Sample cleanup semipreparative

Although sample cleanup on open silica columns or Sep-Pak silica cartridges (and so on) removes parts of the lipids that show different polarity in comparison to vitamin K, many equipolar lipids are collected together with the vitamin fraction. Using silica as stationary phase, only little variability is possible in the choice of solvent, due to the lipophilic character of the vitamin K molecules. Hexane with a content of 3% to 5% diethylether or minimal amounts of acetonitrile, ethyl acetate, or diisopropylether (up to 1%) can be used in these systems, with the consequence of a relative poor separation from interfering lipids. That is why in the last few years adsorption chromatography (inclusive semipreparative silica HPLC) is used only as a cleanup step in sample preparation for re-versed-phase HPLC (see Table 1). 

Cleanup or fractionation procedures that have been used in the more recent fat-soluble vitamin assays include sterol precipitation, open-column chromatography, solid-phase extraction, and high-pressure gel permeation chromatography. High-performance LC has been used on a semipreparative scale in vitamin D and vitamin K assays to obtain purified fractions of sample extracts. This technique is discussed in Sec. V.B.3. 

After extraction into hexane, a solid-phase extraction, using silica and nonpolar eluents, or sometimes reversed-phase extraction with polar solvents, is common in many assays. Some research groups used a semipreparative HPLC method (silica column and acetonitrile [62,67,68] or di-isopropylether [89] in hexane) with UV detection for cleanup. Blanco-Gomis et al. (60,61), introducing narrow-bore HPLC or capillary liquid chromatography for small-sample analysis of vitamins, were able to get appropriate results without solid-phase cleanup by reextracting the hexane layer with methanol water 9 1 to remove interfering lipids, as described elsewhere (24,88,108). 

After hydrolysis of triglycerides, denaturation (ethanol) and extraction (hexane) of milk samples are similar to those procedures used in preparation of plasma samples. EHie to high concentrations of coextracted lipophilic compounds, a semipreparative HPLC cleanup step often is used in these preparations. Indyk et al. (92) and Lambert et al. (99) used adsorption HPLC and hexane/isopropanol mixtures for cleanup and a reversed-phase HPLC for detection, whereas Isshiki et al. (94) used a Cig reversed-phase system with a methanol/acetonitrile mixture for cleanup and a C2 or C3 reversed phase for detection. Canfield et al. (95,96) worked with two open-column chromatography systems (silica) to isolate vitamin K compounds prior to HPLC detection, whereas Schneiderman et al. (98) introduced a completely different method for isolation of these compounds. They used supercritical fluid extraction (SFE) with CO2 as solvent to determine VKl in powdered infant formulas. Details of this particular method will be discussed later in this chapter. 

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