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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

III/VITAMINS/Liquid Chromatography 4443


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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

4444 III/VITAMINS/Liquid Chromatography


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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-soluble vitamins are in general more stablethan fat-soluble vitamins, although vitamin B2and toa lesser extent vitamin B12(folic acid) are light sensi-tive. All manipulations should therefore be performed

in subdued light. No special treatment of samples in



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pharmaceutical preparations is required before chro-matography. In biological Suids or foodstuffs pre-puriRcation and/or derivatization of the compoundsare necessary before LC separation. The methodsinclude acid extraction followed by enzymatic hy-drolysis with takadiastase, papain or acid phos-phatase, sometimes with pre-column or post-column

chromatography. Trichloroacetic, perchloric andmetaphosphoric acids are usually preferred for acidextraction. Depending on the aim of the investigation,vitamins can be determined in their free forms or inboth free and phosphorylated forms. In the latter casethe enzymatic hydrolysis step is omitted.

In blood, plasma and food, vitamin B1 in protein-free extract is oxidized by agents such as [Fe(CN)6]

3,

cyanogen bromide or mercuric chloride to thioch-rome in a pre- or post-column chromatography reac-tor. For pre-column chromatography two differentprocedures are used. In the Rrst, the thiochrome ex-tract is neutralized by concentrated phosphoric acidto ensure a pH level compatible with the C18columnused for the separation and to eliminate possiblepH-dependent alkaline degradation of thiochrome toits disulRde. It is then centrifuged and the supernatantinjected into the HPLC. In the second procedure,isobutyl alcohol is used to extract thiochrome afteralkaline oxidation. Aliquots of the extracts are thenchromatographed.

After acid extraction vitamin B2is readily detectedowing to its intenseSuorescence.

Since vitamin B6

is present in six chemical forms,there are methods for the simultaneous separation ofthe three free forms and the three phosphorylatedforms as well as methods for determining the sum ofall the forms. Pyridoxamine is transformed into py-ridoxal by reaction with glyoxylic acid in the presenceof Fe2# as catalyst. The pyridoxal is then reduced tovitamin B6 pyridoxol by the action of sodiumborohydride in an alkaline medium before LC separ-ation. Semicarbazide is also used for post-columnderivatization of vitamin B6.

In multivitamin}multimineral preparations, vit-

amin B12or cyanocobalamin is extracted with a mix-ture of dimethyl sulfoxide (DMSO) and water, orammonium pyrrolidine dithiocarbamate and citricacid in DMSO and water. The extract is centrifugedand the supernatant is diluted with water before con-centration and clean-up by solid-phase extraction us-ing a quaternary amine and a phenyl column in seriesbefore LC separation. There are few LC methods forthe determination of vitamin B12 in human plasmaand food.

In biological Suids and foodstuffs a treatment forremoving protein is a major requirement for vitamin

C assay. Protein precipitation may be done by organic

reagents (methanol or acetonitrile) or mineral acids(perchloric, metaphosphoric acid, etc.). Aqueoussolutions of vitamin C are rapidly oxidized on expo-sure to air. Stabilizers such as hydrogen sulRde and1,4-dithio-DL-threitol have also been employed. Thedeproteinization may be followed by an enzymaticoxidation of ascorbic acid to dehydroascorbic acid,

which is transformed with 1,2-phenylenediamine toits quinoxaline derivative for Rnal separation.

Biotin, or vitamin H, is very stable. However, thelimitation of HPLC lies in the lack of a suitabledetection system. There are applications of LC topharmaceutical products containing at least 300 gbiotin per tablet. In pharmaceutical products and feedpremix, biotin is extracted from the matrix with buf-fer, followed by puriRcation and concentration bysolid-phase extraction and separation by LC. How-ever, there are few LC methods for the estimation ofbiotin in biological samples.

