Estimation Of Raloxifene Hydrochloride

3.7 ANALYTICAL METHOD FOR ESTIMATION OF RALOXIFENE HYDROCHLORIDE IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING ACETONITRILE:0.05M AMMONIUM ACETATE (33:67) AS MOBILE PHASE
3.7.1 For Plain Drug Estimation
Mode Reverse Phase HPLC
Column C18
Detector UV Spectrophotometer
Mobile Phase 0.05M Ammonium Acetate (pH 4): Acetonitrile
Sample Raloxifene Hydrochloride in Methanol HPLC
Flow Rate 1 mL/min
Injection Volume 20µL
Retention Time 4.6 min
Λmax 289nm
A stock solution of Raloxifene Hydrochloride was prepared in methanol to yield final concentration of 100µg/mL. A series of standard solutions of concentrations in the range of 0.2-75µg/mL were obtained by further dilution of the aliquots with methanol. . 20µLof the solutions prepared was taken, and
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A series of standard solutions of concentrations in the range of 3-60µg/mL were obtained by further dilution of the aliquots with methanol. All the solutions were stored at -20oC and were equilibrated to room temperature prior to use.
To prepare the standard calibration samples, 50µL of standard solution and 100µL of Acetonitrile were added to 100µL of blank plasma. The mixture was vortex-mixed for 10 min, followed by centrifugation at 4000 rpm for 15 min. 20µLof the supernatant was taken, then filtered through 0.22µm size syringe filter, and injected in HPLC for analysis. The final standard Raloxifene Hydrochloride concentrations were 0.6-6µg/mL. [6]

3.7.2 For Rat Liver Homogenate
Mode Reverse Phase HPLC
Column C18
Detector UV Spectrophotometer
Mobile Phase 0.05M Ammonium Acetate (pH 4): Acetonitrile
Sample Raloxifene Hydrochloride in Methanol HPLC+Liver
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MSU/IAEC/2016-17/1647. 1g of liver was washed and minced in 10mL of Phosphate Buffer Saline pH 7.4. The mixture was homogenised. The obtained crude homogenate was centrifuged at 4000 rpm for 10 minutes. Supernatant was taken for further analysis.
3.7.2.2 Calibration Plot
A stock solution of Raloxifene Hydrochloride was prepared in methanol to yield final concentration of 1000µg/mL. A series of standard solutions of concentrations in the range of 100-500µg/mL were obtained by further dilution of the aliquots with methanol. All the solutions were stored at -20oC and were equilibrated to room temperature prior to use.
To prepare the standard calibration samples, 50µL of standard solution were added to 100µL of liver homogenate. The mixture was vortex-mixed for 10 min; followed by centrifugation at 4000 rpm for 15 min. 20µL of the supernatant was taken, then filtered through 0.22µm size syringe filter and injected in HPLC for analysis. The final standard Raloxifene Hydrochloride concentrations were 30-150µg/mL. [6]

3.8 ANALYTICAL INTERFERENCE

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