Development of an Analytical Methodfor the Combined Determination of Water-SolubleVitamins and Minerals Through High-Performance LiquidChromatography–Inductively Coupled Plasma AtomicEmission Spectrometry Hyphenation

E. Paredes & M. S. Prats & S. E. Maestre & J. L. Todolí

Received: 17 April 2011 /Accepted: 25 October 2011 /Published online: 19 November 2011# Springer Science+Business Media, LLC 2011

Abstract The capabilities of inductively coupled plasmaatomic emission spectrometry (ICP-AES) for the determi-nation of water-soluble vitamins after high-performanceliquid chromatography (HPLC) separation have beenevaluated for the first time in the present work. Thanks tothe multielemental capability of ICP-AES, it has beenpossible to develop a method for the joint determination ofseveral water-soluble vitamins and minerals. The vitaminchromatograms were obtained by plotting the carboncorrected emission intensity against time. Meanwhile,minerals were determined through the measurement of theemission intensity at their characteristic wavelengths. Theestablished method was applied to the determination ofthiamine, riboflavin, pantothenic acid, nicotinamide, ascor-bic acid, Cr, Mo, Se, Mn, Zn, Fe, Mg, Ca, and K inmultivitamin complexes. Good linearities were obtained,with correlation coefficients above 0.999 for all thevitamins and metals. The detection limits using ICP-AESfor vitamins were lower than 10 mg L−1 except for biotin(18 mg L−1) and ascorbic acid (35 mg L−1). Moreover, thelimits of detection for metals ranged from 0.3 mg L−1 for Kand 0.02 mg L−1 for Mo. Even though the ICP is lesssensitive than PDA and MS for vitamin determination, theHPLC-ICP-AES allows determination of vitamins andminerals in a period of time not much higher than that

required for the simple determination of the minerals, and itis less sensitive to interferences in trace quantities.

Keywords Water-soluble vitamins .Minerals . Dietarysupplements . Inductively coupled plasma atomicspectrometry . High-performance liquid chromatography

Introduction

The determination of organic compounds by inductivelycoupled plasma atomic emission spectrometry (ICP-AES)has not been extensively used. This is because of the highbackground found for carbon emission lines (Peters andJones 2003) which is detrimental in terms of limits ofdetection (LOD; typically between 1 and 10 mg/L). In spiteof this drawback, several organic compounds are present infoods at concentration levels high enough for theirdetermination by high-performance liquid chromatography(HPLC) coupled to ICP-AES without the need forpreconcentration techniques, among others, amino acids,(Yoshida et al. 1983; Peters et al. 2003, 2004) carbohy-drates, (Jinno et al. 1984; Peters et al. 2001; Paredes et al.2006) alcohols (Jinno et al. 1985; Paredes et al. 2006), andorganic acids (Paredes et al. 2006). From the conclusionsdrawn in these studies, several advantages of the HPLC–ICP-AES hyphenation can be mentioned: (1) similarsensitivities for nonvolatile compounds are obtained whenplotting peak area against carbon concentration; therefore,the calibration line obtained for a given compound can beused for the determination of other analytes (Peters et al.2001, 2003); (2) thanks to this similar sensitivity, acalibration line can be obtained by means of the injection

E. Paredes :M. S. Prats (*) : S. E. Maestre : J. L. TodolíDepartment of Analytical Chemistry,Nutrition and Food Sciences, University of Alicante,P.O. Box 99, Alicante 03080, Spaine-mail: maria.prats@ua.es

J. L. Todolíe-mail: jose.todoli@ua.es

Food Anal. Methods (2012) 5:897–908DOI 10.1007/s12161-011-9327-9

of a single standard containing increasing concentrations ofnonvolatile organic compounds (Paredes et al. 2008), thusshortening the time required for the calibration step; (3)unlike ultraviolet (UV) detectors, the ICP-AES is notsensitive to the presence of interferences at low concentra-tion levels (Peters et al. 2001); and (4) the HPLC–ICP-AEScoupling allows the combined determination of organiccompounds and metals (Paredes et al. 2006).

The determination of water-soluble vitamins in dietarysupplements is usually carried out by high-performanceliquid chromatography using UV (Klejdus et al. 2004; Liand Chen 2001; Almagro et al. 2002; Monferrer-Pons et al.2003; Li 2002; Markopoulou et al. 2002; Wongyai 2000;Chatzimichalakis et al. 2004; Höller et al. 2003), massspectrometry (Chen et al. 2006; Luo et al. 2006),evaporative light scattering (ELS; Spacil et al. 2007), orelectrochemical (Marszall et al. 2005) detection. Some ofthe methods described in the literature have been usedto determine a few number of water-soluble vitamins(Markopoulou et al. 2002; Wongyai 2000; Marszall et al.2005). Meanwhile, other methods are able to provide dataabout the content of a higher number (i.e., >7) of theseanalytes. They are based on gradient elution and detectionby UV (Heudi et al. 2005) or photodiode array (PDA;Chatzimichalakis et al. 2004). As regards the determina-tion of minerals in these products, the current analyticalmethods include complexometric titrations as well asatomic absorption spectrometry (Spacil et al. 2007). Themain disadvantage of these methods is that they are time-consuming and/or the fact that they provide very high limitsof detection and logarithmic calibration curves have to beused. For this reason, other approaches have been proposedfor monitoring the mineral composition of this kind ofsamples. Krampitz and Barnes (1998) determined Ca, Mg,Zn, Mn, and Cr in multivitamin samples by ICP-AES.Accurate results were obtained when a microwave digestionprocedure was used as the sample preparation method.

