American Journal of Analytical Chemistry, 2011, 2, 511-521
doi:10.4236/ajac.2011.25061 Published Online September 2011 (http://www.SciRP.org/journal/ajac)
Liquid Chromatography Mass Spectrometer (LC-MS/MS)
Study of Distribution Patterns of Base Peak Ions and
Reaction Mechanism with Quantification of Pesticides
in Drinking Water Using a Lyophilization Technique
Sukesh Narayan Sinha*
National Institute of Nutrition (ICMR, New Delhi), Jamai-Osmania, Hyderabad, India
Received May 30, 2011; revised July 1, 2011; accepted July 15, 2011
Abstract
In the process of the development of agriculture, pesticides have become an important tool as an insecticide
to kill the insect from plant for boosting food production. Therefore the insecticides/pesticides and herbicides
have been used in India for agriculture setting. In this connection a sensitive method for the quantification of
5 pesticides in drinking water samples to the µg·L–1 level has been developed. The paper also describes the
effect of dissociation energy on ion formation and sensitivity of pesticides in water samples. The structure,
ion formations, distribution of base peak and fragmentation schemes were correlated with the different dis-
sociation energies. The new ion was obtained at different mass to charge ratio, which was the characteristic
ion peak of targeted pesticide. Additionally, a simple solvent lyophilization followed by selective analysis
using a liquid chromatography-mass spectrometry method was used. This method was accurate (≥98%) as it
possesses limits of detection in the 6 - 38 ng·L–1 range, and the percentage relative standard deviations are
less than 8.62% at the low µg·L–1 end of the method‟s linear range. The percentage recovery of all the pesti-
cides at the 0.1 µg ·L–1 levels of detection ranges from 92% - 104%. This method was used for the quantifica-
tion of pesticides in water samples collected from different parts from urban city of Hyderabad, India. In this
study, 13 water samples were analyzed in which all samples showed detectable level of the malathion and
alachlor. The concentration of pesticides ranged from 0.004 µg·L–1 to 0.691 µg·L–1 exceeded to the maxi-
mum residual limit of Indian standard.
Keywords: Water, LC-MS/MS, Lyphilization, Pesticides, Dissociation Energy
1. Introduction
Agricultural development continues to remain the most
important objective of Indian planning and policy. In the
process of development of agriculture, pesticides have
become an important tool as a plant protection agent for
boosting food production. Currently, India is the largest
producer of pesticides in Asia and ranks twelfth in the
world for the use of pesticides [1]. Humans are exposed
to pesticides through soil, water, air and food by different
routes of exposure such as inhalation, ingestion and der-
mal contact [2]. For instance, dietary intake represents
the major source of pesticide exposure to children, and
this exposure may increase pesticide-related health risks
in children in comparison to adults [3]. Increasing inci-
dences of cancer, chronic kidney diseases, suppression of
the immune system, sterility among males and females,
endocrine disorders and neurological and behavioral dis-
orders, especially among children, have been attributed
to chronic pesticide poisoning [1].
The presence of pesticide residues in various compo-
nents of the environment and food commodities is a
matter of concern all over the world [4-6]. In India sev-
eral methods have also been used for pesticide residual
analysis in different food commodities (e.g., vegetables,
fruits and other products of food) using a GC method
[7-9]. We also analyzed pesticide levels using different
method in food and biological samples [10-14]. Fur-
thermore, a method was reported [15] for the analysis of
pesticide residues using a quick, cheap, effective, rugged,

S. N. SINHA ET AL.
512
and safe (QuEChERS) multi-residue method in combina-
tion with gas and liquid chromatography (LC-MS/MS)
and tandem mass spectrometric detection. A mixture of
38 pesticides was quantitatively recovered from spiked
lemon, raisins, wheat and flour using GC-MS/MS, while
42 pesticides were recovered from oranges, red wine, red
grapes, raisins and wheat flour using LC-MS/MS for
determination [15]. A multi-analyte method for the quan-
tification of contemporary pesticides in human serum
and plasma using high-resolution mass spectrometry was
reported [16].
I have used a very accurate, simple and reproducible
LC-MS/MS method for the quantification of pesticide
residue at low levels in drinking water samples collected
from different part of urban areas.
2. Experimental Sections
2.1. Materials
All pesticide standards were purchased from Sigma-Al-
drich, Inc. (USA). Methanol acetonitrile (LC-MS grade),
water (LC-MS grade) were obtained from Sigma Aldrich
GmbH. Formic acid was purchased from Sigma Aldrich
(USA). All reagents were made freshly in LC-MS grade
water or solvent before use.
2.2. Stock Solutions
Individual stock solutions at 1 mgL–1 of pesticides (alach-
lor, malathion, dimethoate, chlorpyrifos and metribuzin)
were prepared in acetonitrile. The stock solutions were
divided into aliquots, sealed in ampoules and stored at
–40˚C.
