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Food Additives & Contaminants: Part A

ISSN: 1944-0049 (Print) 1944-0057 (Online) Journal homepage: http://www.tandfonline.com/loi/tfac20

Multiresidue Method for Simultaneous Analysisof Aflatoxin M1, Avermectins, OrganophosphatePesticides and Milbemycin in Milk by UltraPerformance Liquid Chromatography Coupled toTandem Mass Spectrometry

Marianna Ramos dos Anjos, Izabela Miranda de Castro, Maria de LourdesMendes de Souza, Virgínia Verônica de Lima & Francisco Radler de Aquino-Neto

To cite this article: Marianna Ramos dos Anjos, Izabela Miranda de Castro, Maria deLourdes Mendes de Souza, Virgínia Verônica de Lima & Francisco Radler de Aquino-Neto(2016): Multiresidue Method for Simultaneous Analysis of Aflatoxin M1, Avermectins,Organophosphate Pesticides and Milbemycin in Milk by Ultra Performance LiquidChromatography Coupled to Tandem Mass Spectrometry, Food Additives & Contaminants: PartA, DOI: 10.1080/19440049.2016.1175227

To link to this article: http://dx.doi.org/10.1080/19440049.2016.1175227

Accepted author version posted online: 04May 2016.Published online: 04 May 2016.

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Publisher: Taylor & Francis

Journal: Food Additives & Contaminants: Part A

DOI: 10.1080/19440049.2016.1175227

Multiresidue Method for Simultaneous Analysis of Aflatoxin M1, Avermectins, Or

ganophosphate Pesticides and Milbemycin in Milk by Ultra Performance Liquid

Chromatography Coupled to Tandem Mass Spectrometry

Marianna Ramos dos Anjos a*

Izabela Miranda de Castro a

Maria de Lourdes Mendes de Souza a

Virgínia Verônica de Lima b

Francisco Radler de Aquino Neto b

a Laboratório de Resíduos e Contaminantes, Embrapa Agroindústria de Alimentos, Av.

das Américas, 29501, Guaratiba, Rio de Janeiro, Brasil. Phone: +5521-36229785.

E-mail: izabela .castro@embrapa.br; marialourdes.m.souza@embrapa.br.

*Corresponding author. Email: marianna.anjos@embrapa.br.

b Universidade Federal do Rio de Janeiro, Instituto de Química, LAB-RES/LADETEC,

Rio de Janeiro, Brasil. Email: virginiadelima@iq.ufrj.br; radler@iq.ufrj.br.

Abstract

In this work we describe a method developed for the simultaneous analysis of

aflatoxin M1, abamectin, doramectin, eprinomectin, ivermectin, moxidectin,

acephate, azinphos-ethyl, azinphos-methyl, diazinon, methamidophos,

methidathion, mevinphos, pirimiphos-ethyl and pirimiphos-methyl in whole raw

milk, based on the QuEChERS method for extraction and clean-up, with detection

and quantification by Ultra Performance Liquid Chromatography coupled to

Tandem Mass Spectrometry (UPLC-MS/MS). The method was validated

according to parameters of the Analytical Quality Assurance Manual from

the Brazilian Ministry of Agriculture and Commission Decision 2002/657/EC,

and proved suitable for analysis of these analytes within the proposed working

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range, with recovery values between 77 and 110%, standard deviation lower than

20%, limits of detection between 0.05 to 0.99 µg L-1 and limits of quantification

between 0.15 to 1.98 µg L-1. Samples from animals treated with abamectin,

doramectin, ivermectin and diazinon were analysed by the validated method.

Residues of aflatoxin M1 were also found in field samples at levels below the

established maximum residue limit.

Keywords: aflatoxin M1; macrocyclic lactones; milk; organophosphate pesticides;

QuEChERS; UPLC-MS/MS.

Introduction

Milk quality from the stand point of consumption is linked not only to its nutritional

attributes, but above all, to its safety. If this fundamental food is not obtained

under appropriate conditions, it can become a vehicle for chemical and microbiological

contaminants (Antunes and Pacheco 2009). Among the chemical contaminants in

milk are pesticides, veterinary drug residues and mycotoxins.

Pesticide residues in milk may occur indirectly through the consumption

of contaminated feed and forage, use of disinfectants in stalls and dairy production

sites, or directly by the use of veterinary products to control parasites (Bastos et al.

