PO-CON1674E
Supercritical fluid
chromatography/tandem mass
spectrometry analysis of hundreds
of pesticide residues in food safety
ASMS 2016
TP 222
David Baker1, Christopher Titman1, Neil Loftus1,
Jonathan Horner2
1
Shimadzu, Manchester, UK
2
Scientific Analysis Laboratories (SAL), Cambridge, UK
Supercritical fluid chromatography/tandem
mass spectrometry analysis of hundreds of pesticide residues
in food safety
Introduction
Analysis of pesticides in food commodities is typically
carried out using gas chromatography/mass spectrometry
(GC-MS) and liquid chromatography/mass spectrometry
(LC-MS). These two techniques provide an effective
combination for the analysis of pesticides with a broad
range of physiochemical properties. Supercritical fluid
chromatography (SFC) provides an alternative separation
method which uses compressed carbon dioxide in
supercritical state as its primary mobile phase. The lower
viscosity and high diffusivity of the mobile phase in SFC
provides lower back pressures in comparison to LC
therefore allowing higher flow rates to be used. In
addition SFC has lower aqueous-organic solvent
consumption.
The aim of this work was to demonstrate the application
of SFC-MS/MS for the analysis of a broad range of
pesticides. To do this a method was developed for the
analysis of 338 pesticides commonly analysed by
LC-MS/MS (and GC-MS/MS).
Materials and Methods
Sage samples, extracted using QuEChERS based methods, were provided by SAL. These extracts were then spiked with
pesticides and directly injected into the SFC-MS/MS.
Table 1. LC and MS/MS acquisition parameters used to create the LC-MS/MS method.
Supercritical fluid chromatography
SFC
Analytical column
Column temperature
Flow rate
Modifier
BPR temperature
BPR pressure
Make-up solvent flow rate
Make-up solvent
Binary Gradient
Injection volume
:
:
:
:
:
:
:
:
:
:
Nexera UC system
Restek Ultra AQ C18 (150 x 2.1, 3µm)
40°C
1 mL/minute
2 mmol/L ammonium formate and 0.002% formic acid in methanol
50°C
150 bar
0.1 ml/min
2 mmol/L ammonium formate and 0.002% formic acid in methanol
Time (mins)
%B
8.00
5
10.00
10
11.50
60
12.00
60
12.01
2
17.00
Stop
: 1µL
Mass spectrometry
Mass spectrometer
Pause time/dwell time
Target number of compounds
Source temp. (interface; heat block; DL)
Gas flows (nebulising; heating; drying)
:
:
:
:
:
Shimadzu LCMS-8060
1 msec. / 3 msec.
338
300°C; 400°C; 200°C
3L/min; 10 L/min; 10L/min
2
Supercritical fluid chromatography/tandem
mass spectrometry analysis of hundreds of pesticide residues
in food safety
SFC-MS/MS
In this study a method was developed for the analysis of
338 pesticides by SFC-MS/MS which are commonly
analysed by LC-MS/MS. Method development involved the
evaluation of various analytical columns including; Raptor
biphenyl, Ultra AQ C18, Ultra Silica and the Ultra IBD. The
column selected was the ultra AQ C18 due to its ability to
retain early eluting compounds and its peak capacity.
Intensity 1.0e7
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
min
Figure 1. Chromatogram of 338 pesticides (2 MRMs per compound) spiked into sage extract corresponding to a concentration of
0.10 mg/kg. Peak shape and sensitivity are in broad agreement with the performance of LC-MS/MS methods.
Hexazinone
Furathiocarb
Intensity 1.0e7
Demeton-S-methyl Sulfoxide
Benalaxyl
Karbutilate
Phosphamidon
Dimethoate
Acephate
Atrazine
Thiamethoxam
Mephosfolan
Vamidothion
6-benzyladenine
Azaconazole
Sulfentrazone
Omethoate
Fuberidazole
Thidiazuron
Methacrifos
0.0
1.0
Chloridazon
Fluoxastrobin
2.0
3.0
4.0
5.0
Propamocarb
Forchlorfenuron
6.0
7.0
8.0
9.0
10.0
11.0
Nitenpyram
12.0
Amitraz
13.0
min
Figure 2. MRM chromatograms for 24 pesticides (quantitation ion and primary qualifier ion) selected to show the chromatographic
separation throughout the SFC-MS/MS analysis. Pesticides corresponding to a concentration of 0.10 mg/kg spiked into a
sage extract. As the SFC system has a low dead volume back pressure regulator it suppresses diffusion of peaks and can
transfer the total eluate directly to a mass spectrometer.
3
Supercritical fluid chromatography/tandem
mass spectrometry analysis of hundreds of pesticide residues
in food safety
SFC-MS/MS and polar pesticides
The panel of 338 pesticides included several polar pesticides. In the case of methamidophos, nicotine, cyromazine and
acephate the peak shape in LC-MS/MS typically resulted in split peaks. SFC-MS/MS resulted in well retained, Guassion
peak shapes for these polar pesticides.
