MS/MS Condition Optimization
Achieving maximum sensitivity in mass spectrometry necessitates careful optimization of parameters. In this study, a standard solution was precisely prepared, consisting of a 1 mg/L methanol solution. Both positive and negative ion scanning modes were concurrently established. A first-level full scan was conducted for each individual standard solution of the analytes to be measured, enabling the identification of parent ions with high and stable abundance. Following the determination of parent ions, optimization of the fragmentor in single ion monitoring (SIM) mode was carried out. In single ion monitoring (SIM) mode, the parent ions entered the secondary mass spectrum, undergoing reactions such as breakage or rearrangement to generate distinct ion fragments. Subsequently, in product ion mode, a specific collision energy (CE) was applied to the parent ion of the compound to induce ion fragmentation.
In the multiple reaction monitoring (MRM) mode, the optimal collision energy for the target ion fragment was fine-tuned to obtain the best mass spectrometry parameters. The parameters fine-tuned for enhancing the precision of primary ions (utilized for quantification) and secondary ions (used for identification), along with their respective energies such as Q1 Pre Bias, Collision Energy (CE), and Q3 Pre Bias, have been meticulously optimized (Figs. 1 & 2). The entire optimization process aimed to enhance sensitivity and accuracy in pesticide detection. Table 2 provides a comprehensive overview of the optimized retention time, parent and daughter ions, as well as collision energy (CE) for each target compound, facilitating a detailed understanding of the mass spectrometry parameters employed in this study.
Table 2
Multi reaction monitoring (MRM) parameters for the target analytes with retention time (RT)
S. No.
|
Name of the pesticide
|
M.W
(g/mol)
|
RT
(min)
|
Precursor Ion
|
Product Ion-01
|
Product Ion-02
|
Q1 Pre Bias
|
Collision Energy (CE)
|
Q3 Pre Bias
|
1
|
Acephate
|
183.2
|
2.38
|
184.10
|
143.10
|
49.15
|
-14
|
-21.
|
-18
|
2
|
Acetamiprid
|
227.6
|
6.948
|
223.30
|
126.15
|
56.25
|
-16
|
-14
|
-22
|
3
|
Azoxystrobin
|
403.38
|
9.597
|
404.10
|
372.15
|
344.15
|
-16
|
-23
|
-24
|
4
|
Buprofezin
|
305.44
|
12.513
|
306.10
|
201.20
|
57.20
|
-12
|
-23
|
-22
|
5
|
Carbendazim
|
191.18
|
5.544
|
192.10
|
160.10
|
132.05
|
-14
|
-29
|
-50
|
6
|
Carbofuran
|
221.25
|
8.192
|
222.15
|
165.15
|
123.15
|
-16
|
-20
|
-24
|
7
|
Carbofuran-3-Hydroxy
|
237.25
|
6.934
|
238.30
|
181.15
|
163.10
|
-12
|
-13
|
-18
|
8
|
Chlorantraniliprole
|
483.15
|
9.207
|
484.20
|
452.90
|
285.95
|
-12
|
-13
|
-20
|
9
|
Chlorpyrifos
|
350.59
|
13.163
|
350
|
197.95
|
97.05
|
-18
|
-33
|
-17
|
10
|
Cyantraniliprole
|
473.7
|
8.380
|
475.10
|
285.90
|
443.90
|
-12
|
-13
|
-21
|
11
|
Dimethoate
|
229.26
|
6.885
|
230.10
|
199
|
125.10
|
-12
|
-21
|
-48
|
