Journal of Environmental Science and Health, Part B (2015) 50, 338–345 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2015.1000178
Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides ANDREIA COELHO, M. FATIMA M. LUZ MAIA, LUISA CORREIA-SA, BARROSO, VALENTINA F. DOMINGUES and CRISTINA DELERUE-MATOS REQUIMTE/LAQV, Superior Institute of Engineering of Porto, Polytechnic Institute of Porto R. Dr. Ant onio Bernardino de Almeida, Porto, Portugal
Eruca sativa (rocket salad) has been intensely consumed all over the world, insomuch as, this work was undertaken to evaluate the antioxidant status and the environmental contamination (positive and negative nutritional contribution) of leaves and stems from this vegetable. Antioxidant capacity of rocket salad was assessed by mean of optical methods, such as the total phenolic content (TPC), reducing power assay and DPPH radical scavenging activity. The extent of the environmental contamination was reached through the quantification of thirteen organochlorine pesticides (OCP) by using gas chromatography coupled with electron-capture detector (GC-ECD) and compound confirmations employing gas chromatography tandem mass-spectrometry (GC-MS/MS). The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology.The extent of the environmental contamination was reached through the quantification of thirteen OCP by using gas chromatography coupled with electron-capture detector (GC-ECD) and compound confirmations employing GC-MS/MS. The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology. This demonstrated that leaves presented more antioxidant activity than stems, emphasizing that leaves contained six times more polyphenolic compounds than stems. In what concerns the OCP occurrence, the average recoveries obtained at the three levels tested (40, 60 and 80 mg kg¡1) ranged from 55% to 149% with a relative standard deviation of 11%, (except hexachrorobenzene). Three vegetables samples were collected from supermarkets and analysed following this study. According to data, only one sample achieved 16.21 of b-hexachlorocyclohexane, confirmed by GC-MS/MS. About OCP quantification, the data indicated that only one sample achieved 16.21 mg kg¡1 of b-hexachlorocyclohexane, confirmed by GC-MS/MS, being the QuEChERS a good choice for the of OCPs extraction. Furthermore, the leaves consumption guaranty higher levels of antioxidants than stems. Keywords: Rocket salad, antioxidant capacity, organochlorine pesticides, QuEChERS, GC-ECD, GC-MS/MS.
Introduction Eruca sativa, commonly named rocket salad, is produced mainly in Mediterranean countries and has been intensely used as a source of nutrition, an herb, an aphrodisiac and a medical plant. Its green leaves are used in salads all over the world due to its spicy hot taste and commercial availability since it has a short biological cycle of 45– 60 days.[1,2] Rocket salad is usually consumed fresh and has been described as containing several health promoting agents including fibers, proteins, calcium, iron, magnesium, vitamin A and C, carotenoids and flavonoids,[3–5] Address correspondence to Valentina F. Domingues, Superior Institute of Engineering of Porto, Rua Dr. Ant onio Bernardino de Almeida, 431, Porto 4200-072, Portugal; E-mail: [email protected], [email protected] Received August 27, 2014.
some of these agents are known as powerful antioxidants. Additionally, rocket salad also contains high levels of glucosinolates, flavonoids and phenolics. These compounds are known to have antioxidant and anticancer properties and have been related with the lowering of the risk of cardiovascular and cognitive diseases.[6,7] Previous studies showed that thirteen glucosinolates are present in rocket leaves, being eight aliphatic, one aromatic and four indole compounds.[8] Furthermore, glucosinolates and their derived products have been also reported to be able of inhibiting growth melanoma cells[9] and to present hepatoprotective activity, mediated by antioxidant effect on hepatic injury. Nevertheless, rocket salad seeds have been indicated to present a beneficial antidiabetic effect thought the reduction of the oxidative stress in cases of chemically induced diabetes mellitus.[3] The normal function of cellular metabolism results in a production of reactive oxygen species, which can lead to
Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides cell damage. Therefore, the living beings have developed complex antioxidant defense mechanisms (by using endogenous and/or exogenous antioxidants) to counteract oxidative damages induced by free radicals.[10] Food, such as vegetables is a great source of exogenous antioxidants, which contributes to the first and second defense lines against oxidative stress.[11] Despite the advantages of consuming vegetables, it is necessary to ensure that they are not contaminated by environmental pollutants, even organic food. During decades, various toxic pesticides were claimed to be safe, but several studies contradict this claim, so the use of some pesticides were banned or phased out because they posed risks for people. Organochlorine pesticides (OCP) are pesticides widespread used around the world, they are very persistent in the environment, and are known for accumulating in sediments, plants, animals and humans.[12] These OCP were extensively used for agriculture, which is a major contamination pathway for foodstuff.[13] The OCP has been described to be associated with many health problems, such as neurological damage, birth defects, respiratory illness, breast cancer, lowered sperm counts and immune system dysfunction.[14,15] Due to all of this problematic, the OCPs have been prohibited for agricultural and domestic use in Europe, North America and some countries of South America in accordance with Stockholm convention of 1980. However in Portugal the prohibition to use OCP is more recent (by the EU regulation: lindane was forbidding in 2002, metoxychlor in 2003 and endosulfan in 2007). Despite OCPs have been banned, they can still be detected, nowadays, due to their persistence and lipophilic properties.[16] Considering the high consumption rate of rocket salad, this homemade food cultivation, perhaps its consumption could be the major source of OCP exposure in population that look for healthy food. Therefore, it is necessary to determine OCP levels in rocket salad and to assess OCP exposure. The aim of this work was to evaluate the total antioxidant capacity (TAC) of rocket salad (leaves and stems) and to control the presence of OCPs in this vegetables (that could be a vehicle of these pesticides in human nutrition). The TAC was evaluated by using three conventional optical methods, such as the total phenolic content (TPC), reducing power assay and DPPH radical scavenging activity. The OCP exposure was reached through the quantification of thirteen organochlorine pesticides (OCP) by using gas chromatography coupled with electron-capture detector (GCECD) and compound confirmations employing gas chromatography tandem mass-spectrometry (GC-MS/ MS). The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology.
