Simultaneous determination of eight antiviral drugs and two metabolites in Chinese softshell turtles using pass-through solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry

<p id="p00005">Antiviral drugs (ATVs) are not licensed for the treatment of viral diseases in food-producing animals in China. However, they are reportedly illegally used in the Chinese softshell turtle (<italic>Pelodiscus sinensis</italic>) aquaculture industry; hence, the use of ATVs has become a new aquatic food-safety problem. In this study, a method was developed for analyzing eight ATV residues (amantadine, rimantadine, lamivudine ribavirin, moroxydine, acyclovir, oseltamivir, and imiquimod) and two metabolites (1,2,4-triazole-3-carboxamide and oseltamivir acid) in Chinese softshell turtle muscle using pass-through solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The main factors that affect the sensitivity, precision, and accuracy of the method were optimized during sample pretreatment and instrumental analysis owing to the diverse chemical properties of the analytes. The performance of various chromatographic columns and mobile phases were investigated to eliminate interference from uridine during the detection of ribavirin. The proportion of acetonitrile in extration solution and solid-phase extraction column type were prioritized when optimizing extraction and purification parameter. Samples were hydrolyzed using phosphatase (acid type) in 6 mL ammonium acetate buffer, with optimum results obtained when enzymolysis was performed at 37 ℃, and pH 4.8 for 2 h, after which they were extracted with 14 mL of acetonitrile containing 1% acetic acid. The supernatant (2 mL) was purified using a Wallelut Lipoclean column operated in pass-through solid-phase-extraction mode. The sample was dried under nitrogen at 40 ℃ and the residue was dissolved in 1 mL of methanol-acetonitrile (50∶50, v/v) solution. The sample was finally filtered through a 0.22-μm hydrophilic polytetrafluoroethylene syringe filter for UHPLC-MS/MS. The chromatographic separation of the analytes was performed on a hydrophilic interaction chromatographic column (HILIC-Z, 100 mm×2.1 mm, 2.7 μm) via gradient elution using 2 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) and acetonitrile as mobile phases A and B, respectively. The linear gradient program were as follows: 0-1.0 min, 3%A; 1.0-5.0 min, 3%A-20%A; 5.0-6.0 min, 20%A; 6.0-6.5 min, 20%A-50%A; 6.5-8.0 min, 50%A; 8.0-8.5 min, 50%A-3%A; 8.5-12 min, 3%A. The analytes were detected using a triple quadrupole mass spectrometer operating in positive-ion scanning and multiple-reaction monitoring (MRM) modes, with quantitative analysis performed using the internal standard method. Good linearities (<italic>r</italic>²=0.994-0.998) were obtained for the ten target compounds at experimental concentrations under the optimal conditions. The method detection limits (MDLs) and method quantification limits (MQLs) were in the range of 0.05-1.1 and 0.18-3.8 μg/kg, respectively. The average recoveries of the 10 target compounds ranged from 82.5% to 103%, with relative standard deviations (RSDs) in the range of 3.11%-12.1% at low, medium and high spiked levels. The developed method was applied to 67 Chinese softshell turtle samples obtained from a fish farm in Zhejiang Province. Ribavirin and metabolite were detected in real samples at total contents of 6.02-333.2 μg/kg. The developed method is simple to operate, highly accurate, and suitable for use in risk-monitoring applications owing to its ability to rapidly determine eight ATV residues and two metabolites in Chinese softshell turtle samples.