Determination of Pesticides Residues in Brazilian Grape JuicesUsing GC-MS-SIM

Andréa A. R. Alves & Aline S. Rodrigues &

Elisabete B. Paula Barros & Thaís M. Uekane &

Humberto R. Bizzo & Claudia M. Rezende

Received: 30 November 2013 /Accepted: 3 February 2014# Springer Science+Business Media New York 2014

Abstract The production of grape juice in Brazil is growingat a 30 % rate a year. To increase exports, a full complainmentto food safety protocols, as those in the Codex Alimentarius, ismandatory. Simple and fast analytical methods are needed forquality assurance. The objective of this work was to developand validate an analytical method for detection of pesticidesresidues (organochlorides, organophosphorides, and fungi-cides) in Brazilian whole grape juices. The analyses wereperformed by gas chromatography coupled to mass spectrom-etry, operating in selective ion monitoring. After liquid–liquidextraction, samples were preconcentrate using solid-phase ex-traction in Florisil. The limit of quantification ranged from 3.75to 9.47mg l-1, and recovery varied from 84.0% to 99.7%, withRSD lower than 4.9 %. From a total of 99 Brazilian grapejuices analyzed, pesticide residues were found in 32.

Keywords Pesticides residues . GC-MS-SIM . Braziliangrape juices

Introduction

The Brazilian legislation (Brasil – Ministério da Agricultura,Pecuária e Abastecimento 1988) defines grape juice as anonfermented beverage, obtained by pressing or smashing of

mature, fresh, and healthy grapes, with or without solid parts,with an alcoholic grade not superior than 0.5° GL. The juicemust be obtained exclusively with grapes produced in Brazil.

The grape juice may be classified as whole juice (purejuice, without water removal), concentrated (partiallydehydrated), and nectar (by addition of water and sugar;FSA — Food Standards Agency 2006). As regards color,the juice can be red, rosé, or white. Aroma and taste must becharacteristic from the grapes used. Cultivars most used in redjuice production are Concord, Isabel, and Bordô, from theAmerican native species Vitis labrusca. For the white juice,the V. vinifera cultivars used are mainly Italia, Moscato, andChardonnay (Brasil – Agência de Vigilância Sanitária 1994).

Brazil is one of the world largest producers of whole grapejuice, with an impressive increase of 30 % per year (RizzonandMiele 2012). The USA is its main competitor, because it isone of the few countries producing grape juice made from thespecies V. labrusca (Ibraf — Instituto Brasileiro de Frutas2013). The consumption of whole grape juice in Brazil hasincreased in the last 5 years from 0.390 l per capita in 2008 to0.540 l in 2011. Although Brazilian exports are increasing,mainly to Europe and Latin America, the domestic market isstill its main market, consuming primarily concentrated grapejuice (17 %) and whole juice (26 %) (Ibravin — InstitutoBrasileiro do Vinho 2011).

The grape juice production, particularly the whole grapejuice type, is expanding in Brazil due to improvements ingrape cultivars and juice production processes (Camargo2008). However, the Brazilian quality and safety standardsdo not meet the requirements of the Food and Drug Admin-istration and the World Health Organization (WHO) yet,limiting Brazilian exports expansion.

In addition, Brazil is one of the largest consumers ofpesticides, with imports reaching nearly US$ 2.5 billion peryear (Spadotto and Gomes 2011). Grapes are among the fruits

A. A. R. Alves :A. S. Rodrigues : E. B. P. Barros : T. M. Uekane :C. M. Rezende (*)Instituto de Química, Universidade Federal do Rio de Janeiro,Cidade Universitária, CT, Bloco A, CEP 21941-909 Rio de Janeiro,RJ, Brazile-mail: crezende@iq.ufrj.br

H. R. BizzoEmbrapa Agroindústria de Alimentos, CEP 23020-470 Guaratiba,Rio de Janeiro, RJ, Brazil

Food Anal. MethodsDOI 10.1007/s12161-014-9823-9

with higher exposure to pesticides, usually applied by aerealspraying, which may leave residues in the fruits and theirprocessed products (juices, wines, and jellies; CodexAlimentarius Website 2013).

The increasing use of pesticides on food products is amatter of concern for governments, since the internationalmarket shall follow strict regulations like the CodexAlimentarius. Founded by Food and Agriculture Organizationand WHO in 1963, the Codex Alimentarius Commissionestablishes the maximum residue limits (MRLs) forpesticide/commodity combinations and poses phytosanitarybarriers for a food product whenever these limits are notrespected (Codex Alimentarius Website 2013).