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Material and methods Reagents and materials For the TAC determinations: Gallic acid, 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble analogue of vitamin E) standards were from Sigma-Aldrich and Fluka, respectively. Folin-Ciocalteu reagent and DPPH were obtained from Sigma-Aldrich. All of the chemicals, of the highest quality available (95– 99%), were used without purification. Other compounds of analytical grade, such as sodium carbonate, ethanol and sodium acetate (0.1 mol L¡1, pH 4.3), were from Merck. All solvents used were of HPLC grade. Standard antioxidant solutions were daily prepared and stored in the dark at 4 C when not in use. Water used was deionized. All the spectrophotometric assays were performed in a Synergy HT W/TRF Multi Mode Microplate Reader with Gen5 2.0 software (BioTek Instruments, Winooski, VT, USA). For OCPs evaluation: thirteen OCPs were used in this study: a-, b-, g- and z - hexachlorocyclohexanes (HCH); hexachlorobenzene (HCB); o,p0 -DDT ([ 1,1,1 trichloro-2,2bis-(p-chlorophenyl) ethane]); p,p0 -DDE ([2,2-bis(p-chlorophenyl)-1,1-dichloroethylene]); p,p0 -DDD (dichlodiphenyldichloro-ethane); dieldrin; endrin; a-, b- endosulfan; and methoxychlor. Pesticide standards (purity >97.0%) were obtained from Chemservice (West Chester, PA, USA), Dr. Ehrenstorfer GmbH (Augsburg, Germany) and SigmaAldrich Co. The internal standard (IS) 4.40 - dichlorobenzophenone was purchased from Sigma-Aldrich (Laborche mikalien, Germany). The n-hexane and the acetonitrile (ACN) used were of HPLC grade from Merck (Darmstadt, Germany). The 13 OCPs standard stock solutions were prepared in n-hexane with different concentrations. Working mixed standard solutions containing the IS and the 13 OCPs studied were prepared from stock solutions. The stock solutions were stored at ¡20 C and the working solutions at 4 C. Sample preparation Samples of rocket salad were harvested in the metropolitan area of Porto and three packed samples were bought from three supermarkets brands (SM1, SM2 and SM3). The stems and leaves were separated, crushed and storage at ¡20 C. For antioxidants evaluation the extraction tests were performed with protection from light. About 4 g of each sample was weighed in a beaker and it was added 15 mL of the selected solvent extractor, etanol:water (1:4). The solution was under agitation for 1h after that, the content was filtered with a paper filter and kept at ¡20 C until analysis. For OCPs extraction a QuEChERS procedure was used. 44.2 mL of IS solution was added to a 50-mL polypropylene
340 centrifuge tube, this solution was dried under a nitrogen stream. The homogenized sample was weighed (4 g) into the 50-mL polypropylene centrifuge tube that has previously the IS. 10 mL of ACN was added and the resulting solution was sonicated in an ultrasonic bath working at 50/60 Hz and 100 W from RaypaÒ (Spain) for 5 min. After, the extraction sorbent was added (6 g of anhydrous magnesium sulphate, 1.5 g of sodium chloride, 1.5 g of trisodium citrate dehydrate and 0.75 g of disodium hydrogen citrate sesquihydrate) and this solution was also sonicated in the ultrasonic bath, for 10 min. The extracts were then centrifuged for 10 min at 4000 rpm in a Sartorius 2-16P-Spincontrol Universal model from SigmaÒ . A volume of 1 mL of prepared aliquot was sampled from the upper layer into a 2-mL centrifuge vial containing a clean-up sorbent (150 mg anhydrous magnesium sulphate, 50 mg primary secondary amine, 50 mg C18 and 50 mg graphitized carbon black) from AgilentÒ (Boeblingen, Germany). The tubes were capped tightly and vortexed for 5 min followed by centrifugation at 4000 rpm for 5 min. An aliquot of the supernatant (500 mL) was transferred to a vial, and the extract was concentrated just to dryness using a gentle stream of nitrogen. The sample residue was reconstituted in 500 mL of n-hexane. Finally, the sample was capped, vortexed and placed into an autosampler vial for analysis. TPC–were determined by using a colorimetric assay based on a modified procedure initially described by Singleton and Rossi.[17] Folin-Ciocalteu reagent and the reduced phenols produced a stable blue product at the end of reaction. The reaction mixture (25 mL of sample or standard solution, 75 mL of deionized water and 25 mL of Folin-Ciocalteu reagent) were mixed with 100 mL of 7% Na2CO3. After 90 min. the absorbance was measured at 765 nm in a microplate reader. TPC was expressed as gallic acid equivalents (GAE; mg gallic acid/100 g of sample) through the calibration curve of gallic acid. Calibration curves ranged from 10 to 200 mg of GA mL¡1 (r D 0.99). DPPH Radical Scavenging Activity (DPPH-RSA)[18] of samples against the stable nitrogen radical DPPH was determined spectrophotometrically at 765 nm. DPPH free radical is reduced to the corresponding hydrazine when it reacts with hydrogen donors, such as an antioxidant. In this technique, samples (25 mL) were mixed with 200 mL of ethanolic solution of DPPH (1.86 £ 10¡4 mol L¡1). The mixture, vigorously shaken, was left to stand for 30 min in the dark (until stable absorption values). Lower absorbance values of the reactive mixture indicated higher free radical scavenging activity. The calibration curve was prepared with Trolox solutions ranging from 50 to 500 mg of trolox mL¡1, and the results are given as mg of Trolox per 100 g of sample. Reducing Power Assay (RPA)[19] was obtained by adding 1 mL of sample mixed with 2.5 mL of 0.2 mol L¡1 sodium phosphate buffer (pH 6.6) and 2.5 mL of 1%
Maia et al. potassium ferricyanide. This mixture was incubated for 20 min at 50 C, and then 2.5 mL of 10% trichloroacetic acid (w/v) was added and centrifuged at 1000 rpm for 10 min. The upper layer of the solution (2.5 mL) was mixed with deionized water (2.5 mL) and 0.5 mL of 0.1% ferric chloride, and the absorbance was measured at 765 nm. The calibration curve was prepared with GA solutions ranging from 5 to 100 mg GA mL¡1. Results are given as mg of GAE per 100 g of sample. All measurements were performed in triplicate.
Gas chromatography OCPs were analyzed using a Shimadzu GC-2010 with an ECD detector, equipped with capillary column of 30 m, ZB-XLB (0.25 mm i.d., 0.25 mm film thickness, ZebronPhenomenex). The temperature was programmed starting at 65 C and held for 2 min, followed by increases of 8 C min¡1 to 160 C, then 2 C min¡1 to 235 C and then 15 C min¡1 to 250 C. The injection port was at 250 C splitless mode, and the detection was carried out at 300 C. Helium (Linde Sogas, purity 99.999%) was used as carrier gas at a constant flow rate of 1.3 mL min¡1, whereas nitrogen (Linde Sogas, purity 99.999%) was employed as makeup gas at a flow rate of 30 mL min¡1. The system was operated by GC-Solutions Shimadzu software. Method validation—For OCPs analysis the experimental method was validated according to the European Union SANCO guidelines (EC SANCO/10684/2009 directive) (European Commission 2009). Linearity, sensitivity, precision (repeatability, in terms of percent relative standard deviation) and accuracy (percentage recoveries) were evaluated. Rocket salad without previously detected pesticides was used for the fortification and calibration experiments. The limit of detection (LOD) and limit of quantification (LOQ) were determined by considering the slope of the calibration curve and the residual standard deviation of the regression line.[20] For the fortification study, the samples (4 g of homogenized rocket salad) were contaminated with the standard mixture (of 13 OCPs and IS) solution at levels of spiking 40, 60 and 80 mg kg¡1.