12
|
Emmamectin Benzoate
|
1008.2
|
11.996
|
886.40
|
158.20
|
82.25
|
-22
|
-65
|
-16
|
13
|
Fipronil
|
437.15
|
10.626
|
435
|
330
|
250.10
|
17
|
25
|
13
|
14
|
Fipronil-sulfone
|
453.1
|
11.027
|
451
|
414.90
|
282
|
17
|
26
|
30
|
15
|
Flonicamide
|
229.16
|
5.108
|
230.10
|
203
|
148.05
|
-25
|
-26
|
-28
|
16
|
Flubendiamide
|
682.39
|
10.723
|
681
|
254.05
|
274.15
|
20
|
16
|
34
|
17
|
Fluopyram
|
396.72
|
10.243
|
397
|
145
|
208
|
-12
|
-18
|
-21
|
18
|
Hexaconazole
|
314.21
|
11.226
|
314.10
|
70
|
159
|
-16
|
-30
|
-30
|
19
|
Imidacloprid
|
255.66
|
6.048
|
256.15
|
127.10
|
193.05
|
-20
|
-10
|
-14
|
20
|
Indoxcarb
|
527.84
|
11.760
|
528.15
|
203.10
|
150.15
|
-20
|
-25
|
-16
|
21
|
Malaoxon
|
314.29
|
8.191
|
315.10
|
98.90
|
127.15
|
-16
|
-7
|
-23
|
22
|
Malathion
|
330.35
|
10.147
|
348.10
|
127.05
|
99
|
-10
|
-25
|
-19
|
23
|
Metalaxyl
|
279.33
|
9.022
|
280.10
|
192.05
|
160.10
|
-14
|
-13
|
-30
|
24
|
Metalaxyl-M (Mefenoxam)
|
|
9.043
|
280.20
|
192.05
|
160.20
|
-14
|
-17
|
-28
|
25
|
Methamidophos
|
141.1
|
2.094
|
142.10
|
94
|
125.15
|
-10
|
-17
|
-48
|
26
|
Monocrotophos
|
223.2
|
6.948
|
223.40
|
126.15
|
56.20
|
-16
|
-14
|
-22
|
27
|
Propiconazole
|
342.2
|
11.166
|
342.30
|
159
|
69.20
|
-26
|
-21
|
-26
|
28
|
Spinetoram J
|
748.0
|
11.418
|
748.50
|
142.15
|
98.05
|
-22
|
-55
|
-18
|
29
|
Spinosyn A
|
732.0
|
10.967
|
732.60
|
142.20
|
98.10
|
-28
|
-53
|
-18
|
30
|
Spinosyn D
|
746
|
11.376
|
746.60
|
142.10
|
98.10
|
-28
|
-54
|
-18
|
31
|
Spiromesifen
|
370.48
|
13.014
|
371.20
|
273.10
|
255.20
|
-18
|
-23
|
-27
|
32
|
Spirotetramat
|
373.44
|
10.341
|
374.10
|
302
|
330.05
|
-11
|
-15
|
-24
|
33
|
Tebuconazole
|
307.82
|
10.892
|
308.20
|
70.20
|
203.20
|
-12
|
-22
|
-28
|
34
|
Thiamethoxam
|
291.71
|
5.289
|
292.10
|
211.10
|
181.10
|
-22
|
-23
|
-36
|
35
|
Trifloxystrobin
|
408.37
|
12.088
|
409.10
|
186.10
|
145.10
|
-20
|
-29
|
-29
|
Table 3
(A) % recovery of the 35 pesticides for Tomato and Brinjal matrix
S.No.
|
Pesticide
|
% Recovery (RSD)
|
Tomato
|
Brinjal
|
0.01µg kg− 1
|
0.025µgkg1
|
0.05µg kg− 1
|
0.1µg kg− 1
|
0.5µg kg− 1
|
0.01µg kg− 1
|
0.025µgkg1
|
0.05µg kg− 1
|
0.1µg kg− 1
|
0.5µg kg− 1
|
1
|
Acephate
|
85 (3.53)
|
84 (8.06)
|
84(4.76)
|
83 (2.41)
|
88 (4.55)
|
88 (2.27)
|
87 (5.78)
|
88 (9.09)
|
87 (2.30)
|
82 (7.32)
|
2
|
Acetamiprid
|
80 (7.81)
|
85 (11.76)
|
82(9.76)
|
88 (6.82)
|
86 (6.98)
|
85 (1.18)
|
88 (5.68)
|
82 (4.88)
|
86 (3.49)
|
88 (4.55)
|
3
|
Azoxystrobin
|
81 (1.23)
|
80 (6.25)
|
86(6.98)
|
83 (3.61)
|
88 (9.09)
|
82 (3.66)
|
80 (6.25)
|
86 (2.33)
|
89 (4.49)
|
88 (11.36)
|
4
|
Buprofezin
|
85 (3.53)
|
88 (3.41)
|
86(4.65)
|
84 (5.95)
|
86 (4.65)
|
84 (4.76)
|
86 (6.15)
|
88 (9.09)
|
87 (1.15)
|
88 (9.09)
|
5
|
Carbendazim
|
84 (2.38)
|
89 (3.93)
|
86 (8.39)
|
84 (3.57)
|
88 (9.09)
|
87 (9.20)
|
86 (5.85)
|
86 (4.65)
|
88 (2.27)
|
86 (4.65)
|
6
|
Carbofuran
|
85 (2.35)
|
88 (8.30)
|