To the best of our knowledge, only one study has beenpublished in which one water-soluble vitamin and severalminerals have been determined (Spacil et al. 2007) simulta-neously. In that report, K, Ca, Al, and Mg were determinedtogether with ascorbic acid and aspartate by HPLC using anELS detector. For this detector, minerals and vitamins neededto be separated inside the column prior to their detection.Therefore, the selection of the appropriate separation con-ditions was difficult as a result of the very different chemicalproperties of these species. The limited sensitivity for mineralsand the nonlinear response were other drawbacks associatedto the use of this detector. Some of these problems can besolved using an ICP-AES detector provided that the separa-tion of the minerals is not necessary and the limits of detectionfor these analytes are much lower than those achieved by ELS.There is only one precedent where HPLC–ICP-AES coupling

was used to determine the relative proportions of Co, P, and Cin vitamin B12 (Morita et al. 1980).

In the present work, the capability of the HPLC–ICP-AES hyphenation for the combined determination of fivewater-soluble vitamins and nine minerals has been evalu-ated for the first time. The optimized chromatographicmethod has then been applied for the determination of theseanalytes in commercial dietary supplements.

Material and Methods

Instrumentation and Operating Conditions

The HPLC system consisted of a 150×4.6 mm ZirChrom®-SAX column with 5 μm particle diameter and a 10-mm-length,4-mm i.d. guard column (ZirChromSeparations, Anoka, USA).The column temperature was set at 50 °C bymeans of a Gecko-2000 HPLC column oven (CIL Cluzeau, Sainte-Foy-LA-Grande, France). Mobile phase selected was a 20-mmol L−1

ammonium dihydrogen phosphate solution at a flow rate of1.0 mL min−1 by means of a HPLC pump model PU-2085(Jasco, Tokyo, Japan). Samples and standards containing thewater-soluble vitamins were injected by means of an injectionvalve model 7725i (Rheodyne, Rohnert Park, USA) with a20-μL loop. The exit of the column was connected to aWaters 996 PDA (Waters, Milford, USA) via a 90-cm-length,0.254-mm i.d. stainless steel tubing. When HPLC–ICP-AEScoupling was used, this tubing was connected to an identicalsecond injection valve. Using this second valve, the signalcorresponding to the elements (i.e., Cr, Mo, Se, Mn, Zn, Fe,Mg, Ca, K) was obtained after the injection of the vitamincontaining solutions in the column and before the first vitaminleft the column (Paredes et al. 2006). This second injectionvalve was connected to the sample introduction system of thespectrometer (Optima 4300 DV Perkin-Elmer ICP-AESsystem, Überlingen, Germany) via a 20-cm-length, 0.6-mmi.d. stainless steel tubing coupled to an 8-cm-length, 0.3-mm i.d. poly(tetrafluoroethylene) tubing.

A high-efficiency nebulizer (HEN, Meinhard Glass Prod-ucts, Santa Ana, USA) was adapted to a 20-cm3 inner volumecyclonic spray chamber (Glass Expansion, Melbourne, Aus-tralia). The optimum nebulizer gas flow rate in terms of signalto noise ratio was 0.6 Lmin−1. An ultrasonic nebulizer (U-5000AT+, CETAC Technologies, Omaha, USA) was alsotested. For this nebulizer, the optimum argon flow rate was0.7 Lmin−1. Heating and cooling temperatures of the nebulizerdesolvation system were set at 140 °C and 3 °C, respectively.

The chromatograms for water-soluble vitamins usingICP-AES were obtained by plotting the carbon emissionsignal at 193.03 nm versus elution time. Signals were takenaxially because of the better sensitivity of this plasmaobservation mode. The sampling time was set at 1.2 s so

898 Food Anal. Methods (2012) 5:897–908

that a point was acquired every 1.6 s and, depending on thepeak width, 10–45 points per peak were measured. Table 1shows the experimental conditions employed in thedetection step and emission lines used for the detection.

