Journal of Analytical Methods in Chemistry

Journal of Analytical Methods in Chemistry / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 2170165 | 17 pages |

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

Academic Editor: Jose M. M. Lopez
Received11 Feb 2016
Accepted12 Apr 2016
Published16 May 2016


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 (). 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.

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 100 μg/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 [1518]. 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 (°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 0.45 μm 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, 5 μm 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.

CompoundsPrecursor ionTransition 1Transition 2Ion ratio
()()() (%)

2,4-D (negative)21916012595
Acetamiprid 2231267322
Acrinathrin 56020818175
Alachlor 23816223812
Bentazon (−)23913219778
Bifenazate (−)30025323979
Bitertanol 3397026981
Boscalid 34430714061
Bromacil (−)25920520355
Bromuconazole 3781597066
Bromoxynil (−)274798167
Bupirimate 31710816686
Buprofezin 30711620193
Butocarboxim sulfoxide2077513288
Cadusafos 2721599798
Carbendazim 19216013217
Carbofuran 22216512398
Carbosulfan 38111816090
Carboxin 2341438785
Dimethoate 23019912597
Dimethomorph 38830116558
Dimoxystrobin 32811620599
Diniconazole 3267015965
Dinobuton 32721515266
Dinocap (sum) 29519320989
Dinoterb (−)23920717685
Diphenylamine 1719315217
Disulfoton-sulfoxide 29118521387
Diuron 2337216085
Epoxiconazole 33012110180
Ethiofencarb 22610716481
Ethion 40238519976
Ethirimol 2119814087
Ethofumesate 30412116125
Etoxazole 36114111386
Ethoxyquin 21916017484
Famoxadone 39223833184
Fenamidone 3139223683
Fenamiphos 30421720259
Fenarimol 3312688118
Fenazaquin 30716114780
Fenhexamid 303975563
Fenitrothion 27812510960
Fenpropathrin 36712535033
MCPP (−)21314114318
Metalaxyl-M 28016022085
Mepanipyrim 2251067717
Mesosulfuron-methyl 5051828315
Metazachlor 27921013482
Metosulam 41917514094
Metribuzin 2151878429
Monocrotophos 224127989
Monolinuron 21612614843
Monuron 1997212676
Omethoate 21512512510
Oxadiargyl 34122315187
Oxadiazon 36322017788
Oxadixyl 28021913379
Oxamyl 237729065
Oxasulfuron 40815010789
Oxycarboxin 26917514731
Oxyfluorfen 36231623727
Penconazole 2847015967
Pendimethalin 28221219419
Pethoxamid 29713125062
Phosalone 36818211131
Phenmedipham 30113616854
Phosmet 31816013313
Phosphamidon 30012717428
Picloram (−)23919612356
Pirimicarb 2397293
Thiacloprid 25412618618
Thiamethoxam 29221118139
Thifensulfuron-methyl 38916720514
Thiodicarb 3558810822
Triadimefon 29419722530
Triadimenol 296227709
Triallate 3048614367
Triasulfuron 40316714167
Triazophos 31411916254
Tribenuron-methyl 39715518166
Tributylphosphate 2689867
Trichlorfon 27410922175
Tridemorph 29813011684
Trifloxystrobin 41018620637
Triflumizole 3467327826
Triticonazole 3187012595
Vamidothion 28814611833
Zoxamide 33615918926
Doxycycline hydrate44542841097
Clofentezine 30313810288
Chloridazon 2231049254
Chlorfenvinphos 3591559951
Chlorfluazuron (−)53851835588
Chloroxuron 2927221887
Chlorpyrifos 3501982008
Chlorsulfuron 35914116789
Chlorthiamid 20618915425
Cinidon-ethyl 41234810726
Cyazofamid 32610826135
Cycloate 21615413448
Cymoxanil 19912811159
Cyproconazole 2927012565
Cyprodinil 226937780
Demeton-S-methyl 231896162
Demeton-S-methylsulfoxide 24710916926
Desmedipham 31818213688
Diallate 2718610936
Diazinon 3051699762
Dichlofluanid 35012322416
Dichlorprop (−)23316112587
Dichlorvos 22110912715
Difenoconazole 40625133732
Dimethenamid (sum) 27724416868
Fenthion 27916924733
Flazasulfuron 40918222746
Fludioxonil (−)24712616957
Fluazifop-P-butyl 38528232862
Flufenacet 36519415261
Flufenoxuron (−)48815630499
Flurochloridone 31329214548
Flurtamone 33517824779
Flusilazole 31716524778
Flutolanil 32526224245
Foramsulfuron 45418213954
Fosthiazate 28510422855
Heptenophos 2511271099
Hexythiazox 35322816881
Imazalil 29715920188
Imazamox (−)30425921710
Imazaquin 31319912825
Imazosulfuron 41315626038
Imidacloprid 25620917584
Indoxacarb 5292035684
Ioxynil (−)37012724399
Iprovalicarb 32211920395
Isazofos 31412016234
Isoproturon 2087216599
Isoxaben 33416515048
Lufenuron (−)50932633946
Picolinafen 37823814557
Mecarbam 3312279796
Pirimiphos-methyl 30610816468
Prochloraz 37630826633
Profenofos 3733039760
Prometryn 2421586868
Propamocarb 19010214439
Propanil 21816212766
Propargite 36817523165
Propham 18013812028
Propiconazole 3421596962
Propyzamide 25619017363
Pymetrozine 2191057911
Pyraclostrobin 38919416398
Pyridaben 36530914778
Pyridaphenthion 34120518988
Pyridate 38020735178
Pyriproxyfen 3229618562
Quinalphos 30014716354
Quinoxyfen 30919716297
Rimsulfuron 43318232555
Simazine 20212413270
Spiroxamine 29914410087
Sulfosulfuron 47221126189
Tebuconazole 3087012555
TEPP 2911799996
Terbutryn 2421866851
Tetrachlorvinphos 36712724195
Thiabendazole 20317513112
Oxolinic acid26224420289