2.3. Calibration Standard
From the stock solutions, eleven working standard sets
for alachlor, malathion, dimethoate, chloripyrifos and
metribuzin (0.1, 0.2, 1, 2, 5, 10, 30, 50, 100, 150 and 250
ngml–1) were prepared to encompass the entire linear
range of the method by using serial dilution technique.
These standards were then used for the validation of me-
thod (determination of limit of detection (LOD), limit of
quantification (LOQ), recovery experiment and linearity
experiment). The standard sets were divided into aliquots,
sealed in ampoules and stored at –40˚C until use.
2.4. Laboratory Reagent Blanks
Before extraction of water samples, the purchased water
samples were tested by LC-MS/MS using a similar ex-
traction method that was used for the recovery experi-
ment, and the water was found to be free from pesticide
residues.
2.5. Recovery Experiment by GC-MS/MS
The water sample was spiked with the standard of each
compound, alachlor, malathion, dimethoate, chlorpyrifos
and metribuzin at the different level (0.1, 0.2, 1, 2, 5, 10
and 30 µg·mL–1).
2.6. Sampling
Thirteen water samples were included in the sampling of
water for the purpose of pesticide residue analysis. One
liter water samples were collected from different part of
the urban city. Five ml water has been taken for lyophi-
lization.
3. Sample Preparations
Unknown water and reagent blanks were prepared iden-
tically. Five mL of pure water was pipetted into 20 mL
test tubes. The water was spiked with the mixtures of
different pesticides at different concentrations (0.1, 0.2, 1,
2, 5, 10 and 30 ng·mL–1 of alachlor, malathion, dime-
thoate, chlorpyrifos and metribuzin). Then water was
mixed and allowed to equilibrate for approximately 30
minutes. The tubes were then placed in a methanol bath
and held at –100˚C for at least 15 min. Once the samples
were frozen, they were placed in a lyophilizer at –109˚C.
The vacuum status was checked and the samples were
left for 6 hours to ensure complete dryness. The samples
were then removed from the lyophilizer for extraction.
Four milliliters of acetonitrile was added at neutral pH (7)
to each tube, mixed for 3 min on a vertex shaker and
supernatant was transferred into 20 ml centrifuge tubes.
In the second step, samples were extracted with 4 milli-
liters acetonitrile for 3 min. and supernatant was then
transferred to the first extract. There after all of the ex-
tracted tubes were centrifuged for 10 min at 3000 rpm.
Next, the supernatant solution was transferred into a new
set of 20 ml tubes for drying and placed in a TurboVap at
room temperature under 5-psi nitrogen and completely
dried. The dry residues were reconstituted in 1 mL ace-
tonitrile for analysis.
4. Instrumental Analysis
4.1. Chromatographic Condition
Ten micro-liter of the concentrated extract was analyzed
using, 4000-QTRAP triple-quadrupole hybrid mass spec-
trometer in MRM mode. The analysis of all pesticides
was performed using a liquid chromatograph (LC, Shi-

S. N. SINHA ET AL.
513
madzu, LC 20 AD, binary pump ) interfaced to a 4000-Q
Trap (Applied Biosystems MDS Sciex, USA) mass de-
tector with data analyst software (version 1.4.2) required
for the integration, calibration, collection of LC-MS
spectra and data processing for qualitative and quantita-
tive analysis. The mass spectra operated in the positive
turbo ion spray (ESI) mode. Chromatographic Separation
was achieved on a Phenomenex C18 reversed phase col-
umn with an ID of 5 µm and dimensions of 50 m × 4.68
mm. Ten micro-liter samples were injected using a Shi-
madzu auto-sampler fitted with a Hamilton 100-µl sy-
ringe. Different gradient of mobile phase compositions
of 0.1% formic acid in water and acetonitrile at a flow
rate of 0.5 mL·min–1 were used. The different gradient
compositions have shown in Figure 1. The column oven
temperature was operated at room temperature. The total
running time was 12 min. The spectra of different pesti-
cide were recorded on different dissociation energy (DE)
(10 V - 80 V), injecting similar concentration of analyte
to demonstrate the effect of DE on relative abundance of
molecular ions as well as fragment ions in MS/MS.
4.2. Multiple Reaction Monitoring (MRM) Study
To develop a more sensitive method at the 0.1-µgL–1
level for determining the concentration of these pesti-
cides in water samples, the MRM method was used using
in positive ESI mode with high resolution. The ion-spray
voltage (IS) was used 5500 eV and interface heater was
held at the temperature of 550˚C. A full auto tune of the
mass spectrometer was performed before the analysis of
every set of samples. To select the most abundant ions
(Q1) a full scan of the mass spectra of all pesticides were
recorded by using continuous infusion of each pesticide
in the positive ionization mode of ESI. The daughter mass
spectra were obtained with continuous infusion of each
analyte, so Q1, corresponding to the protonated parent
ion. The most abundant daughter ion for each compound
was then selected for MRM analysis. Besides, this, the
three principle ion criteria was applied for isolation of
two of the most intense product ions: one ion was used
for quantification, whereas the other was used for con-
firmation. The method of isolation of ions were carried
out as per reported method [10,14,18,19]. The optimiza-
tion of the source dependent parameters, such as curtain
gas, heating gas (GS2) and nebulizing gas were carried
out in the flow injection analysis (FIA) mode. The cur-
tain, GS1 and GS2 gas pressures were then maintained at
25, 35 and 40 psi, respectively, during the entire study.