Gas chromatography- tandem mass spectrometry In addition, the samples with positive results by GC-ECD were analyzed using a Thermo Trace-Ultra gas chromatograph coupled to an ion trap mass detector Thermo Polaris (Dreieich, Germany), operated in the electronimpact ionization at 70 eV. The ion source temperature and the MS transfer temperature were at 250 C. The system was operated by Xcalibur v 1.3 software. Confirmation of residues was carried out by GC-MS/MS using also a 30 m, SLB-5ms (0.25 mm i.d., 0.25 mm film thickness, Supelco, Laborche mikalien, Germany). Column previously cut around 2 m in the injector side. The injector was
341
Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides operated in the splitless mode; helium was used as carrier gas at a constant flow rate of 1.3 mL min¡1. The injector was maintained at 240 C. The oven temperature was programmed starting at 40 C and held for 2 min, followed by increases of 30 C min¡1 to 220 C, held for 5 min, then 10 C min¡1 to 270 C, and held for 1 min. For the identification of pesticides, the retention time and three ions, the NIST and Wiley pesticide libraries were used. The MS/ MS conditions were fixed for each compound, trying to select as precursor ion the one with the highest m/z ratio and abundance.[21]
Results and discussion
and 80 mg Trolox E/100 g for rocket stems and leaves, respectively. While, when it was used the RPA assay, the levels for the rocket leaves and steam were 34 and 12 mg GAE/ 100 g, respectively. As it is possible to verify, higher antioxidant activities were obtained with the DPPH-RSA method than with the RPA assay. A previous work found DPPH-RSA values of around 140 mg Trolox E/ 100 g of rocket salad and around 200 mg Trolox E/ 100 g of wild rocket,[26] these values are generally higher than those found in this work. These results indicate that rocket salad is a good source of exogenous antioxidants, namely polyphenols compounds and should be used in the daily diet. OCPs quantification
Antioxidant capacity The assessment of the TAC of rocket salad, leaves and stems, was carried out by the determination of the TPC, DPPH-RSA and RPA. In what concerns the TPC assay, this method can be used to estimate the phenolic compounds present in the samples by the measurement of the chemical reducing capacity of compounds present in the extract relative to gallic acid. TPC of rocket stems and leaves ranged from 43 to 256 mg GAE/ 100 g, respectively. As it is possible to verify leaves presented 6 times more polyphenolic compounds than stems (Table 1). In fact, the antioxidant potential of a particular vegetable depends on the plant part assessed.[22] Some authors had measured the TPC and antioxidant potential of different part of plants, namely leaves, stems, flower buds, roots and seed verifying that different plant part corresponds to different TPC and antioxidant potential.[23,24] Nevertheless, it was found that kale seeds presented higher antioxidant potential than kale leaves, despite the fact that leaves were richer in phenolic compounds than seeds.[24] Heimler and collaborators had found high TPC levels in rocket and wild rocket, namely TPC levels of 208 and 100 mg GAE/100 g for rocket and wild rocket, respectively.,[25] for further, these values are similar to those found in this work. According to a previous work the kaempferol 3,40 -di-O-glucoside, quercetin 3-glucosyl, kaempferol 3-glucosyl, isorhamnetin3-glucosyl, are the major polyphenols compounds present in rocket leaves.[26] Antioxidant activity was achieved using DPPH-RSA and RPA assays (Table 1). However, the different methods tested showed diverse values of activity. Using the DPPH-RSA assay it was found values ranging from 23
Green vegetables are a complex matrix for the OCPs analysis due to their rich composition in vitamins, minerals and especially pigments (carotenoids and chlorophyll). Some authors have described that different vegetable composition (in terms of water, pigments, metabolites and texture) can lead to significantly differences in extraction efficiency.[27] Considering these difficulties and in order to developed an analytical methodology for the OCP identification and quantification, the linearity and calibration curves of OCP were performed and evaluated by injecting OCP standards diluted in n-hexane. It was achieved linearity between 12 and 88 mg L¡1 and the coefficient of correlation with six standards levels were higher than 0.992. QuEChERS optimization In order to optimize the QuEChERS protocol to be used as extraction technique it was important to test different amounts of the rocket sample and QuEChERS composition in order to improve the recoveries of the analytes. QuEChERS-6 g were composed by 6 g of anhydrous magnesium sulphate, 1.5 g of sodium chloride, 1.5 g of trisodium citrate dehydrate and 0.75 g of disodium hydrogen citrate sesquihydrate whereas the QuEChERS-4 g was composed by 4 g of anhydrous magnesium sulphate, 1 g of sodium chloride, 1 g of trisodium citrate dehydrate and 0.5 g of disodium hydrogen citrate sesquihydrate. The use of magnesium sulphate is important, in order to facilitate partitioning to remove water from the organic phase and to improve recovery of polar analytes. Trisodium citrate dehydrate and disodium hydrogen citrate sesquihydrate act as buffers. These QuEChERS compositions were described to be a good extractor source.[28,29] Different
Table 1. TPC, DPPH-RSA and RPA values for rocket salad leaves and stems.
Rocket leaves Rocket stems
TPC (mg GAE 100 g¡1)
DPPH-RSA (mg troloxE 100 g¡1)
RPA (mg GAE 100 g¡1)
256.7 § 2.1 43.4 § 0.9
79.5 § 0.4 23.2 § 1.1
34.2 § 0.1 11.9 § 0.1
342 amounts of the rocket sample (3g, 4g and 5g) were tested in order to observe the perform and the efficiency of QuEChERS chosen. The best recoveries were obtained, when it was used the QuEChERS composition of 6g of magnesium sulphate and 4g of rocket sample. Considering that green vegetables (such as rocket salad) present pigments (chlorophyll) it is required a clean-up step prior to GC analysis in order to remove all the natural pigments that interfere in the GC analysis. The clean-up used contained graphitized carbon black that are a strong sorbent capable to remove hydrophobic interaction-based compounds such as pigments.[30] Nevertheless, the use of a clean-up step affect the recovery of HCB, the best recovery of this analyte was 10% that are very low for 4g of sample and QuEChERS with 6 g. Despite the matrix effect (ME) of lindane, d-HCH and methoxychlor achieved with 4 g of sample and QuEChERS with 6g, this procedure was selected due to the higher recoveries obtained for the other OCPs.
Method validation The validation of the optimized chromatographic procedure was carried out. This experimental procedure was important for the identification and quantification of the 13 OCPs studied. Considering that, the GC-ECD presents the highest selectivity to halogenated compounds, this methodology is often used as detector for the OCPs determination.[28,31] The GC-ECD methodology validation was performed in order to evaluate specificity, precision, linearity, recoveries and limits of detection and quantification. Specificity was assessed by using rocket salad samples from biological culture (pesticides free) as nominated blank samples. Precision of the method was calculated by using
Maia et al.
