tov

ade

LettuceGreen leafy vegetablesNP-HPLC

veor cpplcatithe

n. A

etables, evidencing diverse tocochromanol proles, with variable ratios and amounts of a- and c-tocoph-

of humforms ocotrien-tocophactivit

source of these compounds (Colombo, 2010) but literature data

Indeed, choosing amethod for quantication of vitamin E is com-plicated by an array of variations including food matrix, relativelyanalytical complexity and, more recently, available technologies.

As regards the solvents used, single solvent extraction has beendescribed including direct extraction with ethanol for Brassica veg-etables (Guzman, Yousef, & Brown, 2012), methanol for rapini

minimal interference for chromatographic analysis seemstal lipid quantos, Ponparticularle-Stanley,

food and biological samples.Chromatographic analyses of extracts are usually performed by

RP-HPLC (reversed-phase), which requires solvents compatiblewith the lipophilic characteristics of vitamin E, and the chromato-graphic system does not completely resolve b- and c-isomers(Irakli, Samanidou, & Papadoyannis, 2011). Moreover, co-extractedlipids interfere with the chromatographic resolution, requiringprior saponication (Barba, Esteve, & Frgola, 2011; Annunziataet al., 2012), which is a complex and time-consuming process that

Corresponding author. Tel.: +351 2220428638.

Food Chemistry 141 (2013) 11751180

Contents lists available at

he

lseE-mail address: sucasal@ff.up.pt (S. Casal).and chromatographic approaches are used. which is among the most frequently used methods worldwide inare still scarce, particularly regarding leafy vegetables other thanlettuce, increasingly consumed in fresh salads. Moreover, the re-ported amounts are highly variable among cultivars, growth condi-tions and particularly analytical methods where diverse extraction

the best approach. It has already been used for tocation by several authors (Ferraz, Fiza, dos SCarvalho, & Soares, 2004; Araujo et al., 2013),the classic Folch method (Folch, Lees, & Sloan0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.03.099suringto beanti-tes dey with1957),inhibition of membrane lipid peroxidation (Schneider, 2005) andmaintenance of membrane stability (Munn-Bosch & Falk, 2004).These lipophilic compounds are synthesized by photosyntheticorganisms, occurring mainly in leaves and seeds (Munn-Bosch &Falk, 2004). Green leafy vegetables, like lettuce, represent a good

2001). Recent methods such as supercritical uid extraction orSPME are usually expensive and the highly selective molecularimprinted solid phase extractions are not only expensive but alsolaborious to prepare (Puoci et al. 2007). The use of solvent mixturesof sufcient polarity to ensure full extraction while en1. Introduction

Vitamin E is an essential nutrienttously occurs in eightmain chemicalopherols (a-, b-, c-, and d-) and toAmong the tocochromanols family,aent the most biological antioxidanterol, and other minor compounds. The methodology is adequate for routine analyses, with a reducedchromatographic run (96%). The method was tested in different green leafy veg-Short communication

Validation of a fast and accurate chromaquantication of vitamin E in green leafy

Rebeca Cruz, Susana Casal REQUIMTE, Laboratrio de Bromatologia e Hidrologia, Faculdade de Farmcia, Universid

a r t i c l e i n f o

Article history:Received 23 May 2012Received in revised form 17 March 2013Accepted 19 March 2013Available online 16 April 2013

Keywords:

a b s t r a c t

Vitamin E analysis in greento compare published datasistent method with wide amicro-method for quantiliquid extraction based onwith uorescence detectio

Food C

journal homepage: www.egraphic method for detailedegetables

do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal

getables is performed by an array of different methods, making it difculthoosing the adequate one for a particular sample. Aiming to achieve a con-icability, the current study reports the development and validation of a faston of vitamin E in green leafy vegetables. The methodology uses solidFolch method, with tocol as internal standard, and normal-phase HPLClarge linear working range was conrmed, being highly reproducible, with

SciVerse ScienceDirect

mistry

vier .com/locate / foodchem

induces vitamin E loss (Ruprez et al., 2001). Recent developmentsin chromatographic separation and detection systems (e.g. LCMS)are typically expensive particularly for routine control methods.NP-HPLC (normal-phase) has the advantage of allowing directuse of a polar solvents and, thus, achieving better lipid solubilityand loading capacity (Kamal-Eldin, Gorgen, Peterson, & Lampi,2000), which is essential for the analysis of minor compounds suchas vitamin E without the need for prior hydrolysis. As regards sep-aration efciency, silica columns provide higher chromatographicresolution for tocochromanols, and the use of chemically relatedcompounds as internal standards (e.g. tocol) minimizes potentiallosses during sample preparation, while maintaining or improvingreproducibility.

