A:Ultraviolet Absorption á197Uñ—

B: The retention time of the major peak in the chromatogram of the Test solutioncorresponds 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,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 relative to total carotenoids in the Preparation taken by the formula: in whichriis the individual peak response of zeaxanthin,andrsis 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 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 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 SuitabilityunderChromatography á621ñ).

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

Test solution— Transfer 1.0mLof Test stock solution 1,or 1.0mLof Test stock solution 2,or 2.0mLof Test stock solution 3from the test for the Content of total carotenoidsinto a suitable vial.Evaporate the solvent to dryness under a stream of nitrogen.Add about 1.0mLof 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 relative to total carotenoids in the Preparation taken by the formula: in which riis the individual peak response of lutein,and rsis 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 4.5mg of lutein,to a 100-mLflask.Add about 20mLof warm water,about 60Units of a bacterial alkaline protease preparation,and 1mg of bromelain.Insert the stopper into the flask,and sonicate for 30minutes with occasional swirling.Cool to room temperature,and add 30.0mLof methylene chloride.Shake the flask for 1minute,and place in the dark for 30minutes to allow separation of the layers.Transfer about 10mLof the red lower layer to a test tube containing 2to 3g 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 4.5mg of lutein,to a 100-mLflask.Add about 20mLof warm water,insert the stopper into the flask,and sonicate for 30minutes with occasional swirling.Cool to room temperature,and add 30.0mLof methylene chloride.Shake the flask for 1minute,and place in the dark for 30minutes to allow separation of the layers.Transfer about 10mLof the red lower layer to a test tube containing 2to 3g 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 6mg of lutein,to a 100-mLvolumetric flask,and dilute with Solventto volume.Add a magnetic bar,and stir for 30minutes.

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

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)in the Preparation by the formula: in whichVis the volume of organic solvent,(30.0mLfor Test stock solution 1,30.0mLfor Test stock solution 2,and 100.0mLfor Test stock solution 3)used in preparing the Test stock solution;Dis the dilution factor used in preparing the Test solution;Ais the absorbance of theTest solution;Wis the weight,in mg,of Preparation taken to prepare the Test stock solution;and 255is the absorptivity of the pure lutein.