Fig. 1. GC-ECD chromatogram of a spiked sample at 60 mg kg¡1 with a mixture of 13 OCPs and internal standard. 1 a-HCH, 2 HCB, 3 b-HCH, 4 lindane, 5 d-HCH, 6 IS, 7 a-endosulfan, 8 p,p0 -DDE, 9 dieldrin, 10 endrin, 11 b-Endosulfan, 12 p, p0 -DDD, 13 o,p0 -DDT and 14 methoxychlor.
rocket salad samples spiked at the levels of 40, 60 and 80 mg kg¡1 and analyzed in the GC-ECD with three replicates (Fig. 1). Satisfactory recoveries (from 55 to 149%) were obtained with a relative standard deviation of 11%, for all the compounds with the exception of HCB (Fig. 2). Rocket salad is a green vegetable rich in several compounds such as pigments, so this vegetable is a complex matrix that has a large amount of compounds that can interfere in the analytical signal, providing ME. This ME can cause decreased detection response or increased analytical signal, being a problem in the analysis of pesticide residues. To evaluate the ME in rocket salad samples and to ensure bias-free analytical results, OCPs standard solutions were analyzed in n-hexane (from 12 to 88 mg L¡1) and matrix matched standards (from 10 to 100 mg L¡1). The ME were assessed comparing the responses (which means the areas) obtained with the standard prepared in n-hexane with those prepared in blank rocket salad
Fig. 2. Recovery percentage of the three spiking (40, 60 and 80 mg kg¡1) values used.
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Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides Table 2. Validation parameters of the optimized GC-ECD method. Pesticides
Linearity Range (mg kg¡1)
Linearity (R2)
Regression Equation
Matrix Effect
LOD
LOQ
2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25
0.988 0.989 0.991 0.995 0.995 0.988 0.993 0.992 0.991 0.994 0.999 0.998 0.989
y D 2624.6x C 89799 y D 7287.1x C 97879 y D 3641.1x C 15040 y D 3952.4x C 39430 y D 3410.5x C 30573 y D 35698x C 156744 y D 31692x ¡ 55140 y D 65444x C 64403 y D 51682x ¡ 31456 y D 25249x ¡ 55637 y D 8139.8x ¡ 34882 y D 19092x ¡ 87315 y D 6154.9x ¡ 77696
0.22 1.33 0.31 0.52 0.32 0.95 0.46 0.94 0.96 0.13 2.99 0.74 0.96
3.5 2.9 3.1 2.3 2.1 1.4 2.4 2.6 2.7 2.2 0.9 1.4 1.5
11.6 9.6 10.4 7.7 7 4.8 8.1 8.5 8.9 7.3 3 4.6 4.9
a-HCH HCB b-HCH Lindane d-HCH a-Endosulfan p,p’-DDE Dieldrin Endrin b-Endosulfan p,p’-DDD o,p’-DDT Methoxychlor
extracts. MEs were confirmed for 9 of the 13 OCPs (Table 2). Consequently the complex matrix of the rocket salad sample interfere with the signal of some analytes, so to compensate these effects, matrix-matched standard calibration was used for calibration purposes (Table 2). The performance of the calibration curve on the matrix was obtained using blank rocket salad samples fortified with the standard solutions of the pesticides, at levels ranging from 2.5 to 25 mg kg¡1. All pesticide responses were linear in this concentration range, presenting a correlation coefficient (R2) higher than 0.988 (based on the measurement of each OCP peak areas obtained by GC-ECD). The LODs and LOQs were determined for all OCPs analyzed in rocket salad samples. The LOD values ranged from 0.9 to 3.5 mg kg¡1 and LOQ values ranged from 3 to 11.6 mg kg¡1 (Table 2). Considering that the EU maximum residue limited (MRL) defined is 10–50 mg kg¡1 the method still suits the purpose. Since LODs and LOQs are matrix dependent it was important to perform a matrixmatched calibration for quantitative analysis for the new samples tested with complex matrix such as fruit and vegetables.[32] The values obtained for LODs and LOQs are lower them the ones obtained by other authors,[32–35] but in the same range of values obtained from carrots[29] or in other published studies involving 20 vegetables.[36] In this present study a rapid procedure for the determination of OCPs in rocket salad samples was developed and validated. As described in this section, this method demonstrated good validation parameters and recoveries. The recoveries values obtained in this paper are similar to previous studies.[28,32,34–37] Table 3. Levels of OCPs achieved in rocket salad by GC-ECD. Pesticides b-HCH Lindane p,p’-DDD
SM1 (mg kg¡1) SM2 (mg kg¡1) SM3 (mg kg¡1) 16.27 16.82 5.03
16.21 12.52 9.44
15.83 17.01 10.87
Application to real samples The developed method was applied to quantify 13 OCPs in 3 different brand samples of commercialized rocket salad. The samples were obtained from 3 supermarkets (SM1, SM2 and SM3). The analysis obtained by GC-ECD showed the presence of 3 OCPs (b-HCH, lindane and p,p0 DDD), with concentrations above the LOQ, in all tested samples (Table 3). According with SANCO the OCPs identification should be confirmed by GC-MS. Following the recommendations, only b-HCH was confirmed to be present in levels above the MRL in SM2 rocket salad sample at concentration of 16.21 mg kg¡1. The existence of OCPs in vegetables, even though it was only one (b-HCH), is an alert for a campaign of surveillance that should be established. Although the use of OCPs has been banned for some years ago, the presence of these pesticides remains a reality today.[16]
Conclusions In this study, it was evaluated the TAC of rocket salad leaves and stems and it was verified that the rocket leaves presented 6 times more polyphenolic compounds than rocket stems. It was concluded that rocket salad is a good antioxidant source, the characterization of the antioxidant capacity is an important task in order to assess the quality of daily consumed vegetables. The possible presence of OCPs in commercialized samples of rocket salad from 3 supermarkets was also investigated.. An analytical methodology optimization was carried out to ensure satisfactory recoveries, since rocket salad is a very complex matrix. LODs and LOQs values were determined; the values ranged from 0.9 to 3.5 and from 3 to 11.6 respectively. Recovery percentages obtained presented values between 55 and 149%, with the exception of HCB. According to the obtained results it can be conclude that OCPs are very persistent in the environment, even after being banned for
344 decades. In conclusion these results highlight the importance of monitoring the presence of OCPs in food. The QuEChERS method applied in this study proved to be a good choice for the analysis of OCPs.
Funding Luísa Correia-S a and M. F. Barroso are grateful for the doc fellowship (SFRH/BD/87019/2012) and pos-doc financed by fellowship (SFRH/BPD/78845/2011), respectively, POPH - QREN - Tipologia 4.1 - Forma¸c ~ao Avan¸c ada, subsidized by Fundo Social Europeu and Ministerio da Ci^encia, Tecnologia e Ensino Superior. This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Funda¸c ~ ao para a Ci^encia e Tecnologia) through project Pest-C/EQB/LA0006/2013. The work also received financial support from the European Union (FEDER funds) under the framework of QREN through Project NORTE-07-0124-FEDER-000069 (ALIMENTO). To all financing sources the authors are greatly indebted.