Folic acid (pteroylglutamic acid; also called vit-amin M) and its derivatives are stable substances.Folic acid may be determined simultaneously withother water-soluble vitamins in pharmaceutical prep-arations. In food products folates are extracted fromthe matrix with buffer and enzymes (e.g. hog kidneyand chicken pancreas, or rat plasma conjugase, -amylase and protease together), followed by puriRca-tion and concentration by solid-phase extraction orwith afRnity chromatography beforeRnal separation.

In pharmaceutical preparations panthenol, pan-thotenic and its salt (vitamin B

5

) are extracted witha phosphate solution. The extract is centrifuged, Rl-tered and separated by LC. There are few methods forthe determination of pantothenic acid and its salt infood products and biologicalSuids.

Niacin (or nicotinic acid) and nicotinamide are thetwo different forms of vitamin PP (so called for itspellagra-preventive factor). Nicotinamide is the formof the vitamin generally found in human plasma.Plasma is deproteinated with acetone/chloroform, theorganic layer evaporated to dryness, and the meth-anolic extract of the residue separated by a reversed-

phase HPLC. Isonicotinic acid is used as an internalstandard. Urine is puriRed by extraction with chloro-form, the aqueous phase evaporated, and taken forseparation by LC. In foods vitamin PP is presentmostly in its phosphorylated forms. Hydrolysis isnecessary to break the ester bonds, releasing the totalvitamin PP content of the food for assay. In foodproducts niacin is extracted with buffer and enzyme.The sample extracts are puriRed through an ion ex-change column (e.g. Dowex 1-X8 resin) beforeHPLC.

In multivitamin preparations 0.1 mol L1 hydro-

chloric acid is used to extract the vitamins and



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Figure 3 Example of an HPLC separation of -, -, - and

-tocopherol from a wheat germ oil sample with -tocopherol-

acetate added. Peaks: 1,-tocopherolacetate; 2,-tocopherol; 3,

-tocotrienol; 4, -tocopherol; 5, -tocopherol; 6, -tocopherol.

Experimental conditions: stationary phase, Lichrosorb Si 60,

7m; column dimension, 2504.6 mm; mobile phase, n-

hexane/dioxane (97 : 3 v/v); flow rate, 1.0 mL min1; injection

volume, 20 L; detection, fluorimetric with excitation at 295 nm

and emission at 330 nm. (Reproduced with permission from Fed-

eral Office of Public Health, 1989.)

DMSO containing anhydrous citric acid is used todisperse the multivitamin}multimineral preparation,since vitamin B6is not completely extracted by either0.1 mol L1 hydrochloric acid or DMSO owing toadsorption of the vitamin to the minerals. The extrac-tion of nicotinamide is not impaired by the additionof citric acid to DMSO.

Choline in plant material is extracted with iso-propanol containing internal standards and p-nitro-benzylhydroxylamine hydrochloride for the forma-tion ofp-nitrobenzyl oximes. The extract is puriRedby solid-phase extraction (C18 and ion exchange),after which the choline fraction is benzoylated toyield UV-absorbing derivatives. In biological samplescholine is extracted with formic acid in acetone con-taining an internal standard. After puriRcation thesample is separated by LC.

For the analysis of inositol mono- and diphosphateisomers in foods the method involves extractionof samples with hydrochloric acid and separation ofinositol phosphates by anion exchange chromato-graphy.

Liquid Chromatography

Liquid chromatography is an extremely valuablemethod for separation, identiRcation and quantitat-ion of the different vitamins. Excellent separationscan be achieved in a reasonable time for routineanalysis.

Fat-Soluble Vitamins

For fat-soluble vitamins normal-phase and reversed-phase chromatography are used.

In the normal-phase modes, silica and nonpolarmobile phases containing n-hexane or petroleumether with a small percentage of a more polar solventare used. Addition of a small amount of water oralcohol (e.g. ethanol) regulates the sorbent activity,reduces peak tailing and gives better reproducibilityof retention times. Silica is the adsorbent of choice forthe separation of cis/trans isomers and diastereo-

isomers. Selectivity on silica is determined by thenumber and the nature of the functional groups aswell as the overall steric conRguration (position of thedouble bonds) of the molecule (Figure 3).

