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Available online at www.sciencedirect.com

Journal of Chromatography A, 1185 (2008) 178–184

Single-injection calibration approach for high-performanceliquid chromatography

Eduardo Paredes, Salvador E. Maestre, Ma Soledad Prats, Jose L. Todolı ∗Department of Analytical Chemistry, Nutrition and Food Sciences, University of Alicante, P.O. Box 99, 03080 Alicante, Spain

Received 31 July 2007; received in revised form 10 January 2008; accepted 14 January 2008Available online 31 January 2008

bstract

A new calibration method for high-performance liquid chromatography was validated. The method was called single-injection calibrationpproach (SICA) because it allowed to obtain a complete calibration curve by means of a single injection of a standard solution containingeveral non-volatile and semi-volatile organic compounds at different concentration levels. The compounds studied included carboxylic acids,olyalcohols, carbohydrates and water-soluble vitamins. This method allowed a 1–7-fold reduction in the analysis time with regard to conventionalalibration methods. The method was applied to three different chromatographic detection methods: refractive index (RI) detection, diode arrayetection (DAD) and inductively coupled plasma atomic emission detection (ICP-AED). Good linearity was achieved (r2 > 0.999) for the threeetection methods but signal correction was required for RI detection and DAD. This fact demonstrated that ICP-AES was the most universal

ecause the signal obtained for non-volatile and semi-volatile organic compounds was not a function of the chemical nature of the compound andnly depended on the mass content of carbon. The method was validated by analyzing a reference non-fat milk powder sample as well as severaleal food samples (three fruit juices, four wines, three candies and a multivitamin complex). 2008 Elsevier B.V. All rights reserved.

eywords: HPLC; ICP; AED; Calibration; Single mixture of standards; Determination of carbon content; Food analysis

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. Introduction

Detection in high-performance liquid chromatographyHPLC) is generally based on the use of a relative technique.herefore, in order to quantify a given analyte, a calibrationquation must be applied. According to the IUPAC recommen-ations, to validate a method the calibration should be done witht least a set of six standards by triplicate [1]. That is why calibra-ion in HPLC is time and reagents consuming especially whenmethod has to be validated [2]. In routine analysis, the cali-

ration time can be reduced if the system is stable. In this caseverification of the reliability of the calibration curve can be

one with only one standard used as quality control. The use of

nly one [3,4] or two standards to carry out the calibration pro-ess has also been proposed. However, several conditions muste fulfilled [2]. When two standards are used, the analyte con-

∗ Corresponding author. Tel.: +34 965909775; fax: +34 965903527.E-mail address: jose.todoli@ua.es (J.L. Todolı).

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021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.01.071

entration must be included in the concentration range definedy both standards. If just one standard is used its concentrationust be as close as possible to that of the sample and the blank

ignal must be zero. These conditions are difficult to satisfyhen unknown samples are analysed. Moreover, sample matrix

ffects can influence the signal finally obtained by the detector.n these cases, alternatives to the external calibration can be cho-en as internal calibration [5,6], standard addition methods [5]nd matrix-matching methods.

Other strategies proposed for the shortening of analysis timere based on complicated mathematical procedures. For exam-le, the so-called solute-independent calibration allowed theuantification of the analyte without the use of a calibrationurve [7–10]. The main drawback of this method was that twoifferent chromatographic conditions (e.g., two different mobilehases) were required. Therefore, two chromatographic systems

ere needed to shorten the analysis time. Otherwise, a long timeould be required for the stabilization of the column when theobile phase was changed. The analyte concentration (or vol-

me fraction) in the sample was calculated according to the

atogr

f

C

wca1

dcapom

peaaOrPoshtdtiEtidcd

HooaotpgstOcsc

tidua

So

2

2

iPca(r5Ut2FZrmSva9rsOG

dM2Mewpobiwpe

ing the different organic compounds studied in the present workwere carried out with an Abbe Refractometer model NAR-3T(Atago., Tokyo, Japan).

