VITAMINS - Liquid Chromatography

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    of vitamin K from other lipids. In this way, TLCon silica plates developed with light petroleumether}diethyl ether (85 : 15, by volume) is included inthe sample preparation for the determination of vit-amin K in lipid-rich animal tissues. Although no re-cent publications have been found, TLC on silicaplates is especially suited for the separation of geo-

    metric isomers (cis-transisomers).Silica plates have been impregnated with 5}20%

    silver nitrate. Under these conditions lipids contain-ing unconjugated double bonds in their sidechain form complexes with the silver ions and showa higher retention than the saturated counterparts.Consequently, separation between saturated (K1(20)),partly saturated [MK-n (H

    n)] and fully unsaturated

    homologues (MK-n) becomes possible. On the otherhand, in argentation chromatography the resolutionbetween cis and trans isomers is completely lost.Silver ions are not destructive for vitamin K, so sam-ples can be eluted from the silica afterwards. How-ever, for high molecular weight menaquinones,irreversible adsorption to argentation TLC plates hasbeen reported.

    Unlike in argentation TLC, where retention is cor-related to the degree of unsaturation, in reversed-phase TLC the retention is based on the length of theside chain. Both techniques are thus perfectly com-plementary for the separation of menaquinones.

    In addition to silica plates and argentation TLC,reversed-phase TLC has been applied to vitamin K-related compounds. Typical eluents consist of waterand an organic solvent such as methanol, acetonitrileor tetrahydrofuran. However, because of wettabilityproblems with aqueous solvents, often nonaqueousreversed-phase conditions are used with dichlorome-thane and methanol (70 : 30, by vol.) as eluting solvent.

    Detection

    As with the other fat-soluble vitamins, Suorescencequenching can be applied to localize the position ofvitamin K-related compounds on a TLC plate. Moresensitive but often destructive for the compounds of

    interest include spray reagents such as 70% per-chloric acid (5}10 min at 1053C), a 0.05% solution

    of rhodamine B in ethanol, a 0.2% anilinonaphtha-lene sulfonic acid solution in methanol and a 10%solution of phosphomolybdic acid in ethanol.

    Again densitometry (based on reSectance, trans-mission) has completely replaced visual inspection aswell as the ofSine quantiRcation after elution of thebands. Densitometry allows internal standardization

    and results in a higher degree of sensitivity and speedof analysis.

    General Conclusions

    From the above overview it should be clear that TLCis no longer the method of choice for the analysis offat-soluble vitamins. The major reason for this lies inthe great progress made in HPLC. Newer trends suchas HPTLC and densitometric scanning may give TLCa new momentum but never to the extent that it willagain supersede HPLC as a routine technique for the

    determination of fat-soluble vitamins in foods orbiological materials. Undoubtedly, however, moderninstrumental TLC can offer automation, improvedrepeatability and more accurate quantiRcation com-pared to classical TLC.

    See also: II/Chromatography: Thin-Layer (Planar):

    Spray Reagents.III/Vitamins:Liquid Chromatography.

    Further Reading

    De Leenheer AP, Lambert WE and Nelis HJ (1992) Modern

    Chromatographic Analysis of Vitamins. New York:Marcel Dekker.

    De Leenheer AP and Lambert WE (1996) Lipophilic vit-amins. In: Sherma J and Fried B (eds) Handbook ofThin-layer Chromatography, 2nd edn, pp. 1055}1077.New York: Marcel Dekker.

    Friedrich W (1988)Vitamins. Berlin: Walter de Gruyter.Poole CF and Poole SK (1994) Instrumental thin-layer

    chromatography.Analytical Chemistry66: 27A}37A.Sherma J (1994a) Modern high performance thin-layer

    chromatography. Journal of AOAC International 77:297}306.

    Weins C and Hauck HE (1996) Advances and develop-

    ments in thin layer chromatography. LC-GC Inter-national9: 710}717.

    Liquid Chromatography

    M. H. Bui, Swiss Vitamin Institute,

    University of Lausanne, Lausanne, Switzerland

    Copyright^ 2000 Academic Press

    Introduction

    Vitamins are a group of organic compounds essential

    to life in very low concentrations. They are either

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    Table 1 Different kinds of vitamins

    Fat-soluble vitamins Water-soluble vitamins

    Vitamin A (retionol) Vitamin B1 (aneurin, thiamin)

    Carotenoids Vitamin B2 (riboflavin)

    Vitamin D (ergocalciferol, vitamin D2; cholecalciferol, vitamin D3) Vitamin B6 (pyridoxine, pyridoxal, pyridoxine, pyridoxol)

    Vitamin B12 (cyanocobalamin)

    Vitamin E (-, -, -tocopherols plus tocotrienols) Vitamin C (ascorbic acid dehydroascorbic acid)

    Vitamin K (phylloquinone, vitamin K1; menadione, vitamin K3) Biotin

    Folic acid (vitamin B9)

    Pantothenic acid (vitamin B5, panthenol)

    Niacin (nicotinamide, vitamin PP)

    Choline

    Inositol (myo-inositol)

    insufRciently produced by the body or not at all.Inadequate vitamin intake causes deRciency disordersin both humans and animals. The various vitaminsare not related to each other chemically and havequite different properties. Two main groups, the fat-soluble and the water-soluble vitamins, may be distin-guished.

