# Analysis of Veterinary Drug and Pesticide Residues Using the Ethyl Acetate Multiclass/Multiresidue Method in Milk by Liquid Chromatography-Tandem Mass Spectrometry

Authors: Husniye Imamoglu; Elmas Oktem Olgun
Journal: Journal of Analytical Methods in Chemistry
 (2016)

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## Abstract

A rapid and simple multiclass, ethyl acetate (EtOAc) multiresidue method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) detection was developed for the determination and quantification of 26 veterinary drugs and 187 total pesticide residues in milk. Sample preparation was a simple procedure based on liquid–liquid extraction with ethyl acetate containing 0.1% acetic acid, followed by centrifugation and evaporation of the supernatant. The residue was dissolved in ethyl acetate with 0.1% acetic acid and centrifuged prior to LC-MS/MS analysis. Chromatographic separation of analytes was performed on an Inertsil X-Terra C18 column with acetic acid in methanol and water gradient. The repeatability and reproducibility were in the range of 2 to 13% and 6 to 16%, respectively. The average recoveries ranged from 75 to 120% with the RSD (n
=
18). The developed method was validated according to the criteria set in Commission Decision 2002/657/EC and SANTE/11945/2015. The validated methodology represents a fast and cheap alternative for the simultaneous analysis of veterinary drug and pesticide residues which can be easily extended to other compounds and matrices.


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## Body

## 1. Introduction

Veterinary drugs are widely used in medical and veterinary practices to treat and prevent disease as well as improve feed efficiency and increase animal growth rates [1]. Pesticides are also widely used to enhance food production by protecting food crops from potentially harmful and destructive pests [2]. However, the resulting occurrence of contaminants and/or residues in the human diet represents an issue of high concern.According to the European Union, the maximum residue limit (MRL) in dairy milk is 100μg/kg for tetracycline and sulfenamide, 50 μg/kg for macrolides and quinolones, and 10 μg/kg for pesticides. Sensitive analytic methods have been developed to monitor and detect the MRL values in the dairy milk [3]. There are ultra-high pressure liquid chromatography mass spectrometry (UHPLC-MS/MS) methods reported to detect multiple residues of β-lactams [4, 5], as well as pesticides and mycotoxins [6], and some antihelminthic drugs and phenylbutazone [7].Milk is a complex food that is high in fat and protein, and such ingredients may cause interactions in the analytical processes. Therefore, sample preparation is required, particularly in extraction and cleanup. Formerly, sample preparation methods were based on a few compounds or a single class of such drugs. Applying common extraction procedures and developing chromatographic conditions are difficult in multiclass and multiresidue analyses. Solid phase extraction methods have been applied, after the phases of protein precipitation and centrifugation, in order to observe the fluoroquinolones [8], veterinary drugs [9], mycotoxins, and pesticides in milk [6, 10]. However, these methods are generally found to be time-consuming and require large volumes of organic solvents.Multiresidue veterinary drugs that were developed for milk tests depend on various extraction and cleanup principles. One of the most accepted approaches is to dilute a sample of milk with a solvent like acetonitrile and then to centrifuge and evaporate the obtained supernatant organic extract [11, 12]. Some multiclass analytical method applications by LC-MS/MS or LC-TOF/MS, related to homogenized or raw milk, that have the ability to specify undesirable chemicals, such as tetracycline, quinolone, sulfonamide, peptide, hormone, nonsteroidal anti-inflammatory anthelmintic drugs, mycotoxin, and pesticides, can be found in the literature [7]. Yet most of these methods are unable to offer satisfactory recovery of a large range of compounds of different polarities [13, 14].Most methods for the analysis of veterinary residues have some disadvantages, including high solvent consumption, tedious SPE cleanup steps that require extended time for analysis, and high costs. Therefore, these types of methods are not applied for routine analyses. The Quick Easy Cheap Effective Rugged Safe (QuEChERS) methodology, which was originally developed for pesticide analysis, has recently been proposed for the analysis of veterinary drugs using different matrices [15–18]. However, QuEChERS was found to be inconvenient for the recovery of polar veterinary drugs, including penicillin, tetracycline, and quinolone [13, 18, 19]. Therefore, there is still a great need for simple and rapid multiresidue analytical methods for simultaneously determining veterinary drug and pesticide residues in milk.In this study, we prepared milk samples by using a procedure based on a simple liquid-to-liquid extraction. This method utilized a simple and quick sample preparation procedure using a single extraction step. Through this method, milk samples were analyzed for the determination of both veterinary drugs and pesticide residues by utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, the reduced use of chemicals and steps in the sample preparation phase, together with the avoidance of a sample cleanup step, simplified the sample pretreatment and reduced the overall total cost. Finally, in addition to reducing analyses costs, the method provided a higher recovery of compounds of various polarities and improved the simplicity of detection efforts.


## 2. Materials and Methods

### 2.1. Reagents and Chemicals

HPLC grade acetonitrile (ACN), methanol, ethyl acetate (EtOAc) (Lichrosolv, purity ≥ 99.9), and glacial acetic acid (Emprove, 100%) were purchased from Merck (Darmstadt, Germany). The water used to prepare the solutions was purified in a Milli-Q Plus system (EMD Millipore, Billerica, MA). Magnesium sulfate, sodium chloride, Supelclean™ primary secondary amine (PSA), pure tetracyclines, sulfonamides, quinolones, macrolides, and antibiotics were provided from Sigma Aldrich (St. Louis, Missouri, USA) and the pesticides were provided from Dr. Ehrenstrorfer (Augsburg, Germany).


### 2.2. Samples

All pasteurized whole milk samples were purchased from local markets. Also, raw milk was used for interference and specificity/selectivity as a blank.Standard Solutions. Individual stock solutions of the veterinary drugs and pesticides were prepared in acetonitrile at a concentration of 1000 mg/kg. A mixed intermediate standard solution was prepared by diluting the stock standard solutions of the veterinary drugs and pesticides in acetonitrile at a concentration of 10 mg/kg. Stock and intermediate standard solutions were stored at 4°C in amber flasks and were found stable for at least 6 months.


### 2.3. Extraction Procedures

#### 2.3.1. Ethyl Acetate Extraction without Salting Procedure

Milk samples, upon arrival at our laboratory, were kept at refrigerator temperature (10
±
4°C) until analysis. For the preparation an aliquot of approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 200 mcL acetic acid was added to 10 mL of ethyl acetate. After vortex for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The sample was vortexed vigorously for 10 minutes. The extract was filtered through a 0.45 μm filter prior to LC-MS/MS analysis.


#### 2.3.2. Acetonitrile Extraction without Salting Procedure

Approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 10 mL of acetonitrile and 200 mcL acetic acid were added to milk. After mixing by a vortex stirrer for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The sample was vortexed vigorously for 10 minutes. The extract was filtered through a 0.45μm filter prior to LC-MS/MS analysis.


#### 2.3.3. QuEChERS Extraction Procedure

Approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 2 g of magnesium sulfate and 1 g of sodium acetate were added to milk samples [15]. Then, 10 mL of acetonitrile and 100 mcL acetic acid were added to milk samples. After vortex for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The extract was transferred to a 2 mL Eppendorf microtube containing 50 mg PSA and 200 mg magnesium sulfate. Then, the tube was centrifuged at 4000 rpm during 5 minutes. The extract was filtered through a 0.45 μm filter prior to LC-MS/MS analysis.


### 2.4. LC-MS/MS Analysis

The chromatographic analyses were performed using an HPLC system consisting of a binary pump (Shimadzu UFLC LC-20AD model), Shimadzu automatic injector (Autosampler SIL-20A HT model), and a column oven (CTO-20AC). Analytical columns, Symmetry® C18 2.1 × 150 mm id, 5μm particle size (Waters, Milford, MA), and Waters XTerra C18 150 mm × 2.1 mm id, 5 μm particle size (Waters, Milford, MA), were tested. Chromatographic separation of veterinary drugs and pesticides was carried out on a Waters Symmetry C18 column. The method used a gradient mobile phase containing 0.1% acetic acid water and mobile phase B containing methanol. The column temperature was maintained at 40°C with a flow rate of 0.3 mL/min. The gradient profile was scheduled as follows: initial proportion (98% A and 2% B) for 0.3 minutes, linear increase to 80% (B) until 7 minutes, and hold of 80% (B) for 3 minutes. The injection volume was 50 μL. The chromatographic system was coupled to electrospray ionization (ESI) source followed by an Applied Biosystems MDS SCIEX 4500 Q TRAP mass spectrometer. The MS/MS detector conditions were as follows: curtain gas 20 mL/min, exit potential 10 V, ion source gas 1 and ion source gas 2 set at 50 mL/min, ion spray voltage 5500 V, and turbo spray temperature set at 550°C. MS data were acquired in the positive ion ESI mode using two alternating MS/MS scan events. Two transitions were monitored for each analyte. The selected molecular ion and optimized collision voltages of product ions used for quantification, confirmation, and ion ratio were summarized in Table 1. Applied Biosystems SCIEX Analyst software version 1.6 was employed for data acquisition and processing. Quantification was by comparison with a six-point calibration (0.0, 0.01, 0.025, 0.05, 0.1, and 0.2 mg/kg) in matrix-matched calibration.Table 1
LC-MS/MS ion parameters.
Compounds
Precursor ion
Transition 1
Transition 2
Ion ratio
(m
/
z)
(m
/
z)
(m
/
z)
(%)
2,4-D (negative)
219
160
125
95
2,4,5-T
253
195
197
98
2,4-Dimethylaniline
122
107
80
Acetamiprid
223
126
73
22
Acrinathrin
560
208
181
75
Alachlor
238
162
238
12
Amitraz
294
163
122
75
Atrazine
216
174
104
45
Azoxystrobin
404
372
344
33
Bentazon
(−)239
132
197
78
Bifenazate
(−)300
253
239
79
Bitertanol
339
70
269
81
Boscalid
344
307
140
61
Bromacil
(−)259
205
203
55
Bromuconazole
378
159
70
66
Bromoxynil
(−)274
79
81
67
Bupirimate
317
108
166
86
Buprofezin
307
116
201
93
Butocarboxim sulfoxide
207
75
132
88
Cadusafos
272
159
97
98
Carbaryl
202
145
127
34
Carbendazim
192
160
132
17
Carbofuran
222
165
123
98
Carbosulfan
381
118
160
90
Carboxin
234
143
87
85
Dimethoate
230
199
125
97
Dimethomorph
388
301
165
58
Dimoxystrobin
328
116
205
99
Diniconazole
326
70
159
65
Dinobuton
327
215
152
66
Dinocap (sum)
295
193
209
89
Dinoterb
(−)239
207
176
85
Diphenylamine
171
93
152
17
Disulfoton-sulfoxide
291
185
213
87
Dithianon
(−)296
263
238
86
Diuron
233
72
160
85
Epoxiconazole
330
121
101
80
EPTC
191
128
86
40
Ethiofencarb
226
107
164
81
Ethion
402
385
199
76
Ethirimol
211
98
140
87
Ethofumesate
304
121
161
25
Etoxazole
361
141
113
86
Ethoxyquin
219
160
174
84
Famoxadone
392
238
331
84
Fenamidone
313
92
236
83
Fenamiphos
304
217
202
59
Fenarimol
331
268
81
18
Fenazaquin
307
161
147
80
Fenbuconazole
338
70
125
88
Fenhexamid
303
97
55
63
Fenitrothion
278
125
109
60
Fenoxycarb
302
88
116
25
Fenpropathrin
367
125
350
33
MCPP
(−)213
141
143
18
Metalaxyl-M
280
160
220
85
Mepanipyrim
225
106
77
17
Mesosulfuron-methyl
505
182
83
15
Metazachlor
279
210
134
82
S-Metolachlor
284
252
254
81
Metosulam
419
175
140
94
Metribuzin
215
187
84
29
Monocrotophos
224
127
98
9
Monolinuron
216
126
148
43
Monuron
199
72
126
76
Omethoate
215
125
125
10
Oxadiargyl
341
223
151
87
Oxadiazon
363
220
177
88
Oxadixyl
280
219
133
79
Oxamyl
237
72
90
65
Oxasulfuron
408
150
107
89
Oxycarboxin
269
175
147
31
Oxyfluorfen
362
316
237
27
Penconazole
284
70
159
67
Pendimethalin
282
212
194
19
Pethoxamid
297
131
250
62
Phosalone
368
182
111
31
Phenmedipham
301
136
168
54
Phenthoate
321
163
79
18
Phosmet
318
160
133
13
Phosphamidon
300
127
174
28
Picloram
(−)239
196
123
56
Terbuthylazine
230
174
104
55
Pirimicarb
239
72
93
Thiacloprid
254
126
186
18
Thiamethoxam
292
211
181
39
Thifensulfuron-methyl
389
167
205
14
Thiodicarb
355
88
108
22
Thiophanate-methyl
343
151
192
34
Triadimefon
294
197
225
30
Triadimenol
296
227
70
9
Triallate
304
86
143
67
Triasulfuron
403
167
141
67
Triazophos
314
119
162
54
Tribenuron-methyl
397
155
181
66
Tributylphosphate
268
98
67
Trichlorfon
274
109
221
75
Tridemorph
298
130
116
84
Trifloxystrobin
410
186
206
37
Triflumizole
346
73
278
26
Triticonazole
318
70
125
95
Vamidothion
288
146
118
33
Zoxamide
336
159
189
26
Ciprofloxacin
332
314
288
82
Clindamycin
425
126
82
97
Chlortetracycline
479
462
444
88
Danofloxacin
360
316
342
88
Difloxacin
400
356
299
90
Doxycycline hydrate
445
428
410
97
Flumequine
860
174
109
56
Josamycin
828
109
174
75
Clofentezine
303
138
102
88
Chloridazon
223
104
92
54
Chlorfenvinphos
359
155
99
51
Chlorfluazuron
(−)538
518
355
88
Chloroxuron
292
72
218
87
Chlorpyrifos
350
198
200
8
Chlorsulfuron
359
141
167
89
Chlorthiamid
206
189
154
25
Cinidon-ethyl
412
348
107
26
Cyazofamid
326
108
261
35
Cyclanilide
(−)272
160
228
45
Cycloate
216
154
134
48
Cymoxanil
199
128
111
59
Cyproconazole
292
70
125
65
Cyprodinil
226
93
77
80
Demeton-S-methyl
231
89
61
62
Demeton-S-methylsulfoxide
247
109
169
26
Desmedipham
318
182
136
88
Diallate
271
86
109
36
Diazinon
305
169
97
62
Dichlofluanid
350
123
224
16
Dichlorprop
(−)233
161
125
87
Dichlorvos
221
109
127
15
Difenoconazole
406
251
337
32
Dimethenamid (sum)
277
244
168
68
Fenthion
279
169
247
33
Flazasulfuron
409
182
227
46
Fludioxonil
(−)247
126
169
57
Fluazifop-P-butyl
385
282
328
62
Flufenacet
365
194
152
61
Flufenoxuron
(−)488
156
304
99
Flurochloridone
313
292
145
48
Flurtamone
335
178
247
79
Flusilazole
317
165
247
78
Flutolanil
325
262
242
45
Foramsulfuron
454
182
139
54
Fosthiazate
285
104
228
55
Furathiocarb
384
195
252
62
Heptenophos
251
127
109
9
Hexythiazox
353
228
168
81
Imazalil
297
159
201
88
Imazamox
(−)304
259
217
10
Imazaquin
313
199
128
25
Imazosulfuron
413
156
260
38
Imidacloprid
256
209
175
84
Indoxacarb
529
203
56
84
Ioxynil
(−)370
127
243
99
Iprovalicarb
322
119
203
95
Isazofos
314
120
162
34
Isoproturon
208
72
165
99
Isoxaben
334
165
150
48
Lufenuron
(−)509
326
339
46
Malathion
331
127
99
86
MCPA
(−)199
141
155
94
Picolinafen
378
238
145
57
Mecarbam
331
227
97
96
Pirimiphos-methyl
306
108
164
68
Prochloraz
376
308
266
33
Profenofos
373
303
97
60
Prometryn
242
158
68
68
Propamocarb
190
102
144
39
Propanil
218
162
127
66
Propargite
368
175
231
65
Propham
180
138
120
28
Propiconazole
342
159
69
62
Propyzamide
256
190
173
63
Pymetrozine
219
105
79
11
Pyraclostrobin
389
194
163
98
Pyridaben
365
309
147
78
Pyridaphenthion
341
205
189
88
Pyridate
380
207
351
78
Pyriproxyfen
322
96
185
62
Quinalphos
300
147
163
54
Quinoxyfen
309
197
162
97
Quizalofop-P-ethyl
374
299
56
Rimsulfuron
433
182
325
55
Simazine
202
124
132
70
Spiroxamine
299
144
100
87
Sulfosulfuron
472
211
261
89
Tebuconazole
308
70
125
55
TEPP
291
179
99
96
Terbutryn
242
186
68
51
Tetrachlorvinphos
367
127
241
95
Thiabendazole
203
175
131
12
Oxytetracycline
461
426
443
97
Rifampicin
823
791
151
91
Sarafloxacin
386
368
342
92
Sulfachloropyridazine
285
156
207
75
Sulfaquinoxaline
301
156
108
99
Sulfadiazine
251
156
92
86
Sulfamerazine
265
156
108
89
Sulfathiazole
256
156
92
88
Sulfamethazine
279
186
124
99
Sulfadoxine
311
156
108
97
Sulfapyridine
250
184
156
96
Sulfaclozine
285
156
108
95
Sulfamethoxazole
254
92
108
99
Tilmicosin
869
522
678
97
Tetracycline
445
428
410
85
Lomefloxacin
407
126
82
88
Orbifloxacin
396
352
295
88
Oxolinic acid
262
244
202
89