Methamidophos12.0 142.20>93.95(+)
Methamidophos 142.20>124.90(+)
Methamidophos 142.20>46.95(+)
Cyromazine 167.20>68.00(+)
Cyromazine 167.20>59.90(+)
Methamidophos
LC-MS/MS
Split peaks; poor retention
Methamidophos
SFC-MS/MS
Single peak
Cyromazine
LC-MS/MS
Split peaks; poor retention
SFC-MS/MS
Rt 4 mins
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
min
Nicotine 163.10>130.00(+)
Nicotine 163.10>117.00(+)
Nicotine 163.10>132.10(+)
SFC-MS/MS
Rt 12 mins
0.0
2.0
4.0
6.0
8.0
Nicotine
SFC-MS/MS
Single peak
Acephate
SFC-MS/MS
Single peak
4.0
min
Acephate
SFC-MS/MS
Single peak
SFC-MS/MS
Rt 12 mins
2.0
10.0
Acephate 184.00>49.15(+)
Acephate 184.00>143.00(+)
Acephate 184.00>95.00(+)
Nicotine
LC-MS/MS
Split peaks; poor retention
0.0
Cyromazine
SFC-MS/MS
Single peak
6.0
8.0
10.0
12.0
SFC-MS/MS
Rt 3.6 mins
min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
min
Figure 3. MRM chromatograms for methamidophos, nicotine, cyromazine and acephate determined by SFC-MS/MS and LC-MS/MS. For these early
eluting highly polar pesticides the peak shape on a number of LC systems is often compromised whilst the response on SFC-MS/MS results in a
single Gaussian peak shape. In this example, 2 µL of sage extract spiked at 0.1 mg/kg was injected on both LC and SFC-MS/MS. SFC conditions
are described in the method section. Briefly, the LC-MS/MS conditions were as follows; Raptor ARC-C18 (100x2.1, 2.7µm, 0.4ml/min, 35°C, (A)
2mM ammonium formate with 0.002% formic acid (B) 2mM ammonium formate with 0.002% formic acid in methanol. Gradient: 3.5% to
100% B over 10minutes. MS source parameters were optimised and were found to be similar to those utilised in LC-MS/MS.
4
Supercritical fluid chromatography/tandem
mass spectrometry analysis of hundreds of pesticide residues
in food safety
Quantitative SFC-MS/MS analysis
Linearity was investigated between the range of 0.010 – 0.2 mg/kg; calibration curves for selected compounds are
displayed in Figure 4.
2.6e6
4.5e7
Phorate | Rt 0.821 minutes
R² = 0.9994435 R = 0.9997217
2.4e6
Azaconazole | Rt 4.774 minutes
R² = 0.9978243 R = 0.9989116
4.0e7
2.2e6
2.0e6
3.5e7
1.8e6
3.0e7
1.6e6
2.5e7
1.4e6
1.2e6
2.0e7
1.0e6
1.5e7
8.0e5
6.0e5
1.0e7
4.0e5
2.0e5
0.0e0
0.2
0.4
100
120
0.6
0.8
1.0
5.0e6
1.2
0.0e0
0
20
40
60
80
140
160
180
200
Conc.
1.1e7
40
60
80
100
120
4.6
140
160
4.8
180
5.0
5.2
200
Terbacil | Rt 2.751 minutes
R² = 0.9993297 R = 0.9996648
1.0e7
9.0e6
9.0e6
8.0e6
8.0e6
7.0e6
7.0e6
6.0e6
6.0e6
20
4.4
Conc.
1.0e7
5.0e6
5.0e6
4.0e6
4.0e6
3.0e6
3.0e6
2.0e6
2.0e6
12.4 12.6 12.8 13.0 13.2 13.4
1.0e6
0.0e0
0
1.1e7
Amitraz | Rt 12.951 minutes
R² = 0.9994749 R = 0.9997374
1.2e7
4.2
0
20
40
60
80
100
120
140
160
180
200
Conc.
1.0e6
0.0e0
0
20
40
60
80
100
2.2
2.4
120
140
2.6
160
2.8
180
3.0
3.2
200
Conc.
Figure 4 Calibration curves for selected pesticides (early/mid/late eluting compounds and one negative ESI compound,
terbacil) spiked into the range 0.010 – 0.2 mg/kg. All calibration curves were fitted using a linear curve fit type
and 1/C weighting.
5
Supercritical fluid chromatography/tandem
mass spectrometry analysis of hundreds of pesticide residues
in food safety
Conclusions
In this study a SFC-MS/MS method was developed for the
analysis of 338 pesticides covering a broad range of
physiochemical properties. Excellent peaks were obtained
for all compounds with few exceptions.
All 338 compounds were determined at the 0.01mg/kg
reporting level and the quantitative response was linear in
the range 0.010 – 0.2 mg/kg.
In comparison to reverse phase LC, SFC-MS/MS resulted
in well retained, Guassion peak shapes for several polar
pesticides such as methamidophos, nicotine, cyromazine
and acephate. Using reverse–phase LC these compounds
will typically result in peak splitting.
SFC-MS/MS offers an alternative separation technique for
pesticide analysis, with improved data quality for some
compounds in comparison to LC-MS/MS.
First Edition: June, 2016
For Research Use Only. Not for use in diagnostic procedure.
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