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) (Figure 1).

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 Figure 2. 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 for EtOAc.

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 50 μg/kg by spiking six blank samples at each level for three procedures that provided similar RSD values. These values were within 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 () 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).

CompoundsLOQMRLCCαCCβ% recovery% RSD% recovery% RSD% recovery % RSD% RSDrRelative 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)0.997111021331128906108513320.81
2,4-Dimethylaniline0.99881018267911110897811 350.99
Acetamiprid 0.99710101826979102896711330.92
Acrinathrin 0.9981110182610610888104711350.81
Alachlor 0.997111020301081186492313280.81
Bentazon 0.9981210172311859449448280.85
Bifenazate 0.99811102030109101027111611330.92
Bitertanol 0.9981110172311259839738260.88
Boscalid 0.998121015201156110610157300.99
Bromacil 0.9989102030911610814911113420.97
Bromuconazole 0.997111020301099100895614300.90
Bromoxynil 0.99710101723991410699969330.96
Bupirimate 0.9971210203011651005104312260.90
Buprofezin 0.99791017239016901288810260.81
Butocarboxim sulfoxide0.997111017231079118810277331.06
Cadusafos 0.99711101520110119489556300.85
Carbendazim 0.9971210203011851225113411301.10
Carbofuran 0.9981210172311649049238260.81
Carbosulfan 0.99791021339110106693615320.96
Carboxin 0.99811101826113690489512280.81
Clofentezine 0.99891017238813981093810260.88
Chloridazon 0.9981010182610211114997711301.03
Chlorfenvinphos 0.998111018261128965100310300.86
Chlorfluazuron 0.99811102133107101145108614301.03
Chloroxuron 0.99711101826108131025104510360.92
Chlorpyrifos 0.997111017231089102610767320.92
Chlorsulfuron 0.99710102133961092588515300.83
Chlorthiamid 0.9979102133921710413114913320.94
Cinidon-ethyl 0.997111017231057100611058320.90
Cyazofamid 0.99711101826112794793410280.85
Cycloate 0.998111017231061296118859280.86
Cymoxanil 0.998111017231097112610368321.01
Cyproconazole 0.99810101826961092698412280.83
Cyprodinil 0.99891020309116989110911300.88
Demeton-S-methyl 0.9981010203096169811101411280.88
Demeton-S-methylsulfoxide 0.998101013171008101799611350.91
Desmedipham 0.998101013179989888771400.88
Diallate 0.998101020301001312012110614261.08
Diazinon 0.99810101213959926100512320.83
Dichlofluanid 0.9981010172310391048100812260.94
Dichlorprop 0.9989101826885884953800.82
Dichlorvos 0.99891015209314118111091116351.06
Difenoconazole 0.99891015209212100994411280.90
Dimethenamid (sum) 0.9981110172311213989871010280.94
Dimethoate 0.998111017231138104710069260.94
Dimethomorph 0.99891013179112104897514280.94
Dimoxystrobin 0.998101015201018116597512301.05
Diniconazole 0.998101013179719961587910360.86
Dinobuton 0.998101017231041078692611320.82
Dinocap (sum) 0.99812101317115796590414300.86
Dinoterb 0.99712101826115710049849280.90
Diphenylamine 0.999121013171157108590414300.97
Disulfoton-sulfoxide 0.997111013171091086694513300.82
Diuron 0.999111020301099947101310260.85
Epoxiconazole 0.99711101213105898493411280.88
Ethiofencarb 0.9971210182611579659639260.86
Ethion 0.9981010152010210100898812220.90
Ethirimol 0.998101015209815969111813330.86
Ethofumesate 0.99811101723105119610100713280.86
Etoxazole 0.998111017231148945101411280.85
Ethoxyquin 0.9981010131710013961198411300.86
Famoxadone 0.99811101520112121145117512321.03
Fenamidone 0.99811101317108984891610260.82
Fenamiphos 0.998111017231075106410935320.96
Fenarimol 0.998101013171011199995811230.89
Fenazaquin 0.9971110182611381287113810281.15
Fenhexamid 0.99711101826114780684513280.82
Fenitrothion 0.997101017239912981194911240.88
Fenoxycarb 0.997910131785161001410389350.90
Fenpropathrin 0.9971010182610012100109997250.90
Fenthion 0.99710102336951211211108712331.01
Flazasulfuron 0.9981010304995101188116715301.06
Fludioxonil 0.998121023361157885100513250.82
Fluazifop-P-butyl 0.998121028461188102696511320.92
Flufenacet 0.9981010254010310969100713280.86
Flufenoxuron 0.998101026439581008105411320.90
Flurochloridone 0.9981010182610010102911267280.92
Flurtamone 0.99810102030103690489410320.81