In the reversed-phase mode, hydrophobic columnpackings (C18, C8, etc., bonded to a silica surface) areused together with an aqueous buffered mobile phaseand a water-miscible organic solvent (i.e. methanol,acetonitrile).

Retinol analysis in biological Suids and foodsis performed using both normal-phase and reversed-phase chromatography. For normal-phase (or

liquid}

solid) chromatography there is compatibility

between the sample extraction solvent and the LCmobile phase; this avoids peak artefacts, especiallyfor lipid extracts. Geometrical isomers such as 11-cis,13-cis, 9-cis and all-trans retinol are well resolved.Retinol serum determination by reversed-phasechromatography allows the use of retinol acetate asinternal standard which is well separated from re-tinol, unlike the case for liquid}solid chromatogra-phy. Meanwhile there is an additional step ofevaporation of the extraction solvent in the samplepreparation procedure before LC.

The most appropriate systems for the separation ofpolar and nonpolar carotenoids include the use ofpolymeric C18 or C30 bonded phases without end-capping in conjunction with a moderate pore-sizepacking column and a methanol-based mobile phase(to obtain a good recovery). Cis-isomers of-caro-tene are largely resolved from each other and fromother carotenes. Separation of lutein and zeaxanthineis also obtained with this system. Better separationsof the xanthophylls are also observed. Accurate caro-tenoid measurements require the right selection ofcolumn and mobile phase and, due to the large degree

of variability in the purity of commercial carotenoid



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Figure 4 Chromatogram of a human serum sample. Peaks:

RET, retinol; IS, retinol acetate (internal standard); LUT, lutein;

ZEA, zeaxanthin; -TOC, -tocopherol; -TOC, -tocopherol;-

TOC, -tocopherol; LYC, lycopene; -CAR,-carotene;-CAR,

-carotene; trans-CAR, trans--carotene; a, unidentified caro-

tenoid.

Experimental conditions: stationary phase. Lichrosorb RP-18,

7m; column dimension, 2504.6 mm and 153.2 mm guard

column; mobile phase, acetonitrile/tetrahydrofuran/methanol

(68 : 22 : 7 v/v/v) adjusted to 100% with ammonium acetate; flow

rate, 1.5 mL min1; injectionvolume, 15L; detection, programm-

able and variable-wavelength UV/Vis, 325 nm from 0 to 3.0 min,

450 nm from 3.0 to 4.9 min, 290 nm from 4.9 to 7.4 min, 470 nm

from 7.4 to 12.0 min and 450 nm from 12.0 to 15.0 min. (Repro-

duced with permission from Bui, 1994.)

Figure 5 Chromatogram of HPLC-ECD for 24,25-dihydroxy-

cholecalciferol (24,25-(OH)2D3).

Experimental conditions: stationary phase, Nucleosil C18, 5 m;

column dimension, 3007.5 mm; mobile phase, 5%(v/v) meth-anol in acetonitrile with 0.025 mol L1 HClO4; f low rate,

1.2 mL min1; injection volume, 50 L; detection, dual-electrode

electrochemical detection: detector 1 (#0.20 V); detector

2 (#0.60 V). (Reproduced with permission from Masuda et al.,

1997.)

preparations, special precautions must be taken dur-ing calibration. Simultaneous determination of caro-tenoids, retinoids and tocopherols in serum and foodsis performed on a C18 column using wavelength-programmable UV-visible (Figure 4), Suorescence orelectrochemical detection.

The same separation conditions are used for theseparation of -tocopherol and retinol in biologicalSuids and foods. -, -, - and -Tocopherol as wellas -, -, - and -tocotrienol are well resolved only bynormal-phase chromatography. On an RP-18 column-, - and -tocopherol isomers are all separated,

but not - and -tocopherols. Tocol as internal stan-dard is also used for vitamin E determination inserum.

Retinol and-tocopherol in biologicalSuids, foodsand pharmaceutical preparations can be separatedsimultaneously by normal- or reversed-phase HPLC.