2011). The Boophilus microplus tick (B. microplus) is a major obstacle to

bovine livestock in tropical countries, causing a fall in production of milk and meat.

Organophosphate pesticides (OPs), or their associations, are commonly used in Brazil

as acaricides, due to their relatively low cost and efficient control of cattle ticks (Brito

2007).

Veterinary drug residues in milk occur directly through the administration of

antibiotics to treat mammary gland infections (e.g., mastitis) and reproductive system

diseases, and drugs to control endo and ectoparasites (Beltrane and Machinski Junior

2005). Avermectins, abamectin (ABA), doramectin (DOR), eprinomectin (EPR),

ivermectin (IVR), and the milbemycin, moxidectin (MOX) represent a class of

macrocyclic lactones which have insecticidal, nematicidal and acaricidal activity. These

veterinary drugs are classified as endectocides because they have action against internal

and external (endo and ecto) parasites (Durden 2007).

Aflatoxins are secondary metabolites of some Aspergillus fungi species. The

continuous intake of aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) by lactating

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animals leads to excretion, in milk, of

their hydroxylated metabolites Aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2),

respectively (Biancardi et al. 2013). AFM1 has high genotoxic activity, although it

is about 10 times less carcinogenic than AFB1 (IARC 2002). Most

countries regulate the maximum levels of AFM1 in milk and milk products, including

Brazil (Brazil 2014). The Brazilian maximum level of AFM1 is the same of the Codex

Alimentarius, 0.5 µg L-1 (CODEX ALIMENTARIUS, 2015) but this value is about ten

times higher than the limit established by the European Union, 0.05µg kg-1 (European

Communities 2006).

The knowledge of residues and contaminants in milk is important for the

development of actions to improve handling, storage and control in this production

chain as well to reduce such contaminants. In Brazil, the actions to control residues and

contaminants in milk are described in the National Plan for Control of Residues and

Contaminants (NPCRC) from the Ministry of Agriculture. The following avermectins,

OPs and aflatoxins are monitored by this plan, with their respective MRL (maximum

residue level): ABA (10 µg L-1); DOR (15 µg L-1); EPR (20 µg L-1); IVR (10 µg L-1);

MOX (10 µg L-1); AFM1 (0.5 µg L-1); acephate (20 µg L-1); azinphos-ethyl (50 µg L-1);

azinphos-methyl (50 µg L-1), diazinon (10 µg L-1), methamidophos (10 µg L-1),

methidathion (20 µg L-1), E and Z mevinphos (50 µg L-1), pirimiphos-methyl (50 µg L-

1) and pirimiphos-ethyl (50 µg L-1) (Brasil, 2014).

The development of analytical methods to determine residues and contaminants

in milk presents several challenges due to the complexity of the matrix, and the high

levels of fat and protein which can interfere in these analyses. Consequently, the

extraction methods of these analytes tend to be long, involving several purification steps

to remove the interfering from the matrix (Aguilera-Luiz et al. 2011). The trend in

residue and contaminants analyses has been to use the QuEChERS method (quick, easy,

cheap, effective, rugged and safe), originally developed for pesticides (Anastassiades et

al 2003). This method involves the initial extraction of analytes with acetonitrile,

followed by the addition of salts (sodium chloride and magnesium sulphate), which

promote the removal of proteins and other interfering substances for the aqueous phase.

Then there is a purification step, dispersive solid phase extraction (d-SPE), which

consists of the addition of small quantities of bulk sorbent to the obtained extract

(Prestes et al. 2009). QuEChERS is very flexible, it can be modified for different

purposes depending on the analytes, matrices and analytical instruments. The method is

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also being applied to analyse veterinary drug residues and mycotoxins in food

(Fernandes et al. 2014).

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is

the most sensitive and reliable method for detection and quantification of residues and

contaminants in food. Ultra performance liquid chromatography (UPLC) works

with particles of stationary phase less than 2 micrometres in diameter. The use of these

particles along with the high linear velocities of the mobile phase increases the

resolution and reduces the analytical time (Maldaner and Garden, 2009).

The objective of this work was to develop and validate an analytical method for

simultaneous determination of AFM1, OPs, avermectins and milbemycin in whole

raw milk by ultra performance liquid chromatography coupled to tandem

mass spectrometry (UPLC-MS/MS). The same 16 analytes monitored by NPCRC were

included in this analytical method.