The present work reports the development of a fast and accu-rate method for quantication of tocopherols in fresh green leafyvegetables samples based on the Folch extraction method andNP-HPLC chromatography. It aims to provide a consistent toolresponding to increased interest in tocochromanols characteriza-tion, which are nutritionally important, while making use of stan-dard laboratorial chromatographic equipment.

2. Experimental

2.1. Standards and reagents

Tocopherols (a-, b-, c-, and d-) and tocotrienols (a-, b-, and c-)were acquired from Supelco (USA) and Calbiochem (USA). 2-Methyl-2-(4,8,12-trimethyltridecyl)-chroman-6-ol (tocol) (MatreyaInc., USA) was used as internal standard (IS).

Butylated hydroxytoluene (BHT) was obtained from SigmaAl-drich (Germany), ascorbic acid was purchased from SigmaAldrich(China), n-hexane was HPLC grade from SigmaAldrich (Germany),and 1,4-dioxane was obtained from SigmaAldrich (p.a., USA).Methanol (SigmaAldrich, Germany) and dichloromethane (Sig-maAldrich, Germany) were of analytical grade. Sodium chloridewas purchased from Merck (Germany) and anhydrous sodium sul-fate was obtained from Merck (Germany).

2.2. Standards preparation

Individual stock solutions (5 mg mL1) of each tocopherol andtocotrienol were prepared in n-hexane and stored at 20 C, pro-tected from light, as well as a stock solution of tocol at100 lg mL1. Tocopherol standards purity (a- and c-) was moni-tored by spectrophotometry (UV-1800, Shimadzu, Japan), basedon their E1% 1 cm values (Nesaretnam, Yew, & Wahid, 2007). Amixture of each tocochromanol standard and tocol was prepared,thus obtaining the chromatogram seen in Fig. 1.

2.3. Sampling and extract preparation procedure

Three different lettuce cultivars (Lactuca sativa L. cvs. Butter-head, Green Leaf, and Red Leaf), watercress (Nasturtium ofcinaleL.), chard (Beta vulgaris L. cv. Rhubarb Chard) and corn salad (Vale-rianella locusta L.) were analysed. Butterhead lettuce was used dur-ing method development and validation procedures.

Fresh samples were bought in local supermarkets from Porto,Portugal. On the arrival at the laboratory, the vegetables were

1176 R. Cruz, S. Casal / Food Chemistry 141 (2013) 11751180Fig. 1. NP-HPLC-FLD chromatogram of a standard solution. (1) a-Tocopherol, R(4) c-tocopherol, RT = 3.14 min; (5) b-tocotrienol, RT = 3.45 min; (6) c-tocotrienol, RT =T = 1.74 min; (2) a-tocotrienol, RT = 2.04 min (3) b-tocopherol, RT = 2.79 min;3.92 min; (7) d-tocopherol, RT = 4.77 min; and (8) tocol, RT = 5.88 min.

erol

mistcleaned and washed with deionized water, dried, and the inedibleportion was discarded. Afterward, samples were homogenised ina food processor (Silvercrest, Germany), and immediately sampledfor the chemical analyses. Small portions of nely ground freshsample (

ulti

mistTable 1Method analytical parameters for a- and c-tocopherol content in Butterhead lettuce c

Compounds Retention time Correlation2

1178 R. Cruz, S. Casal / Food Che0.02 lg/g fresh weight and comparatively lower (i.e. more sensi-tive) than published examples (Gmez-Coronado et al., 2004; Irakliet al., 2011; Annunziata et al., 2012; Slavin & Yu, 2012).

In order to evaluate the assay precision, six sample extractswere prepared on non-consecutive days. Intra-day and inter-dayprecisions below 5% (Table 2) were achieved. These values are

coefcient (r )Min CV (%, n = 6)

a-Tocopherol 1.84 1.0 0.9994c-Tocopherol 3.40 0.8 0.9995Tocol 6.15 1.5

CV, coefcient of variation; LOD, limit of detection; LOQ, limit of quantication.

