Research Article Analysis of Veterinary Drug and Pesticide ...downloads.hindawi.com/journals/jamc/2016/2170165.pdf · as pesticides and mycotoxins [], and some antihelminthic drugs

Research ArticleAnalysis of Veterinary Drug and Pesticide Residues Usingthe Ethyl Acetate Multiclass/Multiresidue Method in Milk byLiquid Chromatography-Tandem Mass Spectrometry

Husniye Imamoglu1 and Elmas Oktem Olgun2

1 Istanbul Sabahattin Zaim University, Halkalı, 34303 Istanbul, Turkey2TUBITAK Marmara Research Centre, Food Institute, P.O. Box 21, Gebze, 41470 Kocaeli, Turkey

Correspondence should be addressed to Husniye Imamoglu; [email protected]

Received 11 February 2016; Accepted 12 April 2016

Academic Editor: Jose M. M. Lopez

Copyright © 2016 H. Imamoglu and E. Oktem Olgun. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

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

1. Introduction

Veterinary drugs are widely used in medical and veterinarypractices to treat and prevent disease as well as improve feedefficiency and increase animal growth rates [1]. Pesticides arealso widely used to enhance food production by protectingfood crops frompotentially harmful and destructive pests [2].However, the resulting occurrence of contaminants and/orresidues in the human diet represents an issue of highconcern.

According to the European Union, the maximum residuelimit (MRL) in dairy milk is 100𝜇g/kg for tetracycline andsulfenamide, 50𝜇g/kg for macrolides and quinolones, and10 𝜇g/kg for pesticides. Sensitive analytic methods have beendeveloped to monitor and detect the MRL values in thedairy milk [3]. There are ultra-high pressure liquid chro-matography mass spectrometry (UHPLC-MS/MS) methods

reported to detectmultiple residues of𝛽-lactams [4, 5], aswellas pesticides and mycotoxins [6], and some antihelminthicdrugs and phenylbutazone [7].

Milk is a complex food that is high in fat and protein,and such ingredients may cause interactions in the ana-lytical processes. Therefore, sample preparation is required,particularly in extraction and cleanup. Formerly, samplepreparation methods were based on a few compounds ora single class of such drugs. Applying common extractionprocedures and developing chromatographic conditions aredifficult in multiclass and multiresidue analyses. Solid phaseextraction methods have been applied, after the phases ofprotein precipitation and centrifugation, in order to observethe fluoroquinolones [8], veterinary drugs [9], mycotoxins,and pesticides in milk [6, 10]. However, these methods aregenerally found to be time-consuming and require largevolumes of organic solvents.

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2016, Article ID 2170165, 17 pageshttp://dx.doi.org/10.1155/2016/2170165

2 Journal of Analytical Methods in Chemistry

Multiresidue veterinary drugs that were developed formilk tests depend on various extraction and cleanup prin-ciples. One of the most accepted approaches is to dilute asample of milk with a solvent like acetonitrile and then tocentrifuge and evaporate the obtained supernatant organicextract [11, 12]. Some multiclass analytical method applica-tions by LC-MS/MS or LC-TOF/MS, related to homogenizedor raw milk, that have the ability to specify undesirablechemicals, such as tetracycline, quinolone, sulfonamide, pep-tide, hormone, nonsteroidal anti-inflammatory anthelminticdrugs, mycotoxin, and pesticides, can be found in theliterature [7]. Yet most of these methods are unable tooffer satisfactory recovery of a large range of compounds ofdifferent polarities [13, 14].

Most methods for the analysis of veterinary residues havesome disadvantages, including high solvent consumption,tedious SPE cleanup steps that require extended time foranalysis, and high costs. Therefore, these types of methodsare not applied for routine analyses. The Quick Easy CheapEffective Rugged Safe (QuEChERS) methodology, whichwas originally developed for pesticide analysis, has recentlybeen proposed for the analysis of veterinary drugs usingdifferent matrices [15–18]. However, QuEChERS was foundto be inconvenient for the recovery of polar veterinarydrugs, including penicillin, tetracycline, and quinolone [13,18, 19]. Therefore, there is still a great need for simpleand rapid multiresidue analytical methods for simultane-ously determining veterinary drug and pesticide residues inmilk.

In this study, we prepared milk samples by using aprocedure based on a simple liquid-to-liquid extraction.Thismethod utilized a simple and quick sample preparation pro-cedure using a single extraction step. Through this method,milk samples were analyzed for the determination of bothveterinary drugs and pesticide residues by utilizing liquidchromatography-tandem mass spectrometry (LC-MS/MS).As a result, the reduced use of chemicals and steps in thesample preparation phase, together with the avoidance of asample cleanup step, simplified the sample pretreatment andreduced the overall total cost. Finally, in addition to reducinganalyses costs, the method provided a higher recovery ofcompounds of various polarities and improved the simplicityof 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%) werepurchased from Merck (Darmstadt, Germany). The waterused to prepare the solutions was purified in a Milli-QPlus system (EMD Millipore, Billerica, MA). Magnesiumsulfate, sodium chloride, Supelclean� primary secondaryamine (PSA), pure tetracyclines, sulfonamides, quinolones,macrolides, and antibiotics were provided from SigmaAldrich (St. Louis, Missouri, USA) and the pesticides wereprovided from Dr. Ehrenstrorfer (Augsburg, Germany).

2.2. Samples. All pasteurized whole milk samples were pur-chased from local markets. Also, raw milk was used forinterference and specificity/selectivity as a blank.