### 2.5. Validation Study

The analytical method developed for determination of veterinary drug and pesticide residues in milk was validated according to EU Decision 2002/657/EC [16] and SANTE/11945/2015 [17]. The following parameters were evaluated in the validation procedure: selectivity, sensitivity, linearity, precision (intraday and interday reproducibility), accuracy and CCα and CCβ, LOD, and LOQ.


## 2.1. Reagents and Chemicals

HPLC grade acetonitrile (ACN), methanol, ethyl acetate (EtOAc) (Lichrosolv, purity ≥ 99.9), and glacial acetic acid (Emprove, 100%) were purchased from Merck (Darmstadt, Germany). The water used to prepare the solutions was purified in a Milli-Q Plus system (EMD Millipore, Billerica, MA). Magnesium sulfate, sodium chloride, Supelclean™ primary secondary amine (PSA), pure tetracyclines, sulfonamides, quinolones, macrolides, and antibiotics were provided from Sigma Aldrich (St. Louis, Missouri, USA) and the pesticides were provided from Dr. Ehrenstrorfer (Augsburg, Germany).


## 2.2. Samples

All pasteurized whole milk samples were purchased from local markets. Also, raw milk was used for interference and specificity/selectivity as a blank.Standard Solutions. Individual stock solutions of the veterinary drugs and pesticides were prepared in acetonitrile at a concentration of 1000 mg/kg. A mixed intermediate standard solution was prepared by diluting the stock standard solutions of the veterinary drugs and pesticides in acetonitrile at a concentration of 10 mg/kg. Stock and intermediate standard solutions were stored at 4°C in amber flasks and were found stable for at least 6 months.


## 2.3. Extraction Procedures

### 2.3.1. Ethyl Acetate Extraction without Salting Procedure

Milk samples, upon arrival at our laboratory, were kept at refrigerator temperature (10
±
4°C) until analysis. For the preparation an aliquot of approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 200 mcL acetic acid was added to 10 mL of ethyl acetate. After vortex for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The sample was vortexed vigorously for 10 minutes. The extract was filtered through a 0.45 μm filter prior to LC-MS/MS analysis.


### 2.3.2. Acetonitrile Extraction without Salting Procedure

Approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 10 mL of acetonitrile and 200 mcL acetic acid were added to milk. After mixing by a vortex stirrer for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The sample was vortexed vigorously for 10 minutes. The extract was filtered through a 0.45μm filter prior to LC-MS/MS analysis.


### 2.3.3. QuEChERS Extraction Procedure

Approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 2 g of magnesium sulfate and 1 g of sodium acetate were added to milk samples [15]. Then, 10 mL of acetonitrile and 100 mcL acetic acid were added to milk samples. After vortex for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The extract was transferred to a 2 mL Eppendorf microtube containing 50 mg PSA and 200 mg magnesium sulfate. Then, the tube was centrifuged at 4000 rpm during 5 minutes. The extract was filtered through a 0.45 μm filter prior to LC-MS/MS analysis.


## 2.3.1. Ethyl Acetate Extraction without Salting Procedure

Milk samples, upon arrival at our laboratory, were kept at refrigerator temperature (10
±
4°C) until analysis. For the preparation an aliquot of approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 200 mcL acetic acid was added to 10 mL of ethyl acetate. After vortex for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The sample was vortexed vigorously for 10 minutes. The extract was filtered through a 0.45 μm filter prior to LC-MS/MS analysis.


## 2.3.2. Acetonitrile Extraction without Salting Procedure

Approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 10 mL of acetonitrile and 200 mcL acetic acid were added to milk. After mixing by a vortex stirrer for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The sample was vortexed vigorously for 10 minutes. The extract was filtered through a 0.45μm filter prior to LC-MS/MS analysis.


## 2.3.3. QuEChERS Extraction Procedure

Approximately 5 mL milk sample was pipetted in a 50 mL polypropylene centrifuge tube. Then, 2 g of magnesium sulfate and 1 g of sodium acetate were added to milk samples [15]. Then, 10 mL of acetonitrile and 100 mcL acetic acid were added to milk samples. After vortex for 3 minutes, the mixture was centrifuged at 5000 rpm for 10 minutes. The upper phase was taken in 15 mL centrifuge tube and was dried under a gentle stream of nitrogen, and the residue was reconstituted with 1000 mcL of mobile phase A/mobile phase B (80/20). The extract was transferred to a 2 mL Eppendorf microtube containing 50 mg PSA and 200 mg magnesium sulfate. Then, the tube was centrifuged at 4000 rpm during 5 minutes. The extract was filtered through a 0.45 μm filter prior to LC-MS/MS analysis.


## 2.4. LC-MS/MS Analysis

The chromatographic analyses were performed using an HPLC system consisting of a binary pump (Shimadzu UFLC LC-20AD model), Shimadzu automatic injector (Autosampler SIL-20A HT model), and a column oven (CTO-20AC). Analytical columns, Symmetry® C18 2.1 × 150 mm id, 5μm particle size (Waters, Milford, MA), and Waters XTerra C18 150 mm × 2.1 mm id, 5 μm particle size (Waters, Milford, MA), were tested. Chromatographic separation of veterinary drugs and pesticides was carried out on a Waters Symmetry C18 column. The method used a gradient mobile phase containing 0.1% acetic acid water and mobile phase B containing methanol. The column temperature was maintained at 40°C with a flow rate of 0.3 mL/min. The gradient profile was scheduled as follows: initial proportion (98% A and 2% B) for 0.3 minutes, linear increase to 80% (B) until 7 minutes, and hold of 80% (B) for 3 minutes. The injection volume was 50 μL. The chromatographic system was coupled to electrospray ionization (ESI) source followed by an Applied Biosystems MDS SCIEX 4500 Q TRAP mass spectrometer. The MS/MS detector conditions were as follows: curtain gas 20 mL/min, exit potential 10 V, ion source gas 1 and ion source gas 2 set at 50 mL/min, ion spray voltage 5500 V, and turbo spray temperature set at 550°C. MS data were acquired in the positive ion ESI mode using two alternating MS/MS scan events. Two transitions were monitored for each analyte. The selected molecular ion and optimized collision voltages of product ions used for quantification, confirmation, and ion ratio were summarized in Table 1. Applied Biosystems SCIEX Analyst software version 1.6 was employed for data acquisition and processing. Quantification was by comparison with a six-point calibration (0.0, 0.01, 0.025, 0.05, 0.1, and 0.2 mg/kg) in matrix-matched calibration.Table 1
LC-MS/MS ion parameters.
Compounds
Precursor ion
Transition 1
Transition 2
Ion ratio
(m
/
z)
(m
/
z)
(m
/
z)
(%)
2,4-D (negative)
219
160
125
95
2,4,5-T
253
195
197
98
2,4-Dimethylaniline
122
107
80
Acetamiprid
223
126
73
22
Acrinathrin
560
208
181
75
Alachlor
238
162
238
12
Amitraz
294
163
122
75
Atrazine
216
174
104
45
Azoxystrobin
404
372
344
33
Bentazon
(−)239
132
197
78
Bifenazate
(−)300
253
239
79
Bitertanol
339
70
269
81
Boscalid
344
307
140
61
Bromacil
(−)259
205
203
55
Bromuconazole
378
159
70
66
Bromoxynil
(−)274
79
81
67
Bupirimate
317
108
166
86
Buprofezin
307
116
201
93
Butocarboxim sulfoxide
207
75
132
88
Cadusafos
272
159
97
98
Carbaryl
202
145
127
34
Carbendazim
192
160
132
17
Carbofuran
222
165
123
98
Carbosulfan
381
118
160
90
Carboxin
234
143
87
85
Dimethoate
230
199
125
97
Dimethomorph
388
301
165
58
Dimoxystrobin
328
116
205
99
Diniconazole
326
70
159
65
Dinobuton
327
215
152
66
Dinocap (sum)
295
193
209
89
Dinoterb
(−)239
207
176
85
Diphenylamine
171
93
152
17
Disulfoton-sulfoxide
291
185
213
87
Dithianon
(−)296
263
238
86
Diuron
233
72
160
85
Epoxiconazole
330
121
101
80
EPTC
191
128
86
40
Ethiofencarb
226
107
164
81
Ethion
402
385
199
76
Ethirimol
211
98
140
87
Ethofumesate
304
121
161
25
Etoxazole
361
141
113
86
Ethoxyquin
219
160
174
84
Famoxadone
392
238
331
84
Fenamidone
313
92
236
83
Fenamiphos
304
217
202
59
Fenarimol
331
268
81
18
Fenazaquin
307
161
147
80
Fenbuconazole
338
70
125
88
Fenhexamid
303
97
55
63
Fenitrothion
278
125
109
60
Fenoxycarb
302
88
116
25
Fenpropathrin
367
125
350
33
MCPP
(−)213
141
143
18
Metalaxyl-M
280
160
220
85
Mepanipyrim
225
106
77
17
Mesosulfuron-methyl
505
182
83
15
Metazachlor
279
210
134
82
S-Metolachlor
284
252
254
81
Metosulam
419
175
140
94
Metribuzin
215
187
84
29
Monocrotophos
224
127
98
9
Monolinuron
216
126
148
43
Monuron
199
72
126
76
Omethoate
215
125
125
10
Oxadiargyl
341
223
151
87
Oxadiazon
363
220
177
88
Oxadixyl
280
219
133
79
Oxamyl
237
72
90
65
Oxasulfuron
408
150
107
89
Oxycarboxin
269
175
147
31
Oxyfluorfen
362
316
237
27
Penconazole
284
70
159
67
Pendimethalin
282
212
194
19
Pethoxamid
297
131
250
62
Phosalone
368
182
111
31
Phenmedipham
301
136
168
54
Phenthoate
321
163
79
18
Phosmet
318
160
133
13
Phosphamidon
300
127
174
28
Picloram
(−)239
196
123
56
Terbuthylazine
230
174
104
55
Pirimicarb
239
72
93
Thiacloprid
254
126
186
18
Thiamethoxam
292
211
181
39
Thifensulfuron-methyl
389
167
205
14
Thiodicarb
355
88
108
22
Thiophanate-methyl
343
151
192
34
Triadimefon
294
197
225
30
Triadimenol
296
227
70
9
Triallate
304
86
143
67
Triasulfuron
403
167
141
67
Triazophos
314
119
162
54
Tribenuron-methyl
397
155
181
66
Tributylphosphate
268
98
67
Trichlorfon
274
109
221
75
Tridemorph
298
130
116
84
Trifloxystrobin
410
186
206
37
Triflumizole
346
73
278
26
Triticonazole
318
70
125
95
Vamidothion
288
146
118
33
Zoxamide
336
159
189
26
Ciprofloxacin
332
314
288
82
Clindamycin
425
126
82
97
Chlortetracycline
479
462
444
88
Danofloxacin
360
316
342
88
Difloxacin
400
356
299
90
Doxycycline hydrate
445
428
410
97
Flumequine
860
174
109
56
Josamycin
828
109
174
75
Clofentezine
303
138
102
88
Chloridazon
223
104
92
54
Chlorfenvinphos
359
155
99
51
Chlorfluazuron
(−)538
518
355
88
Chloroxuron
292
72
218
87
Chlorpyrifos
350
198
200
8
Chlorsulfuron
359
141
167
89
Chlorthiamid
206
189
154
25
Cinidon-ethyl
412
348
107
26
Cyazofamid
326
108
261
35
Cyclanilide
(−)272
160
228
45
Cycloate
216
154
134
48
Cymoxanil
199
128
111
59
Cyproconazole
292
70
125
65
Cyprodinil
226
93
77
80
Demeton-S-methyl
231
89
61
62
Demeton-S-methylsulfoxide
247
109
169
26
Desmedipham
318
182
136
88
Diallate
271
86
109
36
Diazinon
305
169
97
62
Dichlofluanid
350
123
224
16
Dichlorprop
(−)233
161
125
87
Dichlorvos
221
109
127
15
Difenoconazole
406
251
337
32
Dimethenamid (sum)
277
244
168
68
Fenthion
279
169
247
33
Flazasulfuron
409
182
227
46
Fludioxonil
(−)247
126
169
57
Fluazifop-P-butyl
385
282
328
62
Flufenacet
365
194
152
61
Flufenoxuron
(−)488
156
304
99
Flurochloridone
313
292
145
48
Flurtamone
335
178
247
79
Flusilazole
317
165
247
78
Flutolanil
325
262
242
45
Foramsulfuron
454
182
139
54
Fosthiazate
285
104
228
55
Furathiocarb
384
195
252
62
Heptenophos
251
127
109
9
Hexythiazox
353
228
168
81
Imazalil
297
159
201
88
Imazamox
(−)304
259
217
10
Imazaquin
313
199
128
25
Imazosulfuron
413
156
260
38
Imidacloprid
256
209
175
84
Indoxacarb
529
203
56
84
Ioxynil
(−)370
127
243
99
Iprovalicarb
322
119
203
95
Isazofos
314
120
162
34
Isoproturon
208
72
165
99
Isoxaben
334
165
150
48
Lufenuron
(−)509
326
339
46
Malathion
331
127
99
86
MCPA
(−)199
141
155
94
Picolinafen
378
238
145
57
Mecarbam
331
227
97
96
Pirimiphos-methyl
306
108
164
68
Prochloraz
376
308
266
33
Profenofos
373
303
97
60
Prometryn
242
158
68
68
Propamocarb
190
102
144
39
Propanil
218
162
127
66
Propargite
368
175
231
65
Propham
180
138
120
28
Propiconazole
342
159
69
62
Propyzamide
256
190
173
63
Pymetrozine
219
105
79
11
Pyraclostrobin
389
194
163
98
Pyridaben
365
309
147
78
Pyridaphenthion
341
205
189
88
Pyridate
380
207
351
78
Pyriproxyfen
322
96
185
62
Quinalphos
300
147
163
54
Quinoxyfen
309
197
162
97
Quizalofop-P-ethyl
374
299
56
Rimsulfuron
433
182
325
55
Simazine
202
124
132
70
Spiroxamine
299
144
100
87
Sulfosulfuron
472
211
261
89
Tebuconazole
308
70
125
55
TEPP
291
179
99
96
Terbutryn
242
186
68
51
Tetrachlorvinphos
367
127
241
95
Thiabendazole
203
175
131
12
Oxytetracycline
461
426
443
97
Rifampicin
823
791
151
91
Sarafloxacin
386
368
342
92
Sulfachloropyridazine
285
156
207
75
Sulfaquinoxaline
301
156
108
99
Sulfadiazine
251
156
92
86
Sulfamerazine
265
156
108
89
Sulfathiazole
256
156
92
88
Sulfamethazine
279
186
124
99
Sulfadoxine
311
156
108
97
Sulfapyridine
250
184
156
96
Sulfaclozine
285
156
108
95
Sulfamethoxazole
254
92
108
99
Tilmicosin
869
522
678
97
Tetracycline
445
428
410
85
Lomefloxacin
407
126
82
88
Orbifloxacin
396
352
295
88
Oxolinic acid
262
244
202
89


