Chapter 21 determination of the water soluble vitamins by HPLC

Cool, dilute to volume with 0.1 M HCl, centrifuge, filter Derivatization: oxidize thiamin to thiochrome with alkaline K 3 FeCN 6 , partition into isobutanol, centrifuge LiChrosorb Si-60 5

exclusion chromatography, and reversed-phase ion-pair (ion interaction)chromatography.

21.1.2.1 Ion Exchange Chromatography

An ion exchange material comprises a porous support bearing fixed genic groups, which, when ionized, function as the ion exchange sites.Depending on their function, ion exchange materials are either anionexchangers or cation exchangers, bearing positively charged and nega-

iono-anion exchangers result from the protonation of basic groups, while thenegative charges of cation exchangers are produced by the protolysis of

within the extensive pore structure of the matrix To preserve electricalneutrality, each fixed ion is paired with an exchangeable counterion of

(Table 21.1)

tively charged functional groups, respectively The positive charges of

Section 20.4.3)

HPLC columns used for the analysis of water-soluble vitamins are of the

chromatography (see Section 20.4.4), ion exchange chromatography, ion

besame type as those used in fat-soluble vitamin assays (see Chapter 20,

opposite charge The type of counterion specifies the “form” of the ionexchanger; for example, a strong anion exchanger is usually supplied inthe chloride form, that is, the counterion is Cl2.

In ion exchange chromatography, the separation of sample ionsdepends on the selectivity at the numerous sorption – desorption cyclesthat take place as the sample material passes through the column Ionshaving a strong affinity for the functional groups will be retained on thecolumn, whereas ions that interact only weakly will be easily displaced

by competing ions and eluted early

Ion exchangers are further classified as strong or weak according to theionization properties of the basic or acidic functional groups (Table 21.1).The degree of ionization depends on the pKaof the functional group and

on the pH of the mobile phase, and is directly proportional to the ionexchange capacity The capacity is maximal when all of the functionalgroups are ionized The maximum exchange capacity for strong anionand cation exchangers is maintained over a wide pH range, whereas forweak exchangers the usable pH range is limited (Table 21.1)

Most classical ion exchange resins are polystyrene-divinylbenzene(PS-DVB) copolymers to which the ionogenic functional groups areattached Such resins exhibit a relatively slow diffusion of soluteswithin the deep pores containing stagnant mobile phase, and thisleads to major band broadening For this reason, such resins wereoften operated at elevated temperatures to speed mass transferthrough a decrease in mobile phase viscosity One way of minimizingthe diffusion path and improving the efficiency of the separation is touse pellicular particles, which have a nonporous, impervious solidcore surrounded by a thin coating of active stationary phase Pellicularpackings have been superseded by totally porous microparticulatesilica-based packings Silica-based packings are stable at temperatures

up to 808C, but strongly acidic (pH , 2) or mildly basic (pH 7.5) ditions destroy the silicon structure, leading to a drastic increase incolumn resistance and loss of efficiency This problem has promptedinvestigation into new supports for a second generation of microparticu-late column packings

con-TABLE 21.1

Characterization of Ion Exchangers

The chief mobile phase parameters that control sample retention andseparation selectivity are ionic strength and pH The role of the buffercomponent is to maintain the pH at the selected value and to providethe desired solvent strength in terms of the appropriate type of counter-ion at the right concentration The ionic strength can be regarded as ameasure of the number of counterions present The sample ions andmobile phase counterions of the same charge compete for the ionexchange sites, and hence an increase in ionic strength will proportion-ately decrease solute retention and vice versa In other words, thesolvent strength increases with increasing ionic strength, accompanied

by a minimal change in solute selectivity The ionic strength of themobile phase can be increased by either increasing the molarity of thebuffer solution while holding the pH constant, or adding a nonbuffersalt such as sodium nitrate when it is undesirable to increase the bufferconcentration The primary effect of pH is to control the ionization ofweak organic acids and bases in the sample Increasing the pH leads to

an increased ionization of weak acids and decreased ionization of weakbases, and vice versa for a decrease in pH An increase in ionization ineach case leads to increased solute retention

Water-miscible organic solvents such as acetonitrile, 2-propanol, andethanol are frequently added as modifiers to the aqueous mobile phase

as a means of lowering the viscosity and improving mass transfer kinetics.Typical amounts of added solvent range between 3 and 10% by volume.The effect of the organic modifier on the ion exchange equilibria is rela-tively minor, and any significant changes that result from such additionsare mainly attributed to hydrophobic mechanisms In weak anionexchange chromatography, an appreciable proportion of an organic acidsolute will exist in the nonionized form, and thus behave differently tothe ionized form (anion) The resultant peak tailing caused by themixed-mode chromatography can be eliminated by use of an organicmodifier, which also decreases the retention time In general, using amodifier can dramatically improve a separation, although the effect isunpredictable and has to be determined empirically It is obviouslyimportant to ascertain beforehand that the column packing material iscompatible with the proposed organic solvent

21.1.2.2 Ion Exclusion Chromatography

In this technique, an ion exchange resin is employed for separating ionicmolecules from nonionic or weakly ionic molecules Ions having thesame charge as the functional groups of the support (i.e., co-ions) arerepelled by the electrical potential across the exchanger– solution inter-face (Donnan potential) and excluded from the aqueous phase within

the pore volume of the resin beads Nonionic or weakly ionic moleculesare not excluded and, provided they are small enough, may freely diffuseinto the matrix, where they can partition between the aqueous phasewithin the resin beads and the aqueous phase between the resin beads.Therefore, ionized sample solutes pass quickly through the column,whereas nonionic or weakly ionic solutes pass through more slowly.The retention mechanisms of the nonionic solutes include polar attrac-tion between the solute and the resin functional groups (i.e., adsorption),van der Waal’s forces between the solute and the hydrocarbon portion ofthe resin (primarily the benzene rings), and size exclusion The overallseparation is accomplished without any exchange of ions, so thecolumn does not require regeneration after use.

Ion exclusion chromatography using a strong cation exchange resin hasbeen successfully applied to the separation of organic acids, includingascorbic acid The technique here is to suppress the ionization of theweak organic acid by adding sulfuric acid to the water mobile phase sothat the highly ionized sulfate ion is excluded and quickly eluted, whilethe undissociated organic acid enters the resin pore structure and isretained The mobile phase pH should be lower than the pKa of theorganic acid to ensure that the acid is undissociated The volume ofaqueous phase within the resin bead must be sufficient to allow partition

of the nonionic solutes to take place and, to achieve optimum separation,must be greater than the sample volume For this reason, PS-DVB types ofresin, which are capable of swelling, are used in preference to silica-basedexchangers

21.1.2.3 Reversed-Phase Chromatography

Ionic compounds cannot be analyzed as such by reversed-phase HPLC,since they elute near the void volumes Ion suppression is a reversed-phase chromatographic technique in which the ionic equilibrium of thesample is controlled by adjusting the pH of the mobile phase to obtainretention and separation of the components according to their pKa

values [1] By buffering of the mobile phase at 1– 2 units below the

pKavalue for a weak acid, and a corresponding amount above the pKb

value for a weak base, the ionization is suppressed and the undissociatedcompound, having a greater affinity for the stationary phase, is retained.Thus, weak acids and weak bases can be retained in the pH regions 2– 5and 7– 8, respectively