    Increased interest in vitamin research, togetherwith the requirements of food and pharmaceuticalquality control, have led to a proliferation of methodsfor vitamin assay, especially by liquid chromato-graphy (LC). Bioassay methods are no longer used,but microbiological methods, physicochemicalmethods and chromatographic procedures (thin-layerchromatography, gas chromatography and liquidchromatography) are commonly employed. Classicalopen-column liquid chromatography is occasion-ally used, but modern high performance liquidchromatography (HPLC) is by far the technique ofchoice for vitamin analysis and is the subject of thisarticle.

    Vitamin analysis is performed to establish the vit-amin status of humans or animals, to determine thepotency of foods and feeds, and to monitor the stor-age stability of vitamin-containing pharmaceuticalpreparations. Information on the physicochemicaland biochemical aspects of vitamins and vitamin in-take is widely available in the literature (see FurtherReading).

    Sample PreparationVitamin A, the carotenoids, and vitamins E, D andK belong to the group of fat-soluble vitamins, whichare soluble in organic solvents. The water-solublevitamins B1, B2, B6, B12, C, biotin, folic acid, pan-tothenic acid, niacin, choline and inositol are solublein water (Table 1). The structures of some fat-solubleand water-soluble vitamins are shown in Figures 1and2.

    Sample preparation prior to the Rnal chromato-graphic analysis is highly dependent upon the natureof the matrix. Minimal preparation is necessary forthe analysis of concentrated solutions. For complexbiological matrices more elaborate sample prepara-tion procedures may be necessary. A recovery testis highly recommended. This consists of adding

    a known amount of pure vitamin, approximativelyequal to the estimated value in the sample, and pro-cessing the fortiRed sample in the same way as thesample itself. Loss of vitamin during analysis shouldnot exceed 6%.

    Fat-Soluble Vitamins

    For fat-soluble vitamin assays all manipulations mustbe carried out in subdued light, in dark glass vessels,and in a nitrogen atmosphere to avoid isomerizationand oxidation.

    In foodstuffs, major interferences in assays for vit-amin A, carotenoids, and vitamins E, D and K arecaused by the large excess of other lipids. The vitaminA, carotenoids, and vitamins E and D contents aremeasured generally after alkaline hydrolysis withethanolic KOH under a nitrogen stream at 60}803Cfor 20}30 min in the presence of an antioxidant.Pyrogallol, hydroquinone, ascorbic acid (vitamin C)and butylated hydroxytoluene (BHT) are the mostcommon antioxidants used during this manipulation.After saponiRcation the free retinol, carotenoids, vit-amin E and vitamin D are extracted into n-hexane orpetroleum ether and evaporated to dryness. VitaminA, carotenoids and vitamin E are redissolved in anorganic solvent compatible with the chromatographicmethod to be employed. Vitamin K needs milderconditions for extraction from protein. Both vitaminsD and K may require further puriRcation beforechromatography.

    In the analysis of serum, vitamin A or retinol isliberated from its binding protein by denaturationwith acetonitrile, ethanol or methanol. An internal

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    Figure 1 Chemical structures of some fat-soluble vitamins.

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    Figure 2 Chemical structures of some water-soluble vitamins.

    standard, e.g. either retinyl acetate or tocol, is gener-ally added for quantitation purposes. After proteinprecipitation the free retinol is extracted with 1%BHT in n-hexane solution, evaporated to drynessunder nitrogen and subjected to chromatographicseparation as above. Vitamin E and carotenoids areextracted in the same manner. Vitamins A, E andcarotenoids can be simultaneously injected for LCseparation. The analysis of vitamin D and trace quant-ities of vitamin D metabolites, e.g. 1,25-dihydroxy-

    cholecalciferol and 25-hydroxycholecalciferol, and

    also the analysis of vitamin K in human plasma,require an additional step for prepuriRcation usingcolumn extraction or a semipreparative LC systemprior to the Rnal HPLC separation.

    Water-Soluble Vitamins

    Water-