## 2.5. Validation Study

The analytical method developed for determination of veterinary drug and pesticide residues in milk was validated according to EU Decision 2002/657/EC [16] and SANTE/11945/2015 [17]. The following parameters were evaluated in the validation procedure: selectivity, sensitivity, linearity, precision (intraday and interday reproducibility), accuracy and CCα and CCβ, LOD, and LOQ.


## 3. Results and Discussion

### 3.1. Optimization of the Extraction Procedures

Ethyl acetate extraction without salt procedure was chosen to be performed in this study because of its advantages. There was no need to use salt and it could give lower detection limit in terms of volatile characteristic of ethyl acetate.Recovery values showed no difference among three different extraction procedures (acetonitrile extraction, QuEChERS extraction, and ethyl acetate extraction without salting procedure) (Figure1).Figure 1
Recovery data for three different extraction procedures.The recovery values expressed as recovery % are all within the reference range of 70–120%. Comparing three procedures, EtOAc without salt provided recoveries between 100% and 120% for a higher number of veterinary drugs and pesticides (26 veterinary drugs and 134 pesticides; total of 160 compounds) than QuEChERS (82 compounds) and ACN (100 compounds), as it can be observed in Figure2. In terms of extraction recoveries, EtOAc was found to be a suitable extraction procedure for all 26 veterinary drugs and most of the pesticides analyzed in this study. Only one analyte (propham) showed R
>
120 for EtOAc.Figure 2
Recovery (%) data obtained using extraction procedures; QuEChERS, ACN, and EtOAc without salt.Accuracy was evaluated in terms of relative standard deviation (RSD) by spiking blank samples with the corresponding volume of the multicompound working standard solution. RSD was evaluated at 50μg/kg by spiking six blank samples at each level for three procedures that provided similar RSD values. These values were within 1
<
R
S
D
<
10 for 75% of each analyte in the three procedures. These results indicated that the EtOAc without salt method was precise, accurate, and reliable for the analysis of the veterinary drug and pesticide compounds in the milk samples as an alternative method.


### 3.2. LC-MS/MS

Mobile phase was acidified with acetic acid in methanol and water. Also, study [18] in the literature was performed for the comparison. Formic acid in acetonitrile and water was used as a mobile phase in [18]. According to analyte intensities, our results gave better peak shapes than chromatograms in [18]. The dried residue was redissolved in a mixture of MeOH/water with 0.1% acetic acid to test different reconstitution solvents. This composition produced better peak shapes for all analytes compared with water-methanol (80 : 20) that gave lower response. Increasing acetic acid to 1% in the mixture did not improve chromatography but caused extra peaks in the background noise.


### 3.3. Validation Study

#### 3.3.1. Selectivity

The selectivity of the method was assessed by duplicate analysis of 10 blank milk samples. No peaks of interfering compounds were observed within the intervals of the retention time of the analytes in any of these samples.


#### 3.3.2. Linearity

Linearity was evaluated from the calibration curves by triplicate analyses of blank milk samples fortified with the analytes at six (0.0, 0.01, 0.025, 0.05, 0.1, and 0.2 mg/kg) concentration levels. Linearity was expressed as the coefficient of linear correlation (r) and from the slope of the calibration curve. The linearity of the analytical response across the studied range was excellent, with correlation coefficients higher than 0.997 for all analytes, which was similar to the findings in [19]. The authors [20] found correlation coefficients higher than 0.992 for all analytes, which was a lower score than ours.


