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Article history:Received 20 December 2011Received in revised form 8 November 2012Accepted 19 April 2013Available online 30 April 2013

Keywords:b-caroteneOn-line sample preparationColumn-switchingHPLCFood supplements

A simple and automated HPLC column-switching method with rapid sample pretreatment has been

the retina, skin and mucous membranes. Biological studies have

Traditionally, the separation and determination of carotenoids(including b-carotene) was often performed using high perfor-mance liquid chromatography (HPLC) with a reversed phase C-18columns and detection at visible part of spectra (Iwase, 2002; Kar-ppi, Nurmi, Alonso, Lorencio, & Nyyssnen, 2008; Pupin, Dennis, &Toledo, 1999). However, most HPLC methods employ time con-

(Birtigh, Johannsen, Brunner, & Nair, 1995; Chuang & Brunner,02; Shen et al.,010), ultraatrix solidbach et al.,

the chromatography system. In addition, using above mentionedsample pretreatment methods could be accompanied with risk ofloss of analytes during sample manipulation and greaterbiohazard.

Nowadays the application of on-line extraction on special SPEsorbents is the growing trend in sample preparation procedures(Sadlek, atnsky, & Solich, 2007). The introduction of specialextraction sorbents, such as restricted access materials (RAM), al-lows the direct and repetitive injection of complex biological

Corresponding author. Tel.: +420 495067228; fax: +420 495518718.

Food Chemistry 141 (2013) 14331437

Contents lists available at

he

lseE-mail address: satinsky@faf.cuni.cz (D. atnsky).harmful free radicals, thus slows down the ageing process. which are carried out in off-line mode before sample injection intoshown that the dietary intake of b-carotene may reduce the riskof cancer and infectious diseases. b-carotene protects plant, humanand animal cells (most mucous membranes and skin) from thedestructive effects of UV radiation. As an antioxidant deactivates

2006), solid phase extraction (SPE) (Iwase, 202009; Snchez, Granados, Serrana, & Martnez, 2assisted extraction (UAE) (Plaza et al., 2011), or mdispersion (MSPD) (Gentili & Caretti, 2011; Putz0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.04.063sound--phase2005),1. Introduction

b-carotene is an important biological compound that is widelydistributed in fruits and vegetables. It is converted in the humanbody to retinol (vitamin A) that is essential for proper function of

suming and laborious sample preparation steps such as pressur-ized liquid extraction (PLE) (Plaza et al., 2011; Sanagi, See,Ibrahim, & Naim, 2005), liquidliquid extraction (LLE) (Burgoset al., 2009; Jackson, Lien, White, Bruns, & Kuhlman, 1998; Karppiet al., 2008; Pupin et al., 1999), supercritical uid extraction (SFE)developed for quantitative determination of b-carotene in food supplements. Commercially samples offood supplements were dissolved in chloroform with help of saponication with 1 M solution of sodiumhydroxide in ultrasound bath. A 20-min sample dissolution/extraction step was necessary before chro-matography analysis to transfer b-carotene from solid state of food supplements preparations (cap-sules, tablets) to chloroform solution. Sample volume 3 lL of chloroform phase was directly injectedinto the HPLC system. Next on-line sample clean-up was achieved on the pretreatment precolumn Chro-molith Guard Cartridge RP-18e (Merck), 10 4.6 mm, with a washing mobile phase (methanol:water,92:8, (v/v)) at a ow rate of 1.5 mL/min. Valve switch to analytical column was set at 2.5 min in aback-ush mode. After column switching to the analytical column Ascentis Express C-18, 30 4.6 mm,particle size 2.7 lm (Sigma Aldrich), the separation and determination of b-carotene in food supplementswas performed using a mobile phase consisting of 100% methanol, column temperature at 60 C and owrate 1.5 mL/min. The detector was set at 450 nm. Under the optimum chromatographic conditions stan-dard calibration curve was measured with good linearity correlation coefcient for b-carotene(r2 = 0.999014; n = 6) between the peak areas and concentration of b-carotene 20200 lg/mL. Accuracyof the method dened as a mean recovery was in the range 96.66102.40%. The intraday method preci-sion was satisfactory at three concentration levels 20, 125 and 200 lg/mL and relative standard devia-tions were in the range 0.901.02%. The chromatography method has shown high sample throughputduring column-switching pretreatment process and analysis in one step in short time (6 min) of thewhole chromatographic analysis.

