Residue analytical method for the determination of trifluoroacetic acid and difluoroacetic acid in plant matrices by capillary electrophoresis tandem mass spectrometry (CE-MS/MS)

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Highlights

  • CE-MS/MS was more sensitive compared to LC-based methods when comparing absolute on-column loading.

  • CE-MS/MS was less affected by sample matrix suppression than LC-based techniques.

  • A limit of quantitation of 0.01 mg/kg, was obtained for all matrices tested meeting legislation requirements.

  • The method was successfully validated and meets GLP validated guidelines.

  • Even though the system ran at very low flow rates CE-MS/MS provided good robustness.

Abstract

In the past years, the technology for trace residue analysis of plant protection compounds in plant and animal matrices, soil, and water has gradually changed to meet changing regulatory demands. Generally, from the '70s to the '90s of the last century, the active compounds and only a few major metabolites had to be determined in a typical "residue definition". Step by step and within the framework of product safety assessments of the enforcement of residues in dietary matrices and in the environment, further metabolites have come into the authorities focus. Many active substances were formerly determined via gas chromatography (GC) based detection techniques. The introduction of liquid chromatography tandem mass spectrometry (LC-MS/MS) technology in the '90s and the acceptance of this technique, by official bodies at the end of the '90s, has led to a major change for residue analytical laboratories all over the world. Most of the medium to non-polar active compounds as well as many of the more polar metabolites can be detected with this technique, and today LC-MS/MS is the "workhorse" in many residue analytical laboratories in the industry as well as official enforcement labs responsible for analyzing registration-related field studies. With the demand to analyze further breakdown products, more and more polar compounds, or even (permanently) charged target compounds, have now come into the focus of the registration authorities. This now brings standard LC-based techniques to their limits and requires the application of approaches such as hydrophilic interaction chromatography (HILIC) MS/MS or ion chromatography, however these techniques often incur related uncertainties and problems with matrix samples.

The aim of this study was to develop a new CE-MS/MS-based approach to reduce the impact of matrix on the separation and detection of trifluoroacetic acid (TFA) and difluoroacetic acid (DFA) in agrochemical field trials. This project used 7 representative examples of fruit, grain and vegetables which had undergone homogenization and extraction with acetonitrile water and filtration before CE-MS/MS analysis. The CE-MS/MS developed reached the limit of quantitation (LOQ) requirement of current legislation for both TFA and DFA (0.01 mg/kg) in all 7 matrices tested. The mean relative standard deviation (RSD) obtained from the repeat analysis of control field trail samples in all matrices, for both TFA and DFA, was less than 10% meeting GLP guidelines. When compared with LC-MS/MS, using on column loading amounts, the CE-MS/MS was 17 - 43 times more sensitive than a standard method and less matrix effects were observed.

The developed method was validated under GLP conditions to provide a GLP-validated residue analytical method for the charged metabolites TFA and DFA in matrix samples from GLP field residue trials.

Introduction

As the use of pesticides increases, the regulation agencies have developed monitoring guidelines not only for the active compound, but also for the degradation products. In the development of a new active pesticide the project plan is very time-consuming, expensive, and consists of many scientific areas. Development of a single pesticide from synthesis to release can take upwards of 10 years and cost $250-300 million and involves the synthesis and purification of the active ingredient and then formulation using inert ingredients to facilitate spraying and coating of target plants. This concentrated final product is then used by farmers or certified applicators who generally dilute it before applying to fields and plants. The objective of Human Safety-related Science is to assess the risk of exposure to consumers and users of the products.

The evaluation of human safety-relevant aspects of a pesticide begins with the pesticide metabolism studies, focusing on the amounts of each degradation product produced. From this data, a residue definition is defined. For example, if 20 metabolites exist, but only 5 are in large quantities, the focus of the residue analysis will be the 5 metabolites of significant yield. Matrices that need to be analyzed include plant material in various stages of growth, from seed to full grown plant, soil, and water.

The residue analysis workflow is based on metabolism results, starting with the validation of analytical methods for the trace analysis of the identified target compounds and is followed by a package of field trials. From these trials, samples are taken to the laboratory where they are homogenized, extracted, analyzed, and reports are generated. Reports are collected into dossiers and these are submitted to the regulatory bodies. These validated GLP studies are required for acceptance and release of pesticides for manufacturing.

Previously, residue analysis was required only on the parent compound and few major metabolites. Since many of the pesticides of the time were non-polar, gas chromatography was the analysis method of choice. As research on the risk of not only the parent compound, but also metabolites, further developed, the analysis moved to the area of liquid chromatography as the polarity of the target compounds increased. However, as the metabolites increase in polarity, other analysis techniques are required. Capillary zone electrophoresis (CZE) is best suited for highly polar analytes and in recent years the development of a CE coupled to MS, has become an attractive alternative for the analysis of charged metabolites, providing not only high-resolution separations but also mass spectrometry confirmation of the targets.

