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QuEChERS adaptability for the analysis of pesticide residues in beehive products seeking the development of an agroecosystems sustainability monitor Silvina Niell, Florencia Jesus, Cecilia Perez Tabarez, Yamandu Mendoza, Rosana Diaz, Jorge Franco, Maria Veronica Cesio, and Horacio Heinzen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00795 • Publication Date (Web): 16 Apr 2015 Downloaded from http://pubs.acs.org on April 21, 2015

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Journal of Agricultural and Food Chemistry

QuEChERS adaptability for the analysis of pesticide residues in beehive products seeking the development of an agroecosystems sustainability monitor Silvina Niell1, Florencia Jesús1, Cecilia Pérez1, Yamandú Mendoza2, Rosana Díaz3, Jorge Franco4, Verónica Cesio5, Horacio Heinzen1,5 1

Universidad de la República, CENUR Noroeste, Departamento de Química del Litoral,

PAAP, Ruta 3 km 363, CP60000 Paysandú, Uruguay 2

INIA La Estanzuela, Apicultura, Colonia, Uruguay

3

Ministerio Ganadería Agricultura y Pesca-DIGEGRA Montevideo, Uruguay

4

Universidad de la República, Facultad de Agronomía, EEMAC, Ruta 3 km 363,

CP60000 Paysandú, Uruguay 5

Universidad de la República, Facultad de Química, Montevideo, Gral. Flores 2124,

CP11800 Montevideo, Uruguay

1

*e-mail address: [email protected]

2

1

ACS Paragon Plus Environment


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Abstract

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Beehive products could be powerful monitors of pesticide residues originated in

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agroecosystems during production cycles. Their ready availability provides enough

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samples to perform analytical determinations but their chemical complexity makes

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residue analysis a real challenge. Taking advantage of the plasticity of QuEChERS

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coupled to LC-MS/MS , validated methodologies were developed for bees, honey,

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beeswax and pollen and applied to real samples for the simultaneous determination of

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19 of the most employed pesticides in intensive cropping fields. Beehives placed in

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Uruguayan agroecosystems accumulated pesticides as thiacloprid, imidacloprid,

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methomyl, carbaryl, hexythiazox, azoxystrobin, pyraclostrobin, tebuconazole and

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haloxyfop-methyl at 0.0001 to 0.01 mg/kg levels. The oscillations on the amount and

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occurrence of residue findings for specific apiaries was correlated statistically with the

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sampling season and the agroecosystem where the beehives were located, showing the

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potential of bees and bee products to record relevant information to survey the

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chemicals applied in their surroundings.

19 20 21

Keywords

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QuEChERS, pesticide residues, beehive matrices, environmental monitor

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Introduction

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Recently, French beekeepers located 4000 m away from an biogas plant that has been

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processing waste from a Mars plant producing M&M’S® found their bees produced

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blue honey, due to the color of the confetti shell cover 1. The striking example shows

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the potential of bees as reporters of environmental information as they look for pollen

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and nectar. The different pollutants and agrochemicals they meet, which were applied in

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the field, are carried and stored in the hive. The final storage point in the hive of these

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xenobiotics depends mainly on their physicochemical properties and their mode of

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action. Beehive products could be powerful tools to monitor pesticide residues

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originated in different agroecosystems in production cycles. Previous reports all over

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the world showed the occurrence of pesticides in bee products trying to find a link

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between agrochemicals and the declining of bees 2-8. Bee declining is a major problem,

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not only for beekeepers but also for farmers and all the producers that depend on

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pollination to succeed in their agricultural activities. These findings have been focused

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on bee health and little attention has been paid to the sentinel behavior of bees as

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reporters of the environmental status of an agroecosystem. The ready availability of bee

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and beehive products provides enough samples to perform such determinations, but

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their chemical complexity makes the detection of residues an analytical challenge. In

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order to systematically analyze bees and bee products, straightforward analytical

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protocols for the determination of pesticide residues in these matrices are needed.

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Among the newest general methods for pesticide residue analysis, QuEChERS, the

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acronym for Quick, Easy, Cheap, Efficient, Rugged and Safe shows the highest

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potential to accomplish such task.

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9

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First developed for pesticide residue determination in produce, QuEChERS method

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spread its applications to a myriad of matrices proving to be a plastic template that can

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be adapted depending on the particular chemical properties of each matrix. This

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plasticity has been acknowledged in the two official methods for pesticide residue

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analysis, namely AOAC 2007.1 and the European Standard EN 15662, where the use of

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sorbents such as C18 and GCB are recommended depending on the matrix type.

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Particularly, QuEChERS variations have been employed for the analysis of pesticide

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residues in bees, bee collected pollen, beeswax and honey

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comprise the use of different strategies for the elimination of coextractives, e.g. in the

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case of bees a hexane partition of the acetonitrile (MeCN) extraction solution is used to

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remove lipids from the extract

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combination of sample amount, extraction temperature and clean up methodology using

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freeze out, PSA, C18 and GCB as sorbents for beeswax pesticide residues analysis 14.