2013 Elsevier Ltd. All rights reserved.a r t i c l e i n f o a b s t r a c tAnalytical Methods

A rapid HPLC column switching methodand determination of b-carotene in food

Ivana Brabcov, Markta Hlavckov, Dalibor atnsDepartment of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovsk

Food C

journal homepage: www.er sample preparationpplements, Petr Solich203, Hradec Krlov 500 05, Czech Republic

SciVerse ScienceDirect

mistry

vier .com/locate / foodchem

leads to the automation, simplication, and speeding up of the

tive, column-switching technique for direct injection, on-line

six-port switching valve and CBM-20A communication module.

mistpreparation and simultaneously analysis of samples is modern ap-proach in implementation of all of analytical process to one step. Incase of analysis of simple matrix such as food supplements sam-ples usual chromatography precolumns can be coupled to col-umn switching system without time-consuming clean-upprotocols and without sorbent destruction.

The main advantage of this technique is strong reduction oftime and sample manipulation during sample preparation steps.One sample pretreatment step is necessary ltration before injec-tion to chromatography system. On the other side method devel-opment process is not simple in some cases and it requiresspecial attention mainly during the rst chromatographic dimen-sion for sample clean up elution power of mobile phase for inter-ferences removal and sufcient recovery of analytes of interest,pretreatment column choice, ow rate of mobile phase and timeof column switch. Moreover, the next complication of this tech-nique is requirement for 2-D chromatography system twopumps, high pressure column switching valve and control software(Sadlek et al., 2007; Souverain, Rudaz, & Veuthey, 2004).

This paper presents for the rst time the development of a newHPLC method for the determination of b-carotene in food supple-ments employing column switching technique using on-line sam-ple preparation and separation in one step. Developed method wasvalidated and successfully applied for the determination of b-caro-tene in food supplements available on local market.

2. Materials and methods

2.1. Chemicals and materials

Standard of b-carotene and organic solvents (HPLC grade)methanol and acetonitrile were obtained from SigmaAldrich Che-mie GmbH, Germany. The ultra-pure water used for mobile phasepreparation was puried through a Milli-Q (Millipore, Bedford, MA,USA). Chloroform and sodium hydroxide were from Merck, Darms-tadt, Germany. All other materials were of analytical grade. Thefood supplements with content of b-carotene (capsules, tabletsand oil suspension) were purchased on local market in CzechRepublic.

2.2. Equipment and chromatographic system

HPLC chromatographic apparatus consisted of a Shimadzusample preparation process. The term RAM sorbents is shared bymaterials used mainly for the analysis of substances with lowmolecular mass (e.g. drugs, endogenous substances, xenobiotics)in complex matrices containing high-molecular substances (mostfrequently proteins). These materials enable direct injection ofthe biological samples into the ow analytical systems withoutprevious sample treatment. They have several different structures,but their mechanism of separation is identical: a hydrophilic bar-rier enables the small molecules to permeate through the hydro-phobic part of the stationary phase, and, at the same time, itexcludes the macromolecules (by physical or chemical means, ora combination). Column-switching technique using these specialsorbents for complex matrix samples can work without the labori-ous and time-consuming clean-up protocols. From this perspec-matrices onto these supports. The coupling of RAM into the col-umn-switching HPLC systems represents a very attractive ap-proach in biological sample preparation. Use of this technique

1434 I. Brabcov et al. / Food CheProminence system (Shimadzu Corporation, Kyoto, Japan),equipped with solvent delivery systems LC-20AD, with a SIL-20AC autosampler, DGU-AS on-line degasser, SPD-M20A DADThe system control, data acquisition and data evaluation were per-formed by Shimadzu LC Lab-Solution software (Shimadzu Corpo-ration, Kyoto, Japan).