Next to wide industrial use and production of TFA [1], it is also known as an atmospheric breakdown product from various hydrofluorocarbons (HFC) cooling agents used in air conditioners and refrigerators [2], which can explain the ubiquitarian occurrence of TFA in almost all environmental areas. The main sources of residues of TFA in the environment are ground water and surface bodies of water and these are under evaluation and assessment of different regulatory bodies [3,4] to those of plant matrices. In recent years, the use of plant protection compounds was identified to be a source for occurrence of DFA and TFA residues. Organization such as the European Food Safety Authority (EFSA) have identified 39 pesticides that are currently approved for use in the EU and contain the trifluoromethyl- moiety. Examples of trifluoromethyl pesticides that could potentially lead to TFA formation [5,6] are fipronil (a trifluoromethylphenyl broad range insecticide belonging to the phenylpyrazole family) or the herbicide flufenacet (an oxyacetamid used in acre crops like cereals, maize/corn or as pre-emergence in potatoes, controlling weeds and grasses). DFA has been identified as a metabolite from the pesticide flupyradifurone, an insecticide used on a wide range of crops to control pests such as aphids, hoppers and whiteflies [7].

Short chain fluorinated acids such as TFA and DFA are not retained on traditional reverse phase C8 or C18 columns [8]. One way to separate TFA and DFA is ion chromatography, for example using Ion Pac ® columns together with a potassium hydroxide gradient from Dionex [9], which has been applied to the detection of TFA in water samples. HILIC LC-MS/MS methods have also been used for the detection of TFA and DFA, for example the use of Obelisc N column where the mobile phase contains 70% acetonitrile [10], but again this has mainly been used to detect TFA in water samples. For a couple of years, TFA and DFA have been monitored in residue enforcement analysis using published multi-residue methods, the QuPPe Method [11], but the matrix from plant extracts causes several challenges including matrix and protein interferences preventing the detection of TFA at low concentrations. This was further reinforced at Bayer where a HILIC separation (Thermo Hypersil GOLD HILIC column using the manufacturers recommended mobile phase conditions) with LC-MS/MS detection was investigated for the determination of TFA in water/acetonitrile plant extracts from residue trials. The results obtained (unpublished) showed strong matrix interferences, the lifetime of the HILIC column used was found to be very short and an extended equilibration time was needed for a stable assay making the method challenging to run. Recently a new approach, utilizing ion mobility to help in the method selectivity, has been tested [12] but still sensitivity was again a challenge for TFA.

The current ubiquitarian occurrence of TFA requires a highly specific, sensitive and precise residue analytical method to allow an accurate background subtraction in process control samples (recovery) and a clear differentiation between control and treated samples from field residue studies. New methods which provide accurate residue levels are needed for this area and this study presents the first GLP-validated method for the analysis of TFA and DFA by capillary electrophoresis with mass spectrometric detection (CE-MS/MS) in plant matrices which has overcome some of the challenges of earlier methods.

Section snippets

Chemicals

Control samples of potato tuber, rape seed, wheat grain, grapefruit, lettuce head, barley green and straw were obtained from different residue trials conducted on Bayer AG trial locations all over Europe or from local food retailers in the Monheim area. Acetonitrile and methanol used for chromatography (LiChrosolv grade), acetic acid (Suprapur grade), ammonium fomate (LiChropur), 1 M sodium hydroxide solution and 0.1 M hydrochloric acid were purchased from Merck (Darmstadt, Germany). Water

Results

In order to comply with GLP regulations, several parameters were tested. These included the linearity as well as the limits of detection in different plant matrices and the % recovery achieved for spiked samples.

Fig. 3 displays the calibration lines for both TFA and DFA. For TFA an R2 = 0.99997 was achieved over the concentration range of 0.2-100 µg/L and for DFA an R2 = 0.99995 was achieved over the same concentration range; indicating that the analytical method meets the required GLP criteria

Discussion

Capillary zone electrophoresis, the separation technique used in this method, is a perfect match for these polar and charged analytes as it separates based on the mass-to-charge ratio of target analytes, so it is well suited for the small polar acidic pesticide metabolites of TFA and DFA.

When comparing the traditional approaches of IC-MS, LC-MS/MS with CE-MS/MS using water samples (Table 5) all approaches provided similar values showing that the CE-MS/MS did not have a sensitivity bias as a

CRediT authorship contribution statement

S. Stuke: Conceptualization, Methodology, Project administration, Supervision, Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Visualization, Resources. P. Bemboom: Validation, Investigation, Data curtion. H. Wirkner: Validation, Investigation, Data curtion. W. Smith: Visualization, Methodology, Writing – original draft. S.J. Lock: Conceptualization, Visualization, Methodology, Writing – review & editing, Writing – original draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors thank Dr. Andreas Stork and Dr. Karl-Josef Haack (both Bayer AG, Crop Science Division) for enabling this work and Dr. Schlett, Dr. Liesener (both Westfälische Wasser- und Umweltanalytik GmbH, D-45891 Gelsenkirchen) and Dr. Ralph Krebber (Bayer AG Crop Science Division), who provided the surface water samples and their analytical results in the independent comparative analyses.

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