**1. Introduction**

Numerous pesticides are used in agriculture worldwide to protect crops and increase harvest yields, with over 1.1 billion pounds used annually in the USA, and nearly six billion pounds (three million tons) usage worldwide [1]. Use of specific pesticides varies by different countries, and certain pesticides may be approved for applications in some countries but banned or no longer used in others. To protect consumer health, maximum residue limits (MRLs), or tolerances in the USA, are established and enforced by regulatory agencies around the world. MRLs depend on pesticide toxicity, physico-chemical properties, and application rates, which can range significantly for the same pesticide in different commodities [2]. Additionally, different countries may have different MRLs for the same pesticide in the same commodity. Hence, reliable identification and accurate measurement of pesticide residues in foods is important in global food trade because false positives and other forms of incorrect results can have detrimental economic and health consequences.

Common approaches for analysis of pesticide residues include liquid chromatography (LC) or gas chromatography (GC) with single or triple quadrupole for tandem mass spectrometry (MS) for separation and identification of targeted contaminants. In recent years, however, high-resolution accurate mass (HRAM) analyzers are becoming more common in routine analysis of pesticides and other contaminants [3,4]. During the MS/MS method development phase, precursor, and fragment/product ions are selected for analyte identification, and often, the most intense ions are selected. Many pesticides belonging to the same chemical classes (triazines, carbamates, *etc.*) produce the same product ions, and analyte/analyte interferences commonly complicate their accurate quantification. To achieve better selectivity and increase confidence in identification, specific fragments should be selected and studied when possible.

The use of HRAM analyzers in comparison with low resolution quadrupole analyzer instruments facilitates ion mass determination with error <5 ppm required for ion formula determination [5]. Combining this information with literature searches, databases, and data interpretation helps with the assignment of product ions structures. Currently, few published manuscripts on the analysis of contaminants report fragment/product ions with elucidation of the corresponding structures [6–10]. Yet, elucidation of ion structures is important to provide further support for elimination of false positive findings. This becomes even more important when dealing with identification of structurally isomeric pesticides, i.e., pesticides with the same molecular formula and weight, but different atom arrangement.

One example of isomeric pesticides are the carbamate herbicides, orbencarb and thiobencarb. Orbencarb (*S*-(2-chlorobenzyl) *N,N*-diethylthiocarbamate) was previously used on cereals, including barley, wheat, rye, maize, soybean, *etc.*, but not currently authorized for use in the European Union (EU) and not registered for use in the USA. Thiobencarb (*S*-(4-chlorobenzyl)-*N,N*-diethylthiocarbamate) differs from orbencarb only by Cl positioning on benzene ring (Table 1). It is used for weed control in paddy fields, mostly on rice, and according to Pesticide Action Network of North America, has an active status in the US products [11], but it is not approved for use in the EU. The MRL for thiobencarb is 10–200 ng/g depending on the country and commodity. As isomeric pesticides possess very similar properties, they are usually difficult or impossible to separate chromatographically. For example, both orbencarb and thiobencarb had the same retention time of 19.38 min and the same ions at *m/z* 258, 125, and 100 when analyzed by LC-MS/MS [12]; thus they were not differentiated by either LC separation or MS identification. MS/MS spectra of isomeric compounds are usually very similar and may have few distinct ions to tell them apart. In order to support these differentiations, it is important to show structures for those specific ions in support of the method of analysis. The use of HRAM is essential for the determination of ion formulas to be able to determine structures and fragmentation pathways [13,14].

In this study, we selected five pairs of isomeric pesticides (see Table 1): Pebulate and vernolate, methiocarb and ethiofencarb, uniconazole and cyproconazole, sebuthylazine and terbuthylazine, and orbencarb and thiobencarb, to test the hypothesis of applying HRAM for finding unique product ions to distinguish between the isomers. A Q-Orbitrap HRAM MS instrument with an error <3 ppm was utilized for identification of products ion formulae and the proposal of structures and pathways of fragmentation to differentiate isomeric pesticides. When necessary, the fragmentation patterns were based on product ions obtained from the MS<sup>2</sup> and their selected ions spectra (MS3) obtained with a triple quadrupole instrument with a linear trap (Q-Trap) mass spectrometer.


**Table 1.** Isomeric structures of studied pesticides.

#### **2. Materials and Methods**
