Development of a matrix solid-phase dispersion method for the simultaneous determination of pyrethroid and organochlorinated pesticides in cattle feed

Journal of Chromatography A, 1216 (2009) 2832–2842

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Development of a matrix solid-phase dispersion method for the simultaneous determination of pyrethroid and organochlorinated pesticides in cattle feed Maria Fernandez-Alvarez a , Maria Llompart a,∗ , J. Pablo Lamas a , Marta Lores a , Carmen Garcia-Jares a , Rafael Cela a , Thierry Dagnac b a Departamento de Quimica Analitica, Nutricion y Bromatologia, Instituto de Investigacion y Analisis Alimentario, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain b INGACAL (Galician Institute for Food Quality)-CIAM (Agrarian and Agronomic Research Centre), Laboratory of Food/Feed Safety and Organic Contaminants, Apartado 10, E-15080 A Coru˜ na, Spain

a r t i c l e

i n f o

Article history: Available online 14 October 2008 Keywords: Matrix solid-phase dispersion Feed analysis Cattle feed Feedingstuff Pyrethroids Organochlorine pesticides Experimental design Gas chromatography

a b s t r a c t A matrix solid-phase dispersion (MSPD) method was developed for the simultaneous extraction of 36 common pesticides and breakdown products (mostly pyrethroids and organochlorines) in cattle feed. Different parameters affecting the extraction efficiency (such as dispersing phase, clean-up adsorbent and elution volume) were investigated. The experimental procedure was optimized using a multivariate statistical approach and the final analyses were carried out by GC–␮ECD. Several protocols for extract purification were also studied. As far as we know, this is the first application of MSPD for the extraction of most of the target pesticides from animal feed. Using the optimized extraction conditions, the method was validated in terms of accuracy, and precision (within-a-day and among-days), using a certified reference material (CRM 115) as well as spiked cattle feedingstuffs at different concentration levels. A matrix effect study was also carried out using various real samples. The recoveries were satisfactory (>75% in most cases) and the quantification limits, at the sub-ng g−1 or low-ng g−1 level, complied with the regulated maximum residue levels (MRLs) in animal feed and in main crops used in the preparation of cattle feeding materials. Finally, the MSPD–GC–␮ECD methodology was applied to the analysis of real cattle feed samples collected in farms of dairy cattle from NW Spain. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Pesticides are broadly used in farming for their economic benefits to fight crop pests and reduce competition from weeds, thus improving yields and protecting the quality, reliability and price of production. The widespread use of these compounds has resulted in contamination of environmental compartments, such as surface water, groundwater, soil and air [1–4]. Also, agricultural plants used for feeding of livestock may be contaminated and, consequently, pesticides may become available for human consumption via animal feed. In recent years, increasing attention has been paid to the risk posed to consumers by chemical contaminants or residues in feedingstuffs [5]. Therefore, rules on undesirable substances in these matrices are needed to ensure agricultural productivity and sustainability and to guarantee public and animal health, animal welfare and environment protection. In this sense, maximum residue limits (MRLs) in feedingstuffs for, among other compounds,

∗ Corresponding author. Tel.: +34 981563100; fax: +34 981595012. E-mail address: [email protected] (M. Llompart). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.10.026

organochlorine pesticides (OCPs), have been established by the European Union in the Directive 2002/32/EC [6]. Besides, maize, barley, soya bean and wheat are the most common crop ingredients in the preparation of cattle feedingstuffs; thus, EU regulations with respect to pesticide residues in these plants should also be considered [7]. Even though most of the OCPs have been forbidden decades ago, recent studies have documented the presence of ␣-, ␤- and ␥-lindane, isomers of DDE, DDD and DDT, endosulfan sulphate and heptachlor epoxide in animal feed (some of them at concentrations far higher than their MRL) [8,9]. For the past three decades, organophosphorus pesticides (OPPs) have been the insecticides most commonly used by both professional pest control bodies and homeowners. Nevertheless, the decision of the US Environmental Protection Agency (EPA) to phase out certain uses of the organophosphate insecticides—because of their potentially toxic effects to humans—has led to their gradual replacement by pyrethroid pesticides [10,11]. Both groups of pesticides have also been detected in animal feed and cereals [9,12]. A total of 36 pesticides and breakdown products have been studied in the present work. Most of them are organochlorine and pyrethroid pesticides, although other main plant protection

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Table 1 Current legislation regarding the residues of the target pesticides in animal feed and some related crops. Class

Pesticide

Organochlorine

␣-Lindane ␤-Lindane

␥-Lindane Heptachlor (sum of heptachlor and of heptachlor epoxide, expressed as heptachlor) Aldrin singly or combined expressed as dieldrin Dieldrin Chlordane (sum of ␣- and ␥-isomers and of oxychlordane expressed as chlordane) DDT (sum of DDT-, TDE- and DDE-isomers, expressed as DDT) Endosulfan (sum of I and II isomers and of endosulfan sulphate expressed as endosulfan)

Endrin (sum of endrin and of delta-ketoi-endrin expressed as endrin)

Legislation in animal feed relative to a feedingstuff with a moisture content of 12% [6]

Legislation in barley, soya bean, maize and wheat [7]

Products intended for animal feed

MRL (ng g−1 )

All feedingstuffs with exception of: fats Compound feedingstuffs with the exception of: feedingstuffs for dairy cattle Feed materials with exception of: fats All feedingstuffs with exception of: fats All feedingstuffs with exception of:

20 200 10 5 10 100 200 2000 10

fats All feedingstuffs with exception of: fats All feedingstuffs with exception of:

200 10 200 20

fats All feedingstuffs with exception of:

50 50

fats All feedingstuffs with exception of:

500 100

maize oilseeds complete feedingstuffs for fish All feedingstuffs with exception of:

200 500 5 10

fats

MRL range (ng g−1 )

10–20

10

10–20 20

50

50–500

10

50

Methoxychlor

10a

Organophosphorus

Chlorpyrifos

50–200

Pyrethroid

␭-Cyhalothrin Permethrin Cyfluthrin Cypermethrin Flucythrinate Fenvalerate Deltamethrin

20–50 50 20–50 50–200 50 20–200 50–1000

a

MRL in soya bean.

compounds (organophosphorus and chloroacetanilides) have also been investigated. Table 1 shows the established MRLs both in animal feed and in main crops used in the preparation of cattle feedingstuffs. Due to the complex nature of the matrices in which the pesticides could be present and the low detection levels required by EU legislation, there is a growing interest in the development of analytical procedures for efficient extraction and trace-level detection of pesticide residues. A suitable method should be capable of separating as much as possible the target analytes from other co-extracted substances (e.g. starch, proteins and fats) that might interfere with the instrument performance. Traditional methods, such as those including classical Soxhlet or solvent extraction, have been applied to the determination of trace pesticides in animal feed [8,9]. These techniques are characteristically timeand solvent-consuming, and their sample throughput is too low. Therefore, alternative techniques such as pressurized liquid extraction (PLE) [13] and microwave-assisted extraction (MAE) [14] have been recently employed for the isolation of different groups of insecticides from feedingstuffs. Gfrerer et al. [15] also applied fluidized-bed extraction (FBE) and ultrasonic extraction (UE) for the analysis of organochlorine pesticides in animal feed. These procedures are often fast, relatively analyte- and matrix-independent and, even with less subsequent clean-up, cleaner extracts are obtained than with classical methods. The QuEChERS (quick, easy,

cheap, effective, rugged and safe) method has also been adapted for simultaneous analysis of several pesticides in cereals and certain feedingstuffs [12]. Matrix solid-phase dispersion (MSPD) is a patented process, first reported in 1989, for conducting simultaneous disruption and extraction of solid and semi-solid samples. The possibility of performing extraction and clean-up at the same time is one of the main advantages of this technique, which reduces sample contamination during the procedure and decreases the amount of organic solvent required [16,17]. Several authors have employed MSPD in the extraction of pesticides from vegetables, fruits and cereals [18–22]. Some of these products might be “feed materials”, i.e. they may be intended for use in animal feeding either directly as such or, after processing, in the preparation of feedingstuffs [6]. At this point, it is important to distinguish a “feed material” from a “feedingstuff” since, as mentioned above, the second one contains additional substances (especially fats) which make extraction of pesticides much more difficult. To our knowledge, there is only one paper dedicated to the analysis of pesticides in animal feed itself by MSPD; in this work, four organochlorine insecticides (among other chlorinated and brominated compounds) were analyzed in aquaculture feed samples [23]. The present article describes a rapid and sensitive procedure based on MSPD coupled to GC–␮ECD detection for the determination of 36 pesticides in cattle feed. Special attention was devoted to

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the optimization of MSPD parameters. The optimized method was validated and applied to the analysis of real feedingstuffs for dairy cattle from farms of the NW Spain. 2. Experimental 2.1. Reagents and materials A standard mix solution containing organochlorinated pesticides and some metabolites (␣-chlordane, methoxychlor, ␥chlordane, endrin ketone, endrin aldehyde, aldrin, ␣-lindane, ␤-lindane, ␥-lindane, ␦-lindane, p,p -DDD, p,p -DDE, p,p -DDT, dieldrin, endosulfan I, endosulfan II, endosulfan sulphate, endrin, heptachlor and heptachlor epoxide isomer B) with a concentration of 2000 ␮g mL−1 of each compound in toluene:hexane (50:50); hexachlorobenzene (HCB), cypermethrin (mixture of isomers) and deltamethrin were supplied by Supelco (Bellefonte, PA, USA). Fenitrotion and alachlor were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Tefluthrin, transfluthrin, allethrin (mixture of stereoisomers), tetramethrin, ␭-cyhalothrin, cyphenothrin (mixture of cis and trans isomers), permethrin (mixture of cis and trans isomers), cyfluthrin (mixture of isomers), flucythrinate, fenvalerate, chlorpyrifos and acetochlor were of Pestanal® grade and were acquired from Riedel-de-Häen (Seelze, Germany). PCB-166 and PCB-195 (employed as internal standard and surrogate, respectively) were received as 10 ␮g mL−1 solutions in isooctane from Dr. Ehrenstorfer (Augsburg, Germany). Ethyl acetate (HPLC grade), n-hexane (GC grade), methanol (gradient grade), isooctane (for organic trace analysis) and toluene (HPLC grade) were obtained from Merck (Mollet del Vallés, Barcelona, Spain). Acetone (pesticide grade) was purchased from Prolabo (VWR, Fontenay-sous-Bois, France). Anhydrous Na2 SO4 was supplied by Panreac (Barcelona, Spain). C18 (70–230 mesh), Florisil (60–100 mesh) and neutral alumina (150 mesh) were obtained from Aldrich (Milwaukee, WI, USA), and silica (230–400 mesh) from Merck. Florisil, alumina and silica were activated at 130 ◦ C for 12 h and then allowed to cool down in a desiccator before being used. Alumina N, Florisil, Silica and C18 Sep-Pak® cartridges were supplied by Waters (Milford, MA, USA). Copper was purchased by Sigma–Aldrich (Madrid, Spain). For activation of copper, firstly, it was washed three times with 20% chlorhydric acid for 15 min in the ultrasounds bath; subsequently, it was washed with deionized water until pH 7 was reached and, finally, it was washed three times with acetone and hexane and stored in the freezer in this last solvent. Individual standard stock solutions of 1.000–10.000 ␮g mL−1 of pyrethroid, chloroacetanilide and organophosphorus pesticides were prepared by exact weighing and dissolution in acetone, methanol, isooctane or ethyl acetate, depending on the compound. Intermediate mixture solutions of 100 ␮g mL−1 in acetone were prepared from stock solutions and commercial organochlorinated pesticide solution. All working solutions containing the target pesticides were prepared by convenient dilutions of the intermediate solutions in acetone (to spike cattle feed samples) or ethyl acetate (to direct injection into the GC). Working solutions of PCB-195 in isooctane were also prepared. All solutions were stored in amber coloured vials at −20 ◦ C. Different cattle feed samples were collected from dairy farms located in NW Spain. These feedingstuffs are very complex samples prepared by mixing several products (more than 10 in most cases) including a high percentage of various cereal meals, vegetal oils, oxides and salts. Cattle feed samples were sieved at 2 mm, ground, and stored at −20 ◦ C for posterior analysis. Optimization was carried out using cattle feed sample A spiked at 100 ng g−1 ; 30 g of this sample was weighed in a big beaker and