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Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides ANDREIA COELHO, M. FATIMA M. LUZ MAIA, LUISA CORREIA-SA, BARROSO, VALENTINA F. DOMINGUES and CRISTINA DELERUE-MATOS REQUIMTE/LAQV, Superior Institute of Engineering of Porto, Polytechnic Institute of Porto R. Dr. Ant onio Bernardino de Almeida, Porto, Portugal
Eruca sativa (rocket salad) has been intensely consumed all over the world, insomuch as, this work was undertaken to evaluate the antioxidant status and the environmental contamination (positive and negative nutritional contribution) of leaves and stems from this vegetable. Antioxidant capacity of rocket salad was assessed by mean of optical methods, such as the total phenolic content (TPC), reducing power assay and DPPH radical scavenging activity. The extent of the environmental contamination was reached through the quantification of thirteen organochlorine pesticides (OCP) by using gas chromatography coupled with electron-capture detector (GC-ECD) and compound confirmations employing gas chromatography tandem mass-spectrometry (GC-MS/MS). The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology.The extent of the environmental contamination was reached through the quantification of thirteen OCP by using gas chromatography coupled with electron-capture detector (GC-ECD) and compound confirmations employing GC-MS/MS. The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology. This demonstrated that leaves presented more antioxidant activity than stems, emphasizing that leaves contained six times more polyphenolic compounds than stems. In what concerns the OCP occurrence, the average recoveries obtained at the three levels tested (40, 60 and 80 mg kg¡1) ranged from 55% to 149% with a relative standard deviation of 11%, (except hexachrorobenzene). Three vegetables samples were collected from supermarkets and analysed following this study. According to data, only one sample achieved 16.21 of b-hexachlorocyclohexane, confirmed by GC-MS/MS. About OCP quantification, the data indicated that only one sample achieved 16.21 mg kg¡1 of b-hexachlorocyclohexane, confirmed by GC-MS/MS, being the QuEChERS a good choice for the of OCPs extraction. Furthermore, the leaves consumption guaranty higher levels of antioxidants than stems. Keywords: Rocket salad, antioxidant capacity, organochlorine pesticides, QuEChERS, GC-ECD, GC-MS/MS.
Introduction Eruca sativa, commonly named rocket salad, is produced mainly in Mediterranean countries and has been intensely used as a source of nutrition, an herb, an aphrodisiac and a medical plant. Its green leaves are used in salads all over the world due to its spicy hot taste and commercial availability since it has a short biological cycle of 45– 60 days.[1,2] Rocket salad is usually consumed fresh and has been described as containing several health promoting agents including fibers, proteins, calcium, iron, magnesium, vitamin A and C, carotenoids and flavonoids,[3–5] Address correspondence to Valentina F. Domingues, Superior Institute of Engineering of Porto, Rua Dr. Ant onio Bernardino de Almeida, 431, Porto 4200-072, Portugal; E-mail: [email protected], [email protected] Received August 27, 2014.
some of these agents are known as powerful antioxidants. Additionally, rocket salad also contains high levels of glucosinolates, flavonoids and phenolics. These compounds are known to have antioxidant and anticancer properties and have been related with the lowering of the risk of cardiovascular and cognitive diseases.[6,7] Previous studies showed that thirteen glucosinolates are present in rocket leaves, being eight aliphatic, one aromatic and four indole compounds.[8] Furthermore, glucosinolates and their derived products have been also reported to be able of inhibiting growth melanoma cells[9] and to present hepatoprotective activity, mediated by antioxidant effect on hepatic injury. Nevertheless, rocket salad seeds have been indicated to present a beneficial antidiabetic effect thought the reduction of the oxidative stress in cases of chemically induced diabetes mellitus.[3] The normal function of cellular metabolism results in a production of reactive oxygen species, which can lead to
Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides cell damage. Therefore, the living beings have developed complex antioxidant defense mechanisms (by using endogenous and/or exogenous antioxidants) to counteract oxidative damages induced by free radicals.[10] Food, such as vegetables is a great source of exogenous antioxidants, which contributes to the first and second defense lines against oxidative stress.[11] Despite the advantages of consuming vegetables, it is necessary to ensure that they are not contaminated by environmental pollutants, even organic food. During decades, various toxic pesticides were claimed to be safe, but several studies contradict this claim, so the use of some pesticides were banned or phased out because they posed risks for people. Organochlorine pesticides (OCP) are pesticides widespread used around the world, they are very persistent in the environment, and are known for accumulating in sediments, plants, animals and humans.[12] These OCP were extensively used for agriculture, which is a major contamination pathway for foodstuff.[13] The OCP has been described to be associated with many health problems, such as neurological damage, birth defects, respiratory illness, breast cancer, lowered sperm counts and immune system dysfunction.[14,15] Due to all of this problematic, the OCPs have been prohibited for agricultural and domestic use in Europe, North America and some countries of South America in accordance with Stockholm convention of 1980. However in Portugal the prohibition to use OCP is more recent (by the EU regulation: lindane was forbidding in 2002, metoxychlor in 2003 and endosulfan in 2007). Despite OCPs have been banned, they can still be detected, nowadays, due to their persistence and lipophilic properties.[16] Considering the high consumption rate of rocket salad, this homemade food cultivation, perhaps its consumption could be the major source of OCP exposure in population that look for healthy food. Therefore, it is necessary to determine OCP levels in rocket salad and to assess OCP exposure. The aim of this work was to evaluate the total antioxidant capacity (TAC) of rocket salad (leaves and stems) and to control the presence of OCPs in this vegetables (that could be a vehicle of these pesticides in human nutrition). The TAC was evaluated by using three conventional optical methods, such as the total phenolic content (TPC), reducing power assay and DPPH radical scavenging activity. The OCP exposure was reached through the quantification of thirteen organochlorine pesticides (OCP) by using gas chromatography coupled with electron-capture detector (GCECD) and compound confirmations employing gas chromatography tandem mass-spectrometry (GC-MS/ MS). The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology.