Table 1Instrumental conditions for the ICP-AED system employed

E. Paredes et al. / J. Chrom

ollowing mathematical relationship [7]:

x = �n1K′1 − �n2K

′2

F2 − F1(1)

here Cx is the analyte volume fraction in the sample, �ni is thehange in refractive index and K′

i and Fi are constants that varyccording to the refractive index of the mobile phase. Subscriptsand 2 indicate the two mobile phases employed.Another procedure was proposed by Comas et al. [11] for

etecting and correcting interferences. In this case, only twohromatographic experiments were required to carry out thenalysis. One of them was for either a pure standard or the sam-le spiked with a known concentration of the analytes. The otherne was for the unknown sample. The main drawback of thisethod was its mathematical complexity.Several works have been published where inductively cou-

led plasma atomic emission detection (ICP-AED) has beenmployed in HPLC determinations. Carbohydrates [12–14],mino acids [15–17], alcohols [14,18] as well as carboxyliccids [14] have been determined by means of this coupling.ne of the characteristics of ICP-AED is its universality with

egard to the determination of non-volatile organic compounds.eters et al. [12] plotted peak area against concentration for a setf carbohydrates (i.e., sucrose, glucose and fructose). The sen-itivity was nearly identical for each carbohydrate, since theyad similar mass content of carbon. Therefore, they claimedhat a calibration curve obtained from several injections of stan-ards containing a single compound could be used to quantifyhese carbohydrates. In another work, a set of standards contain-ng increasing concentrations of amino acids was prepared [17].very standard consisted of a solution of nine amino acids with

he same mass content of carbon. These solutions were injectedn the column and the mean of the peak areas obtained for theifferent amino acids was plotted against the mass content ofarbon. This calibration curve was successfully applied for theetermination of 19 amino acids in different solutions.

In a previous work [14], it was found that calibration inPLC–ICP-AED could be carried out through the injectionf only one standard solution. In this calibration methodol-gy known as single-injection calibration approach (SICA),standard containing a set of non-volatile and semi-volatile

rganic compounds at different concentrations was injected inhe column. Afterwards, the peak areas obtained for these com-ounds were plotted against the mass content of carbon thusiving rise to the calibration curve. This procedure presentedeveral advantages compared to other calibration methods. Onhe one hand, the calibration time was considerably reduced.n the other hand, the number of standards of the calibration

urve was only restricted by the number of non-volatile andemi-volatile organic compounds that could be separated in theolumn.

The aim of the present work was thus to adapt and evaluatehe SICA calibration method to classical detection systems used

n HPLC such as refractive index (RI) detection and diode arrayetection (DAD). Results were compared with those attainedsing ICP-AED. A further goal was to validate the SICA ascalibration method for chromatographic determinations. The

RAAA

. A 1185 (2008) 178–184 179

ICA was finally employed for the analysis of carbohydrates,rganic acids, alcohols and vitamins in foodstuffs.

. Experimental

.1. Apparatus

A 300 mm × 3 mm cation-exchange HPLC column contain-ng 8 �m diameter particles (Rezex RHM Monosaccharide;henomenex, Torrance, CA, USA) was used for the separation ofarboxylic acids, carbohydrates and alcohols. The system waslso equipped with a 4 mm length, 3 mm I.D. guard columnCarbo-H+, Phenomenex). Water-soluble vitamins were sepa-ated using a 150 mm × 4.6 mm ZirChrom-SAX column with�m diameter particles (ZirChrom Separations, Anoka, MN,SA) equipped with a 10 mm × 4 mm I.D. guard column. The

emperature of the columns was controlled by means of a Gecko-000 HPLC column oven (CIL Cluzeau, Sainte-Foy-la-Grande,rance). The temperatures were set at 50 ◦C and 80 ◦C for theirChrom-SAX and the Rezex RHM Monosaccharide columns,

espectively. Mobile phase was pumped through the system byeans of a HPLC pump model PU-2085 (Jasco, Tokyo, Japan).amples and standards were injected by means of an injectionalve (model 7725(i); Rheodyne, Rohnert Park, CA, USA) with20-�l loop. The column was connected to the detectors via a0 cm × 0.254 mm I.D. stainless steel tubing. Detection was car-ied out by means of a Waters 410 RI detector, a Waters 996 DADystem diode array detector (Waters, Milford, MA, USA) or anptima 4300 DV PerkinElmer ICP-AED system (Uberlingen,ermany).When the ICP-AED system was employed, the sample intro-

uction system consisted of a high efficiency nebulizer (HEN;einhard Glass Products, Santa Ana, CA, USA) coupled to a