6 mL of a 500 ng mL−1 solution of the target pesticides in acetone were added. Then, an extra volume of acetone (about 15 mL) was poured all over the sample so that it got completely covered with organic solvent. The obtained slurry was allowed to stand (36 h at room temperature, in a switched off hood) and stirred occasionally until the acetone completely evaporated. Afterwards, 0.5 and 1 g fractions were collected and kept in a freezer at −20 ◦ C until 5–10 min before the analysis. For the analytical performance assessment, aliquots of the same cattle feed sample A spiked at different concentration levels ranging from 5 to 100 ng g−1 were analyzed. Accuracy experiments were carried out with a certified reference material BCR® -115 supplied by the EC Community Bureau of Reference (Brussels, Belgium). It is an animal feed product certified for the content of 10 organochlorine pesticides: HCB (19.4 ± 1.4 ng g−1 ), ␤-lindane (23 ± 3 ng g−1 ), ␥-lindane (21.8 ± 1.9 ng g−1 ), heptachlor (19.0 ± 1.5 ng g−1 ), ␥chlordane (48 ± 5 ng g−1 ), endosulfan I (46 ± 4 ng g−1 ), dieldrin (18 ± 3 ng g−1 ), endrin (46 ± 6 ng g−1 ), o,p -DDT (46 ± 5 ng g−1 ) and p,p -DDE (47 ± 4 ng g−1 ). In all experiments, 10 ␮L of PCB-195 surrogate solution (1 ␮g mL−1 ) was added before extraction. 2.2. MSPD procedure 0.5 or 1 g of cattle feed sample was gently blended with 2 g of dispersing phase (Florisil, alumina or C18) and 200 mg of anhydrous Na2 SO4 into a glass mortar using a glass pestle until a homogeneous mixture was obtained (ca. 5 min). A co-column was employed to simultaneously obtain a further degree of fractionation and sample clean-up. Thus, once the blending process was complete, the mixture was transferred into a column with a polypropylene frit at the bottom and filled with 2 g of adsorbent (Florisil or alumina). A second frit was placed on top of the sample before compression with a syringe plunger. Elution was made by gravity flow with ethyl acetate or hexane. 5 or 10 mL of eluent were collected into a graduated conical tube. In case that extract concentration was required, 1 mL of extract was evaporated to dryness under a nitrogen stream. The dry residue was re-dissolved with 200 ␮L of ethyl acetate and 1 ␮L of PCB-166 internal standard solution (10 ␮g mL−1 ) was poured. Finally, 1 ␮L of the extract was injected directly into the GC system. 2.3. Chromatographic conditions GC analysis was performed in a Hewlett-Packard 6890 GC system fitted with a 63 Ni ␮ECD detector, and equipped with a 7683B auto-sampler and a split/splitless injector. Data were acquired and processed by GC Chemstation software. Injector operated in the splitless mode (2 min) at 280 ◦ C. The separation of compounds was performed in a HP-5MS (30 m × 0.32 mm i.d. and 0.25 ␮m film thickness) column. GC oven temperature was programmed as follows: 80 ◦ C held for 2 min, rate 15 ◦ C min−1 to 200 ◦ C, rate 3 ◦ C min−1 to 235 ◦ C held for 1 min, rate 20 ◦ C min−1 to 300 ◦ C held for 10 min, with a total acquisition programme of 35.92 min. For confirmation of positive results in the analysis of real cattle feed samples, a VF-5MS (30 m × 0.25 mm i.d. and 0.25 ␮m film thickness) column was also employed. With this column, two GC temperature programmes were applied: (a) 80 ◦ C held for 5 min, rate 15 ◦ C min−1 to 100 ◦ C, rate 8 ◦ C min−1 to 160 ◦ C, rate 5 ◦ C min−1 to 285 ◦ C held for 15 min; (b) 80 ◦ C held for 2 min, rate 30 ◦ C min−1 to 200 ◦ C, rate 3 ◦ C min−1 to 210 ◦ C held for 1 min, rate 20 ◦ C min−1 to 230 ◦ C held for 1 min, rate 1 ◦ C min−1 to 235 ◦ C, rate 20 ◦ C min−1 to 280 ◦ C held for 10 min. Helium was used as carrier gas at a constant flow rate of 1 mL min−1 , while nitrogen was employed as make-up gas at the flow of 30 mL min−1 . Detector temperature was set at 300 ◦ C.

