by Related Substance
Determine by HPLC
Note-Prepare the solutions immediately before use.
Test solution. Dissolve 0.2 g of the substance under examination in 2.5 ml of methanol containing 0.46 per cent w/v of tetrabutylammonium hydroxide solution (40 per cent w/v) and dilute to 10.0 ml with the solution containing equal volumes of 1.79 per cent w/v of disodium hydrogen phosphate and 0.78 per cent w/v of sodium dihydrogen phosphate.
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the mobile phase.
Reference solution (c). A solution containing 0.025 per cent w/v each of 4-aminophenol, paracetamol RS and chloroacetanilide in methanol. Dilute 1.0 ml of this solution to 250.0 ml with the mobile phase.
Reference solution (d). Dissolve 20 mg of 4-nitrophenol in 50.0 ml of methanol. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase.
Chromatographic system
– a stainless steel column 25 cm x 4.0 mm, packed with octylsilane bonded to porous silica (Sum) (Such as Zorbax C8), column temperature: 35°,
– mobile phase: a mixture of 37.5 volumes of a 1.79 per cent w/v solution of disodium hydrogen phosphate, 37.5 volumes of a 0.78 per cent w/v solution of sodium dihydrogen phosphate and 25 volumes of methanol containing 0.46 per cent v/v of tetrabutylammonium hydroxide solution (40 per cent w/v),
– flow rate: 1.5 ml per minute,
– spectrophotometer set at 245 nm,
– injection volume: 20 µl.
The relative retention time with reference to paracetamol for 4- aminophenol is about 0.8, for 4-nitrophenol is about 3.0 and for chloroacetanilide is about 7.0. Inject reference solution (c). The test is not valid unless the resolution between the peaks due to 4-aminophenol and paracetamol is not less than 4.0 and the signal-to-noise ratio of the peak due to chloroacetanilide is not less than 50. Inject reference solutions (a), (b), (c), (d) and the test solution. Run the chromatogram 12 times the retention time of the principal peak. In the chromatogram obtained with the test solution the area of the peak due to chloroacetanilide is not more than 0.2 times the area of the corresponding peak in the chromatogram obtained with reference solution (c) (10 ppm) and the area of the peak due to 4-aminophenol is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (50 ppm). The area of peak due to 4-nitrophenol is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (d) (0.05 per cent).The area of any other secondary peak is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). The sum of areas of other secondary peaks is not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent). Ignore any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.01 per cent)
