Action and use
Macrolide antibacterial
DEFINITION
Tylosin Premix contains Tylosin Phosphate.
The premix complies with the requirements stated under Premixes and with the following requirements.
CHARACTERS
Light brown granules.
IDENTIFICATION
A. In the Composition, record the UV spectrum of the principal peak in the chromatograms obtained with solutions (1) and (2) with a diode array detector in the range of 210 to 400 nm.
The UV spectrum of the principal peak in the chromatogram obtained with solution (1) is similar to that of the peak in the chromatogram obtained with solution (2); the retention time of the principal peak in the chromatogram obtained with solution (1) is similar to that of the peak in the chromatogram obtained with solution (2).
B. Shake 2 g of the premix with 20 mL of water and filter. The filtrate, after neutralisation, if necessary, yields reaction B characteristic of phosphates, Appendix VI.
TESTS
Composition
Carry out the method for liquid chromatography, Appendix III D, using the following solutions, prepared immediately before use, and the normalisation procedure.
Solution A: Dissolve 25.82 g of potassium dihydrogen orthophosphate in 800 mL, adjust to pH 5.5 using a 1.32% w/v solution of dipotassium hydrogen orthophosphate and dilute to 1 L.
(1) Dissolve a quantity of the premix in 20% v/v acetonitrile and dilute with the same solvent to produce a solution containing the equivalent of 0.1% w/v of tylosin. Filter through a 0.45 μm filter.
(2) 0.1% w/v of tylosin for system suitability EPCRS in 20% v/v acetonitrile.
(3) Dilute 1 volume of solution (1) to 100 volumes with water. Dilute 1 volume of the resulting solution to 10 volumes.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 μm) (Nucleosil C18 is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 1.0 mL per minute.
(d) Use a column temperature of 60°.
(e) Use a detection wavelength of 280 nm.
(f) Inject 20 μL of each solution.
MOBILE PHASE
Mobile phase A: 100 volumes of solution A, 275 volumes of acetonitrile and 625 volumes of water for chromatography.
Mobile phase B: 100 volumes of solution A, 400 volumes of water for chromatography and 500 volumes of acetonitrile.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comment |
| 0 – 25 | 100 | 0 | isocratic |
| 25 – 45 | 100 → 84 | 0 → 16 | linear gradient |
| 45 – 65 | 84 | 16 | isocratic |
| 65 – 70 | 84 → 44 | 16 → 56 | linear gradient |
| 70 – 82 | 44 | 56 | isocratic |
When the chromatograms are recorded under the prescribed conditions, the relative retentions with reference to tylosin A (retention time about 65 minutes) are: impurity E, about 0.23; tylosin B, about 0.31; impurity A, about 0.38; tylosin C, about 0.60; tylosin D, about 0.78; impurity N, about 0.81; impurity O, about 0.85; impurity R, about 1.17 and impurity S, about 1.20.
Use the chromatogram obtained with solution (2) to identify peaks due to tylosin A, B, C and D and impurities A, E, N, O, R and S.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (2): the resolution between the peaks due to tylosin B and impurity A is at least 2.0; the resolution between the peaks due to impurities N and O is at least 1.5 and; the resolution between the peaks due to impurities R and S is at least 1.3.
LIMITS
In the chromatogram obtained with solution (1), integrate all peaks present with an area greater than the area of the principal peak in the chromatogram obtained with solution (3) to determine the total peak area. Calculate the percentage content of each of the components by normalisation: the content of tylosin A is not less than 80%; the total content of tylosins A, B, C and D is not less than 95%.
Related substances
Carry out the method for liquid chromatography, Appendix III D, as described in the test for Composition.
LIMITS
In the chromatogram obtained with solution (1):
the area of any peak due to impurity A is not greater than 2.0% by normalisation;
the sum of the areas of any peaks eluting between the peak due to impurity A and the peak due to tylosin C is not greater than 2.0% by normalisation;
the area of any peak due to impurity N, O, E, R or S is not greater than 1.0% by normalisation (for each);
the area of any other secondary peak is not greater than 1.0% by normalisation;
the sum of the areas of any secondary peaks is not greater than 5.0% by normalisation.
ASSAY
Carry out the microbiological assay of antibiotics, Appendix XIV A. The precision of the assay is such that the fiducial limits of error are not less than 95% and not more than 105% of the estimated potency.
Calculate the content of tylosin in the premix taking each 1000 IU found to be equivalent to 1 mg of tylosin. The upper fiducial limit of error is not less than 90.0% and the lower fiducial limit of error is not more than 110.0% of the stated content.
IMPURITIES
The impurities limited by the requirements of this monograph include those listed under Tylosin Phosphate.
