Study On The Analysis Methods For Acetylgestagens Multi-residues In Animal Derived Food

Acetylgestagens are a class of synthetic steroid drugs and very important for the physiological function of human and animal by regulate the growth and differentiation of cells.They are highly useful in oestrus regulation,growth-promoting and veterinary practice.However,the residues of AGs in animal derived food will represent potential hazard to human health,such as teratogenic and carcinogenic effect,by entering human body and accumulation.Thus,the use as growth-promoter has been prohibited in livestock by many countries.In this work,anti-acetylgestagens polyclonal antibody was produced and an ELISA method was developed for screening multi-residues of acetylgestagens.A LC-MS/MS method was also established for confirming them in animal fat tissues.Meanwhile,a CEIA with laser-induced fluorescence detection method was discussed.A hapten(3-CMO-MPA) and a conjugate of the hapten and bovine serum albumin were synthesized and identification.An antibody was produced by immunizing New Zealand rabbits with the conjugate,whose affinity constant was 8.1×10⁸ L/mol.The ELISA method against MPA was developed and the IC₅₀ was 5.8μg/L.The antibody was found specific for four acetylgestagens by analyzing cross-activity.The reaction conditions were selected by measuring the effect of them on the ELISA sensitivity.The buffer was 0.01 mol/L PBS(pH 7.5) with 0.02%Tween-20, the incubation time was 60 min at 37℃,the reaction time of enzyme labels was 30 min and the color developing time was 30 min.Three heterologous coating antigens were investigated after they were prepared.The conjugate of 3-CMO-HPA and ovalbumin(3-CMO-HPA-OVA) was found better than the homologous antigen (3-CMO-MPA-OVA) as coating antigen.The new heterologous ELISA method was more sensitive and class-selective than the homologous.The IC₅₀ for MPA,CMA, MEGA and HPA were 1.8,4.5,2.9 and 2.5μg/L,respectively.The sensitivity for MPA was 0.05μg/L and for CMA,MEGA and HPA 0.1μg/L.The linear range was 0.1-15μg/L for MPA and 0.2-30μg/L for CMA,MEGA and HPA,respectively.The cross-activities for MPA,CMA,MEGA and HPA were 100%,40%,62%and 72%, respectively.Pork fat samples were extracted with solvent,de-fatted through freezing and cleaned up with SPE column.The limits of detection(LODs) of the above ELISA method were 0.6,1.2,1.2 and 1.0μg/kg for MPA,CMA,MEGA and HPA, respectively.Mean recoveries were in the range 62.2-83.3%when blank pork fat samples were fortified range from 1 to 10μg/kg.Muscle,liver and kidney samples were extracted with solvent and cleaned up with SPE column.The LODs of the ELISA method were in the range 0.3-1.0μg/kg for the AGs.Mean recoveries were in the range 62.2-91.5%when blank samples were fortified range from 1 to 10μg/kg. After extracted with solvent and de-fatted through freezing,the LODs for in feed samples were 2.0,3.0,3.0 and 2.5μg/kg MPA,CMA,MEGA and HPA,respectively. Fortification at the range 1-10μg/kg,mean recoveries were 72.1-95.4%. Rabbits were administrated with MPA at 0.8 mg/kg.bw successively for 5 days and then withdrawl for 7 days.Then they were slaughtered and the incurred samples, muscle,liver and kidney,were obtained.The mean MPA residue in muscle and liver samples were 7.2 and 5.6μg/kg,respectively.The results by ELISA method had good linear correlation with GC-MS.These showed that the method could satisfy the requirements of AGs screening.The stability of the kits was investigated,and the results showed that they still kept good performance for application after they were stored for 6 months at 4℃or one week at 37℃.After solvent extraction and SPE clean-up,fat samples were detected by liquid chromatography tandem mass spectrometry.A confirming method for five AGs was established,the LODs for five AGs were verified from 0.2 to 0.3μg/kg and the limits of quantification(LOQ) were 0.5μg/kg.When blank fat samples were fortified AGs at 0.5,1.0 and 5.0μg/kg levels,the mean recoveries were in the range 60.5-84.1%and the relative standard deviation(RSD) were in the range 8.9-16.8%.A fluorescein label of MPA was synthesized and the immune reaction activity of the label was identified by capillary electrophoresis immunoassay(CEIA) with laser-introduced fluorescence(LIF) detector.The separation buffer was 0.2 mol/L Tis/0.1 mol/L Boric acid buffer(pH 9.0) containing 20 mM sodium dodecylbenzene sulfonate(SDS).The LOD for MPA in fat was 2.5μg/kg,and mean recovery was 88%with RSD 5.8%at 10μg/kg fortification level.Compared with the above ELISA method,the CEIA could be applied with less solvent consumption after easier sample pretreatment.