*2.8. Practical Screening*

The method was further applied in screening of 21 fishery drug samples (pesticides, water-clean agents and antibacterial agents). Samples were prepared according to sample preparation (4.4) prior to analysis. For the compounds with concentration out of the linear range, samples were re-diluted with the dilution factor of samples adjusted to ensure the concentration to be quantitatively evaluated based on our linear range. The screening was carried out following the home-built database and preset rules. Quantification was conducted through the peak areas of precursor ions in positive samples and was externally calibrated. Based on the database and preset identification rules, 10 out of the 21 fishery drug samples were screened positive with pesticides. 10 samples were detected with unspecified components. As is shown in Table 2, the identified pesticides were chlorpyrifos, ivermectin B1a, phoxim, avermectin B1a, and carbendazim. Three samples contained forbidden drug chlorpyrifos (Figure 5A), and 5 samples contain avermectin and ivermectin (Figure 5B) of more than 3 g/L. Their chromatographic and fragment information was highly identical to the standards, as are shown in Figure 5. Detailed information of the screened positive samples was presented in Table 2.


**Table 2.** Screening results of practical fishery drugs.


**Table 2.** *Cont.*

NA: not available. Listed: active compounds were listed in the label of fishery drugs.

**Figure 5.** Comparison of the spectra of chlorpyrifos ( **A**) and ivermectin B1a (**B**) detected in the positive samples. A1, B1 and A2, B2 are the chromatogram of real samples and standards, respectively. A3, B3 and A4, B4 is the MS/MS spectrum of the positive sample and the standard respectively.

#### **3. Materials and Methods**

#### *3.1. Instruments and Reagents*

The ultrahigh-performance liquid chromatography (UHPLC) system (Dionex UltiMate 3000, Thermo Fisher Scientific, San-Francisco, USA) coupled to quadrupole Orbitrap mass spectrometer with electrospray ionization (Q-Exactive, Thermo Fisher Scientific) was used for data acquisition.

Eight-nine Pesticides were selected for target screening as listed in Table 3. Carbofuran and dichlorvos were obtained from MANHAGE Biotech. Inc. (Beijing, China), thiofanox-sulfone, thiometon, aldicarb-sulfone, phoratoxon sulfoxide, PCP Na were purchased from Accustandard Inc. (New Haven, CT, USA) The other 85 pesticides standards were supplied by Dr. Ehrenstorfer GmbH (Augsburg, Germany). Acetonitrile (MeCN) and Methanol (MeOH) of HPLC grade were obtained from J.T. Baker (Phillipsburg, NJ, USA). Formic acid (FA, 98%, LC-MS grade, Fisher Scientific, Spain, or HPLC grade) was obtained from FLUKA. All the other relevant reagents were purchased from common domestic suppliers. Pure water was obtained through Water Milli Q ELEMENT purification unit (Millipore, Bedford, USA).

## *3.2. Preparation of Standards*

Standards stock solution: c.a. 5 mg solid standards was dissolved with MeOH in 10 mL beaker, and then transferred to a 50 mL flask and diluted with MeOH. For compounds will less solubility in MeOH, 0.1 mL formic acid (98%, HPLC grade) was firstly added and the mixture was sonicated until the solids were completely dissolved. Five microliters of liquid standards were pipetted into a 10 mL-beaker and weighed to ge<sup>t</sup> accurate mass. After that, they were dissolved in methanol following similarly procedure as the solid standards. All these single standards stock solutions were c.a. 100 μg/mL. The purchased standards solutions were not diluted until further preparation of mixed standards solution. Mixed standards solutions were prepared by mixing standards of the same category, which were finally diluted to 5 μg/mL. The standards were categorized into organophosphorus, carbamate, organochlorine, imidazole, pyrethroid, triazole, phenylpyrazole, avermectin, and miscellaneous. All the standards solutions were stored in refrigerator at −42 ◦C.

Matrix-matched standards were used to evaluate the matrix effect, where the standards were dissolved into a matrix of Chinese herbal fishery drug, which was negative for pesticides before spiking.
