Determination of Glyphosate in Rice with HPLC-ICP-MS/MS

Glyphosate (N-(phosphonomethyl)glycine) is a broad-spectrum systemic herbicide and crop desiccant that was approved in 2002 for the first time at the EU level for ten years under a directive replaced in 2011 by Regulation 1107/2009. Its increasing application has fuelled a debate on possible undesirable impacts on the environment and human health. In 2017, the European Commission renewed the approval of glyphosate but only for five years. On 2 December 2022 the Commission adopted an Implementing Regulation, extending the approval of glyphosate until 15 December 2023. In 2013, the European Commission uploaded maximum residue levels (MRLs) for glyphosate in several crops according to European regulation EU No. 293/2013 [8]. The new European MRLs for rice are 0.1 mg kg -1, as the lower limit of analytical determination.

Although glyphosate is one of the most widely used agrochemicals, it is also of the most difficult to measure. Glyphosate and related compounds cannot be sought within the scope of multi-residue methods, but call for a specific single residue method. Several methods are available for the determination of this molecule and its metabolites in different kinds of matrices, such as fruits, cereals, water and body fluids. The majority of methods are based on LC-MS/MS, but the chemical characteristics have been an obstacle with these methods. Sample preparation before chromatographic separation often includes derivatization and purification, making the analysis time-consuming and costly. Matrix effects leading to ionization suppression were found to be dependent on the matrix and on the particle size taken for extraction. For lower glyphosate contents (<1 mg/kg) the signal may even be suppressed by more than 90%. It has been demonstrated that the isotope-labelled standard ¹³C₂-glyphosate undergoes different ionization suppression than glyphosate and is therefore not efficient in compensating for matrix effect.

The new study:
An Italian group of researchers aimed at a rapid and simple method for the determination of glyphosate in rice based on the use of liquid chromatography coupled with inductively coupled plasma tandem mass spectrometry. Extraction of pulverized rice grains was achieved with 30% methanol under mechanical agitation for 30 min, followed by ultrasound irradiation for 15 min. Then, the samples were centrifuged at 2044 x g for 10 min. The supernatant was recovered, filtered through an 0.2 µm nylon filter, and inserted into HPLC vials.

Separation was achieved on a Hamilton PRP-X100 column (250 x 2.1 mm, 5 µm particle size), which was installed in a bio-inert LC system. The column was maintained at 50°C for all analysis. The mobile phase was 2mM malonic acid at pH 5.3, pumped at a flow rate 0f 0.6 ml/min. Sample injection volume was 60 µL.

Detection by ICP-MS/MS was based on the oxygen shift mode using O₂ as the reaction gas in the octopole reaction cell. The obtained detection power for glyphosate in white rice was LOD = 2.7 µg/kg/ LOQ = 9.2 µg/kg and about 5-times higher in brown rice.  Those detection limits are comparable with those obtained by LC-MS/MS methods reported in the literature. External calibration was based on matrix-matched standard solutions. The new method was validated in terms of the linearity, trueness, precision, LOD, LOQ, linearity, and the matrix effect.

The authors emphasized that their new method calls for less sample preparation and is faster than other options.

The original publication:

 Agilent Technologies Inc. - 1260 Infinity Bio-inert Quaternary LC system

January 22, 2020: Quantification of glyphosate and AMPA by HPLC-ICP-MS/MS