2.3. Preparation of stock solution and food supplements samplepreparation

Standard stock solution of b-carotene was prepared by dissolv-ing of substance in chloroform in concentration 2000 lg/mL. Stan-dard stock solution was stored at 18 C in dark for 1 month.Standard stock solution was further diluted with chloroform to ob-tain working standard solution in concentration 100 lg/mL formethod development and validation. The calibration standardsolutions were prepared over the concentration range of 20200 lg/mL, using seven calibration points. All solutions werestored in amber laboratory glassware.

Food supplements samples (capsule, homogenised tablet pow-der or 2 g of oil suspension) were dissolved in 20 ml of 1 M sodiumhydroxide with help of ultrasound bath. Twenty millilitre of chlo-roform was added to sample solution and the mixture was ex-tracted for 20 min. Closed PTFE centrifugation tubes were usedfor sample dissolution and extraction to avoid evaporation of chlo-roform. Three millilitre of chloroform layer was injected directlyinto the HPLC system. Samples with higher concentration of b-car-otene were diluted with pure chloroform to obtain concentrationinside calibration range. All food supplement samples and workingstandard solutions were prepared fresh daily.

2.4. HPLC column switching analysis

The simultaneous sample pretreatment and b-carotene deter-mination was performed using a two column HPLC system. Thesample pretreatment column was precolumn Chromolith GuardCartridge RP-18e (Merck, Darmstadt, Germany) 10 4.6 mm, witha washing mobile phase (methanol:water; 92:8) at a ow rate of1.5 mL/min. The chromatographic separation and determinationof b-carotene was performed on an analytical column Ascentis Ex-press C-18 30 4.6 mm, particle size 2.7 lm (Sigma Aldrich, Ger-many), using a mobile phase consisting of 100% methanol andow rate 1.5 mL/min. The column oven temperature was kept con-stant at 60 C for both columns. The detector was set at 450 nm.

On-line sample pretreatment step (02.5 min) 3 lL chloro-form sample solution of food supplement was directly injectedonto precolumn Chromolith Guard Cartridge RP-18e10 mm 4.6 mm where polar interferences and (tablets, capsules)sample matrix were removed to the waste. Used washing mobilephase consisted of mixture methanol:water; 92:8, (v/v) waspumped for 2.5 min at ow rate of 1.5 mL min1. The analyticalcolumn was equilibrated using the analytical mobile phase at thistime. Valve switch for column was set at 2.5th min.

Separation step and determination of b-carotene was per-formed in interval range from 2.5th to 6th min. Zone of preconcen-trated sample was transported in back-ush direction frompretreatment column onto analytical column Ascentis Express C-18 30 4.6 mm, particle size 2.7 lm, where the analysis was per-formed in isocratic mode. The analytical mobile phase used fordetermination of b-carotene consisted of 100% methanol pumpedat ow rate 1.5 mL/min.

3. Results and discussiondetector, CTO 20AC column oven with FCV-12AH high pressure

ry 141 (2013) 14331437No method for determination of b-carotene in food, foodsupplements and in another plant material with HPLC column

mistswitching technique is reported in the literature so far. Therefore,prior to the method development and validation, the parametersof sample preparation, conditions of mobile phases and stationaryphases in rst and second chromatography dimension wereoptimised.

3.1. Optimisation of rst chromatography dimension

The rst chromatography dimension was used for sample pre-treatment and preconcentration of b-carotene in our case. Samplesolution dissolved in chloroform was injected to rst column,where b-carotene adsorption and matrix component interferenceelution were carried out. Choice of the preconcentration columnand pretreatment mobile phase composition had to fulll require-ments on low interferences absorption on column sorbent andstrong retention of b-carotene. The following preconcentration col-umns were tested in our study: Precolumn sorbent lter Opti-guard C-18-1 mm in tting screw, (Sigma Aldrich, USA), Chromo-lith Guard Cartridge RP-18e 10 4.6 mm and Chromolith GuardCartridge RP-18e 5 4.6 mm (Merck, Darmstadt, Germany).