Table 1 showed the declustering potential (DP), collision
energy (CE), entrance potential (EP) and collision exit
potential (CXP) were used as per the required sensitivity
of the method.
5. Quantifications
5.1. Calibration Curve
Seven different concentrations (0.1, 1, 5, 10, 25, 50, and
150 µgL–1) for each OP pesticide, insecticide or herbi-
cide (alachlor, malathion, dimethoate, chlorpyrifos and
metribuzin) was plotted against the area of the pesticide
to determine the correlation coefficient (Table 3) and
percentage accuracy of this method at µgL–1 level in each
Figure 1. Percentage of acetonitrile in 0.1% formic acid at
different time interval.
Table 1. The isolated precursor and product ions of different pesticides in multiple reaction monitoring (MRM) using differ-
ent energy profiles.
Pesticides
Precursor Ion
(Q-1)
Product ion
(Q-2)
1DP
2CE
3EP
4CXP
Duel
time
5RT
(min)
Alachlor
270.13
238.10
28
15
10
15
30
2.32
Malathion
330.8
127
26
17.80
10
12
30
2.09
Diomethoate
229.80
199.00
16
13
10
6
30
1.36
Chlorpyrifos
350
198
50
25
10
12
30
3.68
Metribuzin
215.3
187.1
31.00
15
7.40
18
30
1.59
2CE = Collision Energy; 3EP = Entrance potential; 4CXP = Collision exit potential; 5RT = Retention time.

S. N. SINHA ET AL.
514
analytical run. Linear regression analyses were performed
on plots of the calculated concentrations versus expected
concentrations. With this analysis, a slope of 0.999 would
be indicative of 99% accuracy (Table 3)
5.2. Recovery Experiment
The recoveries of the method were determined by spik-
ing water samples free of pesticides with different known
concentrations of reference standards. The recovery of
each pesticide was calculated at each of the known con-
centration levels by comparing the measured concentra-
tions with the spiked concentrations, as per the reported
method [17,18,]. A ratio of 1.00 indicated 100% recov-
ery. LC mixtures of 0.10, 0.20, 1, 2, 5, 10, 30 ppb for
alachlor, malathion, dimethoate, chlorpyrifos and me-
tribuzine) in acetonitrile were prepared using the pesti-
cide reference standards previously described. The per-
centage recovery of each pesticide was calculated by
comparing the peak area ratio of the spiked standards
with those of the pure standards. Water samples were
fortified with the mixture of the five pesticides at differ-
ent concentration (0.10, 0.20, 1, 2, 5, 10, 30 µg·L–1) and
allowed to standing for 30 min so that all of the pesti-
cides were absorbed thoroughly by the samples before
making the extraction. Seven un-spiked water samples
and 7 reagent blanks served as the negative control for
quality assurance purposes. All the samples were ex-
tracted as previously described.
5.3. Limit of Detection (LOD)
The point at which the measured value was considered
reliable was when it was larger than the uncertainty as-
sociated with it, also called the LOD. In this method, the
analytical LOD was calculated as per the earlier reported
method [17,18].
5.4. Lower Limits of Method Validation (LLMV)
The LLMV by LC-MS/MS for alachlor, malathion, di-
methoate, chlorpyrifos and metribuzin were 0.1 µg ·L–1.
6. Results and Discussions
This method was developed to confirm and accurately
quantify pesticides in water samples. The lyophilization
followed by extraction process was simple, accurate and
easy. In the case of water samples, several variations of
the extraction procedure were attempted. In many cases
these extractions were not optimal, and good recovery of
the analytes was not achieved due to the polar nature of
OPs pesticides. Therefore, I used a simple lyophilization
process for the complete dryness of the samples to mi-
nimize the matrix effect. The extraction of the analytes
from dry samples was easy and overall good recovery
was achieved. In this method, 5 mL of water samples
was lyophilized and was extracted at neutral pH using 5
mL of a acetonitrile twice with two minutes of shaking
each time. The percentage recoveries and percentage
RSD obtained were well within previously prescribed
analytical method [16]. The acetonitrile resulted in 82% -
104% extraction efficiency for alachlor, malathion, di-
methoate, chlorpyrifos and metribuzin in the water sam-
ples. The different solvent gradient was fixed to accom-
modate the physical and chemical properties of the pesti-
cides (Figure 1). The specificity of 4000 Q-trap mass
spectrometry allows for the elimination of interfering
components in the water sample extracts, which in turn
provided the low detection limits of the method. These
specificity requirements precluded the use of single qua-
drupole mass spectrometry. Thus, this method was applied
in MRM mode to increase the sensitivity for quantifica-
tion at the µg ·L–1 level. The extracted ion chromatograms
of alachlor, malathion, dimethoate, chlorpyrifos and me-
tribuzin (10 µg ·L–1 spikes) are shown in Figure 2.