#### 3.3.3. Decision Limit and Detection Capability

CCα is defined as the limit at and above which it can be concluded with an error probability of α that a sample is noncompliant. CCβ is defined as the smallest content of the substance that may be detected, identified, and/or quantified in a sample with an error probability of β. The CCα and CCβ were determined by analysis of 10 blank milk samples and the signal-to-noise (S/N) ratio is calculated at the time window in which the analyte is expected. The CCα values were calculated as three times the S/N ratio. The CCβ was calculated by analyzing 10 blank samples spiked with concentration at CCα. Then the CCα value was added up to 1.64 times the corresponding standard deviation. Then, a preliminary experiment was conducted to check if all compounds were detected when spiked at their CCα level (Table 2).Table 2
Validation results of the developed method.
Compounds
r
2
LOQ
MRL
CCα
CCβ
% recovery
% RSD
% recovery
% RSD
% recovery
% RSD
% RSDr
Relative uncertainty %
Matrix effect
(µg/kg)
(µg/kg)
(µg/kg)
(µg/kg)
10 (µg/kg)
10 (µg/kg)
25 (µg/kg)
25 (µg/kg)
50 (µg/kg)
50 (µg/kg)
2,4-D (negative)
0.997
11
10
21
33
112
8
90
6
108
5
13
32
0.81
2,4,5-T
0.998
9
10
18
26
85
18
102
17
93
9
11
23
0.92
2,4-Dimethylaniline
0.998
8
10
18
26
79
11
110
8
97
8
11
35
0.99
Acetamiprid
0.997
10
10
18
26
97
9
102
8
96
7
11
33
0.92
Acrinathrin
0.998
11
10
18
26
106
10
88
8
104
7
11
35
0.81
Alachlor
0.997
11
10
20
30
108
11
86
4
92
3
13
28
0.81
Amitraz
0.997
9
10
20
30
86
8
76
4
76
3
14
28
0.81
Atrazine
0.997
11
10
17
23
109
6
94
5
91
3
9
30
0.85
Azoxystrobin
0.998
11
10
21
33
114
9
102
5
110
4
14
30
0.92
Bentazon
0.998
12
10
17
23
118
5
94
4
94
4
8
28
0.85
Bifenazate
0.998
11
10
20
30
109
10
102
7
111
6
11
33
0.92
Bitertanol
0.998
11
10
17
23
112
5
98
3
97
3
8
26
0.88
Boscalid
0.998
12
10
15
20
115
6
110
6
101
5
7
30
0.99
Bromacil
0.998
9
10
20
30
91
16
108
14
91
11
13
42
0.97
Bromuconazole
0.997
11
10
20
30
109
9
100
8
95
6
14
30
0.90
Bromoxynil
0.997
10
10
17
23
99
14
106
9
99
6
9
33
0.96
Bupirimate
0.997
12
10
20
30
116
5
100
5
104
3
12
26
0.90
Buprofezin
0.997
9
10
17
23
90
16
90
12
88
8
10
26
0.81
Butocarboxim sulfoxide
0.997
11
10
17
23
107
9
118
8
102
7
7
33
1.06
Cadusafos
0.997
11
10
15
20
110
11
94
8
95
5
6
30
0.85
Carbaryl
0.997
11
10
17
23
105
10
94
5
89
4
9
28
0.85
Carbendazim
0.997
12
10
20
30
118
5
122
5
113
4
11
30
1.10
Carbofuran
0.998
12
10
17
23
116
4
90
4
92
3
8
26
0.81
Carbosulfan
0.997
9
10
21
33
91
10
106
6
93
6
15
32
0.96
Carboxin
0.998
11
10
18
26
113
6
90
4
89
5
12
28
0.81
Clofentezine
0.998
9
10
17
23
88
13
98
10
93
8
10
26
0.88
Chloridazon
0.998
10
10
18
26
102
11
114
9
97
7
11
30
1.03
Chlorfenvinphos
0.998
11
10
18
26
112
8
96
5
100
3
10
30
0.86
Chlorfluazuron
0.998
11
10
21
33
107
10
114
5
108
6
14
30
1.03
Chloroxuron
0.997
11
10
18
26
108
13
102
5
104
5
10
36
0.92
Chlorpyrifos
0.997
11
10
17
23
108
9
102
6
107
6
7
32
0.92
Chlorsulfuron
0.997
10
10
21
33
96
10
92
5
88
5
15
30
0.83
Chlorthiamid
0.997
9
10
21
33
92
17
104
13
114
9
13
32
0.94
Cinidon-ethyl
0.997
11
10
17
23
105
7
100
6
110
5
8
32
0.90
Cyazofamid
0.997
11
10
18
26
112
7
94
7
93
4
10
28
0.85
Cyclanilide
0.997
12
10
21
33
115
5
104
5
113
5
13
30
0.94
Cycloate
0.998
11
10
17
23
106
12
96
11
88
5
9
28
0.86
Cymoxanil
0.998
11
10
17
23
109
7
112
6
103
6
8
32
1.01
Cyproconazole
0.998
10
10
18
26
96
10
92
6
98
4
12
28
0.83
Cyprodinil
0.998
9
10
20
30
91
16
98
9
110
9
11
30
0.88
Demeton-S-methyl
0.998
10
10
20
30
96
16
98
11
101
4
11
28
0.88
Demeton-S-methylsulfoxide
0.998
10
10
13
17
100
8
101
7
99
6
11
35
0.91
Desmedipham
0.998
10
10
13
17
99
8
98
8
87
7
14
0
0.88
Diallate
0.998
10
10
20
30
100
13
120
12
110
6
14
26
1.08
Diazinon
0.998
10
10
12
13
95
9
92
6
100
5
12
32
0.83
Dichlofluanid
0.998
10
10
17
23
103
9
104
8
100
8
12
26
0.94
Dichlorprop
0.998
9
10
18
26
88
5
88
4
95
3
8
0
0.82
Dichlorvos
0.998
9
10
15
20
93
14
118
11
109
11
16
35
1.06
Difenoconazole
0.998
9
10
15
20
92
12
100
9
94
4
11
28
0.90
Dimethenamid (sum)
0.998
11
10
17
23
112
13
98
9
87
10
10
28
0.94
Dimethoate
0.998
11
10
17
23
113
8
104
7
100
6
9
26
0.94
Dimethomorph
0.998
9
10
13
17
91
12
104
8
97
5
14
28
0.94
Dimoxystrobin
0.998
10
10
15
20
101
8
116
5
97
5
12
30
1.05
Diniconazole
0.998
10
10
13
17
97
19
96
15
87
9
10
36
0.86
Dinobuton
0.998
10
10
17
23
104
10
78
6
92
6
11
32
0.82
Dinocap (sum)
0.998
12
10
13
17
115
7
96
5
90
4
14
30
0.86
Dinoterb
0.997
12
10
18
26
115
7
100
4
98
4
9
28
0.90
Diphenylamine
0.999
12
10
13
17
115
7
108
5
90
4
14
30
0.97
Disulfoton-sulfoxide
0.997
11
10
13
17
109
10
86
6
94
5
13
30
0.82
Dithianon
0.997
11
10
13
17
106
6
108
5
102
3
6
26
0.97
Diuron
0.999
11
10
20
30
109
9
94
7
101
3
10
26
0.85
Epoxiconazole
0.997
11
10
12
13
105
8
98
4
93
4
11
28
0.88
EPTC
0.997
11
10
18
26
113
7
112
4
96
4
10
28
1.01
Ethiofencarb
0.997
12
10
18
26
115
7
96
5
96
3
9
26
0.86
Ethion
0.998
10
10
15
20
102
10
100
8
98
8
12
22
0.90
Ethirimol
0.998
10
10
15
20
98
15
96
9
111
8
13
33
0.86
Ethofumesate
0.998
11
10
17
23
105
11
96
10
100
7
13
28
0.86
Etoxazole
0.998
11
10
17
23
114
8
94
5
101
4
11
28
0.85
Ethoxyquin
0.998
10
10
13
17
100
13
96
11
98
4
11
30
0.86
Famoxadone
0.998
11
10
15
20
112
12
114
5
117
5
12
32
1.03
Fenamidone
0.998
11
10
13
17
108
9
84
8
91
6
10
26
0.82
Fenamiphos
0.998
11
10
17
23
107
5
106
4
109
3
5
32
0.96
Fenarimol
0.998
10
10
13
17
101
11
99
9
95
8
11
23
0.89
Fenazaquin
0.997
11
10
18
26
113
8
128
7
113
8
10
28
1.15
Fenbuconazole
0.997
9
10
13
17
86
16
94
12
97
4
8
32
0.85
Fenhexamid
0.997
11
10
18
26
114
7
80
6
84
5
13
28
0.82
Fenitrothion
0.997
10
10
17
23
99
12
98
11
94
9
11
24
0.88
Fenoxycarb
0.997
9
10
13
17
85
16
100
14
103
8
9
35
0.90
Fenpropathrin
0.997
10
10
18
26
100
12
100
10
99
9
7
25
0.90
Fenthion
0.997
10
10
23
36
95
12
112
11
108
7
12
33
1.01
Flazasulfuron
0.998
10
10
30
49
95
10
118
8
116
7
15
30
1.06
Fludioxonil
0.998
12
10
23
36
115
7
88
5
100
5
13
25
0.82
Fluazifop-P-butyl
0.998
12
10
28
46
118
8
102
6
96
5
11
32
0.92
Flufenacet
0.998
10
10
25
40
103
10
96
9
100
7
13
28
0.86
Flufenoxuron
0.998
10
10
26
43
95
8
100
8
105
4
11
32
0.90
Flurochloridone
0.998
10
10
18
26
100
10
102
9
112
6
7
28
0.92
Flurtamone
0.998
10
10
20
30
103
6
90
4
89
4
10
32
0.81
Flusilazole
0.998
10
10
23
36
104
8
92
6
99
6
11
32
0.83
Flutolanil
0.998
12
10
18
26
115
6
114
7
99
7
10
26
1.03
Foramsulfuron
0.998
11
10
25
40
105
4
86
3
99
4
14
26
0.82
Fosthiazate
0.997
8
10
20
30
82
13
94
5
94
3
13
30
0.85
Furathiocarb
0.997
12
10
21
33
115
5
96
5
90
5
12
33
0.86
Heptenophos
0.997
10
10
31
53
99
15
94
12
90
7
11
28
0.85
Hexythiazox
0.997
10
10
25
40
103
6
100
4
98
5
9
32
0.90
Imazalil
0.997
10
10
28
46
95
12
100
11
88
10
9
28
0.90
Imazamox
0.997
8
10
18
26
84
11
100
9
92
6
12
26
0.90
Imazaquin
0.997
11
10
31
53
112
9
90
5
86
3
12
0
0.81
Imazosulfuron
0.997
10
10
25
40
98
12
95
11
96
8
11
36
0.86
Imidacloprid
0.999
9
10
30
49
87
12
102
9
109
6
15
26
0.92
Indoxacarb
0.999
10
10
23
36
99
15
100
6
93
8
16
30
0.90
Ioxynil
0.999
10
10
20
30
99
4
84
4
81
5
8
30
0.81
Iprovalicarb
0.999
10
10
20
30
97
12
110
8
104
7
15
32
0.99
Isazofos
0.999
12
10
21
33
115
7
94
5
89
11
13
26
0.85
Isoproturon
0.999
12
10
20
30
115
8
90
6
97
6
11
38
0.81
Isoxaben
0.997
11
10
26
43
112
5
110
3
96
5
8
30
0.99
Lufenuron
0.997
10
10
26
43
99
4
98
4
94
4
13
30
0.88
Malathion
0.999
12
10
23
36
115
7
104
5
89
6
13
33
0.94
MCPA
0.997
9
10
25
40
93
9
102
5
103
7
11
28
0.92
Mecarbam
0.998
11
10
31
53
105
16
106
14
94
7
11
30
0.96
MCPP
0.998
10
10
28
46
103
12
84
9
97
9
12
41
0.82
Metalaxyl-M
0.998
10
10
23
36
97
7
122
4
101
5
16
26
1.10
Mepanipyrim
0.997
10
10
31
53
102
16
92
13
107
11
16
33
0.83
Mesosulfuron-methyl
0.997
8
10
28
46
82
15
104
5
93
6
12
35
0.94
Metazachlor
0.997
11
10
23
36
113
5
100
5
93
6
12
16
0.90
S-Metolachlor
0.999
9
10
18
26
93
8
92
7
97
7
11
27
0.83
Metosulam
0.999
11
10
30
49
110
9
94
8
88
8
14
32
0.85
Metribuzin
0.999
11
10
21
33
105
11
72
9
91
6
15
26
0.82
Monocrotophos
0.997
12
10
25
40
115
4
100
3
88
3
14
32
0.90
Monolinuron
0.997
10
10
28
46
104
17
92
14
90
8
13
28
0.83
Monuron
0.997
11
10
31
53
114
8
102
4
94
5
13
35
0.92
Omethoate
0.997
11
10
28
46
105
9
94
9
91
8
13
39
0.85
Oxadiargyl
0.997
11
10
23
36
105
15
110
11
90
11
14
30
0.99
Oxadiazon
0.997
10
10
30
49
96
8
112
5
112
5
6
32
1.01
Oxadixyl
0.997
11
10
21
33
109
12
90
10
88
6
8
35
0.81
Oxamyl
0.997
10
10
30
49
103
14
96
12
89
8
11
33
0.86
Oxasulfuron
0.997
11
10
33
56
108
8
86
8
87
7
12
28
0.82
Oxycarboxin
0.997
12
10
28
46
115
7
112
4
97
6
12
28
1.01
Oxyfluorfen
0.997
9
10
30
49
92
8
101
7
103
6
11
28
0.91
Penconazole
0.997
11
10
23
36
112
7
102
4
96
4
9
28
0.92
Pendimethalin
0.997
11
10
20
30
113
8
108
5
108
4
6
28
0.97
Pethoxamid
0.997
11
10
28
46
108
9
76
4
81
5
14
32
0.83
Phosalone
0.998
10
10
23
36
96
11
112
4
112
5
8
36
1.01
Phenmedipham
0.998
10
10
36
62
95
14
92
13
88
6
11
30
0.83
Phenthoate
0.997
12
10
23
36
115
7
94
5
89
6
12
28
0.85
Phosmet
0.997
10
10
21
33
100
11
94
10
92
9
13
33
0.85
Phosphamidon
0.997
10
10
21
33
99
8
108
7
95
7
10
30
0.97
Picloram
0.997
11
10
21
33
105
12
96
8
91
7
12
36
0.86
Picolinafen
0.997
10
10
25
40
100
12
114
9
104
9
12
25
1.03
Pirimicarb
0.997
12
10
23
36
119
8
94
2
97
3
10
30
0.85
Pirimiphos-methyl
0.997
12
10
23
36
115
7
108
5
94
5
12
30
0.97
Prochloraz
0.999
12
10
21
33
115
5
112
5
94
5
18
30
1.01
Profenofos
0.999
12
10
21
33
115
7
104
5
96
5
8
30
0.94
Prometryn
0.999
11
10
21
33
109
7
94
5
99
6
9
36
0.85
Propamocarb
0.998
10
10
25
40
100
13
102
10
89
9
11
30
0.92
Propanil
0.999
12
10
30
49
115
7
102
5
99
6
12
28
0.92
Propargite
0.999
10
10
26
43
102
14
112
10
113
4
8
30
1.01
Propham
0.999
10
10
21
33
102
13
100
6
96
5
10
28
0.90
Propiconazole
0.999
10
10
30
49
98
6
122
4
111
6
14
30
1.10
Propyzamide
0.999
8
10
28
46
84
10
96
9
95
5
9
33
0.86
Pymetrozine
0.997
10
10
23
36
101
10
96
8
90
7
11
28
0.86
Pyraclostrobin
0.997
12
10
21
33
115
7
130
5
119
4
15
26
1.17
Pyridaben
0.997
9
10
20
30
89
16
80
12
81
4
11
36
0.82
Pyridaphenthion
0.997
10
10
23
36
99
11
120
9
99
10
13
26
1.08
Pyridate
0.998
10
10
31
53
98
5
106
4
98
3
8
32
0.96
Pyriproxyfen
0.997
10
10
21
33
101
11
94
6
86
5
13
22
0.85
Quinalphos
0.997
10
10
23
36
101
5
76
6
90
5
14
23
0.82
Quinoxyfen
0.999
10
10
30
49
99
5
98
1
100
2
8
23
0.88
Quizalofop-P-ethyl
0.997
12
10
30
49
115
7
82
5
85
5
14
26
0.84
Rimsulfuron
0.998
9
10
26
43
88
13
104
3
100
2
14
26
0.94
Simazine
0.997
11
10
23
36
106
16
96
8
99
3
11
23
0.86
Spiroxamine
0.999
8
10
21
33
83
14
84
9
82
2
14
32
0.82
Sulfosulfuron
0.997
8
10
21
33
82
12
110
6
111
6
19
30
0.99
Tebuconazole
0.999
10
10
28
46
99
10
94
6
99
5
9
25
0.85
TEPP
0.997
9
10
23
36
90
7
107
7
105
6
11
29
0.96
Terbutryn
0.997
12
10
26
43
115
8
96
5
102
3
11
28
0.86
Terbuthylazine
0.997
10
10
20
30
104
12
118
5
116
4
6
26
1.06
Tetrachlorvinphos
0.997
11
10
21
33
113
11
78
8
90
4
14
28
0.90
Thiabendazole
0.997
12
10
21
33
118
6
98
4
96
5
13
30
0.88
Thiacloprid
0.997
11
10
18
26
106
13
84
12
89
5
8
35
0.82
Thiamethoxam
0.997
10
10
33
56
104
19
118
11
113
8
11
32
1.06
Thifensulfuron-methyl
0.997
9
10
20
30
91
16
98
8
92
9
15
30
0.88
Thiodicarb
0.997
12
10
31
53
115
7
102
5
97
6
9
35
0.92
Thiophanate-methyl
0.997
11
10
20
30
114
9
112
6
95
5
13
32
1.01
Triadimefon
0.997
10
10
28
46
104
7
106
6
98
5
8
32
0.96
Triadimenol
0.997
11
10
25
40
107
14
106
6
92
7
9
26
0.96
Triallate
0.998
11
10
23
36
106
9
114
3
111
4
12
30
1.03
Triasulfuron
0.998
11
10
20
30
111
8
76
5
91
6
14
30
0.82
Triazophos
0.998
12
10
30
49
115
7
96
5
87
5
9
30
0.86
Tribenuron-methyl
0.998
10
10
35
59
99
9
103
8
101
5
11
30
0.93
Tributylphosphate
0.997
10
10
17
23
104
14
88
12
89
5
8
35
0.81
Trichlorfon
0.998
10
10
25
40
98
10
104
3
100
2
14
27
0.94
Tridemorph
0.997
10
10
21
33
98
17
90
13
100
5
11
30
0.81
Trifloxystrobin
0.997
12
10
25
40
115
7
124
5
114
5
14
30
1.12
Triflumizole
0.997
11
10
20
30
113
14
92
5
99
5
8
28
0.83
Triticonazole
0.998
10
10
17
23
100
11
106
10
98
7
7
32
0.96
Vamidothion
0.997
9
10
21
33
93
13
118
6
102
7
16
28
1.06
Zoxamide
0.997
12
10
23
36
116
7
72
4
85
3
15
25
0.82
Ciprofloxacin
0.997
7
100
124
148
74
25
61
16
97
13
9
38
0.81
Clindamycin
0.997
11
10
12
14
110
23
106
15
109
10
12
34
0.96
Chlortetracycline
0.999
10
100
108
116
101
7
93
7
99
6
9
14
0.84
Danofloxacin
0.999
10
30
40
48
104
14
87
12
88
8
9
24
0.82
Difloxacin
0.999
9
10
11
13
87
8
100
7
96
6
10
15
0.90
Doxycycline hydrate
0.999
10
100
110
119
104
14
93
11
96
8
10
23
0.84
Flumequine
0.998
9
50
54
58
88
12
105
11
93
10
11
22
0.95
Josamycin
0.998
9
10
11
13
88
12
104
11
97
9
12
22
0.94
Lomefloxacin
0.999
10
10
12
13
99
13
89
11
99
8
10
22
0.80
Orbifloxacin
0.999
9
10
11
12
93
14
107
8
94
7
11
20
0.96
Oxolinic acid
0.999
11
10
12
15
110
12
102
10
102
6
15
20
0.92
Oxytetracycline
0.999
11
100
111
122
114
15
104
12
95
6
10
23
0.94
Rifampicin
0.997
10
10
11
13
98
9
85
8
98
5
9
15
0.81
Sarafloxacin
0.997
10
10
12
14
101
10
97
8
102
5
11
16
0.87
Sulfachloropyridazine
0.997
9
100
116
132
88
13
106
12
80
9
10
24
0.96
Sulfaquinoxaline
0.998
10
100
111
122
97
15
99
10
78
9
9
24
0.89
Sulfadiazine
0.997
8
100
108
116
85
12
98
9
98
5
10
18
0.88
Sulfamerazine
0.999
8
100
111
122
85
12
104
8
75
7
9
19
0.94
Sulfathiazole
0.999
9
100
113
126
85
11
109
10
81
10
17
21
0.98
Sulfamethazine
0.999
8
100
114
129
84
13
104
9
104
8
11
21
0.94
Sulfadoxine
0.999
9
100
114
129
88
16
95
12
95
6
10
24
0.86
Sulfapyridine
0.997
8
100
119
138
83
11
108
10
96
8
10
20
0.97
Sulfaclozine
0.998
10
100
111
122
99
10
92
8
98
5
9
16
0.83
Sulfamethoxazole
0.998
10
100
116
132
102
9
95
7
87
5
9
15
0.86
Tilmicosin
0.999
10
50
56
62
98
12
104
9
105
4
10
18
0.94
Tetracycline
0.999
10
100
119
138
100
18
84
8
96
5
9
23
0.82In Figure3, very satisfactory S/N ratios were obtained for all analytes at LOQ level. The lowest LOQ value was 50 μg/kg for tetracyclines and for sulfonamides 20 μg/kg in veterinary drugs in [20] while it was 10 μg/kg for both of them in our study except ciprofloxacin and quinolone. Figure 3 shows MRM chromatograms of milk samples at the lowest validation concentration at LOQ level.Figure 3
MRM chromatograms of milk samples at the LOQ level of tetracyclines, sulfadimethoxine, oxytetracycline, and sulfadiazine (10μg/kg).


