31 Table 2: Quantification ranges of pesticide compounds with derivatisation and without derivatisation
matrix load will typically raise the need for intermediate conditioning with extracts to restore the column [7].
To develop an optimised routine method for the quantification of glyphosate, its metabolite aminomethylphosphonic acid (AMPA), glufosinate and its respective metabolite MPPA in beer samples, the derivatisation method was compared to a non-derivatisation method. A total of 24 different kinds of beer were analysed. The use of a high sensitivity mass spectrometer for both methods (LCMS-8060 coupled to a Nexera UHPLC, both from Shimadzu) avoids the need for an additional sample concentration step such as solid phase extraction (SPE), thereby reducing analysis time and cost.
Sample preparation for LC-MS/MS method and analytical conditions
Figure 2: Calibration curves of glyphosate, glufosinate, AMPA and MPPA determined in duplicate obtained from beer after sample pre-treatment. R² was better than 0.99 for all calibration curves.
technologies for monitoring the steadily increasing number of pesticides [2,3,4].
Pesticide Analysis
The Shimadzu LCMS-8060 triple quadrupole mass spectrometer features high sensitivity and speed for accelerated method development workflows and increased pesticide monitoring programs. Using the Shimadzu Pesticide MRM Library (including information on 766 certified reference materials) a single multi-residue LC-MS/MS method has been developed for the detection of 646 pesticides (3 MRM transitions for over 99% targeted pesticides resulting in 1,919 transitions in total, with a polarity switching time of 5 msec).
Glyphosate is currently one of the most common pesticides used worldwide. Despite its approval by regulatory bodies all over the world, concerns persist regarding its potential harm to humans and the environment. In June 2016, the European Commission extended the license for glyphosate use by 18 months, after member states failed to achieve a qualified majority in favour of or against the executive’s proposal, despite being identified as a possible carcinogen by the International Agency for Research on Cancer (IARC) in March 2015.
Based on new research results, the European Chemical Agency (ECHA) came to a different conclusion on 15th March 2017. The ECHA’s Committee for Risk Assessment (RAC) confirms the current harmonised classification of glyphosate as a substance causing serious eye damage and being toxic to aquatic life with long-lasting effects. But the RAC concluded that the scientific evidence available did not meet the
required criteria to classify glyphosate as a carcinogen, mutagen or as being toxic for reproduction. Glyphosate will therefore still be found in fruit and vegetables as well as in drinking water and beer soon [5].
Determination of Glyphosate and Related Substances using LC-MS/MS
Glyphosate is challenging to analyse chromatographically due to its high polarity, however a well-established method [6] can be employed which includes a derivatisation step with 9-fluorenylmethyl chloroformate (FMOC) followed by LC-MS analysis. Unfortunately, this is time-consuming and expensive and is also susceptible to errors. A further drawback is that not all highly polar and structurally similar pesticides, for example glufosinate’s metabolite 3-methylphosphinico propionic acid (MPPA), can be derivatised with FMOC. On the other hand, a sample pretreatment without derivatisation as described in EURL-SRM QuPPe-Method (5.6.4 Method 1.3) [7] which is not limited to glyphosate detection is therefore desirable since it will be quicker and more cost-efficient and will enable the simultaneous detection of other highly polar pesticides and their metabolites. This method also includes some disadvantages as chromatograms obtained using a new column show poor chromatographic behaviour due to strong interactions of analytes with active sites. The column needs conditioning with QuPPe extracts of e.g. spinach. This masking of the active sites is temporary and the activity of the column gradually increases with the injection of solvent or diluted extracts. Following a sequence of injections with low or no
1 mL methanol (MeOH) was added to 1 mL beer, vortexed and centrifuged for 15 min at 12,000 rpm. 500 µL of the supernatant was used as an underivatised sample. For derivatisation, 25 µL EDTA-borate buffer and 75 µL FMOC were added to 500 µL of the supernatant. After incubating at 50°C for 60 minutes the reaction was stopped by adding 30 µL of 0.2% phosphoric acid. Finally, 125 µL of water was added.
The derivatised and underivatised samples required different chromatographic conditions which are listed in Table 1.
Results of LC-MS/MS Measurements
Two methods (with and without derivatisation) have been evaluated for the quantification of glyphosate and glufosinate in beer, without the use of internal standards AMPA could be quantified using the FMOC derivatisation whereas MPPA could only be quantified with the underivatised method.
Therefore, neither of these methods is suitable for the detection of both metabolites at the same time with adequate sensitivity.
Calibration curves obtained for the compounds are shown in Figure 2, and the corresponding chromatograms in Figure 3. Analytical results for glyphosate in 24 different beer samples are listed in Table 2. Both methods show comparable results. None of the other compounds could be detected in the samples.
Both methods permitted the quantification of all target compounds at or below 0.01 mg/kg (
10 ng/mL), below the European
Union maximum residue levels (MRL) for all compounds. The non-derivatisation method shows comparable sensitivity to the FMOC derivatisation method, but sample preparation is much easier and quicker. Glyphosate was detected at a concentration above LOQ in more than 30% of the commercially available beers tested,
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