Test solutionPrepare as directed in the individual monograph.
Standard solutionWeigh accurately a suitable quantity of the reference substance specified in the individual
monograph, previously dried under the conditions specified in the monograph, and dissolve in a suitable volume of aldehyde-free ethanol. Dilute quantitatively and stepwise with aldehyde-free ethanol to obtain a solution containing about 10 micro gram of the steroid per ml.
Into a glass-stoppered, 50-ml conical flask add 20.0 ml of the test solution. Into two similar flasks add 20.0 ml of the standard solution and 20.0 ml of aldehyde-free ethanol (blank), respectively. To each flask add 2.0 ml of blue tetrazolium solution and mix; to each flask add 2.0 ml of a mixture of tetramethylammonium hydroxide solution (10%) and 90 volumes of aldehyde-free ethanol, mix and allow to stand in the dark at a temperature between 25° and 35°. At the end of exactly 90 minutes add to each flask 1.0 ml of glacial acetic acid and mix. Measure the absorbances of the solutions obtained from the test solution and the standard solution at about 525 nm against the blank.
Calculate the quantity, in mg, of the steroid in the 20-ml aliquot of the test solution from the expression At/ As x Cs where, A is the absorbance of the test solution, As is the absorbance of the standard solution and Cs is the quantity, in mg, of the reference substance in the 20-ml aliquot of the standard solution.
Calculate the quantity of the steroid in the substance under examination on the basis of the aliquot of the test solution taken for the assay and from the declared content of the steroid in the appropriate reference substance.
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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