Food Chemistry 114 (2009) 15101516

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

A validated matrix solid-phase dispersion method for the extraction of organophosphorus pesticides from bovine samples
Martha P. Garca de Llasera *, Mara L. Reyes-Reyes
Laboratorio de Anlisis de Trazas, Departamento de Qumica Analtica, Facultad de Qumica de la Universidad Nacional Autnoma de Mxico (UNAM), Avenida Universidad 3000, 04510 Mxico, DF, Mexico

a r t i c l e

i n f o

a b s t r a c t
A method based on matrix solid-phase dispersion (MSPD) was developed for the quantitative extraction of ve organophosphorus (OPPs) pesticides from bovine samples. The determination was carried out by high performance liquid chromatography (HPLC) with diode array spectrophotometric UV detection. The MSPD extraction with octadecylsilyl (C18) sorbent combined with a silica gel clean-up and acetonitrile elution was optimised for chlorpyrifos, chlorfenvinphos, diazinon, fenitrothion, and parathion-methyl. The method was validated, yielding recovery values higher than 94%, except for chlorfenvinphos in liver (55%), and precision values, expressed as relative standard deviations (RSDs), which were less than or equal to 15% in liver and 11.5% in muscle at spiking levels of 0.25, 2.5 and 5 lg g1. Linearity was studied from 0.5 to 15 lg g1, and the limits of detection (LODs) were found to be lower than 0.1 lg g1. This method was applied to the analysis of real samples with conrmative analyses performed using gas chromatographymass spectrometry (GCMS) in selected ion monitoring mode (SIM). 2008 Elsevier Ltd. All rights reserved.

Article history: Received 9 May 2008 Received in revised form 5 September 2008 Accepted 3 November 2008

Keywords: Matrix solid-phase dispersion Extraction HPLC analysis Organophosphorus pesticides Bovine samples

1. Introduction Organophosphorus pesticides (OPPs) are among the most commonly employed pesticides worldwide. These compounds, even if they are less persistent in the environment than organochlorine pesticides, can also reach the food chain and may therefore represent a risk to human health. Domestic animals can accumulate such substances from contaminated feed and water, or from exposure to insecticides during shed fumigation. Likewise, a source of pesticides to domestic animals is their treatment for the control of pests. Thus, animal-derived products are considered to be an indirect source of OPPs, and the development of analytical methods suitable for their surveillance in these kinds of products is quite important (Juhler, 1998; Leeman, Van Den Berg, & Houben, 2007; Mallatou, Pappas, Kondyli, & Albanis, 1997; Nero et al., 2007; Salas et al., 2003; Schenck & Donoghue, 2000). Organophosphorus pesticide determination in diverse biological samples and food is normally performed by gas chromatography coupled to mass spectrometry (GCMS). High-performance liquid chromatography with diode array detection (HPLCDAD) is sometimes used as an alternative means of analysis (Juan, Pic, & Font, 2003). Sample preparation methods generally involve a solvent- or solid-phase-based extraction technique (Buldini, Ricci, & Sharma, 2002; Juan et al., 2003; Pic, Fernndez, Ruiz, & Font, 2007), although the presence of fat in animal-derived products is
* Corresponding author. Tel./fax: +52 55 56223899 44051. E-mail address: pgcllas@servidor.unam.mx (M.P. Garca de Llasera). 0308-8146/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.11.006

the major interference affecting analyte isolation. Suitable methods for the extraction of OPPs likely to be found in meat were developed using gravimetric fat removal through ice cooling, followed by solid-phase extraction (SPE) (Juhler, 1997; Kuiniven & Bengtsson, 2002). This method is an alternative to gel permeation chromatography (GPC) clean-up, commonly used for the extraction of organophosphorus compounds in bovine tissue (Holstege, Scharberg, Tor, Hart, & Galey, 1994; Pang et al., 2006). Likewise, GPC and SPE clean-up have been used for the extraction of organophosphorus pesticides in other animal (Garrido Frenich, Plaza-Bolaos, & Martnez-Vidal, 2007; Pagliuca, Gazzotti, & Sticca, 2005) and human tissues (Russo, Campanella, & Avino, 2002). The OPPs primary and secondary metabolites were extracted from beef muscle tissue by a liquidliquid extraction with ethylacetate methanol (90:10 v/v) followed by a C18 solid-phase clean-up (Ioerger & Smith, 1993; Coulibaly & Smith, 1994). Due to the complex nature of the animal matrices in which the OPPs and metabolites are present, most of the sample preparation protocols mentioned above are quite complex and require a fairly large sample (1025 g). One alternative that simplies the preparation of fatty tissue samples is matrix solid-phase dispersion (MSPD), introduced by Barker in 1989 (Barker, 2000). This technique integrates sampling, extraction, and preconcentration into a simple, single-step procedure. At present, MSPD has been successfully applied to the extraction of a wide range of drugs, pesticides, naturally occurring constituents, and other compounds from a wide variety of complex plant and animal samples, including the analysis of organophosphorus pesticide residues in products from