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In addition, some of the real samples were analyzed using a Varian 3800 gas chromatograph (Varian Chromatography Systems, Walnut Creek, CA, USA) coupled to an ion trap mass detector Varian Saturn 2000 (Varian Chromatography Systems), operated in the electron impact ionization (EI) positive mode (+70 eV). The mass range was scanned in full scan mode from 80 to 500 m/z at 0.6 s scan−1 . The system was operated by Saturn GC/MS Workstation v5.4 software. 3. Results and discussion 3.1. Optimization of the extraction process Efficiency of MSPD extractions depends on the amount of sample, type and quantity of dispersing phase, and nature and volume of the eluting solvent. As mentioned in Section 2, an adsorbent was packed at the bottom of the MSPD column (co-column) for clean-up purposes; therefore, nature and amount of adsorbent were additional factors to take into account in the optimization of the method. In MSPD, the initial trials were conducted applying the most usual sample/solid support material ratio (1–4), blending 2 g of solid support with 0.5 g of sample [24]. 2 g of clean-up adsorbent was considered as an appropriate quantity since the obtained extracts were clean enough to be directly subjected to chromatographic analysis. The nature of the elution solvent is an important matter since the target analytes should be efficiently desorbed while the remaining matrix components should be retained in the column. Pesticides are often eluted with hexane, ethyl acetate, dichloromethane or mixtures of these solvents [18,19,25–29]. In the present work, two of them were tested for extraction of the target pesticides from cattle feed: hexane (non-polar) and ethyl acetate (polar). Initial

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Table 2 Factors and levels considered in the experimental design. Factor

Code

Low level (−)

Central level

High level (+)

Continuous

Dispersing phase Adsorbent Eluting volume (mL)

A B C

C18 Alumina 5

Florisil

Alumina Florisil 10

No No Yes

experiments demonstrated that hexane was not suitable and most of pesticides were not efficiently recovered, while successful and selective elution of the target compounds was achieved when ethyl acetate was employed (data not shown). Therefore, ethyl acetate was proved to be an adequate solvent and it was selected for all further work. In order to reduce the time to achieve the optimal working conditions, an experimental design was applied to investigate the effect of the remaining variables: nature of dispersing phase (A), nature of clean-up adsorbent (B) and volume of eluting solvent (C). In its original conception, reversed-phase materials such as C18-bonded silica were employed as the solid support in MSPD extractions. More recently, many applications have involved the blending of samples with underivatized silicates or other organic (graphitic fibers) or inorganic (Florisil, alumina, etc.) solids [24]. In this study, dispersing phase was assayed at three levels, C18, alumina and Florisil, while two adsorbents, alumina and Florisil, were tested for clean-up. These levels were selected on the basis of the literature about MSPD extractions of pesticides from vegetable and food matrixes [17–19,25,26]. Eluting volume was studied at two levels, 5 and 10 mL. The selected experimental domain is shown in Table 2.

Table 3 ANOVA results showing the significance of main effects and interactions (in bold significant effects at 90% confidence level). Factors

Interactions

A

␣-Lindane ␤-Lindane ␥-Lindane Tefluthrin ␦-Lindane Acetochlor Transfluthrin Alachlor Heptachlor Fenitrotion Aldrin Chlorpyrifos Heptachlor epoxide Allethrin ␥-Chlordane p,p -DDE Dieldrin Endrin Endosulfan II p,p -DDD Endrin aldehyde Endosulfan sulphate p,p -DDT Endrin ketone Tetramethrin Methoxychlor ␭-Cyhalothrin Cyphenothrin Permethrin Cyfluthrin Cypermethrin Fenvalerate Deltamethrin

B

C

AB

AC

BC

F-value

p-Ratio

F-value

p-Ratio

F-value

p-Ratio

F-value

p-Ratio

F-value

p-Ratio

F-value

p-Ratio

1.90 3.70 8.85 7.20 2.67 0.26 0.23 1.29 4.78 0.04 1.59 0.92 2.40 0.88 0.79 4.23 5.74 0.68 2.00 1.19 0.72 4.02 0.39 0.72 9.34 0.39 0.66 0.14 1.87 2.01 0.15 2.47 0.86

0.35 0.21 0.10 0.12 0.27 0.79 0.82 0.44 0.17 0.96 0.39 0.52 0.29 0.53 0.56 0.19 0.15 0.60 0.33 0.46 0.58 0.20 0.72 0.58 0.10 0.72 0.60 0.88 0.35 0.33 0.87 0.29 0.54

15.34 1.62 1.97 0.68 10.58 2.22 2.97 17.02 0.24 0.03 1.09 17.35 5.78 4.46 2.27 2.31 3.64 1.81 1.35 8.65 351.96 20.85 6.28 8.40 0.01 1.41 0.87 2.52 0.10 33.43 2.79 10.14 120.01

0.06 0.33 0.30 0.50 0.08 0.27 0.23 0.05 0.68 0.87 0.41 0.05 0.14 0.17 0.27 0.27 0.20 0.31 0.36 0.10 0.00 0.04 0.13 0.10 0.92 0.36 0.45 0.25 0.79 0.03 0.24 0.09 0.01

1.76 0.65 0.05 0.00 1.08 3.96 0.02 0.50 0.28 0.05 1.80 5.49 0.20 4.46 0.02 0.42 0.45 0.00 0.00 0.77 275.17 0.18 0.23 0.00 2.93 0.18 1.28 4.03 11.53 1.56 0.52 3.73 4.13