The isolation of ions of OP pesticides was carried out
in a similar fashion as per the reported method [10,14,17,
18]. In first series of experiment the full scan spectra
were recorded, using manual tuning in FIA mode after
that the characteristic stable ions were isolated for MRM
transition for confirmation and quantification of five pes-
ticides in water samples. The detail isolated ion for quan-
tification, different energy parameters (DE, EP, FP and
CE) and retention times (RT) for MRM transitions are
shown in Table 1. The confirmation ions were isolated at
m/z 162, 99, 171, 125 and 131 for alachlor, malathion,
dimethoate, chlorpyrifos and metribuzin, respectively, by
using different energy set up. The percentage recovery
and RSD has been shown in Table 2.
The selected molecular ion, and selected product ion
scan were performed and different collision dissociation
energies were applied in MS/MS mode to obtain different
fragmentation patterns. The ion formation of study sam-
ple of dimethoate is shown in Figure 3 The m/z 198 was
obtained due to the elimination of ethylene (–CH2=CH2–)
molecule from parent ion molecule m/z 229.9, because
the oxy-gen atom donates the lone pair to hydrogen atom
by remote charge mechanism. Similarly, -N=CH2 mole-
cule was removed from m/z 198 leading to the formation
of structure at m/z 170. The dimethoate possesses a suf-
ficient long chain to permit transfer of hydrogen namely
loss due to hydrogen rearrangement mechanism. Similar
pattern noted previously with triazofos, chlorpyrifos and
phenolate ion [10,14]. The structure was formed at m/z
124 due to the removal of –C3H6SNO group from m/z
229.9. This new structure has been isolated due to struc-

S. N. SINHA ET AL.
515
ture reactivity and ion reaction mechanism of dimethoate.
The fragmentation scheme of chlorpyrifos has been shown
in Figure 4. The m/z 321.1 was obtained due to the eli-
mination of ethylene (–CH2=CH2–) molecule from par-
ent ion molecule m/z 350, because the oxy-gen atom
donates the lone pair to hydrogen atom by remote charge
mechanism. Additionally, the phosphorous atom is sta-
bile through dл-pл bonding, therefore ethylene molecule
removed from the m/z 321.1 leading to the formation of
new structure at m/z 293.4. Similarly, –C4H10O2PS mo-
lecule was removed from parent ion m/z 350 leading to
the formation of structure at m/z 197.8. The new stable
structure was formed at m/z 152.8 due to the removal of
–CCl group from m/z 197.8. This new structure has been
isolated due to rearrangement and ion reaction me- chan-
ism of chlorpyrifos.
The ion formation and reaction activity of alachlor has
been shown in Figure 5. The m/z 238 was obtained due
to the elimination of methyl alcohol (–CH3OH) molecule
from parent ion molecule m/z 270, because the oxy-gen
atom donates the lone pair to hydrogen atom by remote
charge mechanism. Additionally, the nitrogen atoms ob-
served steric hindrance, which deactivate the whole mole-
cule and therefore C2HOCl molecule removed from the
m/z ion 238 leading to the formation of new stable mo-
lecule at m/z 162. Similarly, a new structure was formed
at m/z 110 due to expulsion of acetylene molecule from
m/z 162. Additionally, a new structure at m/z 137 was
formed due to removal of C2H molecule from m/z 162.
Figure 2. Extracted ion chromatogram (EIC) of spiked water samples at 10 PPB each (1) alachlor, (2) malathion, (3) dime-
thoate (4) chlorpyrifos (5) metribuzin.

S. N. SINHA ET AL.
516
Table 2. Percentage recovery (mean) and % RSD of pesticides at different spiked concentrations.
