Edition: BP 2025 (Ph. Eur. 11.6 update)
Action and use
Tetracycline antibacterial.
DEFINITION
Oxytetracycline Veterinary Oral Powder is a mixture of Oxytetracycline Hydrochloride and a suitable diluent.
Content of oxytetracycline hydrochloride, C22H24N2O9,HCl
90.0 to 105.0% of the stated amount.
The veterinary oral powder complies with the requirements stated under Veterinary Oral Powders and with the following requirements.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Extract a quantity of the oral powder containing 10 mg of Oxytetracycline Hydrochloride with 20 mL of methanol, centrifuge and use the supernatant liquid.
(2) 0.05% w/v of oxytetracycline hydrochloride BPCRS in methanol.
(3) 0.05% w/v of each of oxytetracycline hydrochloride BPCRS and demeclocycline hydrochloride BPCRS in methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use a silica gel precoated plate (Merck silica gel 60 plates are suitable). Adjust the pH of a 10% w/v solution of disodium edetate to 7.0 with 10M sodium hydroxide and spray the solution evenly onto the plate (about 10 mL for a plate 100 mm × 200 mm). Allow the plate to dry in a horizontal position for at least 1 hour. Before use, dry the plate at 110° for 1 hour.
(b) Use the mobile phase as described below.
(c) Apply 1 µL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry it in a current of air and examine under ultraviolet light (365 nm).
MOBILE PHASE
6 volumes of water, 35 volumes of methanol and 59 volumes of dichloromethane.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with solution (3) shows two clearly separated spots.
CONFIRMATION
The principal spot in the chromatogram obtained with solution (1) is similar in position, colour and size to that in the chromatogram obtained with solution (2).
B. To a quantity of the powder containing 0.4 mg of Oxytetracycline Hydrochloride add 5 mL of a 1% w/v solution of
sodium carbonate, shake and add 2 mL of diazobenzenesulfonic acid solution. A light brown colour is produced.
C. Shake a quantity of the powder containing 0.1 g of Oxytetracycline Hydrochloride with 10 mL of 2M nitric acid and filter. Decolourise the filtrate with activated charcoal and filter again. The filtrate yields the reactions characteristic of chlorides, Appendix VI.
Related substances
Carry out the method for liquid chromatography, Appendix III D, using the following solutions in a mixture of 20 volumes of acetonitrile and 80 volumes of 0.01M oxalic acid (solvent A). Prepare the solutions immediately before use.
(1) Shake a quantity of the oral powder containing 0.16 g of Oxytetracycline Hydrochloride in 150 mL of solvent A and dilute to 200 mL. Filter the resulting solution (Whatman GF/C filter is suitable).
(2) Dilute 1 volume of solution (1) to 100 volumes.
(3) 0.08% w/v of oxytetracycline for system suitability A EPCRS.
(4) Dilute 1 volume of solution (2) to 10 volumes.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 4.6 mm) packed with end-capped octylsilyl silica gel for chromatography (5 µm) (Intertsil C8 is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 1.3 mL per minute.
(d) Use a column temperature of 50°.
(e) Use a detection wavelength of 254 nm.
(f) Inject 10 µL of each solution.
MOBILE PHASE
Mobile phase A 0.05% v/v trifluoroacetic acid.
Mobile phase B 5 volumes of tetrahydrofuran, 15 volumes of methanol and 80 volumes of acetonitrile.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comment |
| 0-5 | 90 | 10 | isocratic |
| 5-20 | 90→65 | 10→35 | linear gradient |
| 20-21 | 65→90 | 35→10 | linear gradient |
| 21-27 | 90 | 10 | re-equilibration |
When the chromatograms are recorded under the prescribed conditions, the relative retentions with reference to oxytetracycline (retention time about 7 minutes) are: impurity A, about 0.9; impurity B, about 1.2; impurity C, about 1.3; impurity D, about 1.4; impurity E, about 2.2; impurity F, about 2.3.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the peak-to-valley ratio is at least 3.0, where Hp is the height above the baseline of the peak due to impurity A and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to oxytetracycline.
The test is not valid unless, in the chromatogram obtained with solution (3), the peak-to-valley ratio is at least 3.0, where Hp is the height above the baseline of the peak due to impurity B and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to oxytetracycline.
LIMITS
Identify any peak corresponding to impurities A, B, C, D, E and F in the chromatogram obtained with solution (1), using the chromatogram obtained with solution (3).
Multiply the areas of the peaks due to Impurity D and E by a correction factor of 0.4.
In the chromatogram obtained with solution (1):
the area of any peaks corresponding to impurity A, impurity B and impurity C are not greater than twice the area of the principal peak in the chromatogram obtained with solution (2) (2.0% of each);
the area of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (1.0%);
the sum of the areas of all the secondary peaks is not greater than 5 times the area of the principal peak in the chromatogram obtained with solution (2) (5.0%).
Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with solution (4) (0.1%).
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions in a mixture of 20 volumes of acetonitrile and 80 volumes of 0.01M oxalic acid (solvent A). Prepare the solutions immediately before use.
(1) Shake, with the aid of ultrasound, a quantity of the powder containing 0.16 g of Oxytetracycline Hydrochloride with 150 mL of solvent A. Dilute to 200 mL and filter (Whatman GF/C filter is suitable). Dilute 1 volume of the filtrate to 10 volumes.
(2) 0.0074% w/v of oxytetracycline BPCRS.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions stated under Related substances may be used.
DETERMINATION OF CONTENT
Calculate the content of C22H24N2O9,HCl in the powder using the declared content of C22H24N2O9 in oxytetracycline BPCRS. Each mg of C22H24N2O9 is equivalent to 1.079 mg of C22H24N2O9,HCl.
IMPURITIES
The impurities limited by the requirements of this monograph include those listed under Oxytetracycline Hydrochloride.
