Test solution: 100mg per mL,in 0.012Nammonium hydroxide.

pHá791ñ— The pHof the solution is between 4.5and 7.0.

Chloride á221ñ— A10-mLportion of the solution shows no more chloride than corresponds to 0.20mLof 0.020Nhydrochloric acid (0.014%).

Iron— To 10mLof the solution add 0.5mLof hydrochloric acid and 3drops of potassium ferrocyanide TS:the solution does not become blue within 1minute.

Reducing sugars— To 2mLof the solution add 5mLof alkaline cupric tartrate TS:no reduction occurs at room temperature,and only slight reduction occurs after 1minute of boiling.

Blue tetrazolium solution— Dissolve 50mg of blue tetrazolium in 10mLof alcohol,and mix.

Tetramethylammonium hydroxide solution— Prepare a mixture of 1volume of tetramethylammonium hydroxide TSand 9volumes of alcohol.

Standard stock solution— Prepare an aqueous solution having a known concentration of about 250µg of USP Fructose RSper mL.Store at about 4.

Standard preparation— On the day of use,quantitatively dilute a portion of theStandard stock solutionwith alcohol to obtain a solution having a known concentration of about 2.5µg per mL.Store at about 4.

Test preparation— Transfer about 2.5g of Inulin,accurately weighed,to a 100-mLvolumetric flask,add about 75mLof water,heat on a steam bath until solution is complete,cool to room temperature,dilute with water to volume,and mix.Pipet 1mLinto a 100-mLvolumetric flask,dilute with alcohol to volume,and mix.If the solution is turbid,pass through fine-porosity filter paper.

Procedure— Pipet 10mLof theTest preparationand 10mLof theStandard preparationinto separate glass-stoppered centrifuge tubes.Into each of the tubes,and into a similar tube containing 10.0mLof alcohol to provide the blank,pipet 1mLofBlue tetrazolium solution,and mix.Then into each tube pipet 1mLofTetramethylammonium hydroxide solution,mix,and allow to stand in the dark for 60minutes.Without delay,concomitantly determine the absorbances of the solutions from theTest preparationand theStandard preparationat 530nm,with a suitable spectrophotometer,against the blank.Calculate the percentage,F,of free fructose in the Inulin taken by the formula: in whichCis the concentration,in µg per mL,of USP Fructose RSin theStandard preparation;Wis the quantity,in g,of Inulin taken;andAUandASare the absorbances of the solutions from theTest preparationand theStandard preparation,respectively.The limit is 2.0%,calculated on the dried basis.

Standard stock solution— Transfer about 50mg of USP Dextrose RS,accurately weighed,to a 100-mLvolumetric flask,dissolve in a solution of benzoic acid (1.7in 1000),dilute with the same solution to volume,and mix.Allow to stand at room temperature for not less than 3hours before using.This solution is stable for 1month at about 4.

Standard preparation— Pipet 7mLofStandard stock solutioninto a 100-mLvolumetric flask,dilute with water to volume,mix,and use at once.

Assay preparation— Transfer about 0.5g of Inulin,accurately weighed,to a 100-mLvolumetric flask,add 5.0mLof water,dissolve by heating on a steam bath,cool to room temperature,add 0.5mLof 8Nhydrochloric acid,and mix.Place the flask in a boiling water bath for 5minutes,cool,dilute with water to volume,and mix.Pipet 2mLof this solution into a 10-mLvolumetric flask,dilute with water to volume,and mix.[NOTE—This solution is used also for preparing theAssay preparationin theAssay for inulin.]

Procedure— Pipet 3-mLportions of glucose oxidase–chromogen TSinto 3separate test tubes,and bring to a temperature of 37±0.5in a water bath.Pipet 2mLofStandard preparationinto one of the tubes,pipet 2mLofAssay preparationinto another,and pipet 2mLof water into the third tube to provide the blank.Maintain at 37±0.5for an additional 10minutes,then remove the tubes,and allow them to cool.Determine the absorbances of the solutions from theAssay preparationand theStandard preparationat about 505nm,with a suitable spectrophotometer,using the reagent blank as a reference.Calculate the percentage of combined glucose,G,in the Inulin taken by the formula: in whichCis the concentration,in mg per mL,of USP Dextrose RSin theStandard preparation;Wis the amount,in g,of Inulin taken;andAUandASare the absorbances of the solutions from theAssay preparationand theStandard preparation,respectively.Not less than 2.0%and not more than 5.0%,calculated on the dried basis,is found.

Thiobarbituric acid solution— Dissolve 250mg of thiobarbituric acid in 100mLof 8Nhydrochloric acid,and mix.This solution is stable for 2weeks at a temperature of about 4.

Standard stock solution— Quantitatively dissolve an accurately weighed quantity of USP Fructose RSin an aqueous solution of benzoic acid (1.7in 1000)to obtain a solution having a known concentration of about 1mg of USP Fructose RSper mL.[NOTE—This solution is stable for 1month at about 4.]

Standard preparation— Quantitatively dilute theStandard stock solutionwith water to one-fiftieth of its concentration.Use immediately.

Assay preparation— Pipet 4mLof theAssay preparationfrom theContent of combined glucoseinto a 200-mLvolumetric flask,add water to volume,and mix.

Procedure— Pipet 1-mLportions of theStandard preparationand theAssay preparationinto separate glass-stoppered tubes.Pipet 1mLof water into a third tube to provide the blank.Pipet 5-mLportions ofThiobarbituric acid solutioninto each tube,and mix.Place all of the tubes simultaneously in a water bath maintained at a temperature of about 83,and allow them to stay immersed for 5minutes,accurately timed.Remove the tubes simultaneously,and allow them to cool in a dark place for 30minutes.Determine the absorbances of the solutions from theAssay preparationand theStandard preparationat about 435nm,with a suitable spectrophotometer,using the reagent blank as a reference.Calculate the percentage of C6H11O5(C6H10O5)nOHin the Inulin taken by the formula: in which 0.900is the ratio of the formula weight of an anhydrofructose unit of inulin to that of fructose;Cis the concentration,in µg per mL,of USP Fructose RSin theStandard preparation;Wis the quantity,in g,of Inulin weighed for theContent of combined glucose;AUandASare the absorbances of the solutions from theAssay preparationand theStandard preparation,respectively;Fis the percentage of free fructose;andGis the percentage of combined glucose.