Materials and Methods

Reagents

Sodium acetate ACS grade was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Acetonitrile and acetic acid HPLC grade were obtained from Tedia (Fairfield, OH,

USA). Formic acid ACS grade was purchased from Merck (Darmstadt, Germany).

Sodium chloride and magnesium sulphate ACS grade were purchased from Vetec (Rio

de Janeiro, Brazil). Ammonium formate HPLC grade was obtained from Fluka (St.

Gallen, Switzerland). Bondesil® PSA and C18 particles were acquired from Agilent

(Santa Clara, CA, USA).Water was purified in an Advantage A10 Millipore System at

18.2 M©.cm-1.

Standards

Avermectins and OPs standards were purchased from Dr. Ehrenstorfer GmbH

(Augsburg, Germany). AFM1 standard was obtained from Sigma-Aldrich (St. Louis,

MO, USA). Stock solutions were prepared from certified

standards with concentration equal to 1000 µg.mL-1. The stock solutions were used

to prepare working solutions containing the 16 analytes in concentrations of 100

times the MRL (MIX 1) and 10 times the MRL (MIX 2).

Method optimization

Chromatographic and mass spectrometer conditions

The analysis was performed using an Acquity UPLC® system coupled to Quattro

Premier XE® (Waters Corp., Ma, USA). The Acquity UPLC® system is composed of a

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mailto:MΩ.cm@25

binary pump, autosampler and column oven. The chromatographic separation was

performed on Waters Acquity BEH UPLC® C18 column (100 x 2.1 mm ID, 1.7 µm).

Compositions of mobile phase A (5 mM ammonium formate + 0.01% formic acid, pH

4.00) and mobile phase B (acetonitrile: mobile phase A, 95: 5), gradient: 0-1 min (10%

B); 1 to 5.5 min (55% B); 5.5 to 10.5 min (100% B); 12 min (10% B). The flow rate

used was 0.3 mL min-1, column oven temperature was 30 °C, the autosampler

temperature was 25 °C. The injector was set to full loop injection of 10 µL and the total

run time was 12 min.

Quattro Premier XE® mass spectrometer was operated with an electrospray

ionization source in the positive mode (ESI+). The operating parameters were adjusted

to the following conditions: capillary voltage: 3.5 kV; ion source temperature: 120 °C,

desolvation temperature: 450 °C; cone gas flow (N2): 20 L h-1; desolvation gas flow

(N2): 500 L h-1; collision gas flow (Ar): 0.15 mL min -1. The cone voltages, collision

energies and quantification and confirmation transitions for each analyte were

established from the direct infusion of 1 µg mL-1 solution. The infusion of analytes was

performed with the mobile phases A and B (1:1) using a flow rate of 0.1 mL min-1 in

full scan mode. After adjusting these parameters the multiple reactions monitoring

method (MRM), used for identification and quantification of analytes, was established.

Extraction and clean-up

Extraction efficiency was evaluated for two QuEChERS methods: original and acetate

buffer from organic milk samples spiked at three levels: 0.5, 1.0 and 1.5 times

the MRL of each analyte. For each method three different types of dispersive solid

phase extraction (d-SPE) cleaning were tested, totalling six different treatments and

eighteen extraction experiments. The experiments carried out are shown in Table 1.

a) Original QuEChERS method: 10 mL of milk was extracted with 10 mL

of acetonitrile, stirred for 1 min, followed by addition of 1 g of NaCl and 4 g of MgSO4,

with stirring at vortex for 1 min and centrifugation at 5000 rpm for 5 min.

b) QuEChERS method with acetate buffer: 15 mL of milk was extracted with 15 mL

of 1% acetic acid in acetonitrile, stirred for 1 min, followed by addition of 6 g

of MgSO4 and 1.5 g of sodium acetate with stirring at vortex for 1 min and

centrifugation at 5000 rpm for 5 min.

Clean-up by d-SPE: 2 mL of the extract obtained was transferred to a centrifuge

tube containing the sorbents, stirred for 30 s and centrifuged at 5000 rpm for 5 min. The

extract was filtered through a PTFE membrane, and then 1 mL of extract

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was transferred to a vial, evaporated to dryness and dissolved in 1 mL

acetonitrile: mobile phase A (2: 8, v / v).

