Chromatography Forum

I am developing a new method for D3. I have a mobile phase consisting of methanol and water, 98:2, and I am using a RP column. The problem I am having is extracting the vitamin completely from the matrix. I do analysis with dietary supplements so the matrices vary from sample to sample. So far I have been using n-Heptane to extract the D3. The process I go through now is to use water and sonicate which gets all the water solubles into solution, then I add the Heptane and get the fat solubles into solution. I heat at approx. 90 degrees for 20 minutes then centrifuge to allow the layers to separate. I then inject the heptane layer. I am able to get a peak but I am not getting full recovery. Any help is much appreciated. Thanks.

jllewis1,

Try more heptane. Or, preferably, take an aliquot of the water extract, dilute it with DI water, and extract with heptane. Just keep track of the math. See what you get.

Best regards,

AICMM

If you can, check out this paper:

A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry

Susan Knox, John Harris, Lisa Calton and A Michael Wallace

Ann Clin Biochem 2009; 46: 226–230. DOI: 10.1258/acb.2009.008206

Hi
vitamin D3 is not so stable at high temprature, you heat your solution containing D3 to 90 degree, and this may be the cause of D3 losing and therefore low racovery.