86 (6.98)
|
88 (2.27)
|
86 (18.60)
|
82 (1.22)
|
80 (6.25)
|
88 (6.82)
|
88 (4.55)
|
88 (9.09)
|
7
|
Carbofuran-3-Hydroxy
|
83 (1.20)
|
84 (6.44)
|
86 (9.30)
|
88 (4.55)
|
82 (4.88)
|
86 (6.98)
|
84 (9.41)
|
84 (11.90)
|
82 (2.44)
|
90 (4.44)
|
8
|
Chlorantraniliprole
|
86 (5.81)
|
88 (8.33)
|
86 (4.65)
|
87 (2.30)
|
82 (2.44)
|
84 (5.95)
|
86 (5.90)
|
82 (4.88)
|
87 (4.60)
|
86 (9.30)
|
9
|
Chlorpyrifos
|
81 (1.23)
|
86 (6.15)
|
90 (11.11)
|
86 (3.49)
|
90 (8.89)
|
87 (3.45)
|
89 (8.32)
|
82 (2.44)
|
90 (2.22)
|
84 (4.76)
|
10
|
Cyantraniliprole
|
85 (4.71)
|
86 (8.43)
|
80 (7.50)
|
89 (4.49)
|
88 (6.82)
|
87 (1.15)
|
86 (4.39)
|
86 (9.30)
|
89 (3.37)
|
80 (17.50)
|
11
|
Dimethoate
|
85 (2.35)
|
89 (5.53)
|
86 (2.33)
|
87 (1.15)
|
90 (2.22)
|
86 (2.33)
|
91 (3.31)
|
84 (4.76)
|
87 (5.75)
|
86 (9.30)
|
12
|
Emmamectin Benzoate
|
83 (3.61)
|
89 (6.74)
|
84 (8.58)
|
84 (3.57)
|
88 (11.36)
|
84 (4.76)
|
86 (4.76)
|
82 (2.44)
|
84 (3.57)
|
82 (2.44)
|
13
|
Fipronil
|
84 (4.76)
|
88 (7.49)
|
88 (6.01)
|
90 (4.44)
|
84 (4.76)
|
82 (1.22)
|
88 (4.10)
|
88 (9.09)
|
80 (12.50)
|
84 (9.52)
|
14
|
Fipronil-sulfone
|
82 (1.22)
|
88 (6.40)
|
86 (4.65)
|
90 (1.11)
|
88 (6.82)
|
85 (2.35)
|
88 (8.57)
|
86 (4.65)
|
90 (1.11)
|
88 (6.82)
|
15
|
Flonicamide
|
83 (6.02)
|
87 (8.73)
|
86 (11.63)
|
90 (2.22)
|
88 (13.64)
|
85 (3.11)
|
89 (6.74)
|
86 (6.98)
|
84 (5.95)
|
88 (2.27)
|
16
|
Flubendiamide
|
81 (2.47)
|
85 (5.92)
|
86 (6.98)
|
84 (4.76)
|
84 (23.81)
|
84 (2.38)
|
88 (7.49)
|
84 (4.76)
|
84 (4.76)
|
88 (6.82)
|
17
|
Fluopyram
|
83 (4.82)
|
89 (8.32)
|
82 (9.76)
|
83 (4.82)
|
84 (7.14)
|
85 (1.18)
|
88 (6.40)
|
82 (9.76)
|
86 (4.65)
|
90 (2.22)
|
18
|
Hexaconazole
|
88 (2.27)
|
89 (5.62)
|
80 (15)
|
82 (2.44)
|
84 (4.76)
|
85 (4.71)
|
87 (8.73)
|
86 (4.65)
|
89 (2.25)
|
88 (11.36)
|
19
|
Imidacloprid
|
85 (1.18)
|
84 (12.11)
|
88 (22.73)
|
90 (1.11)
|
90 (15.56)
|
85 (3.53)
|
90 (5.59)
|
88 (9.09)
|
90 (2.22)
|
84 (4.76)
|
20
|
Indoxcarb
|
82 (3.66)
|
86 (5.85)
|
82 (2.44)
|
90 (2.22)
|
88 (9.09)
|
80 (7.81)
|
89 (8.32)
|
86 (4.65)
|
87 (5.75)
|
80 (15)
|
21
|
Malaoxon
|
84 (4.76)
|
89 (5.62)
|
84 (4.76)
|
89 (3.37)
|
90 (4.44)
|
81 (1.23)
|
89 (5.62)
|
84 (11.90)
|
84 (5.95)
|
88 (6.82)
|
22
|
Malathion
|
87 (9.20)
|
87 (5.78)
|
84 (9.52)
|
87 (5.75)
|
86 (9.30)
|
85 (3.53)
|
84 (12.11)
|
86 (4.65)
|
83 (3.61)
|
90 (8.89)
|
23
|
Metalaxyl
|
82 (1.22)
|
86 (7.74)
|
86 (4.65)
|
84 (2.38)
|
82 (2.44)
|
84 (2.38)
|
86 (5.85)
|
82 (19.51)
|
88 (5.68)
|
82 (2.44)
|
24
|
Metalaxyl-M(Mefenoxam)
|
86 (6.98)
|
80 (6.25)
|
90 (8.89)
|
81 (3.70)
|
90 (11.11)
|
85 (2.35)
|
89 (5.62)
|
86 (6.98)
|
86 (4.65)
|
80 (12.50)
|
25
|
Methamidophos
|
84 (5.95)
|
86 (6.15
|
80 (17.50)
|
84 (5.95)
|
88 (6.82)
|
83 (1.20)
|
84 (8.06)
|
86 (9.30)
|
88 (9.09)
|
82 (14.63)
|
26
|
Monocrotophos
|
87 (3.45)
|
86 (5.85)
|
80 (5)
|
83 (3.61)
|
90 (2.22)
|
86 (5.81)
|
85 (11.76)
|
86 (4.65)
|
83 (7.23.)