Standard Solutions. Individual stock solutions of the veteri-nary drugs and pesticides were prepared in acetonitrile at aconcentration of 1000mg/kg. Amixed intermediate standardsolutionwas prepared by diluting the stock standard solutionsof the veterinary drugs and pesticides in acetonitrile at aconcentration of 10mg/kg. Stock and intermediate standardsolutions were stored at 4∘C in amber flasks and were foundstable 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 atrefrigerator temperature (10 ± 4∘C) until analysis. For thepreparation an aliquot of approximately 5mL milk samplewas pipetted in a 50mL polypropylene centrifuge tube.Then,200mcL acetic acid was added to 10mL of ethyl acetate.After vortex for 3 minutes, the mixture was centrifuged at5000 rpm for 10 minutes. The upper phase was taken in15mL centrifuge tube and was dried under a gentle streamof nitrogen, and the residue was reconstituted with 1000mcLof mobile phase A/mobile phase B (80/20). The sample wasvortexed vigorously for 10 minutes. The extract was filteredthrough a 0.45 𝜇m filter prior to LC-MS/MS analysis.

2.3.2. Acetonitrile Extraction without Salting Procedure.Approximately 5mL milk sample was pipetted in a 50mLpolypropylene centrifuge tube. Then, 10mL of acetonitrileand 200mcL acetic acid were added to milk. After mixing bya vortex stirrer for 3 minutes, the mixture was centrifugedat 5000 rpm for 10 minutes. The upper phase was taken in15mL centrifuge tube and was dried under a gentle streamof nitrogen, and the residue was reconstituted with 1000mcLof mobile phase A/mobile phase B (80/20). The sample wasvortexed vigorously for 10 minutes. The extract was filteredthrough a 0.45 𝜇m filter prior to LC-MS/MS analysis.

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

2.4. LC-MS/MS Analysis. The chromatographic analyseswere performed using an HPLC system consisting of a

Journal of Analytical Methods in Chemistry 3

020406080

100120140

Reco

very

(%)

QuEChERSACN without saltEtOAc without salt

Acrin

athr

inBu

toca

rbox

im su

lfoxi

deCa

dusa

fos

Des

med

ipha

mEp

oxic

onaz

ole

Fost

hiaz

ate

Hep

teno

phos

Lufe

nuro

nO

met

hoat

ePi

colin

afen

Qui

zalo

fop-

P-et

hyl

Rim

sulfu

ron

Sim

azin

eTr

itico

nazo

leVa

mid

othi

onCh

lort

etra

cycli

neD

anofl

oxac

inFl

umeq

uine

Josa

myc

inLo

mefl

oxac

inO

rbifl

oxac

inRi

fam

pici

nSa

raflo

xaci

nSu

lfach

loro

pyrid

azin

eTi

lmic

osin

Tetr

acyc

line

Isox

aben

Figure 1: Recovery data for three different extraction procedures.

binary pump (Shimadzu UFLC LC-20ADmodel), Shimadzuautomatic injector (Autosampler SIL-20A HT model), and acolumn oven (CTO-20AC). Analytical columns, Symmetry�C18 2.1× 150mm id, 5 𝜇mparticle size (Waters,Milford,MA),andWaters XTerraC18 150mm× 2.1mm id, 5 𝜇mparticle size(Waters, Milford, MA), were tested. Chromatographic sepa-ration of veterinary drugs and pesticides was carried out on aWaters Symmetry C18 column. The method used a gradientmobile phase containing 0.1% acetic acid water and mobilephase B containing methanol. The column temperature wasmaintained at 40∘C with a flow rate of 0.3mL/min. The gra-dient profilewas scheduled as follows: initial proportion (98%A and 2% B) for 0.3 minutes, linear increase to 80% (B) until7 minutes, and hold of 80% (B) for 3 minutes. The injectionvolume was 50 𝜇L.The chromatographic system was coupledto electrospray ionization (ESI) source followed by anAppliedBiosystems MDS SCIEX 4500 Q TRAP mass spectrometer.The MS/MS detector conditions were as follows: curtain gas20mL/min, exit potential 10V, ion source gas 1 and ion sourcegas 2 set at 50mL/min, ion spray voltage 5500V, and turbospray temperature set at 550∘C. MS data were acquired inthe positive ion ESI mode using two alternating MS/MS scanevents. Two transitions were monitored for each analyte. Theselected molecular ion and optimized collision voltages ofproduct ions used for quantification, confirmation, and ionratio were summarized in Table 1. Applied Biosystems SCIEXAnalyst software version 1.6 was employed for data acquisi-tion and processing.Quantificationwas by comparisonwith asix-point calibration (0.0, 0.01, 0.025, 0.05, 0.1, and 0.2mg/kg)in matrix-matched calibration.

2.5. Validation Study. The analytical method developed fordetermination of veterinary drug and pesticide residues inmilk was validated according to EU Decision 2002/657/EC[16] and SANTE/11945/2015 [17]. The following parameterswere evaluated in the validation procedure: selectivity, sensi-tivity, linearity, precision (intraday and interday reproducibil-ity), accuracy and CC𝛼 and CC𝛽, LOD, and LOQ.

3. Results and Discussion

3.1. Optimization of the Extraction Procedures. Ethyl acetateextraction without salt procedure was chosen to be per-formed in this study because of its advantages. There was noneed to use salt and it could give lower detection limit interms of volatile characteristic of ethyl acetate.

Recovery values showedno difference among three differ-ent extraction procedures (acetonitrile extraction, QuECh-ERS extraction, and ethyl acetate extraction without saltingprocedure) (Figure 1).

The recovery values expressed as recovery % are allwithin the reference range of 70–120%. Comparing threeprocedures, EtOAc without salt provided recoveries between100% and 120% for a higher number of veterinary drugs andpesticides (26 veterinary drugs and 134 pesticides; total of160 compounds) thanQuEChERS (82 compounds) andACN(100 compounds), as it can be observed in Figure 2. In termsof extraction recoveries, EtOAc was found to be a suitableextraction procedure for all 26 veterinary drugs and mostof the pesticides analyzed in this study. Only one analyte(propham) showed 𝑅 > 120 for EtOAc.