The development of analytical methods for pesticide resi-dues monitoring either in fresh fruits or in processed food,such as grape juice, is important to evaluate the sample ad-herence to international rules. Focusing on fresh fruits (com-plex matrices), there are combined methods that promote abetter cleanup and preconcentration of the pesticides such asthe combination of QuEChERS with dispersive liquid–liquidmicroextraction (DLLME; Melo et al. 2012) and the combi-nation of liquid–liquid extraction (LLE) with solid-phase ex-traction (SPE; Dong et al. 2009; Li and Yuan 2008).

The gas chromatography-mass spectrometry-selective ionmonitoring method (GC-MS-SIM) is a simple alternative forthe analysis of pesticides on different vegetables (Cherta et al.2013a), fruits (Andrade et al. 2011; Dömötörová et al. 2006),fruit juices (Cherta et al. 2013b; Albero et al. 2005), andessential oils (Alves et al. 2012).

The objective of this work was develop and validate ananalytical method to investigate the occurrence oforganochloride (OC) and organophosphoride (OP) pesticides,as well as some fungicides (Fg) in commercial Brazilian grapejuices using LLE/SPE and GC-MS-SIM.

Experimental

Materials

There were 33 commercial and experimental brands of red andwhite whole grape juices obtained from supermarkets in the cityof Niteroi, Rio de Janeiro state (Brazil), and juice companies.Three bottles of each brandwere purchased, totaling 99 samples,15 of them being organic juices (without soluble synthetic fertil-izers, pesticiders, and genetically modified (GM)). The bottleswere opened only on the day of analysis and then kept in afreezer at 5 °C. Pesticides standardswere obtained fromDr.MarkEhrenstorfer GmbH, Germany, Florisil cartridges from J. T.Baker® (lot P2044A1, USA), with 100 mg of adsorbent/cartridge. The solvents used were hexane (Tedia, USA), ethylacetate (Tedia, USA), acetone (Tedia, USA), salts of sodiumchloride (NaCl, Merck), and sodium sulfate (Na2SO4, Merck).

Instrumentation and Chromatographic Conditions

GC-MS was performed in a 6850 gas chromatograph coupledto a 5975C mass selective detector 5975C MSD (AgilentTechnologies) operating in electronic ionization mode at70 eV. Helium was the carrier gas with a flow of 1.0 ml min-1. Separation was achieved in a 5 %-phenyl-95 %-dimethylpolysiloxane (HP5-MS, Agilent 19091S 433E) fusedsilica capillary column (30 m, 0.25 mm i.d., 0.25 film thick-ness). Injections were made in splitless mode and injector waskept at 250 °C. The oven temperature program had an initialtemperature of 50 °C held for 2 min, 10 °C min-1 to 300 °Cheld for 15 min. Transfer line was kept at 310 °C. The massspectrometer was operated in scan mode (50–400 u) to collectmass spectra for components identification and then in SIM toquantify the pesticides.

Standard Stock Solution and Internal Standard Solution

Stock solutions of 1,000 mg l-1 for each pesticide were pre-pared by dissolving 1.0 mg in a 1.0 ml volumetric flask withhexane. These solutions were kept in the freezer at -20 °C andinjected every 15 days to check the pesticides stability. n-Pentacosane was used as internal standard (IS). Stock solutionwas prepared by dissolving 1.0 mg of the IS with hexane in a1.0 ml volumetric flask. This solution was kept at -20 °C andinjected every 15 days to check its stability.

Pesticides Mix

Each pesticide stock solution (50 μl) were transferred to a1.0 ml volumetric flask, and the volume completed withhexane, leading to a mix with 50 mg l-1 of each pesticide.Solutions were kept at -20 °C.

LLE and Cleanup of Fortified Grape Juice

The LLE extraction was performed in an Erlenmeyer flaskwith 15 ml of each grape juice fortified with 1.0 ml of thepesticides mix (with 50 mg l-1 of each pesticide), 4 g of NaCl(Merck, Germany), 15 ml of ethyl acetate–hexane (1:1), and25 μl of the IS solution. After 15 min of sonication (1510ultrasonic bath, Branson), the organic phase was separatedand dried over sodium sulfate.

