A: Infrared Absorption á197Kñ.

B: To 10mLof a solution (1in 50)add 2drops of potassium permanganate TS:the pink color of the permanganate disappears.

Phosphate buffer— Dissolve 1.36g of monobasic potassium phosphate in about 950mLof water,adjust with 1Mpotassium hydroxide to a pHof 7.4,dilute with water to 1000mL,and mix.

Pyridoxal 5-phosphate solution— Dissolve 50mg of pyridoxal 5-phosphate monohydrate in 50mLof Phosphate bufferin a low-actinic flask.Prepare fresh before use.

Standard solutions— Dissolve an accurately weighed quantity of hydroxylamine hydrochloride in water to obtain a final concentration of 2.0mg per mL.To separate 100-mLvolumetric flasks,transfer 5.0,10.0,and 15.0mLof the hydroxylamine stock solution,respectively,dilute each flask with water to volume,and mix.

Test solution— Transfer an accurately weighed quantity of about 1500mg of Acetohydroxamic Acid,previously dried,to a 100-mLbeaker,and dissolve in a sufficient amount of water to cover the electrode of a calibrated pHmeter (about 60mL).While stirring,adjust with 0.05Mpotassium hydroxide to a pHof 7.4.Quantitatively transfer the contents of the beaker,with the aid of small portions of water,to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Procedure— Transfer 2.0mLof each Standard solutionand the Test solutioninto separate 100-mLvolumetric flasks.Pipet 2.0mLof water into a 100-mLvolumetric flask for the reagent blank.To each flask,add 4.0mLof Pyridoxal 5-phosphate solution,and mix.After 8minutes,accurately timed,dilute the contents of each flask with Phosphate bufferto volume.Immediately determine the fluorescence intensities of the solutions from the Standard solutionsand the Test solutionin a fluorometer at an excitation wavelength of 350nm and an emission wavelength of 450nm,setting the instrument to zero with the reagent blank.Determine the best-fit straight line from the fluorescence intensities of the three Standard solutionsversus the hydroxylamine hydrochloride concentrations,in µg per mL.From the best-fit straight line,determine the concentration,in µg per mL,of hydroxylamine hydrochloride in the portion of Acetohydroxamic Acid taken by the formula: in which 33.03and 69.50are the molecular weights of hydroxylamine and hydroxylamine hydrochloride,respectively;Cis the concentration,in µg per mL,of hydroxylamine hydrochloride in the Test solution;and Wis the weight,in mg,of Acetohydroxamic Acid taken.Not more than 0.5%is found.

Ferric chloride solution— Dissolve 4g of ferric chloride in about 50mLof 0.1Nhydrochloric acid,dilute with the same solvent to make 200mL,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Acetohydroxamic Acid RSin 0.1Nhydrochloric acid to obtain a solution having a known concentration of about 500µg per mL.

Assay preparation— Transfer about 250mg of Acetohydroxamic Acid,previously dried and accurately weighed,to a 500-mLvolumetric flask,dilute with 0.1Nhydrochloric acid to volume,and mix.

Procedure— Transfer 10.0mLeach of the Standard preparation,the Assay preparation,and 0.1Nhydrochloric acid to provide the blank,to separate 100-mLvolumetric flasks.To each flask add about 50mLof 0.1Nhydrochloric acid and 10.0mLof Ferric chloride solution,mix,dilute with 0.1Nhydrochloric acid to volume,and mix.Without delay,concomitantly determine the absorbances of the solutions from the Standard preparationand the Assay preparationat the wavelength of maximum absorbance at about 502nm,with a suitable spectrophotometer,using the blank to set the instrument.Calculate the quantity,in mg,of C2H5NO2in the portion of Acetohydroxamic Acid taken by the formula: in which Cis the concentration,in µg per mL,of USP Acetohydroxamic Acid RSin the Standard preparation;and AUand ASare the absorbances of the Assay preparationand the Standard preparation,respectively.