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Development of Detection Methods for Colistin Sulfate, Bacitracin and Virginiamycin M1Residual in Aquatic Products

Author: LuoFangFang
Tutor: WuChengYe
School: Jimei University
Course: Processing and storage of agricultural products
Keywords: CS BTC VGMM1 HPLC-MS/MS HPLC-fluorescence detection Uncertainty
CLC: TS254.7
Type: Master's thesis
Year: 2013
Downloads: 48
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Abstract


Colistin sulfate（CS）, Bacitracin（BTC） and Virginiamycin M1（VGMM1） are peptideantibiotics which are found to be resistant to gram-negative bacteria, gram-positive bacteria, aswell as some fungal and viral infection. They are widely used in animal husbandry for treatmentand prevention of diseases and as feed additives to increase the animal mass. In recent years theirmisuse can pollute environment and leave residues in edible animal tissues, which may affectaquaculture and give rise to public health concern. In this paper, a rapid, sensitive and selectivehigh performance liquid chromatography-tandem mass spectrometric （HPLC-MS/MS） methodand a high performance liquid chromatography （HPLC） with fluorescence detection method forthe determination of these peptide antibiotic residues in aquatic products and in the water wasdeveloped. The study included following sections:1) A new analytical method for simultaneous determination of CS, BTC and VGMM1inaquatic products by HPLC-MS/MS was developed. The samples were extracted withmethanol-water containing0.1%formic acid（5:2, v/v）. The extract was purified with acetonitrilcontaining4%trichloroacetic acid and degreased by n-hexane. Further clean-up was performedby SPE. Data acquisition was achieved by applying selected reaction monitoring （SRM）, andquantification analysis was performed with matrix matched standard calibration curves. Thedetection limit is10μg/kg、10μg/kg、2μg/kg for CS、BTC and VGMM1, the quantificationlimit is20μg/kg、20μg/kg、4μg/kg for CS、BTC and VGMM1。The average recovery of thedrugs fortified in different negative samples at three spiked levels varies from72.3%to103.98%,and the relative standard deviation（RSD） vary from1.10%to10.92%; seven kinds of aquaticproducts have been selected for verification, the average recoveries ranged from76.9%to107.5%and the RSD were less than8.20%.2) A new analytical method for simultaneous determination of BTC in farming waters byHPLC-MS/MS was developed. Based on the analysis of the extract and purificat conditions,different solid-phase extraction columns were compared and the steps of solid-phase extractioncolumn were optimized. The method showed good linear in the range of5ng/mL-5000ng/mL,the detection limit for BTC was5ng/mL and the quantification limit was10ng/mL. Differentplaces waters were selected for verification, the average recoveries varies from79.1%to99.0%and the RSD between1.02%-6.69%.3) A new analytical method for simultaneous determination of CS in aquatic products by HPLC-fluorescence detection was developed. Different derivatizing reagents, pH and time of thederivatization were optimized. And different chromatographic columns of the separation werecompared to establishe this method. The method show good linear between0.03ng/g-6ng/g（R2＞0.999）, the detection limit was30μg/kg. The recoveries of CS in different negativesamples at three spiked levels were ranged from88.8%to97.5%, and the RSD were varied from5.01%to6.91%; six kinds of aquatic products have been selected for verification at150μg/kglevel, the average recoveries ranged from80.7%to96.0%and the RSD were less than10.00%.4) Uncertainty was analyzed for the three detection methods. By analysis we know the mainuncertainty factors were came from the regression equation and the recovery. Therefore, we cantake appropriate measures to minimize the impact of the uncertainty on the results to improve theaccuracy.This paper has established HPLC-MS/MS and HPLC-fluorescence detection for thedetermination of these peptide antibiotic residues in aquatic products and in farming waters. Themethods have the advantages of simplicity, sensitivity and repeatability, based on the verificationexperiments, these three kinds of methods can provide practical reference and application valuesto the drug residual analyses.

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CLC: > Industrial Technology > Light industry,handicrafts > Food Industry > Aquatic products processing industry > Standards and inspection of aquatic products
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