|
90 (2.22)
|
27
|
Propiconazole
|
87 (1.15)
|
80 (6.25)
|
88 (9.09)
|
88 (5.68)
|
90 (8.89)
|
81 (1.23)
|
80 (6.25)
|
86 (6.98)
|
88 (11.36)
|
90 (8.89)
|
28
|
Spinetoram J
|
86 (2.33)
|
84 (9.41)
|
90 (11.11)
|
86 (4.65)
|
84 (4.76)
|
85 (4.71)
|
85 (5.88)
|
80 (7.50)
|
88 (10.23)
|
84 (4.76)
|
29
|
Spinosyn A
|
84 (4.76)
|
86 (5.90)
|
86 (9.30)
|
86 (4.65)
|
88 (6.82)
|
85 (2.35)
|
89 (3.93)
|
86 (2.33)
|
85 (3.53)
|
88 (6.82)
|
30
|
Spinosyn D
|
82 (1.22)
|
89 (8.32)
|
90 (4.44)
|
89 (2.25)
|
80 (25.0)
|
83 (3.61)
|
88 (8.30)
|
86 (11.63)
|
86 (5.81)
|
88 (9.09)
|
31
|
Spiromesifen
|
85 (2.35)
|
86 (4.39)
|
90 (6.67)
|
88 (4.55)
|
90 (4.44)
|
84 (4.76)
|
84 (6.44)
|
90 (8.89)
|
83 (3.61)
|
90 (4.44)
|
32
|
Spirotetramat
|
85 (3.11)
|
85 (6.59)
|
88 (9.09)
|
87 (5.75)
|
82 (14.63)
|
82 (1.22)
|
88 (8.33)
|
86 (4.65)
|
85 (4.71)
|
88 (6.82)
|
33
|
Tebuconazole
|
84 (2.38)
|
86 (4.76)
|
88 (6.82)
|
82 (7.32)
|
82 (12.20)
|
83 (6.02)
|
86 (6.15)
|
84 (7.14)
|
83 (8.43)
|
88 (9.09)
|
34
|
Thiamethoxam
|
85 (1.18)
|
88 (4.10)
|
86 (4.65)
|
83 (7.23)
|
82 (14.63)
|
81 (2.47)
|
86 (8.43)
|
86 (6.98)
|
81 (11.11)
|
80 (25)
|
35
|
Trifloxystrobin
|
85 (4.71)
|
88 (8.57)
|
88 (11.36)
|
88 (11.36)
|
86 (4.65)
|
83 (4.82)
|
89 (5.53)
|
82 (9.76)
|
81 (3.70)
|
80 (10)
|
Table 3 (B) % recovery of the 35 pesticides for Chilli and Okra matrix
S.No.
|
Pesticide
|
%Recovery (RSD)
|
Chilli
|
Okra
|
0.01µg kg-1
|
0.025µgkg1
|
0.05µg kg-1
|
0.1µg kg-1
|
0.5µg kg-1
|
0.01µg kg-1
|
0.025µgkg1
|
0.05µg kg-1
|
0.1µg kg-1
|
0.5µg kg-1
|
1
|
Acephate
|
86 (6.98)
|
84 (6.44)
|
82 (9.76)
|
85 (5.88)
|
82 (24.39)
|
82 (1.22)
|
86 (4.39)
|
90 (6.67)
|
83 (3.61)
|
88 (2.27)
|
2
|
Acetamiprid
|
83 (4.82)
|
89 (5.62)
|
90 (4.44)
|
86 (4.65)
|
84 (19.05)
|
85 (2.35)
|
85 (6.59)
|
84 (11.90)
|
88 (5.68)
|
88 (11.36)
|
3
|
Azoxystrobin
|
87 (3.45)
|
86 (6.15)
|
90 (11.11)
|
87 (8.05)
|
82 (7.32)
|
85 (3.11)
|
86 (7.74)
|
86 (4.65)
|
86 (4.65)
|
84 (4.76)
|
4
|
Buprofezin
|
85 (1.18)
|
88 (7.49)
|
84 (9.52)
|
89 (2.25)
|
88 (4.55)
|
84 (2.38)
|
86 (5.85)
|
86 (6.98)
|
86 (4.65)
|
80 (15)
|
5
|
Carbendazim
|
82 (3.66)
|
88 (6.40)
|
84 (4.76)
|
86 (4.65)
|
84 (7.14)
|
85 (1.18)
|
80 (6.25)
|
84 (11.90)
|
82 (2.44)
|
88 (6.82)
|
6
|
Carbofuran
|
85 (4.71)
|
87 (8.73)
|
82 (19.51)
|
89 (3.37)
|
90 (11.11)
|
85 (4.71)
|
89 (6.74)
|
86 (2.33)
|
84 (2.38)
|
90 (8.89)
|
7
|
Carbofuran-3-Hydroxy
|
85 (2.35)
|
84 (8.06)
|
86 (9.30)
|
87 (5.75)
|
86 (4.65)
|
85 (3.53)
|
86 (5.85)
|
86 (11.63)
|
81 (3.70)
|
82 (2.44)
|
8
|
Chlorantraniliprole
|
83 (3.61)
|
85 (11.76)
|
90 (4.44)
|
84 (2.38)
|
88 (9.09)
|
84 (2.38)
|
89 (5.62)
|
88 (9.09)
|