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different food-producing animals (Barker, 2007; Bogialli & Di Corcia, 2007; Kristenson, Ramos, & Brinkman, 2006). In spite of the great number of MSPD publications, well-described and validated MSPD methods for the extraction of the most used organophosphorus insecticides applied to cattle are scarce. A method for the extraction of organophosphates such as fenthion, crufomate and coumaphos from bovine muscle has been reported in the literature (Barker, Long, & Short, 1989), but no further publications regarding OPP extraction in bovine tissue have been published since then. The published method provides recoveries of 85.6%, 93.6%, and 76.6% for the studied analytes at a range of concentrations between 0.1 and 2 lg g1. MSPD was performed with 0.5 g of tissue dispersed on 2 g of prewashed C18 silica with a co-column clean-up using 0.5 g of C18 sorbent. Elution was performed with hexane or benzene and the extracts were analysed by gas chromatography coupled to nitrogen-phosphorus detection (GCNPD). Recently, a C18-MSPD-based method was developed for 25 pesticides, including some OPPs such as fenthion, isofenphos, chorpyriphos-methyl and -ethyl, pirimiphos-methyl, parathion-ethyl and -methyl, chlorfenvinphos, malathion, methamidophos, and dichlorvos in several kinds of animal liver (chicken, pork and lamb). Good recoveries (7987%) were obtained for the rst six organophosphorus pesticides, but the rest of them were recovered with <30% efciency (Garrido Frenich et al., 2007). The present paper describes a MSPD-based sample preparation procedure for the extraction of some of the most commonly-used OPPs in Mexico (NOM-004-ZOO-1994) for bovine fumigation from bovine liver and muscle: chlorpyriphos, chlorfenvinphos, diazinon, fenithrothion and parathion-methyl. The extraction method involves the use of a C18 dispersant phase followed by clean-up in a silica-packed column. Analytes were eluted with acetonitrile, and the extracts were analysed by HPLCphotodiode array detection. The whole determination method was validated in liver and muscle tissue, and it was subsequently applied to the analysis of these pesticides in two groups of liver samples: (a) brightly-coloured samples with a smooth, healthy aspect, and (b) pale samples with a rugose, unhealthy aspect. GCMS was used for conrmative analysis of positive samples.

a Rheodyne model 7125 injection valve with a 20 lL loop. Quantitative measurements of peak areas were provided by the Varian Star workstation version 4.5. Separation was performed on a 5 lm RES ELUT C18 stainless steel column (150 mm 4.6 mm id) connected to a guard column (13 mm 4.6 mm id, same stationary phase), both from Varian. The elution gradient was programmed linearly from an initial mobile phase composition of 70:30 v/v methanol:water to a nal composition of 100% methanol during 10 min at a ow rate of 1 mL/min. Absorption spectra of the pesticides were obtained by scanning wavelengths in the range of 190350 nm. The individual detection wavelengths selected for each pesticide were as follows: chlorpyrifos, 287 nm; chlorfenvinphos, 244 nm; diazinon, 244 nm; fenithrothion, 268 nm; and parathion-methyl, 273 nm. 2.2.2. Gas chromatography A model 6890N gas chromatograph from Agilent Technologies (Boeblingen, Germany) coupled to a 5973N mass spectrometer detector (MSD) and a MSD Chemstation from Agilent Technologies were employed for gas chromatography conrmative analysis. The MS electron ionisation potential was 70 eV. Compound separation was achieved using a 30.0 m 0.32 mm 0.25 lm DB-5 Hewlett Packard capillary column (J&W Sci., USA) and the following temperature program: from 60 C (holding time 2 min) to 280 C at 20 C/min (holding time 10 min). One microlitre of sample was injected into the GC system in splitless mode (60 s). The injector, transfer and ion source temperatures were set at 280, 230 and 280 C, respectively. Helium (purity P 99.999%) was used as the carrier gas at a ow rate of 1 mL/min. Scan mode (50550m/z) was used for obtaining the pesticide spectra with the standard solutions. Selected ion monitoring mode (SIM) was used for the identication of two pesticides in the bovine samples, with diagnostic fragment ions of m/z (relative abundance) 197 (100), 258 (61) and 314 (80) for chlorpyrifos and m/z (relative abundance) 267 (100), 269 (88), 323 (86) and 325 (86) for chlorfenvinphos (dwell time 100 ms). The analysis of extracts was performed in the Gas Chromatography Laboratory of the Faculty of Chemistry, UNAM. 2.3. Sample preparation