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Material and methods Reagents and materials For the TAC determinations: Gallic acid, 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble analogue of vitamin E) standards were from Sigma-Aldrich and Fluka, respectively. Folin-Ciocalteu reagent and DPPH were obtained from Sigma-Aldrich. All of the chemicals, of the highest quality available (95– 99%), were used without purification. Other compounds of analytical grade, such as sodium carbonate, ethanol and sodium acetate (0.1 mol L¡1, pH 4.3), were from Merck. All solvents used were of HPLC grade. Standard antioxidant solutions were daily prepared and stored in the dark at 4 C when not in use. Water used was deionized. All the spectrophotometric assays were performed in a Synergy HT W/TRF Multi Mode Microplate Reader with Gen5 2.0 software (BioTek Instruments, Winooski, VT, USA). For OCPs evaluation: thirteen OCPs were used in this study: a-, b-, g- and z - hexachlorocyclohexanes (HCH); hexachlorobenzene (HCB); o,p0 -DDT ([ 1,1,1 trichloro-2,2bis-(p-chlorophenyl) ethane]); p,p0 -DDE ([2,2-bis(p-chlorophenyl)-1,1-dichloroethylene]); p,p0 -DDD (dichlodiphenyldichloro-ethane); dieldrin; endrin; a-, b- endosulfan; and methoxychlor. Pesticide standards (purity >97.0%) were obtained from Chemservice (West Chester, PA, USA), Dr. Ehrenstorfer GmbH (Augsburg, Germany) and SigmaAldrich Co. The internal standard (IS) 4.40 - dichlorobenzophenone was purchased from Sigma-Aldrich (Laborche mikalien, Germany). The n-hexane and the acetonitrile (ACN) used were of HPLC grade from Merck (Darmstadt, Germany). The 13 OCPs standard stock solutions were prepared in n-hexane with different concentrations. Working mixed standard solutions containing the IS and the 13 OCPs studied were prepared from stock solutions. The stock solutions were stored at ¡20 C and the working solutions at 4 C. Sample preparation Samples of rocket salad were harvested in the metropolitan area of Porto and three packed samples were bought from three supermarkets brands (SM1, SM2 and SM3). The stems and leaves were separated, crushed and storage at ¡20 C. For antioxidants evaluation the extraction tests were performed with protection from light. About 4 g of each sample was weighed in a beaker and it was added 15 mL of the selected solvent extractor, etanol:water (1:4). The solution was under agitation for 1h after that, the content was filtered with a paper filter and kept at ¡20 C until analysis. For OCPs extraction a QuEChERS procedure was used. 44.2 mL of IS solution was added to a 50-mL polypropylene
340 centrifuge tube, this solution was dried under a nitrogen stream. The homogenized sample was weighed (4 g) into the 50-mL polypropylene centrifuge tube that has previously the IS. 10 mL of ACN was added and the resulting solution was sonicated in an ultrasonic bath working at 50/60 Hz and 100 W from RaypaÒ (Spain) for 5 min. After, the extraction sorbent was added (6 g of anhydrous magnesium sulphate, 1.5 g of sodium chloride, 1.5 g of trisodium citrate dehydrate and 0.75 g of disodium hydrogen citrate sesquihydrate) and this solution was also sonicated in the ultrasonic bath, for 10 min. The extracts were then centrifuged for 10 min at 4000 rpm in a Sartorius 2-16P-Spincontrol Universal model from SigmaÒ . A volume of 1 mL of prepared aliquot was sampled from the upper layer into a 2-mL centrifuge vial containing a clean-up sorbent (150 mg anhydrous magnesium sulphate, 50 mg primary secondary amine, 50 mg C18 and 50 mg graphitized carbon black) from AgilentÒ (Boeblingen, Germany). The tubes were capped tightly and vortexed for 5 min followed by centrifugation at 4000 rpm for 5 min. An aliquot of the supernatant (500 mL) was transferred to a vial, and the extract was concentrated just to dryness using a gentle stream of nitrogen. The sample residue was reconstituted in 500 mL of n-hexane. Finally, the sample was capped, vortexed and placed into an autosampler vial for analysis. TPC–were determined by using a colorimetric assay based on a modified procedure initially described by Singleton and Rossi.[17] Folin-Ciocalteu reagent and the reduced phenols produced a stable blue product at the end of reaction. The reaction mixture (25 mL of sample or standard solution, 75 mL of deionized water and 25 mL of Folin-Ciocalteu reagent) were mixed with 100 mL of 7% Na2CO3. After 90 min. the absorbance was measured at 765 nm in a microplate reader. TPC was expressed as gallic acid equivalents (GAE; mg gallic acid/100 g of sample) through the calibration curve of gallic acid. Calibration curves ranged from 10 to 200 mg of GA mL¡1 (r D 0.99). DPPH Radical Scavenging Activity (DPPH-RSA)[18] of samples against the stable nitrogen radical DPPH was determined spectrophotometrically at 765 nm. DPPH free radical is reduced to the corresponding hydrazine when it reacts with hydrogen donors, such as an antioxidant. In this technique, samples (25 mL) were mixed with 200 mL of ethanolic solution of DPPH (1.86 £ 10¡4 mol L¡1). The mixture, vigorously shaken, was left to stand for 30 min in the dark (until stable absorption values). Lower absorbance values of the reactive mixture indicated higher free radical scavenging activity. The calibration curve was prepared with Trolox solutions ranging from 50 to 500 mg of trolox mL¡1, and the results are given as mg of Trolox per 100 g of sample. Reducing Power Assay (RPA)[19] was obtained by adding 1 mL of sample mixed with 2.5 mL of 0.2 mol L¡1 sodium phosphate buffer (pH 6.6) and 2.5 mL of 1%
Maia et al. potassium ferricyanide. This mixture was incubated for 20 min at 50 C, and then 2.5 mL of 10% trichloroacetic acid (w/v) was added and centrifuged at 1000 rpm for 10 min. The upper layer of the solution (2.5 mL) was mixed with deionized water (2.5 mL) and 0.5 mL of 0.1% ferric chloride, and the absorbance was measured at 765 nm. The calibration curve was prepared with GA solutions ranging from 5 to 100 mg GA mL¡1. Results are given as mg of GAE per 100 g of sample. All measurements were performed in triplicate.
Gas chromatography OCPs were analyzed using a Shimadzu GC-2010 with an ECD detector, equipped with capillary column of 30 m, ZB-XLB (0.25 mm i.d., 0.25 mm film thickness, ZebronPhenomenex). The temperature was programmed starting at 65 C and held for 2 min, followed by increases of 8 C min¡1 to 160 C, then 2 C min¡1 to 235 C and then 15 C min¡1 to 250 C. The injection port was at 250 C splitless mode, and the detection was carried out at 300 C. Helium (Linde Sogas, purity 99.999%) was used as carrier gas at a constant flow rate of 1.3 mL min¡1, whereas nitrogen (Linde Sogas, purity 99.999%) was employed as makeup gas at a flow rate of 30 mL min¡1. The system was operated by GC-Solutions Shimadzu software. Method validation—For OCPs analysis the experimental method was validated according to the European Union SANCO guidelines (EC SANCO/10684/2009 directive) (European Commission 2009). Linearity, sensitivity, precision (repeatability, in terms of percent relative standard deviation) and accuracy (percentage recoveries) were evaluated. Rocket salad without previously detected pesticides was used for the fortification and calibration experiments. The limit of detection (LOD) and limit of quantification (LOQ) were determined by considering the slope of the calibration curve and the residual standard deviation of the regression line.[20] For the fortification study, the samples (4 g of homogenized rocket salad) were contaminated with the standard mixture (of 13 OCPs and IS) solution at levels of spiking 40, 60 and 80 mg kg¡1.
