Simultaneous Determination Of Three Tetracyclines And Two Fluoroquinolones Residues In Chicken And Poultry Eggs By Ultra-high Performance Liquid Chromatography With Fluorescence Detection

The purpose of this study was to establish a method for the simultaneous determination of three tetracyclines and two fluoroquinolones residues in chicken muscle and poultry eggs(chicken whole egg,chicken albumen,chicken yolk,duck whole egg,duck albumen,duck yolk)by ultra-high performance liquid chromatography-fluorescence detection(UPLC-FLD)method.In this experiment,Haiyang yellow chicken and Gaoyou duck were selected as the experimental animals.Liquid-liquid extraction(LLE)combined with solid phase extraction(SPE)was used to extract the targets.Development and optimization of UPLC-FLD method for simultaneous determination of oxytetracycline(OTC),tetracycline(TC),doxycycline(DOX),ciprofloxacin(CIP)and enrofloxacin(ENR)residues in chicken muscle and poultry eggs.The main results of this study are shown as follows:1.A method for simultaneous extraction of the OTC,TC,DOX,CIP and ENR residues in chicken muscle by LLE was established and optimized.That is,acetonitrile-0.1 mol/L citric acid+100 mmol/L magnesium chloride solution(1:1,V/V,and the pH was adjusted to 5.0 with ammonia)was added to the sample.Vortex and ultrasonic extraction,the precipitate was then removed by centrifugation,repeated extraction once.The combined supernatants were purified by Oasis PRiME HLB solid phase extraction column(60 mg/3 mL).The sample solution was dried by nitrogen blowing and then re-dissolved with the initial mobile phase.The recoveries of the five drugs were all over 87.33%,and the extraction method has high recovery rate.2.Amethod for simultaneous extraction of the OTC,TC,DOX,CIP and ENR residues in poultry eggs by LLE was established and optimized.That is,acetonitrile-aqueous solution(90:10,V/V)was added to the sample for vortex and ultra-sonic extraction,the precipitate was then removed by centrifugation,repeated extraction once.The combined supernatants were purified by Oasis PRiME HLB solid phase extraction column(60 mg/3 mL).The sample solution was dried by nitrogen blowing and then re-dissolved with the initial mobile phase.The recoveries of the five drugs were all over 83.50%,and the extraction method has high recovery rate.3.An UPLC-FLD method for simultaneous detection of OTC,TC,DOX,CIP and ENR residues in chicken muscle and poultry eggs was developed and optimized.ACQUITY UPLC BEH C18 column(100 mm×2.1 mm,1.7 ?m)was used for chromatographic separation,acetonitrile-0.1 mol/L malonic acid+50 mmol/L magnesium chloride solution(the pH was adjusted to 5.5 with ammonia)was used as the mobile phase.The targets were separated by gradient elution at a flow rate of 0.2 mL/min,the column temperature was 30?.Double channel detection:the excitation and emission wavelengths of OTC,TC and DOX were all 416 nm and 518 nm,respectively;the excitation and the emission wavelengths of CIP and ENR were all 274 nm and 428 nm,respectively.The results showed that the concentration range of OTC and TC in blank chicken muscle were at the limit of quantitation(LOQ)-500.0 ?g/kg,the concentration range of OTC and TC in blank poultry eggs were at the limit of quantitation(LOQ)-1000.0 ?g/kg,and the concentration range of DOX,CIP and ENR in blank chicken muscle and blank poultry eggs were at the LOQ-300.0?g/kg,the peak area of the targets have a good linear relationship with its concentration,and the determination coefficient(R2)were both greater than 0.999 0.When added the concentrations of OTC,TC,DOX,CIP and ENR were LOQ,0.5 maximum residue limit(MRL),1.0 MRL and 2.0 MRL,the recoveries in blank chicken muscle were 87.33%-96.90%,87.98%-94.58%,87.70%-93.83%,87.68%-90.73%and 90.30%-94.53%,respectively;the intra-day relative standard deviation(RSD)were 2.30%-4.91%,2.06%-4.74%,3.03%-4.36%,2.34%-3.88%and 2.17%-3.84%respectively;the inter-day RSD were 2.43%-5.13%,2.12%-4.90%,4.06%-4.79%,3.03%-4.08%and 2.52%-4.48%,respectively;the limits of detection(LODs)were 6.1 ?g/kg,10.2 ?g/kg,13.1 ?g/kg,0.2?g/kg and 0.1 ?g/kg,respectively;and the LOQs were 20.4 ?g/kg,35.2 ?g/kg,39.4 ?g/kg,0.6 ?g/kg and 0.4 ?g/kg,respectively;the recoveries in blank poultry eggs were 85.30%-91.15%,84.10%-90.20%,83.50%-90.90%,85.53%-92.88%and 86.15%-95.58%,respectively;the intra-day RSD were 2.03%-4.52%,2.24%-4.91%,2.13%-3.98%,1.99%-4.70%and 2.07%-4.67%,respectively;the inter-day RSD were 2.32%-4.96%,2.57%-5.55%,2.37%-5.80%,2.10%-5.33%and 2.81%-6.24%,respectively;the LODs were 5.2-7.7?g/kg,8.9-11.8 ?g/kg,9.6-13.4 ?g/kg,0.2-0.5 ?g/kg and 0.1 ?g/kg,respectively;and the LOQs were 17.4-25.6 ?g/kg,27.3-38.3 ?g/kg,31.9-40.1 ?g/kg,0.6-1.5 ?g/kg and 0.3-0.5?g/kg,respectively.This detection method is fast,efficient and sensitive,and provides a new method for the simultaneous detection of OTC,TC,DOX,CIP and ENR residues in animal derived foods.