**1. Introduction**

Aquaculture is estimated to provide half of aquatic products by 2030 from the farming of freshwater or marine areas [1]. There is inevitably going to be a need for intensive aquaculture developed to supply more products from this industry. According to the "Green food—fishery medicine application guideline (NY/T 755-2013)" in the Agricultural Industry Standards of the People's Republic of China [2], fishery medicine refers to the substances that prevent or treat diseases in aquaculture animals or purposefully regulate the physiology of animals, including chemicals, antibiotics, Chinese herbal medicines and biological products. It is also known as chemical inputs or veterinary medicinal products (VMPs) applied in aquaculture in Europe and the United States [3,4]. Chemical inputs from aquaculture include antifoulants, antibiotics, parasiticides, anesthetics and disinfectants [5], while parasiticides in fishery mainly contain avermectins, pyrethroids, hydrogen peroxide, and organophosphates [5,6].

Based on the Guidelines, ten kinds of fishery drugs originated from pesticide have been banned for aquatic animals and plants. However, the illegal or excessive addition of pesticides in the fishery drug, as well as uncontrollable and uncertain administration during culture process can lead to the accumulation and residue of these pesticides in aquatic product. Illegal and unregulated use of pesticide may occur in many aquaculture areas, and further threaten the food safety for human health. To protect the quality and safety of aquatic products, as well as the sustainable ecosystem, surveillance of pesticides components in fishery drugs should be conducted.

Ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a promising strategy for multi-component screening of pesticides [7–9]. HRMS could record full scan of the precursors or fragmented ions with high-resolution, as well as the relative isotopic abundance, and is virtually able to distinguish unlimited number of compounds from one set of analyzed data [10,11]. In the past, the chromatography coupled to Time of Flight Mass Spectrometry (ToF-MS) was used in the development of multiclass components screening methods [9,12,13]. However, comparing to ToF-MS, the orbitrap mass spectrometer can fast scan and simultaneously switch between positive and negative acquisition modes if there's no need to change mobile phase of chromatography unit [14,15]. The combination of quadrupole and Orbitrap for high-resolution mass spectrometry can acquire data with high throughput, excellent accuracy and better sensitivity, which provides an ideal platform for multiclass risk compound screening [16]. Therefore, more methods of screening detection with Orbitrap MS were developed. With this instrument, the data-dependent data acquisition mode scans the full mass distribution of all precursors and then selectively fragments them sequentially for secondary mass scanning according to their abundance. This scan mode allows the quantification of compounds with precursor ion abundancy and identification with corresponding fragment ions [17]. Moreover, due to the stable and high-resolution mass spectrum recorded at standard data provide enough dependency, the identification of targeted compound can be conducted by comparing their database rather than practically acquire data for standards every time [18,19].

In previous studies, the analysis of 139 pesticide residues in fruit and vegetable commodities was established based on the Q-Orbitrap MS, allowing the retrospective analysis of the data feature which cannot be achieved with QqQ [17]. Jia et al. have developed an untargeted screening method for 137 veterinary drugs and their metabolites (16 categories) in tilapia using UHPLC-Orbitrap MS [20]. Turnipseed established a wide-scope screening method for 70 veterinary drugs in fish, shrimp and eel using LC-Orbitrap MS [7]. Recently, a non-target data acquisition for target analysis workflow based on UHPLC/ESI Q-Orbitrap was examined for its performance in screening pesticide residues in fruit and vegetables [21]. However, there is a lack of works on the multi-component screening detection in fishery drugs, especially for pesticide component screening. A fast screening method for a wide range of pesticides detection can be preferred, as much more reagent, time, and labor can be saved to detect more harmful components for safety evaluation.

Our study aims to develop a more generic screening method for a wider scope of pesticides with a self-built database, which can keep the advantages of robustness, simplicity, and time-e fficiency. In the current work, we investigated 89 possible pesticides that can be used in fishery-related industry and remained in aquatic products. The chromatographic and high-resolution mass spectra for these compounds were acquired with a UHPLC-quadrupole-Orbitrap HRMS after optimizing parameters. The useful fragment ions with high-resolution were explored and selected. Then, a database including the retention time, isotope pattern, ionization mode and adduct, characteristic fragment ions, was established. Identification rules for data comparison with real samples were also investigated. Finally, a fast pesticide screening method for fishery drug was developed in combination with a rapid pretreatment.

#### **2. Results and Discussion**

#### *2.1. Full MS-ddMS<sup>2</sup> Scan for Identification and Qualification*

Full MS-ddMS<sup>2</sup> detection mode was applied on UHPLC-ESI-Q-Orbitrap HRMS system, which is a di fferent data acquisition from single (multiple) reaction monitoring on triple quadrupole mass spectrometry. The Orbitrap analyzer collected accurate mass of all precursor ions as the first identification step of compounds. The precursors of high abundance were isolated through quadrupole in the next round scanning. Each of the precursors can be fragmented sequentially in the HCD multipole, re-collected in C-trap, and analyzed through Orbitrap mass spectrometer. It should be noted that the accurate mass of precursors instead of their fragmentation ions was continuously tracked and can be integrated for peak identification. Therefore, the precursor ions can be used for quantification and their corresponding fragmented ions for each peak of precursor ion can be used for identification in combination.

Under the guideline of European SANTE/11813/2017 and Commission Decision 2002/657/EC [22], identification of the concerned analytes with high-resolution mass spectrometry can be performed. The chromatographic information, their mass information should attain given identification points (IPs) to ge<sup>t</sup> confirmed results. If the high-resolution mass spectrometric data were collected, 2 IPs are earned if the precursor ion match, and 2.5 IPs for each of their product ions [23,24]. For the identification of all compounds, 4.5 IPs are required. In our work, the *m*/*z* of isotope, and its relative abundance for precursors were also identified, which leads to higher IPs for structure identification. Therefore, our identification rule should be stricter and more reliable than current regulations, which can result in less false positive result according to our experiment on fortified samples.