A potential problem with silica-based reversed-phase column packings

is that the siloxane bond linking the alkyl ligand to the silica support isprone to hydrolysis at low pH, resulting in a progressive loss of bonded

phase Although longer-chain ligands such as C18are relatively stable at

pH 3 and below, short-chain bonded phases, including small endcappinggroups, are especially susceptible The problem of loss of column perform-ance due to hydrolysis can be largely overcome by the use of ‘shielded’stationary phases, which are sterically protected from attack by hydrolyz-ing protons One such material is Zorbax SB-C18, which has large, bulkydiisobutyl groups on the silane silicon atom and is nonendcapped Out-standing long-term ruggedness under highly aggressive low-pH con-ditions (pH 2) has been demonstrated using Zorbax SB-C18 [2] Anotherapproach is to use a totally polymeric column packing such as PLRP-S, aPS-DVB copolymer Such materials are not attacked by extremes of pH,but they exhibit appreciably lower separation efficiencies than reversed-phase silica-based packings for small molecules such as vitamins [3]

21.1.2.4 Reversed-Phase Ion-Pair Chromatography

Reversed-phase ion-pair chromatography (also known as ion interactionchromatography) employs the same types of column packing and water/organic mobile phases as those used in conventional reversed-phaseHPLC The pH of the mobile phase is adjusted to encourage ionization

of the ionogenic solutes, and retention is controlled by adding to themobile phase an amphiphilic ion-pairing agent bearing an oppositecharge to that of the analyte The ion-pairing agent should be univalent,aprotic, and soluble in the mobile phase It should ideally give a lowUV-absorbing background, although for special applications a reagentwith a strong chromophore can be used to enhance the response of anabsorbance detector The retention behavior of nonionic solutes is notaffected by the presence of the ion-pairing agent, so both ionized and non-ionized solutes may be resolved in the same chromatographic run Use ofion-pair chromatography is advantageous for determining water-solublevitamins because many polar interferences elute in the dead volume, andhydrophobic compounds would be in low concentration in the aqueousextract of the sample

For the determination of anionic solutes such as ascorbic acid, a variety

of organic amines have been used as ion pairing agents, representingprimary, secondary, tertiary, and quaternary amines One of the morepopular of these is tetrabutylammonium (Bu4Nþ) phosphate, which iscommercially available as a prepared 5 mM solution in pH 7.5 buffer(PIC A reagent, Waters Associates) This aprotic quaternary amine inter-acts with strong and weak acids, and the buffering to pH 7.5 suppressesweak base ions

For the determination of cationic solutes such as thiamin (a protonatedamine), a range of alkyl sulfonates having the formula CH3(CH2)nSO3 2

(n¼ 4–7) predominates Selection of the appropriate reagent is based

on solute retention time, which increases with an increase in the length

of the alkyl chain Prepared 5 mM solutions of the sodium salts in pH3.5 buffer are available from Waters Associates; namely, pentane sulfonicacid (PIC B5), hexane sulfonic acid (PIC B6), heptane sulfonic acid (PICB7), and octane sulfonic acid (PIC B8) These reagents interact withstrong and weak bases, and the buffering to pH 3.5 suppresses weakacid ions

Most ion-pair chromatographic applications reported for water-solublevitamin assays up to the present day have utilized 5- or 10-mm silica-based C18 bonded-phase packings Monomeric phases yield better-shaped peaks than do polymeric phases, and high carbon loadingsensure good retention properties [4] PS-DVB copolymers developed forHPLC have also been utilized for ion-pair chromatography [5]

The practice of ion-pair chromatography has been discussed by Gloorand Johnson [6] Retention and selectivity are optimized mainly by alter-ing the concentration of the ion-pairing agent and the pH of the mobilephase Ionic strength is not a variable for controlling retention and itshould be kept as low as possible, commensurate with satisfactory reten-tion characteristics and reproducibility

Variation of the concentration of ion-pairing agent in the mobilephase provides a simple means of controlling solvent strength Anincrease in the concentration causes an increase in solute retention but,beyond a certain limit, a further increase in concentration causes adecrease in retention A possible explanation for this reversal effect

is that the increased amount of adsorbed surfactant lowers the facial tension between the modified stationary phase and the surround-ing aqueous medium to a point at which solute retention is decreased[7] This nonionic theory also accounts for the observed decrease inretention of neutral solutes with increasing concentration of ion-pairing agent

inter-Alterations in the pH of the mobile phase will have a pronouncedeffect on separation selectivity for weak acids and weak bases because

of the effect of pH on solute ionization Maximal retention is obtainedwhere the solute and ion-pairing agent are completely ionized Thereagents, being strong acids or salts of strong bases, remain completelydissociated over a wide pH range, so that the pH can be adjusted to anoptimal value for the separation Weak acid solutes (pKa 2) are usuallyseparated at a pH of 6 –7.4, and weak bases at pH 2 – 5, using a buffer tohold the pH constant Buffer salts should have poor ion associationproperties, but good solubilities in the mobile phase An excessive con-centration of buffer salt, or the addition of neutral salt to the mobilephase, results in the surplus ions of such salts competing successfullywith analyte ions for association with the adsorbed ion-pairing agent,

thus causing a decrease in retention Solute pKavalues are affected by achange in temperature, so significant changes in selectivity can occurwith relatively small changes in column temperature To ensure repro-ducible separations, it is thus good practice to maintain a constantcolumn temperature with the aid of a column heating oven Ion-pair chromatography is usually carried out at a few degrees aboveambient, although operation at 50 –608C will improve peak resolution(with a slight decrease in retention) by reducing the viscosity of themobile phase.

Increasing the proportion of organic modifier increases the solventstrength, resulting in an overall lowering of solute retention The concen-tration of organic modifier affects the surface potential (and hence soluteretention) by influencing the sorption of the ion-pairing agent onto thestationary phase [8]

The general strategy for separating complex mixtures of nonionic andionic solutes is firstly to adjust the percentage of organic modifier(usually methanol) to obtain optimum retention and separation of non-ionic solutes One then adds a suitable ion-pairing reagent in the appro-priate buffer to the previously established mobile phase to separate theionic compounds isocratically Gradient elution programs usuallyinvolve a decrease in the concentration of ion-pairing agent with time

as a means of decreasing solute retention

Ion-pairing agents may irreversibly adsorb onto the stationary phase,thereby changing the phase chemistry and reducing the apparent porevolume Columns used for ion-pair chromatography should therefore

be reserved exclusively for this purpose

21.1.3 Derivatization

It is sometimes necessary to make a chemical derivative of an analyte

in order to facilitate the use of a more suitable means of detectionand/or a more suitable chromatographic mode Either pre- or post-column derivatization may be employed, depending on whether onewishes to chromatograph the derivatized analyte or the underivatizedanalyte In precolumn derivatization, the reaction is carried out beforethe sample is analyzed by HPLC, so it is the derivatized compoundsthat are actually chromatographed In postcolumn derivatization, thetest solution is injected into the chromatograph, and the separatedcompounds in the column effluent are reacted with the derivatizingagent in a heated reaction coil located between a mixing tee and thedetector [9]

A postcolumn derivatization system requires a second pump to duce the derivatizing agent but, once set up, the system provides an

intro-automatic and standardized means of preparing the derivatives Therewill inevitably be some degree of peak broadening due to the increaseddistance between the HPLC column and the detector Another disadvan-tage is that there is no opportunity to remove or separate excess reagent orimpurities within the reagent that might impair the sensitivity of detec-tion Precolumn derivatization requires manual manipulations, andhence more skill and nonstandardized reaction conditions, unless rigor-ously controlled Advantages are the opportunity to clean up the reactionmixture before injection, and the operation of a simpler and more efficientchromatographic system.