Gas chromatography- tandem mass spectrometry In addition, the samples with positive results by GC-ECD were analyzed using a Thermo Trace-Ultra gas chromatograph coupled to an ion trap mass detector Thermo Polaris (Dreieich, Germany), operated in the electronimpact ionization at 70 eV. The ion source temperature and the MS transfer temperature were at 250 C. The system was operated by Xcalibur v 1.3 software. Confirmation of residues was carried out by GC-MS/MS using also a 30 m, SLB-5ms (0.25 mm i.d., 0.25 mm film thickness, Supelco, Laborche mikalien, Germany). Column previously cut around 2 m in the injector side. The injector was
341
Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides operated in the splitless mode; helium was used as carrier gas at a constant flow rate of 1.3 mL min¡1. The injector was maintained at 240 C. The oven temperature was programmed starting at 40 C and held for 2 min, followed by increases of 30 C min¡1 to 220 C, held for 5 min, then 10 C min¡1 to 270 C, and held for 1 min. For the identification of pesticides, the retention time and three ions, the NIST and Wiley pesticide libraries were used. The MS/ MS conditions were fixed for each compound, trying to select as precursor ion the one with the highest m/z ratio and abundance.[21]
Results and discussion
and 80 mg Trolox E/100 g for rocket stems and leaves, respectively. While, when it was used the RPA assay, the levels for the rocket leaves and steam were 34 and 12 mg GAE/ 100 g, respectively. As it is possible to verify, higher antioxidant activities were obtained with the DPPH-RSA method than with the RPA assay. A previous work found DPPH-RSA values of around 140 mg Trolox E/ 100 g of rocket salad and around 200 mg Trolox E/ 100 g of wild rocket,[26] these values are generally higher than those found in this work. These results indicate that rocket salad is a good source of exogenous antioxidants, namely polyphenols compounds and should be used in the daily diet. OCPs quantification
Antioxidant capacity The assessment of the TAC of rocket salad, leaves and stems, was carried out by the determination of the TPC, DPPH-RSA and RPA. In what concerns the TPC assay, this method can be used to estimate the phenolic compounds present in the samples by the measurement of the chemical reducing capacity of compounds present in the extract relative to gallic acid. TPC of rocket stems and leaves ranged from 43 to 256 mg GAE/ 100 g, respectively. As it is possible to verify leaves presented 6 times more polyphenolic compounds than stems (Table 1). In fact, the antioxidant potential of a particular vegetable depends on the plant part assessed.[22] Some authors had measured the TPC and antioxidant potential of different part of plants, namely leaves, stems, flower buds, roots and seed verifying that different plant part corresponds to different TPC and antioxidant potential.[23,24] Nevertheless, it was found that kale seeds presented higher antioxidant potential than kale leaves, despite the fact that leaves were richer in phenolic compounds than seeds.[24] Heimler and collaborators had found high TPC levels in rocket and wild rocket, namely TPC levels of 208 and 100 mg GAE/100 g for rocket and wild rocket, respectively.,[25] for further, these values are similar to those found in this work. According to a previous work the kaempferol 3,40 -di-O-glucoside, quercetin 3-glucosyl, kaempferol 3-glucosyl, isorhamnetin3-glucosyl, are the major polyphenols compounds present in rocket leaves.[26] Antioxidant activity was achieved using DPPH-RSA and RPA assays (Table 1). However, the different methods tested showed diverse values of activity. Using the DPPH-RSA assay it was found values ranging from 23
Green vegetables are a complex matrix for the OCPs analysis due to their rich composition in vitamins, minerals and especially pigments (carotenoids and chlorophyll). Some authors have described that different vegetable composition (in terms of water, pigments, metabolites and texture) can lead to significantly differences in extraction efficiency.[27] Considering these difficulties and in order to developed an analytical methodology for the OCP identification and quantification, the linearity and calibration curves of OCP were performed and evaluated by injecting OCP standards diluted in n-hexane. It was achieved linearity between 12 and 88 mg L¡1 and the coefficient of correlation with six standards levels were higher than 0.992. QuEChERS optimization In order to optimize the QuEChERS protocol to be used as extraction technique it was important to test different amounts of the rocket sample and QuEChERS composition in order to improve the recoveries of the analytes. QuEChERS-6 g were composed by 6 g of anhydrous magnesium sulphate, 1.5 g of sodium chloride, 1.5 g of trisodium citrate dehydrate and 0.75 g of disodium hydrogen citrate sesquihydrate whereas the QuEChERS-4 g was composed by 4 g of anhydrous magnesium sulphate, 1 g of sodium chloride, 1 g of trisodium citrate dehydrate and 0.5 g of disodium hydrogen citrate sesquihydrate. The use of magnesium sulphate is important, in order to facilitate partitioning to remove water from the organic phase and to improve recovery of polar analytes. Trisodium citrate dehydrate and disodium hydrogen citrate sesquihydrate act as buffers. These QuEChERS compositions were described to be a good extractor source.[28,29] Different
Table 1. TPC, DPPH-RSA and RPA values for rocket salad leaves and stems.
Rocket leaves Rocket stems
TPC (mg GAE 100 g¡1)
DPPH-RSA (mg troloxE 100 g¡1)
RPA (mg GAE 100 g¡1)
256.7 § 2.1 43.4 § 0.9
79.5 § 0.4 23.2 § 1.1
34.2 § 0.1 11.9 § 0.1
342 amounts of the rocket sample (3g, 4g and 5g) were tested in order to observe the perform and the efficiency of QuEChERS chosen. The best recoveries were obtained, when it was used the QuEChERS composition of 6g of magnesium sulphate and 4g of rocket sample. Considering that green vegetables (such as rocket salad) present pigments (chlorophyll) it is required a clean-up step prior to GC analysis in order to remove all the natural pigments that interfere in the GC analysis. The clean-up used contained graphitized carbon black that are a strong sorbent capable to remove hydrophobic interaction-based compounds such as pigments.[30] Nevertheless, the use of a clean-up step affect the recovery of HCB, the best recovery of this analyte was 10% that are very low for 4g of sample and QuEChERS with 6 g. Despite the matrix effect (ME) of lindane, d-HCH and methoxychlor achieved with 4 g of sample and QuEChERS with 6g, this procedure was selected due to the higher recoveries obtained for the other OCPs.
Method validation The validation of the optimized chromatographic procedure was carried out. This experimental procedure was important for the identification and quantification of the 13 OCPs studied. Considering that, the GC-ECD presents the highest selectivity to halogenated compounds, this methodology is often used as detector for the OCPs determination.[28,31] The GC-ECD methodology validation was performed in order to evaluate specificity, precision, linearity, recoveries and limits of detection and quantification. Specificity was assessed by using rocket salad samples from biological culture (pesticides free) as nominated blank samples. Precision of the method was calculated by using
Maia et al.
