2. EXPERIMENTAL
3.4. Chromatogram
2.5. Instrumental conditions
RT: 0.00 - 16.01
MS instrument
Thermo TSQ Vantage triple
Sepax HP-C18, 2.1*100 mm, 3 μm
Restek C18, 2.1*20 mm, 3 μm
Polarity
ESI +
Guard column
Spray Voltage
3000 V
Column temperature
Ambient
Vaporizer Temperature
350 °C
Autosampler
15 °C
Ion Transfer Capillary Temperature
300 °C
Injection volume
10 μL
Sheath Gas Pressure
40 arbitrary units
Mobile phase A
0.1% formic acid in Milli-Q H2O
Auxiliary Gas Pressure
10 arbitrary units
Mobile phase B
0.1% formic acid in MeOH
Q1 and Q3 Peak Width (FWHM)
0.7 Da
Flow rate
200 μL/min
Collision Gas and Pressure
Argon at 1.5 mTorr
Time (min)
Mylar pouch with 4000 mg MgSO4 and 2000 mg NaCl
dSPE cleanup tubes with GCB
(UCT part#: CUMPSC1875CB2CT)
2 mL centrifuge tube with 150 mg MgSO4, 50 mg
PSA, 50 mg C18, and 7.5 mg GCB
dSPE cleanup tubes with ChloroFiltr®
2 mL centrifuge tube with 150 mg MgSO4, 50 mg
PSA, 50 mg C18, and various amounts of ChloroFiltr ®
Dwell time
Gradient program
50 mL polypropylene centrifuge tubes
Extraction salts
(UCT part#: ECQUUS2-MP)
95
95
50
5
5
100 ms
95
95
40
20
5.60
Precursor
ion
192.093
Product ion
1
132.080
29
31
175.070
23
183.140
22
66
77.030
40
93.050
33
88
TPP (IS)
327.093
77.020
37
152.070
33
98
100
80
Diazinone
305.135
153.090
15
169.08
14
89
Pyrazophos
374.103
194.060
20
222.130
20
104
Chlorpyrifos
349.989
96.890
32
197.940
17
7.16 7.35
5.74 6.31
8.27
8.52 8.76 9.28 9.54
9.87
10.38 10.77
10.46
5.27 5.53
6.51
6.70 6.88 7.44
7.94 8.38
8.59 8.80
10.74
9.65 9.84
Cyprodinil
40
20
12.00 12.51
0
0
1
2
4
3
5
6
7
8
Tim e (m in)
9
10
11
69
Relative Abundance
3.1. Comparison of ChloroFiltr® vs. GCB on planar pesticide recovery
Pesticide Spiking Solution
11.40
Thiabendazole
Pyrimethanil
CAS 10605-21-7
pKa = 4.88
CAS 148-79-8
pKa = 4.64
CAS 53112-28-0
pKa = 3.52
O
H
N
OCH3
NH
N
N
N
N
CAS 13457-18-6
pKa not reported
dSPE
tube
O
N
O
Cyprodinil
Diazinone
CAS 121552-61-2
pKa = 4.44
H3C
CAS 333-41-5
pKa = 2.6
Chlorpyrifos
CAS 29121-88-2
pKa = 4.55
H
N
N
N
N
CI
N
N
S
O
P O
O
CI
S N
O
N
A
O
S
P
O
O
MgSO4
(mg)
PSA
(mg)
C18
(mg)
50
GCB
(mg)
ChloroFiltr®
0
7.5
150
50
B
150
50
50
0
15
150
50
50
0
25
D
150
50
50
0
50
E
150
50
50
7.5*
0
*7.5 mg GCB is chosen for the cleanup of dark green extracts as
stated in Method EN 15662.
P
O
Relative Abundance
20
11.22
Relative Abundance
80
CI
2.4. Matrix-matched calibration curves
Figure 1. Chemical structures of the seven pesticides included in this study
WWW.UNITEDCHEM.COM
TIC F: + c ESI SRM
m s 2 349.989
[96.390-97.390,
197.440-198.440]
MS Spike50_4
20
11.35
2
3
4
5
6
7
8
9
10
11
12.19 12.82 12.90
12
13.86
13
14
14.14
15
3.5. Matrix-matched calibration curves
The response of the seven matrix-matched calibration curves were found to be linear over the 2-400 ng/mL concentration range.
The correlation coefficients (R2) were better than 0.9982. The limit of quantification (LOQ) of the method was found to be 2 ng/g for
all seven pesticides.