Reagents, Sample, and Solutions

Nicotinamide, thiamine hydrochloride, riboflavin, pyri-doxine hydrochloride, D-pantothenic acid hemicalciumsalt, and biotin were purchased from Sigma-AldrichChemie (Steinheim, Germany), L(+)-ascorbic acid, ammo-nium molybdate 4-hydrate and methanol of HPLC gradefrom Panreac (Barcelona, Spain), ammonium dihydrogenphosphate, nitric acid (65% w/w), and phosphoric acid ofHPLC grade (85% w/w) from Merck (Darmstadt, Germany),and hydrochloric acid (37% w/w) from Riedel-de Haën(Seelze, Germany).

A stock standard solution of water-soluble vitamins wasprepared by dissolving an appropriate mass of the com-pounds in the mobile phase. This solution contained100 mg L−1 of thiamine, 150 mg L−1 of pyridoxine,1,000 mg L−1 of nicotinamide, 80 mg L−1 of riboflavin,300 mg L−1 of D-pantothenic acid, and 4,000 mg L−1 of L

(+)-ascorbic acid. The rest of the standards were preparedby a proper dilution of this solution in the mobile phase.

As regards the determination of minerals, six standardswere prepared containing the different elements at concen-tration levels ranging from 0.1 to 10 mg L−1. Thesesolutions were prepared from a 1,000-mg L−1 stock multi-elemental solution (Merck IV), a 1,000-mg L−1 Se solution(Merck), and a 1,000-mg L−1 Mo stock solution preparedfrom the corresponding molybdate. The acid concentrationof the standard solutions was matched to that of the sample(see “Reagent, Sample, and Solutions” section). All thestandards were finally spiked with a solution containing Y,

Eu, and Sc used as internal standards. Two commercialdietary complements were analyzed: sample 1 from WyethPharma, S.A. (Madrid, Spain) and sample 2 from Sanofi-Aventis (Barcelona, Spain).

Sample Treatment

For the determination of the water-soluble vitamins,methanol was used as extractant. Firstly, for a givensample, seven tablets were mortar powered and mixed.Then, 0.5 to 1.0 g of the power was exactly weighted and10 mL of extractant was added. The mixture was shaken for10 min using a Promax 1020 agitator (Heidolph Instru-ments, Kelheim, Germany) at room temperature. Thismixture was centrifuged for 10 min at 2,000 rpm using aDigicen centrifuge (Orto Alresa, Ajalvir, Spain), and liquidand solid fractions were separated. The solid residue wasreextracted with 5 mL of extractant and shaken for fivemore minutes. The mixture was then centrifuged for 5 minat 2,000 rpm and liquid and solid fractions were separatedagain. The washing procedure was repeated two moretimes. The different liquid fractions were mixed. Then,methanol was vacuum evaporated at 65 °C by means of arotavapor R-114 (Buchi, Flawil, Switzerland). This solventcould not be introduced into the HPLC–ICP-AES systembecause it interfered with the analytes. The solid residuesobtained were dried under a nitrogen flux.

After that, the residue was dissolved in 20 mmol L−1

ammonium dihydrogen phosphate solution and led up to25 mL with this solution. The extracts were kept at −20 °Cin the freezer until their analysis. Prior to the injection inthe HPLC system, the extracts were filtered through 0.45-μm pore-size nylon filters (Albet, Barcelona, Spain).

Additionally, for minerals determination, two differentsample treatment procedures were tested. In the first one,powder samples were microwave digested using a MARS 5microwave oven (CEM Corporation 2006; CEM Corpora-tion, Matthews, USA). A mass of 0.5 g of sample powderwas introduced inside a PTFE reactor. Then, 3 mL of HCland 8 mL of HNO3 were added. The digestion wasperformed according to the manufacturer user’s manual, a450 W microwave power ramp of 15 min and then 10 minat 200 °C. In the second procedure, the sample was shakenmanually for 10 min with the same acid mixture. Thesolution was centrifuged and the solid residue was washedthree times with 4 mL of water. For both methods, sampleswere placed inside a volumetric flask and milli-Q water wasadded up to 25 ml. These solutions were employed for thedetermination of the minor elements. The rest of theanalytes were determined using 1:100 and 1:1,000 dilutionsof the solutions in milli-Q water. All the samples andstandard solutions were spiked with the same concentrationof internal standards. In the case of the samples, the internal

Table 1 Instrumental conditions for the ICP-AES system andemission lines employed

Radio frequency power (kW) 1.35

Argon outer gas flow rate (L min−1) 15

Argon intermediate gas flow rate (L min−1) 0.2

Emission lines C 193.030 nm

Ca 317.933 nm

Cr 267.716 nm

Fe 238.294 nm

K 766.490 nm

Mg 285.213 nm

Mn 257.610 nm

Mo 202.030 nm

P 178.221 nm

Se 196.026 nm

Zn 206.200 nm

Food Anal. Methods (2012) 5:897–908 899

standard was added before the extraction procedure.Finally, the solutions were filtered through 0.45-μm pore-size nitrocellulose filters (Millipore, Billerica, USA) andkept in the refrigerator until their analysis.

