Edition: BP 2025 (Ph. Eur. 11.6 update)
Action and use
Beta2-adrenoceptor agonist; bronchodilator.
DEFINITION
Clenbuterol Injection is a sterile solution of Clenbuterol Hydrochloride in Water for Injections. It is supplied as a ready-to- use solution. Multi-dose containers may contain a suitable antimicrobial preservative.
The injection complies with the requirements stated under Parenteral Preparations and with the following requirements.
Content of clenbuterol hydrochloride, C12H18Cl2N2O,HCl
95.0 to 105.0% of the stated amount.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
Solution A 0.06% w/v of sodium chloride, adjusted to pH 4.0 with 1M hydrochloric acid.
(1) Dilute the injection with methanol, if necessary, to produce a solution containing 0.003% w/v of Clenbuterol Hydrochloride.
(2) Dilute 1 volume of a 0.06% w/v solution of clenbuterol hydrochloride BPCRS in methanol to 20 volumes with solution A.
CHROMATOGRAPHIC CONDITIONS
(a) Use a silica gel F254 pre-coated plate (Merck silica gel 60 F254 HPTLC plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 5 µL of each solution.
(d) Develop the plate to 6 cm.
(e) After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm).
MOBILE PHASE
0.15 volume of concentrated ammonia, 5 volumes of toluene, and 10 volumes of ethanol.
CONFIRMATION
The principal spot in the chromatogram obtained with solution (1) is similar in position and size to the principal spot in the chromatogram obtained with solution (2).
B. In the Assay, the principal peak in the chromatogram obtained with solution (1) has the same retention time as that in the chromatogram obtained with solution (2).
TESTS
Acidity
pH, 3.5 to 5.0, Appendix V L.
Clarity and colour of solution
The injection is not more opalescent than reference suspension I, Appendix IV A, and is not more intensely coloured than reference solution Y7, Appendix IV B, Method I.
Related substances
Carry out the method for liquid chromatography, Appendix III D, using the following solutions. Solution A 0.06% w/v of sodium chloride, adjusted to pH 4.0 with 1M hydrochloric acid.
(1) Dilute the injection with methanol, if necessary, to produce a solution containing 0.003% w/v of clenbuterol hydrochloride.
(2) Dilute 1 volume of solution (1) to 100 volumes with methanol.
(3) Dilute 1 volume of a solution containing 0.06% w/v of clenbuterol hydrochloride BPCRS and 0.006% w/v of clenbuterol hydrochloride impurity B EPCRS in methanol to 20 volumes with solution A.
(4) Dilute 1 volume of solution (2) to 10 volumes with methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (12 cm × 4.0 mm) packed with octadecylsilyl silica gel for chromatography (5 µm) (Nucleosil C18 is suitable) fitted with a pre-column (0.5 cm × 4.0 mm) packed with octadecylsilyl silica gel for chromatography (5 µm) (Nucleosil C18 is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1 mL per minute.
(d) Use a column temperature of 40°.
(e) Use a detection wavelength of 245 nm.
(f) Inject 20 µL of each solution.
(g) For solution (1) allow the chromatography to proceed for twice the retention time of the peak due to clenbuterol.
MOBILE PHASE
167 volumes of acetonitrile, 333 volumes of methanol and 500 volumes of 0.25% w/v citric acid monohydrate, previously adjusted to pH 4.5 with dilute ammonia solution. Dissolve sodium decanesulfonate in the resulting solution to give a concentration of 0.2% w/v.
When the chromatograms are recorded under the prescribed conditions, the relative retentions with reference to clenbuterol (retention time of about 7.4 minutes) are: impurity A, about 0.5 and impurity B, about 0.8.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the peaks due to impurity B and clenbuterol is at least 2.0.
LIMITS
In the chromatogram obtained with solution (1):
the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (1%);
the sum of the areas of all the secondary peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with solution (2) (2.5%).
Disregard any peak with an area less than 3 times the area of the principal peak in the chromatogram obtained with solution (4) (0.3%).
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
Solution A 0.06% w/v of sodium chloride, adjusted to pH 4.0 with 1M hydrochloric acid.
(1) Dilute the injection with methanol, if necessary, to produce a solution containing 0.003% w/v of clenbuterol hydrochloride.
(2) 0.06% w/v of clenbuterol hydrochloride BPCRS in methanol. Dilute 1 volume of this solution to 20 volumes with solution A.
(3) Dilute 1 volume of a solution containing 0.06% w/v of clenbuterol hydrochloride BPCRS and 0.006% w/v of clenbuterol hydrochloride impurity B EPCRS in methanol to 20 volumes with solution A.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related Substances may be used with an injection volume of 10 µL.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the peaks due to impurity B and clenbuterol is at least 2.0.
DETERMINATION OF CONTENT
Calculate the content of C12H18Cl2N2O,HCl in the injection from the chromatograms obtained using the declared content of C12H18Cl2N2O,HCl in clenbuterol hydrochloride BPCRS
STORAGE
Clenbuterol Injection should be protected from light and stored at a temperature not exceeding 25°.
IMPURITIES
The impurities limited by the requirements of this monograph include impurities A and B listed under Clenbuterol Hydrochloride.
