Hi Dunia,
I did some quick searching online and found a post from roughly 10 years ago that gave this suggestion:
wise_q wrote:
METHOD: HPLC
Mobile Phase:
A mixture of Acetonitrile and methanol in the ratio of 95 : 5,
filter through 0.2µm HPLC filter and sonicate before use.
Sample preparation:
Weigh & powder 50-60 tablets / capsules. Take powder / oil equivalent to 10 µg of Alfacalcidol in 100ml volumetric flask (Amber colour) . Add 10ml of Hexane and mix well. Then add 50-60 mL methanol and sonicate in ultrasonic bath for 10 minutes to dissolve the active ingredient completely. Cool and make volume to 100ml with methanol. Filter through 0.45µm & sonicate before injection. This is final sample solution having known concentration of 0.1 µg /ml of Alfacalcidol.
Standard preparation:
Weigh 05 mg of Alfacalcidol RS in 100ml volumetric flask. Add 10ml of Hexane and mix well. Then add 50-60 mL methanol and sonicate in ultrasonic bath for 02 minutes to dissolve the active ingredient completely. Cool and make volume to 100ml with methanol. Filter through 0.45µm & sonicate before injection. This is final standard solution having known concentration of 50 µg /ml of Alfacalcidol.
Chromatographic Conditions:
Column = C-18, 4.6 x 250 mm, 5 um
Flow rate = 2.0 mL / min
Wavelength (λ) = 285 nm
Injection Vol. = Sample = 50 µL, Std = 10µL
Retention Time = Near 14 minutes
Injection Loop = 100 µL
Procedure:
Make 3 injections from each sample preparations and standard preparation solutions and record the peak areas respectively.
Calculation:
%age = Avg. Peak area of final sample solution x 100
Avg. Peak area of final standard solution