2. Materials and methods 2.1. Chemical and materials HPLC-grade methanol and acetonitrile were obtained from EM Science Merck (Gibb-stown, NJ, USA). Deionised water was obtained from a MilliQ water purication system Millipore (Bedford, MA, USA). The selected pesticides (chlorpyrifos, chlorfenvinphos, diazinon, fenitrothion and parathion-methyl) were supplied by Chem. Service (West Chester, PA, USA) with certied purities of at least 98%. Stock 100 mg/L solutions were prepared in methanol or acetonitrile and stored at 4 C. Working standard solutions were prepared in methanol or acetonitrile at different concentrations. Other chemicals were obtained from J.T. Baker (Phillipsburg, NJ, USA). Supelclean silica and LC-C18 sorbents (particle diameter 40 lm) were purchased from SUPELCO (Bellefonte, PA, USA), and orisil was purchased from J.T. Baker. Six millilitre disposable plastic syringe barrels and plungers were obtained from Varian (Palo Alto, CA, USA). 2.2. Chromatographic analysis 2.2.1. Liquid chromatography A Varian model 9012 liquid chromatographic pump and a Varian model 9065 UV diode array detector (Palo Alto, CA, USA) were employed for HPLC analysis. Manual injection was performed using

Bovine liver samples were obtained directly from abattoirs, and muscle samples were bought in supermarkets in Mexico City. Samples were stored at 20 C until analysis. Twenty grams of liver or 20 g of muscle were homogenised with an Ultraturrax apparatus. Then, 0.5 g of homogenised sample was placed into an agata mortar (50 mL capacity) containing 2.0 g of C18 (previously washed with 2 mL of acetonitrile and vacuum dried). Next, the sample and the solid-phase were gently blended to obtain a homogeneous mixture. This mixture was introduced into a 6 mL polypropylene ltration tube with a polyethylene frit in the bottom and tightly compressed and covered with another polyethylene frit. The resulting MSPD cartridge was washed, conditioned and eluted per the following sequence: (a) 20 mL of deionised water, (b) 3 mL acetonitrile:water (25:75 v/v), (c) 1 mL acetonitrile:water (60:40 v/v), and (d) 5 mL of acetonitrile. Elution was performed using a SPE vacuum manifold at a ow rate of 1 mL/min. The rst, second and third fractions (a, b and c) were discarded. Clean-up of the collected 5 mL of acetonitrile eluate (fraction d) was performed using a 0.5 g silica gel column (previously washed with 15 mL of acetonitrile). This puried extract was evaporated in a rotary evaporator (40 C, 200 mbar). Finally, the residue was re-dissolved in 250 lL of acetonitrile and injected into the HPLC system. The clean-up of the collected 5 mL of acetonitrile (fraction d) was performed using a column packed with 1 g of orisil (activated by heating to 300 C for 8 h) when the samples were prepared for conrmatory GCMS analysis.

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Table 1 Percent recoveries (n = 3) and relative standard deviations (RSDs) obtained from MSPD experiments (2 g of C18 as dispersion sorbent, and 0.5 g of liver sample). Pesticide Recovery (RSD%) 60%a Parathion-methyl Fenitrothion Chlorfenvinphos Diazinon Chlorpyrifos
a b

80%a 99 94 63 85 89 (1.8) (4.4) (7.5) (5.6) (1.0)

100%a 99 98 64 98 97 (1.5) (3.9) (6.8) (5.5) (1.0)

C18b 88 88 50 86 97 (3.2) (3.9) (4.9) (2.5) (2.1)

Florisilb 73 79 51 70 84 (3.5) (4.0) (4.9) (2.6) (2.3)