Flusilazole 0.99810102336104892699611320.83
Flutolanil 0.998121018261156114799710261.03
Foramsulfuron 0.99811102540105486399414260.82
Fosthiazate 0.9978102030821394594313300.85
Heptenophos 0.997101031539915941290711280.85
Hexythiazox 0.99710102540103610049859320.90
Imazalil 0.9971010284695121001188109280.90
Imazamox 0.99781018268411100992612260.90
Imazaquin 0.9971110315311299058631200.81
Imazosulfuron 0.997101025409812951196811360.86
Imidacloprid 0.999910304987121029109615260.92
Indoxacarb 0.999101023369915100693816300.90
Ioxynil 0.999101020309948448158300.81
Iprovalicarb 0.9991010203097121108104715320.99
Isazofos 0.999121021331157945891113260.85
Isoproturon 0.99912102030115890697611380.81
Isoxaben 0.99711102643112511039658300.99
Lufenuron 0.9971010264399498494413300.88
Malathion 0.999121023361157104589613330.94
Mecarbam 0.99811103153105161061494711300.96
Metalaxyl-M 0.998101023369771224101516261.10
Mepanipyrim 0.997101031531021692131071116330.83
Mesosulfuron-methyl 0.99781028468215104593612350.94
Metazachlor 0.997111023361135100593612160.90
Metosulam 0.99911103049110994888814320.85
Metribuzin 0.999111021331051172991615260.82
Monocrotophos 0.997121025401154100388314320.90
Monolinuron 0.9971010284610417921490813280.83
Monuron 0.997111031531148102494513350.92
Omethoate 0.99711102846105994991813390.85
Oxadiargyl 0.997111023361051511011901114300.99
Oxadiazon 0.99710103049968112511256321.01
Oxadixyl 0.997111021331091290108868350.81
Oxamyl 0.9971010304910314961289811330.86
Oxasulfuron 0.99711103356108886887712280.82
Oxycarboxin 0.997121028461157112497612281.01
Oxyfluorfen 0.99791030499281017103611280.91
Penconazole 0.99711102336112710249649280.92
Pendimethalin 0.997111020301138108510846280.97
Pethoxamid 0.99711102846108976481514320.83
Phosalone 0.998101023369611112411258361.01
Phenmedipham 0.998101036629514921388611300.83
Phosmet 0.9971010213310011941092913330.85
Phosphamidon 0.99710102133998108795710300.97
Picloram 0.997111021331051296891712360.86
Picolinafen 0.99710102540100121149104912251.03
Pirimicarb 0.99712102336119894297310300.85
Pirimiphos-methyl 0.997121023361157108594512300.97
Prochloraz 0.999121021331155112594518301.01
Profenofos 0.99912102133115710459658300.94
Prometryn 0.9991110213310979459969360.85
Propamocarb 0.99810102540100131021089911300.92
Propanil 0.999121030491157102599612280.92
Propargite 0.99910102643102141121011348301.01
Propham 0.9991010213310213100696510280.90
Propiconazole 0.999101030499861224111614301.10
Propyzamide 0.999810284684109699559330.86
Pymetrozine 0.997101023361011096890711280.86
Pyraclostrobin 0.9971210213311571305119415261.17
Pyridaben 0.99791020308916801281411360.82
Pyridaphenthion 0.9971010233699111209991013261.08
Pyridate 0.9981010315398510649838320.96
Pyriproxyfen 0.997101021331011194686513220.85
Quinalphos 0.99710102336101576690514230.82
Quinoxyfen 0.9991010304999598110028230.88
Rimsulfuron 0.998910264388131043100214260.94
Simazine 0.997111023361061696899311230.86
Spiroxamine 0.9998102133831484982214320.82
Sulfosulfuron 0.997810213382121106111619300.99
Tebuconazole 0.9991010284699109469959250.85
TEPP 0.99791023369071077105611290.96
Terbutryn 0.997121026431158965102311280.86
Tetrachlorvinphos 0.997111021331131178890414280.90
Thiabendazole 0.99712102133118698496513300.88
Thiacloprid 0.997111018261061384128958350.82
Thiamethoxam 0.997101033561041911811113811321.06
Thifensulfuron-methyl 0.9979102030911698892915300.88
Thiodicarb 0.99712103153115710259769350.92
Triadimefon 0.99710102846104710669858320.96
Triadimenol 0.997111025401071410669279260.96
Triallate 0.9981110233610691143111412301.03
Triasulfuron 0.99811102030111876591614300.82
Triazophos 0.9981210304911579658759300.86
Tribenuron-methyl 0.998101035599991038101511300.93
Tributylphosphate 0.997101017231041488128958350.81
Trichlorfon 0.9981010254098101043100214270.94
Tridemorph 0.9971010213398179013100511300.81
Trifloxystrobin 0.9971210254011571245114514301.12
Triflumizole 0.99711102030113149259958280.83
Triticonazole 0.9981010172310011106109877320.96
Vamidothion 0.997910213393131186102716281.06
Zoxamide 0.99712102336116772485315250.82
Doxycycline hydrate0.9991010011011910414931196810230.84
Oxolinic acid0.999111012151101210210102615200.92