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This approach was more effective than Matrix Solid Phase Dispersion (MSPD) that

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gave a high load of coextractives that spoiled the chromatographic system after a few

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injections. Pesticide residues in bees were analyzed using the MeCN based extraction

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followed by different combinations of sorbents that optimized the performance of the

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method for the investigated analytes

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honey is a simpler matrix, and due to its importance as food, it has been widely

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analyzed following different protocols. CEN 15662 QuEChERS has been smoothly

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applied to pesticide residue analysis in honey by our group

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extensive surveys of pesticide residues in bees and beehives in North America and other

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parts of the world have shown the presence of important amounts of pesticide residues

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in beehives, looking for possible causes of bee declining

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classes found were insecticides and fungicides, being the latter the most ubiquitous

2, 7, 10-13

. The methods

11

. Recently, our group reported an optimized

10, 114

. Comparatively with pollen, bees and wax,

15

. As pointed out above,

2-85

. The major pesticide

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among North American apiaries. Chlorothalonil, strobilurins as well as some azoles

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accounted for the more frequent findings. The relationship of these pesticide cocktails to

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bee health is an open question that deserves a multiple approach to solve it, but at the

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same time, the vast array of residue findings in beehives supports the idea that bees

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collect what is in the environment, an ideal characteristic for an environmental monitor

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3, 16-24

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In the present communication, the application of QuEChERS protocols for pesticide

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residue analysis in bees and bee products is reported. After studying the data gathered, a

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novel, first approach to check the recording of season variations in pesticide residues

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profiles in beehives within an agroecosystem is explored along with other statistical

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analysis in order to check the viability of beehives as environmental monitors.

.

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Materials and methods

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Chemicals and standards

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MeCN and n-Hexane of HPLC quality was from Pharmco Products Inc. (Brookfield,

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CT, USA). Water was deionized in the laboratory using a Thermo Scientific (Marietta,

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OH, USA) EASYpure RoDi Ultrapure water purification system. Magnesium sulfate

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anhydrous, reagent grade was from J.T. Mallinckrodt Baker Inc. (Phillipsburg, NJ,

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USA) and formic acid p.a. 88% was purchased from Macron chemicals (Netherlands).

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A solution of 5% formic acid (V/V) was prepared in MeCN. The bulk amino sorbent

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(PSA, 40-60 µm), RP-C18 and graphitized carbon black (GCB) were from Scharlab

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(Barcelona, Spain). Analytical standards, of purity ≥95%, were from Dr. Ehrenstorfer

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(Augsburg, Germany).

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Stock standard solutions of 1 mg/mL were prepared in MeCN and working standard

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mixtures were prepared by appropriately diluting multiple stock solutions with MeCN.

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The pesticides studied were all LC amenable pesticides, no GC amenable pesticides

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were included. They were selected among the most used in extensive plantations in

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Uruguay aiming to check the model suitability. All working solutions were stored in the

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dark at 4 °C.

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Apparatus

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LC–MS/MS was performed with an Agilent 1200 LC system coupled to a 4000

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QTRAP® LC/MS/MS System from AB SCIEX™ run in the Scheduled® MS/MS-

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mode. LC-Separation was performed on a ZORBAX Eclipse XDB-C18 (150 mm×4.6

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mm, 5 µm) column. The operation of the LC gradient involved the following elution

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programme: A: water/HCOOH 0.1% (V/V); B: MeCN. It was run at 600 µL min−1

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starting with 10% component B at injection time during 1 min and gradually changing

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to 100% B over 15 min. This mobile phase was kept for 10 min and then shifted back to

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the starting conditions (10% component B) and kept there until 35 min after injection.

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The injection volume was 5 µL. MS/MS detection was performed in the multiple

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reaction monitoring (MRM) mode using an ESI interface in the positive ion mode. The

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ionization voltage was 4500 V, the nebulizer gas was synthetic air at 70 psi, and the

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curtain gas was nitrogen at 30 psi. The solvent evaporation in the source was assisted by

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a drying gas (heated synthetic air at 425 °C/ 50 psi). The optimal MRM transitions,

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collision energies and declustering potentials for each investigated compound, were

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determined infusing with a syringe directly the standard solutions to the instrument at a

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constant flow. The MS/MS settings used in this study are listed as Supplementary

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Information. 6


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Methodology

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Sampling design

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Seven apiaries located in different production zones of Uruguay were selected based on

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their health history and management. All the apiaries have at least 20 beehives. From

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each apiary, samples of bees and honeycombs were taken randomly from five beehives

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in fall, winter and spring. Then the samples were driven to the laboratory where they

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were joined, obtaining one composed sample per beehive product representing each

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apiary. The beehive matrices were obtained allowing honey to drain. Once the wax was

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honey free, the pollen inside the cells was collected digging into them one by one.