Silicabott.b 87 87 52 94 95 (4.0) (4.2) (5.6) (2.8) (2.3)

Silicab 96 (3.0) 97 (3.5) 55 (5.2) 97 (2.0) 100 (1.9)

99 91 60 48 24

(1.3) (3.9) (6.8) (6.5) (1.5)

Acetonitrile percent; elution volume: 10 mL; previous application of 20 mL water + 3 mL acetonitrile 25%; level of fortication: 20 lg/g; non-evaporated. Sorbent amount: 0.5 g; elution volume: 5 mL pure acetonitrile; previous application of 20 mL water + 3 mL acetonitrile 25% + 1 mL acetonitrile 60%; level of fortication: 10 lg/g; evaporated.

2.4. Recovery studies and validation Recovery studies were carried out on liver and muscle samples (0.5 g) known to be free of pesticide residues, and then spiked at six concentration levels: 0.5, 1.0, 2.5, 5.0, 10.0 and 15.0 lg g1. Three replicates were performed at each spiking level to determine the relative standard deviation (RSD). Accuracy was determined by comparing the amount of OPP added vs. the amount recovered with the slope of the added vs. recovered curve representing the average recovery of each pesticide. Method linearity (peak area vs. concentration) was evaluated using linear regression analysis at the spike levels mentioned above. Precision was determined in terms of repeatability, by running ve extractions of liver or muscle sample spiked at three different levels (0.25, 2.5 and 5 lg g1) on a single day, and reproducibility was quantied using ve or three replicate samples spiked at the same levels on ve or three different days. The limits of detection (LODs) and the limits of quantication (LOQs) of the method were obtained at the kmax of each pesticide. A signal-to-noise (S/N) ratio of three was dened as the LOD for each OPP, and the LOQ was dened as S/N = 10. 3. Results and discussion 3.1. Optimisation of conditions for matrix solid-phase dispersion MSPD conditions were carefully selected to yield extracts with the highest recovery for the pesticides and the lowest amount of matrix compounds. C18-bonded silica was chosen as the dispersion sorbent because its non-polar character provides the best afnity for the studied compounds, and because it causes the complete disruption and distribution of the lipophilic entities commonly existing in animal tissues such as liver or muscle (Barker, 2000, 2007). Typically, a sample:sorbent ratio of 1:4 was used in this study. Thus, preliminary assays for the optimisation of the extraction method were made with 100 mg of sample and 400 mg of C18. Optimisation of the nal protocol was performed with 0.5 g of sample to enhance sensitivity and 2 g of C18. Preliminary elution experiments were performed with pure methanol or methanol mixed with water in different ratios. Prewashing of the dispersion column with 5 mL of water followed by 4 mL of a poorly-eluting 30:70 methanolwater mixture was necessary to eliminate the most polar fraction of sample (typically consisting of pigments and proteinaceous compounds), without breakthrough of the most polar pesticides. Four millilitre of pure methanol eluent provided the best recovery efciency but a light yellow colouration was present, perhaps as a result of lipid and/ or coloured matrix compounds co-elution. For this reason, methanol was rejected as an eluent, and acetonitrile was used to optimise the nal elution sequence. Acetonitrile was expected to produce non-coloured extracts.