Accuracywas evaluated in terms of relative standard devi-ation (RSD) by spiking blank samples with the correspondingvolume of the multicompound working standard solution.RSD was evaluated at 50 𝜇g/kg by spiking six blank samplesat each level for three procedures that provided similar RSDvalues. These values were within 1 < RSD < 10 for 75% ofeach analyte in the three procedures. These results indicatedthat the EtOAcwithout saltmethodwas precise, accurate, andreliable for the analysis of the veterinary drug and pesticidecompounds in the milk samples as an alternative method.

3.2. LC-MS/MS. Mobile phase was acidified with acetic acidin methanol and water. Also, study [18] in the literature wasperformed for the comparison. Formic acid in acetonitrileand water was used as a mobile phase in [18]. According toanalyte intensities, our results gave better peak shapes than


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) 219 160 125 952,4,5-T 253 195 197 982,4-Dimethylaniline 122 107 80Acetamiprid 223 126 73 22Acrinathrin 560 208 181 75Alachlor 238 162 238 12Amitraz 294 163 122 75Atrazine 216 174 104 45Azoxystrobin 404 372 344 33Bentazon (−)239 132 197 78Bifenazate (−)300 253 239 79Bitertanol 339 70 269 81Boscalid 344 307 140 61Bromacil (−)259 205 203 55Bromuconazole 378 159 70 66Bromoxynil (−)274 79 81 67Bupirimate 317 108 166 86Buprofezin 307 116 201 93Butocarboxim sulfoxide 207 75 132 88Cadusafos 272 159 97 98Carbaryl 202 145 127 34Carbendazim 192 160 132 17Carbofuran 222 165 123 98Carbosulfan 381 118 160 90Carboxin 234 143 87 85Dimethoate 230 199 125 97Dimethomorph 388 301 165 58Dimoxystrobin 328 116 205 99Diniconazole 326 70 159 65Dinobuton 327 215 152 66Dinocap (sum) 295 193 209 89Dinoterb (−)239 207 176 85Diphenylamine 171 93 152 17Disulfoton-sulfoxide 291 185 213 87Dithianon (−)296 263 238 86Diuron 233 72 160 85Epoxiconazole 330 121 101 80EPTC 191 128 86 40Ethiofencarb 226 107 164 81Ethion 402 385 199 76Ethirimol 211 98 140 87Ethofumesate 304 121 161 25Etoxazole 361 141 113 86Ethoxyquin 219 160 174 84Famoxadone 392 238 331 84Fenamidone 313 92 236 83Fenamiphos 304 217 202 59Fenarimol 331 268 81 18Fenazaquin 307 161 147 80


Table 1: Continued.


Fenbuconazole 338 70 125 88Fenhexamid 303 97 55 63Fenitrothion 278 125 109 60Fenoxycarb 302 88 116 25Fenpropathrin 367 125 350 33MCPP (−)213 141 143 18Metalaxyl-M 280 160 220 85Mepanipyrim 225 106 77 17Mesosulfuron-methyl 505 182 83 15Metazachlor 279 210 134 82S-Metolachlor 284 252 254 81Metosulam 419 175 140 94Metribuzin 215 187 84 29Monocrotophos 224 127 98 9Monolinuron 216 126 148 43Monuron 199 72 126 76Omethoate 215 125 125 10Oxadiargyl 341 223 151 87Oxadiazon 363 220 177 88Oxadixyl 280 219 133 79Oxamyl 237 72 90 65Oxasulfuron 408 150 107 89Oxycarboxin 269 175 147 31Oxyfluorfen 362 316 237 27Penconazole 284 70 159 67Pendimethalin 282 212 194 19Pethoxamid 297 131 250 62Phosalone 368 182 111 31Phenmedipham 301 136 168 54Phenthoate 321 163 79 18Phosmet 318 160 133 13Phosphamidon 300 127 174 28Picloram (−)239 196 123 56Terbuthylazine 230 174 104 55Pirimicarb 239 72 93Thiacloprid 254 126 186 18Thiamethoxam 292 211 181 39Thifensulfuron-methyl 389 167 205 14Thiodicarb 355 88 108 22Thiophanate-methyl 343 151 192 34Triadimefon 294 197 225 30Triadimenol 296 227 70 9Triallate 304 86 143 67Triasulfuron 403 167 141 67Triazophos 314 119 162 54Tribenuron-methyl 397 155 181 66Tributylphosphate 268 98 67


Table 1: Continued.


Trichlorfon 274 109 221 75Tridemorph 298 130 116 84Trifloxystrobin 410 186 206 37Triflumizole 346 73 278 26Triticonazole 318 70 125 95Vamidothion 288 146 118 33Zoxamide 336 159 189 26Ciprofloxacin 332 314 288 82Clindamycin 425 126 82 97Chlortetracycline 479 462 444 88Danofloxacin 360 316 342 88Difloxacin 400 356 299 90Doxycycline hydrate 445 428 410 97Flumequine 860 174 109 56Josamycin 828 109 174 75Clofentezine 303 138 102 88Chloridazon 223 104 92 54Chlorfenvinphos 359 155 99 51Chlorfluazuron (−)538 518 355 88Chloroxuron 292 72 218 87Chlorpyrifos 350 198 200 8Chlorsulfuron 359 141 167 89Chlorthiamid 206 189 154 25Cinidon-ethyl 412 348 107 26Cyazofamid 326 108 261 35Cyclanilide (−)272 160 228 45Cycloate 216 154 134 48Cymoxanil 199 128 111 59Cyproconazole 292 70 125 65Cyprodinil 226 93 77 80Demeton-S-methyl 231 89 61 62Demeton-S-methylsulfoxide 247 109 169 26Desmedipham 318 182 136 88Diallate 271 86 109 36Diazinon 305 169 97 62Dichlofluanid 350 123 224 16Dichlorprop (−)233 161 125 87Dichlorvos 221 109 127 15Difenoconazole 406 251 337 32Dimethenamid (sum) 277 244 168 68Fenthion 279 169 247 33Flazasulfuron 409 182 227 46Fludioxonil (−)247 126 169 57Fluazifop-P-butyl 385 282 328 62Flufenacet 365 194 152 61Flufenoxuron (−)488 156 304 99Flurochloridone 313 292 145 48Flurtamone 335 178 247 79


Table 1: Continued.


