Oxytetracycline HCl Injection Assay (MOA) By HPLC
Mobile Phase - Mobile Phase A : Mobile Phase B (80:20 v/v)
Mobile Phase A - Buffer ( 0.05% v/v Trifluroacetic acid in water)
Mobile Phase B - Tetrahydrofuran: Methanol : Acetonitrile (5:15:80 v/v/v)
Chromatographic Condition
Column -
Waters Sun fire C-18 250 x 4.6 mm, 5µ or equivalent.
Wavelength - VWD
and 254 nm
Flow rate - 1.0 ml/min
Injection volume - 10 µl
Column temperature - 40°C +
2°C
Auto sampler temperature - 15°C + 1°C
Mode - Isocratic
Run Time
- 10 min
Retention Time
- About 5.0 min
Diluent and wash vial - 0.1% Hydrochloric acid in water.
Standard Preparation
Accurately weigh and transfer about 50.0 mg of Oxytetracycline HCl WS in 50 ml volumetric flask and add about 35 ml of diluent, Sonicate to dissolve and make up the volume 50 ml with diluent. Transfer 5 ml of above solution to 25 ml volumetric flask and volume with diluent.
Sample Preparation
Weigh and transfer sample equivalent to 200 mg of Oxytetracycline HCl in 200 ml
volumetric flask, add about 140 ml of diluent, sonicate for 15 min with
intermittent shaking in every after 5 min and make up the volume 200 ml with diluent.
Transfer 5 ml of above solution to 25 ml volumetric flask and volume with diluent. Filter this solution with 0.45µm nylon syringe filter and inject.
Procedure: Inject one blank, five replicate injection of standard solution, two replicate injection of sample solution & two bracketing standard solution.
System Suitability :
1) RSD of five replicate injection of standard
solution should not be more than 2.0%
2) RSD of five replicate injection of
standard solution and two bracketing standard solution should not be more
than 5.0%
3) Tailing factor should not be more than 2.0 and Theoretical plates should not be less than 2000.
Calculation