3.1.1. Elution sequence Final optimisation of the elution sequence was performed using pure acetonitrile and acetonitrilewater mixtures (60:40 v/v and 80:20 v/v) as eluting solvents rather than methanol or methanol water mixtures. Acetonitrile and methanol present similar polarities, but acetonitrile demonstrated higher efciency for pesticide extraction (Garrido Frenich et al., 2007). For this reason, acetonitrile was considered to be more suitable for desorbing the analytes from the nal MSPD-column that was four times larger than the preliminary MSPD-column. Additionally, the use of a larger MSPD-column required that the water volume be scaled-up, from 5 to 20 mL, to eliminate the polar and coloured matrix components during the washing step. Likewise, the percent of acetonitrile in the acetonitrilewater clean-up mixture was reduced from 30% (as used in the methanol elution preliminary studies) to 25% with an elution volume of 3 mL (instead of 4 mL) to avoid breakthrough of methyl-parathion. Finally, the elution volume of the pesticides was established at 10 mL to ensure reproducible results, even for the most retained pesticides. Recoveries from these assays in liver samples (Table 1) indicate that desorption with 60% acetonitrile was poor for diazinon and chlorpyrifos, whereas the 80% and 100% acetonitrile eluents attained quantitative recovery of all analytes, except chrorfenvinfos, which showed a 6364% recovery value. This low recovery efciency was also observed in subsequent assays with liver samples, but not in muscle samples. This fact could be due to stronger interactions of chrorfenvinfos with the MSPD-column or co-eluting matrix. MSPD interactions involving the stationary phase, eluting phase, and all of the matrix components may affect the recoveries and results even though they are not well-understood (Barker, 2000). Taking into consideration the overall recovery results, it is important to note that the 100% acetonitrile fraction produces better recoveries and non-coloured extracts than the methanolic eluents, indicating a lower level of co-extracted interferents than the methanol extract. In addition, the acetonitrilewater (25:75 v/v) washing eluent could eliminate an important fraction of interferences without breakthrough of the most polar parathion-methyl pesticide as observed in Table 1. Collection and subsequent analysis of 10 1 mL of each of the percolated eluents was performed to determine the optimum elution volume for quantitative recovery of all compounds. Fig. 1 presents the cumulative recoveries of pesticides with acetonitrile and the two acetonitrilewater mixtures tested. Fig. 1A clearly shows that the 10 mL of 60% acetonitrile quantitatively eluted only the most polar pesticides, parathionmethyl and fenitrothion. Larger amounts of eluting solvent would be needed to completely extract all the analytes. Fig. 1B shows that a 9 mL volume of 80% acetonitrile was enough to completely elute all analytes from the cartridge (refer to the chorpyrifos elution prole) and Fig. 1C shows that a 4 mL volume of pure acetonitrile allows the complete desorption of all ve analytes. As a result, pure acetonitrile was selected as the eluting solvent of pesticides in this

M.P. Garca de Llasera, M.L. Reyes-Reyes / Food Chemistry 114 (2009) 15101516

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100

75

50

25

of 25:75 v/v acetonitrilewater solution. In summary, the nal elution sequence was: (a) 20 mL of water, (b) 3 mL of 25:75 v/v acetonitrilewater, (c) 1 mL of 60:40 v/v acetonitrilewater, and (d) 5 mL of pure acetonitrile. Once optimisation of the elution sequence was complete, the next step in method optimisation was reduction of the extract volume by evaporation. One consequence of this evaporation step was the concentration of analytes. At the same time, however, perceptible co-eluted interferents were also concentrated. Given that even non-coloured, acetonitrile extract could contain a great number of interferences, especially lipidic compounds, the acetonitrile extract was cleaned up with sorbents. 3.1.2. Clean-up with sorbents Three sorbents, silica, C18, and orisil, of which 0.5 g of each was packed in different cartridges, were considered as a means of cleaning the extract. At the same time, another assay was made with the same amount of silica packed in the bottom of the MSPD cartridge. Recoveries obtained after rotary evaporation of the extract at 40 C are shown in Table 1, where it can be observed that the use of the silica sorbent packed in an independent cartridge gave the best recoveries, whereas both the silica sorbent packed on the bottom of the MSPD cartridge and the C18 co-column produced lower recoveries than the silica sorbent packed in an independent cartridge for all compounds. On the other hand, the use of silica in the bottom of the MSPD phase was not advantageous in this case, perhaps because this sorbent receives and retains a lot of interferences that are subsequently desorbed from the MSPD phase during the washing step. In contrast, the orisil clean-up gave a much cleaner extract and consequently better chromatograms; however this clean-up produced the lowest recoveries. The orisil sorbent, on which polar analytes are retained too strongly, has often been preferred for the clean-up of apolar pesticides in fatty matrices because of its potential to retain lipids (Kristenson et al., 2006). As the current study illustrates, the use of orisil may result in poor recoveries of certain OPPs in fatty animal matrices (Garrido Frenich et al., 2007; Juhler, 1997). For the reasons mentioned above, a silica clean-up made on an independent column was selected in the nal protocol. 3.2. Performance of the analytical procedure 3.2.1. Regression analysis and recovery The method was validated using uncontaminated liver and muscle samples processed according to the nal MSPD extraction conditions described above. Linear calibration curves were prepared at six levels of fortication in triplicate measurements and plotted by a least-squares regression of concentration vs. the relative peak area for each analyte. Adequate linearity was observed in the concentration range from 0.5 to 15 lg g1, with correlation coefcients higher than 0.99 achieved for most of the OPP compounds in both liver and muscle. The exception was chlorfenvinphos, which had a regression coefcient of 0.95 in the liver sample. Recovery efciencies were determined and accuracy was evaluated using the added vs. recovered amount curves. The linear parameters and correlation coefcients are presented in Table 2, and they show good linearity. The optimised extraction procedure is highly efcient with recoveries higher than 94% in liver and 95% in muscle, except chlorfenvinphos in liver (55%). The condence interval is <5.7% for all samples. Global recovery efciency can be deduced from the slope of the recovery curve parameters from Table 2. 3.2.2. Precision and lower limit values The precision of the whole method was evaluated in terms of single-day repeatability and inter-day precision. Table 3 compiles

