APPLICATION NOTE #20147

Determination of Pesticide Recovery Rates
from Fruit and Vegetables using QuEChERS Extraction andBead Mill Homogenization on the Bead Mill 24
Shari Garrett and James Atwood PhD

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 andEN 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 placedin a sealed tube with beads and vigorously
shaken to produce a final homogenate of
sub micron particle sizes (2). The advantageof this 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 the startingsolvent for a standard QuEChERs extraction.
Herein, we evaluate the effectiveness ofbead mill homogenization on the Bead Mill
24 for multiple pesticide extraction using
the QuEChERS AOAC 2007.01 method. The
newly designed 50 mL tube carriage for theBead Mill 24 supports simultaneous
homogenization of up to three samples in astandard 50 mL polypropylene centrifuge
tube and provides sufficient force tohomogenize even extremely hard samples
such as seeds and roots.

Materials & Methods
Reagents

17 component pesticide standard wasobtained from Restek (Cat#31155). Q-SepQuEChERs salts and dispersive solid phaseextraction (dSPE) materials were purchasedfrom Restek (Cat#26238 and 26222). HPLC
grade acetonitrile was purchased fromFisher.

Table 1 Product Typeand Bead Mill 24 Settings
Produce

Speed(m/s)

Processing
Time (sec)Strawberry

4.230

Standards

Soybean

5.030

The 17 component pesticide standards wereobtained at 300 µg/mL concentration foreach component. A 7.5 µg/mL spiking
solution was created by diluting 5 µL of thestock solution in 195 µL of acetonitrile(0.1% acetic acid).

Apple5.030

Post processing, the samples were visuallyinspected and determined that complete
homogenization was achieved (Figure 1).

Sample Preparation
Certified organic strawberries, soybeans(shells removed) and apples were purchasedfrom a local grocery store. The produce wasmanually diced and ~15 grams of material
was placed in a 50 mL polypropylene
centrifuge tube containing 15 g of yttriastabilized zirconium oxide ceramic beads .
200 µL of pesticide spiking solution was
gently pipetted over each produce sample tocreate a final pesticide concentration of 100
ppb and the tubes were rocked for one
minute to disperse the pesticides. The spiked
samples were incubated at 4°C overnight.
Comminution
The spiked produce samples were diluted
with 5 mL of acetonitrile (0.1% acetic acid)and homogenized on the Bead Mill 24
using the settings outlined in Table 1. It wasdetermined in a previous study that theoptimum amount of buffer for 15 g ofstarting material was between 4-6 mL in a50 mL centrifuge tube. Increased amountsof buffer in excess of 6 mL resulted in poor
homogenization efficiency and increased
processing time requirements.

Figure 1 Produce Samples Pre andPost Homogenization. 15 g samples ofstrawberry, soybeans, and apples werehomogenized on the Bead Mill 24 with
15 g of ceramic beads in 30 seconds.

12Materials & Methods (continued)
Pesticide Extraction
10 mL of acetonitrile was added to eachhomogenate followed by the additionof one Q-Sep AOAC Method packet
containing 6.0 g magnesium sulfate and1.5 g sodium acetate. The samples werereturned to the Bead Mill 24 and gently
shaken for 1 minute at a speed of 0.8 m/s.
The samples were centrifuged at 3000rpm for 2 min in a Hettich Rotanta HP
centrifuge. The supernatant was removed
and placed in a 15 mL centrifuge tube
containing 1.2 g magnesium sulfate, 400mg PSA, 400 mg C18 and 400 mg
graphitized carbon (Restek Cat#26222),
shaken for 1 minute then centrifuged
to pellet solid material. 500 µL of thesupernatant was extracted and placedin an autosampler vial for GC/MS.

Figure 2
GC-MS Total Ion
Chromatogram –100 ppb Spiked
Strawberry
Sample.
TIC displays theelution profile ofthe 17 pesticide
standards
extraction fromthe 100 ppb spiked
strawberry sample
post QuEChERs
extraction.