21.2 Applications of HPLC

In this section, applications are arbitrarily divided into single vitaminanalyses and multiple vitamin analyses The requirement to determinethe naturally occurring vitamin of a foodstuff allows little scope for deter-mining more than one vitamin at a time This is because of difficulties

of quantitatively extracting the vitamins from their various bound forms,the need to measure low indigenous concentrations in the presence of acomplex matrix, and the requirement to determine several vitamers ofsome vitamins

21.2.1 Thiamin

21.2.1.1

The absorption spectrum of thiamin hydrochloride is pH-dependent, as

the 1 value at this wavelength is 11,305 At pH 5.5 two maxima occur at

234 and 264 nm, which correspond to the substituted pyrimidine andthiazole moieties, respectively

esters can be reacted with alkaline potassium hexacyanoferrate(III) assium ferricyanide, K3Fe(CN)6] to form the corresponding thiochrome

[pot-fluorescence excitation and emission spectra of thiochrome possess

wave-molar amounts of the thiochrome derivatives of thiamin, TMP, TDP,and TTP produce different fluorescence intensities [10]

shown in Figure 21.1 At pH 2.9 a single maximum at 246 nm occurs;

Detection

Thiamin itself does not fluoresce, but the vitamin and its phosphate

length maxima at 375 and 432 –435 nm, respectively (Figure 21.2) compound (Figure 7.2), which displays a strong blue fluorescence The

Equi-A B

21.2.1.2 Methodology

and Young [15]

When determining the total thiamin content of a food commodity, thetest material is extracted by autoclaving with dilute mineral acid(usually 0.1 N hydrochloric acid) followed by enzymatic hydrolysis, inorder to convert protein-bound and phosphorylated forms of thevitamin to free thiamin Although thiamin exhibits a rather low molarabsorptivity (1¼ 11,305 at lmax 246 nm), absorbance detection has ade-quate sensitivity for fortified foods [16,17] and also for foods that arerelatively rich in the vitamin, such as legumes and pork muscle [14].For other food commodities, absorbance detection is inadequate, and it

is necessary to employ the more sensitive fluorescence detection after dation of the thiamin to thiochrome by pre- or postcolumn reaction withalkaline hexacyanoferrate(III)

oxi-Precolumn derivatization allows the relatively nonpolar thiochrome to

be determined using conventional reversed-phase chromatography, withits attendant ease of operation and long-term stability Some workers[18 –20] added orthophosphoric acid 45 sec after treatment with alkalinehexacyanoferrate(III) to minimize formation of thiamin disulfide, a pH-dependent side reaction of the thiamin to thiochrome oxidation.Cleanup of the reaction mixture prior to HPLC has been effected using

C18solid-phase extraction cartridges [18,19,21] An alternative approach

is to selectively extract the thiochrome into isobutanol, and then toinject an aliquot of the organic solution onto an HPLC column of under-ivatized silica eluted with chloroform/methanol (80:20) [11] An on-column fluorescence detection limit of 0.05 ng thiamin was reportedusing this approach [22]

If the derivatization is carried out postcolumn, it is actually thiaminthat is being chromatographed, and this compound in the ionized state

is not retained under simple reversed-phase conditions However,reversed-phase columns can be utilized for thiamin assay by means ofion-pair chromatography using hexane (or heptane) sulfonic acid asthe ion-pairing reagent, either after postcolumn derivatization ofthiamin and fluorescence detection, or without derivatization, using

UV detection Reversed-phase columns can also be used with ion pression [23,24]

sup-Postcolumn derivatization is not only more reproducible and venient than precolumn derivatization, but the alkaline pH of the effluent

con-is more conducive to the fluorometric detection of thiochrome Thcon-is con-isbecause the fluorescence intensity of thiochrome is pH-dependent andreaches a steady state at pH above 8 [10]

HPLC methods used for determining thiamin per se are summarized in

Acid and enzymatic hydrolysis:

digest homogenized sample with 0.1 N HCl at 95–1008C for

1 h Cool, adjust pH to 4.5.

Incubate with Takadiastase and papain at 45 –508C for 3 h Cool, dilute to volume with 0.1 M HCl, centrifuge, filter Derivatization: oxidize thiamin to thiochrome with alkaline

K 3 Fe(CN) 6 , partition into isobutanol, centrifuge

LiChrosorb Si-60

5 mm

250  4 mm

CHCl 3 /MeOH, 80:20

Cooked sausages Acid and enzymatic hydrolysis:

autoclave ground sample with 0.1 N HCl at 1208C for 20 min.

Cool, adjust pH to 4.0–4.5.

Incubate with Claradiastase at 508C for 3 h Precipitate proteins by heating at 90 8C with 50% (w /v) TCA, dilute to volume with water, filter

Spherisorb C 8 (octyl)

5 mm

250  4 mm Column temperature 358C

5 mM phosphate buffer,

pH 7.0/MeCN, 70:30

TABLE 21.2 Continued

Quantitative HPLC

Compounds

Derivatization: oxidize thiamin to thiochrome with alkaline

K 3 Fe(CN) 6

Cleanup and concentration: pass oxidized extract through a C 18

solid-phase extraction cartridge, wash cartridge with

5 mM phosphate buffer, pH 7.0 /MeOH (95:5) Elute thiamin with 3 ml MeOH, dilute to 5 ml with MeOH

Reversed-phase ion-pair chromatography

to 4.0–4.2 Dilute to volume with water, filter

mBondapak C 18

10 mm

300  3.9 mm Column temperature 508C

Water/MeOH (80:20) containing 0.15% sodium hexane sulfonate, 0.75% acetic acid and 0.1%

pH of extract to 5.0 –5.5 and keep at 08C Pass aliquot of extract through an Amberlite CG-50 ion exchange column, wash column bed with water, elute thiamin with 0.15 M HCl Evaporate eluate to dryness, dissolve residue in water, adjust pH to 5.5 –6.0.

Pass aliquot of solution through a C 18 solid-phase extraction cartridge, wash cartridge with aqueous 5 mM sodium hexane sulfonate, elute thiamin with methanolic 5 mM sodium hexane sulfonate

10 mm

300  3.9 mm Column temperature 308C (legumes) or 508C (pork and milk products)

(69:31) containing

5 mM sodium hexane sulfonate/5 mM sodium heptane sulfonate (75 þ 25) and 0.5% acetic acid

Note: K 3 Fe(CN) 6 , potassium hexacyanoferrate(III); MeOH, methanol; MeCN, acetonitrile; CHCl 3 , chloroform; trichloroacetic acid, TCA; EDTA, lenediaminetetraacetic acid.