Recovery (%) A

0 0 1 2 3 4 5 6 7 8 9 10 11

Acetonitrile 60% (mL)

100

75

Recovery (%) B

50

25

0 0 1 2 3 4 5 6 7 8 9 10 11

Acetonitrile 80% (mL)

100

Recovery (%) C

75

50

25

Acetonitrile (mL)

parathion-methyl chlorfenvinphos chlorpyrifos

fenitrothion diazinon

Fig. 1. The cumulative recoveries of pesticides as a function of eluent volume (previous washing step: 20 mL water + 3 mL acetonitrile:water 25:75 v/v). Eluting solvent (A) acetonitrile:water (60:40 v/v); (B) acetonitrile:water (80:20 v/v) and (C) pure acetonitrile. MSPD was performed with 2 g of C18 and 0.5 g of liver.

work because it produced the best recoveries with the least elution volume. The nal elution volume was established at 5 mL to ensure reproducible results, even for the most retained pesticides. Finally, an additional clean-up step was considered to remove polar interferents. The elution prole in Fig. 1A shows that no breakthrough of pesticides was observed with 1 mL of 60% acetonitrile eluent. Thus, one millilitre of a 60:40 v/v acetonitrilewater mixture was included in the nal elution sequence after the application of 3 mL

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Table 2 Regression analysis parameters from the added vs. recovered amount curve (linearity range of spiked samples: 50015,000 ng). Pesticide Parameters r Liver Parathion-methyl Fenitrothion Chlorfenvinphos Diazinon Chlorpyrifos 0.995 0.991 0.946 0.994 0.997 Muscle 0.997 0.995 0.997 0.997 0.998 b (100) Liver 93.5 2.3 96.3 4.0 54.8 5.7 94.8 1.0 100.7 2.3 Muscle 98.5 3.3 101.2 3.3 97.8 2.5 95.4 2.1 99.2 1.8 a Liver 19 55 16 95 63 99 11 75 37 54 Muscle 4 43 22 43 7 32 12 27 10 23

y = a + bx (a = intercept; b = slope and r = correlation coefcient).

Table 3 Method precision expressed as the relative standard deviation (RSD), limits of detection (LODs) and limits of quantication (LOQs) of pesticides extracted from bovine samples. Pesticide RSD% 5 lg g Liver Parathion-methyl Fenitrothion Chlorfenvinphos Diazinon Chlorpyrifos
a b c 1

LOQs (ng g1) 2.5 lg g Muscle 6.7 7.0b 15.0b 8.5b 4.5b
b 1

LODs (ng g1)

0.25 lg g Muscle Liver 6.6 6.6b 5.5b 3.4b 3.2b

b

Liver 5.6 4.5b 2.7b 3.2b 3.1b

b

Muscle 9.4 11.8b,c 14.9b,c 13.6b,c 13.0b,c

b,c

Liver 7.6 7.9b,c 7.1b,c 11.5b,c 9.9b,c

b,c

Muscle 75 75 150 75 200

Liver 50 50 75 50 100

Muscle 25 25 50 25 50

5.0 5.1a 10.0a 8.0a 3.0a

4.1 3.9a 2.5a 1.6a 1.9a

4.7 6.2a 10.5a 8.6a 3.8a

7.6 7.5b 14.8b 10.1b 4.6b

5.2 4.5a 4.3a 2.5a 2.6a

7.1 10.5a 11.8a 9.3a 12.5a

6.8 6.5a 6.0a 10.2a 8.5a

150 150 200 150 300

Repeatability (single-day) n = 5. Reproducibility (inter-day) n = 5. n = 3.

