A: The retention time of the isoniazid peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.

B: Transfer a portion of finely powdered Tablets,equivalent to about 50mg of isoniazid,to a 500-mLvolumetric flask.Add water to volume,mix,and filter a portion of the mixture.Proceed as directed in Identificationtest Bunder Isoniazid,beginning with “Transfer 10.0mLof the resulting solution to a 100-mLvolumetric flask.”

Medium: 0.01Nhydrochloric acid;900mL.

Apparatus 1: 100rpm.

Time: 45minutes.

Procedure— Determine the amount of C6H7N3Odissolved by employing UVabsorption at the wavelength of maximum absorbance at about 263nm on filtered portions of the solution under test,suitably diluted with Dissolution Medium,in comparison with a Standard solution having a known concentration of USP Isoniazid RSin the same Medium.

Tolerances— Not less than 80%(Q)of the labeled amount of C6H7N3Ois dissolved in 45minutes.

Procedure for content uniformity— Transfer 1finely powdered Tablet to a 500-mLvolumetric flask with the aid of 200mLof water.Shake by mechanical means for 30minutes,add water to volume,and mix.Filter,and discard the first 20mLof the filtrate.Dilute a portion of the filtrate quantitatively and stepwise,if necessary,with a 3in 100mixture of 0.1Nhydrochloric acid and water to obtain a solution containing about 10µg per mL.Dissolve an accurately weighed quantity of USP Isoniazid RSin a volume of water corresponding to that used to dissolve a similar amount of isoniazid from the Tablet,and dilute quantitatively and stepwise,if necessary,with a 3in 100mixture of 0.1Nhydrochloric acid and water to obtain a Standard solution having a known concentration of about 10µg per mL.Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 263nm,with a suitable spectrophotometer,using water as the blank.Calculate the quantity,in mg,of isoniazid (C6H7N3O)in the Tablet taken by the formula: in which Tis the labeled quantity,in mg,of isoniazid in the Tablet;Cis the concentration,in µg per mL,of USP Isoniazid RSin the Standard solution;Dis the concentration,in µg per mL,of isoniazid in the solution from the Tablet,based on the labeled quantity per Tablet and the extent of dilution;and AUand ASare the absorbances of the solution from the Tablet and the Standard solution,respectively.

Buffer solution— Prepare a 0.1Mmonobasic potassium phosphate solution,adjust with 10Nsodium hydroxide to a pHof 6.9,add sufficient triethanolamine to obtain a solution having a known concentration of 0.2mMof triethanolamine,and mix.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand methanol (95:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Isoniazid RSin Mobile phase,and dilute quantitatively,and stepwise,if necessary,with Mobile phaseto obtain a solution having a known concentration of about 0.32mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to 32mg of isoniazid,to a 100-mLvolumetric flask,add 40mLof Mobile phase,and sonicate for 10minutes.Cool to room temperature,dilute with Mobile phaseto volume,and centrifuge for 5minutes.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the capacity factor,k¢,is not less than 2.35;the column efficiency is not less than 1800theoretical plates;the tailing factor is not more than 1.5;and the relative standard deviation for replicate injections is not more than 1.0%.

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 quantity,in mg,of isoniazid (C6H7N3O)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Isoniazid RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.