21.2.2 Vitamin B2

21.2.2.1 Detection

The positions of the maxima and their absorbance coefficients vary what according to the nature of the solvent The 373-nm band is the mostaffected by solvents, generally shifting to shorter wavelengths withdecreasing solvent polarity The position of the visible 445-nm band isnot greatly affected The spectra of riboflavin and FMN are practicallyidentical to one another under similar conditions, but the spectrum ofFAD is slightly different All three flavins lose their absorbance in the

forms, known as leuco bases Molar absorptivity (1) values of flavins

Riboflavin, FMN, and FAD in aqueous solution exhibit an intenseyellowish-green fluorescence with an emission maximum at around

530 nm when excited at 440 –500 nm The fluorescence spectra of

A B

riboflavin are shown in Figure 21.4 The fluorescence of flavins is a teristic of uncharged neutral forms of isoalloxazines; anionic and cationicforms do not fluoresce Riboflavin shows a maximum and equal fluor-escence in the pH range between 3.5 and 7.5 The same is true for FMN,but FAD displays maximal intensity at pH 2.7 –3.1 In equimolar neutral

Source: Yagi, K., Chemical determination of flavins, in Methods of

Biochemical Analysis, Glick, D., Ed., Vol 10, John Wiley & Sons,

New york, 1962, p 319 With Permission.

A

Wavelength (nm) 300

Fluorescence intensity ex. em.

600 550 500 450 400 350

FIGURE 21.4

Fluorescence excitation and emission spectra of riboflavin dissolved in water (pH 7.4) ( lmax

of peak A ¼ 360 nm; B ¼ 465 nm; C ¼ 521 nm).

aqueous solutions, riboflavin and FMN exhibit practically the same escence intensity, whereas the intensity of FAD is only about 15% of that ofriboflavin The fluorescence property of riboflavin depends on the3-imino group being free Because protein binding takes place via the3-imino group, protein-bound forms of the vitamin do not fluoresce It

fluor-is therefore essential to release flavins from proteins if the quantification

is to rely on fluorescence detection

21.2.2.2

HPLC methods for determining riboflavin per se are summarized in

A standard extraction technique suitable for HPLC, and applicable toany kind of foodstuff, is to convert the bound flavins to free riboflavin

by autoclaving the sample with dilute mineral acid, followed by matic digestion at the optimum pH In some applications, the hydrolyzedextract is heated with 50% (w/v) trichloroacetic acid to deactivate andprecipitate the previously added enzyme The total vitamin B2 content(excluding covalently bound flavins) is then calculated, based onmeasurement of the riboflavin peak The chromatography is capable ofseparating FMN and riboflavin, so a valid estimate of the total vitamin

enzy-B2using this technique depends on the conversion of FMN to riboflavinbeing complete If the hydrolysis is incomplete, as indicated by the pre-sence of an FMN peak in the sample chromatogram, an alternativeenzyme preparation and/or a longer incubation period can be tried Ifthese steps fail to achieve a complete hydrolysis, the FMN peak must

be measured in addition to the riboflavin peak Summation of FMN andriboflavin peak heights provides a total vitamin B2content without sig-nificant error, provided that the FMN peak height is ,25% of the sum

of the FMN and riboflavin peak heights [37] An unavoidable source oferror is the formation during acid digestion of biologically active isomericriboflavin monophosphates, which are chromatographically separatedfrom FMN

The above extraction technique has been used for the determination oftotal vitamin B2in a wide range of foods, and the results compared with a

importance of the enzyme treatment in converting FMN to riboflavin is

two assay techniques for vitamin-enriched samples such as baby foods,gruels, porridges, breakfast cereals, and flour (r¼ 0.9996) and for milkproducts (r¼ 0.9999) This work was reported from Sweden, whereFMN, rather than riboflavin, is generally used for enrichment In contrast,the riboflavin values found with the HPLC method in nonenriched floursand flours with a high rate of extraction were 25 –50% lower than those

Table 21.4

Methodology

illustrated inFigure 21.5 High correlations were obtained between thestandard microbiological assay using Lactobacillus rhamnosus [34] The

HPLC Methods Used for the Determination of Riboflavin and Other Flavins in Food

Italian cheeses Homogenize sample with

water/MeOH, 2:1 Add acetic acid, mix, centrifuge.

Resuspend the pellet three times in water/MeOH/acetic acid, 65:25:10 Dilute pooled supernatants to volume with the same solvent, centrifuge

LiChrosorb RP-18

5 mm

250  4 mm

Water/MeCN, 80:20

Milk, yoghurt,

cheese

Milk: pass sample through C 18

solid-phase extraction cartridge, wash column bed twice with water, elute riboflavin with 20 mM acetate buffer (pH 4.0) /MeOH, 1 þ 1 Yoghurt and cheese: blend sample with acetate buffer, filter, then solid-phase extraction as for milk

Food Sample Preparation Column Mobile Phase

Compounds

Pasta (enriched) Acid hydrolysis: autoclave

ground sample with 0.1 N HCl at 1218C for 30 min, cool, centrifuge Resuspend the pellet twice in 0.1 N HCl.

Dilute pooled supernatants

acetic acid, 50 /

49 /1

(1) Riboflavin (2) Lumichrome

Fluorescence (1) ex

450 nm

em 510 nm (filters) (2) ex 300–

350 nm

em 479 nm (filters)

[28]

Wines, beers,

fruit juices

Filter (0.22 mm), inject directly

or after dilution with water

Hypersil C 18 5 mm

200  2.1 mm Gradient of solvent(A) 0.05 M

phosphate buffer,

pH 3.0 and (B) MeCN

FAD, FMN, riboflavin

Fluorescence

ex 265 nm

em 525 nm with a

500 nm cut-off filter

Homogenize sample with 6%

formic acid containing 2 M urea, centrifuge Mix a 2-ml aliquot with sorboflavin (internal standard) Cleanup and concentration: pass solution through C 18 solid- phase extraction column, wash column bed with 10%

formic acid, elute the flavins with 10% formic acid / MeOH, 4:1

LC-18 3 mm (Supelco)

75  4.6 mm

14% MeCN in 0.1 M

KH 2 PO 4 (final pH 2.9)

FAD, FMN, sorboflavin (internal standard), riboflavin

pyrophosphatase Digest with buffered pronase (pH 6.8) for 1 h at 458C to simultaneously release the flavins bound to milk proteins and deproteinize.

