Preparation Of Immunoaffinity Stir Bar And Its Application In Determination Of Quinolones Residue

Quinolones(QNs)have been widely used in human and animals for treatment of infectious diseases due to its good antibacterial effect.Its residues in edible animal products pose serious threats to consumers’health.There is an urgent need to develop a highly specific,sensitive and efficient method to quantify QNs in milk.In this study,SPME was combined with IAC by immobilization of the Mabs on a glass bar,the resultant immunoaffinity stir bar is more convenient to use and is highly selective,permitting the direct extraction and purification of QNs from milk.A specific immunoaffinity stir bar was firstly developed using broad-specificity Mabs(anti-QNs)to selectively extract and purify 11 QNs(MAR,DAN,NOR,CIP,LEV,ORB,ENR,LOM,OFL,FLE and FLU).The preparation procedures were as follows,the lower halves of glass bars were cleanedand etched by immersing in a mixture of 70 m L 98%sulfuric acid and 30 m L 30%hydrogen peroxide.After the mixture lowered toroom temperature,heating mixture in water bath at 80?C for 1 h.Bars were then thoroughly rinsed with water,absolute ethanoland water.Particular care was taken in case of contamination.The cleaned parts of bars were silanized with ethanolic APTES solution(5m L APTES,5 m L water and 90 m L absolute ethanol)for 24 h at room temperature,followed by rinsing with water and absolute ethanol.The bars were placed in a vacuum oven with nitrogen flushed at 70?C overnight or 15 h.Bars were activated by placing in2.5%GA solution for 7 h,followed by rinsing with water.After rinsing,the GA activated surface were immersedin 10 m L m Ab PBS solution(0.5 mg/m L)to a depth of 2.5 cm and stirred for 24 h at 4?C using a magnetic stirrer.Extraction time,stirring rate,proportions of elution solution,desorption time and volume of desorption solvent were optimized.The column capacities of 11 QNs were0.35-2.15 pmol/cm~2.The column capacities were about 29.3%-40.2%of the original capacity after 15 cycles of use in 45 days.An immunoaffinity stir bar sorptive microextraction(SBSME)-HPLC-FLD method was developed for residual determination of 11 QNs in bovine milk.Analytes were extracted by placing stir bar in milk and shaking on a rotary shaker for 30 min at 30 r/min,then the bars were washed with water,the compound was eluted in 900μL Me OH and PBS mixture(8:2,v/v),which was gently shaken at room temperature for 20 min.followed by liquid chromatography and fluorescence detection.At a spiked concentration of 0.1 ng/g,the mean recoveries of the 11 QNs ranged from 11.8%to 40%.The newly developed method has limits of detection for each QN from 0.05 to 0.1 ng/g with intra-day and inter-day precision ranging from 3.2%to 11.9%and from 5.2%to 12.5%,respectively.