Chromatography Forum

hi everyone

i got the problem with the fat soluble vitamin analysis by using hplc.
my sample is coconut meat.
i extracted the fat soluble vitamin by solid-liquid extraction.
the problem here is the sample cannot totally dissolved in MeOH because still can see the fat globular.
then i tried to dissolved in hexane. the sample could dissolved but the solvent peak eluted out in the middle of those wanted compound.
since the mobile phase is 95%MeOH which not miscible with hexane.
the baseline drifted since i changed the wavelength of each different fat soluble vitamin.
if that a problem to quantify the fat soluble vitamin?

thank you.

THF or acetonitrile may be better choices for dissolving your sample.

tried already but the acetonitrile also cannot dissolved.
stil got fat globular.
i used the isopropanol, DMSO, n-butanol and petroleum ether but the elution very bad.

ok so you've got problem with dissolving your sample in RP solvents but not in NP solvents

i don't know too much (=nothing) about fat soluble but why don't you consider NP separation

i can see that you are fixed on RP HPLC but NP can be useful sometimes

Reextration with Acetonitrile for Vit. D3 and 25(OH)D3 we descibed
in J. Anal. Toxicol (Lukaszkiewicz J., Kozlowski K., Lorenc R).

Acetonitril precipitate yellow impurities and is very well solvent for
fat soluble vitamins.

Reextration with Acetonitrile for Vit. D3 and 25(OH)D3 we descibed
in J. Anal. Toxicol (Lukaszkiewicz J., Kozlowski K., Lorenc R).

Acetonitril precipitate yellow impurities and is very well solvent for
fat soluble vitamins.

i cannot access above journal from here.
can you send to me???
thank you.
ACN is a good solvent but it cannot totally dissolved the fat content in sample.this the problem annoying me right now.

Here is correct detailed bibliographic data concerning article:

Lukaszkiewicz J, Kozłowski K, Lorenc R.
Rapid high-performance liquid chromatographic method for the estimation of toxicological levels of 25-hydroxycholecalciferol (25-OH D3) in human serum.
J Appl Toxicol. 1983 Dec;3(6):269-71.

A reversed-phase HPLC method for determination of 25-hydroxycholecalciferol (25-OH D3) in serum samples is described. The method involves extraction of 25-OH D3 with hexane, followed by differential resolubilization in acetonitrile. The sample is then applied directly on the C18 bonded phase column of the liquid chromatograph, and developed in a acetonitrile-water solvent system. In the range 12.5-400 ng, a linear relationship was observed between detector response and the amount of 25-OH D3 placed on the column. Also, a very good correlation was observed between the amounts of 25-OH D3 added to serum samples, and the calculated amounts. The method can be used for screening for toxicological levels of 25-OH D3 in patients treated with high doses of vitamin D3.
If you need copy of article I have one own xero copy (k.kozlowski@czd.pl)

Hi guys,
Vitamin is soluble in fat solvents and oils (lipo-soluble). Fat-soluble vitamins include vitamins A, D, E, K, F. Absorbed with ingested dietary fat, fat-soluble vitamins are stored in moderate amounts from the gastrointestinal tract. Present in minute amounts in various foods, these vitamins are essential to maintaining normal metabolism and biochemical functions; fat malabsorption may result in fat-soluble vitamin deficiencies.

Hi Lobak

You could try extracting the fat soluble vitamins into hexane, then dry the hexane, and re-dissolve the residue in acetonitrile. The acetonitrile will mix with your methanol mobile phase ok.

This works for me for extracting vitamins A and E from plasma

Good luck!

Craig

Did you try saponification using, say 50% KOH in ethanol/water mixture? Overnight extraction at room temperature may extract the fat soluble vitamins in lipid samples.

Hi!
I do not know how heat sensitive is your vitamin, but maybe you could use GC instead of LC.
Good luck