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Sample preparation

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Pesticide residue analysis in beeswax

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The methodology employed for the pesticide residue analysis in beeswax was the

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already described in a previous work by our group 8. Shortly, 2 g of beeswax is

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extracted with 10 mL MeCN at ∼80 °C. Then, the extract is freezed-out and cleaned-up

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with 25 mg of PSA primary−secondary amine (PSA) and 25 mg of C18 sorbent per

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milliliter of extract. Finally the extract is acidified with 5% formic acid solution in

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MeCN (v/v) (10 µL/mL extract) and injected in LC-MS/MS.

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Pesticide residue analysis in honey

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The methodology employed for the pesticide residue analysis in honey was the already

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described in a previous work by our group 9. Shortly, 5 g of honey are extracted with 10 7



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mL water and 10 mL MeCN. Then the mixture of citrate buffer salts is added. The

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extract is cleaned-up with PSA 25 mg per milliliter and MgSO4 150 mg per milliliter.

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Finally the extract is acidified with 5% formic acid solution in MeCN (v/v) (10 µL/mL

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extract) and injected in LC-MS/MS.

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Pesticide residue analysis in bees

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50 g of freezed honeybees were thoroughly comminuted and homogeneized with a hand

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blender, then 2 g were weighed in a 50 mL centrifuge tube and 5 mL of water; 10 mL

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MeCN were added and shaken vigorously 1 min, 0.5g disodium hydrogencitrate

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sesquehydrate, 1 g trisodium citrate dihydrate, 4 g anhydrous magnesium sulphate, and

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1 g sodium chloride were added, shaken again and the tube was centrifuged 5 min at

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3000 U/min. The extract was freezed-out overnight and cleaned-up with dispersive

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Solid Phase Extraction: PSA and C18 25 mg/mL and MgSO4: GCB (59:1) 150 mg/mL,

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vortexed 30 s, centrifuged 5 min 3000 U/min. The aliquot for LC-MS/MS analysis is

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acidified with HCOOH 5% in MeCN 10 µL/mL.

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Pesticide residue analysis in pollen

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Pollen isolated from the sampled portions of honeycomb was thoroughly comminuted

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and homogeneized with a hand blender, then 5 g were weighed in a 50 mL centrifuge

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tube and 5 mL of water; 10 mL MeCN were added and shaken vigorously 1 min, 1 g

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sodium acetate, 4 g anhydrous magnesium sulphate, and 100 µL acetic acid were added,

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shaken again and the tube was centrifuged 5 min at 3000 U/min. The extract was

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cleaned-up with dispersive Solid Phase Extraction: PSA and C18 25 mg/mL and 8


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MgSO4 150 mg/mL, vortexed 30 s, centrifuged 5 min 3000 U/min. Finally the cleaned

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extract is transferred into a screw cap vial and injected in LC-MS/MS.

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Statistical analysis

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Statistical analysis were performed using R, which is a free software environment for

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statistical computing and graphics 25.

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Besides the statistical description by average values and standard errors two kind of

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statistical analysis were done: a Multiple Factorial Analysis (MFA, Scofier and Pages,

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1998) 26, 27 and the calculus of the Gower (1971) 28 distance. The MFA analysis is done

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in two steps: first a principal components analysis (PCA) is made per group of variables

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(groups being bees, wax, pollen and honey) and the original values are standardized by

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the square root of the first eigenvalue from each group analysis, second a new global

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PCA is made using the standardized values; that kind of analysis allows to show the

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relation between concentration of products per group in a two dimensions figure in such

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a way that the groups near in the figure are more similar in concentration of products.

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The Gower distance is a distance based in the absolute values of differences between

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statistical samples, in this case the samples are the apiaries in each season and what is

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compared is the pesticide residues profile obtained in the four matrixes analyzed.

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Calculus:(value – minimum) / range.

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This distance allows the presence of missing values in some comparisons and results are

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in the (0-1) interval allowing an easy interpretation.

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For all the calculations, the concentration of coumaphos found for the different apiaries,

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was not considered as the acaricide is an agrochemical commonly employed in

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apiculture, and has no meaning when considering the agroecosystem under study.

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Results and Discussion

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The extracts produced had a significant load of co-extractives. To overcome this

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difficulty and obtain low LODs an LC-MS/MS using the scheduled option in the

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multiple reaction monitoring (MRM) mode with optimized transitions, collision

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energies and declustering potentials for the 19 LC amenable currently employed

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pesticides in Uruguayan agroecosystems was used, as continuous scanning of the whole

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mass range lead to higher LODs and LOQs.

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Methods validation

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In agreement with the analytical quality control procedures document by DG-SANCO 29

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five replicates of spiked blanks of bees at different levels (0.2; 0.1; 0.05; 0.01; 0.001;

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0.0001 mg/kg) and of pollen (0.05; 0.01; 0.001; 0.0001 mg/kg) were analyzed to assess

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accuracy (% recovery) and repeatability (% RSD) of the procedure (Tables 2 and 3).

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Limits of Quantification (LOQs) were considered as the lowest successfully validated

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levels, i.e. the levels where acceptable recoveries (70-120 %) and RSDs (

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