Method validation

The best extraction method obtained was validated according to the Analytical Quality

Assurance Manual from the Brazilian Ministry of Agriculture (Brasil 2011). The values

established in this Manual also comply with the requirements of Commission Decision

2002/657/EC (European Communities 2002). The following parameters were

evaluated: selectivity; matrix effect; linearity; decision limit (CC±); detection capability

(CC² ); recovery; limit of detection (LOD); limit of quantification

(LOQ), and repeatability. The calculations were performed by the

software MassLynx® and Microsoft Excel®.

In the proposed method, the selectivity was assessed by analysing six replicates

of the sample extracts of organic whole raw milk.

Evaluation of linearity involved plotting an analytical solvent curve from the

MIX 1, working solution containing the16 analytes, with five points corresponding to 0,

0.5, 1.0, 1.5 and 2.0 times the MRL established for each analyte. The Cochran test was

used to assess the homogeneity of variances obtained for each concentration level.

Calibration data were evaluated by ordinary linear regression in case of

homoscedasticity, or weighted linear regression in case of heteroscedasticity. The

matrix effect was evaluated by comparing the slope of the analytical curve in matrix

extract with the slope of the analytical curve in solvent, through the F-test (Fisher-

Snedecor). Then the Student t- test was applied to determine the statistical equivalence

between the slopes of analytical curves in solvent and matrix.

CC± and CC² were calculated from the standard deviation of the method

repeatability value at the MRL concentration. The LOD and LOQ were calculated by

signal to noise ratio of the equipment. LOD was the concentration equivalent to three

times the noise and LOQ was the concentration equivalent to six times the noise.

The method recovery and repeatability were carried out using organic milk

samples spiked at three levels: 0.5, 1.0 and 1.5 times the MRL of each analyte, with six

replicates for each level. The average recovery and relative standard deviation (RSD)

were calculated for each level.

Samples Analysis

Field samples kindly provided by producers in the state of Rio de Janeiro, Brazil were

analysed using the validated method. The milk samples were obtained from animals that

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were being treated with diazinon, ABA, DOR and IVR. The animals were crossbred

cows, between nine and ten years old and average weight of 400 kg. The samples were

collected between the 1st and 30th day after drug administration. For each drug

six samples were analysed from three different animals, totalling eighteen samples.

These milk samples were not intended for human consumption.

Results and Discussion

Method optimization

Chromatographic and mass spectrometric conditions

The choice of the mobile phase, ionization mode (positive ESI), and transitions of

quantification and confirmation was made according to the literature (Aguilera-Luiz et

al., 2011; Ortelli et al., 2009; Rubensam et al., 2011) and the chemical nature of the

analytes. The presence of an ammonium salt in the mobile phase is important because

ABA, DOR and IVR tend to form adducts, and the formation of ammonium adducts is

better than sodium adducts, which produce no linear response at the detector due to the

presence of sodium traces in the matrix extracts or derived from the analytical process

(Durden, 2007). Some of the parameters used in the Quattro Premier XE® system, such

as the capillary voltage; ion source temperature; desolvation temperature, among others,

were established during calibration of the instrument by the manufacturer.

The precursor ions of each analyte were observed by direct infusion. In the majority of

cases the protonated ion [M + H] + was observed. Ammonium adducts [M + NH4]

+ were observed for ABA, DOR and IVR. The mass spectrometry parameters for

positive ion MS/MS analysis are shown in Table 2.

Extraction and clean-up

The results of the experiments to evaluate the best method for extraction and clean-up

are shown in Table 3. The original QuEChERS method was better than the QuEChERS

method with acetate buffer for most of the analytes, especially for LM, diazinon,

methamidophos, pirimiphos-ethyl and pirimiphos-methyl. Although the best treatment

for AFM1 was 6 (QuEChERS method with acetate buffer and d-SPE using MgSO4,

PSA and C18), the recovery values obtained with the others treatments were within the

acceptable range 50-120% (Brasil, 2011).