Recovery Study

Sample 1 power was spiked with a known amount ofthiamine, nicotinamide, riboflavin, pantothenic acid, pyri-doxine, biotin, and ascorbic acid for the vitamins recoveryand with a know amount of Ca, Mg, Cr, Mo, Se, Mn, Zn,Mg, Fe, and K. The spiked sample was then extracted asdescribed before in the “Reagents, Sample, and Solutions”section. Recovery percentage (R%) was calculated accord-ing to the following equation R%=[(ms−mo)×100/ma],where ms is the analyte mass determined in the spikedsample, mo is the analyte mass found in the sample, and ma

is the analyte mass added to the spiked sample. Allrecovery assays were done in triplicate.

Results and Discussion

Selection of the Mobile Phase

Organic solvents cannot be used as mobile phases when theICP-AES is employed for the detection of organic com-pounds because the baseline signal would be too high, thusdegrading the detection limits. Therefore, these solventswere not considered as possible mobile phase constituents.Solutions containing 1, 0.1, and 0.01 mmol L−1 phosphoricacid concentrations were instead tested. With these mobilephases, the retention times for pantothenic acid, biotin, andascorbic acid were higher than 20 min. Therefore, the useof phosphoric acid mobile phases was ruled out. The rest of

the mobile phases tested consisted of ammonium dihydro-gen phosphate aqueous solutions at different concentrationlevels (i.e., 10, 20, 30, 40, and 50 mmol L−1). Whenretention time was plotted against the concentration ofammonium dihydrogen phosphate in the mobile phase, twodifferent behaviors were observed depending on the water-soluble vitamin considered. The retention time for thiamine,pyridoxine, nicotinamide, and riboflavin remained unal-tered (Fig. 1), while the retention times for ascorbic acid,pantothenic acid, and biotin decreased as the concentrationof ammonium dihydrogen phosphate increased. Thesedifferent trends could be explained by taking into accountthe chemical properties of vitamins and the separationmechanism in the column. For the column employed, themechanisms responsible for the separation of the analyteswere anionic exchange, hydrophobic and Lewis acid–baseinteractions. Biotin and pantothenic acid have carboxylategroups. On the other hand, ascorbic acid has acidichydrogen. For this reason, these vitamins were presentpartially in anionic form in the solution, and the mainmechanism responsible for their retention seemed to beanionic exchange. Dihydrogen phosphate present in themobile phase competed with these vitamins for the anionicexchange sites of the stationary phase. Therefore, anincrease in its concentration caused a drop in the retentiontime of these vitamins. As regards the rest of the vitamins,the mechanisms responsible for their retention werehydrophobic and Lewis acid–base interactions since thesevitamins were not present in anionic form. Therefore, theirretention times did not change with mobile phase compo-sition. Finally, the 20-mmol L−1 ammonium dihydrogenphosphate solution was the selected mobile phase because itrepresented a compromise between reduction in the analysistime and ICP-AES background signal stability as it will bediscussed in the following sections.

Fig. 1 Effect of ammoniumdihydrogen phosphateconcentration in the mobilephase on the retention times ofdifferent water-soluble vitamins

900 Food Anal. Methods (2012) 5:897–908

Detection of Water-Soluble Vitamins by ICP-AES

ICP-AES experimental conditions were selected in order toachieve suitable detection conditions for the determinationof vitamins. In a previous work, three different sampleintroduction systems were compared, and the combinationbetween a HEN and a Cinnabar spray chamber was selectedfor the determination of different organic compounds(Paredes et al. 2006). This sample introduction system hasbeen compared against an ultrasonic nebulizer equippedwith a desolvation system. The last one is usually employedto improve the sensitivity and limits of detection for metals(Goulden and Anthony 1984). However, its use for thedetermination of carbon-containing compounds throughHPLC–ICP-AES has not been evaluated. Figure 2 showsthe chromatograms obtained with both sample introductionsystems. Three main differences were observed: (1) the

ultrasonic nebulizer provided lower sensitivities, (2) base-line intensity was higher for the ultrasonic nebulizer. Thiscould be attributed to the release of carbon dioxide presentin the mobile phase caused by the heating in thedesolvation system, thus enhancing its transport to theplasma, and (3) the resolution was degraded when theultrasonic nebulizer was used. This decrease in resolutionwas due to the desolvation system and also to the 85-cmtube which connects the exit of the ultrasonic nebulizer tothe anchor of the ICP. Therefore, a significant peakbroadening was observed for the former and also higherretention times (Fig. 2b). Therefore, the HEN–Cinnabarcombination was selected as the sample introductionsystem.

