Beruflich Dokumente
Kultur Dokumente
This project describes an efficient and effective analytical method to screen pesticides in fruits and
vegetable samples using liquid chromatography tandem mass spectrometry (LC-MS/MS). A quick, easy,
cheap, effective, rugged, and safe (QuEChERS) method with acetate buffering (AOAC Official Method
2007.01) was used for sample preparation, which has been previously shown to yield high-quality
results for hundreds of pesticide residues in foods.
Key words: Liquid chromatography/mass spectrometry (LC/MS), pesticide residues, pesticides, fruits,
vegetables.
INTRODUCTION
Plant protection products (more commonly known as
pesticides) are widely used in agriculture to increase the
yield, improve the quality, and extend the storage life of
food crops (Fern ndez-Alba and Garc a-Reyes 2008).
Pesticide residues are the deposits of pesticide active
ingredient, its metabolites or breakdown products present
in some component of the environment after its application, spillage or dumping. Residue analysis provides a
measure of the nature and level of any chemical
contamination within the environment and of its
persistence. The pesticides must undergo extensive
efficacy, environmental, and toxicological testing to be
registered by governments for legal use in specified
applications. The applied chemicals and/or their degradation products may remain as residues in the agricultural
products, which becomes a concern for human exposure.
Selected sampling programmes can be used to
investigate residual levels of pesticide in the environment,
their movement and their relative rates of degradation.
The maximum residue levels (MRLs) (or tolerances in
the United States) limit the types and amounts of
residues that can be legally present on foods are set by
regulatory bodies worldwide. Pesticide residue analysis is
tremendously an important process in determining the
safety of using certain pesticides. Pesticides polluting the
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volume GC-MS injection for the analysis of organophosphorus and organochlorine pesticides in vegetables
using a miniaturised ethyl acetate extraction) for the
measurement of only ten organophosphorus and
organochlorine pesticides by GC-MS, but over the past
decade, the number of pesticides typically included in
methods has increased dramatically. The sample
preparation techniques have also advanced to
complement the analytical techniques depending on the
types of analytes and matrices monitored.
Anastassiades et al. (2003) described the quick, easy,
cheap, effective, rugged, low solvent consumption, wide
pesticide range (Polar, pH dependent compounds) and
safe (QuEChERS) method for pesticide residues in food
as an example of a method that takes advantage of the
powerful features of nearly universal selectivity and high
sensitivity of modern GC- and LC-MS(/MS) instruments.
The QuEChERS approach has been extensively
validated for hundreds of pesticide residues in many
types of foods, and has become Association of Analytical
Communities (AOAC) Official Method 2007.01 (Lehotay
et al., 2007) and CEN (2008). The QuEChERS method
has several advantages over most traditional methods of
analysis. High recoveries (greater than 85%) are
achieved for a wide polarity and volatility range of
pesticides, including notoriously difficult analytes. Very
rugged because extract clean up is done to remove
organic acids.
The most common approach is to use matrix-matched
calibration standards. However, it can be difficult to find a
blank matrix from which to prepare the calibration
standards and compensation from one sample to another
(even for the same matrix) may not be the same. A
method of standard additions in the sample extract may
be an alternative approach.
Sample preparation
The acetate-buffered QuEChERS sample preparation method for
pesticides (AOAC Official Method 2007.01) was applied to all the
samples. After homogenization with a house-hold mill (equipped
with stainless steel knives), a 15 g portion of the homogenized
sample was weighed into a 50 ml polytetra fluoro ethylene (PTFE)
tube and 100 ml of 50 mg/ml triphenyl phosphate (TPP) surrogate
standard solution in acetonitrile was added followed by 15 ml of
acetonitrile containing 1% acetic acid (v/v not accounting for purity).
Then, 6 g MgSO4 and 2.5 g sodium acetate trihydrate (equivalent
to 1.5 g of anhydrous form) were added, and the sample was
shaken forcefully for 4 min. The sample was then centrifuged at
4000 rpm for 5 min and 5 ml of the supernatant were transferred to
a 15 ml PTFE tube to which 750 mg MgSO4 and 250 mg PSA were
added. The extract was shaken using a vortex mixer for 20 s and
centrifuged at 4000 rpm again for 5 min. Approximately 3ml of the
supernatant were filtered through a 0.45 mm PTFE filter (13 mm
diameter), and 800 ml portions were transferred to autosampler
vials. The extracts were evaporated to dryness under a stream of
argon and reconstituted in 800 ml acetonitrile/water (20/80, v/v) for
the LC-MS/MS analysis.
For the matrix-matched and standard addition calibrations, 4 to
80 ml of reconstituted samples were transferred into autosampler
glass inserts and 20 ml portions of 0, 250, 500 and 1250 ng/ml
standard mix solutions containing pesticides in 25/75 acetonitrile/
water (v/v) were added to reach the final additional concentrations
of 0, 50, 100 and 250 ng/ g equivalents, respectively.
LC-MS analysis
MATERIALS AND METHODS
Sampling
For the present project random sampling was done from the various
markets in the twin cities of Hyderabad, Secunderabad and R. R.