In biological Suids analyses of vitamin D and itsmetabolites are performed using solid-phase extrac-tion (SPE) cartridge coupled to semipreparative HPLCon a silica column and analytical HPLC on an oc-tadecyl (C18) column with UV or electrochemical de-tection (Figure 5). They can also be puriRed by one or

two preparative HPLC steps on silica and quantiRed

by HPLC on a C18 column with UV detection. VitaminD2or D3may be used as internal standard (Figure 6).

The analysis of vitamin K (phylloquinone,menaquinone and epoxides), like vitamin D, usesnormal-phase semipreparative LC followed by ananalytical reversed-phase column with UV or electro-chemical detection (Figure 7). Water-soluble mena-dione sodium bisulRte or vitamin K3 in animal feeds isdetermined by reversed-phase HPLC with UV detec-tion. To improve vitamin K3 detection limits, post-column reaction Suorimetric detection is used.Menadione is hydrogenated by sodium borohydrideto 2-methyl-1,4-dihydroxynaphthalene, which is de-tected Suorimetrically.

Water-Soluble Vitamins

Water-soluble vitamins are separated using ion ex-

change (IEC), normal-phase or reversed-phase chro-matography.Ion exchange chromatography is the preferred

method of separationfor the analysis of strongly ioniccompounds. The chromatographic separation may beoptimized by altering the pH or ionic strength of themobile phase. Reversed-phase chromatography is themethod of choice for water-soluble vitamins. Rever-sed-phase columns such as C18 and mobile phase NH2have been employed. The mobile phase is a mixture ofmethanol or acetonitrile with an acetate or phosphatebuffer. For ionic compounds the reversed-phase

ion pair mode is generally used. Unlike conventional



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Figure 6 Analytical HPLC chromatograms of (A) standard mix-

ture of ergocalciferol (D2) and cholecalciferol (D3); (B) D2(internal

standard, IS) and D3in chicken sample; and (C) D2(IS) and D3in

pork liver.

Experimental conditions: stationary phase, Zorbax ODS#

Vydac 201 TP 54 5 m; column dimension, 2504.6 mm; mobile

phase, 4% water in methanol; flow rate, 1.0 mL min1; injection

volume, 50L; detection, UV at 264 nm. (Reprinted from Horvath

CsG (ed.) (1980) High PerformanceLiquid Chromatography,New

York: Academic Press. Copyright ^ 1980 by Academic Press.)

Figure 7 Analytical HPLC chromatogram of a kiwi fruit (A) with-

out purification and (B) with purification with semipreparative

HPLC.

Experimental conditions: stationary phase, Vydac 201 TP 548

5m; column dimension, 2504.6mm; mobile phase, 96%

methanol/0.05mol of sodium acetate buffer (pH 3); flow rate,

1.5 mL min1

; injection volume, 30L; detection, dual-electrodeelectrochemical detection: upstream electrode (!1.1 V); down-

stream electrode (0 V). (Reproduced with permission from Koivu

et al., 1997.)

Figure 8 Chromatogram of thiamin (thiochrome) and riboflavin

in a skimmed milk sample using fluorescence detection.

Experimental conditions stationary phase, Ultrasphere ODS

5m; column dimension, 2504.6 mm; mobile phase, meth-

anol/water (20#80) containing 0.005 mol L1 tetrabutylammo-

nium phosphate pH 7.5; detection, fluorimetric with excitation at

360 nm and emission 425 nm. (Reproduced with permission from

Augustin, 1984.)

IEC, this technique can separate nonionic and ioniccompounds simultaneously. The chromatographicseparation may be optimized by altering the ion pairreagent, pH and ionic strength of the mobile phase.

Water-soluble vitamins (vitamin B1, thiamin, B2,

riboSavin or riboSavin 5-monophosphate; B6, py-ridoxine and nicotinamide) in commercial vitaminpreparations can be separated using either strongcation exchange resins or reversed-phase chromatog-raphy using an ion pair reagent (e.g. sodium alkane -sulfonate, dioctyl sodium sulfosuccinate, tetrabutyl am-monium phosphate) in the eluent with UV detection.