0.32 0.53 0.84 0.95 0.41 0.18 0.91 0.55 0.65 0.84 0.31 0.14 0.70 0.17 0.89 0.58 0.57 0.97 0.98 0.47 0.04 0.71 0.68 0.95 0.23 0.71 0.38 0.18 0.08 0.34 0.55 0.19 0.18

0.29 0.03 0.35 0.03 0.27 0.39 0.06 1.59 0.04 0.11 0.85 0.40 0.28 1.26 0.32 0.05 0.20 0.05 0.38 0.46 1.89 0.45 0.11 0.10 0.42 0.03 0.16 0.89 0.04 7.30 0.20 0.22 39.33

0.77 0.97 0.74 0.97 0.79 0.72 0.94 0.39 0.96 0.90 0.54 0.71 0.78 0.44 0.76 0.95 0.83 0.95 0.73 0.69 0.35 0.69 0.90 0.91 0.71 0.97 0.86 0.53 0.97 0.12 0.83 0.82 0.02

0.42 0.04 0.02 0.10 0.25 0.07 0.14 0.64 0.02 0.11 1.67 0.39 0.13 0.36 0.18 0.23 0.33 0.05 0.32 0.31 3.82 0.29 0.00 0.20 1.80 0.04 0.30 0.23 0.48 0.74 0.07 0.13 0.67

0.70 0.96 0.98 0.91 0.80 0.94 0.88 0.61 0.98 0.90 0.37 0.72 0.89 0.73 0.85 0.81 0.75 0.95 0.76 0.77 0.21 0.77 1.00 0.83 0.36 0.96 0.77 0.81 0.67 0.58 0.94 0.88 0.60

0.09 0.12 0.95 0.91 0.69 0.30 0.00 0.00 0.06 0.24 1.00 0.12 0.01 0.21 0.09 0.01 0.11 0.11 0.08 0.09 182.86 0.71 0.43 0.06 0.65 0.30 2.85 1.48 0.03 0.24 0.02 0.05 5.16

0.80 0.76 0.43 0.44 0.49 0.64 0.99 0.98 0.83 0.67 0.42 0.76 0.92 0.69 0.79 0.92 0.77 0.78 0.81 0.80 0.01 0.49 0.58 0.83 0.50 0.64 0.23 0.35 0.88 0.67 0.91 0.84 0.15

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Fig. 1. Mean plots of the main factors studied in the multi-factor categorical design: (a) dispersing phase; (b) adsorbent type; (c) eluting volume.

M. Fernandez-Alvarez et al. / J. Chromatogr. A 1216 (2009) 2832–2842

Since it was intended to study multiple non-quantitative factors with several levels of each, a multi-factor categorical design (3 × 2 × 2) was proposed [30]. This design is a standard factorial, consisting of all combinations of the levels of the factors (12 runs). It has resolution V, allowing the evaluation of the main effects as well as the two-factor interactions of the investigated parameters. In each experiment, 0.5 g of spiked cattle feed sample (100 ng g−1 ) were blended with 2 g of dispersing phase and 200 mg of anhydrous Na2 SO4 . When blending was complete, the sample was packed into an MSPD column with 2 g of adsorbent at the bottom and analytes were eluted with ethyl acetate by gravity flow. An analysis of variance (ANOVA) was used to assess whether factors or interactions showed a statistically significant contribution to the variance of the response. Results of ANOVA are shown in Table 3, where F-ratios and p-values are given. The Fratio measures the contribution of each factor or interaction on the variance of the response, while the p-value tests the statistical significance of each factor and interaction. At a 90% confidence level, a main factor or a factor interaction is statistically significant when its p-value is lower than 0.10. The results showed that the adsorbent type (B) was the most important variable affecting extraction efficiency (see Table 3), and this factor was significant for several pesticides (␣-lindane, ␦-lindane, alachlor, chlorpyrifos, endrin aldehyde, endosulfan sulphate, cyfluthrin, fenvalerate and deltamethrin) and at the limit of significance for p,p -DDD and endrin ketone. On the other hand, the dispersing phase (A) was not significant for any of the target compounds, while eluting volume (C) was significant only for endrin aldehyde and permethrin. As regards the interactions between factors, adsorbent–eluting volume (BC) and dispersing phase–adsorbent (AB) interactions were significant only for endrin aldehyde and deltamethrin, respectively. The mean plots are useful graphs that illustrate the effect of the variables by showing the mean values as well as the confidence intervals for each level. These plots (see Fig. 1 for some representative compounds) were employed to identify which level of each factor led to higher responses for each specific analyte. Generally, most of the pesticides showed a similar behaviour related to the most suitable extraction conditions. Analyzing the mean plots for the dispersing phase factor, alumina was found to be the most suitable dispersant phase for the majority of the studied compounds (see Fig. 1a for ␦-lindane and transfluthrin) although, in general, equivalent responses were obtained whatever the solid phase. Nevertheless, it was observed that the use of C18 increased the chromatographic artefacts with higher baselines than Florisil and alumina; moreover, this solid support led to abnormal high recoveries (close to or even higher than 200%) for some pesticides such as aldrin. Dórea and Lanc¸as [18] reported similar drawbacks after employing C18 as dispersant in the MSPD extraction of OPPs and pyrethroids from cashew nut and passion fruit. Regarding adsorbent type, Florisil provided the most effective recoveries for all target analytes (see some examples in Fig. 1b). In relation to eluting volume, in general, no differences or a slight increase in the chromatographic signal was appreciated when 10 mL of extract was collected instead of 5 mL (see Fig. 1c) although, as mentioned above, this response improvement was only significant for endrin aldehyde and permethrin. As it was already deduced from Table 3, only two interactions were statistically significant and, as an example, the BC interaction plot for endrin aldehyde is illustrated in Fig. 2. This plot shows the average response at each combination of adsorbent (B) and eluting volume (C). As can be deduced from Fig. 2, when alumina is used, a significant response improvement was observed after elution of 10 mL, while if Florisil is employed as adsorbent, the elution of 10 mL led to similar results than with 5 mL.

