Oxytetracycline
Powder Assay (MOA) By HPLC
Mobile phase A: 0.05 % V/Vsolution
of trifluoroacetic acid.
Mobile phase B:Tetrahydrofuran for
chromatography , Methanol , Acetonitrile (5:15:80 V/V/V)
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Time Mobile Phase A Mobile Phase B
( Min ) (% v/v) (% v/v)
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0 - 5 90 10
5 - 20 90→65 10→35
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Chromatographic Condition
Column - size: l = 0.15 m, Ø = 4.6 mm
Stationary
phase - end
capped octylsilyl silica gel for
chromatography(5 µm)
Temperature - 50 °C.
Flow Rate - 1.3 ml/min.
Detection - Spectrophotometer at 254 nm.
Injection - 10 µl of the test solution
(b) and reference solutions (a).
Test
solution (a): Dissolve 20.0 mg of the substance to be
examined in a 1 g/L solution of hydrochloric acid and dilute to
25.0 ml with the same solution.
Test
solution (b): Dilute 5.0 ml of test solution (a) to 50.0 ml
with a 1 g/L solution of hydrochloric acid.
Reference
solution (a): Dissolve 20.0 mg of oxytetracycline WS in a 1 g/L solution
of hydrochloric acid and dilute to
25.0 ml with the same acid. Dilute 5.0 ml of the solution to
50.0 ml with a 1 g/L solution of hydrochloric acid.
Procedure:
Inject
one blank, five replicate injection of standard solution, two replicate
injection of sample solution & two bracketing standard solution.
Calculate the percentage
content of C22H25ClN2O9 taking into
account the assigned content of oxytetracycline WS and a conversion factor
of 1.079.
Calculation :