0-cm3 inner volume cyclonic spray chamber (Glass Expansion,elbourne, Australia). The former transformed the solution

merging the column into an aerosol whose finest fractionas selected by the spray chamber and directed towards thelasma. The detection of the organic compounds was carriedut by means of the measurement of the emission line of car-on at 193.09 nm. Signals were taken axially, because of thencreased sensitivity. The sampling time was set at 1 s so a pointas acquired every 1.6 s. A total number of 15–20 points pereak were measured. Table 1 shows the experimental conditionsmployed in the detection step.

The measurements of refractive indices of solutions contain-

adio frequency power (kW) 1.35rgon outer gas flow rate (l min−1) 15rgon intermediate gas flow rate (l min−1) 0.2rgon nebulizer gas flow rate (l min−1) 0.6

1 matog

2

f(aaG(Fg

aa((os

2

NwinS

(wwJitopsfi

2

2

ptthvCD

2

oa9o

Af1oasraaScdwo

2s1p

2

wlcIpsOqt

3

3q

tgbrT8F3rihtncw

80 E. Paredes et al. / J. Chro

.2. Reagents and solutions

All reagents were of analytical grade. d(+)-Glucose, d(−)-ructose and succinic acid were purchased from FluckaBuchs, Switzerland), d-sorbitol, nicotinamide, riboflavin, thi-mine hydrochloride, d-pantothenic acid hemicalcium saltnd pyridoxine hydrochloride from Sigma–Aldrich (Steinheim,ermany), dl-malic acid and ethylene glycol from Panreac

Barcelona, Spain), citric acid from Prolabo (Val de Fontenay,rance), l(+)-tartaric acid from Carlo Erba (Milan, Italy) andlycerol from Scharlau (Barcelona, Spain).

Mobile phase employed for the separation of carboxyliccids, carbohydrates and alcohols consisted of a 0.001 M nitriccid solution prepared from a 65% (w/w) nitric acid solutionSuprapure; Merck, Darmstadt, Germany) and Milli-Q water<18 M�). The separation of water-soluble vitamins was carriedut using a 50-mM ammonium dihydrogenphosphate (Merck)olution in Milli-Q water.

.3. Sample treatment

A non-fat milk powder standard reference material (USational Institute of Standards and Technology (NIST) 1549)as analysed. Twenty milligrams of this sample were dissolved

n 100 ml of water. This solution was finally filtered through aylon syringe filter with 0.45 �m pore size (Filalbet, Barcelona,pain).

Several food samples purchased from local supermarketsi.e., fruit juices, wines, candies and a multivitamin complex)ere analysed. Liquid samples were 10-fold or 20-fold dilutedhile candies were dissolved in water (1 g of sample in 100 ml).

uice samples and those candies containing sucrose were acid-fied by adding 0.2 ml of a 65% nitric acid solution in order tootally hydrolyze sucrose [19]. For these samples, the contentf glucose and fructose was the sum of that present in the sam-le and that generated from sucrose hydrolysis. The resultingolutions were filtered through 0.45 �m pore size nylon syringelters.

.4. Procedure

.4.1. Conventional calibration methodSix or seven standard solutions containing the different com-

ounds were prepared by dissolving the appropriate amount ofhe component in water. The concentrations ranged from 10o 1500 mg l−1 for carboxylic acids, carbohydrates and alco-ols solutions or from 0.9 to 80 mg l−1 for vitamins. In order toalidate the method, these solutions were injected three times.olumn effluents were monitored by ICP-AED, RI detection andAD and a calibration curve was obtained for every compound.