Fig. 3. Examples of NP-HPLC-FLD chromatographic prole of several grevar.

Working1

Limits

ry 141 (2013) 11751180comparable with data found in the literature including those ofAnnunziata et al. (2012) (intra-day precision

day precision

terms of effectiveness and protein preservation. Journal of Biochemical andBiophysical Methods, 58, 187193.

Folch, J., Lees, M., & Sloane-Stanley, G. H. (1957). A simple method for the isolationand purication of total lipids from animal tissues. The Journal of BiologicalChemistry, 226, 497509.

Gleize, B., Steib, M., & Reboul, E. (2012). Simple and fast HPLC method forsimultaneous determination of retinol, tocopherols, coenzyme Q10 andcarotenoids in complex samples. Food Chemistry, 134, 25602564.

Gmez-Coronado, D. J. M., Ibnez, E., Ruprez, F. J., & Barbas, C. (2004). Tocopherolmeasurement in edible products of vegetable origin. Journal of ChromatographyA, 1054, 227233.

Guzman, I., Yousef, G. G., & Brown, A. F. (2012). Simultaneous extraction andquantitation of carotenoids, chlorophylls, and tocopherols in brassicavegetables. Journal of Agricultural and Food Chemistry, 60, 72387244.

Irakli, M. N., Samanidou, V. F., & Papadoyannis, I. N. (2011). Development andvalidation of an HPLC method for the simultaneous determination oftocopherols, tocotrienols and carotenoids in cereals after solid-phaseextraction. Journal of Separation Science, 34, 13751382.

Kamal-Eldin, A., Gorgen, S., Peterson, J., & Lampi, A. (2000). Normal-phase highperformance liquid chromatography of tocopherols and tocotrienols:Comparison of different chromatographic columns. Journal of ChromatographyA, 881, 217227.

Kim, Y., Giraud, D. W., & Driskell, J. A. (2007). Tocopherol and carotenoid contents ofselected Korean fruits and vegetables. Journal of Food Composition and Analysis,20, 458465.

Malheiro, R., Casal, S., Lamas, H., Bento, A., & Pereira, J. A. (2012). Can tea extractsprotect extra virgin olive oil from oxidation during microwave heating? FoodResearch International, 48, 148154.

Munn-Bosch, S., & Falk, J. (2004). New insights into the function of tocopherols inplants. Planta, 218, 323326.

Nesaretnam, K., Yew, W. W., & Wahid, M. B. (2007). Tocotrienols and cancer:Beyond antioxidant activity. European Journal of Lipid Science and Technology,109, 445452.

Puoci, F., Cirillo, G., Curcio, M., Iemma, F., Spizzirri, U. G., & Picci, N. (2007).Molecularly imprinted solid phase extraction for the selective HPLCdetermination of a-tocopherol in bay leaves. Analytica Chimica Acta, 593,164170.

Ruprez, F. J., Martn, D., Herrera, E., & Barbas, C. (2001). Chromatographic analysisof a-tocopherol and related compounds in various matrices. Journal ofChromatography A, 935, 4569.

Samuoliene, G., Sirtautas, R., Brazaityte, A., & Duchovskis, P. (2012). LED lighting andseasonality effects antioxidant properties of baby leaf lettuce. Food Chemistry,134, 14941499.

Schneider, C. (2005). Chemistry and biology of vitamin E.Molecular Nutrition & FoodResearch, 19, 730.

Slavin, M., & Yu, L. (2012). A single extraction and HPLC procedure for simultaneousanalysis of phytosterols, tocopherols and lutein in soybeans. Food Chemistry,135, 27892795.

1180 R. Cruz, S. Casal / Food Chemistry 141 (2013) 11751180

Validation of a fast and accurate chromatographic method for detailed quantification of vitamin E in green leafy vegetables1 Introduction2 Experimental2.1 Standards and reagents2.2 Standards preparation2.3 Sampling and extract preparation procedure2.4 HPLC equipment and chromatographic separation

3 Results and discussion3.1 Extraction performance and chromatographic conditions3.2 Method validation3.3 Method application to green leafy vegetables

4 ConclusionsAcknowledgmentsReferences