Free betamethasone and other derivatives: Carry out the method for thin-layer chromalography

same manner as solution (1) but use 20 ml of 0.01% w/v of solution of hydrocortisone in methanol (50%) in place of the 20 ml of melhanol (50%). Carry out the chromatographic procedure described under Uniformity of content. Calculate the content of C22H29FO5 in the tablets from the declared content of C22H29FO5 in betamethasone RS. BETAMETHASONE SODIUM PHOSPHATEBelamethasone Sodium Phosphate is disodium 9α-fluoro11 β,17a-dihydroxy-l6p-methyl-3,20-dioxo-l,4-pregnadien21-yl phosphate. Category: Adrenocortical steroid. Dose: The equivalent of 0.5 to 5 mg of betamethasone daily, in divided doses. In the treatment of acute adrenal insufficiency, by intravenous or intramuscular injection, the equivalent of 10 to 80 mg of betamethasone daily, in divided doses. (6.5 mg of Betamethasone Sodium Phosphale is approximately equivalent to 5 mg of betamethasone) Description: White or almost white powder, odourless, very hygroscopic Solubility: Freely soluble in water, slightly soluble in ethanol (95%); practically insoluble in chloroform, in dichloromethane and in ether. Storage: Store in tightly-closed, light-resistant containers. STANDARDSBetamethasone Sodium Phosphate contains not less than 96.0 percent and not more than 103.0 percent of C22H28FNa2O8P, calculated with reference to the anhydrous substance Identification: A: To 2 ml of a 0.013% w/v solution in ethanol (95%) in a stoppered tube add 10 ml of phenylhydrazine-sulphuric acid solution, mix, warm in a water-bath at 60° for 20 minutes and cool immediately, Absorbance of the resuting solution at the maximum at about 450 nm, not more than 0.13, Appendix 5 .5.B: Carry out the method for thin-layer chromatography, Appendix 4.6, using silica gel G as the coaling substance and a freshly prepared mixture of 30 volumes of 1-butanol, 10 volumes of acetic anhydride and 10 volumes of water as the mobile phase. Apply separately to the plate 2 µl of each of the following solutions in methanol containing (1) 0.25% w/v of the substance being examined and (2) 0.25% w/v of betamethasone sodium phosphate RS. Solution (3) is a mixture of equal volumes of solutions (1) and (2) Solution (4) is a mixture of equal volumes of solution (1) and a 0.25% w/v solution of prednisolone sodium phosphate RS in melhanol. After removal of the plate, allow it to dry in air until the odour of solvents is no longer detectable, spray with ethanolic sulphuric acid (20%), heat at 120° for 10 minutes, allow to cool, and examine under ullra-violet light (365 nm) The principal spot in Ihe chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2) The principal spot in the chromatogram obtained with solution (3) appears as a single, compact spot and the chromatogram obtained with solution (4) shows two closely running spots. C: Complies with test C described under Betamethasone. D: Dissolve 2 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes, no red colour or yellowish-green fluorescence is produced (distinction from prednisolone sodium phosphate and hydrocortisone sodium phosphate) E Heat gently 40 mg with 2 ml of sulphuric acid until while fumes are evolved, add nitric acid dropwise until oxidation is complete and cool Add 2 ml of water, heat until while fumes are again evolved, cool, add 10 ml of water and neutralise to litmus paper with dilute ammonia solution. The solution gives the reactions of sodium salts and of phosphates, Appendix 3.1 Specific optical. relation Between +98° and +104°, determined in a 1 % w/v solution, Appendix 8.9 Light absorption: Ratio of the absorbance of the solution prepared as directed under Assay at the maximum at about 241 run to that at about 263 run, 1.70 to 1.90, Appendix 5.5 pH: Between ?. 5 and 90, determined in a 0.5% w/v solution, Appendix 8.11 Clarity and colour solution A 2.0% w/v solution is clear, Appendix 6.1, and colourless, Appendix 6.2 Inorganic phosphate Not more than 0.5%, calculated as PO,, determined by the following method Weigh accurately about 25 mg, dissolve in 10 ml of water, add 4 ml of dilule sulphuric acid, 1 ml of ammonium molybdate solution and 2 ml of methylammophenol with sulphite solution and allow to stand for 15 minutes. Add sufficient water to produce 25.0 ml, allow to stand for further 15 minutes and measure the absorbance of the resulting solution at about 730 nm, Appendix 5 5 Calculate the content of phosphate from a calibration curve prepared by treating suitable aliquots of a 0.00143%, w/v solution of potassium dihydrogen phosphate in a similar manner. Free betamethasone and other derivatives: Carry out the method for thin-layer chromalography, Appendix 4.6, using silica gel GF254 as the coaling substance and methanol as the mobile phase. Apply separately to the plate 2 µl of each of three solutions in methanol containing (1) 1.0% w/v of the substance being examined, (2) 1.0% w/v of betamethasone sodium phosphate RS. and (3) 0.02% w/v of betamethasone RS. After removal of the plate, allow it to dry in air for 5 minutes and examine under ultra-violet light (254 nm). Any spot in the chromatogram obtamed with solution (1) other than that corresponding to betamethasone sodium phosphate RS is not more intense than the spot in the chromatogram obtained with solution (3) Water: Not more than 8.0% w/w, determined on 0.5 g, Appendix 3.24. Assay: Weigh accurately about 0.2 g and dissolve in sufficient water to produce 200.0 ml.