Since the proposed multiresidue method includes three classes of substances, the

best extraction was treatment 3 (original QuEChERS method with d-SPE using MgSO4,

PSA and C18), which showed the best recovery values for nine analytes. Treatment 2

(original QuEChERS method with d-SPE using MgSO4 and PSA) also showed good

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recovery results. Treatment 2 had only one recovery result outside the acceptable range

80-110% (Brasil, 2011), for diazinon (78%), and treatment 3 for MOX (77%).

However, the extract obtained with treatment 3 was clearer than the one treated with

only MgSO4 and PSA, since the C18 particles were able to remove some pigments.

Therefore, the extraction method chosen to be validated was the original QuEChERS

method with d-SPE using MgSO4, PSA and C18. The clean-up step by d-SPE was

necessary because the first extract obtained was not clear, probably because the whole

raw milk has high contents of fat.

Method validation

There was no interfering with the same m/z and retention time of the analytes in the

six replicates performed with the unspiked matrix extract, demonstrating the selectivity

of the method. The chromatograms obtained for unspiked matrix extract for each

analyte are shown in Figure 1. The chromatograms obtained for spiked matrix

extract for each analyte are shown in Figure 2.

Performance characteristics of the optimized method are shown in Table 4,

namely the working range, the r2 values for analytical curves, LOD and LOQ obtained

for each analyte, CC± and CC² calculated for LM. The Analytical Quality Assurance

Manual does not require calculation of CC± and CC² for pesticide residues and

mycotoxins in milk (Brasil, 2011).

For the majority of analytes, the coefficients of determination (r2) were close to

one, showing good linearity. The C values calculated were smaller than the tabulated

(0.684, k=5,n=3), indicating a homoscedastic dispersion profile for the majority of

analytes, allowing the standard curves to be evaluated by linear regression using the

ordinary least squares method. The exceptions were AFM1(C=0.928), azinphos-ethyl

(C=0.764) and methidathion (C=0.804), for these analytes weighted linear fits (1/x)

were performed using the MassLynx® software.

In terms of the matrix effect, the Student-t values calculated were greater than

the critical value for most of analytes, so the curves in solvent and matrix did

not have the same inclinations and could not be considered equivalent, confirming the

existence of a matrix effect (the exception being ABA, methamidophos and pirimiphos-

methyl. Thus, the curve in matrix extract was used to quantify the samples,

including the analytes for which no matrix effect was observed.

The values obtained for LOD and LOQ were much smaller

than the MRLs established for these analytes, demonstrating that the method is

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adequate to meet the current Brazilian legislation. To meet the European legislation, the

LOD and LOQ obtained for AFM1 need to be reviewed, since they are very close to the

maximum level established (European Communities 2006). The CC² value must not be

greater than 14 for ABA, IVR and MOX, and not greater than 21 for DOR, and 28 for

EPR (Brasil, 2011). The CC± and the CC² values obtained for EPR and MOX

were within acceptable limits, while for ABA, DOR and IVR, values are above

the acceptance criteria adopted.

The method recoveries and repeatability are shown in Table 5. The

recovery results are within the acceptable range (70-110%). The method showed good

repeatability with RSD values lower than 20%.

Sample Analysis

ABA residues in milk samples ranged from 57.3 to 6.2 µg L-1. Residues above the MRL

(10 µg L-1) were found up to 14 days after treatment. Even after 29 days, ABA residues

in milk were also found, although these values were below the MRL. The IVR residues

ranged from 32.3 to 1.6 µg L-1 and after the 29th day they were not found. DOR residues

were found in the range of 27.8 to 11.5 µg L-1. In Brazil, products whose

active ingredient is ABA or DOR are not recommended for lactating cows, and milk

obtained from animals under treatment is not allowed for human consumption.

For IVR, some products have the same recommendations, however, there are

products formulated with 1% ivermectin that are intended for dairy cattle, without a

grace period for milk collection (Sindan, 2015).

For animals treated with diazinon ear tags, residues below the MRL (10µg L-

1) were found and remained in the range of 1 to 6.7 µg L-1 from the 1st to the

30th day. The ear tags allow a slow release of diazinon and the deadline for their

removal is 150 days. The use of diazinon in dairy cattle is not prohibited in Brazil, and

there is no grace period for the collection of milk when it is administered in the form of

ear tags (Sindan, 2015).