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Fig. 2. Interaction between the adsorbent type and the eluting volume for endrin aldehyde (BC).

Although interaction effects between the studied factors were not generally noticed, interaction plots were very useful to display the most favourable extraction conditions. Fig. 3 illustrates some examples showing general behaviours. AB (dispersing phase–adsorbent) interaction plot (Fig. 3a) clearly shows that using alumina as dispersant lead to more efficient extractions, being Florisil the most adequate adsorbent in order to obtain higher chromatographic responses. The same conclusion regarding the most satisfactory dispersant is drawn from AC (dispersing phase–eluting volume) plots (Fig. 3b), in which it is confirmed that elution with 10 mL instead of 5 mL does not change the obtained responses. Finally, BC (adsorbent–eluting volume) interaction plot (Fig. 3c) shows again that Florisil is the most convenient clean-up adsorbent while 10 mL of eluting volume does not lead to an important improvement in the recoveries. In this sense, it is important to underline that elution with 5 mL not only leads to less diluted extracts, but also reduces the use of solvent and the unintended coelution of potential interferences, as well as the time of analysis. As a conclusion from these observations, the proposed method for the simultaneous MSPD extraction of the investigated pesticides was blending of 0.5 g of feed sample with 2 g of alumina as dispersing phase and 200 mg of anhydrous Na2 SO4 , clean-up with a co-column of 2 g of Florisil and elution with 5 mL of ethyl acetate. 3.2. Influence of sample size Experiments were performed to test the validity of the developed MSPD method when increasing the quantity of sample. 1 g of spiked cattle feed sample (100 ng g−1 ) was extracted employing the optimized conditions and the results were compared with those obtained when 0.5 g of sample was analyzed. Responses for the target pesticides were duplicated without detecting a considerable worsening in the chromatographic background. Therefore, in order to improve the sensitivity of the method, aliquots of 1 g of sample were analyzed in the subsequent experiments. 3.3. Purification of extracts The eluates obtained using the selected MSPD conditions were adequately clean for their direct analysis in the GC–␮ECD system. Nevertheless, the corresponding chromatograms showed some peaks attributed to matrix ECD-sensitive substances. Although most of these peaks did not compromise the detection and quantization of analytes, some protocols for further extract purification were tried. Firstly of all, classical SPE approaches by using Alumina N, Florisil, Silica and C18 Sep-Pak® cartridges preconditioned with ethyl acetate were tested. Afterwards, dispersive-SPE (dSPE) procedures using alumina, Florisil and silica as adsorbents were assessed. Activated copper was also investigated in order to remove possible interferences from elemental sulphur.

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Fig. 3. Interaction plots for some representative compounds: (a) dispersing phase–adsorbent type; (b) dispersing phase–eluting volume; (c) adsorbent type–eluting volume.

No significant differences in the recoveries and no evident improvement in the chromatographic profiles were found with any of these purification protocols. Just in the case of dSPE with alumina, permethrin and deltamethrin showed a slightly improvement in their chromatographic resolution. In conclusion, it was not worthwhile including an extra stage for further cleaning of the MSPD extracts. 3.4. Concentration of extracts With the purpose of improving method sensibility, two concentration strategies were tested. Aliquots of 1 mL of MSPD extracts were concentrated to dryness under nitrogen and the dry residues were re-dissolved with 200 ␮L of ethyl acetate. Several experiments concentrating 1 mL of eluate directly to 200 ␮L were also performed. Responses obtained from the two sets of experiments were equivalent, observing a fivefold increase in the response of the analytes compared to the results achieved with the GC–␮ECD analysis of the corresponding extracts without further concentration. Therefore, it was demonstrated that no loss of target pesticides occurred by volatilization during solvent evaporation. In this way, it was confirmed that concentration of extracts was a proper resort to enhance the sensitivity of the developed procedure. 3.5. Validation of the method: linearity, precision and limits of detection and quantification With the aim of verifying that the MSPD–GC–␮ECD developed method was suitable for the quantitative determination of pesti-

cides in cattle feed, method quality parameters were estimated (Table 4). The instrumental linearity was evaluated at a concentration range between 1 and 100 ng mL−1 (including six concentration levels) using standard solutions prepared in ethyl acetate. Each concentration level was injected in triplicate and the response function was found to be linear with correlation coefficients (R) higher than 0.9901. Precision and LODs and LOQs were assessed by analyzing spiked cattle feedingstuff samples containing known concentrations of the investigated pesticides. Method precision was studied within-aday and among-days at two fortification levels (20 and 100 ng g−1 ) by calculating the relative standard deviation (RSD). Results are summarized in Table 4. As regards the intra-day precision, RSDs ranged from 0.2 to 16% with an average of 3.3%, while the interdays variability showed an average of 5.8%, ranging from 1.2 to 14%. The limits of detection (LOD) and limits of quantification (LOQ) of the overall method were calculated as the concentration giving a signal-to-noise ratio of three (S/N = 3) and ten (S/N = 10), respectively. These limits were estimated using the MSPD extract of a feedingstuff spiked at 5 ng g−1 . LODs and LOQs values were between the sub-ng g−1 and low-ng g−1 level and both were lower than the maximum residue levels (MRLs) for pesticide residues in cereals and animal feed set by the European legislation (see Tables 1 and 4). Furthermore, these limits were well bellow those reported in the previous works dealing with the extraction of pesticides from animal feed [13,14,31] and grain [12,32]. Fig. 4 shows the overlapped chromatograms obtained for a spiked cattle feed product containing all the target pesticides at