Spiked concentration (ngmL–1)
0.10
0.20
1.00
2.0
5.0
10.0
30.00
Diomethoate
SD
2.60
2.96
2.85
6.23
4.65
4.30
0.89
% RSD
2.54
2.83
2.86
6.21
5.01
4.36
0.88
% Recovery
102
104
99
100
92
98
101
N
5
5
5
5
5
5
5
Alachlor
SD
3.54
4.77
1.63
5.92
8.17
3.43
4.72
% RSD
3.47
4.76
1.02
5.84
8.62
3.45
4.60
% Recovery
102
100
102
101
94
99
102
N
5
5
5
5
5
5
5
Malathion
SD
2.95
3.33
2.94
2.11
4.37
2.74
1.60
% RSD
2.81
3.26
2.92
2.10
4.54
2.77
1.60
% Recovery
102
102
101
99
96
99
100
N
5
5
5
5
5
5
5
Chlorpyrifos
SD
7.09
1.25
5.97
5.67
6.24
4.63
0.504
%RSD
6.94
1.27
6.26
6.26
6.23
4.45
0.508
% Recovery
102
98
95
102
100
104
100
N
5
5
5
5
5
5
5
Metribuzin
SD
2.30
2.56
3.39
4.10
7.29
6.22
3.19
% RSD
2.26
2.60
3.31
4.25
7.28
6.06
3.21
% Recovery
101
98
102
96
100
102
99
N
7
7
7
7
7
7
7
RSD = Relative Standard Deviation; SD = Standard Deviation; N= Number of replicate
The fragmentation schemes and ion reaction mechan-
ism were observed in case of metribuzin, which has been
shown in Figure 6. The m/z 186 was obtained due to the
elimination of ethylene (–CH2=CH2–) molecule from
parent ion molecule m/z 215, because the oxy-gen atom
donates the lone pair to hydrogen atom by remote charge
mechanism. Additionally, the three nitrogen atoms are
present in aromatic ring, which deactivate the whole
molecule and therefore ethylene molecule removed from
the parent ion molecule [14]. Similarly, C5H9N2O mole-
cule was removed from m/z 215 leading to the formation
of structure at m/z 72.1. The new structure was formed at
m/z 117 due to the removal of –C5H12O group from m/z
215. This new structure has been isolated due to structure
reactivity and ion reaction mechanism of metribuzin.
The removal of ions and formation of new structure
was observed in this study due to remote charge me-
chanism, rearrangement, nucleophilic and electrophilic
reaction. Similar pattern noted previously with triazofos,
chlorpyrifos and phenolate ion [10,14].
The pesticide-free water samples were spiked with dif-
ferent concentrations of standard (i.e., alachlor, malathion,
dimethoate, chloripyrifos and metribuzin,). The inter-day
percentage recoveries, the relative standard deviation
(RSD), limit of quantification (LOQ) and the limit of
detection (LOD) were determined as per the reported
methodology [17,18], and the results are shown in Table
4. The obtained percent recoveries for all these pesticides
were found to be in the range of 96% - 103% of the
standard value (Table 2) [19,20]. The obtained RSD was
below 8% for all compounds, which further reinforced
the importance, sensitivity, precision and selectivity of
this method.
The different behaviours of base peak pattern recorded
on different dissociation energy (DE) are illustrated in
Figure 7. These results reveal the m/z at 350 was ob-
tained at DE 10 V, while m/z at 197, 125 and 97 were
obtained at DE 20, 30, and 40, respectively for chlorpy-
rifos. The result clearly indicates that the m/z 125 and
m/z 97 were used for confirmation of chlorpyrifos in
water samples. Additionally, the m/z at 270,110, 83 and
70 were obtained at DE 10, 30, 60 and 70 respectively

S. N. SINHA ET AL.
517
for alachlor. The 83, 110 and 70 were used as confirma-
tory ions. In similar fashion the base peak of dimethoate
were observed. The m/z at 229, 88, 124, 79 and 63 were
obtained at DE 10, 20, 40, 70 and 80, respectively. The
ions 88, 79 and 63 were confirmatory ions. Similarly,
m/z at 215, 116, 72, 70 and 60 were obtained at DE 10,
20, 30, 40 and 80, respectively. The ions obtained at 72,
79 and 60 were used as confirmatory ions of metribuzin.
From this study we conclude that the MS/MS recorded at
different DE showed that the distribution pattern of base
peak ions of different compounds depends upon used DE,
in which some ions were used for confirmation and
structure illustration and also some ion was used for
quantification of compounds.
At least seven-point calibration curves were prepared
using an area count plotted against different concentra-
tions, and these curves were evaluated by linear square
regression analysis (Table 3). Correlation coefficients of
r > 0.999 were obtained for all these pesticides through-
out the study within the acceptable range [16]. The me-
thod„s accuracy was indistinguishable from 99%, which
is indicative of a high degree of accuracy. These data are
shown in Table 3.
Figure 3. Fragmentation schemes of dimethoate.
Figure 4. Fragmentation schemes of chlorpyrifos.
Figure 5. Fragmentation schemes of alachlor.

S. N. SINHA ET AL.
518
Figure 6. Fragmentation schemes of metribuzin.
Table 3. Accuracy determination using the correlation coefficient of spiked samples at different concentrations with uncer-
tainties parameter (slope, intercept and standard error in slope).
Compounds
Concentrations (ngmL-1)
ar
slope
intercept
br2 (mean)
N
%RSD
% accuracy
cSES
Dimethoate
0.1, 1, 5, 10, 25, 50, 150
1
0.9999
–0.01297
0.999
7
0.320
100
0.002
Alachlor
0.1, 1, 5, 10, 25, 50, 150
0.9999
1.000
–0.00236
0.999
7
0.0264
99.99
0.001
Malathion
0.1, 1, 5, 10, 25, 50, 150
1
0.9993
–0.19779
0.999
7
0.0248
100
0.030
Metrobuzin
0.1, 1, 5, 10, 25, 50, 150
1
0.9993
–0.19779
0.999
7
0.0248
100
0.030
Chlorpyrifos
0.1,, 5, 10, 25, 50, 150
0.9999
0.9999
–0.19779
0.999
0.0263
99.99
0.030
ar = correlation coefficient; br2 = Determinations of coefficient; RSD = Relative Standard Deviation; N = Number of replicate; cSES = Standard error in slope
Table 4. LOD, LOQ, % accuracy, and coefficient of deter-
mination for eight pesticides.