Flusilazole 317 165 247 78Flutolanil 325 262 242 45Foramsulfuron 454 182 139 54Fosthiazate 285 104 228 55Furathiocarb 384 195 252 62Heptenophos 251 127 109 9Hexythiazox 353 228 168 81Imazalil 297 159 201 88Imazamox (−)304 259 217 10Imazaquin 313 199 128 25Imazosulfuron 413 156 260 38Imidacloprid 256 209 175 84Indoxacarb 529 203 56 84Ioxynil (−)370 127 243 99Iprovalicarb 322 119 203 95Isazofos 314 120 162 34Isoproturon 208 72 165 99Isoxaben 334 165 150 48Lufenuron (−)509 326 339 46Malathion 331 127 99 86MCPA (−)199 141 155 94Picolinafen 378 238 145 57Mecarbam 331 227 97 96Pirimiphos-methyl 306 108 164 68Prochloraz 376 308 266 33Profenofos 373 303 97 60Prometryn 242 158 68 68Propamocarb 190 102 144 39Propanil 218 162 127 66Propargite 368 175 231 65Propham 180 138 120 28Propiconazole 342 159 69 62Propyzamide 256 190 173 63Pymetrozine 219 105 79 11Pyraclostrobin 389 194 163 98Pyridaben 365 309 147 78Pyridaphenthion 341 205 189 88Pyridate 380 207 351 78Pyriproxyfen 322 96 185 62Quinalphos 300 147 163 54Quinoxyfen 309 197 162 97Quizalofop-P-ethyl 374 299 56Rimsulfuron 433 182 325 55Simazine 202 124 132 70Spiroxamine 299 144 100 87Sulfosulfuron 472 211 261 89Tebuconazole 308 70 125 55


Table 1: Continued.


TEPP 291 179 99 96Terbutryn 242 186 68 51Tetrachlorvinphos 367 127 241 95Thiabendazole 203 175 131 12Oxytetracycline 461 426 443 97Rifampicin 823 791 151 91Sarafloxacin 386 368 342 92Sulfachloropyridazine 285 156 207 75Sulfaquinoxaline 301 156 108 99Sulfadiazine 251 156 92 86Sulfamerazine 265 156 108 89Sulfathiazole 256 156 92 88Sulfamethazine 279 186 124 99Sulfadoxine 311 156 108 97Sulfapyridine 250 184 156 96Sulfaclozine 285 156 108 95Sulfamethoxazole 254 92 108 99Tilmicosin 869 522 678 97Tetracycline 445 428 410 85Lomefloxacin 407 126 82 88Orbifloxacin 396 352 295 88Oxolinic acid 262 244 202 89

0

20

40

60

80

100

120

140

160

180

100 < R < 120 R > 120

Num

ber o

f ana

lyte

s

Recoveries (%)

QuEChERSACN

EtOAc without salt

70 < R < 100

Figure 2: Recovery (%) data obtained using extraction procedures;QuEChERS, ACN, and EtOAc without salt.

chromatograms in [18].The dried residuewas redissolved in amixture ofMeOH/water with 0.1% acetic acid to test differentreconstitution solvents. This composition produced betterpeak 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 causedextra peaks in the background noise.

3.3. Validation Study

3.3.1. Selectivity. The selectivity of the method was assessedby duplicate analysis of 10 blank milk samples. No peaks ofinterfering compounds were observed within the intervals ofthe retention time of the analytes in any of these samples.

3.3.2. Linearity. Linearity was evaluated from the calibrationcurves by triplicate analyses of blank milk samples fortifiedwith the analytes at six (0.0, 0.01, 0.025, 0.05, 0.1, and0.2mg/kg) concentration levels. Linearity was expressed asthe coefficient of linear correlation (𝑟) and from the slope ofthe calibration curve. The linearity of the analytical responseacross the studied range was excellent, with correlation coef-ficients higher than 0.997 for all analytes, which was similarto the findings in [19]. The authors [20] found correlationcoefficients higher than 0.992 for all analytes, which was alower score than ours.

3.3.3. Decision Limit and Detection Capability. CC𝛼 isdefined as the limit at and above which it can be concludedwith an error probability of 𝛼 that a sample is noncompliant.CC𝛽 is defined as the smallest content of the substance thatmay be detected, identified, and/or quantified in a samplewith an error probability of 𝛽. The CC𝛼 and CC𝛽 were


0.0

15.50

15.71

15.96

15.61

5.0e4

0.0

3.3e4

0.0

3.0e5

0.0

1.5e4

1.0e4

1.0e51.4e5

555045403530252015105

555045403530252015105

555045403530252015105

555045403530252015105

Time (min)

Time (min)

Time (min)

Time (min)

XIC of +MRM (40 pairs): 445.302/410.200 Da ID: tetracycline from sample 3(std) of 2201 vet.wiff (turbo spray)

XIC of +MRM (40 pairs): 311.104/156.100 Da ID: sulfadimethoxine from sample 3(std) of 2201 vet.wiff (turbo spray)

XIC of +MRM (40 pairs): 461.172/426.000Da ID: oxytetracycline from sample 3(std) of 2201 vet.wiff (turbo spray)

XIC of +MRM (40 pairs): 251.124/156.000 Da ID: sulfadiazine from sample 3(std) of 2201 vet.wiff (turbo spray)

Max. 3.0e5 cps.

Max. 1.5e4 cps.

Max. 3.3e4 cps.

Max. 1.4e5 cps.

Figure 3: MRM chromatograms of milk samples at the LOQ level of tetracyclines, sulfadimethoxine, oxytetracycline, and sulfadiazine(10𝜇g/kg).

determined by analysis of 10 blank milk samples and thesignal-to-noise (S/N) ratio is calculated at the timewindow inwhich the analyte is expected.TheCC𝛼 valueswere calculatedas three times the S/N ratio. The CC𝛽 was calculated byanalyzing 10 blank samples spiked with concentration atCC𝛼. Then the CC𝛼 value was added up to 1.64 timesthe corresponding standard deviation. Then, a preliminaryexperiment was conducted to check if all compounds weredetected when spiked at their CC𝛼 level (Table 2).