Cool, adjust volume with phosphate buffer, adjust pH

to 5.5 Centrifuge, filter

5 mm

250  4.6 mm Column temperature 408C

(Solvent A) and 0.01 M phosphate buffer, pH 5.5 (Solvent B) Linear gradient from 35% of A and 65%

of B to 95% of A and 5% of B over

sodium azide, rehomogenize, filter

2 PLRP-S 5 mm (Polymer Laboratories) columns in series

250  4.6 mm Column temperature 408C

MeCN/0.1% sodium azide in 0.01 M citrate-phosphate buffer (pH 5.5) in multistep gradient elution program

FAD, FMN, riboflavin, 7-Et-8- Me-RF (internal standard)

Spherisorb ODS-2

5 mm

250  4.6 mm

Initial: 94% of 0.1 M sodium acetate buffer (pH 4.8) and 6% of water /

(50 þ 40 þ 10) Linear gradient elution:

proportion of water/MeCN/

MeOH mixture

ex 450 nm

em 520 nm (cut-off filter

30 min Baby foods,

pH to 4.5 Incubate with acid phosphatase at 458C overnight Precipitate proteins by heating at 1008C with 50% (w/v) TCA for

5 min Adjust pH to 4.5, dilute

to volume with water, filter

Hypersil-ODS 5 mm

250  4.6 mm Water/MeOH(3 þ 2) adjusted

to pH 4.5 with acetic acid

Riboflavin (representing total vitamin B 2 )

pH to 4.5 Incubate with Claradiastase at 458C overnight Cool, dilute to volume, filter

Cleanup and concentration: pass aliquot of filtrate through C 18

cartridge with water, elute the riboflavin with 40–70%

MeOH Milk- and soy-

Solid samples and CRMs:

homogenize with MeCN, add 10 mM phosphate buffer (pH 5), homogenize, centrifuge, dilute to volume Liquid samples: filter (0.45 mm nylon)

Discovery RP-AmideC 16

(hexadecyl) 5 mm

150  4.6 mm

10 mM phosphate buffer, pH 5 / MeCN, 90:10

FAD, FMN, riboflavin

Fluorescence;

two channels at wavelengths

of 270/516 and 452/

516 nm (ex/em)

[36]

Reversed-phase ion-pair chromatography

Flour, bread, raw

121 8C for 30 min Adjust pH

to 6.0, dilute to volume, filter

LiChrosorb RP-8 (octyl) 10 mm

250  4.0 mm

Water/MeOH, 60:40 containing 5 mM sodium hexane sulfonate

FMN, riboflavin (representing total vitamin B 2 )

Fluorescence

ex 440 nm (filter),

em 565 nm (filter)

[37]

Fresh fruit and

vegetables

Acid and enzymatic hydrolysis:

digest ground dry sample with 0.1 N HCl at 998C for

Ultrasphere-ODS

5 mm

250  4.6 mm

MeOH/water (40:60) containing

5 mM sodium

Riboflavin (representing total vitamin B 2 )

TABLE 21.4 Continued

Quantitative HPLC

Compounds

30 min, cool Incubate with buffered Mylase at 388C overnight Precipitate proteins by heating at 608C with 50% (w/v) TCA for

5 min Adjust pH to 4.0, dilute

to volume with water, filter

heptane sulfonate;

adjusted to pH 4.5 with H 3 PO 4

Cooked

sausages

Acid and enzymatic hydrolysis:

autoclave ground sample with 0.1 N HCl at 1208C for

20 min Cool, adjust pH to 4.0–4.5; incubate with Claradiastase at 508C for 3 h.

Precipitate proteins by heating at 908C with 50%

(w/v) TCA, dilute to volume with water, filter

Spherisorb ODS-2

5 mm

250  4 mm Column temperature 358C

5 mM heptanesulfonic acid adjusted to

pH 2.7 with phosphoric acid/

MeCN, 75:25

Riboflavin (representing total vitamin B 2 )

Acid and enzymatic hydrolysis:

autoclave ground sample with dilute HCl at 121 8C for 15 min.

Riboflavin (representing total vitamin B 2 )

Cool, dilute to volume with water, filter

Cleanup and concentration:

readjust pH of extract to 4.0 – 4.5 Pass through Florisil column, wash column bed with water, elute riboflavin with triethylamine/MeOH/

water, 7:30:13 Evaporate eluate to dryness, dissolve residue in MeOH/water (same proportion as mobile phase) Pass aliquot of solution through C 18 solid- phase extraction cartridge, wash cartridge with aqueous

5 mM sodium hexane sulfonate, elute riboflavin with methanolic 5 mM sodium hexane sulfonate

acetic acid (68.5:31:0.5) containing 5 mM sodium heptane sulfonate/5 mM sodium hexane sulfonate, 25:75

Note: TCA, trichloroacetic acid; MeOH, methanol; MeCN, acetonitrile.

found with the microbiological assay A possible reason for thisdisparity was the loss of fluorescence by the inner filter effect Thisrefers to the absorption of incident or emitted radiation by substances

in the complex food matrix which coeluted with riboflavin Further fication of the filtered extract using C18 solid-phase extraction failed toincrease the fluorescence signal, and unchanged results were obtained.Vidal-Valverde and Reche [40] reported that C18 solid-phase extractionwas inadequate for removing all interfering substances present in hydro-lyzed food extracts These authors employed a cleanup procedure invol-ving adsorption chromatography on a Florisil (fuller’s earth) column,followed by C18solid-phase extraction, which removed the major interfer-ing substances present in legumes, and made absorbance detection at

puri-254 nm sufficiently sensitive for these products (detection limit: 0.4 ngper injection)

An alternative technique for determining total vitamin B2is to measurethe FMN and riboflavin liberated during autoclaving with dilute mineralacid, after precipitation of proteinaceous material by pH adjustment toabout 4.5 (as in the AOAC fluorometric procedure) The enzymatichydrolysis step is omitted For acid extracts containing significant

foodstuff by treatment with (a) 0.1 N hydrochloric acid and (b) acid and enzyme.

H and Branzell, C., Int J Vitam Nutr Res., 57, 53, 1987 With permission.)

Operating parameters as in Table 21.4 [34] Peaks: (1) FMN; (2) riboflavin (From Johnsson, Reversed-phase HPLC with fluorescence detection of the flavins extracted from a typical

amounts of FMN, that is, where the FMN peak height is.25% of thesum of the FMN and riboflavin peak heights (such as were obtainedfrom raw beef, corned beef, fresh and cooked liver, and canned mush-rooms), FMN and riboflavin were calculated separately, using theircorresponding response factors (ratio of concentration of standard-to-peak height), and the results were summed to obtain total vitamin

B2 For smaller amounts of FMN, the total vitamin B2could be obtained

by summation of FMN and riboflavin peak heights without significanterror [37] The necessity of applying a correction factor to compensatefor the lack of purity of commercial FMN preparations has been stressed

by Russell and Vanderslice [32] It is important to understand thatthe quantity of FMN found does not represent the original FMNcontent of the food sample, as the FMN peak originates largely fromhydrolyzed FAD

For the analysis of milk, eggs, and dairy products, in which free orloosely bound riboflavin is considered to be the predominant naturallyoccurring flavin present, riboflavin can be determined specificallywithout the need for acid and enzyme hydrolysis A common extractionprocedure for milk entails precipitation of the protein to below pH 4.5(the isoelectric point of most proteins) and filtration This procedurehas been employed using acetic acid [41,42], trichloroacetic acid[43,44], and acidified lead acetate solution [45] as the deproteinizingagent

