Characterization of Impurities in the Fungicide Flutriafol Using UNIFI and UPLC-ToF-MS^E

Flutriafol is a triazole fungicide that is used in the treatment of a broad spectrum of diseases in cereal crops. This application note describes the analysis of technical-grade flutriafol material using  the ACQUITY UPLC I-Class System coupled to the Xevo G2-XS QTof and PDA Detector

Benefits

• A comprehensive approach to the characterization of structurally related components in technical grade flutriafol.
• A rapid chromatographic separation that provides maximum component resolution.
• Accurate mass precursor and product ion information collected simultaneously increase confidence in the compound assignments.

The profiling, identification, and quantification of impurities plays a critical role at all stages of both the pesticide development and manufacturing processes, and are essential requirements for the registration of crop protection products. Impurities in agrochemical products are regulated in order to ensure product safety and compliance. The structure of impurities present at or greater than 0.1% in the technical grade active substance must be identified to ensure that the overall safety of the formulated product is well understood.¹ Impurities of toxicological significance are of particular interest.

During the development process, different routes of synthesis may be evaluated, each producing its own distinct impurity profile. It is critical that these synthesis routes are thoroughly investigated with an aim to identify (or elucidate) relevant impurities alerted by the threshold criteria. Analytical investigation is often performed using a combination of LC-MS and other techniques that provide structural information, such as NMR spectroscopy. Modern, high-sensitivity exact mass instruments with time-of-flight (Tof) technology are capable of extremely fast acquisition rates and offer the opportunity to dramatically simplify the process of impurity data capture and analysis. These high acquisition rates allow mass spectrometry to be coupled together with UltraPerformance Liquid Chromatography (UPLC), thereby greatly facilitating the analysis of complex samples. Additionally, Waters’ proprietary Q-Tof technology enables the simultaneous collection of low and high collision energy (CE) data, otherwise referred to as “MS^E”,² allowing the capture and interpretation of precursor and product ion data from a single analytical injection. As this data is collected with a high degree of mass accuracy, elemental compositions can be obtained for both intact molecular ions (e.g., M+H⁺) and structurally significant fragments greatly aiding structural elucidation.

In this application note, Waters ACQUITY UPLC I-Class System coupled to the Xevo G2-XS QTof and the ACQUITY UPLC PDA Detector were used to analyze technical-grade flutriafol material. Flutriafol, shown in Figure 1, is a triazole fungicide that is used in the treatment of a broad spectrum of diseases in cereal crops.³ The Screening Platform Solution with UNIFI was used to screen the sample and expedite the discovery of unknown components of interest, as well as to confirm the presence of the active ingredient (AI). Using the data evaluation tools available in UNIFI, relationships between the unknown components were easily visualized and the impurity relationship to flutriafol was hypothesized. The MS data from the impurity profile can be later be used to further refine the synthetic strategy.

UPLC separation using the CORTECS C₁₈ Column provided maximum component resolution. Three minor chromatographic peaks (peaks 1–3) and flutriafol (peak 4) were detected in the UV at 220 nm, Figure 2. PDA detection allowed the area% contributions of peaks 1–3 to be determined relative to the AI, where they each exceeded the threshold of >0.1%. MS detection was used to perform the initial structural elucidation of the impurities.

In the component summary of the UNIFI review window, candidate masses were tabulated (Figure 3) and the identified components are listed.

The precursor and product MS^E spectral data for each candidate mass are automatically extracted and can be viewed simultaneously in the spectrum window (Figure 3). If adducts for the precursor ion are present they are automatically assigned. UNIFI also performs a fragment match when a structure is available, intelligently assigning likely structures to high CE fragments.

The flutriafol was identified and represented as an extracted ion chromatogram (XIC) using specific information such as compound name, retention time (tR), mol files, and fragment ions that were generated from prior analyses of the fungicide and entered into the UNIFI Scientific Library. This approach can be used to build the analysis method prior to data acquisition (Figure 4). UNIFI’s Scientific Library can store structures and begin retaining information associated with the individual library entries. Parent compounds and impurities of interest can be stored in the library for later searching and retrieval.