In Figure 3, very satisfactory S/N ratios were obtainedfor all analytes at LOQ level. The lowest LOQ value was50 𝜇g/kg for tetracyclines and for sulfonamides 20𝜇g/kg inveterinary drugs in [20] while it was 10 𝜇g/kg for both of themin our study except ciprofloxacin and quinolone. Figure 3shows MRM chromatograms of milk samples at the lowestvalidation concentration at LOQ level.

3.3.4. Accuracy and Precision. The accuracy was evaluated byrecovery tests, analyzing fortified blank samples at the sameconcentration levels used in the precision tests (0.01, 0.025,and 0.05mg/kg). The accuracy and precision of the method

results (Table 2) confirmed the values given in Decision2002/657/EC [16]. Thus, the mean accuracy values obtainedin the recovery tests were between 61 and 130%.The precisionof the method was determined in two stages: repeatability(intraday) and intermediate precision (interday). Repeatabil-ity was expressed by the RSD of the results from six replicatesanalyzed on the same day by the same analyst using thesame instrument. The intermediate precision was expressedby the RSD of the results of eighteen analyses performed onthree different days (𝑛 = 3), six analyses/day, by the sameanalyst using the same instrument. The relative standarddeviation (RSD) of interday values of veterinary drugs andpesticides analyzed by the present method was 2 to 13% andfor the intraday test 5–19% (Table 2), while relative standarddeviation (RSDr) of intraday values was 4–26% in [20].

3.3.5. Matrix Effects. Evaluation of matrix effect is importantduring validation of analytical methods using the LC-MS/MStechnique. The ionization efficiency of the analytes in ESIsource may be affected by matrix interference. In order toevaluate the degree of ion suppression or signal enhancement,


Table2:Va

lidationresults

ofthed

evelop

edmetho

d.

Com

poun

ds𝑟2

LOQ

MRL

CC𝛼

CC𝛽

%recovery

%RS

D%recovery

%RS

D%recovery

%RS

D%RS

Dr

Relativ

eMatrix

(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)

uncertainty%

effect

2,4-D(negative)

0.997

1110

2133

112

890

6108

513

320.81

2,4,5-T

0.998

910

1826

8518

102

1793

911

230.92

2,4-Dim

ethylanilin

e0.998

810

1826

7911

110

897

811

350.99

Acetam

iprid

0.997

1010

1826

979

102

896

711

330.92

Acrin

athrin

0.998

1110

1826

106

1088

8104

711

350.81

Alachlor

0.997

1110

2030

108

1186

492

313

280.81

Amitraz

0.997

910

2030

868

764

763

1428

0.81

Atrazine

0.997

1110

1723

109

694

591

39

300.85

Azoxystrobin

0.998

1110

2133

1149

102

5110

414

300.92

Bentazon

0.998

1210

1723

1185

944

944

828

0.85

Bifenazate

0.998

1110

2030

109

10102

7111

611

330.92

Bitertanol

0.998

1110

1723

112

598

397

38

260.88

Boscalid

0.998

1210

1520

115

6110

6101

57

300.99

Brom

acil

0.998

910

2030

9116

108

1491

1113

420.97

Brom

ucon

azole

0.997

1110

2030

109

9100

895

614

300.90

Brom

oxyn

il0.997

1010

1723

9914

106

999

69

330.96

Bupirim

ate

0.997

1210

2030

116

5100

5104

312

260.90

Buprofezin

0.997

910

1723

9016

9012

888

1026

0.81

Butocarboxim

sulfo

xide

0.997

1110

1723

107

9118

8102

77

331.0

6Ca

dusafos

0.997

1110

1520

11011

948

955

630

0.85

Carbaryl

0.997

1110

1723

105

1094

589

49

280.85

Carbendazim

0.997

1210

2030

118

5122

5113

411

301.10

Carbofuran

0.998

1210

1723

116

490

492

38

260.81

Carbosulfan

0.997

910

2133

9110

106

693

615

320.96

Carboxin

0.998

1110

1826

1136

904

895

1228

0.81

Clofentezine

0.998

910

1723

8813

9810

938

1026

0.88

Chlorid

azon

0.998

1010

1826

102

11114

997

711

301.0

3Ch

lorfe

nvinph

os0.998

1110

1826

112

896

5100

310

300.86

Chlorfluazuron

0.998

1110

2133

107

10114

5108

614

301.0

3Ch

loroxu

ron

0.997

1110

1826

108

13102

5104

510

360.92

Chlorpyrifo

s0.997

1110

1723

108

9102

6107

67

320.92

Chlorsulfuron

0.997

1010

2133

9610

925

885

1530

0.83

Chlorthiam

id0.997

910

2133

9217

104

13114

913

320.94


Table2:Con

tinued.

