Polar pesticide analysis by CESI-MS
Stephen Lock and Jianru Stahl-Zeng, Sciex, Warrington, UK
INTRODUCTION
RESULTS
Glyphosate is a common, broad-spectrum, systemic herbicide widely used to kill weeds especially annual
broadleaf weeds and grasses known to compete with crops. Usually native glyphosate is analyzed after
derivatization with fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before analysis. This derivatization step
complicates the analysis. Thus, there is a growing need for a method which can detect not only glyphosate
but also its major metabolites, for example glufosinate, in their underivatized state.
In CESI-MS, separation of components is based on their charge state and therefore by their isoelectric
points (pI). This enables the separation of very similar species which produce identical fragmentation
ions in LC-MS/MS (Figure 3 and 4).
Standard HPLC methods which have been used to analyze underivatized polar pesticides usually involve
HILIC- or Hypercarb-based separations and are still prone to some technical challenges. In the case of
Hypercarb-based methods new HPLC columns require conditioning before analysis to stabilize retention
times and response. Other HILIC based methods can be subject to matrix interference effects which can
cause retention time drifts and analyte suppression.
A
B
B
A
A
C
C
Figure 4:- CESI-MS/MS electropherogram of a 10ppb
standard 10ppb standard for Glyphosate (A),
Glufosinate (B) and AMPA (C) in under 7 minutes.
AMPA is well separated from Fosetyl Alumina as
required by QuPPe method.
Figure 3:- CESI-MS/MS electropherogram of a 10ppb
standard for HPO3- (A), Fosetyl Alumina (B) and
H2PO4- (C) showing Separation of these three anions
in under 6 minutes.
Figure 1. CESI
Sprayer
Calibration for Fosetyl Alumina 2: y = 4518.96007 x + 2608.67856 (r = 0.99769) (weighting: None)
Calibration for Glyphosate 2: y = 2233.11640 x + 2710.04942 (r = 0.99730) (weighting: None)
4.5e5
4.0e5
2.0e5
R = 0.9977
3.5e5
R = 9973
3.0e5
Capillary electrophoresis (CE) is a separation technique
designed to separate polar constituents based on their
charge and size and is well suited for the separation of
small polar contaminants such as glyphosate. CE takes
advantage of ultra-low flow rates and although loading
amounts are lower than LC based methods, this is
offset by the vast gains in ion generation and reduction
in ion suppression at these low flow rates. In this poster
we will show how CE integrated with electrospray
ionization into one dynamic process within the same
devise coupled to mass spectrometry1 (CESI-MS,
Figure 1) , has been used to analyze polar pesticides in
food extracts. This technique helps reduce the effects
of matrix and achieves low limits of detection through
separation of a selection of underivitized polar
pesticides and in combination with the QTRAP® 5500
LC-MS/MS system (Figure 2) offers a new approach to
analyze these contaminants.
2.0e5
1.0e5
1.5e5
1.0e5
5.0e4
5.0e4
0.0e0
0.0e0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
Concentration
Concentration
Figure 5:- Example of a CESI-MS/MS Calibration line
Figure 6:- Example of a CESI-MS/MS calibration line
for fosetyl Alumina (1 – 100ppb) with no internal
for glyphosate (1 – 100ppb) with no internal standard
standard used.
used.
Onion
Grape
Onion
Grape
Wheat
Rice
Wheat
Rice
Figure 2. QTRAP® 5500 LC/MS/MS
system
All chemicals were Reagent Grade and were purchased from Sigma Aldrich.
Sample Preparation:
For linearity and sensitivity tests, calibration standards at ranging from 1 – 100 part per billion (ppb) were
prepared in water from at 1 – 100 ppb. Matrix samples were prepared by spiking the polar pesticides into
50% aqueous methanol extracts of onion, wheat, rice and grapes (prepared as per the QuPPe-Method 2
from the EU Reference Laboratories for Residues of Pesticides). These extracts were then diluted 1 in 10
with water before injection.
The sample was injected by pressure (5 psi, 20 s) onto a 30 μm ID x 91 cm
bare-fused-silica capillary (including porous spray tip) housed in an OptiMS CESI cartridge thermostatted
using recirculating liquid coolant regulated at 25 ºC. For this analysis, the SCIEX QTRAP® 5500 LCMS/MS system was fitted with the NanoSpray® III source. Gas 1 and 2 were not used and temperature
set low (50 ⁰C) as ionization at these very low flow rates occurs by simply applying the ion-spray voltage
which was set to -2100 V. The curtain gas was set low (5psi) and other MS conditions are shown in Table
1. The MS method was split into 3 periods to cover the CE separation, in the first and last period (1
minute each) the ionspray voltage was set to zero, this was the only difference in the MS conditions. The
total MS acquisition time (summation of all 3 periods) was 11 minutes with the MS resolution setting for
Q1 and Q3 set to unit. The CE separation used the conditions shown in Table 2 with a background
electrolyte (BGE) composed of a mixture of 5% Acetic acid : 10% Methanol: 85% Water. The voltage used
for the separation was in reverse polarity as the ions are negatively charged. The total injection to
injection time was less than 25 minutes with MS acquisition initiated by an AUX/IO trigger 0.1 minutes into
the CE separation.