Fig. 1. GC-ECD chromatogram of a spiked sample at 60 mg kg¡1 with a mixture of 13 OCPs and internal standard. 1 a-HCH, 2 HCB, 3 b-HCH, 4 lindane, 5 d-HCH, 6 IS, 7 a-endosulfan, 8 p,p0 -DDE, 9 dieldrin, 10 endrin, 11 b-Endosulfan, 12 p, p0 -DDD, 13 o,p0 -DDT and 14 methoxychlor.
rocket salad samples spiked at the levels of 40, 60 and 80 mg kg¡1 and analyzed in the GC-ECD with three replicates (Fig. 1). Satisfactory recoveries (from 55 to 149%) were obtained with a relative standard deviation of 11%, for all the compounds with the exception of HCB (Fig. 2). Rocket salad is a green vegetable rich in several compounds such as pigments, so this vegetable is a complex matrix that has a large amount of compounds that can interfere in the analytical signal, providing ME. This ME can cause decreased detection response or increased analytical signal, being a problem in the analysis of pesticide residues. To evaluate the ME in rocket salad samples and to ensure bias-free analytical results, OCPs standard solutions were analyzed in n-hexane (from 12 to 88 mg L¡1) and matrix matched standards (from 10 to 100 mg L¡1). The ME were assessed comparing the responses (which means the areas) obtained with the standard prepared in n-hexane with those prepared in blank rocket salad
Fig. 2. Recovery percentage of the three spiking (40, 60 and 80 mg kg¡1) values used.
343
Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides Table 2. Validation parameters of the optimized GC-ECD method. Pesticides
Linearity Range (mg kg¡1)
Linearity (R2)
Regression Equation
Matrix Effect
LOD
LOQ
2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25 2.5–25
0.988 0.989 0.991 0.995 0.995 0.988 0.993 0.992 0.991 0.994 0.999 0.998 0.989
y D 2624.6x C 89799 y D 7287.1x C 97879 y D 3641.1x C 15040 y D 3952.4x C 39430 y D 3410.5x C 30573 y D 35698x C 156744 y D 31692x ¡ 55140 y D 65444x C 64403 y D 51682x ¡ 31456 y D 25249x ¡ 55637 y D 8139.8x ¡ 34882 y D 19092x ¡ 87315 y D 6154.9x ¡ 77696
0.22 1.33 0.31 0.52 0.32 0.95 0.46 0.94 0.96 0.13 2.99 0.74 0.96
3.5 2.9 3.1 2.3 2.1 1.4 2.4 2.6 2.7 2.2 0.9 1.4 1.5
11.6 9.6 10.4 7.7 7 4.8 8.1 8.5 8.9 7.3 3 4.6 4.9
a-HCH HCB b-HCH Lindane d-HCH a-Endosulfan p,p’-DDE Dieldrin Endrin b-Endosulfan p,p’-DDD o,p’-DDT Methoxychlor
extracts. MEs were confirmed for 9 of the 13 OCPs (Table 2). Consequently the complex matrix of the rocket salad sample interfere with the signal of some analytes, so to compensate these effects, matrix-matched standard calibration was used for calibration purposes (Table 2). The performance of the calibration curve on the matrix was obtained using blank rocket salad samples fortified with the standard solutions of the pesticides, at levels ranging from 2.5 to 25 mg kg¡1. All pesticide responses were linear in this concentration range, presenting a correlation coefficient (R2) higher than 0.988 (based on the measurement of each OCP peak areas obtained by GC-ECD). The LODs and LOQs were determined for all OCPs analyzed in rocket salad samples. The LOD values ranged from 0.9 to 3.5 mg kg¡1 and LOQ values ranged from 3 to 11.6 mg kg¡1 (Table 2). Considering that the EU maximum residue limited (MRL) defined is 10–50 mg kg¡1 the method still suits the purpose. Since LODs and LOQs are matrix dependent it was important to perform a matrixmatched calibration for quantitative analysis for the new samples tested with complex matrix such as fruit and vegetables.[32] The values obtained for LODs and LOQs are lower them the ones obtained by other authors,[32–35] but in the same range of values obtained from carrots[29] or in other published studies involving 20 vegetables.[36] In this present study a rapid procedure for the determination of OCPs in rocket salad samples was developed and validated. As described in this section, this method demonstrated good validation parameters and recoveries. The recoveries values obtained in this paper are similar to previous studies.[28,32,34–37] Table 3. Levels of OCPs achieved in rocket salad by GC-ECD. Pesticides b-HCH Lindane p,p’-DDD
SM1 (mg kg¡1) SM2 (mg kg¡1) SM3 (mg kg¡1) 16.27 16.82 5.03
16.21 12.52 9.44
15.83 17.01 10.87
Application to real samples The developed method was applied to quantify 13 OCPs in 3 different brand samples of commercialized rocket salad. The samples were obtained from 3 supermarkets (SM1, SM2 and SM3). The analysis obtained by GC-ECD showed the presence of 3 OCPs (b-HCH, lindane and p,p0 DDD), with concentrations above the LOQ, in all tested samples (Table 3). According with SANCO the OCPs identification should be confirmed by GC-MS. Following the recommendations, only b-HCH was confirmed to be present in levels above the MRL in SM2 rocket salad sample at concentration of 16.21 mg kg¡1. The existence of OCPs in vegetables, even though it was only one (b-HCH), is an alert for a campaign of surveillance that should be established. Although the use of OCPs has been banned for some years ago, the presence of these pesticides remains a reality today.[16]
Conclusions In this study, it was evaluated the TAC of rocket salad leaves and stems and it was verified that the rocket leaves presented 6 times more polyphenolic compounds than rocket stems. It was concluded that rocket salad is a good antioxidant source, the characterization of the antioxidant capacity is an important task in order to assess the quality of daily consumed vegetables. The possible presence of OCPs in commercialized samples of rocket salad from 3 supermarkets was also investigated.. An analytical methodology optimization was carried out to ensure satisfactory recoveries, since rocket salad is a very complex matrix. LODs and LOQs values were determined; the values ranged from 0.9 to 3.5 and from 3 to 11.6 respectively. Recovery percentages obtained presented values between 55 and 149%, with the exception of HCB. According to the obtained results it can be conclude that OCPs are very persistent in the environment, even after being banned for
344 decades. In conclusion these results highlight the importance of monitoring the presence of OCPs in food. The QuEChERS method applied in this study proved to be a good choice for the analysis of OCPs.
Funding Luísa Correia-S a and M. F. Barroso are grateful for the doc fellowship (SFRH/BD/87019/2012) and pos-doc financed by fellowship (SFRH/BPD/78845/2011), respectively, POPH - QREN - Tipologia 4.1 - Forma¸c ~ao Avan¸c ada, subsidized by Fundo Social Europeu and Ministerio da Ci^encia, Tecnologia e Ensino Superior. This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Funda¸c ~ ao para a Ci^encia e Tecnologia) through project Pest-C/EQB/LA0006/2013. The work also received financial support from the European Union (FEDER funds) under the framework of QREN through Project NORTE-07-0124-FEDER-000069 (ALIMENTO). To all financing sources the authors are greatly indebted.
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