Thiabendazole
Diazinone
Y = -0.00589376+0.00846086*X R^2 = 0.9991 W: 1/X
Y = -0.00975634+0.0224835*X R^2 = 0.9986 W: 1/X
5
3.0
0.6
0.4
8
4
2.5
Area Ratio
Area Ratio
0.8
2.0
1.5
6
4
0.2
2
0.5
0.0
0.0
0
100
200
ng/ml
300
400
0
100
200
ng/ml
300
400
dSPE cleanup C
(25 mg ChloroFiltr®)
dSPE cleanup D
(50 mg ChloroFiltr®)
dSPE cleanup E
(7.5 mg GCB)
Figure 3. Visual comparison of extract cleanliness after dSPE cleanup with
different amounts of ChloroFiltr®, and 7.5 mg GCB
3.3. Recovery study
The spinach sample that confirmed the absence of the target pesticides was used for recovery study at three spiking levels: 10,
50, and 100 ng/mL. The fortified spinach samples were extracted using a QuEChERS approach and cleaned up with dSPE containing
150 mg MgSO4, 50 mg PSA, 50 mg C18 and 50 mg ChloroFiltr®,. The recoveries ranged from 87.1 to 108.7% with relative standard
deviations (RSD) less than 6.1%, indicating that this method is suitable for the analysis of planar pesticides in spinach samples.
Fortified at 10 ng/g
RSD%
(n=4)
94.9
6.1
5.8
Fortified at 50 ng/g
RSD%
(n=4)
87.1
1.0
Recovery%
93.2
1.9
Fortified at 100 ng/g
RSD%
(n=4)
95.1
2.0
0
0
100
94.2
Pyrimethanil
100.0
4.1
97.3
1.2
94.8
2.6
Cyprodinil
98.6
1.0
91.2
0.5
98.2
87.2
3.1
1.7
Diazinone
108.7
3.6
104.5
2.3
100.2
1.0
Pyrazophos
99.7
2.9
92.0
0.9
87.7
2.1
Chlorpyrifos
100.9
5.5
95.6
2.5
94.9
1.8
99.6
4.1
94.4
1.5
94.0
2.0
200
ng/ml
300
400
Pyrimethanil
Cyprodinil
Chlorpyrifos
Y = -0.00184134+0.00285847*X R^2 = 0.9990 W: 1/X
Y = -0.00154409+0.00703027*X R^2 = 0.9991 W: 1/X
Y = -0.000204927+0.00100318*X R^2 = 0.9982 W: 1/X
1.2
3.0
1.0
2.5
0.8
2.0
0.6
0.4
0.40
0.35
1.5
1.0
200
ng/ml
300
400
0.25
0.20
0.15
0.05
0.00
0.0
100
0.30
0.10
0.5
0
100
200
ng/ml
300
400
0
4. CONCLUSIONS
A simple, sensitive, and effective method was developed for the determination
of planar pesticides in spinach samples. Spinach samples were extracted
using the original QuEChERS approach and cleaned up by dSPE containing
MgSO4, PSA, endcapped C18 and ChloroFiltr®. ChloroFiltr® is a novel
sorbent that effectively removes chlorophyll without sacrificing the recovery
of planar pesticides, offering a successful substitute for GCB in cleanup of
samples containing high chlorophyll levels.
Recovery%
Thiabendazole
2
1
0
Figure 5. Calibration curves of the seven pesticides in this study
dSPE cleanup B
(15 mg ChloroFiltr®)
3
1.0
0
dSPE cleanup A
(7.5 mg ChloroFiltr®)
Pyrazophos
Y = -0.00303411+0.012131*X R^2 = 0.9997 W: 1/X
3.5
0.0
Mean
NL: 2.44E4
40
0.2
Carbendazim
14.68
GCB
After undergoing cleanup with a variety of different sorbent combinations (Table 2), spinach extracts were visually assessed for
sample cleanliness and chlorophyll removal. As can be seen in Figure 3, the spinach extract decreases in chlorophyll content as the
quantity of ChloroFiltr®, is increased (A-D). Although GCB (E) is effective in removing chlorophyll, it contains slightly more chlorophyll than
that cleaned with 50mg ChloroFiltr®,, and results in low recovery of planar pesticides. Therefore, dSPE tube containing 150 mg
MgSO4, 50 mg PSA, 50 mg C18 and 50 mg ChloroFiltr® (UCT part #: CUMPSGGC182CT) was selected for spinach cleanup as it
gave efficient chlorophyll removal and excellent pesticide recoveries.
Compound
13.06 13.50 14.04
13.56
ChloroFiltr
Recovery%
Matrix-matched calibration curves were generated from the blank spinach extracts that were prepared by QuEChERS extraction and
dSPE cleanup with 150 mg MgSO4, 50 mg PSA, 50 mg C18 and 50 mg ChloroFiltr®. Appropriate volumes of the 0.5 and 5 ppm pesticide
standard solutions were spiked into the sample extracts to generate 6-point calibration curves with concentrations of 2, 10, 40, 100,
200, and 400 ng/mL.