The organic phase was added to a Florisil SPE cartridgepreconditioned with 5 ml of acetone. The cartridge was theneluted with 5 ml of ethyl acetate–hexane (1:1). Both fractionswere added to a flask and solvent evaporated at room temper-ature under a flux of nitrogen and redissolved in 1.0 ml ofhexane; 1 μl of this extract was injected in the GC-MS-SIM.Injections were made in triplicate.

Food Anal. Methods

GC-MS Calibration Solutions

To establish the analytical curve, it was used the AU juice,because it contained few compounds and was free from resi-dues, and themix of pesticides at a concentration of 1.0, 5.0, 10,15, 20, 25, and 50 mg l-1 of each pesticide. The solutions wereextracted, purified, and concentrated as previously described.

Pesticides solutions at a concentration of 15, 30, 45, and60 mg l-1 of each pesticide were prepared for repetitivityevaluation. Once again, extraction, purification, and concen-tration procedures were repeated.

The limits of detection (LOD) and quantification (LOQ)werecalculated from the analytical curve according to the formula:

LOD ¼ 3:3 s=Sð ÞandLOQ ¼ 10:0 s=Sð Þ;

where s and S are the estimates of the standard deviation andthe linear and angular coefficients of the analytical curve,respectively (Miller and Miller 1988; Box et al. 1987).

Samples

Each of the 99 samples of grape juice were extracted, purified,and concentrated as previously described, excluding the forti-fication procedure.

Results and Discussion

Qualitative Analysis of Pesticides

Only 18 volatile compounds were detected in AU juice,among them 1-hexanol, furfural, α-ionone and α-terpineol. No pesticides were detected. Due to the poorcomposition, the AU juice was chosen for method val-idation, since less interference was expected. In theother samples, circa 45 volatiles were found, mainlyethyl esters (anthranilate, 2-hexenoate, and benzoate),primary alcohols (hexanol, octanol, 2-ethyl-hexanol,and 2-pheny l e thano l ) , and t e rpen i c a l coho l s(α-terpineol and linalool). The AU juice was fortifiedwith the mix of pesticides (50 mg l-1) and screened byGC-MS-SIM to determine the retention times and thecharacteristic ions (Table 1) used for qualitative andquantitative analyses.

Figure 1 shows the chromatogram of the AU grape juicefortified with pesticides standards from GC-MS-SIM.

Quantitative Analysis of Pesticides

Analytical curves were obtained from fortified AU juice, atseven different concentrations (1, 5, 10, 15, 20, 25, and50 mg l-1), injected in triplicate. The IS concentration was

25mg l-1 for all analyses, and the resulting pattern was observedto be homoscedastic according to Grubbs and Cochran statisti-cal methods (Miller andMiller 1988). In Table 2, it is shown thecalibration parameters of the pesticides standards.

R2 values might be above 0.990 for a method valida-tion for pesticide residues in food matrices (Miller andMiller 1988; Ribani et al. 2004). Considering all con-centrations tested, R2 ranged from 0.990 to 0.997, whichcan be considered satisfactory.

The LOD values ranged from 1.20 to 6.25 mg l-1 and theLOQ varied from 3.75 to 9.47 mg l-1, within the range for fruitjuices (Albero et al. 2005).

The method precision was evaluated by repeatability usingthe AU juice fortified with four pesticides concentrationsinjected in triplicate (15, 30, 45, and 60 mg l-1, n=12). Accu-racy was calculated by recovery. Both repeatability and re-covery values are presented in Table 3.

The relative standard deviation (RSD%) was below 4.3 %,within the acceptance limit of the Brazilian Health Surveil-lance Agency (ANVISA 2013) and the International Confer-ence on Harmonization (ICH — International Conference onHarmonisation 1995), which established an RSD up to 20 %for trace level compounds, particularly in a complex matrix,such as food products.

The recovery ranged from 84.0% to 99.7%. In the analysisof pesticide residues, the range from 70 % to 120 % isacceptable (Miller and Miller 1988; Box et al. 1987).

