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Quantitation of Underivatized Glyphosate and Other Polar Pesticides using LC-MS/MS
and LC-HR-MS/MS in Food and Beer Samples
André Schreiber 1, Jen Win1, and Paul Winkler 2
1SCIEX Concord, ON, Canada; 2 SCIEX Redwood City, CA, USA
ABSTRACT
Here we present results of using LC-MS/MS and LC-HR-MS/MS method to identify and quantify underivatized
glyphosate and other polar pesticide in food and beer samples.
Table 1. MRM transitions and compound dependent parameters
Compound
Q1 (amu)
Q3 (amu)
DP (V)
CE (V)
Compound
Q1 (amu)
Q3 (amu)
DP (V)
CE (V)
Glyphosate
168
63
-30
-26
Glufosinate
180
63
-50
-66
INTRODUCTION
Glyphosate (N-(phosphonomethyl)glycine) is a widely used broad-spectrum systemic herbicide and crop
desiccant. Glyphosate is a topic with an extraordinary degree of public attention and concerns since the
International Agency for Research on Cancer (IARC), a branch of the World Health Organization, classified
glyphosate as a probable human carcinogen. Traces of glyphosate have been found in surface water, many
foods (i.e. bread, breakfast cereals, and beer) and also in human urine and breast milk.1,2
Usually Glyphosate, as it is very polar, undergoes FMOC derivatization before analysis. This derivatization step
complicates the analysis and there is a growing need for a method which can detect not only Glyphosate (and
its major metabolite AMPA) but also Glufosinate and similar highly polar compounds, in their underivatized
states. Anion exchange, HILIC, porous graphitized carbon and mixed-mode columns were used with LC-MS/MS
to determine underivatized polar pesticides with limited success.
Here we compare different published LC methods with respect to sensitivity, selectivity and routine use. The
most promising method using a mixed-mode column was successfully applied to the analysis of QuPPe extracts
of food and to the direct injection analysis of beer.
AMPA
168
150
-30
-14
180
95
-50
-24
168
124
-30
-16
180
136
-50
-22
168
81
-30
-20
180
85
-50
-24
110
63
-15
-26
151
133
-10
-18
110
79
-15
-36
151
63
-10
-44
110
81
-15
-16
151
107
-10
-20
110
80
-15
-24
151
78
-10
-28
MMPA
RESULTS
Linearity for quantitation was evaluated over 3 orders of magnitude. Good linearity was achieved over 3 orders
of magnitude from 1 to 1000 ng/mL with accuracies between 80 and 120% and coefficients of regression >
0.999 using linear regression and 1/x weighting Figure 3).
Glyphosate r = 0.9993
AMPA r = 0.9995
Glufosinate r = 0.9998
MMPA r = 0.9999
Recently scientists from the Munich Environmental Institute released results of the presence of Glyphosate in
14 popular German beers (0.5 to 30 µg/L). Testing was performed using ELISA and only a few beer samples
with higher levels of glyphosate were confirmed based on LC-MS/MS testing. The developed method with an
injection volume of 50 µL had an instrument LOQ for glyphosate of 0.1 µg/L and a method LOQ of 0.2 µg/L in
beer after 2x dilution. Figure 5 shows examples of beer tested positive for Glyphosate. The results correlate
very well with concentrations reported in Germany.
a
d
b
e
c
f
Figure 3. Linearity of Glyphosate, AMPA, Glufosinate, MMPA using 4 MRM transitions (1 to 1000 ng/mL)
QuPPe matrix extracts were spiked at a concentration of 100 ng/mL and diluted 10x. Results for corn and soy
are shown in Figure 4. All target compounds were positively identified using at least 2 MRM transitions,
however, background interference can vary depending on the matrix and the monitoring of more than 2
transitions is recommended.
Evaluation of Different LC Methods
Three different LC Methods were evaluated in their performance with respect to selectivity, sensitivity, and ease
of use in routine environment. Figure 1 shows example chromatograms of all three methods. Method 2 was
selected for all future studies because of good peak shape, best signal-to-noise (S/N), good separation to allow
use of MRM scheduling.