.4.2. SICA methodA solution containing 9 mg l−1 of succinic acid, 15 mg l−1

f glycerol, 30 mg l−1 of citric acid, 36 mg l−1 of tartariccid, 45 mg l−1 of ethylene glycol, 600 mg l−1 of malic acid,00 mg l−1 of sorbitol, 1200 mg l−1 of glucose and 1500 mg l−1

f fructose was prepared for SICA experiments with ICP-

r

Ir

r. A 1185 (2008) 178–184

ED and RI detection. Other two solutions were preparedor SICA experiments with DAD. The first one contained0 mg l−1 of citric acid, 50 mg l−1 of tartaric acid, 150 mg l−1

f malic acid, 500 mg l−1 of fructose, 1000 mg l−1 of sorbitolnd 1500 mg l−1 of succinic acid. The second one consisted of aolution containing 0.9 mg l−1 of pantothenic acid, 5 mg l−1 ofiboflavin, 24.7 mg l−1 of pyridoxine, 44.6 mg l−1 of thiaminend 80 mg l−1 of nicotinamide. These solutions were injectednd column effluents were monitored with the three detectors.ubsequently, peak areas were calculated and plotted against theompound concentration for the refractive index and diode arrayetectors or against the mass content of carbon when ICP-AEDas employed. The data obtained corresponded to the averagef five replicates.

For DAD, the detection was carried out at 236 nm for fructose,47.2 nm for citric acid, 244 nm for malic acid, 270.5 nm fororbitol, 246 nm for tartaric acid, 238.9 nm for succinic acid,92 nm for pantothenic acid, 283.5 nm for riboflavin, 284 nm foryridoxine, 233 nm for thiamine and 228.8 nm for nicotinamide.

.4.3. Lower limit of determinationA solution containing 20 mg l−1 of the compounds studied

as diluted and analysed several times in order to estimate theowest concentration of a compound which provided a peak thatould be discriminated from the base line for RI detection andCP-AED systems. According to the criterion selected in theresent work, a peak could be detected when 10 consecutiveignal measurements were higher than the background average.n the other hand, limits of detection (LODs) and limits ofuantification (LOQs) were calculated using the 3 and 10 signal-o-noise ratio criteria, respectively.

. Results and discussion

.1. Evaluation of the HPLC–ICP-AED coupling foruantitative analysis

When the refractive index detector was used, depending onhe compound the peaks were distinguishable from the back-round (10 consecutive signal measurements were above theackground average) at concentrations from 2 to 5 mg l−1. Theseesults did not agree with LODs and LOQs calculated above.hus, for glycerol and ethylene glycol the LODs were 5 andmg l−1 whereas the LOQs were 16 and 28 mg l−1, respectively.or the rest of compounds, the LODs were included within the–4 mg l−1 range whereas the LOQs were close to 10 mg l−1. Asegards the ICP-AED system, the minimum concentration to bencluded in the calibration graph for the studied compounds wasigher than that for the refractive index one. Thus, it was foundhat the peaks were clearly distinguished from the backgroundoise for concentrations from 5 to 20 mg l−1, depending on theompound. LODs obtained in ICP-AED for all the compoundsere included within the 20–30 mg l−1 range while the LOQs

anged from 70 to 100 mg l−1.Fig. 1 shows a typical chromatogram obtained with either

CP-AED (Fig. 1a) or RI detection (Fig. 1b). The former cor-esponded to a solution of several organic compounds with the

E. Paredes et al. / J. Chromatogr. A 1185 (2008) 178–184 181

Fig. 1. Chromatograms obtained for a solution containing a set of non-volatileand semi-volatile organic compounds: (a) 200 mg of C l−1 for every compoundats

sfcmo(ocTthgmpAiwwRci

3

s

Fig. 2. Calibration curves obtained by (a) RI detection using the conventionalmethod; (b) RI detection with refractive index correction using the conventionalmethod; (c) DAD using the SICA method. (�) Citric acid; (�) tartaric acid;(g

Topamdtta

nd ICP-AED; (b) 500 mg of compound l−1 for every compound and RI detec-ion. 1: citric acid; 2: tartaric acid; 3: malic acid; 4: glucose; 5: fructose; 6:orbitol; 7: succinic acid; 8: glycerol; 9: ethylene glycol.

