The Study Of Detection Methods Of Macrolides Residual In Aquatic Products

Macrolide antibiotics (MALs) are a kind of weak alkaline and lipophilic antibiotics used forhuman and animal, the chemical structures of these drugs containing a large ring lactonegroup and several amino sugars which are connected with glycoside bond, especially containingthe alkaline tertiary amine structure in their structure. MALs are prominently active againstGram-positive bacteria and Mycoplasma. MALs were used widely in animal bacteria andmycoplasma infection of the chemical treatment, these drugs were also used as growth promotersadded in animal drugs and feed additives. However, the widely use of MALs including someunreasonable application may leave antibiotics residues phenomenon, it’s a serious threat on thesafety of aquatic products, and then possibly lead to adverse effects on consumers. In this paper,spiramycin(SPI), kitasamycin(KIT), tylosin(TYL), tilmicosin(TIL), erythromycin(ERM),oleandomycin(OLD) and Josamycin(JOS) were selected as target analytes, to establish a highperformance liquid chromatographic(HPLC) method and a high performance liquidchromatography tandem mass spectrometric(HPLC-MS/MS) method for determination of theseantibiotic residues in aquatic products. The study included following sections:1) An HPLC method has been developed for simultaneous determination of SPI, TIL, TYL,KIT and JOS in aquatic products. Chromatographic conditions such as chromatography column,column temperature, flow rate, gradient elution procedures and pretreatment method wereoptimized, respectively. Samples were extracted with acetonitrile, and the extracts wereevaporated to near dryness and redissolved in phosphate buffer solution. The extracts werecleaned up on Oasis HLB column. The standard curves showed good linear between the peakareas ratio and concentrations of five macrolides in the range of0.1~10μg/mL, and thecorrelation coefficients were not less than0.9990. The mean recoveries of the five macrolidesfrom different samples fortified at three spiked levels of0.1,0.25,0.5μg/kg were varied from75.2%to110%, the relative standard deviations were varied from2.74%to11.0%. The detectionlimits for SPI, TIL, TYL, KIT and JOS were10,10,20,50,20μg/kg, respectively. This methodis accurate and reliable with good application.2) An HPLC-MS/MS method has been developed for simultaneous determination of theseseven macrolide antibiotics in aquatic products. Chromatographic conditions and massspectrometric parameters such as sheath gas pressure, electrospray ionization voltage wereoptimized, respectively. Samples were extracted with acetonitrile, cleaned up by neutral alumina and n-hexane, quantification analysis was performed with matrix matched standard calibrationcurves. It showed good linear between the peak areas and concentrations of seven macrolides in1~100ng/mL, and the correlation coefficients were not less than0.995. The mean recoveries ofthe drugs fortified in different negative samples at three spiked levels of4,20and40μg/kg werevaried from75.4%to108%, the relative standard deviations were varied from0.665%to12.9%.The detection limits of the drugs in aquatic products were1μg/kg and the quantification limitswere4μg/kg. This method is simple, sensitive and reproducible, therefore, it can providetechnical support for the determination of seven macrolide antibiotic residues in aquaticproducts.3) Uncertainty was anlyzed for the HPLC and HPLC-MS/MS method which established fordetermination of macrolide residues in aquatic products, we clearly know main source ofuncertainty which influence the experimental results uncertainty through analytical results ofuncertainty in measurement, including the preparation of standard solution, recovery and theregression equation. Therefore, we can take corresponding measures to reduce the effect in orderto improve the accuracy of experimental results in the actual detection work.4) The HPLC-MS/MS method for simultaneous determination of these seven macrolideantibiotics in aquatic products has been constituted as industry standard. The method will beverified by other aquatic product quality supervision and inspection and testing Centers ofMinistry of agriculture.This paper have established the HPLC and HPLC-MS/MS method for simultaneousdetermination of macrolides in aquatic products. Sample pretreatment is simple, the methods canbe applied for determination of the drugs residues in aquatic products. Based on the verificationexperiments, these two kinds of methods can provide practical reference and application valuesfor daily drug residual analyses.