Table 1 Retention times (tR) and three selected characteristic ions forGC-MS

Pesticide tR (min) Characteristic ions (m/z)

Organochlorine (OC)

Aldrin 20.8 263, 265, 293

Dicofol 20.6 197, 286, 314

Chlordane* 21.9, 22.3 237, 272, 373

Dieldrin 22.7 139, 141, 250

Quintozene 18.3 237, 249, 295

Organophosphorus (OP)

Dimethoate 17.4 87, 125, 229

Methyl parathion 19.4 109, 125, 153

Methyl chlorpyrifos 19.6 237, 263, 345

Methidathion 21.7 85, 145, 302

Dithianon 25.2 264, 268, 296

Fungicides (Fg)

λ-Cyhalothrin 26.1 181, 197, 225

Carbaryl 14.9 115, 144, 202

Tebuconazole 24.3 131, 175, 201

Thiabendazole 21.2 125, 153, 307

Prochloraz 27.1 180, 266, 308

n-Pentacosane (IS) 25.7 197, 239, 352

*For chlordane, these are the two isomers frequently monitored

Food Anal. Methods

Determination of Pesticide Residues in Brazilian Commercialand Experimental Grape Juices

The validated method was applied to samples from commer-cial and experimental juices from 33 different brands pro-duced in 2012. For each brand, three different samples, namedG1, G2, and G3, were tested (n=99). Among them, 32 werecontaminated, sometimes with more than one pesticide. Thequantitative data of these juices are presented in Table 4.Quintozene was the pesticide most commonly found. From

the 15 pesticides tested, only dicofol, dithianone, andtebuconazol are allowed in grapes (Codex AlimentariusWebsite 2013). However, the quantities found were wellabove the MRLs.

The most contaminated juices were SP_G1, with60.17 mg l-1 of dieldrin; AL_G3, with 34.52 mg l-1 ofquintozene; 34.38 mg l-1 and 21.38 mg l-1 of dicofol inDC_G3 and DC_G2, respectively; and for CL_G1 residues,13.20 mg l-1 of aldrin, 34.00 mg l-1 of dicofol, and 21.66 mg l-1 of quintozene were detected.

Table 2 Calibration equation, correlation coefficient (R2), limit of detection (LOD), limit of quantification (LOQ), and maximum residues limits (MRL)for each pesticide analyzed

Pesticide R2a GC-MS-SIM MRLb (ml)

Equation LOD (ml) LOQ (ml)

Aldrin 0.991 y=0.051x+0.011 1.42 4.16 –c

Dicofol 0.996 y=0.105x-0.018 1.70 6.86 8.50

Chlordane 0.992 y=0.014x+0.005 5.89 7.14 –c

Dieldrin 0.990 y=0.156x+0.078 6.60 9.00 –c

Quintozene 0.997 y=0.147x+0.004 5.39 8.16 –c

Dimethoate 0.991 y=0.042x-0.013 5.11 9.29 –c

Methyl parathion 0.990 y=0.008x+0.006 1.24 3.75 1.40

Methyl chlorpyrifos 0.992 y=0.182x+0.075 1.36 4.12 1.70

Methidathion 0.997 y=0.101x-0.018 1.47 3.56 1.70

Dithianon 0.994 y=0.011x+0.004 1.20 3.64 5.10

λ-cyhalothrin 0.994 y=0.042x+0.003 5.89 7.14 5.10

Carbaryl 0.990 y=0.095x-0.009 6.25 9.47 –c

Tebuconazole 0.994 y=0.015x+0.005 5.50 8.33 10.20

Thiabendazole 0.990 y=0.008x+0.006 6.19 9.00 –c

Prochloraz 0.991 y=0.024x+0.008 5.50 8.33 –c

a Determination coefficientb Reference Codex Alimentarius Internationalc No reference value

Fig. 1 GC-MS-SIM chromatogram of the AU grape juice fortified withpesticides: 1Carbaryl, 2Dimethoate, 3Quintozene, 4Methyl parathion, 5Methyl chrorpyrifos, 6 Dicofol, 7 Aldrin, 8 Thiabendazole, 9

Methidathion, 10 Chlordane, 11 Dieldrin, 12 Tebuconazole, 13Dithianon, 14 λ- cyhalothrin, and 15 Prochloraz and n-pentacosane asinternal standard (IS)

Food Anal. Methods

It is worth noting that dieldrin has been banished fromgrape plantations in Brazil since the 1990s, but it is a persistentcontaminant due to its high chemical stability (Milhomeet al. 2011). Quintozene is also a forbidden pesticide ingrape cultivation.

