*HELP** on Serum Vitamin D3/ D2 Measurement

[BHORJING]

Can anyone please help me with my project on Vitamin D3 and D2 HPLC? I am asked to prepare my solutions for the solid phase extraction. At the moment, i am using acetonitrile, butanol, TCA as combinations for SPE (precipitation reagent, wash buffers, elution buffer). What is the best flow rate on Agilent 1100?
Thanks a bunch!

[JGK]

How in the world do you expect an answer to that question?

Nobody could give an educated response without knowing what other conditions you're using such as column (type & dimensions) and mobile phase(s).

[BHORJING]

Hi JGK,
here are the conditions of my Vit D3/D2:
Mobile phase: CH3CN
HPLC analyser: HP1100
Column: C18

SPE:
Precipitation reagent: CH3CN:Butanol:TCA (75:24:1)
Wash buffer 1: CH3CN:H20 (50:50)
Wash buffer 2: CH3CN:H20:CH3OH (80:17.5:2.5)
Elution buffer: CH3CN (100%)

I am not contented with peaks i am getting because the D3 and D2 are so tiny that they blend with the background when the solvent front peak has a 'tall' height.

Does anyone have other SPE preparation that I can try to make this project work? Thanks!!

[tom jupille]

First question: when you do the HPLC with standards (ignoring the SPE part for now), do you see separate peaks for D2 and D3 ?

Next question: can you get reasonable calibration curves for D2 & D3?

If the answer to the first two is "yes", then your next step is to run solutions of standards through your existing SPE procedure (by the way, you didn't tell us what type of SPE cartridge you are using -- that *does* matter!) and determine your recovery.

If the answer to either of the first two questions is "no", then SPE is not your problem (at least, not yet!) and you need to get the HPLC separation working properly.