M. Fernandez-Alvarez et al. / J. Chromatogr. A 1216 (2009) 2832–2842

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Fig. 4. MSPD–GC–␮ECD overlapped chromatograms obtained for a spiked cattle feed sample at 20 ng g−1 (solid line) and 5 ng g−1 (dotted line) following the recommended procedure: 1–36 target pesticides (see number code in Table 4); (a) PCB-166; (b) PCB-195.

a concentration level of 20 ng g−1 (solid line) and 5 ng g−1 (dotted line) 3.6. Accuracy evaluation: application to real samples Recovery studies were carried out by applying the optimized MSPD method to the extraction of five cattle feed samples (A–E) spiked at 100 ng g−1 with the target pesticides. Sample A was also fortified at 20 ng g−1 . None of the target compounds was detected in the non-spiked samples. As can be seen in Table 5, recoveries were higher than 75% for most compounds in all samples, excluding endrin aldehyde. The lower recovery for this compound may

be attributed to its strong retention on the solid adsorbents. In fact, other authors have also observed a similar behaviour for this metabolite of endrin [14,25]. The observed variability (RSD) between the analyzed spiked feedingstuff samples was in general lower than 10% that can be attributed to the experimental error (see Table 4). These results demonstrate that the developed MSPD–GC–␮ECD method allows the quantification of the target compounds in cattle feed samples of different composition. Method accuracy was also evaluated by analyzing a certified reference material (BCR® -115) containing some of the target pesticides. Table 6 shows the estimated concentrations and the recovery

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Table 4 Instrumental linearity, precision and limits of detection (LODs) and quantification (LOQs) of the proposed method. Code

Pesticide

Linearity, R

Repeatability (RSD, %) Within-a-day

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

␣-Lindane ␤-Lindane ␥-Lindane Tefluthrin ␦-Lindane Acetochlor Transfluthrin Alachlor Heptachlor Fenitrotion Aldrin Chlorpyrifos Heptachlor epoxide Allethrin ␥-Chlordane Endosulfan I ␣-Chlordane p,p -DDE Dieldrin Endrin Endosulfan II p,p -DDD Endrin aldehyde Endosulfan sulphate p,p -DDT Endrin ketone Tetramethrin Methoxychlor ␭-Cyhalothrin Cyphenothrin Permethrin Cyfluthrin Cypermethrin Flucythrinate Fenvalerate Deltamethrin

0.9978 0.9976 0.9976 0.9976 0.9961 0.9996 0.9979 0.9995 0.9962 0.9986 0.9981 0.9993 0.9986 0.9982 0.9984 0.9986 0.9984 0.9967 0.9986 0.9970 0.9982 0.9978 0.9985 0.9978 0.9925 0.9969 0.9992 0.9962 0.9969 0.9943 0.9974 0.9901 0.9927 0.9914 0.9916 0.9963

LOD (ng g−1 )

LOQ (ng g−1 )

0.03 0.11 0.08 0.08 0.08 1.20 0.08 1.50 0.08 0.06 0.08 0.11 0.06 0.30 0.05 0.09 0.10 0.10 0.09 0.06 0.05 0.05 0.12 0.09 0.30 0.12 0.75 1.05 0.21 0.42 0.36 0.90 0.54 0.90 0.90 1.50

0.10 0.38 0.25 0.28 0.26 4.00 0.26 5.00 0.28 0.21 0.26 0.38 0.20 1.00 0.18 0.30 0.32 0.32 0.30 0.20 0.18 0.18 0.40 0.30 1.00 0.40 2.50 3.50 0.70 1.40 1.20 3.00 1.80 3.00 3.00 5.00

Among-days

20 ng g−1 (n = 3)

100 ng g−1 (n = 4)

20 ng g−1 (n = 2 days)

100 ng g−1 (n = 3 days)

1.9 1.5 1.5 0.9 1.9 7.3 3.7 1.0 0.9 0.6 2.5 1.0 1.9 3.6 3.1 1.6 2.6 2.2 3.8 7.3 7.1 2.6 7.3 1.6 6.7 2.0 3.9 5.6 0.8 1.9 0.3 0.2 9.4 4.5 3.4 3.3

3.7 2.7 3.5 3.0 3.3 2.3 2.8 1.1 2.0 2.3 3.6 2.3 3.3 2.1 3.3 1.9 6.1 3.9 4.5 5.0 3.6 3.0 4.5 2.2 5.1 3.7 16.3 5.1 3.0 1.5 3.2 3.2 3.4 2.9 2.0 1.6

4.2 5.4 4.7 3.5 2.7 5.0 3.7 3.6 5.5 2.0 3.0 1.2 3.4 3.7 3.6 7.3 4.5 4.3 4.0 4.4 8.1 5.1 8.6 4.0 13.7 7.3 9.0 12.6 10.1 7.3 13.3 1.9 13.5 4.1 5.2 14.0

4.0 3.5 3.9 4.1 3.9 3.3 4.8 1.4 3.4 3.6 4.6 4.6 4.2 3.4 4.5 10.5 7.5 4.8 4.6 5.4 5.0 5.6 10.9 3.2 10.8 4.8 14.2 10.9 5.2 3.4 5.5 5.3 2.7 5.7 6.7 8.9

values for the studied organochlorine pesticides. The estimated values were in good agreement with the certified ones. Some of the target analytes (␣-lindane, aldrin, heptachlor epoxide and p,p -DDT) were also found in this sample although its certified concentration

is not provided. Nevertheless, these values were in general in good agreement with the ones reported in the previous papers. Thus, the suitability of the optimized method for the analysis of pesticides at trace levels in cattle feed samples is demonstrated [33,13,15].