Besides the effect of the sample introduction system intothe ICP-AES, the influence of mobile phase composition onthe chromatograms’ characteristics was studied. Figure 3

Fig. 2 Chromatogramsobtained (absolute retentiontime vs. signal) for a solutioncontaining a set of water-solublevitamins using as the sampleintroduction system: aHEN–Cinnabar combination, bultrasonic nebulizer. 1 thiamine,2 pyridoxine, 3 nicotinamide,4 riboflavin, 5 pantothenic acid,6 biotin, 7 ascorbic acid

Food Anal. Methods (2012) 5:897–908 901

shows the chromatograms obtained for a solution contain-ing five water-soluble vitamins using two different mobilephases. When a 0.1-mmol L−1 phosphoric acid solution wasemployed (Fig. 3a), the pantothenic acid peak was notobserved. As has been previously indicated, the retentiontime for this compound was higher than 20 min. Anyway,phosphoric acid-based mobile phases can be useful whenpantothenic acid, biotin, and ascorbic acid are not going tobe determined. Even more, these mobile phases were

preferred against a 50-mmol L−1 ammonium dihydrogenphosphate solution provided that they gave rise to a morestable baseline (see Fig. 3b). In order to correct theinstability of the baseline, an internal standard procedurewas proposed. The phosphorous emission line at178.221 nm was proposed as internal standard for severalmain reasons: (1) this element was present in the mobilephase, (2) both P 178.221 nm and C 193.030 nm are atomicemission lines and their excitation energies are relatively

Fig. 3 Effect of mobilephase composition on thecharacteristics of thechromatograms obtained byHPLC–ICP-AES: a H3PO4

0.0001 mol L−1, b (NH4)H2PO4

50 mmol L−1, c (NH4)H2PO4

50 mmol L−1 after signalcorrection with P emission lineat 178,221 nm; 1 thiamine, 2pyridoxine, 3 nicotinamide, 4riboflavin, 5 pantothenic acid

902 Food Anal. Methods (2012) 5:897–908

close (i.e., 6.95 and 6.22 eV, respectively), and (3)sensitivities were similar. Therefore, this emission linewas expected to reflect both changes in the aerosoltransport process and plasma excitation conditions. Whena conventional internal standard procedure (i.e., by multi-plying the net signal obtained for the carbon peak at time tiby the ratio between the internal standard signals measuredat times 0 and ti, respectively) was used, the use of theinternal standard did not improve appreciably baselinestability.

For this reason, a different approach was proposed basedon the following equation:

SCð Þcorrected;ti ¼ SCð Þti � Si:s:ð Þti � Si:s:ð Þ0h i

fh i

ð1Þ

where SCð Þcorrected;ti was the corrected carbon signal at time

ti, SCð Þti was the signal measured for carbon at time ti,

Si:s:ð Þtiand (Si.s.)0 were the signals measured for the internal

standard at times ti and 0, respectively, and f was thecorrection factor that was given by:

f ¼

Ptni¼t0

ΔSC;iΔSi:s:;i

n

0BBB@

1CCCA ð2Þ

This factor was obtained by measuring the change in theemission signal with respect to the signal obtained at timet=0 for both carbon (ΔSC,i) and internal standard (ΔSi,s,,i)emission lines. The ratio between both changes wasobtained for n measurements, and the mean value (correc-tion factor) was calculated. The chromatogram regionemployed to obtain this ratio is framed in Fig. 3b. In thepresent work, 20 measurements were used to obtain thecorrection factor. If both carbon and the internal standardemission lines were time correlated, f remained nearlyconstant after 15–20 measurements (i.e., the change of fwith a new measurement was lower than 2%) and a goodcorrection was obtained. The result was an improvement inthe baseline stability (Fig. 3c).

In order to determine whether random signal changeswere only caused by the plasma source or they could alsobe attributed to other causes (i.e., column, aerosol genera-tion, and transport), the background emission signal wasmeasured at both sides of the spectral peak of carbon.Specifically, background signal was measured at 193.001and 193.057 nm. The mean background measurement wasthen used to correct for the net emission signal obtained forthe carbon peak. Two procedures were used for thesepurposes: conventional internal standard correction andcorrection using Eq. 1. The former method allowed a bettersignal correction, but worse results were obtained thanthose achieved with P 178.221 nm emission line. Therefore,

it could be concluded that the random changes in thecarbon emission intensity were partially due to othersources than the plasma itself.

Analytical Figures of Merit

Unlike other chromatographic detectors, when nonvolatileorganic compounds are determined, ICP-AES can beconsidered as a universal detector. If peak area is plottedagainst carbon concentration, the slope of the calibrationline remains the same irrespective of the analyte (Yoshida etal. 1983; Peters et al. 2001, 2003; Paredes et al. 2006).Figure 4a shows the calibration curves obtained for thewater-soluble vitamins studied. Similar sensitivities wereobtained for all the vitamins except for ascorbic acid. Inorder to find the source of this behavior, 20 μL ofindependent standards containing four of the studiedvitamins at different concentrations was injected at the exitof the column and steered towards the ICP-AES. Thevitamins considered were thiamine, pyridoxine, nicotin-amide, and ascorbic acid. When peak area was plottedagainst carbon concentration, the points fitted well to astraight line at a significant level <0.05 (Fig. 4b). Theseresults suggested that the low sensitivity obtained forascorbic acid was due to either the partial degradation ofthis compound inside the column, formation of a complex,or its greater dispersion in the column. Good linearity wasobtained for all metals with regression coefficients (r2)greater than 0.999 independently of using external calibra-tion or internal calibration.