Similar LC methods are used for the separation ofthiamin in foods and biological Suids, usually withSuorescence detection. In pre-column procedures, sil-ica, C18, NH2 and poly(styrene}divinyl benzene)phases are used. Since the intensity of thiochrome

Suorescence depends on pH and reaches a steady

level at pH'8, the mobile phase should containa buffer. Polymeric C18 packings are more suitable forthese high pH conditions (Figure 8).

C18columns are used for the determination of ribo-Savin and its derivatives, Savin mononucleotide(FMN) and Savin}adenine dinucleotide (FAD). Thecompounds are separated isocratically with a mixture



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Figure 9 Chromatograms of B1, B2and B6vitamers in standard

and in whole blood.

Experimental conditions: stationary phase, Nova Pack

C18 5 m; column dimension, 1254.6 mm; mobile phase, 20%

methanol in ion pair solution (at least 70 L of di-N-butylamine

solution per litre of the eluent); flow rate, 1.0 mL min1, detection,

UV at 254 nm for B1; fluorimetric with excitation at 290 nm and

emission at 395 nm for B2. (Reprinted from Setrell WH Jr and

Harris RS (eds) (1967) The Vitamins, 2nd edn. New York/London:

Academic Press. Copyright ^ 1967 by Academic Press.)

Figure 10 (A) Representative chromatogram of standard vit-

amin B6 vitamer, 4-deoxypyridoxine (dPN) and 4-pyridoxic acid

(4-PA). Peaks: A, pyridoxal phosphate PLP (8 pmol); B, 4-PA

(10 pmol); C, pyridoxamine phosphate PMP (5.5 pmol); D, py-

ridoxal PL (10 pmol); E, pyridoxine PN (10 pmol); F, dPN

(12 pmol); G, pyridoxamine PM (6 pmol). (B) Vitamin B6vitamer

profile of human plasma.

Experimental conditions: stationary phase, Ultramex C18guard

column (304.6 mm) 3 m and Ultramex C18 column

(1504.6 mm) 3 m; mobile phase, (A) 0.033 mol L1

phosphoricacid containing 0.01 mol L1 1-octanesulfonic acid adjusted to pH

2.2 with 6 mol L1 potassium hydroxide; (B) 0.33 mol L1 phos-

phoric acid in 10% (v/v) 2-propanol adjusted to pH 2.2 with

6 mol L1 potassium hydroxide; flow rate, 1.2 mL min1; injection

volume, 25 L; detection, fluorimetric with excitation at 328 nm

and emission at 393 nm. (Reprintedfrom Setrell WH Jr and Harris

RS (eds) (1968)The Vitamins, 2nd edn. New York/London: Aca-

demic Press. Copyright ^ 1967 by Academic Press.)

Figure 11 Chromatographic separation of vitamin B6vitamers in yeast. (A) With deletion of the deamination and reduction steps; (B)

with deletion of the reduction step: (C) without deletion of either step.

Experimental conditions: stationary phase, Lichrospher 60 RP Select B octylsilyl 5 m; column dimension, 2505 mm; mobile

phase, acetonitrile/0.05 mol L1 potassium dihydrogen phosphate (4 : 96) containing 0.5103 mol L1 sodium heptanesulfonate

adjusted to pH 2.5 with phosphoric acid; flow rate, 1.0 mL min1; injection volume, 20L; detection, fluorimetric with excitation at

290 nm and emission at 395 nm. (Reprinted from Setrell WH Jr and Harris RS (eds) (1972)The Vitamins, 2nd edn. New York/London:

Academic Press. Copyright ^ 1972 by Academic Press.)

of methanol and water (or buffer solution) usingSuorimetric detection (Figure 9).