84 (5.95)
|
80 (12.50)
|
9
|
Chlorpyrifos
|
84 (4.76)
|
80 (6.25)
|
82 (14.63)
|
81 (3.70)
|
90 (4.44)
|
85 (2.35)
|
87 (5.78)
|
84 (4.76)
|
90 (1.11)
|
82 (14.63)
|
10
|
Cyantraniliprole
|
82 (1.22)
|
85 (5.88)
|
86 (9.30)
|
84 (5.95)
|
82 (4.88)
|
83 (1.20)
|
85 (5.92)
|
84 (23.81)
|
90 (2.22)
|
90 (2.22)
|
11
|
Dimethoate
|
83 (6.02)
|
89 (3.93)
|
84 (4.76)
|
83 (3.61)
|
82 (2.44)
|
86 (5.81)
|
89 (8.32)
|
92 (4.35)
|
89 (3.37)
|
90 (8.89)
|
12
|
Emmamectin Benzoate
|
85 (3.53)
|
88 (8.30)
|
86 (23.26)
|
88 (5.68)
|
84 (9.52)
|
81 (1.23)
|
89 (5.62)
|
88 (9.09)
|
87 (5.75)
|
84 (4.76)
|
13
|
Fipronil
|
80 (7.81)
|
86 (4.76)
|
86 (6.98)
|
83 (2.41)
|
84 (4.76)
|
85 (4.71)
|
84 (12.11)
|
88 (6.82)
|
89 (2.25)
|
88 (6.82)
|
14
|
Fipronil-sulfone
|
80 (2.50)
|
88 (4.10)
|
86 (4.65)
|
82 (12.20)
|
88 (6.82)
|
80 (7.81)
|
89 (8.32)
|
82 (9.76)
|
90 (2.22)
|
88 (9.09)
|
15
|
Flonicamide
|
85 (3.53)
|
88 (8.57)
|
84 (11.90)
|
80 (8.75)
|
88 (13.64)
|
81 (1.23)
|
85 (2.96)
|
90 (8.89)
|
87 (5.75)
|
90 (4.44)
|
16
|
Flubendiamide
|
87 (11.49)
|
86 (8.43)
|
86 (6.98)
|
84 (5.95)
|
90 (4.44)
|
85 (3.53)
|
80 (6.25)
|
84 (7.14)
|
82 (7.32)
|
88 (6.82)
|
17
|
Fluopyram
|
87 (1.15)
|
89 (5.53)
|
86 (9.30)
|
84 (3.57)
|
88 (6.82)
|
85 (2.35)
|
86 (6.15)
|
86 (6.98)
|
83 (7.23)
|
88 (9.09)
|
18
|
Hexaconazole
|
86 (2.33)
|
89 (6.74)
|
86 (4.65)
|
88 (2.27)
|
92 (10.87)
|
83 (3.61)
|
84 (9.41)
|
82 (9.76)
|
88 (11.36)
|
80 (25)
|
19
|
Imidacloprid
|
84 (4.76)
|
86 (5.85)
|
86 (6.98)
|
88 (4.55)
|
88 (6.82)
|
84 (4.76)
|
86 (5.90)
|
90 (11.11)
|
83 (3.61)
|
80 (10)
|
20
|
Indoxcarb
|
82 (1.22)
|
89 (5.62)
|
84 (11.90)
|
87 (6.90)
|
90 (11.11)
|
83 (6.02)
|
88 (7.49)
|
82 (2.44)
|
84 (5.95)
|
82 (7.32)
|
21
|
Malaoxon
|
85 (2.35)
|
87 (5.78)
|
86 (2.33)
|
90 (2.22)
|
88 (9.09)
|
81 (2.47)
|
86 (6.15)
|
88 (13.64)
|
84 (3.57)
|
88 (4.55)
|
22
|
Malathion
|
85 (3.11)
|
85 (5.92)
|
86 (11.63)
|
87 (5.75)
|
86 (4.65)
|
83 (4.82)
|
84 (6.44)
|
86 (6.98)
|
88 (2.27)
|
88 (11.36)
|
23
|
Metalaxyl
|
84 (2.38)
|
89 (8.32)
|
82 (9.76)
|
82 (7.32)
|
88 (9.09)
|
84 (4.76)
|
89 (5.62)
|
90 (4.44)
|
88 (4.55)
|
88 (9.09)
|
24
|
Metalaxyl M(Mefenoxam)
|
85 (1.18)
|
89 (5.62)
|
86 (4.65)
|
89 (7.87)
|
90 (4.44)
|
88 (2.27)
|
88 (6.40)
|
82 (14.63)
|
87 (2.30)
|
86 (4.65)
|
25
|
Methamidophos
|
85 (4.71)
|
84 (12.11)
|
84 (7.14)
|
88 (11.36)
|
82 (12.20)
|
85 (1.18)
|
87 (8.73)
|
86 (9.30)
|
86 (3.49)
|
88 (9.09)
|
26
|
Monocrotophos
|
81 (1.23)
|
89 (8.32)
|
86 (6.98)
|
87 (2.30)
|
86 (6.98)
|
82 (3.66)
|
89 (3.93)
|
84 (4.76)
|
89 (4.49)
|
90 (4.44)
|
27
|
Propiconazole
|
84 (4.76)
|
86 (4.39)
|
82 (9.76)
|
86 (3.49)
|
82 (4.88)