With regard to the limits of detection, these wereincluded within the 5 to 10 mg L−1 for ICP-AES (Table 2).However, for the water-soluble vitamins that showed longerretention times, higher limits of detection were reached(i.e., 18 and 35 mg L−1 for biotin and ascorbic acid,respectively). These limits of detection are low enough forthe quantification of thiamine, pyridoxine, nicotinamide,riboflavin, pantothenic acid, and ascorbic acid in most ofthe multivitamin dietary supplements present in themarket. However, biotin was not quantified as itsconcentration was under its LOD. For biotin determina-tion, a preconcentration step would be advisable beforethe analysis. On the other hand, all metals’ LOD (Table 3)were lower than 0.3 mg L−1 when ICP-AES was used.These limits are at least three orders of magnitude lowerthan ELSD ones (Table 2).

Moreover, repeatability and intermediate precision wereestimated considering within- and between-day variationswhen ICP-AES was used as detector. The precision within aday obtained after six repetitions of the same sample waslower than 5% for all the vitamins included in the analysis,except for riboflavin and pantothenic acid which showedvalues near 7% (Table 2). Meanwhile, the precision

Food Anal. Methods (2012) 5:897–908 903

obtained for a sample analyzed during seven consecutivedays showed relative standard deviation (RSD)% lowerthan 6% in all the cases. Repeatability of metals was alsoacceptable when three digestions of a mineral complexwere analyzed (Table 3).

With regard to the accuracy, a recovery test wasperformed for the water-soluble vitamins. Samples werespiked so as to reach a vitamin mass 1.5 times thatindicated in the label. Recovery assay was done intriplicate, and the mean recovery values obtained togetherwith the RSD% were acceptable with values near 100%(Table 2) in all cases except for pyridoxine.

Concerning the evaluation of the sample treatment forminerals determination, two extraction methods wereassayed, and it was verified that for Cr, Mo, Se, Mn, Zn,Fe, and K, the mean concentrations found when micro-wave digestion was employed were not different to the

declared quantities shown in the label of the dietarysupplement with 95% of probability, while the meanvalues obtained for the same elements when the roomtemperature extraction was applied were 60%, 20%, 19%,22%, 23%, 31%, and 28% lower than the declaredquantities in the label of the sample, respectively.Moreover, the recovery obtained for the spiked sampleswhen the microwave digestion was employed was near100% in all the cases (Table 3). Therefore, the formermethod was selected to dissolve the samples (Krampitzand Barnes 1998).

Combined Determination of Water-Soluble Vitaminsand Minerals

The experimental setup used for the combined determina-tion of water-soluble vitamins and minerals was based on

Fig. 4 Calibration curves (peakarea vs. carbon concentration)obtained a for differentwater-soluble vitamins byHPLC–ICP-AES: blackdiamond thiamine, white squarepyridoxine, black trianglenicotinamide, white diamondriboflavin, white circlepantothenic acid, x biotin, blacksquare ascorbic acid; b After theinjection in the column of foursolutions of different vitamins

904 Food Anal. Methods (2012) 5:897–908

the injection of vitamin solutions in the column and theinjection of mineral solutions at the exit of the columnbetween the time elapsed from the first injection to the time

at which the first vitamin was eluted (Paredes et al. 2006).Figure 5 shows a typical chromatogram obtained for amultivitamin and mineral supplement using the ICP-AES

Table 2 Comparison of validation parameters with literature data for vitamins

Sample Sample preparation HPLCdetector

Vitamins LODa

(μg L−1)%RSDinterday

R% Reference

Pharmaceuticalpreparation,fortified powdereddrinks and foodsamples

1 g of powder samplewas dissolved in10 mL of 0.010%TFA/methanol (50:50)solution and stirredon Vortex

PDA (280 nm) (μg L−1) 2–8 Near 100 Klejdus et al. 2004Water-solublevitamins

12–800

B12 in multivitamintablets

Extraction with 50 mMsodium acetate buffer(pH 4.0), 1 mL of sodiumcyanide (1%) and 0.25 gof amylase