Simultaneous determination of the six chemicalforms of vitamin B

6

in foods and biological samples isperformed by IEC or reversed-phase chromatography,



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Figure 12 Chromatogram of multivitamin}multimineral tablets

at (A) 50 nm and (B) 360 nm after preconcentration and clean-up

by solid-phase extraction. Peaks: 1, vitamin B12; 2, vitamin B2.

Experimental conditions: stationary phase, Bondapack

C18 10 m; column dimension, 15039 mm; mobile phase, (A)

methanol/water (10 : 90); (B) methanol/water (90 : 10); gradient

elution, linear gradient to 50% B for the first 15 min, followed by

100% B for the next 2 min and maintained isocratically for 10 min;

flow rate, 1.0 mL min1; injection volume, 200 L; detection, UV

at 550 nm. (Reproduced with permission from Dalbacke and

Dahlquist, 1991.)

Figure 13 Chromatogram of the main folate forms found in fortified white bread.

Experimental conditions: stationary phase, Phenomenex Ultramex C18 5 m; column dimension, 2504.6 mm; mobile phase,

33 mmol L1 phosphoric acid, pH 2.3 with increasing acetonitrile; gradient elution, 5% (v/v) acetonitrile maintained isocratically for the

first 8 min, linear gradient to 17.5% (v/v) within 25 min; flow rate, 1.0 mL min1; detection, UV at 280 nm. (Reproduced with permission

from Pfeifferet al., 1997.)

with or without ion pair reagents; detection is bySuorimetry (Figure 10). The vitamin B6 content offoods can also be determined by ion pair HPLC after

pre-column derivatization of the free and phos-phorylated vitamin into pyridoxol (Figure 11).

Vitamin B12is separated from other water-solublevitamins in pharmaceutical preparations by reversed-phase using a methanol/water gradient with detectionat 550 nm (Figure 12).

Reversed-phase chromatography is mostly used for

ascorbic acid determination. In foods total vitaminC (ascorbic acid and its oxidized form, dehydroascor-bic acid) are determined using ion pair chromatogra-phy with UV detection. In biologicalSuids and foodstotal vitamin C, as its quinoxaline derivative, is separ-ated on a C18 column withSuorescence detection. Thedetermination of ascorbic acid in plasma can also beachieved using a C18column and electrochemical de-tection. Another procedure for vitamin C determina-tion consists of Rrst measuring the ascorbic acidpresent, then reducing the dehydroascorbic acid, atneutral pH, with dithiothreitol, and Rnally measuringthe total ascorbic acid. The dehydroascorbic acid isdetermined by difference. The separation is on a C18column with electrochemical detection.

After a clean-up procedure, biotin in pharmaceut-ical products is assayed using a C18 column withmethanol/water as the mobile phase and UV detec-tion. Extracts of folates (folate monoglutamates andfolic acid) in food and biological samples after puriR-cation are separated by gradient elution and UV orSuorescence detection (Figure 13).

Pantothenic acid is separated from other water-soluble vitamins with an isocratic system on anaminopropyl bonded phase using a mixture ofacetonitrile/phosphate buffer as mobile phase and UVdetection (Figure 14). Panthenol in multivitamin



11/12

Figure 14 Chromatogram of a high potency B complex tablet

extract. Peaks: A, niacinamide; B, vitamin B6; C, vitamin B2; D,

vitamin B12; E, unknown; F, pantothenic acid.

Experimental conditions: stationary phase, Hibar II Lichrosorb

NH2 10m; column dimension, 2504.6 mm; mobile phase,

0.005 mol L1 monobasic potassium phosphate (pH

4.5)/acetonitrile (13 : 87 v/v) 0.01 mol L1 1-octanesulfonic acid;

flow rate, 2.0 mL min1; injection volume, 10 L; detection, UV at

210 nm. (Reproduced with permission from Hudson and Allen,

1984.)

preparations is determined on a C18 column witha gradient system using an ion pair reagent (e.g.sodium hexanesulfonate) and UV detection. Niacinand nicotinamide are also separated on a C18columnusing an ion pair reagent with UV detection.Isonicotinic acid is used as an internal standard.