Different pretreatment mobile phases with methanol rangingfrom 90% to 100% (v/v) with water and different ow rates rangingfrom 1.0 to 3.0 ml min1 were tested and optimised for all pre-treatment columns. Washing times for rst column dimensionranging from 30 s to 3 min were tested. Volume of methanol lowerthan 90% resulted in decreasing of recovery (decreasing of peakarea) due to poor solubility of b-carotene in mobile phase. On theother side, 100% of methanol was not found to be suitable for b-carotene preconcentration on pretreatment column due to theweak retention of b-carotene and washing out it with ballast ma-trix of food supplements. Optimisation of column-switch timeand ow rate were estimated as the time and ow rate sufcientfor washing ballast matrix components for each food supplementpreparation. Optimal pretreatment mobile phase was found as ra-tio methanol water (92:8, v/v) pumped for 2.5 min at ow rate1.5 mL min1 through the preconcetration column ChromolithGuard Cartridge RP-18e 10 4.6 mm. Mentioned conditions werefound to be optimal for sample preparation and preconcentrationprior to b-carotene separation in the analytical column. Precolumnsorbent lter Opti-guard C-18-1 mm was found to be sorbentwithout sufcient adsorption capacity. Partially elution of b-caro-tene after the sample zone injection was observed. This effectwas caused by chloroform (strong eluent) in sample solution andsmall amount of sorbent (1 mm length) in tting screw. On theother hand, there was possible to say that monolithic precolumnswere suitable for the desired purpose of sample pretreatment. Bothmonolithic precolumns showed ability of sample pretreatment andb-carotene preconcentration under the tested and optimised con-ditions. Chromolith Guard Cartridge RP-18e 10 4.6 mm was -nally chosen for validation of the method.

3.2. Optimisation of second chromatography dimension

Two analytical chromatographic columns with totally differentstructure of stationary phases core shell particle column AscentisExpress C-18 30 4.6 mm, particle size 2.7 lm (Sigma Aldrich,Germany) and monolithic column Chromolith SpeedRod RP-18e50 4.6 mm (Merck, Darmstadt, Germany) were tested. The iso-cratic separation of b-carotene was optimised with methanol/water mobile phases ranging from 95% to 100% (v/v) of methanolin water. Addition of water to mobile phase resulted in b-caroteneretention times increasing and peak symmetry started to be out ofvalidation range (higher than 1.5) for both columns. Successful

I. Brabcov et al. / Food Chepeak symmetry and faster analysis was achieved on Ascentis Ex-press C-18 column. The column temperature optimisation was car-ried out in the range 3060 C for both columns (precolumn andanalytical column) together. Decreasing time of analysis and betterpeak symmetry was observed with increasing temperature of col-umn oven. Therefore the nal separation conditions for methodvalidation on Ascentis Express C-18 column were chosen at 60 Cand ow rate of mobile phase 1.5 mL/min. The chromatogram ofb-carotene determination in food supplement sample with usingof column switching technique is given in Fig. 1.

3.3. Validation of the method

Validation of the method includes the evaluation of followingperformance parameters such as linearity, precision, accuracyand selectivity in order to evaluate the reliability of the results pro-vided by the method. Within the method validation the parametersof the HPLC system suitability test (SST) were measured and eval-uated in accordance with ICH guidelines International Confer-ence on harmonisation (ICH, 2005). The samples of standardsolution were six times injected into the chromatographic system.Mean values and standard deviations of retention time, number oftheoretical plates, peak asymmetry, resolution, and repeatability ofanalytical run were calculated according to the European Pharma-copoeia recommendations (European Pharmacopoeia, 2007). Ob-tained validation results and chromatography system suitabilityparameters are summarized in Tables 1 and 2.

3.3.1. Linearity, selectivity and sensitivitySeven test solutions ranging 20200 lg/mL of b-carotene were

prepared by dilution of stock solution of 2000 lg/mL and testedfor linearity test. The detector response at 450 nmwas found linearin whole range of calibration curve. The linear regression curvewas obtained by plotting peak area count of b-carotene at each le-vel against the concentration of each injection. The detaileddescriptions of regression curve are depicted in Table 2. Statisticaldata of calibration curve parameters were computed from sevenconcentrations levels in mentioned range (200, 175, 150, 100,50, 20 lg/mL). Good linearity (coefcient of correlationr2 = 0.999014) was achieved in the investigated range for b-caro-tene. Linear regression parameters were described by the followingequation: A = (7726.6 153.6)c (24263.5 20244.9), where A isthe peak area at 450 nm and c b-carotene concentration. All sam-ples were measured in triplicates.