ame mass content of carbon whereas the latter was obtainedor a solution containing the same concentration in milligram ofompound per liter. Note that ICP-AED is an elemental detectionethod whereas the analytical signal for RI detection depends

n the refractive index of the analyte solution. For ICP-AEDFig. 1a) the maximum difference between the peak heightsbtained for the different compounds was 38%. However, in thease of RI detection this difference was close to 56% (Fig. 1b).herefore, ICP-AED seemed to be more universal than RI detec-

ion. For DAD, a chromatogram is not presented because of theigh dependence of the analyte response with the wavelengthiving rise to higher differences than for the other detectionethods. In fact, within the concentration range studied in the

resent work only six of the compounds tested could be detected.n interesting feature of the chromatograms showed in Fig. 1

s that the overlapping observed between two consecutive peaksas similar for both detectors. In fact, the mean baseline peakidths for the compounds in Fig. 1 were 24.1 and 24.4 s forI detection and ICP-AED, respectively. Therefore, it was con-luded that the dispersion of the analytes in the ICP-AED samplentroduction system was negligible.

.2. Conventional calibration

In order to apply the SICA all the compounds present in thetandard as well as the analytes must show the same sensitivity.

dpti

�) malic acid; (�) glucose; (�) fructose; (©) sorbitol; (+) succinic acid; ( )lycerol; (�) ethylene glycol.

he calibration curves for a set of non-volatile and semi-volatilerganic compounds were obtained. For ICP-AED, the nine com-ounds evaluated showed nearly the same sensitivity when peakrea was plotted against the mass content of carbon in agree-ent with the results previously reported [12,17]. Note that the

ifference in peak area was lower than in peak height becausehose compounds that provided the lowest peak heights showedhe widest peaks. Thus the maximum difference in terms of peakrea was close to 15%.

On the contrary, when RI detection was employed (Fig. 2a),ifferences in sensitivity as high as 40% were observed. This

roblem could be solved considering the refractive indices ofhe compounds. Thus, refractive indices of solutions contain-ng the same concentration for the different compounds studied

1 matog

wf

(

wi(ra

cct

flstwtsmnc

3

ttwIc(t(tlwmfaHcwuu

tdpcbA

t

C

wp

hrluHtcappstIaomiti

3

opMbTbvsbwrcmowFdcco

3

82 E. Paredes et al. / J. Chro

ere measured. Finally, corrected peak areas were calculated asollows:

Acorrected)i = (Ameasured)i(�RI)reference

(�RI)i(2)

here (Acorrected)i is the corrected peak area for compound, (Ameasured)i is the measured peak area for compound i and�RI)reference and (�RI)i are the changes in refractive index withespect to water for a solution containing the reference (glucose)nd compound i, respectively.

Fig. 2b shows the corrected calibration curves for RI. Asan be seen, the dispersion was lower when the correction wasarried out, being the maximum difference in sensitivity closeo 10%.

As regards DAD, sensitivities not statistically different wereound for the compounds studied by selecting different wave-engths depending on the compound. To achieve this, theensitivity of the six compounds detected (i.e., citric acid, tar-aric acid, malic acid, fructose, sorbitol and succinic acid)as registered through a 210–300 nm wavelength range. Then

he compound showing the lowest maximum sensitivity waselected as reference. Finally, the rest of the compounds wereeasured at wavelengths that provided sensitivities that were

ot significantly different from that obtained for the referenceompound at a 95% confidence level.

.3. SICA method

From the results previously obtained it could be concludedhat a single-injection was appropriated to carry out the calibra-ion procedure for these detectors. Calibration graphs obtainedith SICA were plotted for the different detection methods. For

CP-AED, a good correlation between peak area and the massontent of carbon was obtained irrespective of the compoundr2 = 0.9999). If the peak areas were corrected, for the refrac-ive index detector a well-fitted straight line could be drawnr2 = 0.9997) as well. A similar conclusion could be drawn fromhe plot obtained with DAD (r2 = 0.9994) when suitable wave-engths were selected. An example of the well fitting obtainedith the SICA method is shown in Fig. 2c for this detectionethod. The SICA method was also satisfactorily employed

or the quantification of compounds for which DAD is usu-lly employed such as water-soluble vitamins (r2 = 0.9992).owever, it must be considered that in some instances SICA

ould not be employed with this detection method (e.g.hen the analytes do not show significant absorbance val-es in the whole UV–vis range covered by the instrumentsed).