Most published riboflavin HPLC assays utilize C18stationary phases,either with aqueous/organic mobile phases in the reversed-phase mode

or using ion-pairing agents (hexane or heptane sulfonic acid) rescence monitoring is the preferred means of detection in most cases.The limits of fluorescence detection at 450/522 nm (excitation/emission)for the biologically active flavins at a signal-to-noise ratio of 3:1 were0.55 pmol or 0.21 ng riboflavin; 1.96 pmol or 0.89 ng FMN; 14.9 pmol or11.15 ng FAD [32] The sensitivity can be increased even further toachieve a detection limit of 0.02 ng per injection by irradiating the ribo-flavin at high pH to form lumiflavin [22], but this technique has notbeen widely adopted

Fluo-The use of a 254-nm fixed-wavelength absorbance detector provided aminimum on-column detection limit of 0.4 ng riboflavin [40], which wasadequate for the analysis of legumes and full cream milk powder.Absorbance monitoring at 270 nm was also sufficiently sensitive forthe analysis of milk and milk products after cleanup and concentrationusing C18 solid-phase extraction [27] and has the advantage in simul-taneously detecting riboflavin decomposition products [42] Stancherand Zonta [26] utilized visible absorbance detection at 446 nm toavoid detection interference in the analysis of Italian cheeses

The on-column detection limit of 2.5 ng (cf 0.1 ng by fluorescence) wasmore than adequate for determining the relatively high concentration ofriboflavin (at least 100 mg/100 g) present in the cheese commoditiesanalyzed.

For samples with low riboflavin content (,10 mg/100 g), Lumley andWiggins [46] employed a trace-enrichment technique based on the extre-mely long retention time of riboflavin on a reversed-phase column whenpure water was used as a mobile phase Concentration was achieved byloading successive 100-ml volumes of sample extract onto the guardcolumn The riboflavin, which concentrated at the column head, couldthen be eluted as a tight band by changing the eluent from water to themethanol/water mobile phase Jaumann and Engelhardt [47] described

an online enrichment technique for separation of riboflavin in foodusing a column-switching device

Greenway and Kometa [33] developed an online sample preparationmethod for the determination of riboflavin and FMN in milk and cerealsamples by reversed-phase HPLC The online system consisted of micro-wave extraction followed by dialysis and trace enrichment with a C18

mini-column Sample preparation was minimal, with milk samples beingdirectly introduced into the system and cereal samples only needing to beground prior to analysis During the microwave extraction, the FAD wasconverted into FMN, and 15% of the FMN was converted into riboflavin.The full analysis time on the ground samples was about 20 min Resultswere found to be in agreement with those obtained using the AOAC fluoro-metric method and a previously reported HPLC method

Russell and Vanderslice [32] employed a nondegradative two-step tion procedure for the simultaneous quantification of riboflavin, FMN and

extrac-was added at the start of the extraction procedure, and separation of theflavins and internal standard was accomplished with a polymer-based

Appropriate correction factors were applied to account for the impurities

in the commercial FMN and FAD standards Russell et al [48] employed

a robotic system to automate the extraction, duplicating the manual steps

as closely as possible The robotic method was faster and generally duced slightly higher results than the manual process Higher molar con-centrations of FMN and FAD relative to riboflavin indicated that lessdegradation or interconversion of the individual vitamers was takingplace The advantage of operating the robotic system in complete darkesscould be responsible for this protective effect Bilic and Sieber [30] extractedriboflavin, FMN and FAD from dairy products by homogenization with 6%formic acid containing 2 M urea in the presence of sorboflavin added as aninternal standard Sorboflavin contains a glucityl side chain instead of aribityl chain on the isoalloxazine ring

pro-column packing and a multistep gradient elution program (Figure 21.6).FAD in a variety of foods An internal standard, 7-ethyl-8-methyl-riboflavin,

Vin˜as et al [36] used a C16 stationary phase with embedded amidegroups and trimethylsilyl endcapping for the separation of riboflavin,FMN, and FAD by isocratic elution A two-channel fluorescence detectorallowed two excitation wavelengths to be selected: 270 nm was optimalfor maximum fluorescence and 425 nm overcame interferences from thematrix Fluorescence spectra were continuously measured duringpassage of the solute through the flow cell The standard additionsmethod was used to investigate the possibility of interference by thematrix Slopes of the standard additions calibration graphs for the foodsamples were similar to those of aqueous standards, confirming that thematrix did not interfere The procedure was applied to the determination

of B2vitamers in milk- and soy-based infant formulas, beer, fruit juices,and honey of different types On-column detection limits were

1 2

Operating parameters as in Table 21.4 [32] Arrow indicates change of detector attenuation.

0.03, 0.05, and 0.24 ng for riboflavin, FMN, and FAD, respectively Themethod was validated using two certified reference materials, andresults within the certified range were obtained.

21.2.3 Niacin

21.2.3.1 Detection

Nicotinic acid and its amide exhibit similar absorption spectra in the UVregion The absorptivity is strongly affected by pH, being higher in anacidic than in an alkaline solution, but the lmax remains almostunchanged at 261 nm (Figure 21.7) The A1 cm1% value for nicotinamide in0.1 N sulfuric acid at 261 nm is 478 The presence of electrolytes alsohas a marked effect on the absorbances of the solutions [49]

Nicotinic acid and nicotinamide do not fluoresce, but fluorescentderivatives can be formed by treatment with a mixture of cyanogenbromide and p-aminoacetophenone [50] Ultraviolet irradiation of niacinsolutions in the presence of hydrogen peroxide and copper(II) ions alsoinduces fluorescence [51]

HPLC Methods Used for the Determination of Niacin in Food

Quantitative HPLC

Compounds

Anion exchange chromatography

to 6.5 then immediately to

pH  1.0 Filter through paper, dilute to volume with water

Cleanup: apply 3.0-ml aliquot

of filtrate to pretreated ArSCX solid-phase extraction cartridge Flush column bed with water, elute nicotinic acid with

2  6.0 ml 0.25 M acetate buffer, pH 5.6 Bring to final volume with 0.5 M acetate buffer

PRP-X100

250  4.1 mm 0.1 M acetatebuffer, pH 4.0

Nicotinic acid (representing total niacin)

TABLE 21.5 Continued

Quantitative HPLC

Compounds

Strong cation exchange chromatography

Fresh meat (to test for

added nicotinic acid)

Homogenize sample in water then boil, cool, and filter

Partisil SCX 10 mm

250  4.6 mm 0.05 Mphosphate

buffer, pH 3.0

Nicotinic acid, nicotinamide (free forms) ascorbic acid, sorbic acid

Reversed-phase ion-pair chromatography

cool, dilute to volume, filter Pass through C 18

solid-phase extraction cartridge

Spherisorb ODS-2

150  5 mm MeOH/water(8:92)

containing

5 mM tetrabutyl- ammonium hydroxide (final pH adjusted to 7.0)

Nicotinic acid (free)

Beef, pork, tuna (to test

for added nicotinic

acid)

Add H 3 PO 4 to homogenized sample, extract with MeOH, filter

Shim-Pack FLC ODS 50  4.6 mm Column

temperature 508C

1 mM sodium dodecyl sulfate and 0.02 M

H 3 PO 4 / MeOH, 7:3 (pH 2.4)