Com

poun

ds𝑟2

LOQ

MRL

CC𝛼

CC𝛽

%recovery

%RS

D%recovery

%RS

D%recovery

%RS

D%RS

Dr

Relativ

eMatrix

(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)

uncertainty%

effect

Cinido

n-ethyl

0.997

1110

1723

105

7100

6110

58

320.90

Cyazofam

id0.997

1110

1826

1127

947

934

1028

0.85

Cycla

nilid

e0.997

1210

2133

115

5104

5113

513

300.94

Cyclo

ate

0.998

1110

1723

106

1296

1188

59

280.86

Cymoxanil

0.998

1110

1723

109

7112

6103

68

321.0

1Cy

procon

azole

0.998

1010

1826

9610

926

984

1228

0.83

Cyprod

inil

0.998

910

2030

9116

989

110

911

300.88

Dem

eton

-S-m

ethyl

0.998

1010

2030

9616

9811

101

411

280.88

Dem

eton

-S-m

ethylsu

lfoxide

0.998

1010

1317

100

8101

799

611

350.91

Desmedipham

0.998

1010

1317

998

988

877

140

0.88

Diallate

0.998

1010

2030

100

13120

12110

614

261.0

8Diazino

n0.998

1010

1213

959

926

100

512

320.83

Dichlofl

uanid

0.998

1010

1723

103

9104

8100

812

260.94

Dichlorprop

0.998

910

1826

885

884

953

80

0.82

Dichlorvos

0.998

910

1520

9314

118

11109

1116

351.0

6Difeno

conazole

0.998

910

1520

9212

100

994

411

280.90

Dim

ethenamid

(sum

)0.998

1110

1723

11213

989

8710

1028

0.94

Dim

etho

ate

0.998

1110

1723

1138

104

7100

69

260.94

Dim

etho

morph

0.998

910

1317

9112

104

897

514

280.94

Dim

oxystro

bin

0.998

1010

1520

101

8116

597

512

301.0

5Dinicon

azole

0.998

1010

1317

9719

9615

879

1036

0.86

Dinob

uton

0.998

1010

1723

104

1078

692

611

320.82

Dinocap

(sum

)0.998

1210

1317

115

796

590

414

300.86

Dinoterb

0.997

1210

1826

1157

100

498

49

280.90

Diphenylamine

0.999

1210

1317

115

7108

590

414

300.97

Disu

lfoton-sulfo

xide

0.997

1110

1317

109

1086

694

513

300.82

Dith

iano

n0.997

1110

1317

106

6108

5102

36

260.97

Diuron

0.999

1110

2030

109

994

7101

310

260.85

Epoxicon

azole

0.997

1110

1213

105

898

493

411

280.88

EPTC

0.997

1110

1826

113

7112

496

410

281.0

1Ethiofencarb

0.997

1210

1826

115

796

596

39

260.86

Ethion

0.998

1010

1520

102

10100

898

812

220.90

Ethirim

ol0.998

1010

1520

9815

969

111

813

330.86

Etho

fumesate

0.998

1110

1723

105

1196

10100

713

280.86

Etoxazole

0.998

1110

1723

1148

945

101

411

280.85


Table2:Con

tinued.

Com

poun

ds𝑟2

LOQ

MRL

CC𝛼

CC𝛽

%recovery

%RS

D%recovery

%RS

D%recovery

%RS

D%RS

Dr

Relativ

eMatrix

(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)

uncertainty%

effect

Etho

xyqu

in0.998

1010

1317

100

1396

1198

411

300.86

Famoxadon

e0.998

1110

1520

112

12114

5117

512

321.0

3Fenamidon

e0.998

1110

1317

108

984

891

610

260.82

Fenamipho

s0.998

1110

1723

107

5106

4109

35

320.96

Fenarim

ol0.998

1010

1317

101

1199

995

811

230.89

Fenazaqu

in0.997

1110

1826

1138

128

7113

810

281.15

Fenb

ucon

azole

0.997

910

1317

8616

9412

974

832

0.85

Fenh

exam

id0.997

1110

1826

1147

806

845

1328

0.82

Fenitro

thion

0.997

1010

1723

9912

9811

949

1124

0.88

Feno

xycarb

0.997

910

1317

8516

100

14103

89

350.90

Fenp

ropathrin

0.997

1010

1826

100

12100

1099

97

250.90

Fenthion

0.997

1010

2336

9512

11211

108

712

331.0

1Flazasulfuron

0.998

1010

3049

9510

1188

1167

1530

1.06

Flud

ioxonil

0.998

1210

2336

115

788

5100

513

250.82

Fluazifop-P-bu

tyl0.998

1210

2846

118

8102

696

511

320.92

Flufenacet

0.998

1010

2540

103

1096

9100

713

280.86

Flufenoxuron

0.998

1010

2643

958

100

8105

411

320.90

Flurochloridon

e0.998

1010

1826

100

10102

9112

67

280.92

Flurtamon

e0.998

1010

2030

103

690

489

410

320.81

Flusilazole

0.998

1010

2336

104

892

699

611

320.83

Flutolanil

0.998

1210

1826

115

6114

799

710

261.0

3Fo

ramsulfu

ron

0.998

1110

2540

105

486

399

414

260.82

Fosthiazate

0.997

810

2030

8213

945

943

1330

0.85

Furathiocarb

0.997

1210

2133

115

596

590

512

330.86

Hepteno

phos

0.997

1010

3153

9915

9412

907

1128

0.85

Hexythiazox

0.997

1010

2540

103

6100

498

59

320.90

Imazalil

0.997

1010

2846

9512

100

1188

109

280.90

Imazam

ox0.997

810

1826

8411

100

992

612

260.90

Imazaquin

0.997

1110

3153

112

990

586

312

00.81

Imazosulfuron

0.997

1010

2540

9812

9511

968

1136

0.86

Imidacloprid

0.999

910

3049

8712

102

9109

615

260.92

Indo

xacarb

0.999

1010

2336

9915

100

693

816

300.90

Ioxynil

0.999

1010

2030

994

844

815

830

0.81

Iprovalicarb

0.999

1010

2030

9712

1108

104

715

320.99

Isazofos

0.999

1210

2133

115

794

589

1113

260.85

Isop

roturon

0.999

1210

2030

115

890

697

611

380.81

Isoxaben

0.997

1110

2643

112

5110

396

58

300.99

Lufenu

ron

0.997

1010

2643

994

984

944

1330

0.88

Malathion

0.999

1210

2336

115

7104

589

613

330.94


Table2:Con

tinued.