Cation exchange chromatography has been used todetermine with UV or electrochemical detection. Sep-arations of inositol mono- and diphosphate isomersin foods is performed on an anion exchange columnusing a sodium acetate in sodium hydroxide gradient

with electrochemical detection.

Future Developments

Liquid chromatography is the method of choice forvitamin analysis in pharmaceutical products, foods,feeds and especially in biologicalSuids. In biologicalsample analysis LC affords separation of the vit-amins, their related compounds and various metab-

olites for nutrition research. The main problemencountered in biological materials is the detectionlimit, particularly for water-soluble vitamins. Thereare two main areas where developments are necessaryfor future vitamin LC. First, automatic sample prep-aration techniques involving vitamin puriRcation andenrichment, e.g. automating solid-phase extraction,need to be improved. Second, the coupling LC andmass spectrometric (MS) detection needs to be furtherdeveloped. These techniques may become leadingmethods in vitamin analysis in the future.

See also: II/Chromatography: Liquid: Mechanisms:Normal Phase; Mechanisms: Reversed Phases. III/Carot-

enoid Pigments: Supercritical Fluid Chromatography.

Food Technology: Supercritical Fluid Chroma-

tography. Vitamins: Fat-Soluble: Thin-Layer (Planar)Chromatography; Water-Soluble: Thin-Layer (Planar)Chromatography.

Further Reading

Augustin J (1984) Simultaneous determination of thiamineand riboSavine by liquid chromatography. Journal

of the Association of OfTcial Analytical Chemists67(5):1012}1015.Ball GFM (ed.) (1988)Fat-Soluble Vitamin Assays in Food

Analysis. London and New York: Elsevier AppliedScience.

Ball GFM (ed.) (1994) Water-Soluble Vitamin Assays inHuman Nutrition. London: Chapman & Hall.

BoK tticher B and BoK tticher D (1987) A new HPLC-methodfor the simultaneous determination of B1-, B2- andB6-vitamers in serum and whole blood. InternationalJournal for Vitamin and Nutrition Research 57:273}278.

Bui MH (1994) Simple determination of retinol, -tocoph-

erol and carotenoids (lutein, all-trans-lycopene, - and-carotene) in human plasma by isocratic liquidchromatography. Journal of Chromatography B 654:129}133.

Dalbacke J and Dahlquist I (1991) Determination of vit-amin B12 in multivitamin}multimineral tablets by high-performance liquid chromatography after solid-phaseextraction.Journal of Chromatography541: 382}394.

De Leenheer AP, Lambert WE and Nelis HJ (eds) (1992)Modern Chromatographic Analysis of Vitamins, 2ndedn. New York: Marcel Dekker.

Federal OfRce of Public Health (1989)Vitaminbestimmun-gen in Lebensmitteln und Kosmetica Kapitel62. Bern,

Switzerland: The Federal OfRce of Public Health.



12/12

Gaby SK, Bendich A, Singh VN and Machlin LJ (eds)(1991)Vitamin Intake and Health. New York: MarcelDekker.

Hudson TJ and Allen RJ (1984) Determination of pan-tothenic acid in multivitamin pharmaceutical prepara-tions by reversed-phase high performance liquidchromatography. Journal of Pharmaceutical Sciences

73: 113}

115.Koivu TJ, Piironen VI, Henttonen SK and Mattila PH(1997) Determination of phylloquinone in vegetables,fruits and berries by high performance liquidchromatography with electrochemical detection. Jour-nal of Agricultural and Food Chemistry 45: 4644}4649.

Masuda S, Okano T, Kamao M, Kanedai Y and KobayashiT (1997) A novel high-performance liquid chromato-graphic assay for vitamin D metabolites using a coulo-metric electrochemical detector.Journal of Pharmaceut-ical and Biomedical Analysis 15 (9}10): 1497}1502.

Mattila PH, Piironen VI, Uusi-Rauva EJ and KoivistoinenPE (1995) Contents of cholecalciferol, ergocalciferol,

and their 25-hydroxylated metabolites in milk productsand raw meat and liver as determined by HPLC.Journalof Agricultural and Food Chemistry 43: 2394}2399.