Dicofol is an authorized pesticide for many cultures, grapesincluded. However, residues of this compound were found inDC_G2 and DC_G3 samples, labeled as organic products.The dicofol contamination was also well above the MRL forgrapes (8.50 mg l-1) and was probably caused by a contami-nated water supply, as it is a water soluble pesticide.

Most of contaminated juices were produced with red grapes,except the three bottles of PE_B juice (G1, G2, and G3),produced with white grapes and contaminated with dicofol.

The contamination with dicofol and tebuconazole may becaused by excessive usage, while thiabendazol and dimetho-ate may occur due to soil or wind contamination or illegal use,since they are forbidden products.

Conclusion

A combined LLE and SPE (Florisil cartridge) allowed thevalidation of an analytical method to determine the residuesof multiclass pesticides in Brazilian whole grape juices. Thelimit of quantification of the method ranged from 3.75 to9.47 mg l-1, and the recovery ranged from 84.0 to 99.7 %,with a coefficient less than 4.9 %.

Table 3 Repeatability and recovery of the pesticides in GC-MS-SIM

Pesticide GC-MS-SIM

RSDa (%) Recoveryb (%)

Aldrin 1.9 93.9

Chlordane 4.9 94.9

Dicofol 3.5 86.5

Dieldrin 3.1 91.5

Quintozene 0.4 99.7

Dimethoate 0.5 86.3

Methidathion 0.2 94.8

Methyl chlorpyrifos 2.9 99.7

Methyl paration 0.5 89.3

Dithianon 0.9 93.9

λ-cyhalothrin 0.6 92.3

Carbaryl 4.4 87.8

Tebuconazole 1.8 91.2

Thiabendazole 3.9 84.0

Prochloraz 2.4 90.4

a Averages of repeatability for n=12bAverages of recovery for the seven concentrations used in the analyticalcurve

Table 4 Grape juices contaminated with pesticide residues

Pesticide residues Juice_bottle Concentration (mg l-1)

Aldrin CL_G1 13.20

Chlordane SB*_G1 13.29

SB*_G2 12.11

EP_G3 13.36

Dicofol CL_G1 34.00

DC*_G1 17.13

DC*_G2 21.38

DC*_G3 34.38

GA_G3 16.95

PE_B_G1 18.25

PE_B_G2 14.57

PE_B_G3 18.20

SA_isabelB_G3 15.28

Dieldrin SP*_ G1 60.17

Dimethoate AM_G1 14.50

AM_G2 17.00

AM_G3 16.20

SA_coraM_G3 15.58

Dithianon GB_G2 15.23

Quintozene AL_G1 15.85

AL_G2 18.71

AL_G3 34.52

CL_G1 21.66

DC*_G1 11.80

DC*_G2 12.55

DC*_G3 15.34

GA_G1 16.33

GA_G2 15.90

GA_G3 16.80

MT_G1 17.83

PE_B_G1 12.67

PZ_G1 16.67

PZ_G2 15.01

PZ_G3 18.33

SI_G3 13.34

Tebuconazole GA_G1 16.65

GA_G2 16.63

GA_G3 16.60

PE_ G1 13.53

PE_ G3 13.25

PZ_G1 16.65

PZ_G2 18.62

PZ_G3 19.11

SN_G2 13.34

Thiabendazole GB_G1 13.33

G1, G2, and G3 refer to the juice bottle sample

*Organic juice: soluble synthetic fertilizers, pesticides, and transgenicwere not used

Food Anal. Methods

The validated method was used to test 99 grape juices, and32 were found to be contaminated with pesticides residueswithin the method limits. All the pesticides found were eitherabove the MRL allowed by the Codex Alimentarius or werecompounds with unauthorized use on grape culture.

These findings point out the need for a more rigorouscontrol of grape juices. The contamination of six productslabeled as organic in a universe of 15 organic juices clearlystates that analytical monitoring rather than simply audit in-spections are needed. The method developed could be an aidin food safety actions.

Acknowledgments The authors thank CAPES for research fellowshipsand CNPq and FAPERJ for financial support.

Conflict of Interest Andréa A.R. Alves declares that he has no conflictof interest. Aline S. Rodrigues declares that he has no conflict of interest.Elisabete Paula Barros declares that he has no conflict of interest. ThaísM. Uekane declares that he has no conflict of interest. Humberto R. Bizzodeclares that he has no conflict of interest. Claudia M. Rezende declaresthat he has no conflict of interest. This article does not contain any studieswith human or animal subjects.

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