Food and environmental

Improving Identification and Quantification ofPolar Herbicides by CESI-MS
Achieving better differentiation of glyphosate, fosetyl aluminum and their degradation products
Bryan Fonslow,1 Stephen Lock,2 Wiley Hall3 and Spencer Walse4
1 SCIEX Separations, Brea, CA
2 SCIEX Separations, Warrington, UK
3 Safe Food Alliance, Fresno, CA
4 USDA-ARS, Parlier, CA

Overview
Who should read this: Senior scientists, lab directors
Focus: Demonstrate the advantages of CESI-MS for separating,identifying and quantifying the polar herbicides glyphosate andfosetyl aluminum as well as their degradation products.
Goals: Develop an effective CESI-MS method for separating,identifying and quantifying polar herbicides and compare theselectivity, accuracy and reproducibility of that method to thoseof an approved, currently used LC-MS method.
Problem: Concerns about the safety of glyphosate-basedherbicides (GBHs) have made it essential to be able to detect
glyphosate in foods (especially fruits and nuts) and distinguishit from other alternative herbicides such as fosetyl aluminum.
Current LC-MS methods have significant limitations, including
ion suppression, retention time instability and problems in
distinguishing between degradation products of these herbicides

The CESI 8000 Plus System(Figure 1). Both glyphosate and fosetyl aluminum are regulated,but false positive identification (ID) and inaccurate quantificationof their degradation products, phosphate and phosphonate, ispossible using current LC-MS methods. Therefore, a method
is needed that provides accurate ID and quantification of thesedegradation products.
Results: The developed CESI-MS method demonstrated anexcellent ability to distinguish between glyphosate and its
degradants, and between similar degradation products of anotherwidely used herbicide, fosetyl aluminum. It also demonstrated
better migration/retention time stability and quantitative linearity
than the LC-MS method.

Key challenges:• Separation of highly polar molecules by LC requires either
time-consuming analyte derivatization prior to reverse-phaseLC, or reliance on less reliable LC techniques, such as HILIC
or anion exchange chromatography• LC-MS methods suffer from a variety of issues, includingderivatization selectivity, ion suppression due to matrix
effects and retention time instability• LC-MS methods frequently have difficulty resolving phosphate
and phosphonate, the final degradation products of glyphosate
and fosetyl aluminum, respectively

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Key features:• Capillary electrophoresis is well suited to the separation ofpolar herbicides

Differentiating glyphosate, other herbicides andtheir degradation products
Glyphosate is a common, broad-spectrum, systemic herbicide• The CESI-MS method provided excellent specificity, easily
resolving and identifying glyphosate, fosetyl aluminum andmany of their degradation products

widely used to kill weeds that compete with crops. Concerns• The CESI-MS method demonstrated very good migration
time stability over more than 160 runs

recently in the European Union (EU).3 As such, it is increasingly• The CESI-MS method exhibited excellent quantitative
linearity when analyzing phosphonate, the degradation
product of fosetyl aluminum, in matrices

about the safety of glyphosate1,2 have led to increasing
restrictions on glyphosate-based herbicides (GBHs), mostimportant to develop robust analytical methods with the sensitivity
and selectivity to identify and quantify glyphosate and its
degradation products in foods and differentiate them from otherherbicides, such as fosetyl aluminum.

Figure 1. Degradation
pathways for (a) glyphosateand (b) fosetyl aluminum.

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Limitations of LC-MS methods

Advantages of CESI-MS

Several LC-MS-based methods are currently used to analyze

Capillary electrophoresis (CE) is well suited to the analysis

glyphosate and its degradation products, with many listed in theof polar ions and has already proven useful in the analysis ofQuick Polar Pesticides (QuPPe) Method document created bypesticides.6 The mechanism of separation is by differences

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the EU Reference Laboratories for Residues of Pesticides. The

in pKa and hydrodynamic radii. In the case of phosphate

methods use anion-exchange, porous-graphitized carbon orand phosphonate, they differ in pKa by 0.6 units (FigureHILIC liquid chromatography coupled with mass spectrometry.

1). Integration of capillary electrophoresis and electrospray

Reverse-phase LC methods have also been used but require

ionization (CESI) into a single dynamic process facilitates theanalyte derivatization with fluorenylmethyloxycarbonyl chloride

mass spectrometric use of CE detection and analysis. The(FMOC-Cl) before sample analysis.5 While LC-MS methods

developed CESI-MS method readily differentiated glyphosate,

generally can differentiate glyphosate and fosetyl aluminum

fosetyl aluminum and their degradation products (Figure 2). It(Figure 1), these methods can suffer from derivatization

demonstrated far better migration/retention time stability thanselectivity, matrix effects, ion suppression and poor retention

a corresponding LC-MS method (Figure 3). Finally, quantitative

time reproducibility. Additionally, LC-MS methods are generally

CESI-MS/MS analysis of phosphonate in real-world nut

not suitable for resolving phosphate and phosphonate, theextracts showed exceptional linearity while the corresponding

final degradation products of glyphosate and fosetyl aluminum,

anion-exchange LC-MS/MS method exhibited significant ion

especially in real-world matrices.

suppression due to matrix effects (Figure 4).

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Figure 2. CESI-MS provides clear separation and detection of glyphosate and 3 of its degradation products (glufosinate, AMPA and phosphate) alongwith fosetyl aluminum and 1 of its degradation products (phosphonate).

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Food and environmentalReferences
1. Myers, J.P. et al. Concerns over use of glyphosate-basedherbicides and risks associated with exposures: a consensus
statement. Environmental Health, 2016, 15:19.
2. World Health Organization’s International Agency for Research
on Cancers Monographs on the Evaluation of CarcinogenicRisks to Humans. Glyphosate, 2015, Volume 112.
3. http://europa.eu/rapid/press-release_MEMO-16-2012_en.htm.
4. 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), 2016,
Version 9.1. http://www.eurl-pesticides.eu/.

Figure 3. Migration/retention times for phosphonate and phosphate
across more than 160 analyses, with sample electropherograms(CESI-MS/MS) and chromatograms (LC-MS/MS). CESI-MS proved
far more stable over time and baseline separation was achieved onlyin the CESI-MS analysis.

5. Schreiber, A. and Cabrices, O.G. Automated Derivatization,SPE Cleanup and LC-MS/MS Determination of Glyphosate
and Other Polar Pesticides. 2013, SCIEX publication number:
8013813-01.
6. El Rassi, Z. Capillary Electrophoresis of Pesticides.
Electrophoresis, 1997, 18, 2465–2481.

Figure 4. CESI-MS/MS and anion-exchange LC-MS/MS quantificationof phosphonate spiked into 0.1% formic acid (solvent) and nut extracts.
Significant ion suppression due to matrix effects is clearly visible in theLC-MS/MS results, but not in the CESI-MS/MS results. Fit lines are forthe solvent curves only.

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