Application of zirconium dioxide nanoparticle sorbent for the clean-up step in post-

harvest pesticide residue analysis Ana Uclés Moreno1, Sonia Herrera López1, María Dolores Hernando2, Roberto Rosal3 Carmen Ferrer1 and Amadeo R. Fernandez-Alba1

1 European Union Reference Laboratory for Pesticide Residues in Fruits and Vegetables. Pesticide Residue Research Group. University of Almeria. 04120 (Spain); e-mail:

anauclesm@ual.es 2 National Institute for Agriculture and Food Research and Technology, INIA, 28040, Madrid, Spain.

3 Department of Chemical Engineering, University of Alcalá, 28771 Alcalá de Henares, Spain.

INTRODUCTION

Acknowledgements: The authors wish to acknowledge the Spanish Ministry of Science and Innovation for its economic support (NANOAQUAL project, Ref. CTM2008-04239)

EXPERIMENTAL

The complexity of certain matrices can cause problems with the ion production efficiency and with the analytical instruments’ detection systems . Consequently, there is interest in the introduction of any effective

new extraction modification for matrix-dependent clean-up combinations. A possible alternative to these clean up combinations is to use yttria-stabilized zirconium dioxide nanoparticles (ZrO2/Y2O3), which have

a high surface area (>100 m2/g), in the clean-up system to simultaneously obtain excellent purification effects and satisfactory results for multiple pesticides. Sorbents containing ZrO2 can be used as the clean-up

material for pesticide analysis in difficult matrices; these are better than PSA or C18 at removing fatty acids, esters of fatty acids, sterols and carboxylic acids.

Orange and pear blanks extracts were spiked with 18 pesticides, diluted 10 times and analyzed by UPLC-QqQ-MS/MS. a reversed-phase C8 column of 2.1 mm × 100 mm and 1.8 µm was used. Data were used to

evaluate recoveries at 10 and 100 µg Kg-1, linearity (r2), LOQs, matrix effects (%), precision (RSD%), repeatability and reproducibility . To evaluate the amount of matrix compounds in the final extract with the

different clean-up combinations, a LC-QTOF in full-scan mode was used. To characterize and determine the particle size of the ZrO2 nanoparticles, a scanning electron miscroscopy (SEM) was used. An

application of the method was carried out analyzing 20 real orange and pear samples

RESULTS

CONCLUSIONS

Sample handling UPLC-MS/MS

UPLC parameters:

- Injection volume: 5µL

- Flow rate: 0.3 mL/min

- Column: reversed-phase C8 1.8 μm 2.1 × 100 mm

- Mobile Phases and gradient :

· A H2O 0.1% formic acid

· B AcN 0.1% formic acid and 5% of water

Agilent 6490 QqQ

MS parameters: - Ion source: ESI

- Polarity: Positive and Negative

- Dynamic MRM software features

LC-QqQ-MS/MS total ion chromatograms of orange and pear extracts spiked at 10 µg Kg-1 and diluted 5 times.

Total Ion Chromatogram

%A

%B

Citrate buffered

QuEChERS

modification

Blank extracts

Spiking at appropriate level

Diluted 5 times

Clean up: d-SPE

MgSO4+ ZrO2

Agilent 6550 QTOF

- Full-scan mode

Scanning electron microscopy

HITACHI S-3500N

- High vacuum

- Acceleration voltage 5kV

LC-QToF-MS full scan chromatograms of blank orange and pear extracts diluted 5-

times obtained by using QuEChERS methodology and a different clean-up.

x105

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

0.1 0.5 0.9 1.3 1.7 2.1 2.5 2.9 3.3 3.7 4.1 4.5 4.9 5.3 5.7 6.1 6.5 6.9

Counts vs. Acquisition Time (min)

Orange

Pear

7 x10

0.2 0.4 0.6 0.8

1 1.2 1.4 1.6 1.8

2 2.2 2.4 2.6 2.8

3 3.2 3.4 3.6 3.8 1 1

Counts vs. Acquisition Time (min) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5

7 x10

0.3 0.4 0.5 0.6 0.7 0.8 0.9

1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

2 2.1 1 1

Counts vs. Acquisition Time (min) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5

MgSO4:PSA

MgSO4:ZrO2-Y2O3

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12

%

Time (min)

Gradient

Nº of compounds: 8,647 Nº of compounds: 7,938 MgSO4:PSA

MgSO4:ZrO2

Co-eluting matrix compounds of orange extracts using LC-QTOF-MS with the different clean-up procedures. The x-axis represents the retention time and the y-axis

the m/z.

APPLICATION OF THE METHOD

No. of sample

Orange samples Carbendazim Imazalil Pyrimethanil Thiabendazole

1 <LOQ nd nd <LOQ

2 <LOQ nd nd <LOQ

3 <LOQ <LOQ <LOQ <LOQ

4 <LOQ <LOQ <LOQ <LOQ

5 <LOQ nd <LOQ <LOQ

6 <LOQ 1006.9 <LOQ <LOQ

Pear samples

1 <LOQ <LOQ <LOQ <LOQ

2 <LOQ 755.2 892.6 <LOQ

3 63.9 1175.4 nd <LOQ

4 <LOQ 14.5 nd <LOQ

Concentration: µg kg−1; nd: not detected. <LOQ: detected but at lower concentration (<10 µg Kg-1).

Selected linear response

Linearity

• Linearity was evaluated at 7 calibration levels, from 1 to 500 μg/Kg (diluted 5 times).

• All the compounds had a R2 > 0.99.

CHARACTERIZATION BY SEM

Determination of the particle size of the ZrO2 nanoparticles by scanning electron miscroscopy (SEM)

-

10

20

30

40

50

60

70

80

90

100

nd <40% 40-70% 70-120%

% o

f co

mp

ou

nd

s

10 µg/Kg

Orange

Pear

-

10

20

30

40

50

60

70

80

90

100

nd <40% 40-70% 70-120%

% o

f co

mp

ou

nd

s

100 µg/Kg

Orange

Pear

Recoveries

METHOD VALIDATION

0

500000

1000000

1500000

2000000

2500000

0 100 200 300 400 500 600

Are

a

Concentration level (µg/Kg)

Diphenylamine

Solvent

Pear

Orange

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

0 100 200 300 400 500 600

Are

a

Concentration level (µg/Kg)

Methomyl

Solvent

Pear

Orange

EVALUATION OF MATRIX COMPOUNDS

• The method’s validation parameters in terms of recovery, quantification limits (LOQs), linearity (r2), matrix effects and intra-day and inter-day precision showed that the

proposed method meets the pesticide analysis requirements.

• It was applied for the determination of selected fungicides in 20 real orange and pear samples. Four different pesticide residues were detected in 10 of these commodities; 40%

of the samples contained pesticide residues at a quantifiable level (equal to or above the LOQs) for at least one pesticide residue.

• It has been demonstrated that yttria-stabilized zirconium dioxide can be used as an effective d-SPE material with the QuEChERS method acting as a suitable alternative material

to PSA in the extract cleanup of various matrices.