Identification—
Tablets meet the requirement of the
Thin-layer Chromatographic Identification Test á201ñ,the test solution being the filtrate obtained by shaking a quantity of powdered Tablets,equivalent to about 200mg of azathioprine,with 10mLof 6Nammonium hydroxide,the Standard solution being a solution of
USP Azathioprine RSin 6Nammonium hydroxide containing 20mg per mL,and 5-µLportions of each solution being spotted on a thin-layer chromatographic plate coated with a 0.25-mm layer of microcrystalline cellulose,and butyl alcohol,previously saturated with 6Nammonium hydroxide,being used for developing.
Dissolution á711ñ—
Medium:
water;900mL.
Apparatus 2:
50rpm.
Time:
30minutes.
Procedure—
Determine the amount of C
9H
7N
7O
2Sdissolved from UVabsorbances at the wavelength of maximum absorbance at about 280nm of filtered portions of the solution under test,suitably diluted with
Medium,if necessary,in comparison with a Standard solution having a known concentration of
USP Azathioprine RSin the same medium.
Tolerances—
Not less than 75%(Q)of the labeled amount of C9H7N7O2Sis dissolved in 30minutes.
Assay—
Mobile phase—
Dissolve 1.1g of sodium 1-heptanesulfonate in 700mLof water,add 300mLof methanol,and mix.Adjust the solution with 1Nhydrochloric acid to a pHof 3.5.Filter the solution through a 0.8-µm solvent-resistant membrane,and degas,making adjustments if necessary (see
System Suitabilityunder
Chromatography á621ñ).
Standard preparation—
Transfer about 25mg of
USP Azathioprine RS,accurately weighed,to a 50-mLvolumetric flask.Add about 15mLof methanol and 0.5mLof ammonium hydroxide to the flask,swirl,and sonicate for 2minutes.Dilute with methanol to volume,and mix.Transfer 10.0mLof this solution to a 50-mLvolumetric flask,dilute with water to volume,and mix.
Assay preparation—
Weigh and finely powder not fewer than 20Tablets.Accurately weigh a portion of the powder,equivalent to about 50mg of azathioprine,and transfer to a 100-mLvolumetric flask.Add 25mLof methanol and 1.0mLof ammonium hydroxide to the flask,swirl,and sonicate for 2minutes.Dilute with methanol to volume,and mix.Allow the excipients to settle,transfer 10.0mLof the supernatant to a 50-mLvolumetric flask,dilute with water to volume,and mix.
Chromatographic system
(see
Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the
Standard preparation,and record the peak responses as directed for
Procedure:the column efficiency is not less than 800theoretical plates,the tailing factor for the azathioprine peak is not more than 1.5,and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 10µL)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 azathioprine (C
9H
7N
7O
2S)in the portion of Tablets taken by the formula:
500(C)(rU/rS),
in which
Cis the concentration,in mg per mL,of
USP Azathioprine RSin the
Standard preparation,and
rUand
rSare the peak responses for azathioprine obtained from the
Assay preparationand the
Standard preparation,respectively.