The limit of detection (LOD) and limit of quantication (LOQ)were calculated by the comparison of the threefold (3r) and 10-fold (10r) variation, respectively, of base-line noise and signalsof b-carotene. The detection limit of b-carotene determinationwas 0.50 lg/mL. The limit of quantication was estimated to be1.67 lg/mL.

The selectivity of the method was evaluated by conducting theexperiments using batches of different food supplements samplesto check the peak purity of b-carotene onto the chromatogram.No interfering peaks in retention time of b-carotene were observedand peak purity index reached the values greater than 0.9999 forall food supplement samples. Ballast matrix components from cap-sules and tablets dissolved in chloroform were eluted in the frontof the chromatogram of the rst dimension to the waste.

3.3.2. Repeatability, precision and accuracyRepeatability of the proposed method was characterised by rel-

ative standard deviation (RSD, (%)), which was calculated for sixconsecutive measurements at three concentration levels 20, 125and 200 lg/mL of b-carotene standard solutions at the same day.The results in form of RSD were determined in the range 0.901.02%.

ry 141 (2013) 14331437 1435To validate the precision of the method a number of 6 food sup-plement sample solutions were used, which were prepared fromthe same batch and analysed consecutively. This approach

mist1436 I. Brabcov et al. / Food Cheprovides a means of covering the precision of the entire method,from sample preparation to data handling. The intra-day precisionvalues of measured concentration of b-carotene in different foodsupplement samples were calculated as RSD values which werein the range 1.432.59%. In both situations (parameters of repeat-ability and precision), the relative standard deviation values werein the range recommended in accordance with European Pharma-copoeia recommendations.

The accuracy of the method was carried out measuring of thefood supplement samples fortied with known quantity of b-caro-tene (addition of 100% amount of the standard to food supplement

Fig. 1. Representative chromatogram of b-carotene determination in food su

Table 1HPLC column switching system suitability parameters.

Retentiontime (min)

Retention timerepeatabilityRSD (%)a

Repeatabilityof peak areasRSD (%)a

Number oftheoreticalplates

Peaksymmetry

b-Carotene 4.86 0.17 1.01 2630 1.374

a Made in six replicates at one concentration level 125 lg/mL.

Table 2Analytical characteristics of the validated HPLC column switching method.

Parameters b-carotene

Standard calibration range (lg/mL)a 20200Correlation coefcient 0.9990LOD (lg/mL) 0.50LOQ (lg/mL) 1.67Repeatability RSD (%)b 0.901.02Method precision intra-day RSD (%)c 1.432.59Accuracy spike recovery (%) SDd 96.66102.40 (1.712.92)

a Each concentration of calibration standard was measured in triplicate.b Relative standard deviation (RSD) was calculated from six injections of b-car-

otene standard at three concentration levels 20, 125 and 200 lg/mL.c Relative standard deviation (RSD) for repeated injections of multiple prepara-

tions of different food supplement samples (n = 6), three injections of each samplepreparation.

d Accuracy was determined as a method recovery using spiked food supplementsamples at one concentration level (added amount 100% of b-carotene content foreach food supplement) in six samples from one batch (minimal and maximalstandard deviation of recovery determination).preparation). Fortied sample solutions and un-fortied samplesolutions were compared for recovery evaluation. The methodaccuracy results were tested for different food supplement prepa-rations and values of the recoveries were found in the range 96.66102.40% (1.712.92%) for b-carotene. Assay values of recoveriesshow that the HPLC column switching method allows direct deter-mination of b-carotene in commercial food supplement prepara-tions in the presence of other antioxidants and excipients.