The peaks labeled 1 and 2 from Figure 2 eluted at 2.94 min and 3.41 min respectively. Both components were extracted as XICs, as the two peaks have an m/z that is within the flutriafol target mass tolerance of 3 ppm set in the processing method (Figure 3).

Candidate masses can be investigated using the elucidation toolset in UNIFI which contains a comprehensive suite of structural elucidation tools that can be utilized by selecting any candidate mass from the component summary list. The elucidation toolset allows precursors and fragment ions to be interrogated more intensively. This includes ChemSpider search (www.chemspider.com, The Royal Society of Chemistry), isotope modeling, and elemental composition tools. Discovery tools for related ions using mass defect, common fragment, and neutral losses are also available. 

The structural elucidation tools indicated that both components 1 and 2 had identical elemental composition to the flutriafol, C₁₆H₁₃F₂N₃O (Figure 5). In addition the high energy MS^E data indicated that the components had common fragment ions at different ratios compared with the AI (Figure 3). The existence of the high energy product ion data along with the intact precursor increased the confidence in the identifications.

Based on these observations it is likely that components 1 and 2 are isomers of the AI.

Using UNIFI’s common fragment search function, components that share common structural features can be efficiently extracted from the data. By selecting prominent fragments in the spectrum of component 1, including the signature fragment originating from the triazole ring m/z 70.0399 and m/z 109.0450, we can see an additional chromatographic peak at t_R 3.49 minutes was extracted using the same product ions, as shown in Figure 6.

This component (peak 3), has an m/z of 314.1303 and has been tabulated in the component summary, it was investigated in the same way as the previous example (Figure 7).

The elemental composition of the unknown mass was determined to be C₁₇H₁₆FN₃O₂. The common fragment search function indicated the component shared common structural characteristics with the flutriafol. The elemental composition of component 3 was searched using the ChemSpider Search function, which is embedded in the elucidation toolset.

A suitable match was not made based on the inability to assign all fragments, including the common fragments simultaneously within the same structure proposal. Therefore, an alternative route to assign the structure was carried out, using a combination of elemental composition for the individual fragmentation spectra of component 3, as well as confirmation of the fragments using MS/MS, knowledge of the synthetic route, and a comparison with the elemental composition of the fragmentation spectra for the flutriafol. The proposed structure was then used to assign probable structures to the product ions (Figure 8).

Based on the general structural proposal (inset) the fragments were assigned using the UNIFI fragment match tool. Absolute confirmation of the structure would require scale up, trace isolation followed characterization using appropriate techniques, such as NMR spectroscopy.

The components can be added to the UNIFI Scientific Library, including any known information (retention time, known fragments, etc.) so that they can be tracked across further syntheses.

During chemical synthesis residual starting products, intermediates, or reaction by-products can lead to impurities in the final technical grade materials. Identification of the components can help to optimize the synthetic process. Minor components present in the technical grade flutriafol were detected in the PDA at 220 nm. The unidentified components were characterized using UPLC coupled with ToF MS^E and the UNIFI Screening Platform Solution. Two components were tentatively identified as isomers of the active ingredient, and a third unknown component was also observed. Using the elucidation toolset within UNIFI, the unknown component was found to have structural features that were common to flutriafol. Based on the elemental composition of the fragments, knowledge of the synthetic route, and the structure of the active ingredient, a structure for the unknown component having m/z 314.1303 was tentatively proposed. This tentative structure is in agreement with all of the data that iscurrently available. Future impurity isolation studies will allow for the definitive  determination of the structures of these impurities.

1. Commission Regulation (EU) No 544/2011. (2011) [cited February 13, 2015 ]. Available from: http://eur-lex.europa. eu/LexUriServ/LexUriServ.do uri=OJ:L:2011:155:0001: 0066:EN:PDF
2. An overview of the principles of MS^E, The engine that drives MS performance. Waters White Paper, p/n 720004036en. October, 2011.
3. Chang, M, Kim, TH, Kim, HD. Stereoselective synthesis of flutriafol. Tetrahedron: Asymmetry. 19: 1503–1507, 2008.