Packer L and Fuchs J (eds) (1993) Vitamin E in Health andDisease. New York: Marcel Dekker.

Pfeiffer CM, Rogers LM and Gregory III JF (1997) Deter-mination of folate in cereal-grain food products using

trienzyme extraction and combined afRnity and rever-sed-phase liquid chromatography. Journal of Agricul-tural and Food Chemistry45: 407}413.

Reitzer-Bergaentzle M, Marchioni E and HasselmannC (1993) HPLC determination of vitamin B6 in foodsafter pre-column derivatization of free and phos-phorylated vitamers into pyridoxol. Food Chemistry 48:321}324.

Sebrell WH Jr and Harris RS (eds) (1972) The Vitamins,2nd edn. New York/London: Academic Press.

Sharma SK and Dakshinamurti K (1992) Determination ofvitamin B6 vitamers and pyridoxic acid in biologicalsamples.Journal of Chromatography 578: 45}51.

Water-Soluble: Thin-Layer (Planar) Chromatography

J. C. Linnell, Royal Free and University College Medi-

cal School, London, UK

Copyright^ 2000 Academic Press

As a tool, chromatography has long been importantfor the separation of vitamins from complex mixturesand their initial isolation and identiRcation wouldhave been greatly hampered without the use of paper,column or thin-layer chromatography (TLC). Whilemore sophisticated chromatographic techniques arenow widely available, TLC has great advantages interms of its simplicity and Sexibility of use.

The vitamins classiRed as water-soluble are allcompounds important in human metabolism either ascoenzymes or their precursors which the body cannotmake for itself (Figure 1). The recommended dailyallowance (RDA) of each vitamin ranges from hun-dreds of milligrams to just a few micrograms a day

(Table 1). These compounds have few properties incommon apart from their water-solubility, but thisfact alone makes TLC an excellent technique for theirseparation, particularly in pharmaceutical prepara-tions and food products. Even at physiological con-centrations, TLC is widely used after extraction of thevitamins from tissues or body Suids. This generallyneeds to be under acid conditions. Since most of thesecompounds are unstable at high pH. Some are inaddition very light-sensitive. Following TLC separ-ation, special methods of detection may also berequired, since tissue levels of most water-soluble

vitamins are low or very low.

Thiamin (Vitamin B1)

Thiamin occurs in plant and animal tissues and therichest sources are seeds and nuts, peas and beans,cereals and yeast. Fish and meat, notably pork, arealso good sources. Thiamin is commonly available asits monohydrochloride, but it also forms acid saltsand esters with nitric and phosphoric acids. Meta-bolically, thiamin is required as the coenzymethiamin pyrophosphate for the mitochondrial meta-bolism of glucose and pyruvate.

Thiamin may be extracted from tissues, foodstuffsor pharmaceutical preparations with aqueous alcoholmixtures at a pH of 4}6 and separated from closelyrelated compounds and metabolites by TLC oncellulose or silica gel. Various mobile phases havebeen successfully used, including isopropanol}water}trichloracetic acid}ammonia (71 : 9 : 20 :0.3) and butan-1-ol}acetic acid}water (40 : 10 : 50).

Thiamin may be separated from its hydrolysis andoxidation products by TLC/densitometry and otherchromatographic techniques have been reviewed.Sandwich-type chambers afford rapid separation ofthiamin from other water-soluble vitamins by TLCon silica gel GF254 and the spots then located underUV light. An alternative technique for the quantitat-ion of thiamin in pharmaceutical products involvesthe use of high performance TLC (HPTLC) and post-separation derivatization with a hexacyanoferrate(III)-sodium hydroxide reagent and Suoroden-sitometry, sensitive down to 500 pg per spot. Other

modiRcations include the use of a Rbreoptic probe

4454 III/VITAMINS/Water-Soluble: Thin-Layer (Planar) Chromatography