Vitamin direct solvent extraction

Methods of extracting the fat-soluble vitamin from food matrices include alkaline hydrolysis, enzymatic hydrolysis, alcoholysis, direct solvent extraction, and supercritical fluid extraction of the total lipid component. 

Also, direct solvent extraction [84] is used for the isolation of vitamers susceptible to degradation in alkaline media (retinyl esters, vitamers K), but it is ineffectual for removing interfering fatty substances. In some cases, ultrasonication has been employed to break up the lipoproteic complex encapsulating fat-soluble vitamins [85]. 

For the determination of vitamin E in seed oils by HPLC, the oils can simply be dissolved in hexane and analyzed directly. Solid-food samples demand a more rigorous method of solvent extraction. In a modified Rose-Gottlieb method to extract vitamin E from infant formulas (86), dipotassium oxalate solution (35% w/v) was substituted for ammonia to avoid alkalizing the medium, and methyl tert-butyl ether was substituted for diethyl ether because of its stability against the formation of peroxides. 

The stabilization of charge in the primary reaction of PSI is promoted by rapid electron transfer reactions involving the five redox centers AqAiF F Fb (1). Aq is monomeric Chi a and FxF Fb are 4Fe 4S clusters. Fx is associated with the PSI core and F Fb are localized on an 8-9 kDa polypeptide. Provisional identification of Al as a quinone was based upon the EPR signal of Aj (2,3) and comparison of the electron spin polarized flash-induced EPR K-band spectrum of the species Pvoo Ar with that of PgTO Q" Fe-depleted bacterial reaction centers (4) Direct support for the participation of quinone followed a study of flash-induced optical transients at low temperature (5) and the specific reconstitution of room temperature electron transfer reactions in solvent-extracted cyanobacterial PSI using phylloquinone (vitamin K ) (6). The reconstitution studies have been recently confirmed using an ether-extracted preparation of PSI derived from spinach (7). 

In the presence of vitamin A the vitamin may be extracted with organic solvent either directly or after saponification with ethanolic potassium hydroxide. Both vitamins Dg and D3 have identical spectra with broad bands having maxima at 265 m in hexane but the extinction coefficients in this solvent are comparatively low, being 459 for and 474 for D3, and the more intense absorption of vitamin A, with its peak at 328 m//, may completely overshadow the absorption of vitamin D. The spectro-photometric method therefore requires separation of vitamin D from vitamin A and other absorbing materials and can only be applied to comparatively high-potency materials. 

Silica columns can tolerate relatively heavy loads of triglyceride and other nonpolar material. Such material is not strongly adsorbed and can easily be washed from the column with 25% diethyl ether in hexane after a series of analyses (83). Procedures for determining vitamins A and E have been devised in which the total lipid fraction of the food sample is extracted with a non-aqueous solvent, and any polar material that might be present is removed. An aliquot of the nonpolar lipid extract containing these vitamins is then injected into the liquid chromatograph without further purification. Direct injection of the lipid extract is possible because the lipoidal material is dissolved in a nonpolar solvent that is compatible with the predominantly nonpolar mobile phase. Procedures based on this technique are rapid and simple, because there is no need to saponify the sample. 

Saponification of the sample simplifies the analysis by converting the vitamin A esters to retinol. The unsaponifiable material is extracted with hexane, or a predominantly hexane solvent mixture, which is compatible with the nonpolar mobile phase (146,153,156). In vitamin A-fortified foods there is no need to concentrate the unsaponifiable extract—an aliquot can be injected directly into the chromatograph (153). 

The direct measurement of plasma phylloquinone is probably the best indicator of vitamin K status and has been shown to correlate well with intalce. HPLC methods have been reviewed, and typically require 0.5 to 2,0mL of serum or plasma. Protein precipitation and lipid extraction (often into hexane), followed by solvent evaporation preparative HPLC (to isolate vitamin K from other lipids) reevaporation of the vitamin K-rich fraction dilution in the mobile phase and further HPLC, with either electrochemical or fluorometric detection often after postcolumn reduction, are required. Typical between-batch imprecision values are coefficient of variation (CV)s of 11% to 18% with limits of detection of lower than 50pmol/L. An external quality assessment scheme (EQAS) is avafiable in the UK. 

Figure 19 Mass spectrum of 25-OH-D3 purified from Hep 3B cells (upper panel) compared to the mass spectrum of synthetic 25-OH-D3 (lower panel). Hep 3B cells were incubated with vitamin D3 (50 pM) for 48 h. Flasks were then extracted and the lipid extract dried under nitrogen and purified on HPLC (conditions Zorbax SIL [6.2 mm X 25 cm], solvent HIM 96/3/3, flow rate 2 mL/min). A metabolite peak possessing the vitamin D chromophore and comigrating with synthetic 25-OH-D3 was collected, purified further on a different HPLC system (conditions Zorbax CN [4.6 mm X 25 cm], solvent HIM 94/ 5/1, flow rate 1 mL/min), and then dried under nitrogen and subjected to direct probe mass spectrometry using electron impact EI(+) ionization. The putative 25-OH-D3 gave the expected molecular ion with m/z 400 the other ions observed were consistent with the molecule being 25-hydroxylated (see inset fragmentation pattern). (From Ref. 207.)...

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