Test solution: 10mg per mL,in 1Nhydrochloric acid.

Standard stock solution— Transfer an accurately weighed quantity of about 254mg of sodium chloride,previously dried at 105for two hours,to a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix to obtain a solution having a known concentration of about 10µg of sodium per mL.

Standard solutions— Into separate 100-mLvolumetric flasks,pipet 2.0,5.0,and 10.0mLof the Standard stock solution.To each flask add 2.0mLof potassium chloride solution (1in 5)and 1.0mLof hydrochloric acid,dilute with water to volume,and mix to obtain Standard solutionshaving known concentrations of about 0.2,0.5,and 1.0µg of sodium per mL.

Test solution— Transfer about 1.0g of Selenomethionine,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask.Add 2.0mLof potassium chloride solution (1in 5)and 1.0mLof hydrochloric acid,dilute with water to volume,and mix.

Procedure— Concomitantly determine the absorbances of the Standard solutionsand the Test solutionat the sodium emission line of 589nm with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-scattering á851ñ)equipped with a sodium hollow-cathode lamp and an oxidizing air–acetylene flame.Plot the absorbances of the Standard solutionsversus their concentrations,in µg per mL,of sodium,and draw the straight line best fitting the plotted points.From the graph so obtained,determine the concentration of sodium,in µg per mL,in the Test solution.Calculate the percentage of sodium in the portion of the Selenomethionine taken by the formula: in which Cis the concentration,in µg per mL,of sodium in the Test solution,and Wis the weight,in g,of Selenomethionine taken for the Test solution:not more than 0.1%is found.

Developing solvent— Prepare a mixture of butanol,glacial acetic acid,and water (80:20:20).

Spray reagent— Prepare a solution containing 200mg of ninhydrin in 100mLof alcohol.

Standard solution— Dissolve about 50mg of USP Selenomethionine RS,accurately weighed,in 2mLof water,warming if necessary,dilute with methanol to 10.0mL,and mix to obtain a solution having a known concentration of about 5mg per mL.

Diluted standard solution— Quantitatively dilute a portion of the Standard solutionwith methanol to obtain a solution having a known concentration of about 50µg per mL.

Test solution— Dissolve about 50mg of Selenomethionine,accurately weighed,in 2mLof water,warming if necessary,dilute with methanol to 10.0mL,and mix.

Procedure— Separately apply 10µLportions of the Test solution,the Standard solution,and the Diluted standard solutionto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in the Developing solventuntil the solvent front has traveled about three-fourths of the length of the plate.Remove the plate from the chromatographic chamber,mark the solvent front,and allow the solvent to evaporate.Spray the plate with Spray reagent,and dry it at 110for 10minutes.The RFvalue of the principal spot obtained in the chromatogram of the Test Solutioncorresponds to that obtained from the chromatogram of the Standard solution,and no spot,other than the principal spot,in the chromatogram of the Test solutionis larger or more intense than the principal spot obtained from the Diluted standard solution(1.0%).

Selenium stock solution— Dissolve 1g of metallic selenium,accurately weighed,in a minimum volume of nitric acid.Evaporate to dryness,add 2mLof water,and evaporate to dryness.Repeat the addition of water and evaporation to dryness three times.Dissolve the residue in 3Nhydrochloric acid,transfer to a 1000-mLvolumetric flask,dilute with 3Nhydrochloric acid to volume,and mix.This solution contains about 1000µg of selenium per mL.

Standard solutions— To separate 100-mLvolumetric flasks,transfer 2.0,5.0,and 10.0mLof the Selenium stock solution,dilute the contents of each flask with water to volume,and mix to obtain Standard solutionscontaining 20,50,and 100µg of selenium per mL,respectively.

Test solution— Transfer about 250mg of Selenomethionine,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Procedure— Concomitantly determine the absorbances of the Standard solutionsand the Test solutionat the selenium emission line of 196nm,with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-scattering á851ñ)equipped with a selenium hollow-cathode lamp and an air–acetylene flame,using water as the blank.Plot the absorbances of the Standard solutionsversus their concentrations,in µg per mL,of selenium,and draw the straight line best fitting the three plotted points.From the graph so obtained,determine the concentration of selenium,in µg per mL,in the Test solution.Calculate the percentage of Se in the portion of Selenomethionine taken by the formula: in which Cis the concentration,in µg per mL,of Se in the Test solution;and Wis the weight,in g,of Selenomethionine taken for the Test solution.

Mobile phase— Prepare a filtered and degassed solution of 6.8g of monobasic potassium phosphate in one liter of water.Adjust with phosphoric acid to a pHof 2.75±0.25.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability preparation— Dissolve suitable quantities of USP Methionine RSand USP Selenomethionine RSin Mobile phaseto obtain a solution containing about 0.8mg per mLand 0.16mg per mL,respectively.

Standard preparation— Dissolve an accurately weighed quantity of USP Selenomethionine RSin Mobile phaseand dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 0.16mg per mL.

Assay preparation— Transfer about 40mg of Selenomethionine,accurately weighed,to a 250-mLvolumetric flask,dissolve in Mobile phasewith sonication,dilute with Mobile phaseto volume,and mix.Filter through a 0.45-µm membrane.

Chromatographic system (seeChromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm detector and 4.6-mm ×25-cm column that contains packing L1with polar end-capping.The flow rate is about 1.0mLper minute.Chromatograph the System suitability preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.8for methionine and 1.0for selenomethionine;the resolution,R,between methionine and selenomethionine is not less than 3.0;the tailing factor is not more than 2;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of C5H11NO2Se in the portion of Selenomethionine taken by the formula: in which Cis the concentration,in mg per mL,of USP Selenomethionine RSin the Standard preparation;Wis the weight,in g,of the portion of Selenomethionine taken to prepare the Assay preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.