APPLICATION NOTE #20149

Pesticide Detection in Food Products through Bead Mill
Homogenization and QuEChERS Extraction
Narong Chamkasem PhD1, Southeast Regional Laboratory, FDA

The QuEChERS method was introduced
in 2003 for “Quick, Easy, Cheap, Effective,Rugged and Safe” extraction of multiple
pesticides and has since been modified
and optimized to support a wide rangeof analytes (1). The QuEChERS method
is widely used and is formalized in two
documented methods, AOAC 2007.01
and EN 156223. Prior to the QuEChERS
extraction the sample must first be
comminuted. The comminuting process
can be performed in a number of ways
including, manual chopping, blending
and milling. A more recent approach has
been to homogenize the plant material
through bead beating, a process in which
the plant material is placed in a sealed
tube with beads and vigorously shaken
to produce a final homogenate of sub
micron particle sizes (2). The advantage ofthis approach is that the homogenization
can be performed in a 50 mL centrifuge
tube containing 15 g of plant material
and 5 to 15 mL of acetonitrile which is thestarting solvent for common QuEChERSextractions.
Herein, we describe the applicationof bead mill homogenization andQuEChERS extraction for the detectionof pesticides in food products. The 50 mL
tube carriage for the Bead Mill 24
supports simultaneous homogenizationof up to three samples in a standard 50
mL polypropylene centrifuge tube andprovides sufficient force to homogenize
even extremely hard samples such as
seeds and roots.

Materials & Methods
Materials
Apples, grapes, carrots and wholesoybeans were obtained from a local
supermarket.
Equipment•Bead Mill 24 Homogenizer(Cat # 15340163)•4000 Q-Trap mass spectrometer(AB Sciex) interfaced to a LC-20AD
HPLC (Shimadzu) with a 100 x 2.1 mm
ultra aqueous C18 column (Restek).

minute linear gradient from 60 to 95%
Buffer B. Buffer A was 4 mM ammoniumformate, 0.1% formic acid and Buffer B
was methanol, 4 mM ammonium formate
and 0.1% formic acid. Samples were
electrosprayed at a voltage of 4500 V
and source temp of 350°C. Twenty six
transitions were monitored for detectionof the thirteen pesticides as shown in
Table 1.
continued

Table 1
PeakQ1

Q2

RT

Pesticides

192.2

160.24

Carbendazim.1

192.2

132.1

4

Carbendazim.2

202.1

145

6

Carbaryl.1

202.1

127

6

Carbaryl.2

3

318

160

6.8

Phosmet.1

318

133

6.8

Phosmet.2

4

412.1

328.1

7

Mandipropamide 1

412.1

356.1

7

Mandipropamide 2

331

99.1

7.2

Malathion.1

331

127

7.2

Malathion.2

249.1

160

7.2

Linuron.1

249.1

182.1

7.2

Linuron.2

343

307

7.3

Boscalid.1

343

140

7.3

Boscalid.2

200

107

7.3

Pyrimethanil.1

200

82

7.3

Pyrimethanil.2

325

1088.1

Cyazofamid.1

325

261.1

8.1

Cyazofamid.2

388

194

9.8Pyraclostrobin.1388

163

9.8

Pyraclostrobin.2#1Sample Preparation
The produce was manually diced and ~15grams of material was placed in a 50 mL
polypropylene centrifuge tube containing
15 g of yttria-stabilized zirconium oxide
ceramic beads.
The produce samples were diluted with
15 mL of acetonitrile with 0.1 ppm d10chlorpyrifos (as an internal standard) andhomogenized on the Bead Mill 24 at 6 m/s
for 30 seconds. 6 g MgSO4 and 1.5 g NaCl
was then added to the sample posthomogenization. The tubes were returnedto the Bead Mill 24 and shaken for 10
seconds at 6 m/s then centrifuged for 10
minutes at 3500 rpm. 0.5 mL of the top
layer containing acetonitrile and extracted
pesticides was removed and mixed with
0.5 mL of methanol.
Mass Spectrometry
1 µL of the mixture was analyzed by LCMS/MS on a 4000 Q-Trap tandem mass
spectrometer. Pesticides were separated
by reverse phase chromatography on aRestek Ultra Aqueous C18 column (100 x2.1 mm) during a 4 minute linear gradientfrom 5 to 60% buffer B followed by a 7.525678910111312IS

406.1

251.1

11.2

Difenoconazole.1

408.2

253.1

11.2

Difenoconazole.2

748.5

142.2

12

SpinetoramA.1
SpinetoramA.2

748.5

98.1

12308.1

197.1

12.6

Quinoxyfen.1

308.1

162.1

12.6

Quinoxyfen.2

324

260

11.6

d10-chlorpyrifos

Results

Figure 2 Pesticides were extracted and analyzed by LC-MS/MS operated in MRM mode

Pesticide extraction efficiency is insome part dependent on the ability
to effectively homogenize thesample. As pesticides are presenton both the fruit and vegetableskin and the interior flesh, it isadvantageous to reduce the sample
to a homogeneous mixture prior to
pesticide extraction. However, thisprocess can be challenging due to thevast diversity of sizes and densities oftarget plant and food products thatneed to be analyzed. Furthermore,
many pesticides are volatile or semivolatile and are not amendable tohomogenization methods which
generate large amounts of heat.

Grape

Apple

SoybeanCarrots

In this study we evaluated theuse of bead milling for sample
comminution on four common fruits
and vegetables prior to pesticide
extraction using the QuEChERS.
Samples were homogenized in thepresence of acetonitrile on the Bead
Mill 24 outfitted with a 50 mL tube
carriage. Full homogenization wasachieved in 30 seconds forall sample types. Figure one is animage of the grape sample pre andpost homogenization (Figure 1).
Pesticides were then extracted andanalyzed by LC-MS/MS operated in
MRM mode (Figure 2).
Of the twelve pesticide transitions
monitored two were detected in
the apple, eight in the grape, one in
the soybean, and four in the carrot
samples (Figure 1). The internal
standard d10-chlorpyrifos was also
detected in each sample.

Figure 1
Grape sample
pre and post
homogenization

References
1.

M. Anastassiades, S. J. Lehotay,
Fast and easy multiresidue
method employment
acetonitrile extraction/partitioning and “dispersivesolidphase extraction” fordetermination of pesticide
residues in produce. J. AOAC Int.
2003, 86, 412-431.

Bead Mill 24
2.

J. Wong et al, Development
and interlaboratory validationof QuEChERS-based liquid
chromatography-tandem
mass spectrometry method for
multiresidue pesticide analysis.
J. Agric. Food. Chem. 2010, 58,5897-5903.

Part Numbers Referenced
Bead Mill 24 Motor Unit: 15340163