MS (μg L−1) 2.5 93 Luo et al. 20066–150Vitamin B12

K, Al, Ca, Mg,and vitamin C inpharmaceuticalformulations

Analytes extracted withmobile phase

ELSD (mg L−1) 1–4 95–105 Spacil et al. 2007Vitamin C 17

K 11

Mg 1.3

Group B vitaminsand vitamin C

Vitamins extracted withmethanol and redissolvedin mobile phase

ICP-AES (mg L−1)a (n=6) Present workThiamine 5.3 3 95.6±0.3

Piridoxine 5.0 4 75.4±0.5

Nicotinamide 4.8 5 100±0.4

Riboflavin 7.5 7 98.1±0.3

Pantothenic acid 9.8 7 97.6±0.5

Biotin 18 – 102.1±0.5

Vitamin C 35 1.7 96.1±0.4

Group B vitaminsand vitamin C

Vitamins extracted withmethanol and redissolvedin mobile phase

PDA (μg L−1)a (n=6) Present workThiamine 3 0.9

Piridoxine 10 –

Nicotinamide 4 1

Riboflavin 6 0.8

Pantothenic acid 150 0.8

Vitamin C 60 1.4

a LOD (calculated using the criterion of 3× standard deviation of the residuals of the calibration line)

Table 3 Limits of detection, linearity (r 2), slope and intercept of the regression equation, recovery, and RSD for the minerals analyzed

Element LODa (mg/L) r 2 (with i.s., without i.s.) Slope±ts (p<0.05) Intercept±ts (p<0.05) R% RSD (%) (n=3)

Cr 0.06 0.99990, 0.99996 (100±2)×103 (−2±9)×103 105±2 3.6

Mo 0.02 0.9998, 0.9998 (827±15)×10 (2±7)×102 101±2 3.5

Se 0.19 0.997, 0.9990 (61±4)×10 (1±2)×102 96±3 15

Mn 0.005 0.99990, 0.999990 (199±3)×103 (5±13)×103 93.6±0.5 2.7

Zn 0.3 0.998, 0.9997 (227±11)×102 (1±5)×103 98±.3 8.4

Fe 0.15 0.997, 0.9998 (52±5)×103 (0±2)×104 97.9±0.3 1.7

Mg 0.03 0.998, 0.9998 (216±15)×103 (1±3)×104 102±2 5.2

Ca 0.2 0.995, 0.998 (143±4)×103 (1±2)×104 98.1±0.4 6.5

K 0.3 0.998, 0.9990 (26±2)×104 (0±4)×104 102±2 5.3

a LOD (calculated using the criterion of 3× standard deviation of the residuals of the calibration line)

Food Anal. Methods (2012) 5:897–908 905

detector. As can be observed, minerals are detectedsimultaneously by monitoring different emission lines,whereas water-soluble vitamins were detected using thecarbon emission line at 193.03 nm. Using this method, ithas been possible to determine nine minerals (i.e., Cr, Mo,Se, Mn, Zn, Fe, Mg, Ca, and K) and five water-solublevitamins (i.e., thiamine, nicotinamide, riboflavin, panto-thenic acid, and ascorbic acid). Biotin and vitamin B12could not be quantified since their concentrations wereslightly below the limits of detection, whereas pyridoxinewas interfered by different compounds present in thesupplements and traces of methanol (see peak betweenthiamine and nicotinamide in Fig. 5). This problem couldbe solved using a preconcentration extraction method suchas SPE or SPME for vitamins, but this point was out of theaim of this manuscript.

In spite of the fact that ICP-AES has higheroperation cost than common HPLC detectors, it showsseveral advantages (Spacil et al. 2007). Firstly, the ICP-AES detector does not require prior separation ofminerals. Therefore, the selection of the separationconditions is easier than for ELS. Secondly, the limits ofdetection for the minerals are lower than those obtainedby ELS (i.e., 1–3 mg L−1; Spacil et al. 2007). In fact,some elements like Se, Cr, and Mo were present atconcentrations of 0.35 mg L−1 in the solutions analyzedand were accurately determined. Therefore, HPLC–ICP-AES allows the determination of a higher number ofminerals than ELS without the preconcentration of thesample.

Application of the Method to the Analysis of DietarySupplements

Table 4 shows the masses of two water-soluble vitaminsdetermined using ICP-AES and PDA detectors as well asthe declared content. For most of the cases, the mean valuesobtained by HPLC–ICP-AES for thiamine, riboflavin,pantothenic acid, and nicotinamide are not significantlydifferent to the values claimed by the manufacturer after theapplication of the Student’s t criteria for a sample (note thatthe manufacturers did not provide confidence intervals forthe masses of the analytes). However, the ascorbic acidmasses obtained for two of the three samples were clearlylower than the value declared. As it was shown before,nearly a total recovery was obtained for this compoundwhen a spiked sample was analyzed so the low results mustbe due to the form in which the vitamin C is found in thetablet, which maybe associated to other molecules. Thisfact confirms that an alternative extraction process forvitamins in those samples should be studied. In Table 4, themean values obtained using PDA are also shown. In mostof the cases, the values obtained when two detectors arecompared are not significantly different; however, there aresome exceptions. For example, nicotinamide was notdetected in sample 2 with the PDA detector. This resultcould be attributed to vitamin B3 which can be present inseveral chemical forms. The standard employed for thedetermination of this vitamin was nicotinamide. However,the label for sample 2 indicated that vitamin B3 was presentas niacin (probably as nicotinic acid). Therefore, the

