Gentamicin Sulfate Injection Assay (MOA) By HPLC
Mobile phase: 0.025M sodium heptanesulfonate monohydrate in a mixture of 5 volumes of glacial acetic acid, 25 volumes of water and 70 volumes of methanol.
Chromatographic
Condition
Column - C18 (12.5
cm × 4.6 mm) (5 µm)
Flow Rate - 1.5 ml/minute
Temperature - Ambient column temperature
Detection wavelength - 330 nm
Injection Volume -
20 µL of each
Solution (1): Add 5 ml of methanol to 10 ml of a solution
prepared by diluting a suitable volume of the injection with water to contain the
equivalent of 0.045% w/v of gentamicin. Swirl and add 4 ml of phthalaldehyde reagent, mix and add
sufficient methanol to produce 25 ml, heat in
a water bath at 60° for 15 minutes and cool. If the solution is not
used immediately, cool to 0° and use within 4 hours.
Solution (2): Prepare in the same manner as solution (1) but using 10 ml
of a 0.065% w/v solution of (2) gentamicin sulfate WS in place of 10 ml of the
preparation being examined.
The retention time
of component C2 is 10 to 20 minutes. The
retention times relative to component C2 are: about
0.13 (reagent); about 0.27 (component C1); about 0.65
(component C1a); about 0.85 (component C2a).
System Suitability
The test is not
valid unless, in the chromatogram obtained with solution (2), the resolution factor between the peaks due to
components C2a and C2 is at least
1.3.