AFM1 residues were detected in all 72 milk samples, but only 20 samples

had values greater than the LOQ (0.04 µg L-1). Values found above LOQ, ranged from

0.16 to 0.48 µg L-1, below the Brazilian limit (0.5 µg L-1), but above the limit

established by the EU (0.05 µg kg-1). These results were expected since animal feed

was supplemented with cereals (a mixture of maize, rice and wheat bran). These

cereals were probably contaminated with AFB1.

Conclusion

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The multiresidue method developed proved to be selective, accurate and precise

in the studied range, enabling the simultaneous analysis of four classes of substances

(aflatoxin, organophosphate pesticides, avermectins and milbemycin) that are included

in the Brazilian official milk monitoring program.

The QuEChERS method, with minor changes, was suitable for multiresidue

extraction of the selected analytes (AFM1, abamectin, doramectin, eprinomectin,

ivermectin, moxidectin, acephate, azinphos-ethyl, azinphos-methyl, diazinon,

methamidophos, methidathion, mevinphos, pirimiphos-ethyl and pirimiphos-methyl) in

whole raw milk, with extracts clear and free from interfering. Ultra performance liquid

chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was adequate

for detection and quantification of these analytes in milk matrix, with recovery values

between 77 and 110%, standard deviation lower than 20%, limits of detection between

0.05 to 0.99 µg L-1 and limits of quantification between 0.15 to 1.98 µg L-1, appropriate

to meet current legislation.

The results of the field trial showed that the method is suitable for quantitative

analysis of analytes evaluated in milk within the working range. One advantage of this

method is the ability to analyse AFM1 together with other classes of analytes commonly

present in milk.

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Figure 1: Chromatograms obtained for unspiked matrix extract.

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Figure 2: Chromatograms obtained for spiked matrix extract.

Table 1 Summary of extraction experiments

Treatment QuEChERS Method Clean-up (d-SPE)

1 Original 150 mg MgSO4 + 50 mg PSA

2 Original 150 mg MgSO4 + 50 mg C18

3 Original 150 mg MgSO4 + 50 mg PSA + 50 mg C18

4 Acetate Buffer 150 mg MgSO4 + 50 mg PSA

5 Acetate Buffer 150 mg MgSO4 + 50 mg C18

6 Acetate Buffer 150 mg MgSO4 + 50 mg PSA + 50 mg C18

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Table 2 Parameters for Mass Spectrometry Analysis

Analyte Retention time

(min.)

Precursor ion

Transitions (m/z) Cone (eV)

Collision energy (eV)

ABA 8.10 [M + NH4]+ 890.5 ’ 567.2 (q) 20 15

890.5 ’ 305.2 (c) 35 Acephate 0.90 [M + H]+ 184 ’ 142.7 (q) 20 8

184 ’ 94.7 (c) 25 AFM1 4.32 [M + H]+ 329 ’ 272.9 (q) 50 26

329 ’ 229 (c) 44 Azinphos-ethyl 6.64 [M + H]+ 346 ’ 137 (q) 18 30

346 ’ 97 (c) 20 Azinphos-methyl 6.31 [M + H]+ 318 ’ 132 (q) 22 15

318 ’ 124.8 (c) 20 Diazinon 6.96 [M + H]+ 305 ’ 169 (q) 25 22

305 ’ 153.9 (c) 20 DOR 8.60 [M + NH4]

+ 916.3 ’ 331.2 (q) 916.3 ’ 593.3 (c)

20 30 15

EPR 7.53 [M + H]+ 914.5 ’ 186 (q) 20 18 914.5 ’ 144.1 (c) 40

IVR 9.70 [M + NH4]+ 892.4 ’ 569.3 (q) 25 20

892.4 ’ 307.3 (c) 40 Methamidophos 0.90 [M + H]+ 142 ’ 94 (q) 28 15

142 ’ 125 (c) 14 Methidathion 6.32 [M + H]+ 303 ’ 145 (q) 20 12

303’ 85 (c) 20 Mevinphos 3.70 (E)

4.20 (Z) [M + H]+ 225’ 192.9 (q) 18 8

225 ’ 126.8 (c) 15 MOX 8.88 [M + H]+ 640.4 ’ 528.2 (q) 18 8

640.4 ’ 498.2 (c) 15 Pirimiphos-ethyl 7.65 [M + H]+ 334.2 ’ 198(q) 35 25

334.2 ’ 306.1 (c) 18 Pirimiphos-methyl 7.12 [M + H]+ 306 ’ 108(q) 30 30

306 ’ 67 (c) 40

q- quantification transition; c- confirmation transition

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Table 3 Average recoveries obtained for each treatment Analyte Spiked range