12.64
60
Table 6. Accuracy and Precision Data
O
11.86
Chlorpyrifos
56
(mg)
C
NL: 4.14E5
TIC F: + c ESI SRM
m s 2 374.103
[193.560-194.560,
221.630-222.630]
MS Spike50_4
Figure 4. Chromatogram of spinach sample fortified with 50 ng/g pesticides and cleaned up by dSPE with ChloroFiltr®.
3.2. Visual assessment of extract cleanliness
Table 2 Sorbent
.
components in dSPE tubes for spinach cleanup
14.67
40
Carbendazim
Spinach extract
without cleanup
14.25
Tim e (m in)
Pyrazophos
NH
13.44
12.49
12.28
Pyrazophos
60
Y = -0.00144338+0.0024999*X R^2 = 0.9986 W: 1/X
1. Transfer 1 mL of the supernatant into 2 mL dSPE tubes containing different amounts of sorbents (listed in Table 2) and
shake for 30 sec.
2. Centrifuge at 10,000 rpm for 5 min.
3. Transfer 0.5 mL of the cleaned extracts into 2-mL autosampler vials.
4. The samples are ready for LC/MS/MS analysis.
N
S
500 g of fresh spinach purchased from a local grocery store was homogenized using a food processor.
10 g of the homogenized spinach sample was weighed into 50 mL centrifuge tubes.
Spike all fortified and blank control samples with 100 μL of the 50 ppm TPP IS solution.
Add appropriate amounts of the 5 ppm pesticide spiking solution to the fortified samples.
Vortex for 30 sec and equilibrate for 15 min.
Add 10 mL of MeCN and vortex for 1 min.
Add salts packed in a Mylar pouch containing 4000 mg MgSO4 and 2000 mg NaCl, shake vigorously for 1 min.
Centrifuge at 5000 rpm for 5 min. The supernatant is ready for cleanup.
NL: 9.34E5
TIC F: + c ESI SRM
m s 2 305.135
[152.590-153.590,
168.580-169.580]
MS Spike50_4
11.47
80
2.3.2. dSPE cleanup
Carbendazim
H
N
1.
2.
3.
4.
5.
6.
7.
8.
14.61
20
Area Ratio
To overcome the problems caused by use of GCB and to avoid the use of toluene, UCT has developed a novel polymeric sorbent, named
ChloroFiltr®, for the removal of chlorophyll from highly pigmented samples without sacrificing the recovery of planar analytes. This poster
details a study that compared the recovery of planar pesticides and extract cleanliness of fortified spinach samples that underwent dSPE
cleanup with ChloroFiltr® and GCB.
2.3.1. QuEChERS extraction
13.51 13.98
40
1.0
2.3. Procedure
13.20
60
1
A 50 ppm TPP internal standard (IS) solution was prepared by mixing 50 μL of the 5000 ppm TPP solution with 5 mL of MeCN.
All standards were stored in amber glass vials with Teflon lined caps in freezer (-20ºC) until use.
12.24
11.92
Diazinone
80
96 96
Figure 2. Comparison of pesticide recovery from extracted spinach samples
purified by dSPE with ChloroFiltr®, and GCB
Internal Standard triphenyl phosphate (TPP)
NL: 8.58E5
TIC F: + c ESI SRM
m s 2 327.093
[76.520-77.520,
151.570-152.570]
MS Spike50_4
20
0
92 93
16
TPP (IS)
Area Ratio
The two most commonly used sorbents in the dispersive solid-phase extraction (dSPE) cleanup step of the QuEChERS method include
primary secondary amine (PSA) and endcapped C18. PSA is effective for removal of acids and polyphenolic anthocyanin, while C18 effectively
removes lipids, carotenoid and xanthophyll. An alternative sorbent widely used for the effective cleanup of highly pigmented matrices,
including those containing chlorophyll, is graphitized carbon black (GCB). However, while GCB is effective for purifying samples, a major
disadvantage is its adsorption of target analytes that contain planar structures, resulting in low recovery of these compounds. Examples
of planar pesticides include carbendazim, thiabendazole, pyrimethanil, and cyprodinil. To release planar analytes from the GCB sorbent,
toluene can be added to the sample extract before dSPE cleanup. However, additional evaporation and reconstitution steps are needed
to remove toluene prior to LC analysis, as toluene causes solvent immiscibility that leads to peak shape issues. Many laboratories have
reduced their use of toluene due to its health risks.