CESI-MS method:
Compound
Area
Area
2.5e5
METHODS
Chemicals:
1.5e5
Q1 Q3 transition Q3 transition Dwell
(amu)
1 (amu)
2 (amu)
(s)
DP CE transition CE transition
(V)
1 (V)
2 (V)
AMPA
110
79
63
40
-100
-28
-26
Ethephon
143
79
109
40
-45
-30
-12
Glyphosate
168
79
63
40
-85
-58
-34
Glufosinate
180
79
95
40
-45
-40
-24
Fosetyl
alumina
Maleic
hydrazide
109
79
63
40
-100
-28
-28
111
82
42
40
-100
-22
-22
Table 1:- MS/MS conditions used
for detection of polar pesticides.
Figure 7:- CESI-MS/MS electropherograms of different
Figure 8:- CESI-MS/MS electropherograms of different
extracts prepared by the QuPPe method which had been
extracts prepared by the QuPPe method which had
spiked with fosetyl alumina (100 ppb) and diluted 1 in 10
been spiked with glyphosate (100 ppb) and diluted 1
with water.
in 10 with water.
The linearity of response was tested for these polar pesticides and examples of the results obtained are
shown in Figure 5 and 6. The pesticides gave a linear response over the range tested (1-100 ppb) as
shown by the R value of 0.99796 obtained for fosetyl alumina (Figure 5) and R value of 0.99730 for
glyphosate (Figure 6), even though no internal standards were used.
Spiked matrix samples were then assessed. Figures 7 and 8 compare spiked extracts prepared using
the QuPPe method for four different foods. Both fosetyl alumina and glyphosate were easily detected in
the 100 ppb spike, in all matrices, even though the extract was diluted 10 fold. Migration time was also
not affected by the matrix type even though extracts were only diluted. However, some evidence of
matrix suppression was observed for fosetyl alumina with the overall signal height varying with matrix.
This suppression effect is still being investigated as the method used a pressure separation to speed up
the analysis, but also reduced ion generation, and without the use of internal standards it is difficult to
verify if this variation in peak height was injection or matrix related.
CONCLUSIONS
In this early study it has been demonstrated that CESI-MS/MS can be used for quantitation and detection
of several similar underivatised polar pesticides, known to be difficult to analyze by LC-MS/MS using
extracts prepared using the QuPPe method. These pesticides were easily detected at the current
required EU limits of detection.
Future studies are planned to assess the robustness of this method and to investigate the use of neutral
capillaries, sample preparation, injection techniques such as isotachophoresis (ITP) and the effect of
organic additives to the BGE to increase the overall sensitivity of this approach for these compounds.
We are also looking into reducing the overall injection to injection time by reducing the conditioning time.
Action
Time
(mins)
Pressure
(psi)
Direction Voltage
(kV)
Solution
Description
Rinse
0.5
100
Forward
0
Water
Wash
Rinse
4
100
Forward
0
0.1 Molar NaOH
Condition
Rinse
0.5
100
Forward
0
Water
Wash
Rinse
2
100
Forward
0
0.1 Molar HCl
Condition
Rinse
2
100
Forward
0
BGE
Capillary Fill
Rinse
0.5
75
Reverse
0
10% Acetic acid
Injection
0.3
5
Forward
0
Sample Vial
Conductive
Fill
Injection
Injection
0.5
0.5
Forward
0
BGE
Push
Separation
11
10
Forward
30
BGE
Separation
Voltage
1
5
Forward
1
BGE
Ramp down
Table 2:- CESI separation method.
REFERENCES
1.
2.
Busnel, J-M., et. al., “High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry:
Coupling a Porous Sheath less Interface with Transient-Isotachophoresis”, Anal. Chem. 82 9476-9483 2010.
Anastassiades, M., et. al., “Quick Method for the Analysis of numerous Highly Polar Pesticides in Foods of
Plant Origin via LC-MS/MS involving Simultaneous Extraction with Methanol (QuPPe-Method)”. EU
Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM), CVUA Stuttgart,
Germany.
TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic procedures.
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