Chromatography Forum

Hi!!

I'm trying to develop a HPLC-fluorescence method for the determination of vitamin B6 in plasma. I'm using a C18 and a precipitation method for extraction. But I'm not having good results.... I'm facing with a lot of problems!!!!!!!!!

Maybe somebody could give me an easy and home-made method to do it????

I'd appreciat a lot!!!!

Thanks!!!!

Does a simple ACN/buffer (buffer = 0.1% H3PO4 in water) mobile phase not provide what you need? What is the problem you are facing?

You could add an ion pairing agent such as hexanesulfonate to increase retention.

What excitation (395nm?) and emission (295nm?) wavelengths are you using?

There would be a good chance that B6 would be retained on a decent, end-capped C18 column using the following isocratic mobile phase conditions; 95% phosphate buffer, pH 2.5 : 5% MeCN. If you still don't get the desired retention switch to a polar end-capped or embedded column and run 99% phosphate buffer, pH 2.5 : 1% MeCN.

If you're looking retain / separate other water soluble vitamins a shallow gradient from the above starting conditions to 30% MeCN should do the trick.

Cheers,
Scott

Hi!

I'm using a C18 column.
As a mobile phase: 4.18g of semicarbazide + 3.4g KH2PO4 in 500 mL of Water and adjusted at ph=2.85

As excitation wavelenght: 325nm and emission: 418nm

I'm not sure where is the problem.... Maybe on the extraction procedure?????

What is the purpose of the semicarbazide in the mobile phase? Are you analyzing pyridoxine or pyridoxal?