(µg L-1) Recovery (%)*

1 2 3 4 5 6 ABA 5-15 86 101 92 89 62 63

Acephate 10-30 116 83 103 72 60 80 AFM1 0.25-0.75 76 76 78 85 72 88

Azinphos- ethyl 25-75 101 101 99 98 87 92 Azinphos- methyl 25-75 112 92 96 103 100 101

Diazinon 10-30 97 78 102 51 53 49 DOR 7.5-22.5 103 96 97 82 52 69 EPR 10-30 94 88 89 93 67 80 IVR 5-15 103 83 84 98 64 75

Methamidophos 5-15 120 103 105 58 57 52 Methidathion 10-30 93 92 89 83 74 76

Mevinphos (E) 15-45 92 95 96 83 74 90 Mevinphos (Z) 15-45 99 95 99 85 67 101

MOX 5-15 100 100 77 91 83 83 Pirimiphos-ethyl 25-75 95 86 97 60 50 48

Pirimiphos-methyl 25-75 121 91 83 74 67 75 * Average values for three levels of fortification.

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Table 4 Validated parameters for the proposed method

Analyte Linear

range a r2 solvent

curve

r2 matrix

curve

LOD a LOQ a CC± a CC² a

ABA 0-20 0.982 0.976 0.43 0.86 14,02 18,04

Acephate 0-40 0.992 0.991 0.56 1.12 _ _

AFM1 0-1.0 0.997 0.993 0.02 0.04 _ _

Azinphos- ethyl 0-110 0.999 0.982 0.67 1.34 _ _

Azinphos- methyl 0-104 0.996 0.994 0.99 1.98 _ _

Diazinon 0-20 0.998 0.992 0.13 0.25 _ _

DOR 0-30 0.979 0.965 0.28 0.56 20.22 26.09

EPR 0-40 0.996 0.993 0.84 1.68 22.84 25.67

IVR 0-20 0.992 0.974 0.42 0.84 13.76 17.51

Methamidophos 0-20 0.990 0.994 0.42 0.84 _ _

Methidathion 0-40 0.996 0.995 0.05 0.10 _ _

Mevinphos (E) 0-60 0.998 0.996 0.16 0.32 _ _

Mevinphos (Z) 0-40 0.997 0.996 0.11 0.22 _ _

MOX 0-20 0.991 0.976 0.28 0.56 11.03 12.05

Pirimiphos-ethyl 0-100 0.993 0.996 0.27 0.54 _ _

Pirimiphos-methyl 0-100 0.995 0.995 0.08 0.16 _ _

a µg L-1

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Table 5 Method recovery and repeatability

Analyte Level 1 (0.5 x MRLb)

Recovery(%)/RSD(%)a

Level 2 (MRLb)

Recovery(%)/RSD(%)a

Level 3 (1.5 x MRLb)

Recovery(%)/RSD(%)a

ABA 77 /13 81/6.7 91/8.9 Acephate 99 /4.5 87/1.6 100/3.6

AFM1 85 /16.2 97/14.5 91/14.8 Azinphos-ethyl 105 /5.9 97/3.6 101/1.7

Azinphos-methyl 101 /2.5 94/2.2 97/1.1 Diazinon 102 /7.9 90/12.6 85/12

DOR 73 /3.4 80/6.7 80/7.8 EPR 98 /13.7 110/6.6 108/6.1 IVR 87 /9.8 107/10.6 109/5.6

Methamidophos 108 /5.6 102/2.3 98/3.8 Methidathion 97 /12.6 81/8.0 87/5.6

Mevinphos (E) 107 /3.4 105/0.9 99/1.1 Mevinphos (Z) 83 /9.7 81/1.7 94/2.4

MOX 77 /2.8 91/2.4 87/2.5 Pirimiphos-ethyl 108 /6.2 83/11.5 81/9.6

Pirimiphos-methyl 95/7.2 93/4.2 99/1.8 a- Relative standard deviation (n=6).

b- Maximum residue limit established by Brazilian legislation (µg L-1).

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Introduction