Figure 5. Glyphosate findings in beer a: German Pilsner 21.6 µg/L (4 MRMs), b: American Light beer 3.8 µg/L
(3 MRMs), c: Irish Stout 16.2 µg/L (4 MRMs), d: Canadian Craft IPA 9.5 µg/L (3 MRM), e: German Weissbier
0.2 µg/L (1 MRM), f: Homemade Ale brewed in Canada with Barley malted in Germany 0.7 µg/L (2 MRMs)
MATERIALS AND METHODS
Figure 6. Identification of Glyphosate using LC-HRMS/MS, after sample-control comparison TOF-MS and
MS/MS data was used for empirical formula finding, the
MS and MS/MS mass error and the ChemSpider hit
count was used to find the correct formula, which
subsequently was searched against the ChemSpider
database. Structures found in ChemSpider are
automatically compared against the HR-MS/MS
spectrum for identification.
• Food samples obtained from a local supermarket
- QuPPe (Quick Polar Pesticides) extraction: 10 g sample extracted after adjustment of water content with
10 mL methanol + 1% formic acid, centrifugation
- 10x dilution to minimize possible matrix effects
- Injection of 10 µL
• Beer samples purchased at Liquor Control Board of Ontario (LCBO)
- Direct injection of 50 µL after degassing and 2x dilution
• Evaluation of 3 different LC methods using a SCIEX ExionLC™ AD system
• Method 1:3
- Hypercarb (50 x 2.1 mm, 3µm), modified from the method published by M. Anastassiades et al.
- Gradient of water/methanol (95/5) + 1% acetic acid and methanol + 1% acetic acid at flow rate 0.2 to
0.4 mL/min
• Method 2:2
- Acclaim Trinity Q1 (100 x 3 mm, 3µm)
- Gradient of water + 50 mM ammonium formate/formic acid (pH=2.9) and acetonitrile at flow rate
0.5 mL/min
• Method 3:4
- Phenomenex LUNA NH2 50 x 2 mm 3µm
- Gradient of water + 10 mM ammonium bicarbonate (pH=10) at flow rate 0.4 mL/min
• Injection volume of 10 µL and 50 µL to analysis food and beverage samples, respectively
• SCIEX QTRAP® 6500+ system with IonDrive™ source
- Negative polarity ESI
- Multiple Reaction Monitoring (MRM) of 4 transition per analyte with (Table 1)
- Data processing in MultiQuant™ software
• SCIEX X500R QTOF system with Turbo V™ source
- Negative polarity ESI
- Continuous recalibration between injections using the Calibrant Delivery System (CDS) using a
TwinSpray setup (dual ESI needle)
- TOF-MS-IDA-MS/MS with DP = -40 V and CE = -35 V and CES = 15 V
- Data processing in SCIEX OS software
REFERENCES
1
2
Figure 1. Comparison of three LC methods to identify and quantify Glyphosate (yellow), AMPA (blue),
Glufosinate (red), MMPA (light blue)
3
Method performance
Figure 2 shows chromatograms of all 4 target analytes at a concentration of 10 ng/mL using an injection volume
of 10 µg/L. All 4 MRM transitions were detected allowing a 10x dilution of sample extracts while achieving the
target LOQ of 100 µg/kg and allowing confident identification based on multiple ion ratios. Excellent
repeatability was achieved with %CV below 5% (n = 5).
Figure 4. Identification of Glyphosate, AMPA, Glufosinate, MMPA in QuPPe extracts of corn and soy at
100 ng/mL (10 ng/mL in the 10x diluted extract)
Additional studies (not presented here) show that background interferences can be reduced using SelexION®
Differential Mobility Separation (DMS) Technology to further improve selectivity and data quality.
4
5
A. Vass et al.: ‘Study of different HILIC, mixed-mode, and other aqueous normal-phase approaches
for the liquid chromatography mass spectrometry-based determination of challenging polar pesticides’
Anal. Bioanal. Chem. 2016 May 13. [Epub ahead of print]
N. Chamkasem et al.: ‘Direct Determination of Glyphosate, Glufosinate, and AMPA in milk by
Liquid chromatography/tandem mass spectrometry’ Journal of Regulatory Science 02 (2015) 20-26
M. Anastassiades 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’ version 9 (2016)
Phenomenex Application ‘Underivatized glyphosate on a Luna 3 µm NH2’ #22767
http://www.umweltinstitut.org/aktuelle-meldungen/meldungen/umweltinstitut-findet-glyphosat-indeutschem-bier.html
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Figure 2. Sensitivity and repeatability of Glyphosate, AMPA, Glufosinate, MMPA using 4 MRM transitions
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