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TFJD448-05-163454 703.

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Journal of Environmental Science and Health Part B, 41:1–6, 2006


Copyright C Taylor & Francis Group, LLC
ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/03601230600634745

1 Analysis of Organophosphorus
2 Pesticides in Whole Milk by
3 Solid Phase Microextraction
4 Gas Chromatography Method
5 Zenilda de Lourdes Cardeal and Claudia Maria Dias Paes
6 Chemistry Department, ICEx, Federal University of Minas Gerais, Belo Horizonte, M.G.,
7 Brazil
8 Solid phase microextraction (SPME) was used for the extraction of residual coumaphos
9 and dichlorvos in whole milk. The residues were analyzed by capillary gas chromatog-
10 raphy equipped with nitrogen phosphorus detector (GC-NPD). A manual SPME holder
11 with a 100-µm polyacrylate fiber was used. The optimized conditions for extraction
12 by SPME method were: sample agitation, absorption temperature of 30◦ C, absorp-
13 tion time of 40 min, desorption time of 10 min, and sample volume was 16.0 mL in
14 the vial. Under these conditions, the calibration graphs were linear in the range of
15 0.17 µgL−1 to 1.75 µgL−1 for coumaphos and 0.69 µgL−1 to 6.90 µgL−1 for dichlorvos.
16 Precision was good with RSD values of 13% for coumaphos and 14% for dichlorvos.
17 The detection limits (LOD) were 0.060 µgL−1 for dichlorvos and 0.052 for coumaphos.
18 The quantification limits (LOQ) were 0.086 µgL−1 for dichlorvos and 0.066 µgL−1 for
19 coumaphos. The results obtained in this study suggest that SPME is a suitable tech-
20 nique for residual pesticide analysis of milk. The data demonstrate that particular
21 OP pesticides used in dairy farming in the region of Minas Gerais were found to
22 contaminate cow whole milk, and the residues are not removed by treating the milk
23 by boiling.

24 Key Words: Milk analysis; OP residues; Solid phase microextraction; Gas chromatogra-
25 phy.

26 INTRODUCTION
27 The external application of organophosphorus (OP) pesticides as insecticides
28 directly on dairy cattle for ectoparasite control within dairy farms may add to

Received January 18, 2005.


Address correspondence to Zenilda Cardeal, Chemistry Department, University of
Minas Gerais, Belo Horizonte, M.G., C.P. 702, 31270-901, Brazil; E-mail: zenilda@
ufmg.br or x03201@ems.rmit.edu.au or Zenilda@qui.ufmg.br

1
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Analysis of Organophosphorus Pesticides in Milk 3 Q3

72 MATERIALS AND METHODS

73 Chemicals, Materials, and Sample Preparation


74 The pesticide standards were acquired from PolyScience, Niles, USA;
75 coumaphos (99.4%) and dichlorvos (93%). The stock solution of each pesticide
76 was first prepared in methanol HPLC grade (Merck, Darmstadt, Germany)
77 with concentrations of 2748.41 mg L−1 for coumaphos and 3864.15 mg L−1 for
78 dichlorvos. A range of different dilutions was prepared in water purified by a
79 Milli-Q system (Millipore, Milford, MA, USA) and in pasteurized UHT milk (a
80 control milk sample free of pesticide) purchased in different supermarkets of
81 Belo Horizonte City. Milk samples were obtained from dairy farmers around
82 the cities of Belo Horizonte, Betim, Pedro Leopoldo, Contagem, Lagoa Santa e
83 Ponte Nova. Twenty samples of 1 L were collected in glass vials with Teflon tops
84 and held at the temperature of 3–6◦ C. Analyses were performed in the period
85 of 1 to 5 days after collection.
86 The whole milk samples were mixed with a buffer solution (acetic acid and
87 sodium acetate pH 4.85) with a 1:9 (v/v) ratio. At this pH value the milk protein
88 precipitates and the upper layer becomes clear. The samples were centrifuged,
89 and a 16-mL aliquot of the upper layer was transferred to 20-mL headspace
90
vial.
91 A manual SPME holder and 100-µm thickness polydimethylsiloxane
92 (PDMS) fiber film assembly was purchased from Supelco (Bellefonte, PA, USA).
93 The fiber was immersed directly into the glass vial containing the sample, which
94 was held for 40 min at 30◦ C and stirred with Teflon magnetic stirring bars. Af-
95 ter the extraction, the fiber was retracted into the needle and inserted into the
96 GC injector at 240◦ C for 10 min for thermal desorption and analysis. A blank
97 run of the SPME fiber was carried out before each sample analysis to check
98 memory effect and also to condition the SPME fiber for the next sample.

99 Instrumentation
100 The gas chromatographic analyses were performed on a 3800 Varian gas
101 chromatograph (Walnut Creek, CA, USA) equipped with a HP-5 capillary col-
102 umn (30 m × 0.32 mm × 0.25 µm film thickness, Hewlett Packard Company,
103 Avondale, PA, USA). The split/splitless injector was used in splitless mode at
104 240◦ C for 5 min. The oven temperature was programmed from 150◦ C held for
105 4 min, 20◦ C min−1 up to 280◦ C held for 30 min. A nitrogen-phosphorus (NPD)
106 detector was used with temperature set at 290◦ C. The gas carrier used was
107 helium at a flow rate of 0.8 mL min−1 .

