sample preparation

[Holly]

Dear members,

I'm experiencing problems on extracting vitamin D from calcium tablets. Does anyone have a solution? I'm also having problems on filtering the samples. I rather follow the BP method, by reversed phase chromatography.

Thanks in advance.

Holly

[unmgvar]

Hello Holly,

Of the top of my head i can at least advise you according to march's article in LCGC to try and centrifuge the samples. If that doesn't help then check the type of filter you use. the inside composition of them is very crucial. also for many filters you must flush them first with at least 3-10 ml otherwise you get unwanted peaks.

As for the rest when you say you rather follow does it means that you have made changes to it or do you follow it to the letter?

[Holly]

I had tryed some changes in sample preparation. I prefer to follow BP method because its a RP method, and USP method is normal phase.

[unmgvar]

Have you made sur that you use sun protected glass?
vitamin D is affected by sun light and heat.
make sure that your samples in the HPLC are in a cooles autosampler tray. at least 15 degrees. better go to 10 if you can, although not too much under that because i have seen it also deposit incide the vials after a while if cooled to close to 4-5 degrees.
do you get any problems when injecting a standard solution? like RSD problems?

[Dea Hilton]

I have been experiencing the same problems Holly had experienced. I prepare my samples of calcium wuth vitamin D in a mixture of MeOH : water (90:10), extract the vitamin with sonication and mechanical agitation and then, centrifuge the samples. Then I try to filtrate them, but I have some resistance of the filter abd its very difficult to filter. When I use larger pore membranes, the filtrate is not clear. I have tryed to treat my samples with acid to solubilize calcium, but vitamine suffer degradation. Does anyone has any suggestions???

Have you made sur that you use sun protected glass?
vitamin D is affected by sun light and heat.
make sure that your samples in the HPLC are in a cooles autosampler tray. at least 15 degrees. better go to 10 if you can, although not too much under that because i have seen it also deposit incide the vials after a while if cooled to close to 4-5 degrees.
do you get any problems when injecting a standard solution? like RSD problems?

[unmgvar]

Hy Dea Hilton.
Can you specify the way you centrifuge? It looks akward that you needed to filter some more after it.
Generally when put to 3000-5000 rpm for 10-30 minutes samples come out clear and the filtration becomes only a safety procedure more then anything else.
if you still need to filter because the sample is trully not clear then at least from my experience I can tell you that we had more then one ocasion when the filtration procedure had to be done dropwise until the vial was filled enough (from the top of my head i remember an impurity sample preparation for Gemfibrozil).
just make sure that it is not the filter that absorb or react with the vitamin D.

[yangz00g]

I would suggect to try SPE or MSPD (Matrix solid phase dispersion).

I used to use those two methods to extract trace active ingredients from phamarcutical tablets and tropic creams. You can expect a very good recovery and clean extract if the phase material is selected right. Try C-18 or DVB (Divinylbenzene).