|
84 (4.76)
|
88 (8.30)
|
86 (23.26)
|
87 (1.15)
|
86 (9.30)
|
28
|
Spinetoram J
|
87 (9.20)
|
85 (6.59)
|
86 (2.33)
|
89 (4.49)
|
88 (6.82)
|
87 (9.20)
|
86 (4.76)
|
86 (6.98)
|
84 (3.57)
|
84 (4.76)
|
29
|
Spinosyn A
|
82 (1.22)
|
86 (7.74)
|
82 (2.44)
|
87 (1.15)
|
82 (14.63)
|
82 (1.22)
|
88 (4.10)
|
86 (9.30)
|
90 (4.44)
|
80 (17.50)
|
30
|
Spinosyn D
|
86 (5.81)
|
80 (6.25)
|
82 (2.44)
|
84 (3.57)
|
84 (4.76)
|
86 (6.98)
|
88 (8.57)
|
82 (9.76)
|
90 (1.11)
|
86 (9.30)
|
31
|
Spiromesifen
|
88 (6.82)
|
86 (6.15)
|
84 (4.76)
|
90 (4.44)
|
90 (6.67)
|
84 (5.95)
|
86 (8.43)
|
90 (4.44)
|
90 (2.22)
|
82 (2.44)
|
32
|
Spirotetramat
|
85 (2.35)
|
86 (5.85)
|
88 (4.55)
|
89 (6.74)
|
88 (11.36)
|
87 (3.45)
|
89 (5.53)
|
90 (11.11)
|
84 (4.76)
|
84 (9.52)
|
33
|
Tebuconazole
|
83 (1.20)
|
80 (6.25)
|
92 (4.35)
|
90 (2.22)
|
82 (19.51)
|
82 (1.22)
|
85 (11.76)
|
84 (9.52)
|
83 (4.82)
|
88 (6.82)
|
34
|
Thiamethoxam
|
86 (6.98)
|
84 (9.41)
|
88 (9.09)
|
84 (4.76)
|
90 (2.22)
|
87 (1.15)
|
80 (6.25)
|
80 (17.50)
|
87 (5.75)
|
86 (13.95)
|
35
|
Trifloxystrobin
|
84 (5.95)
|
86 (5.90)
|
88 (6.82)
|
84 (5.95)
|
90 (8.89)
|
86 (2.33)
|
85 (5.88)
|
88 (15.91)
|
86 (5.81)
|
88 (11.36)
|
Optimization chromatographic condition
Extensive optimization was performed on the high-performance liquid chromatography (HPLC) method after the best multiple reaction monitoring (MRM) parameters were identified. The 3 µm C18 column retained analyte better and showed superior peak shape and response, thus it was selected for analyte analysis. Subsequently, two mobile phase containers were employed in the chromatographic system. containers A, designated as Mobile Phase A, consisted of a 0.3153gm ammonium formate solution in water with formic acid, while containers B, denoted as Mobile Phase B, contained a 0.3153gm ammonium formate solution in methanol with formic acid. The concentration of formic acid was systematically varied at levels of 0.1% and 0.5%. Upon evaluation, it was observed that the peak shape for each pesticide was notably improved when the concentration of formic acid was set at 0.1%. This optimized condition was consequently selected for the subsequent chromatographic analysis. For the purpose of dilution, three different solvents were considered: a) 100% acetonitrile, b) 100% methanol, and c) a mixture of mobile phase A and B in a 1:1 ratio. During elution of the targeted pesticide from the column, it was observed that the 1:1 ratio of mobile phase A and B provided the best results in terms of signal response. Subsequently, various injection volumes were compared to optimize sensitivity and peak shape.