108 Analytical Methods


109 For this work, some SPME parameters used were optimized previously
110 by Silva et al.[9] and Capobiango and Cardeal.[1] Extractions were at room
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4 Cardeal and Dias Paes

temperature because SPME extraction is an exothermic process; consequently, 111


the higher the temperature, the higher the efficiency. The polymeric phase of the 112
fiber chosen was PDMS because of the efficiency of pesticide extractions with 113
this fiber. A extraction/absorption time of 40 min was chosen, since it presented 114
high signal area values and an acceptable time of analysis. A desorption time 115
of 10 min of fiber exposure in the injector at 240◦ C meant that analytes were 116
completely desorbed without carryover effect. The sample volume of 16 mL 117
of sample in a headspace 22-mL vial presented good sensitivity and did not 118
interfere with fiber mass uptake. 119
Standard solutions of coumaphos (range 0.17 to 1.75 µg L−1 ) and dichlorvos 120
(range 0.69 to 6.90 µg L−1 ) were prepared in pasteurized UHT milk (without 121
pesticides residue). These were analyzed in triplicate by SPME-GC method 122
for construction of analytical curves. Precision studies were carried out using 123
a coumaphos solution at 2.07 µg L−1 and dichlorvos at 0.52 µg L−1 . Eleven 124
injections were done for a between-day test. 125

RESULTS AND DISCUSSION 126

Precision, linearity, detection, and quantification limits were studied under 127
optimal extraction and desorption conditions. Retention times in the chro- 128
matogram of whole milk (Fig. 1A) identified the dichlorvos at 3.97 min and the 129
coumaphos at 13.85 min. The values of relative standard deviation (%RSD), 130
also known as variation coefficient, were 14% RSD for dichlorvos and 13% 131
RSD for coumaphos, indicating that this method has good precision. Regres- 132
sion equations and correlation coefficients calculated were: Y = 2.77 + 196.05 X 133
and R2 = 0.996 for dichlorvos and Y = 82.87 + 9729.15 X and R2 = 0.999 for 134
coumaphos. The values of correlation coefficients show that the equations have 135

Q4

Figure 1: Chromatograms of standard solutions of dichlorvos and coumaphos and whole


milk samples: (a) chromatogram of control whole milk sample; (b) chromatogram of
control whole milk sample spiked with standard solution of dichlorvos and coumaphos at
0.24 µg L−1 ; (c) chromatogram of whole milk sample without boiling; (d) chromatogram of
whole milk sample boiled in heater; (e) chromatogram of milk sample boiled in microwave.
TFJD448-05-163454 703.cls March 13, 2006 21:10

Analysis of Organophosphorus Pesticides in Milk 5


136 good linearity in the range of concentrations studied, and therefore it is possible
137 to quantify these pesticides.
138 Limits of detection (LOD) and quantification (LOQ) were determined ac-
139 cording to IUPAC recommendations.[10] Twenty experimental repetitions were
140 performed for the calculation of the blank standard deviation (σ B). The limits
141 of detection and quantitation were calculated by 3.29 × σ B and 16.67 × σ B, re-
142 spectively. The results obtained were: LOD of 0.060 µg L−1 for dichlorvos and
143 0.052 µg L−1 for coumaphos, LOQ of 0.086 µg L−1 for dichlorvos and 0.066 µg
144 L−1 for coumaphos.
145 The analysis of the pesticide contaminated whole milk in the region of
146 Minas Gerais was carried out with triplicate analyses of different samples col-
147 lected in six different cities. A previous study[11] showed that dichlorvos and
148 coumaphos have been widely used on dairy cattle in this region. However,
149 dichlorvos was not detected in any of the samples analyzed. Coumaphos was
150 found in 17 samples, as shown in Table 1. Generally, the household treatment
151 used to eliminate contamination in whole milk is boiling. Thus, the samples of
152 milk were boiled in a heater and in a microwave system and analyzed. Results
153 demonstrate that particular OP pesticides used in dairy farming in the region
154 of Minas Gerais were found to contaminate cow whole milk, and they are not
155 removed by treating the milk by boiling (Table 1 and Fig. 1).
156 The FAO/WHO Joint Committee on Pesticide (JMPR) has recommended
157 guideline levels for residues of coumaphos of 0.02 mg Kg−1 in milk.[11,12] The
158 values obtained in this work were in the range of 0.061 µg L−1 to 0.511 µg L−1 ,
159 which are lower than the limit recommended.

Table 1: Analysis of coumaphos in samples of cow whole milk.

Concentration of coumaphos (µg L−1 )


Sample Without boiling Boiling in heater Boiling in microwave
1 0.133 ± 0.018 0.174 ± 0.024 0.428 ± 0.058
2 0.392 ± 0.006 0.371 ± 0.048 <LOQ
3 0.213 ± 0.029 0.211 ± 0.028 0.511 ± 0.069
4 0.067 ± 0.009 <LOQ 0.192 ± 0.026
5 0.300 ± 0.041 0.202 ± 0.026 0.345 ± 0.046
6 0.090 ± 0.012 0.221 ± 0.029 <LOQ
7 0.147 ± 0.019 0.426 ± 0.058 0.447 ± 0.060
8 0.100 ± 0.014 <LOQ 0.253 ± 0.034
9 0.189 ± 0.026 0.151 ± 0.021 0.240 ± 0.032
10 0.138 ± 0.019 0.218 ± 0.029 0.181 ± 0.024
11 0.071 ± 0.009 0.220 ± 0.029 0.136 ± 0.018
12 0.061 ± 0.008 0.212 ± 0.028 0.198 ± 0.027
13 0.115 ± 0.016 0.307 ± 0.042 0.093 ± 0.013
14 0.205 ± 0.028 0.036 ± 0.005 0.029 ± 0.004
15 0.235 ± 0.031 0.264 ± 0.036 <LOQ
16 0.301 ± 0.041 0.674 ± 0.092 0.447 ± 0.061
17 0.210 ± 0.029 0.356 ± 0.048 0.217 ± 0.029

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