#### 3.3.4. Accuracy and Precision

The accuracy was evaluated by recovery tests, analyzing fortified blank samples at the same concentration levels used in the precision tests (0.01, 0.025, and 0.05 mg/kg). The accuracy and precision of the method results (Table2) confirmed the values given in Decision 2002/657/EC [16]. Thus, the mean accuracy values obtained in the recovery tests were between 61 and 130%. The precision of the method was determined in two stages: repeatability (intraday) and intermediate precision (interday). Repeatability was expressed by the RSD of the results from six replicates analyzed on the same day by the same analyst using the same instrument. The intermediate precision was expressed by the RSD of the results of eighteen analyses performed on three different days (n
=
3), six analyses/day, by the same analyst using the same instrument. The relative standard deviation (RSD) of interday values of veterinary drugs and pesticides analyzed by the present method was 2 to 13% and for the intraday test 5–19% (Table 2), while relative standard deviation (RSDr) of intraday values was 4–26% in [20].


#### 3.3.5. Matrix Effects

Evaluation of matrix effect is important during validation of analytical methods using the LC-MS/MS technique. The ionization efficiency of the analytes in ESI source may be affected by matrix interference. In order to evaluate the degree of ion suppression or signal enhancement, calibration curves were established with and without matrix. Matrix-induced effects were assessed by comparing the slopes of these calibration curves using the following formula: matrix effect (ME) =1
-
a
m
a
t
r
i
x
/
a
s
t
a
n
d
a
r
d
×
100, where a
m
a
t
r
i
x and a
s
t
a
n
d
a
r
d are the slopes of calibration straight lines for standard and matrix-matched calibration graphs. The matrix-matched calibration curves were constructed using milk samples (5 g/mL matrix equivalent) prepared in MeOH-water solution with 0.1% acetic acid and spiked with veterinary drug and pesticides at concentration levels of 0.01, 0.025, and 0.05 mg/kg. Matrix effect was further evaluated for ion suppression between the standards prepared in pure solvent and standards prepared in matrix and the matrix effect was found to be in a range of 15–25%. These results showed that standard calibration which was simpler and less time-consuming compared with matrix-matched calibration can effectively be used for quantitation of veterinary drug and pesticides in milk (Table 2).


### 3.4. Real Samples

The method used analyzed more than 220 milk samples submitted to the laboratory for veterinary drug and pesticide residues by the local markets. Two transition ion pairs were monitored for each of the analytes and the ion ratios of detected samples were compared well with those of standards. Retention times of analytes were also confirmed by addition of known standards in detected samples. Eight samples out of 220 milk samples were found to contain residues of veterinary drug and pesticide residues (4% incidence was positive). Sulfadiazine (veterinary drug) residue amount was found between 0.075 and 0.125 mg/L in 2 samples and tetracycline (veterinary drug) amount was found to be 0.015–0.100 mg/L in 4 samples. Carbaryl (pesticide) residue concentration level was 0.005–0.025 mg/L in 2 samples.


## 3.1. Optimization of the Extraction Procedures

Ethyl acetate extraction without salt procedure was chosen to be performed in this study because of its advantages. There was no need to use salt and it could give lower detection limit in terms of volatile characteristic of ethyl acetate.Recovery values showed no difference among three different extraction procedures (acetonitrile extraction, QuEChERS extraction, and ethyl acetate extraction without salting procedure) (Figure1).Figure 1
Recovery data for three different extraction procedures.The recovery values expressed as recovery % are all within the reference range of 70–120%. Comparing three procedures, EtOAc without salt provided recoveries between 100% and 120% for a higher number of veterinary drugs and pesticides (26 veterinary drugs and 134 pesticides; total of 160 compounds) than QuEChERS (82 compounds) and ACN (100 compounds), as it can be observed in Figure2. In terms of extraction recoveries, EtOAc was found to be a suitable extraction procedure for all 26 veterinary drugs and most of the pesticides analyzed in this study. Only one analyte (propham) showed R
>
120 for EtOAc.Figure 2
Recovery (%) data obtained using extraction procedures; QuEChERS, ACN, and EtOAc without salt.Accuracy was evaluated in terms of relative standard deviation (RSD) by spiking blank samples with the corresponding volume of the multicompound working standard solution. RSD was evaluated at 50μg/kg by spiking six blank samples at each level for three procedures that provided similar RSD values. These values were within 1
<
R
S
D
<
10 for 75% of each analyte in the three procedures. These results indicated that the EtOAc without salt method was precise, accurate, and reliable for the analysis of the veterinary drug and pesticide compounds in the milk samples as an alternative method.


## 3.2. LC-MS/MS

Mobile phase was acidified with acetic acid in methanol and water. Also, study [18] in the literature was performed for the comparison. Formic acid in acetonitrile and water was used as a mobile phase in [18]. According to analyte intensities, our results gave better peak shapes than chromatograms in [18]. The dried residue was redissolved in a mixture of MeOH/water with 0.1% acetic acid to test different reconstitution solvents. This composition produced better peak shapes for all analytes compared with water-methanol (80 : 20) that gave lower response. Increasing acetic acid to 1% in the mixture did not improve chromatography but caused extra peaks in the background noise.


## 3.3. Validation Study

### 3.3.1. Selectivity

The selectivity of the method was assessed by duplicate analysis of 10 blank milk samples. No peaks of interfering compounds were observed within the intervals of the retention time of the analytes in any of these samples.


### 3.3.2. Linearity

Linearity was evaluated from the calibration curves by triplicate analyses of blank milk samples fortified with the analytes at six (0.0, 0.01, 0.025, 0.05, 0.1, and 0.2 mg/kg) concentration levels. Linearity was expressed as the coefficient of linear correlation (r) and from the slope of the calibration curve. The linearity of the analytical response across the studied range was excellent, with correlation coefficients higher than 0.997 for all analytes, which was similar to the findings in [19]. The authors [20] found correlation coefficients higher than 0.992 for all analytes, which was a lower score than ours.