Larger injection volumes yielded higher sensitivity, while smaller injection volumes resulted in better peak shapes. The experiment assessed injection volumes of 1, and 5 µL for each target analyte. Notably, at an injection volume of 5 µL, some target analytes exhibited flat-topped and pre-delayed peaks. Contrastingly, optimal peak shapes were observed at injection volumes of 1 and 5 µL for all analytes. To strike a balance between high sensitivity and optimal peak shape, 5 µL was chosen as the preferred injection volume for the subsequent analysis. This decision was made based on a thorough evaluation of the trade-off between sensitivity and peak shape considerations. Under optimized conditions, all targeted compounds eluted within 13.16 minutes. Malaoxon eluted first at 8.19 minutes, and Chlorpyrifos last at 13.16 minutes. This rapid elution confirms the safety of the chosen run time and highlights the efficiency of the optimized instrumental conditions for compound separation.
Extraction and clean-up
Samples were initially chopped and crushed in a mixer grinder, but this method as such doesn’t lead to complete homogenization, leading to poor precision (n = 6) and low recovery (< 66%). Adding water in a 1:1 ratio with the sample improved homogeneity and increased recovery to over 79% for all 35 targeted pesticides, meeting international standards for multi-residue analysis in vegetables. Homogenized samples weighing 10 g were extracted with 10 mL of 1% acetic acid in acetonitrile. The resulting acetonitrile supernatant was filtered through a 0.2 µm nylon syringe filter and directly injected into the instrument, causing signal suppression in some compounds possibly due to interfering co-extracts from the matrix. As a result, two cleanup sets were made: Set A included 150 mg MgSO4 + 25 mg PSA per mL, and Set B contained 150 mg MgSO4 + 25 mg PSA + 50 mg GCB per mL. Without cleanup, there was no significant improvement in signal response for Set A, but for the 35 selected compounds in Set B, both signal response and peak shape were excellent. We hypothesize that this could be because GCB adsorbed the color co-extract preferentially, interfering with its adsorption. Consequently, B was the clean-up agent that was ultimately chosen, and it was employed in the cleanup phase of the analysis of the tomato, brinjal, chilli and okra samples.
Method Validation
Pesticides exhibited good linearity in MRM mode, with correlation coefficients ranging from 0.998 to 0.999 in the calibration curves. Recovery of pesticides at spike levels of 0.001, 0.0025, 0.005, 0.01 and 0.05 µg kg− 1 ranged from 80–90%, demonstrating satisfactory precision (RSD < 20%) as shown in Table 3A & 3B). Repeatability was also confirmed, with all pesticides displaying RSD < 20% at each spiking level (n = 5). These results align with the criteria outlined in the SANTE Guideline 2021. The suggested method's limits of quantification (LOQs) were determined using the Sanco Guideline; at the lowest spiked concentration, mean recovery was between 70 and 120 percent, RSD was less than 20, and the method's LOQ was 0.01 µg/g. The matrix effect was minimized by combining various cleaning agents in different ways. The matrix effects of the target compounds in the four matrix were found to be less than 20%. Calibration curves for each pesticide exhibited linearity within the concentration range of 0.0025 to 0.1 µg mL− 1, with correlation coefficients ranging from 0.993 to 0.999 (Fig. 3)
Application of the method for the analysis of market samples
The technique was used to analyze the tomato, brinjal, chilli and okra samples that were collected from various local markets in Varanasi, India, including Sunderpur, Chanuwa satti, and Raja talab market. Chlorpyrifos residue status was found most prominent in all the samples. 2412.94, 2578.56, 1183.25 and 2027.9 ppb chloropyrifos residue were detected in tomato, brinjal, chilli and okra respectively collected from Chanuwa setti market and in case of Raja talab market the chloropyrifos residue were detected is 1676.23, 1291.14, 982.86 and 518.78 ppb in tomato, brinjal, chilli and okra respectively. Similarly, in the market of Sunderpur it was 2913.6, 2299.84, 1648.72 and 1276.88 ppb in tomato, brinjal, chilli and okra, respectively (Table 4). Improved pest control that is grounded in ecological principles is necessary to reduce or eliminate the limitations of pesticides in contemporary agriculture and agricultural products. Integrated Pest Management (IPM), which minimizes the use of pesticides to targeted pests, is viewed as a means of achieving sustainable agricultural production with less harm to the environment. Additionally, Varanasi, India is located in the indo-gangetic plains, which have abundant vegetable crops. Since the area has been designated as the nation's export hub, farmer awareness programs are necessary to ensure the safe and prudent use of pesticides in vegetable production systems.