pesticides
a r
b LOQ
c LOD
% accuracy
Diomethoate
0.982
0.128
0.038
98.2
Alachlor
0.986
0.039
0.011
98.6
Malathion
0.986
0.081
0.024
98.6
Chlorpyrifos
0.999
0.044
0.014
99.9
Metribuzin
0.999
0.0021
0.006
99.9
LOD = Limit of Determination; LOQ = Limit of Quantification
The mean concentration of chloripyrifos Malathion,
Alachlor, dimethoate and metribuzin in bore water were
0.283 (ranged from 0.029 to 0.691 µg·L–1), 0.246 (ranged
from 0.032 to 0.566), 0.157 (ranged from 0.038 to 0.231
µg ·L–1), 0.102 (ranged from 0.041 to 0.233 µg ·L–1), 0.227
(ranged from 0.051 to 0.51 µg ·L–1) µg ·L–1, respectively.
The averaged concentration of chlorpyrifos malathion,
alachlor, dimethoate and metribuzin in MC water is 0.095
(ranged from 0.054 to 0.19 µg ·L–1), 0.100 (ranged from
0.057 to 0.21 µg ·L–1), 0.0986 (ranged from 0.05 to 0.162
µg ·L–1), 0.092 (ranged from 0.083 to 0.105 µg ·L–1), 0.027
(ranged from 0.004 to 0.051 µg·L–1) µg ·L–1 , respectively.
The percentage of pesticide showed in Figure 8.

S. N. SINHA ET AL.
519
0
10
20
30
40
50
60
70
80
90
100
110
120
350 198 125
97 97 97 97 97
270 270 110 110 110
83
70.1 70.1
215 116
72.1 70.1 70.1 70.1 70.1
60
229
88
125 125
79 79 79
63.1
Fragmented Ion
% RA
0
10
20
30
40
50
60
70
80
90
100
110
120
DE
% RADE
Chlorpyrifos
Alachlor
Metribuzin
Dimethoate
Figure 7. Distribution pattern of base peak with dissociation energy of different compounds.
Pesticides
Figure 8. Percentage of pesticides in water samples.
The organochlorine pesticides were reported in the water
off the central west coast of India using anin-situ sampler.
The γ-BHC (ranged 0.26 to 9.4 ng·L–1) and the two cyc-
lodiene compounds, aldrin and dieldrin (ranged from 1.4
to 9.8 and 2.1 to 50.9 ng·L−1, respectively) were found to
be more consistent than the compounds of the DDD.
Among the metabolites of DDT, pp′-DDE was found to
be present in every alternate station with increasing con-
centration (2.5 - 20.39 ng·L−1) whereas op′-DDE could
be detected occasionally in the northern part of the re-
gion [21]. The study was reported the pesticide contami-
nation in wheat flour and drinking water from Jaipur City,
Rajasthan, India using Gas Chromatograph. The water
samples were found to be contaminated with various
organochlorine pesticide residues of DDT and its meta-
bolites, HCH and its isomers, heptachlor and its exp-
oxide and aldrin [22]. The high concentrations of both
organochlorine and organophosphorous pesticides in the
surface and ground water samples in Kanpur, northern
India were reported. In this study liquid–liquid extraction
followed by GC-ECD was used for the determination of
these compounds. The high levels of γ-HCH (0.259
μg·L−1) and malathion (2.618 μg·L−1) were detected in
the surface water samples collected from the river
Ganges in Kanpur. In the ground water samples beside
from γ-HCH and malathion, dieldrin was also detected.
The maximum concentration values of γ-HCH, malathion
and dieldrin were 0.900, 29.835 and 16.227 μg·L−1, re-
spectively [23]. Our study showed that the MC water,
which has been used for drinking purposes, is safe as
compare to bore water in urban City.
7. Conclusions
We used a highly sensitive and selective method for
quantifying pesticide residues in drinking water samples
at low levels. Our method employs a simple lyophiliza-
tion followed by solvent extraction analysis using LC-
MS/MS. The lower limit of method validation and limit
of determination was in the μg·L−1 range with coefficient
of variation values of typically < 8%. Additionally, the
effect of DE on ions formation and distribution of base
peak were studied. The fragmentation schemes were well
illustrated. These results reveal the m/z at 350 was ob-
tained at DE 10 V, while m/z at 197and 97 were obtained

S. N. SINHA ET AL.
520
at DE 20, and 40, respectively for chlorpyrifos. The re-
sult clearly indicates that the m/z 125 and m/z 97 were
used for confirmation of chlorpyrifos in water samples.