Nicotinic acid, nicotinamide (free forms)

added nicotinic acid) with MeOH

Cleanup: alumina N phase extraction cartridge, eluting nicotinamide with MeOH and nicotinamide with 0.1 M NaHCO 3

solid-buffer (pH 5), 2:10

containing 0.1 M sodium acetate and

10 mM tetrabutyl ammonium hydroxide

nicotinamide (free forms in separate chromato- grams)

Fresh beef and pork,

fresh fish, fish

products (to test for

added nicotinic acid)

Homogenize sample with water, dilute to volume with water, centrifuge, filter through paper Precipitate proteins by successive addition of saturated zinc sulfate solution and 1 N NaOH Dilute to volume with water, let stand for

30 min, filter through paper, refilter (0.45 mm)

Radial-PAK mBondapak C 18

10 mm

100  8 mm

(1) 5 mM tetrabutyl- ammonium phosphate acid (PIC-A)

in MeOH / water, 1 þ 9 (2) 10 mM heptane sulfo- nic acid (PIC- B7) in water

(1) Nicotinic acid, (2) Nicotin- amide (free forms)

5 mm 250  4 mm Column

temperature 35 8C

5 mM heptane sulfonic acid adjusted to pH 3.3 with phos phoric acid / MeCN, 75:25

(1) Nicotinic acid, (2) Nicotin- amide (free forms)

Autoclave at 1218C for

15 min Cool to 08C, filter, adjust pH to 6.5– 7.5 with oxalic acid, filter Cleanup and concentration: pass

10 ml of filtrate through C 18

solid-phase extraction cartridge Discard first 6 ml

of eluate and collect next 3.5 ml Add one drop 85%

H 3 PO 4 , mix

C 18 LC-18-DB 5 mm (Supelco)

150  4.6 mm

23% MeCN, 0.10% H 3 PO 4 , 0.10% sodium dodecyl sulfate in water (final

pH 2.8)

Nicotinic acid (representing total niacin)

Cool, adjust pH to 4.0 –4.5.

Incubate with Takadiastase

at 488C for 3 h Cool, filter, dilute to volume with water

mBondapak C 18

10 mm

300  3.9 mm

0.01 M sodium acetate buffer,

pH 4.66/

MeOH (9:1) containing

Nicotinic acid (representing bioavailable niacin)

readjust pH of extract to 4.7 + 0.02 Pass an aliquot through a Dowex 1-X8 (Bio- Rad) anion exchange column Wash column bed with water, elute nicotinic acid with 0.15 M HCl.

Evaporate eluate to dryness.

Dissolve residue in MeOH/

0.1 M acetate buffer of the required pH to obtain a final

pH of 4.7–4.9

ammonium bromide (final

H 2 SO 4 at 1 bar for 1 h, cool.

Incubate with buffered Clarase at 458C for 3 h Cool, dilute to volume, filter Alkaline hydrolysis: heat sample with Ca(OH) 2 suspension at

95 –100 8C for 30 min.

Autoclave at 1 bar for

30 min Cool, dilute to volume Refrigerate overnight, centrifuge

Nicotinic acid fraction transferred from reversed-phase column (Nucleosil C 18

5 mm) to anion exchange column (Nucleosil SB

5 mm) using automatic column switching Both columns

150  4.6 mm

A 0.57% acetic acid adjusted

to pH 3.0 with NaOH to elute the nicotinic acid from the

C 18 column and to place it

on to the anion exchange column.

B Mobile phase A/MeOH, 5:95 to flush the C 18

column

Nicotinic acid (representing bioavailable niacin after acid hydrolysis and total niacin after alkaline hydrolysis)

to pH 3.0 with NaOH to elute the nicotinic acid from the ion exchange column Beef liver, fruit juice,

brewer’s yeast,

tomato, biscuits,

green peas, peanuts,

crystallized fruit,

beer, pork, veal

Acid and alkaline hydrolysis: add

30 ml 0.1 N HCl to 1–5 g ground sample, heat suspension in a water bath

at 100 8C for 1 h Cool, dilute

to volume with water, filter.

Autoclave at 120 8C in a medium of 0.8 N NaOH for

1 h Cool adjust pH to 4.5, dilute to volume with water, filter through paper then through cellulose nitrate filter (0.45 mm)

Lichrospher 100 RP 18

5 mm

250  5 mm

0.07 M KH 2 PO 4 , 0.075 M hydrogen peroxide,

5 mM copper (II) sulfate

Nicotinic acid (representing bioavailable niacin)

Fluorescence

ex 322 nm

em 380 nm, after postcolumn derivatization

(peas, spinach,

French beans), fresh

meat (beef, pork),

sweet corn, yeast,

wheat flour, wheat

germs, rice, peanuts

50 mM sodium acetate (pH 4.5) and NADase solution to ground sample, incubate at 378C for 18 h Dilute to volume with water

HDO (Interchim)

5 mm

150  4.6 mm

preceding entry

and nicotinamide (representing bioavailable niacin)

preceding entry

Fortified foods (infant

to volume with water, filter

Inertsil ODS 3 5 mm

250  4.6 mm As inpreceding

entry

As in preceding entry

As in preceding entry

In determining the naturally occurring niacin in foodstuffs, it must bedecided whether to estimate total niacin or free (available) niacin, orboth The terms “total” and “free” niacin are defined by the extractionmethods employed, as discussed in Section 17.3 Ingested NAD andNADP are readily converted to nicotinamide in the body, and so theextraction must be capable of at least hydrolyzing these compounds

to nicotinamide Enzymatic hydrolysis using an NADase yieldslower niacin values than does mildly acidic extraction (boiling 0.1 Nhydrochloric acid for 1 h) for certain foods that contain large amounts

of bound nicotinic acid [63] This suggests that the acid conditionshydrolyze some of the bound nicotinic acid, and that the enzymetreatment yields a more reliable estimate of bioavailable niacin

The free nicotinamide and nicotinic acid naturally present in freshmeat, as well as that possibly added, can be extracted quantitativelywith water or methanol and determined by HPLC with reasonable pre-cision Strong cation exchange chromatography permits the separation

of the two vitamers, together with ascorbic acid and sorbic acid, inaqueous extracts of meat [53] Other investigators have used reversed-phase ion-pair chromatography for determining the niacin vitamers.Takatsuki et al [57] found it necessary to use two different mobilephases for determining each vitamer in deproteinized aqueous extracts

of meat The detection limit for a 10-g sample was 1 mg/100 g for nicotinicacid and nicotinamide Values greater than 20 mg/100 g indicated theillegal addition of either vitamer in Japan Tsunoda et al [55] reportedthe simultaneous determination of the two vitamers in methanolicextracts of meat and fish using sodium dodecylsulfate as the ion-pairing agent Oishi et al [56] obtained nicotinic acid and nicotinamidefractions from methanolic extracts of fresh meat by selective elutionfrom an alumina solid-phase extraction cartridge, and chromatographedeach fraction separately