### 3.3.3. Decision Limit and Detection Capability

CCα is defined as the limit at and above which it can be concluded with an error probability of α that a sample is noncompliant. CCβ is defined as the smallest content of the substance that may be detected, identified, and/or quantified in a sample with an error probability of β. The CCα and CCβ were determined by analysis of 10 blank milk samples and the signal-to-noise (S/N) ratio is calculated at the time window in which the analyte is expected. The CCα values were calculated as three times the S/N ratio. The CCβ was calculated by analyzing 10 blank samples spiked with concentration at CCα. Then the CCα value was added up to 1.64 times the corresponding standard deviation. Then, a preliminary experiment was conducted to check if all compounds were detected when spiked at their CCα level (Table 2).Table 2
Validation results of the developed method.
Compounds
r
2
LOQ
MRL
CCα
CCβ
% recovery
% RSD
% recovery
% RSD
% recovery
% RSD
% RSDr
Relative uncertainty %
Matrix effect
(µg/kg)
(µg/kg)
(µg/kg)
(µg/kg)
10 (µg/kg)
10 (µg/kg)
25 (µg/kg)
25 (µg/kg)
50 (µg/kg)
50 (µg/kg)
2,4-D (negative)
0.997
11
10
21
33
112
8
90
6
108
5
13
32
0.81
2,4,5-T
0.998
9
10
18
26
85
18
102
17
93
9
11
23
0.92
2,4-Dimethylaniline
0.998
8
10
18
26
79
11
110
8
97
8
11
35
0.99
Acetamiprid
0.997
10
10
18
26
97
9
102
8
96
7
11
33
0.92
Acrinathrin
0.998
11
10
18
26
106
10
88
8
104
7
11
35
0.81
Alachlor
0.997
11
10
20
30
108
11
86
4
92
3
13
28
0.81
Amitraz
0.997
9
10
20
30
86
8
76
4
76
3
14
28
0.81
Atrazine
0.997
11
10
17
23
109
6
94
5
91
3
9
30
0.85
Azoxystrobin
0.998
11
10
21
33
114
9
102
5
110
4
14
30
0.92
Bentazon
0.998
12
10
17
23
118
5
94
4
94
4
8
28
0.85
Bifenazate
0.998
11
10
20
30
109
10
102
7
111
6
11
33
0.92
Bitertanol
0.998
11
10
17
23
112
5
98
3
97
3
8
26
0.88
Boscalid
0.998
12
10
15
20
115
6
110
6
101
5
7
30
0.99
Bromacil
0.998
9
10
20
30
91
16
108
14
91
11
13
42
0.97
Bromuconazole
0.997
11
10
20
30
109
9
100
8
95
6
14
30
0.90
Bromoxynil
0.997
10
10
17
23
99
14
106
9
99
6
9
33
0.96
Bupirimate
0.997
12
10
20
30
116
5
100
5
104
3
12
26
0.90
Buprofezin
0.997
9
10
17
23
90
16
90
12
88
8
10
26
0.81
Butocarboxim sulfoxide
0.997
11
10
17
23
107
9
118
8
102
7
7
33
1.06
Cadusafos
0.997
11
10
15
20
110
11
94
8
95
5
6
30
0.85
Carbaryl
0.997
11
10
17
23
105
10
94
5
89
4
9
28
0.85
Carbendazim
0.997
12
10
20
30
118
5
122
5
113
4
11
30
1.10
Carbofuran
0.998
12
10
17
23
116
4
90
4
92
3
8
26
0.81
Carbosulfan
0.997
9
10
21
33
91
10
106
6
93
6
15
32
0.96
Carboxin
0.998
11
10
18
26
113
6
90
4
89
5
12
28
0.81
Clofentezine
0.998
9
10
17
23
88
13
98
10
93
8
10
26
0.88
Chloridazon
0.998
10
10
18
26
102
11
114
9
97
7
11
30
1.03
Chlorfenvinphos
0.998
11
10
18
26
112
8
96
5
100
3
10
30
0.86
Chlorfluazuron
0.998
11
10
21
33
107
10
114
5
108
6
14
30
1.03
Chloroxuron
0.997
11
10
18
26
108
13
102
5
104
5
10
36
0.92
Chlorpyrifos
0.997
11
10
17
23
108
9
102
6
107
6
7
32
0.92
Chlorsulfuron
0.997
10
10
21
33
96
10
92
5
88
5
15
30
0.83
Chlorthiamid
0.997
9
10
21
33
92
17
104
13
114
9
13
32
0.94
Cinidon-ethyl
0.997
11
10
17
23
105
7
100
6
110
5
8
32
0.90
Cyazofamid
0.997
11
10
18
26
112
7
94
7
93
4
10
28
0.85
Cyclanilide
0.997
12
10
21
33
115
5
104
5
113
5
13
30
0.94
Cycloate
0.998
11
10
17
23
106
12
96
11
88
5
9
28
0.86
Cymoxanil
0.998
11
10
17
23
109
7
112
6
103
6
8
32
1.01
Cyproconazole
0.998
10
10
18
26
96
10
92
6
98
4
12
28
0.83
Cyprodinil
0.998
9
10
20
30
91
16
98
9
110
9
11
30
0.88
Demeton-S-methyl
0.998
10
10
20
30
96
16
98
11
101
4
11
28
0.88
Demeton-S-methylsulfoxide
0.998
10
10
13
17
100
8
101
7
99
6
11
35
0.91
Desmedipham
0.998
10
10
13
17
99
8
98
8
87
7
14
0
0.88
Diallate
0.998
10
10
20
30
100
13
120
12
110
6
14
26
1.08
Diazinon
0.998
10
10
12
13
95
9
92
6
100
5
12
32
0.83
Dichlofluanid
0.998
10
10
17
23
103
9
104
8
100
8
12
26
0.94
Dichlorprop
0.998
9
10
18
26
88
5
88
4
95
3
8
0
0.82
Dichlorvos
0.998
9
10
15
20
93
14
118
11
109
11
16
35
1.06
Difenoconazole
0.998
9
10
15
20
92
12
100
9
94
4
11
28
0.90
Dimethenamid (sum)
0.998
11
10
17
23
112
13
98
9
87
10
10
28
0.94
Dimethoate
0.998
11
10
17
23
113
8
104
7
100
6
9
26
0.94
Dimethomorph
0.998
9
10
13
17
91
12
104
8
97
5
14
28
0.94
Dimoxystrobin
0.998
10
10
15
20
101
8
116
5
97
5
12
30
1.05
Diniconazole
0.998
10
10
13
17
97
19
96
15
87
9
10
36
0.86
Dinobuton
0.998
10
10
17
23
104
10
78
6
92
6
11
32
0.82
Dinocap (sum)
0.998
12
10
13
17
115
7
96
5
90
4
14
30
0.86
Dinoterb
0.997
12
10
18
26
115
7
100
4
98
4
9
28
0.90
Diphenylamine
0.999
12
10
13
17
115
7
108
5
90
4
14
30
0.97
Disulfoton-sulfoxide
0.997
11
10
13
17
109
10
86
6
94
5
13
30
0.82
Dithianon
0.997
11
10
13
17
106
6
108
5
102
3
6
26
0.97
Diuron
0.999
11
10
20
30
109
9
94
7
101
3
10
26
0.85
Epoxiconazole
0.997
11
10
12
13
105
8
98
4
93
4
11
28
0.88
EPTC
0.997
11
10
18
26
113
7
112
4
96
4
10
28
1.01
Ethiofencarb
0.997
12
10
18
26
115
7
96
5
96
3
9
26
0.86
Ethion
0.998
10
10
15
20
102
10
100
8
98
8
12
22
0.90
Ethirimol
0.998
10
10
15
20
98
15
96
9
111
8
13
33
0.86
Ethofumesate
0.998
11
10
17
23
105
11
96
10
100
7
13
28
0.86
Etoxazole
0.998
11
10
17
23
114
8
94
5
101
4
11
28
0.85
Ethoxyquin
0.998
10
10
13
17
100
13
96
11
98
4
11
30
0.86
Famoxadone
0.998
11
10
15
20
112
12
114
5
117
5
12
32
1.03
Fenamidone
0.998
11
10
13
17
108
9
84
8
91
6
10
26
0.82
Fenamiphos
0.998
11
10
17
23
107
5
106
4
109
3
5
32
0.96
Fenarimol
0.998
10
10
13
17
101
11
99
9
95
8
11
23
0.89
Fenazaquin
0.997
11
10
18
26
113
8
128
7
113
8
10
28
1.15
Fenbuconazole
0.997
9
10
13
17
86
16
94
12
97
4
8
32
0.85
Fenhexamid
0.997
11
10
18
26
114
7
80
6
84
5
13
28
0.82
Fenitrothion
0.997
10
10
17
23
99
12
98
11
94
9
11
24
0.88
Fenoxycarb
0.997
9
10
13
17
85
16
100
14
103
8
9
35
0.90
Fenpropathrin
0.997
10
10
18
26
100
12
100
10
99
9
7
25
0.90
Fenthion
0.997
10
10
23
36
95
12
112
11
108
7
12
33
1.01
Flazasulfuron
0.998
10
10
30
49
95
10
118
8
116
7
15
30
1.06
Fludioxonil
0.998
12
10
23
36
115
7
88
5
100
5
13
25
0.82
Fluazifop-P-butyl
0.998
12
10
28
46
118
8
102
6
96
5
11
32
0.92
Flufenacet
0.998
10
10
25
40
103
10
96
9
100
7
13
28
0.86
Flufenoxuron
0.998
10
10
26
43
95
8
100
8
105
4
11
32
0.90
Flurochloridone
0.998
10
10
18
26
100
10
102
9
112
6
7
28
0.92
Flurtamone
0.998
10
10
20
30
103
6
90
4
89
4
10
32
0.81
Flusilazole
0.998
10
10
23
36
104
8
92
6
99
6
11
32
0.83
Flutolanil
0.998
12
10
18
26
115
6
114
7
99
7
10
26
1.03
Foramsulfuron
0.998
11
10
25
40
105
4
86
3
99
4
14
26
0.82
Fosthiazate
0.997
8
10
20
30
82
13
94
5
94
3
13
30
0.85
Furathiocarb
0.997
12
10
21
33
115
5
96
5
90
5
12
33
0.86
Heptenophos
0.997
10
10
31
53
99
15
94
12
90
7
11
28
0.85
Hexythiazox
0.997
10
10
25
40
103
6
100
4
98
5
9
32
0.90
Imazalil
0.997
10
10
28
46
95
12
100
11
88
10
9
28
0.90
Imazamox
0.997
8
10
18
26
84
11
100
9
92
6
12
26
0.90
Imazaquin
0.997
11
10
31
53
112
9
90
5
86
3
12
0
0.81
Imazosulfuron
0.997
10
10
25
40
98
12
95
11
96
8
11
36
0.86
Imidacloprid
0.999
9
10
30
49
87
12
102
9
109
6
15
26
0.92
Indoxacarb
0.999
10
10
23
36
99
15
100
6
93
8
16
30
0.90
Ioxynil
0.999
10
10
20
30
99
4
84
4
81
5
8
30
0.81
Iprovalicarb
0.999
10
10
20
30
97
12
110
8
104
7
15
32
0.99
Isazofos
0.999
12
10
21
33
115
7
94
5
89
11
13
26
0.85
Isoproturon
0.999
12
10
20
30
115
8
90
6
97
6
11
38
0.81
Isoxaben
0.997
11
10
26
43
112
5
110
3
96
5
8
30
0.99
Lufenuron
0.997
10
10
26
43
99
4
98
4
94
4
13
30
0.88
Malathion
0.999
12
10
23
36
115
7
104
5
89
6
13
33
0.94
MCPA
0.997
9
10
25
40
93
9
102
5
103
7
11
28
0.92
Mecarbam
0.998
11
10
31
53
105
16
106
14
94
7
11
30
0.96
MCPP
0.998
10
10
28
46
103
12
84
9
97
9
12
41
0.82
Metalaxyl-M
0.998
10
10
23
36
97
7
122
4
101
5
16
26
1.10
Mepanipyrim
0.997
10
10
31
53
102
16
92
13
107
11
16
33
0.83
Mesosulfuron-methyl
0.997
8
10
28
46
82
15
104
5
93
6
12
35
0.94
Metazachlor
0.997
11
10
23
36
113
5
100
5
93
6
12
16
0.90
S-Metolachlor
0.999
9
10
18
26
93
8
92
7
97
7
11
27
0.83
Metosulam
0.999
11
10
30
49
110
9
94
8
88
8
14
32
0.85
Metribuzin
0.999
11
10
21
33
105
11
72
9
91
6
15
26
0.82
Monocrotophos
0.997
12
10
25
40
115
4
100
3
88
3
14
32
0.90
Monolinuron
0.997
10
10
28
46
104
17
92
14
90
8
13
28
0.83
Monuron
0.997
11
10
31
53
114
8
102
4
94
5
13
35
0.92
Omethoate
0.997
11
10
28
46
105
9
94
9
91
8
13
39
0.85
Oxadiargyl
0.997
11
10
23
36
105
15
110
11
90
11
14
30
0.99
Oxadiazon
0.997
10
10
30
49
96
8
112
5
112
5
6
32
1.01
Oxadixyl
0.997
11
10
21
33
109
12
90
10
88
6
8
35
0.81
Oxamyl
0.997
10
10
30
49
103
14
96
12
89
8
11
33
0.86
Oxasulfuron
0.997
11
10
33
56
108
8
86
8
87
7
12
28
0.82
Oxycarboxin
0.997
12
10
28
46
115
7
112
4
97
6
12
28
1.01
Oxyfluorfen
0.997
9
10
30
49
92
8
101
7
103
6
11
28
0.91
Penconazole
0.997
11
10
23
36
112
7
102
4
96
4
9
28
0.92
Pendimethalin
0.997
11
10
20
30
113
8
108
5
108
4
6
28
0.97
Pethoxamid
0.997
11
10
28
46
108
9
76
4
81
5
14
32
0.83
Phosalone
0.998
10
10
23
36
96
11
112
4
112
5
8
36
1.01
Phenmedipham
0.998
10
10
36
62
95
14
92
13
88
6
11
30
0.83
Phenthoate
0.997
12
10
23
36
115
7
94
5
89
6
12
28
0.85
Phosmet
0.997
10
10
21
33
100
11
94
10
92
9
13
33
0.85
Phosphamidon
0.997
10
10
21
33
99
8
108
7
95
7
10
30
0.97
Picloram
0.997
11
10
21
33
105
12
96
8
91
7
12
36
0.86
Picolinafen
0.997
10
10
25
40
100
12
114
9
104
9
12
25
1.03
Pirimicarb
0.997
12
10
23
36
119
8
94
2
97
3
10
30
0.85
Pirimiphos-methyl
0.997
12
10
23
36
115
7
108
5
94
5
12
30
0.97
Prochloraz
0.999
12
10
21
33
115
5
112
5
94
5
18
30
1.01
Profenofos
0.999
12
10
21
33
115
7
104
5
96
5
8
30
0.94
Prometryn
0.999
11
10
21
33
109
7
94
5
99
6
9
36
0.85
Propamocarb
0.998
10
10
25
40
100
13
102
10
89
9
11
30
0.92
Propanil
0.999
12
10
30
49
115
7
102
5
99
6
12
28
0.92
Propargite
0.999
10
10
26
43
102
14
112
10
113
4
8
30
1.01
Propham
0.999
10
10
21
33
102
13
100
6
96
5
10
28
0.90
Propiconazole
0.999
10
10
30
49
98
6
122
4
111
6
14
30
1.10
Propyzamide
0.999
8
10
28
46
84
10
96
9
95
5
9
33
0.86
Pymetrozine
0.997
10
10
23
36
101
10
96
8
90
7
11
28
0.86
Pyraclostrobin
0.997
12
10
21
33
115
7
130
5
119
4
15
26
1.17
Pyridaben
0.997
9
10
20
30
89
16
80
12
81
4
11
36
0.82
Pyridaphenthion
0.997
10
10
23
36
99
11
120
9
99
10
13
26
1.08
Pyridate
0.998
10
10
31
53
98
5
106
4
98
3
8
32
0.96
Pyriproxyfen
0.997
10
10
21
33
101
11
94
6
86
5
13
22
0.85
Quinalphos
0.997
10
10
23
36
101
5
76
6
90
5
14
23
0.82
Quinoxyfen
0.999
10
10
30
49
99
5
98
1
100
2
8
23
0.88
Quizalofop-P-ethyl
0.997
12
10
30
49
115
7
82
5
85
5
14
26
0.84
Rimsulfuron
0.998
9
10
26
43
88
13
104
3
100
2
14
26
0.94
Simazine
0.997
11
10
23
36
106
16
96
8
99
3
11
23
0.86
Spiroxamine
0.999
8
10
21
33
83
14
84
9
82
2
14
32
0.82
Sulfosulfuron
0.997
8
10
21
33
82
12
110
6
111
6
19
30
0.99
Tebuconazole
0.999
10
10
28
46
99
10
94
6
99
5
9
25
0.85
TEPP
0.997
9
10
23
36
90
7
107
7
105
6
11
29
0.96
Terbutryn
0.997
12
10
26
43
115
8
96
5
102
3
11
28
0.86
Terbuthylazine
0.997
10
10
20
30
104
12
118
5
116
4
6
26
1.06
Tetrachlorvinphos
0.997
11
10
21
33
113
11
78
8
90
4
14
28
0.90
Thiabendazole
0.997
12
10
21
33
118
6
98
4
96
5
13
30
0.88
Thiacloprid
0.997
11
10
18
26
106
13
84
12
89
5
8
35
0.82
Thiamethoxam
0.997
10
10
33
56
104
19
118
11
113
8
11
32
1.06
Thifensulfuron-methyl
0.997
9
10
20
30
91
16
98
8
92
9
15
30
0.88
Thiodicarb
0.997
12
10
31
53
115
7
102
5
97
6
9
35
0.92
Thiophanate-methyl
0.997
11
10
20
30
114
9
112
6
95
5
13
32
1.01
Triadimefon
0.997
10
10
28
46
104
7
106
6
98
5
8
32
0.96
Triadimenol
0.997
11
10
25
40
107
14
106
6
92
7
9
26
0.96
Triallate
0.998
11
10
23
36
106
9
114
3
111
4
12
30
1.03
Triasulfuron
0.998
11
10
20
30
111
8
76
5
91
6
14
30
0.82
Triazophos
0.998
12
10
30
49
115
7
96
5
87
5
9
30
0.86
Tribenuron-methyl
0.998
10
10
35
59
99
9
103
8
101
5
11
30
0.93
Tributylphosphate
0.997
10
10
17
23
104
14
88
12
89
5
8
35
0.81
Trichlorfon
0.998
10
10
25
40
98
10
104
3
100
2
14
27
0.94
Tridemorph
0.997
10
10
21
33
98
17
90
13
100
5
11
30
0.81
Trifloxystrobin
0.997
12
10
25
40
115
7
124
5
114
5
14
30
1.12
Triflumizole
0.997
11
10
20
30
113
14
92
5
99
5
8
28
0.83
Triticonazole
0.998
10
10
17
23
100
11
106
10
98
7
7
32
0.96
Vamidothion
0.997
9
10
21
33
93
13
118
6
102
7
16
28
1.06
Zoxamide
0.997
12
10
23
36
116
7
72
4
85
3
15
25
0.82
Ciprofloxacin
0.997
7
100
124
148
74
25
61
16
97
13
9
38
0.81
Clindamycin
0.997
11
10
12
14
110
23
106
15
109
10
12
34
0.96
Chlortetracycline
0.999
10
100
108
116
101
7
93
7
99
6
9
14
0.84
Danofloxacin
0.999
10
30
40
48
104
14
87
12
88
8
9
24
0.82
Difloxacin
0.999
9
10
11
13
87
8
100
7
96
6
10
15
0.90
Doxycycline hydrate
0.999
10
100
110
119
104
14
93
11
96
8
10
23
0.84
Flumequine
0.998
9
50
54
58
88
12
105
11
93
10
11
22
0.95
Josamycin
0.998
9
10
11
13
88
12
104
11
97
9
12
22
0.94
Lomefloxacin
0.999
10
10
12
13
99
13
89
11
99
8
10
22
0.80
Orbifloxacin
0.999
9
10
11
12
93
14
107
8
94
7
11
20
0.96
Oxolinic acid
0.999
11
10
12
15
110
12
102
10
102
6
15
20
0.92
Oxytetracycline
0.999
11
100
111
122
114
15
104
12
95
6
10
23
0.94
Rifampicin
0.997
10
10
11
13
98
9
85
8
98
5
9
15
0.81
Sarafloxacin
0.997
10
10
12
14
101
10
97
8
102
5
11
16
0.87
Sulfachloropyridazine
0.997
9
100
116
132
88
13
106
12
80
9
10
24
0.96
Sulfaquinoxaline
0.998
10
100
111
122
97
15
99
10
78
9
9
24
0.89
Sulfadiazine
0.997
8
100
108
116
85
12
98
9
98
5
10
18
0.88
Sulfamerazine
0.999
8
100
111
122
85
12
104
8
75
7
9
19
0.94
Sulfathiazole
0.999
9
100
113
126
85
11
109
10
81
10
17
21
0.98
Sulfamethazine
0.999
8
100
114
129
84
13
104
9
104
8
11
21
0.94
Sulfadoxine
0.999
9
100
114
129
88
16
95
12
95
6
10
24
0.86
Sulfapyridine
0.997
8
100
119
138
83
11
108
10
96
8
10
20
0.97
Sulfaclozine
0.998
10
100
111
122
99
10
92
8
98
5
9
16
0.83
Sulfamethoxazole
0.998
10
100
116
132
102
9
95
7
87
5
9
15
0.86
Tilmicosin
0.999
10
50
56
62
98
12
104
9
105
4
10
18
0.94
Tetracycline
0.999
10
100
119
138
100
18
84
8
96
5
9
23
0.82In Figure3, very satisfactory S/N ratios were obtained for all analytes at LOQ level. The lowest LOQ value was 50 μg/kg for tetracyclines and for sulfonamides 20 μg/kg in veterinary drugs in [20] while it was 10 μg/kg for both of them in our study except ciprofloxacin and quinolone. Figure 3 shows MRM chromatograms of milk samples at the lowest validation concentration at LOQ level.Figure 3
MRM chromatograms of milk samples at the LOQ level of tetracyclines, sulfadimethoxine, oxytetracycline, and sulfadiazine (10μg/kg).