Additionally, the m/z at 270,110, 83 and 70 were ob-
tained at DE 10, 30, 60 and 70 respectively for alachlor.
The 83, 110 and 70 were used as confirmatory ions. In
similar fashion the base peak of dimethoate were ob-
served. The m/z at 229, 88, 124, 79 and 63 were obtained
at DE 10, 20, 40, 70 and 80, respectively. The ions 88,
79 and 63 were confirmatory ions. Similarly, m/z at 215,
116, 72, 70 and 60 were obtained at DE 10, 20, 30, 40
and 80, respectively. The ions obtained at 72, 79 and 60
were used as confirmatory ions of metribuzin. Thirteen
water samples were collected from the different parts of
the urban city, and they were each analyzed showing
residual pesticide at detectable concentrations. These
data indicate that drinking water (MC) is less contami-
nated with pesticide residues than that of bore water at
lower levels. We plan to further explore pesticide residue
analysis in marketed water samples. Additionally, we
will apply this method for measuring pesticides in water
samples collected from different places in India.
8. Acknowledgements
The authors are thankful to the Indian Council of Medi-
cal Research for financial assistance. They would also
like to take this great opportunity to express their heart-
felt gratitude to the Director General of the Indian Coun-
cil of Medical Research for granting an opportunity to
work on this project. They are extremely thankful to the
Director of the National Institute of Nutrition (Hydera-
bad) for giving the necessary facilities and kind support
to carry out this work at the National Institute of Nutri-
tion, Hyderabad. The authors are thankful to all the tech-
nical staff especially Mr Vasudev, scientific staff and the
statistician for the technical and statistical support during
this work.
9. References
[1] P. C. Abhilash and N. Singh, “Pesticide Use and Applica-
tion: An Indian Scenario,” Journal of Hazardous Mate-
rials, Vol. 165, No. 1-3, 2009, pp. 1-12.
HHUdoi:10.1016/j.jhazmat.2008.10.061U
[2] V. K. Bhatnagar, “Pesticides Pollution: Trends and Pers-
pectives,” ICMR Bulltin, Vol. 31, 2001, pp. 87-88.
[3] D. Atkinson, F. Burnett, G. N. Foster, A. Litterick, M.
Mullay and C. A. Watson, “The Minimization of Pesti-
cide Residues in Food: A Review of the Published Lite-
rature,” Food Standards Agency, London, 2003.
[4] B. Kumari, R. Gulati, T. S. Kathpal, “Monitoring of Pes-
ticidal Contamination in Honey,” The Korean Journal of
Apiculture, Vol. 18, No. 2, 2003, pp. 155-160.
[5] B. Kumari, V. K. Madan, J. Singh, S. Singh and T. S.
Kathpal, “Monitoring of Pesticidal Contamination of
Farmgate Vegetables from Hisar Environmental Moni-
toring and Assessment,” Earth and Environmental
Science, Vol. 90, No. 1-3, 2004, pp. 65-77.
HHUdoi:10.1023/b:emas.0000003566.63111U
[6] B. Kumari, J. Singh, S. Singh and T. S. Kathpal, “Moni-
toring of Butter and Ghee (Clarified Butter Fat) for Pesti-
cidal Contamination from Cotton Belt of Haryana, India,”
Environmental Monitoring and Assessment, Vol. 105. No.
1-3, 2005, pp. 111-120.
HHUdoi:10.1007/s10661-005-3159-2U
[7] B. Kumari, T. S. Kathpal and E. M. Assess. “Monitoring
of Pesticide Residues in Vegetarian Diet,” Environmental
Monitoring Assessment, Vol. 151, 2009, p. 1926.
[8] B. Kumari, V. K. Madan and T. S. Kathpal, “Monitoring
of Pesticide Residues in Fruits,” Environmental Monitor-
ing and Assessment, Vol. 123, No. 1-3, 2006, pp.
407-412. HHUdoi:10.1007/s10661-006-1493-7U
[9] B. Kumari, V. K. Madan, R. Kumar and T. S. Kathpal,
“Monitoring of Seasonal Vegetables for Pesticide Resi-
dues,” Environmental Monitoring and Assessment, Vol.
74, No. 3, 2002, pp. 263-270.
HHUdoi:10.1023/A:1014248827898U
[10] S. N. Sinha, R. Pal, A. Dewan, M. M. Mansuri and H. N.
Saiyed, “Effect of Dissociation Energy on Ion Formation
and Sensitivity of An Analytical Method for Determina-
tion of Chlorpyrifos in Human Blood, Using Gas Chro-
matography-Mass Spectrometer (GC-MS in MS/MS),”
International Journal of Mass Spectrometry, Vol. 253,
No. 1-2, 2006, pp. 48-57.
HHUdoi:10.1016/j.ijms.2006.02.020U
[11] V. K. Dua, G. Rosy, S. N. Sinha and A. P. Das, “Alleth-
rin in the Air during the Use of a Heated Mosquito Re-
pellent Mat,” Bulletin of Environmental Contamination
and Toxicology, Vol. 75, No. 4, 2005, pp. 747-750.
