Sulfasalazine 

C₁₈H₁₄N₄O₅S                                                                                      Mol. Wt. 398.4

Sulfasalazine is 2-hydroxy-5-[2-[4-(pyridin-2-ylsulfamoyl)phenyl]diazenyl]benzoic acid. 

Sulfasalazine contains not less than 97.0 per cent and not more than 101.5 per cent of C₁₈H₁₄N₄O₅S, calculated on the dried basis.

Category.  Ulcerative colitis,                                                                                                                                                                                              

Dose. 3 to 4 g per day orally in evenly divided doses.                                                                                                          

Description. A bright yellow or brownish-yellow, fine powder.

Identification

Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulfasalazine RS or with the reference spectrum of sulfasalazine. 

Tests

Related substances. Determine by liquid chromatography (2.4.14).

Test solution Dissolve 25 mg of the substance under examination in dilute ammonia and dilute to 25.0 ml with the same solvent.

Reference solution (a) Dilute 1.0 ml of the test solution to 100.0 ml with dilute ammonia.

Reference solution (b) Dissolve 1 mg of sulfasalazine derivative for resolution RS in 10.0 ml of reference solution (a). Dilute 1.0 ml of this solution to 10.0 ml with reference solution (a).

Chromatographic system

      –  a stainless steel column 25 cm × 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm),

–   mobile phase A. dissolve 1.13 g of sodium dihydrogen phosphate and 2.5 g of sodium acetate in 900 ml of water ; adjust to pH 4.8 with glacial acetic acid  and dilute to 1000 ml with water;

1.   B. a mixture of 80 volumes of methanol and 20 volumes of mobile phase A,

      –  a gradient programme using the condition given below,

      –  flow rate: 1 ml per minute,

      –  spectrophotometer set at 320 nm,

      –  injection volume: 20 µl.          

              Time           Mobile phase A          Mobile phase B

           (in min.)          (per cent v/v)               (per cent v/v)

                  0                          60                                  40

                15                         45                                  55

                25                         45                                  55

                60                          0                                  100

                65                          0                                  100

                70                         60                                  40

Name                                              Relative                   

                                                    retention time              

Sulfasalazine impurity H8             0.16                       

Sulfasalazine impurity I9               0.28                       

Sulfasalazine impurity C3             0.80                       

Sulfasalazine impurity F6              0.85                       

Sulfasalazine                                    1.00                       

Sulfasalazine impurity G7             1.39

Sulfasalazine impurity E5              1.63

Sulfasalazine impurity B2             1.85

Sulfasalazine impurity D4             1.90

Sulfasalazine impurity A1             2.00 

¹ 4,4′-[(4-hydroxy-1,3-phenylene)bis(diazenediyl)]bis[N-(pyridin-2-yl)benzenesulfonamide],

²2-hydroxy-3,5-bis[2-[4-(pyridin-2-ylsulfamoyl)phenyl]diazenyl]benzoic acid,

³2-hydroxy-5-[2-[4-(2-iminopyridin-1(2H)-yl)phenyl]diazenyl]benzoic acid,

⁴4-[2-(2-hydroxyphenyl)diazenyl]-N-(pyridin-2-yl)benzenesulfonamide,

⁵2-hydroxy-4′-(pyridin-2-ylsulfamoyl)-5-[2-[4-(pyridin-2-ylsulfamoyl)phenyl]diazenyl]biphenyl-3-carboxylic acid,

⁶2-hydroxy-3-[2-[4-(pyridin-2-ylsulfamoyl)phenyl]diazenyl]benzoic acid,

⁷5-[2-[4′,5-bis(pyridin-2-ylsulfamoyl)biphenyl-2-yl]diazenyl]-2-hydroxybenzoic acid,

⁸2-hydroxybenzenecarboxylic acid (salicylic acid),

⁹2-hydroxy-5-[2-(4-sulfophenyl)diazenyl]benzoic acid, 

Inject reference solutions (a), (b) and the test solution.

Inject reference solution (b). The test is not valid unless the resolution between the peaks due to sulfasalazine and its derivatives is not less than 3.0.

Inject reference solution (a) and the test solution. In the chromatogram obtained with the test solution, the area of each peak due to impurities A, B, C, D, E, F, G and I is not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (1.0 per cent). The sum of areas of all the secondary peaks is not more than four times the area of the principal peak in the chromatogram obtained with reference solution (a) (4.0 per cent). Ignore any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). And also ignore any peak with a retention time less than 6 min (due to impurities H and J).

Impurities H and J. Determine by liquid chromatography (2.4.14).

Test solution. Dissolve 25 mg of the substance under examination in dilute ammonia and dilute to 25.0 ml with the same solvent.

Reference solution (a). Dissolve 5 mg of salicylic acid  RS (impurity H) and 5 mg of sulfapyridine RS (H₂N-R: 4-amino-N-(pyridin-2-yl)benzenesulfonamide: impurity J) in dilute ammonia and dilute to 10.0 ml with the same solvent.

Reference solution (b). Dilute 2.0 ml of reference solution (a) to 100.0 ml with dilute ammonia. 

Chromatographic system

      –  a stainless steel column 25 cm × 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm),

–  mobile phase: a mixture of 30 volumes of Mobile phase B and 70 volumes of mobile phase A (as described under related substances),

–  flow rate: 1 ml per minute,

      –  spectrophotometer set at 300 nm,

      –  injection volume: 20 µl.

Inject reference solution (b) and the test solution.

Inject reference solution (b). The test is not valid unless the resolution between the peaks due to sulfasalazine impurity H and sulfasalazine impurity J is not less than 2.0.

Inject reference solution (b) and the test solution. In the chromatogram obtained with the test solution, the area of each peak due to impurities H and J is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Ignore any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).

Chlorides (2.3.12). Dissolve 1.25 g in 50 ml of water heat at about 70° for 5 minutes, cool and filter (solution A). To 20 ml of the filtrate add 1 ml of nitric acid, allow to stand for 5 minutes and filter to obtain a clear solution. The solution complies with the limit test for chlorides (140 ppm).

Sulphates (2.3.17). 20 ml of solution A add 1 ml of dilute hydrochloric acid and allow to stand for 5 minutes and filter, complies with the limit for sulphates (400 ppm).

Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method D (10 ppm)

Sulphated ash (2.3.18). Not more than 0.5 per cent, determined on 1 g.

Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1 g by drying in an oven at 105° for 2 hours.

Assay.

Dissolve 150 mg in 100.0 ml of 0.1 M sodium hydroxide. Dilute 5.0 ml of this solution with 750 ml of water add 20 ml of 0.1M acetic acid and dilute to 1000 ml with water. Measure the absorbance of the resulting solution at the maximum at about 359 nm (2.4.7) using sulfasalazine RS prepared as the test solution.  

Calculate the content of C₁₈H₁₄N₄O₅S.