Fig. 5. MSPD–GC–␮ECD chromatogram obtained for a real cattle feed sample contaminated with organochlorine pesticides.

M. Fernandez-Alvarez et al. / J. Chromatogr. A 1216 (2009) 2832–2842 Table 5 Recovery of pesticides from different cattle feed samples. Pesticide

sulfan sulphate) were detected, at concentration of 7.4, 4.1, 3.7 and 4.5 ng g−1 , respectively.

Cattle feed sample A

B −1

␣-Lindane ␤-Lindane ␥-Lindane Tefluthrin ␦-Lindane Acetochlor Transfluthrin Alachlor Heptachlor Fenitrotion Aldrin Chlorpyrifos Heptachlor epoxide Allethrin ␥-Chlordane Endosulfan I ␣-Chlordane p,p -DDE Dieldrin Endrin Endosulfan II p,p -DDD Endrin aldehyde Endosulfan sulphate p,p -DDT Endrin ketone Tetramethrin Methoxychlor ␭-Cyhalothrin Cyphenothrin Permethrin Cyfluthrin Cypermethrin Flucythrinate Fenvalerate Deltamethrin

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−1

C

D

E

77 77 72 82 78 83 82 80 73 76 79 77 78 83 80 75 91 81 82 84 73 76 37 79 73 73 81 76 71 78 74 80 94 72 78 69

79 79 75 85 78 85 87 82 77 76 81 80 80 80 83 84 90 84 86 90 81 79 33 84 81 78 134 88 82 83 80 88 96 85 90 77

−1

20 ng g

100 ng g

100 ng g

96 94 96 125 94 97 102 76 87 100 95 88 92 98 96 107 121 95 97 100 91 81 62 91 102 98 85 116 82 86 99 93 103 66 107 86

90 90 90 97 91 95 92 88 84 92 91 86 91 92 93 85 92 93 93 96 94 84 58 95 111 86 96 109 81 85 86 87 94 91 99 86

80 79 77 85 80 89 88 87 77 80 80 83 81 78 83 86 93 84 88 91 80 78 41 85 79 81 88 85 79 83 77 86 79 81 91 76

88 88 86 91 90 92 92 90 84 98 89 91 88 91 91 87 95 92 93 98 89 90 32 90 85 83 90 88 84 87 86 91 92 88 92 85

4. Conclusions In this work, an efficient, fast and easy to perform analysis method based on MSPD was successfully applied to the analysis of 36 pesticides and breakdown products in cattle feed. To our knowledge, this is the first time that most of the target pesticides have been extracted from animal feed by MSPD. The developed method uses a co-column so that extraction and clean-up were performed in a single step. A multi-factor categorical design was employed for the optimization of the extraction/clean-up stage and the type of adsorbent in the co-column was the most important variable. The recommended analytical procedure was blending of 1 g of feed sample with 2 g of alumina and 200 mg of anhydrous Na2 SO4 , cleaning-up with a co-column of 2 g of Florisil and elution with 5 mL of ethyl acetate. The obtained extracts were clean enough to be directly injected in the GC–␮ECD even after concentration. The analytical results confirmed that the described MSPD– GC–␮ECD procedure provides good recoveries (in general >75%), with quantification limits well below those set by the international regulations for pesticide residues in this kind of matrix. The proposed method was applied to the analysis of real complex cattle feed samples obtained from different farms of Galicia (NW Spain). Acknowledgements This research was supported by FEDER funds and projects CTQ2006-03334 (CICYT, Ministerio de Ciencia y Tecnologia, Spain) and PGIDIT05RAG50302PR (Xunta de Galicia). M.F.-A. would like to acknowledge her FPU doctoral grant to Ministerio de Ciencia y Tecnologia. References

Real cattle feed samples collected from dairy farms located in NW Spain were analyzed using the developed MSPD–GC–␮ECD method. Some of these samples were analyzed by GC–MS for confirmation. In all analyzed samples none of the target compounds were found at concentration levels above the MRLs (see Table 1). As an example, Fig. 5 shows the GC–␮ECD chromatogram obtained for one of these samples where four of the target pesticides, an organophosphorus pesticide (fenitrotion), two organochlorine pesticides (endosulfan I and II) and one degradation product (endo-

Table 6 Validation of the method: analysis of a certified reference material (BCR® -115). Pesticide

Estimated value (ng g−1 )

HCB ␣-Lindanea ␤-Lindane ␥-Lindane Heptachlor Aldrina Heptachlor epoxidea ␥-Chlordane Endosulfan I 4,4 -DDE Dieldrin Endrin p,p -DDTa

17.8 28.4 22.2 16.4 18.7 13.0 18.2 44.3 30.2 41.8 21.2 40.7 52.7

a

Not certified.

± ± ± ± ± ± ± ± ± ± ± ± ±

4.8 3.0 2.9 3.0 1.4 2.2 4.8 4.5 2.8 2.5 3.7 5.7 3.1

Recoveries (%) 92 96 75 99

92 66 89 118 89

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