Propionating reagent— Mix 10mLof pyridine with 20mLof propionic anhydride.

Internal standard solution— Dissolve a suitable quantity of tributyrin,accurately weighed,in chloroform,and dilute quantitatively with chloroform to obtain a solution having a concentration of about 0.2mg per mL.

Standard preparation— Transfer about 15mg of glycerin and about 50mg of tributyrin,both accurately weighed,to a glass-stoppered,25-mLconical flask,add 3mLof Propionating reagent,and heat at 75for 30minutes.Volatilize the reagents with the aid of a stream of nitrogen at room temperature,add about 12mLof chloroform,and mix.Dilute about 1mLof this mixture with chloroform to about 20mL,and mix.

Test preparation— Transfer about 50mg of Glyceryl Monostearate,accurately weighed,to a glass-stoppered,25-mLconical flask,add by pipet 5mLof Internal standard solution,and mix to dissolve.Immerse the flask in a water bath maintained at a temperature between 45and 50,and volatilize the chloroform with the aid of a stream of nitrogen.Add 3mLof Propionating reagent,and heat at 75for 30minutes.Volatilize the reagents with the aid of a stream of nitrogen at room temperature,add about 5mLof chloroform,and mix.

Chromatographic system— Under typical conditions,the instrument is equipped with a flame-ionization detector,and contains a 4-mm ×2.4-m borosilicate glass column packed with 2%liquid phase G16on 80-to 100-mesh support S1A.The column is maintained isothermally at a temperature between 190and 200,the injection port and detector block are maintained at about 300and 310,respectively,and helium is used as the carrier gas at a flow rate of about 70mLper minute.

System suitability— Chromatograph six to ten injections of the Standard preparationas directed for Procedure:the resolution factor,R,between the peaks for the derivatized glycerin and the tributyrin is not less than 4.0,and the relative standard deviation of the ratio of their peak areas is not more than 2.0%.

Procedure— Inject a suitable portion of the Standard preparationinto a suitable gas chromatograph,and record the chromatogram.Measure the areas under the peaks,and record the values of the areas under the tripropionin and tributyrin peaks as ASand AD,respectively.Calculate the response factor,F,by the formula: in which WSand WDare the weights,in mg,of glycerin and tributyrin,respectively,in the Standard preparation.Similarly inject a suitable portion of the Test preparation,and record the chromatogram.Measure the areas under the peaks,and record the values of the areas under the tripropionin and tributyrin peaks as aUand aD,respectively.Calculate the percentage of glycerin by the formula: in which wDis the weight,in mg,of tributyrin in 5mLof Internal standard solution;and wUis the weight,in mg,of Glyceryl Monostearate in the Test preparation:the limit is 1.2%.

Propionating reagent— Mix 10mLof pyridine and 20mLof propionic anhydride.

Internal standard solution— Transfer about 400mg of hexadecyl hexadecanoate,accurately weighed,to a 100-mLvolumetric flask,dissolve in chloroform,dilute with chloroform to volume,and mix.

Standard preparation— Transfer about 50mg of USP Monoglycerides RS,accurately weighed,to a 25-mLconical flask,add by pipet 5mLof Internal standard solution,and mix.When solution is complete,immerse the flask in a water bath maintained at a temperature between 45and 50,and volatilize the chloroform with the aid of a stream of nitrogen.Add 3.0mLof Propionating reagent,and heat on a hot plate at 75for 30minutes.Evaporate the reagents with the aid of a stream of nitrogen and gentle steam heat.Add 15mLof chloroform,and swirl to dissolve the residue.

Assay preparation— Transfer about 50mg of Glyceryl Monostearate,accurately weighed,to a 25-mLconical flask,and proceed as directed for Standard preparation,beginning with “add by pipet 5mLof Internal standard solution.”

Chromatographic system— Under typical conditions the instrument is equipped with a flame-ionization detector,and contains a 4-mm ×2.4-m borosilicate glass column packed with 2%liquid phase G27on 80-to 100-mesh support S1A.The column is maintained isothermally at a temperature between 270and 280,the injection port and detector block temperature are maintained at about 310,and helium is used as the carrier gas at a flow rate of about 70mLper minute.

System suitability— Chromatograph six to ten injections of the Standard preparationas directed for Procedure:the resolution factor,R,between the peaks for the derivatized glyceryl hexadecanoate and glyceryl octadecanoate is not less than 2.0,and the relative standard deviation of the ratio of the peak area of the derivatized glyceryl octadecanoate to that of the hexadecyl hexadecanoate is not more than 2.0%.

Procedure— Inject a suitable portion of the Standard preparationinto a suitable gas chromatograph,and record the chromatogram.Measure the areas under the peaks,and record the values of the sum of the areas under the derivatized monoglyceride peaks and of the area under the hexadecyl hexadecanoate peak as ASand AD,respectively.Calculate the response factor,F,by the formula: in which WDand WSare the weights,in mg,of hexadecyl hexadecanoate and USP Monoglycerides RS,respectively,in the Standard preparation.Similarly inject a suitable portion of the Assay preparation,and record the chromatogram.Measure the areas under the peaks,and record the values of the sum of the areas under the derivatized monoglyceride peaks and of the area under the hexadecyl hexadecanoate peak as aUand aD,respectively.Calculate the quantity,in mg,of monoglycerides in the amount of Glyceryl Monostearate taken by the formula: in which the terms are as defined therein.