ICP-AED showed some advantages with respect to RI detec-ion and DAD. First, when using ICP-AED the signal of theetector could be used directly to obtain the amount of analyte

resent in a sample using the calibration curve. In this case, thealibration equation allowed to obtain the mass content of car-on (Ccarbon) from peak area measured for a given compound.nalyte concentration (Canalyte) was then calculated according

mbT

r. A 1185 (2008) 178–184

o the following equation:

analyte = CcarbonManalyte

Mcarbon(3)

here Manalyte and Mcarbon are the masses of analyte and carboner mole of analyte, respectively.

However, for the other two detection methods additional stepsad to be performed previously to the quantification, as theefractive index correction or the selection of the optimal wave-ength for each compound. This fact indicates that the settingp of the SICA method was time consuming mainly for DAD.owever, once the appropriate corrections had been performed,

he analysis time was shortened. Hence, the calibration processould be carried out daily in a short period of time (i.e. 17 minpproximately). Note that with the compounds studied in theresent work, the time required to carry out a full calibrationrocedure (i.e., taking the signals by triplicate and using seventandards) would be about 50 and 350 min for the SICA andhe conventional method, respectively. The second advantage ofCP-AED was that it allowed obtaining quantitative informationbout unknown organic compounds present in the sample. Nonef the detectors employed in the present work allowed to deter-ine the concentration of a given compound in the sample unless

t had been previously identified. However, ICP-AED allowedo calculate the mass content of carbon for any compound evenf its identity was unknown.

.4. Comparison of calibration methods

Table 2 shows the parameters of the calibration equationsbtained by conventional calibration for the different com-ounds studied with the three detection methods employed.oreover, the parameters of the calibration equations obtained

y the SICA method are presented. As can be observed inable 2, for ICP-AED there were no significant differencesetween the values of the slope and intercept obtained with con-entional and SICA methods. As regards RI detection, Table 2ummarizes the parameters of the calibration equations obtainedy taking into account the refractive index correction (glucoseas selected as the reference compound, see Section 3.2). The

esults shown in Table 2 for DAD indicate that no signifi-ant differences were obtained between conventional and SICAethods when suitable wavelengths were selected. Finally, in

rder to assess the linearity of the calibration graphs obtainedith the SICA method, an F-test was carried out. The calculatedvalues were 0.20, 0.78 and 2.68 for ICP-AED, DAD and RI

etection, respectively. Tabulated value was 2.57. Only in thease of RI detection a lack of fit was obtained probably as aonsequence of the low RSD values found in the measurementf peak areas (i.e., lower than 2%).

.5. Analysis of real samples

In order to validate the developed method, a certified non-fatilk powder sample was analysed. The analysis was carried out

y means of the SICA method using ICP-AED and RI detection.he certified content of lactose in the sample was 49 ± 3% and

E. Paredes et al. / J. Chromatogr. A 1185 (2008) 178–184 183

Tabl

e2

Para

met

ers

(b=

slop

e;a

=in

terc

ept)

ofth

eca

libra

tion

equa

tions

obta

ined

for

seve

ralo

rgan

icco

mpo

unds

with

both

conv

entio

nala

ndSI

CA

met

hods

usin

gIC

P-A

ED

,RI

dete

ctio

nan

dD

AD

Met

hod

Com

poun

dIC

P-A

ED

RI

DA

D

b±

tsb

(cou

nts

lmg−

1

ofca

rbon

)a

±ts

a(c

ount

s)b

±ts

bco

rrec

ted

(�V

slm

g−1

ofco

mpo

und)

a±

tsa

corr

ecte

d(�

Vs)

b±

tsb

(�V

slm

g−1

ofco

mpo

und)

a±

tsa

(�V

s)

SIC

A61

4±

5(6

7±

4)×

1021

1±

3(1

13±

7)×

1043

.7±

1.5

(8±

7)×

102

Citr

icac

id62

5±

12(2

±3)

×10

324

2±

3(−

18±

5)×

102

44±

4(8

±7)

×10

2

Tart

aric

acid

(60

±7)

