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The initial method development was conducted on pure drug using working standards solution protected from light.
The reference compounds were accurately weighed and dissolved in methanol, the mixed standards solution was obtained by mixing the stock solutions.
To assess stability, the peak area was measured by applying the mixed standards solution, developing the plate, and scanning each band every 5 min for 30 min. Instrumental precision was checked by scanning the same spot of the investigated compound in mixed standards solution (10 μL) six times.
Identical plate precision was determined by analyzing six spots of the mixed standards solution (10 μL) on one plate, while different plate precision was tested by determining one spot of the mixed standards solution (10 μL) on six TLC plates, respectively.
In comparison with the individual standard solutions, a combined standards solution showed two additional peaks under acidic (1N HCl) condition at Rf 0.78 and 0.81 while three degraded products of prednisolone acetate at Rf 0.03, 0.19 and 0.73 were missing.
HPLC chromatograms of the standards solution and sample (under the optimal conditions) are shown in Fig. 7. To determine the repeatability of the novel extraction method, six samples of the same weight (10 mg) were processed under the optimum extraction conditions.
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These standards were used to prepare stock standards solutions.
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