3.3.3. Robustness and stabilityThe robustness of the method was determined by measuring

the effect of small and deliberated changes in the analytical param-eters on retention time and peak area counts. The parameters thatwere taken into consideration were ow rate of mobile phase and

pplement sample (capsule) with using of column switching technique.

ry 141 (2013) 14331437temperature in column oven. Mobile phase composition in secondchromatography dimension was kept constant at 100% of metha-nol. At a time only one parameter was changed while the secondwas kept constant. The relative standard deviations of peak areacounts of b-carotene were calculated for both parameters. Differ-ences in peak area due to the changes in column temperature were2.00% for 50 C and 7.90% for 70 C. Variations in peak area due tothe changes in ow rate (tested range 1.0 and 2.0 mL/min) werehigher than 10%. The higher variations of peak areas are in agree-ment that the optimised conditions of the method must be strictlykept to avoid impression during analysis.

Stability of b-carotene working standard solution was tested at4 C and 20 C in autosampler for 24 h. Variations (RSD (%)) in peakareas of b-carotene (range 1st24th h) were at 4 C and 20 C 2.88%and 1.60%, respectively.

3.3.4. Determination of b-carotene in food supplementsThe method developed has been applied to the determination of

b-carotene in capsules, tablets and oil suspension. The sampleswere commercially available on the local market. The disturbingeffect of excipients (saccharine, gelatin, starch, shellac, matrix ofoil suspension and other antioxidants) was not observed. Peak pur-ity in sample run and high accuracy of the analysis showed that nointerference was observed in the quantitative analysis of b-caro-tene. The optimal extraction procedure was reached due to columnswitching technique. The suggested procedure of samplepreparation was simple, fast and achieving high precision and

Table 3Determination of b-carotene content in various food supplement samples (n = 4).

Food supplement Type Declared amount of b-carotene inof food supplement (mg)a

Found amount in (%) RSD

Walmark Beta carotene Capsules 6.00 98.76 1.77GS Betacarotene Forte Capsules 15.00 100.07 1.41Bioactive carotene Capsules 9.00 97.00 1.91Betacarotene farmax Capsules 6.00 96.66 1.71Max Beta carotene Capsules 6.00Carovite Tablets 6.00

g o

I. Brabcov et al. / Food Chemistry 141 (2013) 14331437 1437low sample and reagent consumption. The average amounts of b-carotene determined in food supplement preparations are givenin Table 3. Obtained results in range 95.0105.0% for the activecompound content in preparations were fullled for four commer-cially products only.

4. Conclusion

The HPLC column switching methodology described in this pa-per provides a simple, rapid and rugged approach for qualitativeand quantitative analysis of b-carotene in food supplements sam-ples. The determination of b-carotene with using HPLC columnswitching method as sample pretreatment is reported here forthe rst time. The sample preparation procedure involved sonica-tion followed by dissolution in chloroform (20 min in ultrasoundbath) and direct injection into HPLC system eliminated usual timeconsuming sample preparation steps such as off-line solid phaseextraction or liquidliquid extraction. Real application of the newmethod was carried out and the procedure was applied for thequantitation of b-carotene in different commercial food supple-ments with high recovery. It can be concluded that because ofthe simple column switching extraction procedure, high precision,accuracy, simplicity and short chromatography analysis time of6 min, the method may be useful for fast screening or quality con-trol testing of b-carotene content in different food supplementssuch as tablets, capsules or oil suspensions.

Acknowledgements

The authors acknowledge the nancial support of the CharlesUniversity in Prague project SVV 267 002 and project UNCE No.204026/2012.

Selzink plus Tablets 6.00Primrose oil Oil suspension 30.00

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A rapid HPLC column switching method for sample 1 Introduction2 Materials and methods2.1 Chemicals and materials2.2 Equipment and chromatographic system2.3 Preparation of stock solution and food supplements sample preparation2.4 HPLC column switching analysis

3 Results and discussion3.1 Optimisation of first chromatography dimension3.2 Optimisation of second chromatography dimension3.3 Validation of the method3.3.1 Linearity, selectivity and sensitivity3.3.2 Repeatability, precision and accuracy3.3.3 Robustness and stability3.3.4 Determination of -carotene in food supple

4 ConclusionAcknowledgementsReferences