Fig. 5 A typical chromatogram obtained for a multivitamin and mineral supplement; 1 minerals, 2 thiamine, 3 nicotinamide, 4 riboflavin, 5pantothenic acid, 6 ascorbic acid

906 Food Anal. Methods (2012) 5:897–908

inaccurate results obtained by PDA for this compoundcould be attributed to the different response of thesechemical forms at the wavelength selected. It must bepointed out that the use of a different standard would notgive rise to inaccurate results for ICP-AES detector since thepercentage of carbon would be similar for both compounds.

Table 5 shows the masses obtained for the mineralsusing external and internal calibration methods. Dataprovided by the manufacturer are included in this table aswell. Among the different internal standards tested, Sc361.384 nm provided the best results for Se, Mo, and Cr,whereas Y 360.073 nm allowed a better correction ofrandom changes of signal for the rest of the elements. Theselines have been proposed in different works as internalstandards in ICP-AES (Grotti and Frache 2003; Garden etal. 1991). In general terms, the use of an internal standardallowed the reduction of the confidence intervals, thusimproving the precision of the method. Results obtainedwere not different with 95% of probability to the masseslabeled for most of the elements. However, significantdifferences were observed in some cases. In the case of Ca,the results obtained with internal standardization deviatedmore significantly from the labeled concentrations thanthose found with external calibration. This was likely due tothe bad selection of the internal standards. For example,

previous studies have shown that neither Sc nor Y can beefficiently used as internal standards to correct for interfer-ences caused on Cu (Grotti et al. 2008). These results couldbe improved by selecting a different internal standard suchas Al. However, this was not within the scope of the presentstudy.

Conclusions

The HPLC–ICP-AES coupling is advantageous with regardto other analytical methods since it allows the determinationof both minerals and vitamins in a short period of timeusing a single analytical method. Thus, minerals and water-soluble vitamins could be determined after their extractionin a sample in less than 30 min carrying out the analysis intriplicate. This time is not much higher than that requiredfor the determination of the minerals in these samples byICP-AES in a conventional analysis. The only drawback isthat ICP-AES is less sensitive than other possible detectorsfor vitamins like PDA and MS, but LOD could beimproved if a preconcentration step, as for example solidphase microextraction, is applied to the sample preparationstep. This aspect was not the scope of this work, and it willbe developed in the future by our research group.

Table 5 Masses of minerals (milligrams) per tablet and confidence intervals (α=0.05) obtained for the dietary supplements analyzed

Sample Cr Mo Se Mn Zn Fe Mg Ca K

Sample 1 0.025 0.025 0.03 2.5 7.5 14 100 162 40

0.029±0.011 0.028±0.003 0.04±0.02 2.5±0.4 7.5±0.3 14±2 96±11 160±20a 37±4

0.025±0.006 0.027±0.003 0.03±0.02 2.13±0.02 6.8±0.8 13.5±0.4 106±4 189±12b 41±6

Sample 2 0.2 1 1.5 15 5 5

0.21±0.09 1.2±0.2 1.9±0.9 16±3 4.7±0.6 5.5±0.3

0.21±0.08 0.94±0.04 1.5±0.6 15.2±0.7 5.4±0.2 5.16±0.06

For every sample, the first row indicates the labeled mass, whereas the second and third rows indicate the masses obtained by HPLC–ICP-AESusing external and internal calibration, respectively

Table 4 Masses of water-soluble vitamins (milligrams) per tablet and confidence intervals (α=0.05) obtained for the dietary supplementsanalyzed

Sample Thiamine Riboflavin Pantothenic acid Nicotinamide Ascorbic acid

Sample 1 1.4 1.6 6 18 60

1.8±0.3 1.7±0.2 6.4±0.6 22±2 35±4

1.5±0.2 1.5±0.2 6.2±0.3 20±2 31±4

Sample 2 10 5 10 30 100

9.2±0.7 4.6±0.2a 11.4±1.0 33±3a 54±5

8.5±0.8 3.8±0.2b 11±2 ND 51±4

For every sample, the first row indicates the labeled mass, whereas the second and third rows indicate the masses obtained using ICP-AES andPDA detectors, respectively. Mean values with different lower case letters are significantly different

ND not detected

Food Anal. Methods (2012) 5:897–908 907

Acknowledgments The authors would like to thank to the SpanishEducation Ministry (Projects PETRI95-0980-OP and CTQ2009-14063) and to the Vicerrectorado de Investigación of the Universityof Alicante for the financial support. E.P. also thank the GeneralitatValenciana for the FPI grant.

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