In Figure 3, 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.

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 (Table 2) 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 (), 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) = , where and 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.

Competing Interests

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


  1. Y. Zhang, X. Li, X. Liu et al., “Multi-class, multi-residue analysis of trace veterinary drugs in milk by rapid screening and quantification using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry,” Journal of Dairy Science, vol. 98, no. 12, pp. 8433–8444, 2015. View at: Publisher Site | Google Scholar
  2. Rekha, S. N. Naik, and R. Prasad, “Pesticide residue in organic and conventional food-risk analysis,” Journal of Chemical Health and Safety, vol. 13, no. 6, pp. 12–19, 2006. View at: Publisher Site | Google Scholar
  3. European Union, “Commission Regulation No 37/2010 of 22nd December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin,” Official Journal of the European Communities International, vol. 15, pp. 1–72, 2010. View at: Google Scholar
  4. M. M. Aguilera-Luiz, J. L. M. Vidal, R. Romero-González, and A. G. Frenich, “Multi-residue determination of veterinary drugs in milk by ultra-high-pressure liquid chromatography-tandem mass spectrometry,” Journal of Chromatography A, vol. 1205, no. 1-2, pp. 10–16, 2008. View at: Publisher Site | Google Scholar
  5. S. B. Turnipseed, W. C. Andersen, C. M. Karbiwnyk, M. R. Madson, and K. E. Miller, “Multi-class, multi-residue liquid chromatography/tandem mass spectrometry screening and confirmation methods for drug residues in milk,” Rapid Communications in Mass Spectrometry, vol. 22, no. 10, pp. 1467–1480, 2008. View at: Publisher Site | Google Scholar
  6. M. M. Aguilera-Luiz, P. Plaza-Bolaños, R. Romero-González, J. L. M. Vidal, and A. G. Frenich, “Comparison of the efficiency of different extraction methods for the simultaneous determination of mycotoxins and pesticides in milk samples by ultra high-performance liquid chromatography-tandem mass spectrometry,” Analytical and Bioanalytical Chemistry, vol. 399, no. 8, pp. 2863–2875, 2011. View at: Publisher Site | Google Scholar
  7. A. Kaufmann, P. Butcher, K. Maden, S. Walker, and M. Widmer, “Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to orbitrap and liquid chromatography coupled to tandem mass spectrometry,” Talanta, vol. 85, no. 2, pp. 991–1000, 2011. View at: Publisher Site | Google Scholar
  8. A. A. M. Stolker, P. Rutgers, E. Oosterink et al., “Comprehensive screening and quantification of veterinary drugs in milk using UPLC-ToF-MS,” Analytical and Bioanalytical Chemistry, vol. 391, no. 6, pp. 2309–2322, 2008. View at: Publisher Site | Google Scholar
  9. M. Gaugain-Juhel, B. Delépine, S. Gautier et al., “Validation of a liquid chromatography-tandem mass spectrometry screening method to monitor 58 antibiotics in milk: a qualitative approach,” Food Additives and Contaminants—Part A, vol. 26, no. 11, pp. 1459–1471, 2009. View at: Publisher Site | Google Scholar
  10. Y.-Y. Tang, H.-F. Lu, H.-Y. Lin, Y.-C. Shih, and D.-F. Hwang, “Multiclass analysis of 23 veterinary drugs in milk by ultraperformance liquid chromatography-electrospray tandem mass spectrometry,” Journal of Chromatography B, vol. 881-882, pp. 12–19, 2012. View at: Publisher Site | Google Scholar