Tyler and Genzale [59] reported a simple, yet efficient, means of ing alkaline digests of three major food representatives (beef, semolina,and cottage cheese) After autoclaving with calcium hydroxide solution,digests were cooled in an ice bath, filtered, and then adjusted to pH6.5 –7.0 with oxalic acid to precipitate the excess calcium A 10-mlaliquot of the filtered suspension was loaded onto a C18 solid-phaseextraction cartridge, the first 6 ml of effluent was discarded, and theremaining effluent collected and acidified with phosphoric acid.Analysis of the purified extracts was performed by reversed-phase ion-pair chromatography A feature of this method is the unusually sharp nic-

purify-analyte in a large volume (200 ml) of dilute phosphoric acid, and whichled to a detection limit of ca 0.5 mg/100 g The high efficiency of thesingle cleanup step is attributable to the amphoteric nature of nicotinicotinic acid peak (Figure 21.8), which resulted from the injection of the

was

acid At pH 7, nicotinic acid is not retained on the C18cartridge, but a largenumber of pigmented compounds are retained Conversely, nicotinic acid

is strongly retained on the C18analytical column at acidic pH in the sence of an ion-pairing agent, whereas many polar interferences elute inthe dead volume Tyler and Genzale [59] compared HPLC results withresults obtained from a microbiological assay Agreement between thetwo methods was obtained for the analysis of beef, semolina andcottage cheese, but certain food samples (e.g., instant coffee) gavehigher niacin values using HPLC

pre-van Niekerk et al [61] did not include a separate cleanup step in theirassay procedure for the analysis of cereal products and mushroomssubjected to acid or alkaline hydrolysis They employed, instead, inlinetwo-dimensional HPLC, whereby the nicotinic acid fraction was trans-ferred from a reversed-phase column to an anion exchange column.The HPLC method gave lower values for the acid extracts than for thealkaline extracts (except for the standard), whereas results obtained

Retention time (min)

0 10

Nicotinic acid

FIGURE 21.8

after cleanup by Sep-Pak C 18 solid-phase extraction and acidification of Sep-Pak effluent.

J AOAC Int., 73, 467–469, Copyright 1990 by AOAC International With permission.) Operating parameters as in Table 21.5 [59] (Reprinted from Tyler, T.A and Genzale, J.A., Reversed-phase ion-pair HPLC with UV detection of nicotinic acid extracted from semolina

microbiologically on both acid and alkaline extracts showed no difference.This suggests that part of the nicotinic acid in the acid extract was present

in a bound form that was available to the assay organism (Lactobacillusplantarum)

For the estimation of free (available) niacin in legumes and meat [60],samples (1– 10 g, containing 10– 100 mg of niacin) were mixed with hydro-chloric acid (30 ml of 0.1 Nþ 1 ml of 6 N HCl) and autoclaved at 1218C for

15 min The acid digest was then adjusted to pH 4.0– 4.5 and incubatedwith Takadiastase to hydrolyze the starch present in the legumes Theextracts were purified by strong anion exchange chromatography, andthen analyzed by reversed-phase ion-pair HPLC Each different foodrequired a trial-and-error procedure to ascertain the column temperaturerequired for optimum separation

LaCroix and Wolf [52] presented the following method for the nation of total niacin in milk- and soy-based infant formula Samples areautoclaved at 1218C for 45 min in a medium of ca 2.5 N sulfuric acid tofree endogenous niacin from protein and to convert nicotinamide tonicotinic acid The digest is adjusted to pH 6.5 and then immediately

determi-to pH ,1.0 to precipitate the proteins The clarified solution is filteredthrough paper and the filtrate is diluted to volume with water Samplecleanup is achieved by passing an aliquot of the filtrate through astrong cation exchange solid-phase extraction cartridge The columnbed is flushed with water and the nicotinic acid is eluted with two6-ml portions of 0.25 M acetate buffer (pH 5.6) HPLC is performedusing an anion exchange polystyrene– divinylbenzene column packingand UV detection at 260 nm Results obtained from the analysis ofSRM 1846 Infant Formula were consistent with the certified niacinvalue The method has achieved AOAC recognition as Peer-VerifiedMethod (PVM) 1:2000 [65]

Most of the published HPLC methods to date have used UV absorbancedetection of nicotinamide and nicotinic acid at 261 nm (lmax) or 254 nm.Because absorbance detection is not very selective, sample extracts havehad to be purified to remove interfering UV-absorbing material Kral[66] used pulsed amperometric detection for the analysis of fruit juicessubjected to acid hydrolysis, but, despite the improved selectivity, it wasstill necessary to employ open-column cation exchange chromatography

as a cleanup step In 1991, Mawatari et al [51] described an ingeniousway of converting nicotinamide and nicotinic acid to fluorescent deriva-tives postcolumn, thereby increasing the selectivity and sensitivity ofdetection The derivatives are formed instantaneously by UV irradiation ofthe column effluent in the presence of hydrogen peroxide and copper(II)ions, and the fluorescence is monitored by an inline detector Apostcolumn pump is not required as the reagents are components of the

mobile phase The derivatization method, originally applied to the sis of human serum, has been applied to food samples subjected toacid-alkaline hydrolysis [62], acid hydrolysis [64], and enzymatic(NADase) hydrolysis [63] Typical chromatograms obtained after acid

analy-and enzymatic hydrolysis are shown in Figure 21.9 analy-and

tion over UV absorbance detection eliminated the need to purify thehydrolysates The detection limit for a 5-g food sample was 20 mg/100 g[62] compared to 500 mg/100 g by HPLC -UV [61] Rose-Sallin et al [64]checked the accuracy of their HPLC-fluorimetric method by analyzingtwo standard reference materials, SRM 1846 Infant Formula and afortified cereal, VMA 195 Results were in good accordance with the certi-fied values

acid hydrolysis of a cereal-based reference material Operating parameters as in Table 21.5

Figure 21.10, respectively The increased selectivity of fluorescence hydrolysis

detec-21.2.4 Vitamin B6

21.2.4.1 General Considerations

HPLC has the ability to isolate pyridoxine (PN), pyridoxal (PL), amine (PM), and their 50-phosphate esters (PNP, PLP, and PMP), and alsoglycosylated pyridoxine (PN-glucoside) The proportion of PN-glucoside

pyridox-in a food sample is a determpyridox-inant of the bioavailability of vitampyridox-in B6

in that sample The analyst can obtain values for total or bioavailablevitamin B6by using the appropriate sample pretreatment One approach

is to hydrolyze the phosphate esters and PN-glucoside in one step,and measure the PN, PL, and PM that together represent total vitamin

B6 This approach is unable to determine bioavailable vitamin B6

for plant foods that contain significant amounts of b-glucoside gates However, by using selective enzymes (acid phosphatase andb-glucosidase), differential hydrolysis of the phosphate esters andPN-glucoside can be achieved Taking the difference between PN dataobtained with both enzymes (total PN) and with acid phosphatase only(free and phosphorylated PN) gives the content of PN-glucoside.Another approach is to preserve the phosphorylated and glycosylatedforms and measure all six vitamers, together with PN-glucoside

conju-Retention time (min)

0 5 10 15 20 25

1

2

FIGURE 21.10

Reversed-phase HPLC with fluorescence detection of nicotinic acid and nicotinamide after

Peaks: (1) nicotinic acid; (2) nicotinamide (Reprinted from Ndaw, S., Food Chem., 78, 129–134, 2002 With permission from Elsevier.)

Table 21.5