### 3.3.4. Accuracy and Precision

The accuracy was evaluated by recovery tests, analyzing fortified blank samples at the same concentration levels used in the precision tests (0.01, 0.025, and 0.05 mg/kg). The accuracy and precision of the method results (Table2) confirmed the values given in Decision 2002/657/EC [16]. Thus, the mean accuracy values obtained in the recovery tests were between 61 and 130%. The precision of the method was determined in two stages: repeatability (intraday) and intermediate precision (interday). Repeatability was expressed by the RSD of the results from six replicates analyzed on the same day by the same analyst using the same instrument. The intermediate precision was expressed by the RSD of the results of eighteen analyses performed on three different days (n
=
3), six analyses/day, by the same analyst using the same instrument. The relative standard deviation (RSD) of interday values of veterinary drugs and pesticides analyzed by the present method was 2 to 13% and for the intraday test 5–19% (Table 2), while relative standard deviation (RSDr) of intraday values was 4–26% in [20].


### 3.3.5. Matrix Effects

Evaluation of matrix effect is important during validation of analytical methods using the LC-MS/MS technique. The ionization efficiency of the analytes in ESI source may be affected by matrix interference. In order to evaluate the degree of ion suppression or signal enhancement, calibration curves were established with and without matrix. Matrix-induced effects were assessed by comparing the slopes of these calibration curves using the following formula: matrix effect (ME) =1
-
a
m
a
t
r
i
x
/
a
s
t
a
n
d
a
r
d
×
100, where a
m
a
t
r
i
x and a
s
t
a
n
d
a
r
d are the slopes of calibration straight lines for standard and matrix-matched calibration graphs. The matrix-matched calibration curves were constructed using milk samples (5 g/mL matrix equivalent) prepared in MeOH-water solution with 0.1% acetic acid and spiked with veterinary drug and pesticides at concentration levels of 0.01, 0.025, and 0.05 mg/kg. Matrix effect was further evaluated for ion suppression between the standards prepared in pure solvent and standards prepared in matrix and the matrix effect was found to be in a range of 15–25%. These results showed that standard calibration which was simpler and less time-consuming compared with matrix-matched calibration can effectively be used for quantitation of veterinary drug and pesticides in milk (Table 2).


## 3.3.1. Selectivity

The selectivity of the method was assessed by duplicate analysis of 10 blank milk samples. No peaks of interfering compounds were observed within the intervals of the retention time of the analytes in any of these samples.


## 3.3.2. Linearity

Linearity was evaluated from the calibration curves by triplicate analyses of blank milk samples fortified with the analytes at six (0.0, 0.01, 0.025, 0.05, 0.1, and 0.2 mg/kg) concentration levels. Linearity was expressed as the coefficient of linear correlation (r) and from the slope of the calibration curve. The linearity of the analytical response across the studied range was excellent, with correlation coefficients higher than 0.997 for all analytes, which was similar to the findings in [19]. The authors [20] found correlation coefficients higher than 0.992 for all analytes, which was a lower score than ours.


## 3.3.3. Decision Limit and Detection Capability

CCα is defined as the limit at and above which it can be concluded with an error probability of α that a sample is noncompliant. CCβ is defined as the smallest content of the substance that may be detected, identified, and/or quantified in a sample with an error probability of β. The CCα and CCβ were determined by analysis of 10 blank milk samples and the signal-to-noise (S/N) ratio is calculated at the time window in which the analyte is expected. The CCα values were calculated as three times the S/N ratio. The CCβ was calculated by analyzing 10 blank samples spiked with concentration at CCα. Then the CCα value was added up to 1.64 times the corresponding standard deviation. Then, a preliminary experiment was conducted to check if all compounds were detected when spiked at their CCα level (Table 2).Table 2
Validation results of the developed method.
Compounds
r
2
LOQ
MRL
CCα
CCβ
% recovery
% RSD
% recovery
% RSD
% recovery
% RSD
% RSDr
Relative uncertainty %
Matrix effect
(µg/kg)
(µg/kg)
(µg/kg)
(µg/kg)
10 (µg/kg)
10 (µg/kg)
25 (µg/kg)
25 (µg/kg)
50 (µg/kg)
50 (µg/kg)
2,4-D (negative)
0.997
11
10
21
33
112
8
90
6
108
5
13
32
0.81
2,4,5-T
0.998
9
10
18
26
85
18
102
17
93
9
11
23
0.92
2,4-Dimethylaniline
0.998
8
10
18
26
79
11
110
8
97
8
11
35
0.99
Acetamiprid
0.997
10
10
18
26
97
9
102
8
96
7
11
33
0.92
Acrinathrin
0.998
11
10
18
26
106
10
88
8
104
7
11
35
0.81
Alachlor
0.997
11
10
20
30
108
11
86
4
92
3
13
28
0.81
Amitraz
0.997
9
10
20
30
86
8
76
4
76
3
14
28
0.81
Atrazine
0.997
11
10
17
23
109
6
94
5
91
3
9
30
0.85
Azoxystrobin
0.998
11
10
21
33
114
9
102
5
110
4
14
30
0.92
Bentazon
0.998
12
10
17
23
118
5
94
4
94
4
8
28
0.85
Bifenazate
0.998
11
10
20
30
109
10
102
7
111
6
11
33
0.92
Bitertanol
0.998
11
10
17
23
112
5
98
3
97
3
8
26
0.88
Boscalid
0.998
12
10
15
20
115
6
110
6
101
5
7
30
0.99
Bromacil
0.998
9
10
20
30
91
16
108
14
91
11
13
42
0.97
Bromuconazole
0.997
11
10
20
30
109
9
100
8
95
6
14
30
0.90
Bromoxynil
0.997
10
10
17
23
99
14
106
9
99
6
9
33
0.96
Bupirimate
0.997
12
10
20
30
116
5
100
5
104
3
12
26
0.90
Buprofezin
0.997
9
10
17
23
90
16
90
12
88
8
10
26
0.81
Butocarboxim sulfoxide
0.997
11
10
17
23
107
9
118
8
102
7
7
33
1.06
Cadusafos
0.997
11
10
15
20
110
11
94
8
95
5
6
30
0.85
Carbaryl
0.997
11
10
17
23
105
10
94
5
89
4
9
28
0.85
Carbendazim
0.997
12
10
20
30
118
5
122
5
113
4
11
30
1.10
Carbofuran
0.998
12
10
17
23
116
4
90
4
92
3
8
26
0.81
Carbosulfan
0.997
9
10
21
33
91
10
106
6
93
6
15
32
0.96
Carboxin
0.998
11
10
18
26
113
6
90
4
89
5
12
28
0.81
Clofentezine
0.998
9
10
17
23
88
13
98
10
93
8
10
26
0.88
Chloridazon
0.998
10
10
18
26
102
11
114
9
97
7
11
30
1.03
Chlorfenvinphos
0.998
11
10
18
26
112
8
96
5
100
3
10
30
0.86
Chlorfluazuron
0.998
11
10
21
33
107
10
114
5
108
6
14
30
1.03
Chloroxuron
0.997
11
10
18
26
108
13
102
5
104
5
10
36
0.92
Chlorpyrifos
0.997
11
10
17
23
108
9
102
6
107
6
7
32
0.92
Chlorsulfuron
0.997
10
10
21
33
96
10
92
5
88
5
15
30
0.83
Chlorthiamid
0.997
9
10
21
33
92
17
104
13
114
9
13
32
0.94
Cinidon-ethyl
0.997
11
10
17
23
105
7
100
6
110
5
8
32
0.90
Cyazofamid
0.997
11
10
18
26
112
7
94
7
93
4
10
28
0.85
Cyclanilide
0.997
12
10
21
33
115
5
104
5
113
5
13
30
0.94
Cycloate
0.998
11
10
17
23
106
12
96
11
88
5
9
28
0.86
Cymoxanil
0.998
11
10
17
23
109
7
112
6
103
6
8
32
1.01
Cyproconazole
0.998
10
10
18
26
96
10
92
6
98
4
12
28
0.83
Cyprodinil
0.998
9
10
20
30
91
16
98
9
110
9
11
30
0.88
Demeton-S-methyl
0.998
10
10
20
30
96
16
98
11
101
4
11
28
0.88
Demeton-S-methylsulfoxide
0.998
10
10
13
17
100
8
101
7
99
6
11
35
0.91
Desmedipham
0.998
10
10
13
17
99
8
98
8
87
7
14
0
0.88
Diallate
0.998
10
10
20
30
100
13
120
12
110
6
14
26
1.08
Diazinon
0.998
10
10
12
13
95
9
92
6
100
5
12
32
0.83
Dichlofluanid
0.998
10
10
17
23
103
9
104
8
100
8
12
26
0.94
Dichlorprop
0.998
9
10
18
26
88
5
88
4
95
3
8
0
0.82
Dichlorvos
0.998
9
10
15
20
93
14
118
11
109
11
16
35
1.06
Difenoconazole
0.998
9
10
15
20
92
12
100
9
94
4
11
28
0.90
Dimethenamid (sum)
0.998
11
10
17
23
112
13
98
9
87
10
10
28
0.94
Dimethoate
0.998
11
10
17
23
113
8
104
7
100
6
9
26
0.94
Dimethomorph
0.998
9
10
13
17
91
12
104
8
97
5
14
28
0.94
Dimoxystrobin
0.998
10
10
15
20
101
8
116
5
97
5
12
30
1.05
Diniconazole
0.998
10
10
13
17
97
19
96
15
87
9
10
36
0.86
Dinobuton
0.998
10
10
17
23
104
10
78
6
92
6
11
32
0.82
Dinocap (sum)
0.998
12
10
13
17
115
7
96
5
90
4
14
30
0.86
Dinoterb
0.997
12
10
18
26
115
7
100
4
98
4
9
28
0.90
Diphenylamine
0.999
12
10
13
17
115
7
108
5
90
4
14
30
0.97
Disulfoton-sulfoxide
0.997
11
10
13
17
109
10
86
6
94
5
13
30
0.82
Dithianon
0.997
11
10
13
17
106
6
108
5
102
3
6
26
0.97
Diuron
0.999
11
10
20
30
109
9
94
7
101
3
10
26
0.85
Epoxiconazole
0.997
11
10
12
13
105
8
98
4
93
4
11
28
0.88
EPTC
0.997
11
10
18
26
113
7
112
4
96
4
10
28
1.01
Ethiofencarb
0.997
12
10
18
26
115
7
96
5
96
3
9
26
0.86
Ethion
0.998
10
10
15
20
102
10
100
8
98
8
12
22
0.90
Ethirimol
0.998
10
10
15
20
98
15
96
9
111
8
13
33
0.86
Ethofumesate
0.998
11
10
17
23
105
11
96
10
100
7
13
28
0.86
Etoxazole
0.998
11
10
17
23
114
8
94
5
101
4
11
28
0.85
Ethoxyquin
0.998
10
10
13
17
100
13
96
11
98
4
11
30
0.86
Famoxadone
0.998
11
10
15
20
112
12
114
5
117
5
12
32
1.03
Fenamidone
0.998
11
10
13
17
108
9
84
8
91
6
10
26
0.82
Fenamiphos
0.998
11
10
17
23
107
5
106
4
109
3
5
32
0.96
Fenarimol
0.998
10
10
13
17
101
11
99
9
95
8
11
23
0.89
Fenazaquin
0.997
11
10
18
26
113
8
128
7
113
8
10
28
1.15
Fenbuconazole
0.997
9
10
13
17
86
16
94
12
97
4
8
32
0.85
Fenhexamid
0.997
11
10
18
26
114
7
80
6
84
5
13
28
0.82
Fenitrothion
0.997
10
10
17
23
99
12
98
11
94
9
11
24
0.88
Fenoxycarb
0.997
9
10
13
17
85
16
100
14
103
8
9
35
0.90
Fenpropathrin
0.997
10
10
18
26
100
12
100
10
99
9
7
25
0.90
Fenthion
0.997
10
10
23
36
95
12
112
11
108
7
12
33
1.01
Flazasulfuron
0.998
10
10
30
49
95
10
118
8
116
7
15
30
1.06
Fludioxonil
0.998
12
10
23
36
115
7
88
5
100
5
13
25
0.82
Fluazifop-P-butyl
0.998
12
10
28
46
118
8
102
6
96
5
11
32
0.92
Flufenacet
0.998
10
10
25
40
103
10
96
9
100
7
13
28
0.86
Flufenoxuron
0.998
10
10
26
43
95
8
100
8
105
4
11
32
0.90
Flurochloridone
0.998
10
10
18
26
100
10
102
9
112
6
7
28
0.92
Flurtamone
0.998
10
10
20
30
103
6
90
4
89
4
10
32
0.81
Flusilazole
0.998
10
10
23
36
104
8
92
6
99
6
11
32
0.83
Flutolanil
0.998
12
10
18
26
115
6
114
7
99
7
10
26
1.03
Foramsulfuron
0.998
11
10
25
40
105
4
86
3
99
4
14
26
0.82
Fosthiazate
0.997
8
10
20
30
82
13
94
5
94
3
13
30
0.85
Furathiocarb
0.997
12
10
21
33
115
5
96
5
90
5
12
33
0.86
Heptenophos
0.997
10
10
31
53
99
15
94
12
90
7
11
28
0.85
Hexythiazox
0.997
10
10
25
40
103
6
100
4
98
5
9
32
0.90
Imazalil
0.997
10
10
28
46
95
12
100
11
88
10
9
28
0.90
Imazamox
0.997
8
10
18
26
84
11
100
9
92
6
12
26
0.90
Imazaquin
0.997
11
10
31
53
112
9
90
5
86
3
12
0
0.81
Imazosulfuron
0.997
10
10
25
40
98
12
95
11
96
8
11
36
0.86
Imidacloprid
0.999
9
10
30
49
87
12
102
9
109
6
15
26
0.92
Indoxacarb
0.999
10
10
23
36
99
15
100
6
93
8
16
30
0.90
Ioxynil
0.999
10
10
20
30
99
4
84
4
81
5
8
30
0.81
Iprovalicarb
0.999
10
10
20
30
97
12
110
8
104
7
15
32
0.99
Isazofos
0.999
12
10
21
33
115
7
94
5
89
11
13
26
0.85
Isoproturon
0.999
12
10
20
30
115
8
90
6
97
6
11
38
0.81
Isoxaben
0.997
11
10
26
43
112
5
110
3
96
5
8
30
0.99
Lufenuron
0.997
10
10
26
43
99
4
98
4
94
4
13
30
0.88
Malathion
0.999
12
10
23
36
115
7
104
5
89
6
13
33
0.94
MCPA
0.997
9
10
25
40
93
9
102
5
103
7
11
28
0.92
Mecarbam
0.998
11
10
31
53
105
16
106
14
94
7
11
30
0.96
MCPP
0.998
10
10
28
46
103
12
84
9
97
9
12
41
0.82
Metalaxyl-M
0.998
10
10
23
36
97
7
122
4
101
5
16
26
1.10
Mepanipyrim
0.997
10
10
31
53
102
16
92
13
107
11
16
33
0.83
Mesosulfuron-methyl
0.997
8
10
28
46
82
15
104
5
93
6
12
35
0.94
Metazachlor
0.997
11
10
23
36
113
5
100
5
93
6
12
16
0.90
S-Metolachlor
0.999
9
10
18
26
93
8
92
7
97
7
11
27
0.83
Metosulam
0.999
11
10
30
49
110
9
94
8
88
8
14
32
0.85
Metribuzin
0.999
11
10
21
33
105
11
72
9
91
6
15
26
0.82
Monocrotophos
0.997
12
10
25
40
115
4
100
3
88
3
14
32
0.90
Monolinuron
0.997
10
10
28
46
104
17
92
14
90
8
13
28
0.83
Monuron
0.997
11
10
31
53
114
8
102
4
94
5
13
35
0.92
Omethoate
0.997
11
10
28
46
105
9
94
9
91
8
13
39
0.85
Oxadiargyl
0.997
11
10
23
36
105
15
110
11
90
11
14
30
0.99
Oxadiazon
0.997
10
10
30
49
96
8
112
5
112
5
6
32
1.01
Oxadixyl
0.997
11
10
21
33
109
12
90
10
88
6
8
35
0.81
Oxamyl
0.997
10
10
30
49
103
14
96
12
89
8
11
33
0.86
Oxasulfuron
0.997
11
10
33
56
108
8
86
8
87
7
12
28
0.82
Oxycarboxin
0.997
12
10
28
46
115
7
112
4
97
6
12
28
1.01
Oxyfluorfen
0.997
9
10
30
49
92
8
101
7
103
6
11
28
0.91
Penconazole
0.997
11
10
23
36
112
7
102
4
96
4
9
28
0.92
Pendimethalin
0.997
11
10
20
30
113
8
108
5
108
4
6
28
0.97
Pethoxamid
0.997
11
10
28
46
108
9
76
4
81
5
14
32
0.83
Phosalone
0.998
10
10
23
36
96
11
112
4
112
5
8
36
1.01
Phenmedipham
0.998
10
10
36
62
95
14
92
13
88
6
11
30
0.83
Phenthoate
0.997
12
10
23
36
115
7
94
5
89
6
12
28
0.85
Phosmet
0.997
10
10
21
33
100
11
94
10
92
9
13
33
0.85
Phosphamidon
0.997
10
10
21
33
99
8
108
7
95
7
10
30
0.97
Picloram
0.997
11
10
21
33
105
12
96
8
91
7
12
36
0.86
Picolinafen
0.997
10
10
25
40
100
12
114
9
104
9
12
25
1.03
Pirimicarb
0.997
12
10
23
36
119
8
94
2
97
3
10
30
0.85
Pirimiphos-methyl
0.997
12
10
23
36
115
7
108
5
94
5
12
30
0.97
Prochloraz
0.999
12
10
21
33
115
5
112
5
94
5
18
30
1.01
Profenofos
0.999
12
10
21
33
115
7
104
5
96
5
8
30
0.94
Prometryn
0.999
11
10
21
33
109
7
94
5
99
6
9
36
0.85
Propamocarb
0.998
10
10
25
40
100
13
102
10
89
9
11
30
0.92
Propanil
0.999
12
10
30
49
115
7
102
5
99
6
12
28
0.92
Propargite
0.999
10
10
26
43
102
14
112
10
113
4
8
30
1.01
Propham
0.999
10
10
21
33
102
13
100
6
96
5
10
28
0.90
Propiconazole
0.999
10
10
30
49
98
6
122
4
111
6
14
30
1.10
Propyzamide
0.999
8
10
28
46
84
10
96
9
95
5
9
33
0.86
Pymetrozine
0.997
10
10
23
36
101
10
96
8
90
7
11
28
0.86
Pyraclostrobin
0.997
12
10
21
33
115
7
130
5
119
4
15
26
1.17
Pyridaben
0.997
9
10
20
30
89
16
80
12
81
4
11
36
0.82
Pyridaphenthion
0.997
10
10
23
36
99
11
120
9
99
10
13
26
1.08
Pyridate
0.998
10
10
31
53
98
5
106
4
98
3
8
32
0.96
Pyriproxyfen
0.997
10
10
21
33
101
11
94
6
86
5
13
22
0.85
Quinalphos
0.997
10
10
23
36
101
5
76
6
90
5
14
23
0.82
Quinoxyfen
0.999
10
10
30
49
99
5
98
1
100
2
8
23
0.88
Quizalofop-P-ethyl
0.997
12
10
30
49
115
7
82
5
85
5
14
26
0.84
Rimsulfuron
0.998
9
10
26
43
88
13
104
3
100
2
14
26
0.94
Simazine
0.997
11
10
23
36
106
16
96
8
99
3
11
23
0.86
Spiroxamine
0.999
8
10
21
33
83
14
84
9
82
2
14
32
0.82
Sulfosulfuron
0.997
8
10
21
33
82
12
110
6
111
6
19
30
0.99
Tebuconazole
0.999
10
10
28
46
99
10
94
6
99
5
9
25
0.85
TEPP
0.997
9
10
23
36
90
7
107
7
105
6
11
29
0.96
Terbutryn
0.997
12
10
26
43
115
8
96
5
102
3
11
28
0.86
Terbuthylazine
0.997
10
10
20
30
104
12
118
5
116
4
6
26
1.06
Tetrachlorvinphos
0.997
11
10
21
33
113
11
78
8
90
4
14
28
0.90
Thiabendazole
0.997
12
10
21
33
118
6
98
4
96
5
13
30
0.88
Thiacloprid
0.997
11
10
18
26
106
13
84
12
89
5
8
35
0.82
Thiamethoxam
0.997
10
10
33
56
104
19
118
11
113
8
11
32
1.06
Thifensulfuron-methyl
0.997
9
10
20
30
91
16
98
8
92
9
15
30
0.88
Thiodicarb
0.997
12
10
31
53
115
7
102
5
97
6
9
35
0.92
Thiophanate-methyl
0.997
11
10
20
30
114
9
112
6
95
5
13
32
1.01
Triadimefon
0.997
10
10
28
46
104
7
106
6
98
5
8
32
0.96
Triadimenol
0.997
11
10
25
40
107
14
106
6
92
7
9
26
0.96
Triallate
0.998
11
10
23
36
106
9
114
3
111
4
12
30
1.03
Triasulfuron
0.998
11
10
20
30
111
8
76
5
91
6
14
30
0.82
Triazophos
0.998
12
10
30
49
115
7
96
5
87
5
9
30
0.86
Tribenuron-methyl
0.998
10
10
35
59
99
9
103
8
101
5
11
30
0.93
Tributylphosphate
0.997
10
10
17
23
104
14
88
12
89
5
8
35
0.81
Trichlorfon
0.998
10
10
25
40
98
10
104
3
100
2
14
27
0.94
Tridemorph
0.997
10
10
21
33
98
17
90
13
100
5
11
30
0.81
Trifloxystrobin
0.997
12
10
25
40
115
7
124
5
114
5
14
30
1.12
Triflumizole
0.997
11
10
20
30
113
14
92
5
99
5
8
28
0.83
Triticonazole
0.998
10
10
17
23
100
11
106
10
98
7
7
32
0.96
Vamidothion
0.997
9
10
21
33
93
13
118
6
102
7
16
28
1.06
Zoxamide
0.997
12
10
23
36
116
7
72
4
85
3
15
25
0.82
Ciprofloxacin
0.997
7
100
124
148
74
25
61
16
97
13
9
38
0.81
Clindamycin
0.997
11
10
12
14
110
23
106
15
109
10
12
34
0.96
Chlortetracycline
0.999
10
100
108
116
101
7
93
7
99
6
9
14
0.84
Danofloxacin
0.999
10
30
40
48
104
14
87
12
88
8
9
24
0.82
Difloxacin
0.999
9
10
11
13
87
8
100
7
96
6
10
15
0.90
Doxycycline hydrate
0.999
10
100
110
119
104
14
93
11
96
8
10
23
0.84
Flumequine
0.998
9
50
54
58
88
12
105
11
93
10
11
22
0.95
Josamycin
0.998
9
10
11
13
88
12
104
11
97
9
12
22
0.94
Lomefloxacin
0.999
10
10
12
13
99
13
89
11
99
8
10
22
0.80
Orbifloxacin
0.999
9
10
11
12
93
14
107
8
94
7
11
20
0.96
Oxolinic acid
0.999
11
10
12
15
110
12
102
10
102
6
15
20
0.92
Oxytetracycline
0.999
11
100
111
122
114
15
104
12
95
6
10
23
0.94
Rifampicin
0.997
10
10
11
13
98
9
85
8
98
5
9
15
0.81
Sarafloxacin
0.997
10
10
12
14
101
10
97
8
102
5
11
16
0.87
Sulfachloropyridazine
0.997
9
100
116
132
88
13
106
12
80
9
10
24
0.96
Sulfaquinoxaline
0.998
10
100
111
122
97
15
99
10
78
9
9
24
0.89
Sulfadiazine
0.997
8
100
108
116
85
12
98
9
98
5
10
18
0.88
Sulfamerazine
0.999
8
100
111
122
85
12
104
8
75
7
9
19
0.94
Sulfathiazole
0.999
9
100
113
126
85
11
109
10
81
10
17
21
0.98
Sulfamethazine
0.999
8
100
114
129
84
13
104
9
104
8
11
21
0.94
Sulfadoxine
0.999
9
100
114
129
88
16
95
12
95
6
10
24
0.86
Sulfapyridine
0.997
8
100
119
138
83
11
108
10
96
8
10
20
0.97
Sulfaclozine
0.998
10
100
111
122
99
10
92
8
98
5
9
16
0.83
Sulfamethoxazole
0.998
10
100
116
132
102
9
95
7
87
5
9
15
0.86
Tilmicosin
0.999
10
50
56
62
98
12
104
9
105
4
10
18
0.94
Tetracycline
0.999
10
100
119
138
100
18
84
8
96
5
9
23
0.82In Figure3, very satisfactory S/N ratios were obtained for all analytes at LOQ level. The lowest LOQ value was 50 μg/kg for tetracyclines and for sulfonamides 20 μg/kg in veterinary drugs in [20] while it was 10 μg/kg for both of them in our study except ciprofloxacin and quinolone. Figure 3 shows MRM chromatograms of milk samples at the lowest validation concentration at LOQ level.Figure 3
MRM chromatograms of milk samples at the LOQ level of tetracyclines, sulfadimethoxine, oxytetracycline, and sulfadiazine (10μg/kg).