71
100
79
Area Ratio
Spinach is a highly pigmented matrix containing chlorophyll, carotenoid, xanthophyll and anthocyanin pigments. Among these, chlorophyll
pigments have the greatest adverse effect on GC systems due to their non-volatile characteristics. When a sample containing chlorophyll
is injected into a GC, chlorophyll will accumulate in the GC inlet and on the front end of the column, generating active sites and affecting
GC performance. Therefore, it is essential to remove chlorophyll prior to analysis.
A 5 ppm pesticide spiking solution was prepared by adding 500 μL of the seven 100 ppm pesticide standards into a 10-mL volumetric
flask and diluting to volume with MeCN. A 0.5 ppm mixed pesticide standard solution was prepared by adding 1 mL of the 5 ppm
pesticide spiking solution into a 10-mL volumetric flask and diluting to volume with MeCN.
91
Area Ratio
1. INTRODUCTION
85
15.71
15
40
Area Ratio
2.2. Preparation of standard solutions
104
97
93
14
60
0
100
X I A O YA N WA N G , WAY N E K I N G , B R I A N K I N S E L L A A N D M I C H A E L J . T E L E P C H A K
87
13
11.78
0
100
Spinach samples fortified with 50 ng/g pesticides were extracted and cleaned up with dSPE tubes containing 150 mg MgSO4, 50 mg
PSA, 50 mg C18, and either 50 mg ChloroFiltr®, or 7.5 mg GCB. ChloroFiltr®, gave good recoveries for all 7 pesticides. The recoveries of
carbendazim, cyprodinil, pyrimethanil and thiabendazole were adversely affected by GCB, with thiabendazole in particular obtaining
much lower recovery with GCB (56% vs. 93% with ChloroFiltr®,). Diazinone, pyrazophos, and chlorpyrifos were unaffected by GCB
due to the presence of non-planar side chains in their structures.
110
100
90
80
70
60
50
40
30
20
10
0
14.25 14.93 15.19
13.10 13.54
12
RT: 0.00 - 15.02
3. RESULTS
U C T, 2 7 3 1 B A R T R A M R O A D , B R I S T O L PA 1 9 0 0 7 U S A
NL: 1.92E5
TIC F: + c ESI SRM
m s 2 226.122
[76.530-77.530,
92.550-93.550] MS
Spike50_4
11.57
60
0
100
COMPARISON OF GRAPHITIZED CARBON BLACK
AND A NOVEL SORBENT IN DISPERSIVE-SPE
CLEANUP OF SPINACH EXTRACT
NL: 8.74E4
TIC F: + c ESI SRM
m s 2 200.116
[106.560-107.560,
182.640-183.640]
MS Spike50_4
Pyrimethanil
80
107.060
81
NL: 1.93E5
TIC F: + c ESI SRM
m s 2 202.059
[130.560-131.560,
174.570-175.570]
MS Spike50_4
20
131.060
103
10.81
40
200.116
31
9.68 10.06
60
202.059
17
8.89
80
226.122
S-Lens
8.26 8.49
Thiabendazole
20
Cyprodinil
Thiabendazole
CE 2
6.95 7.69
6.88
40
Pyrimethanil
Carbendazim
6.78
6.85
60
Product ion
2
160.080
CE 1
5.89 6.16
80
0
100
Compound
TIC F: + c ESI SRM
m s 2 192.093
[131.580-132.580,
159.580-160.580]
MS Spike50_4
Carbendazim
6.55
Table 5. SRM transitions
5
5
50
95
95
5
5
NL: 6.96E4
6.48
60
0
100
Mobile phase B
Mobile phase
0
1
3
8
14
14.2
16
6.45
80
0
100
Relative Abundance
LC Column
Relative Abundance
50 mL tubes
(UCT part#: RFV0050CT)
Thermo Accela 1250 LC equipped with a PAL
Relative Abundance
Table 1. UCT materials for QuEChERS extraction and dSPE cleanup
HPLC system
Table 4. MS Parameters
Relative Abundance
100 ppm carbendazim, thiabendazole, pyrimethanil, cyprodinil, and pyrazophos standards in methanol were purchased from Ultra Scientific
(N. Kingstown, RI). 100 ppm chlorpyrifos and diazinone standards in methanol were purchased from Chem Service (West Chester, PA).
A 5000 ppm solution of triphenyl phosphate (TPP) in methyl tert -butyl ether was purchased from Cerilliant (Round Rock, TX). HPLC
grade acetonitrile (MeCN) and methanol (MeOH) were purchased from Spectrum (New Brunswick, NJ). The QuEChERS
extraction and dSPE cleanup materials were supplied by UCT (Bristol, PA) and are listed in the table below.
Table 3. HPLC conditions
Relative Abundance
100
2.1. Materials
QR
Code
100
200
ng/ml
300
400
0
100
200
ng/ml
300
400