Com

poun

ds𝑟2

LOQ

MRL

CC𝛼

CC𝛽

%recovery

%RS

D%recovery

%RS

D%recovery

%RS

D%RS

Dr

Relativ

eMatrix

(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)

uncertainty%

effect

MCP

A0.997

910

2540

939

102

5103

711

280.92

Mecarbam

0.998

1110

3153

105

16106

1494

711

300.96

MCP

P0.998

1010

2846

103

1284

997

912

410.82

Metalaxyl-M

0.998

1010

2336

977

122

4101

516

261.10

Mepanipyrim

0.997

1010

3153

102

1692

13107

1116

330.83

Mesosulfuron-methyl0.997

810

2846

8215

104

593

612

350.94

Metazachlor

0.997

1110

2336

113

5100

593

612

160.90

S-Metolachlor

0.999

910

1826

938

927

977

1127

0.83

Metosulam

0.999

1110

3049

1109

948

888

1432

0.85

Metrib

uzin

0.999

1110

2133

105

1172

991

615

260.82

Mon

ocrotoph

os0.997

1210

2540

1154

100

388

314

320.90

Mon

olinuron

0.997

1010

2846

104

1792

1490

813

280.83

Mon

uron

0.997

1110

3153

1148

102

494

513

350.92

Ometho

ate

0.997

1110

2846

105

994

991

813

390.85

Oxadiargyl

0.997

1110

2336

105

15110

1190

1114

300.99

Oxadiazon

0.997

1010

3049

968

112

5112

56

321.0

1Oxadixyl

0.997

1110

2133

109

1290

1088

68

350.81

Oxamyl

0.997

1010

3049

103

1496

1289

811

330.86

Oxasulfu

ron

0.997

1110

3356

108

886

887

712

280.82

Oxycarboxin

0.997

1210

2846

115

7112

497

612

281.0

1Oxyflu

orfen

0.997

910

3049

928

101

7103

611

280.91

Pencon

azole

0.997

1110

2336

1127

102

496

49

280.92

Pend

imethalin

0.997

1110

2030

113

8108

5108

46

280.97

Pethoxam

id0.997

1110

2846

108

976

481

514

320.83

Phosalon

e0.998

1010

2336

9611

112

4112

58

361.0

1Ph

enmedipham

0.998

1010

3662

9514

9213

886

1130

0.83

Phenthoate

0.997

1210

2336

115

794

589

612

280.85

Phosmet

0.997

1010

2133

100

1194

1092

913

330.85

Phosph

amidon

0.997

1010

2133

998

108

795

710

300.97

Piclo

ram

0.997

1110

2133

105

1296

891

712

360.86

Picolin

afen

0.997

1010

2540

100

12114

9104

912

251.0

3Pirim

icarb

0.997

1210

2336

1198

942

973

1030

0.85

Pirim

ipho

s-methyl

0.997

1210

2336

115

7108

594

512

300.97

Prochloraz

0.999

1210

2133

115

5112

594

518

301.0

1Profenofos

0.999

1210

2133

115

7104

596

58

300.94

Prom

etryn

0.999

1110

2133

109

794

599

69

360.85

Prop

amocarb

0.998

1010

2540

100

13102

1089

911

300.92

Prop

anil

0.999

1210

3049

115

7102

599

612

280.92

Prop

argite

0.999

1010

2643

102

14112

10113

48

301.0

1Prop

ham

0.999

1010

2133

102

13100

696

510

280.90

Prop

icon

azole

0.999

1010

3049

986

122

4111

614

301.10

Prop

yzam

ide

0.999

810

2846

8410

969

955

933

0.86


Table2:Con

tinued.

Com

poun

ds𝑟2

LOQ

MRL

CC𝛼

CC𝛽

%recovery

%RS

D%recovery

%RS

D%recovery

%RS

D%RS

Dr

Relativ

eMatrix

(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)

uncertainty%

effect

Pymetrozine

0.997

1010

2336

101

1096

890

711

280.86

Pyraclo

strobin

0.997

1210

2133

115

7130

5119

415

261.17

Pyrid

aben

0.997

910

2030

8916

8012

814

1136

0.82

Pyrid

aphenthion

0.997

1010

2336

9911

120

999

1013

261.0

8Py

ridate

0.998

1010

3153

985

106

498

38

320.96

Pyrip

roxyfen

0.997

1010

2133

101

1194

686

513

220.85

Quinalpho

s0.997

1010

2336

101

576

690

514

230.82

Quino

xyfen

0.999

1010

3049

995

981

100

28

230.88

Quizalofop-P-ethyl

0.997

1210

3049

115

782

585

514

260.84

Rimsulfu

ron

0.998

910

2643

8813

104

3100

214

260.94

Simazine

0.997

1110

2336

106

1696

899

311

230.86

Spiro

xamine

0.999

810

2133

8314

849

822

1432

0.82

Sulfo

sulfu

ron

0.997

810

2133

8212

110

6111

619

300.99

Tebu

conazole

0.999

1010

2846

9910

946

995

925

0.85

TEPP

0.997

910

2336

907

107

7105

611

290.96

Terbutryn

0.997

1210

2643

115

896

5102

311

280.86

Terbuthylazine

0.997

1010

2030

104

12118

5116

46

261.0

6Tetrachlorvinp

hos

0.997

1110

2133

113

1178

890

414

280.90

Thiabend

azole

0.997

1210

2133

118

698

496

513

300.88

Thiaclo

prid

0.997

1110

1826

106

1384

1289

58

350.82

Thiametho

xam

0.997

1010

3356

104

19118

11113

811

321.0

6Th

ifensulfuron-methyl0.997

910

2030

9116

988

929

1530

0.88

Thiodicarb

0.997

1210

3153

115

7102

597

69

350.92

Thioph

anate-methyl

0.997

1110

2030

1149

112

695

513

321.0

1Triadimefon

0.997

1010

2846

104

7106

698

58

320.96

Triadimenol

0.997

1110

2540

107

14106

692

79

260.96

Triallate

0.998

1110

2336

106

9114

3111

412

301.0

3Triasulfu

ron

0.998

1110

2030

111

876

591

614

300.82

Triazoph

os0.998

1210

3049

115

796

587

59

300.86

Tribenuron

-methyl

0.998

1010

3559

999

103

8101

511

300.93

Tributylph

osph

ate

0.997

1010

1723

104

1488

1289

58

350.81


Table2:Con

tinued.

