36 May / June 2019
LC-MS/MS Method for Determination of Glyphosate, AMPA, and Glufosinate in Cereals
by Olga I. Shimelis, R&D Manager, MilliporeSigma, 595 N. Harrison Rd., Bellefonte, PA 16823, USA
A simplified LC-MS/MS method for the determination of glyphosate, (aminomethyl)phosphonic acid (AMPA), and glufosinate in cereals is described. The method enables the analysis of glyphosate and its metabolites without sample derivatisation. The samples are prepared utilising an extraction method based upon the Quick Polar Pesticides Method and separated by high-performance liquid chromatography with MS detection. A polymer-based, ion-exchange chromatography column allowed retention of the analytes while water, methanol and ammonium carbonate mobile phase system insured proper ionisation under negative ESI conditions. The use of isotopically labelled standards allowed the use of solvent-based calibration curves.
Introduction
Glyphosate is one of the most commonly used herbicides in the world with more than 1.4 billion pounds of glyphosate applied to fields per year [1]. Its usage increased after the introduction of genetically modified, glyphosate tolerant crops such as corn, soybeans and cotton. In the USA, US Environmental Protection Agency (EPA) regulation document Code of Federal Regulations (CFR)-title 40-volume 24 sets the tolerance levels for the occurrence of glyphosate in food commodities and produce [2]. The EPA tolerance for glyphosate residues in cereal grains (also called crop group 15) are set at 30 ppm; this limit excludes rice, soy, and corn. In rice the tolerance is 0.1 ppm whereas in sweet corn it is 3.5 ppm [2]. For glufosinate, a herbicide that is often included with glyphosate in analytical methods, the tolerance values are 0.4 ppm for cereal and 1.0 ppm for rice. These tolerance values include metabolites and degradants. Therefore, a glyphosate metabolite, AMPA, was also included into this study (Figure 1).
Since glyphosate is widely used during production of soybeans and corn, it was expected to be found in these commodities. In this application, the presence of glyphosate in other grains such as oats and wheat used in the production of breakfast cereals, including infant cereal, was explored.
Various methods for glyphosate analysis were developed over the last 30 years. Some required derivatisation of analytes
for HPLC with fluorescence detection with o-phthalaldehyde [3]. A method with glyphosate derivatisation using fluorenylmethyloxycarbonyl chloride (FMOC) and fluorescence detection has also been proposed and widely used [4]. Recently, with the advent of modern, more sensitive and rugged LC-MS/MS instruments, it has become possible to analyse glyphosate and its metabolites without derivatisation as illustrated in this work with direct analysis of glyphosate by MS/MS.
Experimental
All reagents were purchased from Sigma- Aldrich, St. Louis, MO, USA unless indicated otherwise. Glyphosate, AMPA and ammonium glufosinate were of analytical standard grade. Isotopically labelled internal standards were used including Glyphosate- 2-13
C,15 N, AMPA-13 C,15 N,D2 . Glufosinate-D3
was obtained from Toronto Chemicals, North York, ON, Canada. Solutions of internal standards and non-isotopically labelled standards were prepared in water at 1 mg/mL and used for spiking the grain matrices.
Organic instant oatmeal and organic whole wheat flour were selected as test matrices during method development. These foods were scanned for the presence of glyphosate using the methods described below and glyphosate was not found. Whole wheat flour was used as received while the quick oats were ground prior to use. For the method development study, both matrices were spiked to contain 100 ppb of
Glyphosate
AMPA Glufosinate
Figure 1. Structures of Glyphosate, AMPA and Glufosinate
glyphosate and 100 ppb of glufosinate. The oats were spiked in addition to contain 100 ppb of AMPA.
For studies of glyphosate occurrence in grain products, the following matrices were tested for glyphosate using the developed methods: wheat white flour, instant oatmeal, infant rice cereal, infant oat cereal, and infant mixed grain cereal. All of these products were purchased in the local grocery store and were not labelled as organic.
Supel™-Select HLB Solid Phase Extraction (SPE) in 1 ml/30 mg size and Amicon® Ultra –0.5 centrifugal filter units with 3kDa Molecular Weight Cut Off (MWCO) were purchased from Sigma-Aldrich (St. Louis, USA).
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