District local fields and markets. Depending upon the nature of the
vegetation (size, shape, etc.), samples were enclosed in a clean
blotting paper and wrapped inside a clean, paper envelope. The
addition of a small sachet of silica gel to the envelope helps to
reduce the moisture content of the system. However, samples were
provided to the analytical laboratory to check for possible coextractives, which could interfere with the analysis.
Statistical analysis
1. An Analysis Of Variance (ANOVA) test and a t-Test were
conducted between the 'no wash' and 'salted lukewarm water wash'
Dasika et al.
21
Apples
Guava
Vegetables
Egg plant or Brinjal
No Wash: Eggplants showed the most pesticides that
have residue levels over the MRL. They ranged from 100
to 500% above MRL.
Lukewarm Water Wash: The overall residue levels of all
pesticides tested showed significant improvement with
values going down anywhere from 50 to 80% when
compared against no wash readings. However, at this
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S/N
Pesticide
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Pyraclostrobin
Dithiocarbamates
Dodine
Acetamiprid
Diphenylamine
Dithiocarbamates
Imazalil
Phosmet
Pirimicarb
Chlorpyrifos
Captan
Dithiocarbamates
Malathion
Thiacloprid
Thiabendazole
S/N
1
2
3
4
5
Pesticide
Diphenylamine
Phosmet
Chlorpyrifos
Malathion
Thiabendazole
S/N
1
2
3
4
5
Pesticide
Diphenylamine
Phosmet
Chlorpyrifos
Malathion
Thiabendazole
Figure 1. Average pesticide residues (Pink Lady, Granny Smith, Gold Del Apples).
Dasika et al.
23
S/N
1
2
3
4
Pesticide
Imazalil
Thiabendazole
Phosmet
Decamethrin
Green grapes
No wash
Lukewarm water wash
8.6
6.9
21.4
17
11.8
8.9
0.03
ND
S/N
1
2
3
4
Pesticide
Imazalil
Thiabendazole
Phosmet
Decamethrin
Black grapes
No wash
Lukewarm water wash
9.3
8.2
32
27.4
16.7
11.3
0.05
0.04
24
Pear
S/N
1
2
3
4
5
6
7
Pesticide
Diphenylamine
Imazalil
Phosmet
Pirimicarb
Chlorpyrifos
Captan
Thiabendazole
Max residue
limit (mg/kg)
5
5
10
1
0.5
3
10
No wash
6.1
3
11
1.8
0.6
0.06
26.4
Deviation from
MRL (%)
-14
-58
-11
20
-40
ND
78
Salted lukewarm
Water Wash
3.4
1.8
8.8
0.6
0.2
ND
14.3
Deviation
from MRL (%)
-32
-64
-12
-40
-60
ND
43
Guava
No Wash Lukewarm water wash
4.8
2.2
4.2
3.6
9.3
7.9
0.43
0.28
0.02
ND
Conclusions
The developed combination of the two methods
described above permitted the fast and easy
qualitative screening of target pesticides in a 45
min LCMS/ MS run.
Although the manual evaluation of the given
chromatograms increased the analysis time by an
additional 15 min per sample, very little time, cost
and labour was spent on sample preparation. In
the case of dirty samples, some false indications
were observed, but these were caught by the use
of the MRM confirmatory and quantitative method
for the more common pesticides. The construction
Dasika et al.
25
S/N
1
2
3
4
5
6
7
8
Pesticide
Diphenylamine
Imazalil
Phosmet
Chlorpyrifos
Thiabendazole
Chlorothalonil
Endosulfan
Acephate
S/N
1
2
3
Pesticide
Diphenylamine
Imazalil
Phosmet
5
6
7
8
9
Chlorpyrifos
Thiabendazole
Chlorothalonil
Endosulfan
Acephate
5.2
4.2
2.1
33.2
0.6
2.8
7.8
1.7
24.1
0.45
2.3
5.7
Apples
Grapes
Test revealed increased residue levels of Chlorpyrifos,
Thiabendazole, Malathion and Diphenylamine. Most
26
S/N
1
2
3
4
S/N
1
2
3
4
Pesticide
Chloropyrifos
Imazilil
Thiabendazole
Diphenylamine
Pesticide
Chloropyrifos
Imazilil
Thiabendazole
Diphenylamine
No
Wash
3.5
4.8
11.4
8.7
No
Wash
4.1
3.5
14.6
9.8
Deviation from
MRL (%)
35
-26
0
38
Salted lukewarm
water wash
2.2
3.3
9.7
6.3
Deviation from
MRL (%)
10
-34
-3
26
Deviation from
MRL (%)
45
-52
23
58
Salted lukewarm
water wash
2.3
1.8
11.5
7.2
Deviation from
MRL (%)
15
-64
15
44
Dasika et al.
27
Figure 6. Average pesticide residue analysis (green and red bell peppers).
Statistical Tests
Pesticide tested
Diphenylamine
Chlorpyrifos
Imazalil
Thiabendazole
Phosmet
-value
Degrees of freedom
t-value
Look-up t-value
p-value
0.05
0.05
0.05
0.05
0.05
15.86
12.56
18.74
17.4
12.96
4.05
1.88
2.13
2.31
3.45
1.753
1.782
1.74
1.74
1.771
0.00097
0.085
0.046
0.033
0.0043
Statistically significant?
yes
yes
yes
yes
yes
Green pears
Guava
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