Fig. 2. HPLC chromatograms of the extracts from a unhealthy bovine liver sample. Detection at two wavelengths (244 and 287 nm). Sample spiked at 1 lg/g (A: 244 nm and B: 287 nm); non-spiked sample (a: 244 nm and b: 287 nm). Chromatographic conditions were as explained in Section 2. Peak identications: 1 = parathion-methyl; 2 = fenitrothion; 3 = chlorfenvinphos; 4 = diazinon; and 5 = chlorpyrifos. Arrows indicate chlorfenvinphos (peak 3) and chorpyrifos (peak 5).

the recovery values and the RSDs obtained from these assays. Additionally, the limits of detection (LODs) and limits of quantication (LOQs) were obtained. Satisfactory results were found for all pesticides, with RSD values no greater than 15% in liver and 11.5% in muscle. LODs and LOQs were calculated at the wavelength of maximum absorption for each pesticide using the minimum accepted signal-to-noise (S/N) values of 3 and 10, respectively. The LODs of pesticides were below the regulatory tolerance levels in Mexico (NOM-004-ZOO-1994), but they are a little high compared with the maximum residue levels (MRLs) established by the European Union (Commission Regulation (EC) 149/2008).

3.3. Application of the method to real samples The proposed method was applied to the analysis of 20 real liver samples: 10 samples of healthy appearance and ten samples of unhealthy appearance. The results from the real samples show that only two pesticides, chlofenvinphos and chlorpyrifos, were detected in two different unhealthy samples. Fig. 2 shows the HPLC chromatogram of one of these samples. These positive samples did not exceed the LOD concentrations of these pesticides using the developed method. As a result, subsequent analysis of the extracts using GCMS with acquired ion signals using time-sched-

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noma de Mxico) for their cooperation in collecting samples. Also, the authors would like to thank A. Pea (Facultad de Qumica de la Universidad Nacional Autnoma de Mxico) for her technical assistance in gas chromatography. This work was supported by the Direccin General de Asuntos de Personal Acadmico from the Universidad Nacional Autnoma de Mxico (DGAPA-UNAM Research Project IN203302).

References
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Fig. 3. Selected ion monitoring (SIM) gas chromatograms of (a) a standard solution at 0.1 mg/L, and (b) the extract from the unhealthy bovine liver sample. Chromatographic conditions were as explained in Section 2. Detection: collection time 013.2 min, monitored ions m/z (relative abundance) 197 (100), 258 (61), 314 (80); collection time 13.213.4 min, monitored ions m/z (relative abundance): 267 (100), 269 (88), 323 (86) and 325 (86). Peak identication: 3 = chlorfenvinphos and 5 = chlorpyrifos.

uled multiple-ion SIM mode (Fig. 3) was able to conrm the presence of these pesticides. GCMS was more sensitive than HPLCDAD, but in this case, an exhaustive MSPD clean-up with 1 g of orisil sorbent was necessary to eliminate the lipidic interference from the samples. Consequently, recoveries were low, less than 30% for both compounds. 4. Conclusions A simple and reproducible analytical MSPDHPLC method for determining residues of organophosphorus pesticides in bovine samples was developed. The MSPD extraction procedure using C18 sorbent followed by a silica clean-up was carefully optimised to maximise recovery of the pesticides contained in bovine samples while eliminating most of the interfering matrix components. Acceptable recoveries for pesticides P94% were obtained, except for chlorvenvinphos in liver, for which the recovery seems to be a function of the analysed tissue (recovery is 55% in the liver samples and 98% in the muscle sample). The results demonstrate that the accuracy, precision and selectivity of the proposed method are satisfactory for analysis of the OPPs examined in this study. The limits of detection (LODs) were between 25 and 100 ng g1, allowing application of the procedure for detection below the levels imposed by existing regulations. Acknowledgements The authors would like to thank L. Garca and I. Rangel (Facultad de Estudios Superiores Cuautitln de la Universidad Nacional Aut-

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Russo, M. V., Campanella, L., & Avino, P. (2002). Determination of organophosphorus pesticide residues in human tissues by capillary gas chromatographynegative chemical ionisation mass spectrometry analysis. Journal of Chromatography B, 780, 431441. Salas, J. H., Gonzlez, M. M., Noa, M., Prez, N. A., Daz, G., Gutierrez, R., et al. (2003). Organophosphorus pesticide residues in Mexican commercial

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