151651393142711610817

Mass Spectrometry
Extracted pesticides were analyzed on aHP 5890 Series II GC fitted with a Rtx-5 30m x 0.25 mm x 0.25 um column, hydrogen
carrier gas and injection temperature at250°C. Injection volume was 1 µL withcolumn heating increasing from 100 to330°C and the MS scan range set from50 – 600 m/z.

Results
The sensitivity of any QuEChERs based
pesticide analysis is in some part
dependent on the ability to effectively
homogenize the sample. As pesticides arepresent on both the sample surface andinterior, the sample must first be reduced
to a single homogeneous mixture prior
to extraction. However, this process canbe challenging due to the vast diversityof sizes and densities of target plant andfood products that need to be analyzed.
Furthermore, many pesticides are volatileor semi-volatile and are not amendable tohomogenization methods which generatelarge amounts of heat.
Bead milling is an attractive method forfront end pesticide sample prep due toits ability to rapidly homogenize multiple
hard or soft samples in a variety of sizes
while maintaining a relatively low levelof temperature rise. In this study, three
produce samples with varying degreesof density were spiked with 17 standard
pesticides at a concentration of 100 ppb(100 ng/g) (Table 2).

14

Table 2 17 Pesticide Compounds in Spiked Mixture
Percent Recovery 100 ng/g Fortified Sample#Pesticide

Strawberry

Soybeans

Apple1

Vinclozolin

69.5

75.4

68.9

2

Carbaryl

71.4

80.7

69.6

3

Metalaxyl

82.4

88.1

78.9

4

Methiocarb

90.9

83.4

74.1

5

Cyprodinil

78.1

84.875.1

6

Thiabendazole

87.9

81

83.2

7

Captan

65.4

60

72.5

8

Folpet

58.7

59.6

69.9

9

Imazalil

86.9

85

83.3

10

Myclobutanil

91.5

80.4

75.8

11

Fenhexamid

75

85.3

69.9

12

Iprodione

82.5

77.5

58.4
96.4

13

Bifenthrin

89.4

86.7

14

Fenpropathrin

91.7

76.4

83.7

15

cis-Permethrin

104.3

94.8

88.8

16

trans-Permethrin

95.7

80.9

89.5

17

Deltamethrin

76.4

88.9

91.7

Pesticides were extracted from three produce samples and analyzed by GC-MSto determine percent recoveries

Results (continued)

References

Homogenization of the three produce
samples was accomplished in 30 secs withno detectable heat increase (Figure 1).
While the strawberry sample had intact
seeds visible post homogenization, boththe apple flesh and peel were completely
homogenized with no visible peel present
after processing.

1.

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

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.

Following extraction, each pesticide
mixture was analyzed by GC-MS to quantify
the pesticide yields generated from the
combined bead milling and QuEChERs
process (Figure 2). Individual peak areas
were calculated and compared to thestock mixture of known concentration
to determine recovery values. Pesticide
recoveries ranged from 58.4 to 104.3%
based on the analyte and produce material
with the highest recovery being seenfor strawberry and apple samples. Thismay be due to differences in the sample
composition between soybeans and thefruits analyzed in this study.

Conclusion
The Bead Mill 24 supports bead milling involumes up to 50 mL and has been
demonstrated to effectively disrupt both
soft and hard samples. When combinedwith the QuEChERs pesticide extraction
method, high recoveries can be achieved
from a variety of produce types. Futurestudies will aim at expanding
to a wider range of sample matrices
and target analytes to further evaluate
the effectiveness of the bead milling
approach as a comminution solution.

Part Numbers Referenced
Bead Mill 24 Homogenizer:
15340163

935-C Cobb Place Blvd. NW
Kennesaw, GA 30144
800.776.4431 • 770.421.0058
www.omni-inc.com