  11. J. Zhan, X.-J. Yu, Y.-Y. Zhong et al., “Generic and rapid determination of veterinary drug residues and other contaminants in raw milk by ultra performance liquid chromatography–tandem mass spectrometry,” Journal of Chromatography B, vol. 906, pp. 48–57, 2012. View at: Publisher Site | Google Scholar
  12. S. B. Turnipseed, J. M. Storey, S. B. Clark, and K. E. Miller, “Analysis of veterinary drugs and metabolites in milk using quadrupole time-of-flight liquid chromatography-mass spectrometry,” Journal of Agricultural and Food Chemistry, vol. 59, no. 14, pp. 7569–7581, 2011. View at: Publisher Site | Google Scholar
  13. R. Romero-González, M. M. Aguilera-Luiz, P. Plaza-Bolaños, A. G. Frenich, and J. L. M. Vidal, “Food contaminant analysis at high resolution mass spectrometry: application for the determination of veterinary drugs in milk,” Journal of Chromatography A, vol. 1218, no. 52, pp. 9353–9365, 2011. View at: Publisher Site | Google Scholar
  14. A. Garrido Frenich, M. D. M. Aguilera-Luiz, J. L. Martínez Vidal, and R. Romero-González, “Comparison of several extraction techniques for multiclass analysis of veterinary drugs in eggs using ultra-high pressure liquid chromatography–tandem mass spectrometry,” Analytica Chimica Acta, vol. 661, no. 2, pp. 150–160, 2010. View at: Publisher Site | Google Scholar
  15. S. Lehotay, M. O'Neil, J. Tully et al., “Determination of pesticide residues in foods by acetonitrile extraction and partitioning with magnesium sulfate: collaborative study,” Journal of the European Communities International, vol. 90, no. 2, pp. 485–520, 2007. View at: Google Scholar
  16. European Commission Decision, “2002/657/EC, implementing Council Directive 96/23/EC, concerning the performance of analytical methods and interpretation of the results,” Official Journal of the European Communities International, vol. L221, pp. 8–36, 2002. View at: Google Scholar
  17. European Commission Decision SANTE/11945/2015 implementing Council Directive Supersedes SANCO/12571/2013 concerning guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed, April 2016,
  18. A. Kaufmann, P. Butcher, K. Maden, S. Walker, and M. Widmer, “Multi-residue quantification of veterinary drugs in milk with a novel extraction and cleanup technique: salting out supported liquid extraction (SOSLE),” Analytica Chimica Acta, vol. 820, pp. 56–68, 2014. View at: Publisher Site | Google Scholar
  19. S. J. Lehotay, “Determination of pesticides residues in food by acetonitrile extraction and partitioning with magnesium sulfate: Collaborative study,” The Journal of AOAC International, vol. 90, no. 2, pp. 485–520, 2007. View at: Google Scholar
  20. F. Gao, Y. Zhao, B. Shao, and J. Zhang, “Determination of residues of pesticides and veterinary drugs in milk by ultra performance liquid choromatography coupled with quadrupole time of fligt mass spectrometry,” Chinese Journal of Chromatography, vol. 30, no. 6, pp. 560–567, 2012. View at: Google Scholar

Copyright © 2016 Husniye Imamoglu and Elmas Oktem Olgun. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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