformamide or dimethyl sulfoxide.

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 SuitabilityunderChromatography á621ñ).

Standard solution— Dissolve a suitable quantity of USP Lutein RSinMobile phaseto obtain a solution containing about 150µg per mL.

Test solution— Transfer about 1mLof Test stock solution from the test for Content of total carotenoids,and evaporate under a stream of nitrogen to dryness.Add 1mLof Mobile phase,and sonicate to dissolve.

Chromatographic system (see Chromatography á621ñ)— 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 1.5mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 1.05for zeaxanthin and 1.0for 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 10µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the peak responses.Calculate the percentage of Lutein taken by the formula: in which Tis the content,in percentage,of total carotenoids as determined in the test for Content of total carotenoids;riis the individual peak response of lutein in the Test solution;and rsis the sum of the responses of all the peaks:not less than 75.0%of lutein is found.

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

Test stock solution— Transfer about 15mg of Lutein to a 100-mLvolumetric flask,and dissolve in and dilute with Solventto volume.

Test solution— Quantitatively dilute the Test stock solution (1in 100)with dehydrated alcohol to obtain a solution having a final concentration of about 1.5µg per mL.

Procedure— Determine the absorbance of the Test solutionat the wavelength of maximum absorbance at about 446nm,with a suitable spectrophotometer,using dehydrated alcohol as a blank.Calculate the percentage of total carotenoids as lutein (C40H56O2)by the formula: in which Ais the absorbance of the Test solution;Wis the weight,in g,of Lutein taken to prepare the Test stock solution;and 255is the absorptivity of the pure lutein.