Com

poun

ds𝑟2

LOQ

MRL

CC𝛼

CC𝛽

%recovery

%RS

D%recovery

%RS

D%recovery

%RS

D%RS

Dr

Relativ

eMatrix

(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)

uncertainty%

effect

Trichlorfon

0.998

1010

2540

9810

104

3100

214

270.94

Tridem

orph

0.997

1010

2133

9817

9013

100

511

300.81

Trifloxystro

bin

0.997

1210

2540

115

7124

5114

514

301.12

Triflum

izole

0.997

1110

2030

113

1492

599

58

280.83

Triticonazole

0.998

1010

1723

100

11106

1098

77

320.96

Vamidothion

0.997

910

2133

9313

118

6102

716

281.0

6Zo

xamide

0.997

1210

2336

116

772

485

315

250.82

Ciprofl

oxacin

0.997

7100

124

148

7425

6116

9713

938

0.81

Clindamycin

0.997

1110

1214

110

23106

15109

1012

340.96

Chlortetracycline

0.999

10100

108

116101

793

799

69

140.84

Danofl

oxacin

0.999

1030

4048

104

1487

1288

89

240.82

Difloxacin

0.999

910

1113

878

100

796

610

150.90

Doxycyclin

ehydrate

0.999

10100

110119

104

1493

1196

810

230.84

Flum

equine

0.998

950

5458

8812

105

1193

1011

220.95

Josamycin

0.998

910

1113

8812

104

1197

912

220.94

Lomefloxacin

0.999

1010

1213

9913

8911

998

1022

0.80

Orbifloxacin

0.999

910

1112

9314

107

894

711

200.96

Oxolin

icacid

0.999

1110

1215

110

12102

10102

615

200.92

Oxytetracyclin

e0.999

11100

111

122

11415

104

1295

610

230.94

Rifampicin

0.997

1010

1113

989

858

985

915

0.81

Saraflo

xacin

0.997

1010

1214

101

1097

8102

511

160.87

Sulfachloropyrid

azine

0.997

9100

116132

8813

106

1280

910

240.96

Sulfaqu

inoxaline

0.998

10100

111

122

9715

9910

789

924

0.89

Sulfadiazine

0.997

8100

108

116

8512

989

985

1018

0.88

Sulfamerazine

0.999

8100

111

122

8512

104

875

79

190.94

Sulfathiazole

0.999

9100

113

126

8511

109

1081

1017

210.98

Sulfamethazine

0.999

8100

114129

8413

104

9104

811

210.94

Sulfado

xine

0.999

9100

114129

8816

9512

956

1024

0.86

Sulfapyrid

ine

0.997

8100

119138

8311

108

1096

810

200.97

Sulfaclo

zine

0.998

10100

111122

9910

928

985

916

0.83

Sulfametho

xazole

0.998

10100

116

132

102

995

787

59

150.86

Tilm

icosin

0.999

1050

5662

9812

104

9105

410

180.94

Tetracyclin

e0.999

10100

119138

100

1884

896

59

230.82


calibration curves were established with and without matrix.Matrix-induced effectswere assessed by comparing the slopesof these calibration curves using the following formula:matrix effect (ME) = 1− (𝑎matrix/𝑎standard) × 100, where 𝑎matrixand 𝑎standard are the slopes of calibration straight lines forstandard andmatrix-matched calibration graphs.Thematrix-matched calibration curves were constructed using milksamples (5 g/mLmatrix equivalent) prepared inMeOH-watersolution with 0.1% acetic acid and spiked with veterinarydrug and pesticides at concentration levels of 0.01, 0.025,and 0.05mg/kg. Matrix effect was further evaluated for ionsuppression between the standards prepared in pure solventand standards prepared in matrix and the matrix effect wasfound to be in a range of 15–25%. These results showedthat standard calibration which was simpler and less time-consuming compared with matrix-matched calibration caneffectively be used for quantitation of veterinary drug andpesticides in milk (Table 2).

3.4. Real Samples. Themethod used analyzed more than 220milk samples submitted to the laboratory for veterinary drugand pesticide residues by the local markets. Two transitionion pairs were monitored for each of the analytes and the ionratios of detected samples were compared well with those ofstandards. Retention times of analytes were also confirmedby addition of known standards in detected samples. Eightsamples out of 220 milk samples were found to containresidues of veterinary drug and pesticide residues (4% inci-dence was positive). Sulfadiazine (veterinary drug) residueamountwas found between 0.075 and 0.125mg/L in 2 samplesand tetracycline (veterinary drug) amount was found to be0.015–0.100mg/L in 4 samples. Carbaryl (pesticide) residueconcentration level was 0.005–0.025mg/L in 2 samples.

4. Conclusions

Amulticlass/multiresidue procedure with LC-MS/MS detec-tion has been developed and validated to determine andquantify veterinary and pesticide residues in milk. A simplesample preparationmethod involved liquid extraction saltingout procedures in ethyl acetate system, without cleanup steps,and shortening the sample preparation time. Validation of themethod was performed according to Commission Decision2002/657/EC. The method was characterized by good resultsin terms of recovery, reproducibility, and repeatability allow-ing the detection of veterinary drug and pesticide residuesbelow the recommended analytical level. Based on theseresults, LC-MS/MS method with ethyl acetate extractionshowed the suitability for sensitive quantification of veteri-nary and pesticide residues in milk samples for food safetyapplications. The validated method was applied on 220 realcommercial samples. This short protocol can be applicableto a large number of samples for routine analysis and rapiddetection.

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper.

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Research Article Analysis of Veterinary Drug and Pesticide ...downloads.hindawi.com/journals/jamc/2016/2170165.pdf · as pesticides and mycotoxins [], and some antihelminthic drugs