Table 4
Detection of the pesticide’s residue found in vegetables sample collected from market
Pesticide
|
RT
(min)
|
Chanua Satti
|
Rajatalab
|
Sunderpur
|
Residue detection (ppb)
|
Residue detection (ppb)
|
Residue detection (ppb)
|
Tomato
|
Brinjal
|
Chilli
|
Okra
|
Tomato
|
Brinjal
|
Chilli
|
Okra
|
Tomato
|
Brinjal
|
Chilli
|
Okra
|
Acephate
|
2.38
|
BDL
|
BDL
|
44.50
|
0.25
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Acetamiprid
|
6.95
|
0.21
|
0.06
|
12.65
|
0.18
|
0.68
|
0.07
|
BDL
|
2.21
|
1.91
|
0.63
|
BDL
|
0.31
|
Azoxystrobin
|
9.624
|
BDL
|
BDL
|
BDL
|
BDL
|
2.19
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Buprofezin
|
12.642
|
0.59
|
0.62
|
0.612
|
0.617
|
0.58
|
0.6
|
BDL
|
0.63
|
0.59
|
BDL
|
BDL
|
0.82
|
Carbendazim
|
5.611
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
3.32
|
BDL
|
1.97
|
BDL
|
Carbofuran
|
9.089
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Carbofuran-3-Hydroxy
|
6.947
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Chlorantraniliprole
|
9.213
|
30.46
|
10.45
|
133.26
|
14.47
|
14.06
|
10.46
|
31.79
|
43.83
|
20.26
|
13.66
|
25.72
|
23.28
|
Chlorpyrifos
|
14.438
|
2412.94
|
2578.56
|
1183.25
|
2027.9
|
1676.23
|
1291.14
|
982.86
|
518.78
|
2913.6
|
2299.84
|
1648.72
|
1276.88
|
Cyantraniliprole
|
8.394
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Dimethoate
|
6.943
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Emmamectin Benzoate
|
12.02
|
0.38
|
0.42
|
0.45
|
0.37
|
0.37
|
0.37
|
0.58
|
0.54
|
0.4
|
BDL
|
1.02
|
0.46
|
Fipronil
|
10.710
|
1.07
|
0.54
|
1.4
|
0.46
|
BDL
|
BDL
|
BDL
|
BDL
|
13.28
|
2.13
|
1.84
|
24.62
|
Fipronil-sulfone
|
11.104
|
1.24
|
0.77
|
2.84
|
0.92
|
BDL
|
BDL
|
BDL
|
BDL
|
5.3
|
1.75
|
3.51
|
17.04
|
Flonicamide
|
5.203
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Flubendiamide
|
10.804
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Fluopyram
|
10.319
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Hexaconazole
|
11.294
|
BDL
|
BDL
|
4.32
|
BDL
|
BDL
|
BDL
|
0.81
|
BDL
|
BDL
|
BDL
|
BDL
|
2.32
|
Imidacloprid
|
6.10
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Indoxcarb
|
11.876
|
2.41
|
2.23
|
3.39
|
2.31
|
10.28
|
2.69
|
2.63
|
2.35
|
26.97
|
2.43
|
3.25
|
4.02
|
Malaoxon
|
8.236
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Malathion
|
10.201
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Metalaxyl
|
9.07
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
7.5
|
BDL
|
BDL
|
BDL
|
Metalaxyl-M(Mefenoxam)
|
9.063
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Methamidophos
|
1.946
|
BDL
|
BDL
|
28.32
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Monocrotophos
|
6.95
|
BDL
|
BDL
|
12.51
|
BDL
|
0.02
|
BDL
|
BDL
|
1.47
|
BDL
|
BDL
|
BDL
|
BDL
|
Propiconazole
|
11.228
|
2.46
|
0.21
|
0.63
|
0.01
|
0.04
|
0.69
|
BDL
|
BDL
|
BDL
|
BDL
|
1.89
|
BDL
|
Spinetoram J
|
11.404
|
BDL
|
12.77
|
170.31
|
197.30
|
18.99
|
16.39
|
10.36
|
5.78
|
33.87
|
BDL
|
BDL
|
1.93
|
Spinosyn A
|
10.954
|
0.35
|
BDL
|
0.37
|
0.41
|
0.48
|
0.35
|
0.34
|
BDL
|
0.37
|
BDL
|
0.34
|
0.34
|
Spinosyn D
|
11.419
|
BDL
|
BDL
|
BDL
|
0.14
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Spiromesifen
|
13.061
|
549.47
|
509.19
|
712.45
|
501.9
|
477.17
|
469.6
|
522.81
|
562.2
|
BDL
|
BDL
|
BDL
|
BDL
|
Spirotetramat
|
10.394
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
Tebuconazole
|
11.976
|
BDL
|
0.5
|
BDL
|
BDL
|
7.63
|
BDL
|
BDL
|
BDL
|
0.11
|
0.64
|
BDL
|
BDL
|
Thiamethoxam
|
5.39
|
4.12
|
9.2
|
6.13
|
12.93
|
5.41
|
7.13
|
5.2
|
77.46
|
2.03
|
19.61
|
46.05
|
18.57
|
Trifloxystrobin
|
12.155
|
BDL
|
BDL
|
BDL
|
2.3
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|
BDL
|