| Purpose Establish a QuEChERS ultra high performance liquid chromatography tandem mass spectrometry method for the determination of diazepam,oxazepam,and MS-222 sedative residues in freshwater fish.Optimize the QuEChERS extraction method for three sedatives in freshwater fish products using single factor experiments and five factor four level orthogonal experiments.Three sedatives commonly found in freshwater fish markets across Yunnan Province were tested and analyzed.Discussions and analysis were conducted on the detection results.Method A QuEChERS pre-treatment method for three sedatives was established using a highperformance liquid chromatography four stage time-of-flight tandem mass spectrometer.The mass spectrometry conditions were optimized by comparing the chromatographic column,column temperature,flow rate,mobile phase,and its elution gradient to establish a detection method.On the basis of single factor analysis,5 factors were selected using the range method.Orthogonal experiments were designed using orthogonal tables to evaluate factors such as material liquid ratio,PSA powder amount,C8 powder amount,ultrasound time,and ultrasound temperature.The recovery rate was used as the evaluation index for comprehensive evaluation to determine the optimal extraction method for the sample.The established detection method was used to qualitatively and quantitatively detect three sedatives in freshwater fish samples from various states in Yunnan Province.Result 1.A method was established for the determination of diazepam,oxalazepam,and MS-222 sedative residues in freshwater fish using QuEChERS sample pretreatment combined with ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The method can complete the detection within 8 minutes,and the three target compounds have good linear relationships within the linear range.The linear correlation coefficient(r2)of diazepam is 0.9986,the detection limit and quantification limit are 0.24 and 0.81 μ g/kg,respectively.The linear correlation coefficient(r2)of oxazepam is 0.9990,the detection limit and quantification limit are 0.27 and 0.91 μ g/kg,respectively.The linear correlation coefficient(r2)of MS-222 is 0.9989,the detection limit and quantification limit are 0.03 and 0.12 μ g/kg,respectively,The absolute values(%)of mechanism effect ME are 10.1,12.3,and 8.6,respectively.The precision of the instrument is good,and the relative standard deviations of the three compounds at low,medium,and high concentrations are 1.28%,2.13%,and 4.02%,respectively;1.39%,1.97%,3.88%,and 1.18%,2.09%,3.83%.This method is fast,stable,reliable,and has a lower and more accurate detection limit compared to traditional methods.2.Using single factor experiments and orthogonal experiments to compare the extraction efficiency of different sample weights and extraction reagent volume ratios,different amounts of PSA powder,different amounts of C18 powder,different ultrasound times,and different ultrasound temperatures,the optimal sample conditions were determined as follows: the volume ratio of sample weight to extraction reagent was 2:5,the amount of PSA powder was 0.1g,the amount of C18 powder was 0.1g,the ultrasound temperature was 50 ℃,and the ultrasound time was 10 min,Extract the supernatant using polytetrafluoroethylene(PTFE)with a diameter of 13 mm and a pore size of 0.2 μ m)Filter membrane.3.A total of 40 samples were collected from freshwater fish markets in various states of Yunnan Province.The test results showed that none of the three sedatives were detected in the 40 samples collected from various states of Yunnan Province.Conclusion The QuEChERS liquid chromatography tandem mass spectrometry method established in this article has high sensitivity,good stability,low detection line,and is more accurate.It can simultaneously detect diazepam,oxazepam,and MS-222 sedatives,and the loss of its target substance in the recovery experiment is less,which can meet the detection requirements of three target sedatives in large-scale freshwater fish products.2.The optimal pre-treatment conditions for the detection of three sedatives in this experiment were determined as follows: a volume ratio of 2:5 for sample weighing and extraction reagents,0.1g for PSA powder,0.1g for C18 powder,50 ℃ for ultrasound temperature,and 10 minutes for ultrasound time.The supernatant was extracted through a polytetrafluoroethylene membrane.3.Three types of sedatives were tested in freshwater fish markets in various regions of Yunnan Province,and the results of the three sedatives in 40 samples collected from various states in Yunnan Province were all undetected. |
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