## 3.3.4. Accuracy and Precision

The accuracy was evaluated by recovery tests, analyzing fortified blank samples at the same concentration levels used in the precision tests (0.01, 0.025, and 0.05 mg/kg). The accuracy and precision of the method results (Table2) confirmed the values given in Decision 2002/657/EC [16]. Thus, the mean accuracy values obtained in the recovery tests were between 61 and 130%. The precision of the method was determined in two stages: repeatability (intraday) and intermediate precision (interday). Repeatability was expressed by the RSD of the results from six replicates analyzed on the same day by the same analyst using the same instrument. The intermediate precision was expressed by the RSD of the results of eighteen analyses performed on three different days (n
=
3), six analyses/day, by the same analyst using the same instrument. The relative standard deviation (RSD) of interday values of veterinary drugs and pesticides analyzed by the present method was 2 to 13% and for the intraday test 5–19% (Table 2), while relative standard deviation (RSDr) of intraday values was 4–26% in [20].


## 3.3.5. Matrix Effects

Evaluation of matrix effect is important during validation of analytical methods using the LC-MS/MS technique. The ionization efficiency of the analytes in ESI source may be affected by matrix interference. In order to evaluate the degree of ion suppression or signal enhancement, calibration curves were established with and without matrix. Matrix-induced effects were assessed by comparing the slopes of these calibration curves using the following formula: matrix effect (ME) =1
-
a
m
a
t
r
i
x
/
a
s
t
a
n
d
a
r
d
×
100, where a
m
a
t
r
i
x and a
s
t
a
n
d
a
r
d are the slopes of calibration straight lines for standard and matrix-matched calibration graphs. The matrix-matched calibration curves were constructed using milk samples (5 g/mL matrix equivalent) prepared in MeOH-water solution with 0.1% acetic acid and spiked with veterinary drug and pesticides at concentration levels of 0.01, 0.025, and 0.05 mg/kg. Matrix effect was further evaluated for ion suppression between the standards prepared in pure solvent and standards prepared in matrix and the matrix effect was found to be in a range of 15–25%. These results showed that standard calibration which was simpler and less time-consuming compared with matrix-matched calibration can effectively be used for quantitation of veterinary drug and pesticides in milk (Table 2).


## 3.4. Real Samples

The method used analyzed more than 220 milk samples submitted to the laboratory for veterinary drug and pesticide residues by the local markets. Two transition ion pairs were monitored for each of the analytes and the ion ratios of detected samples were compared well with those of standards. Retention times of analytes were also confirmed by addition of known standards in detected samples. Eight samples out of 220 milk samples were found to contain residues of veterinary drug and pesticide residues (4% incidence was positive). Sulfadiazine (veterinary drug) residue amount was found between 0.075 and 0.125 mg/L in 2 samples and tetracycline (veterinary drug) amount was found to be 0.015–0.100 mg/L in 4 samples. Carbaryl (pesticide) residue concentration level was 0.005–0.025 mg/L in 2 samples.


## 4. Conclusions

A multiclass/multiresidue procedure with LC-MS/MS detection has been developed and validated to determine and quantify veterinary and pesticide residues in milk. A simple sample preparation method involved liquid extraction salting out procedures in ethyl acetate system, without cleanup steps, and shortening the sample preparation time. Validation of the method was performed according to Commission Decision 2002/657/EC. The method was characterized by good results in terms of recovery, reproducibility, and repeatability allowing the detection of veterinary drug and pesticide residues below the recommended analytical level. Based on these results, LC-MS/MS method with ethyl acetate extraction showed the suitability for sensitive quantification of veterinary and pesticide residues in milk samples for food safety applications. The validated method was applied on 220 real commercial samples. This short protocol can be applicable to a large number of samples for routine analysis and rapid detection.



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*Source: 2170165-2016-05-16.xml*