HHUdoi:10.1007/s00128-005-0814-9U
[12] A. Dewan, V. K. Bhatnagar, M. L. Mathur, T. Chakma, R.
Kashyap and G. H. Sadhu, et al., “Repeated Episodes of
Endosulfan Poisoning,” Journal of Toxicology-Clinical
Toxicology, Vol. 42, No. 4, 2004, pp. 1-7.
HHUdoi:10.1081/CLT-120039542UHH
[13] S. N. Sinha, T. S. Patel, N. M. Desai, M. M. Mansuri, A.
Dewan and H. N. Saiyed, “GC-MS Study of Endosulfan
in Biological Samples,” Asian Journal of Chemistry, Vol.
16, No. 3-4, 2004, pp. 1685-1690.
[14] S. N. Sinha, “Effect of Dissociation Energy: Signal to
Noise Ratio on Ion Formation and Sensitivity of Analyti-
cal Method for Quantification and Confirmation of Tria-
zofos in Blood Samples Using Gas Chromatography-Mass
Spectrometer (GC-MS/MS),” International Journal of
Mass Spectrometry, Vol. 296, No. 1-3, 2010, pp. 47-52.
HHUdoi:10.1016/j.ijms.2010.08.014U
[15] P. Paya, M. Anastassiades, D. Mack, I. Sigalova, B. Tas-
delen and J. Oliva, et al., “Analysis of Pesticide Residues
Using the Quick Easy Cheap Effective Rugged and Safe
(QuEChERS) Pesticide Multiresidue Method in Combi-
nation with Gas and Liquid Chromatography and Tandem
Mass Spectrometric Detection,” Analytical Bioanalytical

S. N. SINHA ET AL.
521
Chemistry, Vol. 389, No. 6, 2007, pp. 1697-1714.
HHUdoi:10.1007/s00216-007-1610-7U
[16] D. B. Barr, J.R. Barr, V. L. Maggio, Jr. R. D. Whitehead,
M. A. Sadowski and R. Whyatt, et al. “A Multi-Analyte
Method for the Quantification of Contemporary Pesti-
cides in Human Serum and Plasma Using High-Resolution
Mass Spectrometry,” Journal of Chromatography B, Vol.
778, 2002, pp. 9-11.
[17] S. N. Sinha, K. Vasudev, M. V. Rao and M. Odetokun,
“Quantification of Organophosphate Insecticides in Drin-
king Water in Urban Areas Using Lyophilization and
High-Performance Liquid Chromatography-Electrospray
Ionization-Mass Spectrometry Techniques,” International
Journal of Mass Spectrometry, Vol. 300, 2011, pp. 12-20.
HHUdoi:10.1016/j.ijms.2010.11.006U
[18] S. N. Sinha, V. K. Bhatnagar, P. Doctor, G. S. Toteja, N.
P. Agnihotri and R. L. Kalra, “A Novel Method for Pesti-
cide Analysis in Refined Sugar Samples Using a Gas
Chromatography-Mass Spectrometer (GC-MS/MS) and
Simple Solvent Extraction Method,” Food Chemistry,
Vol. 126, No. 1, 2011b, pp. 379-386.
HHUdoi:10.1016/j.foodchem.2010.10.110U
[19] K. D. Miller and P. Milne, “Determination of Low-Level
Pesticides Residues in Soft Drinks and Sport Drinks by
Liquid Chromatography with Tandem Mass Spectrometry:
Collaborative Study,” Journal of AOAC International,
Vol. 91 No. 1, 2008, pp. 181-201.
[20] K. D. Miller and P. Milne, “Determination of (0.5 µg/L)
in Soft Drinks and Sport Drinks by Gas Chro Low-Level
Pesticides Residues Monograph with Mass Spectrometry:
Collaborative Study,” Journal of AOAC International,
Vol. 91, No. 1, 2008b, pp. 202-236.
[21] A. Sarkar and R. Gupta Sen, “Determination of Orga-
nochlorine Pesticides in Indian Coastal Water Using a
Mooredin-Situ Sampler,” Water Research, Vol. 23, No. 8,
1989, pp. 975-978.
HHUdoi:10.1016/0043-1354(89)90170-XU
[22] N. Bakore, P. J. John and P. Bhatnagar, “Organochlorine
Pesticide Residues in Wheat and Drinking Water Samples
from Jaipur, Rajasthan, India,” Environmental Monitor-
ing and Assessment, Vol. 98, No. 1-3, 2002, pp. 381-389.
HHUdoi:10.1023/B:EMAS.0000038197.76047.83U
[23] N. Sankararamakrishnan, A. K. Sharma and R. Sanghi,
“Organochlorine and Organophosphorous Pesticide Re-
sidues in Ground Water and Surface Waters of Kanpur,
Uttar Pradesh, India,” Environment International, Vol. 31,
No. 1, 2005, pp. 113-120.
HHUdoi:10.1016/j.envint.2004.08.001U