×10

(2±

11)×

103

226

±3

(19

±5)

×10

244

±2

(2±

2)×

103

Mal

icac

id(5

9±

4)×

10(3

±7)

×10

321

8±

5(8

±10

)×10

242

.5±

1.5

(12

±12

)×10

2

Con

vent

iona

lG

luco

se(6

0±

3)×

10(5

±7)

×10

322

2±

8(−

1±

2)×

103

Fruc

tose

(63

±2)

×10

(3±

3)×

103

229

±3

(8±

6)×

102

45±

2(1

1±

7)×

102

Sorb

itol

(62

±2)

×10

(1±

4)×

103

219

±3

(−8

±5)

×10

244

.2±

0.8

(−4

±7)

×10

2

Succ

inic

acid

(58

±5)

×10

(1±

10)×

103

241

±1

(−4

±2)

×10

244

.1±

0.2

(0±

4)×

102

Gly

cero

l(6

0±

3)×

10(1

±5)

×10

321

5.4

±0.

6(−

2±

1)×

102

(18

±9)

×10

Eth

ylen

egl

ycol

(52

±7)

×10

(3±

14)×

103

216.

6±

0.5

(−1

±1)

×10

2

FAdo

tftv

cmnvpfumaIoydcacctatIdiSa

4

oMsetsb

ig. 3. Comparison of the results obtained with the SICA method using ICP-ED, RI detection or DAD (x-axis) and the conventional method with RIetection or DAD (y-axis) in the determination of non-volatile and semi-volatilerganic compounds in different food samples.

he concentration found with the method developed was 49 ± 2%or ICP-AED and 46 ± 4% for RI detection. This result supportshe use of the SICA method to carry out the analysis of non-olatile and semi-volatile organic compounds.

To further validate the developed calibration method the con-entrations of a set of non-volatile (i.e., citric acid, tartaric acid,alic acid, glucose, fructose, maltose, maltotriose, maltitol,

icotinamide, riboflavin, thiamine and pyridoxine) and semi-olatile (i.e., succinic acid and glycerol) organic compoundsresent in the different food samples analysed (i.e., candies,ruit juices, wines and a multivitamin complex) were determinedsing both the SICA method and a conventional calibrationethod. For the quantification of carbohydrates, carboxylic

cids and sugar alcohols, the SICA method was used with eitherCP-AED or RI detection while the reference method consistedf an external calibration method using RI detection. The anal-sis of vitamins, in turn, was carried out using the diode arrayetector for both the SICA and the conventional method. In thisase, the analysis by the conventional method was carried out atdifferent wavelength (i.e. the absorbance maximum for each

ompound) than that selected for the SICA one. In Fig. 3 theoncentrations determined with the SICA are plotted againsthose obtained with the reference method. The data fit well tostraight line (r2 = 0.9995) and the regression equation shows

hat the slope and intercept are close to 1 and 0, respectively.n fact, these values are included within their respective confi-ence intervals thus indicating the absence of systematic errorsn the method developed. Therefore, it can be concluded that theICA method allows an accurate determination of non-volatilend semi-volatile organic compounds in food samples.

. Conclusions

The method developed showed several advantages in termsf reduction of analysis time with respect to a conventional one.oreover, sometimes it is necessary to carry out the analysis of

amples of different nature. These samples could contain differ-

nt analytes being similarly retained by the stationary phase. Inhis case, these compounds could not be included in the sametandard because their peaks would overlap. So a double num-er of injections would be required giving rise to an additional

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84 E. Paredes et al. / J. Chro

ncrease in the analysis time. This problem could be solved withhe SICA method because it did not require to use standardsontaining the same compounds as the analytes present in theample.

The versatility of the SICA has allowed its application tohree different chromatographic detection methods, ICP-AEDeing the method that allowed a more significant reduction ofnalysis time.

cknowledgements

The authors would like to thank the Spanish Education Min-stry (Projects PETRI95-0980-OP and CTQ2006-01377/BQU)nd to the Vicerrectorado de Investigacion of the Universityf Alicante for the financial support. E.P. also thanks to theeneralitat Valenciana for the FPI grant.

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