alfacalcidol 0.25 microgram capsules

[fuad19752010]

hello everybody,
I have a problem in the analysis of alfacalcidol capsules bu HPLC-UV.
These capsules contain 0.25 microgram of alfacalcidol (in 100mg of oil). so the problem is the very low concentration of the active (alfacalcidol) and that the oil is not soluble in acetonitrile or methanol mixthure, it is soluble in n-hexane.
so any one have an idea, or a method for analysis by HPLC of this capsules (preferred Reversed phase over the normal-phase mode)

Fuad Al-Rimawi
Birzeit Pharm. Company
Palestine

[wise_q]

METHOD: HPLC

[b]Mobile Phase: [/b]

A mixture of Acetonitrile and methanol in the ratio of 95 : 5,
filter through 0.2µm HPLC filter and sonicate before use.

[b]Sample preparation:[/b]
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.

[b]Standard preparation:[/b]
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.

[b]Chromatographic Conditions:[/b]

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

[b]Procedure:[/b]

Make 3 injections from each sample preparations and standard preparation solutions and record the peak areas respectively.

[b]Calculation:[/b]

%age = [u]Avg. Peak area of final sample solution [/u] x 100
Avg. Peak area of final standard solution