A Novel And Efficient Method For Monitoring Multiple Drug Residues In Animal-Derived Foods

With the continuous improvement of people’s living standards and optimization of dietary structure,the consumption of animal-derived foods,including meat,eggs and milk,has increased,and the problem of drug residues in animal-derived foods has become increasingly prominent.In the breeding process of animals,in order to maximum the survival rate and production,veterinary drugs(VDs)are used frequently and widely,and even multi-class VDs are applied in combination for more effective broad-spectrum anti-bacterial and anti-virus capabilities,resulting in the widespread presence of multiple drug residues in animal-derived food.The complex matrixes of animal-derived food,the large number of samples,the various types of targets are the emphases and difficulties of food safety monitoring.Therefore,the establishment of rapid and efficient monitoring methods is the inevitable trend of detection technology,and is also the key to meet the guarantee food safety requirements.The most difficult and critical part is the sample preparation,and solid phase extraction(SPE)is the most common sample preparation method for VDs residues detection.In addition to commercial SPE cartridge,some SPE methods based on new advanced adsorbents are more and more popular because of their high-efficiency and low-costing.Due to its large specific surface area and high porosity,NFsM as a superior adsorbent for SPE has great potential for sample preparation.In addition,the functionalized NFsM modified with specific functional groups can significantly improve the extraction efficiency of the target compounds and the removal effect for sample matrix.In this paper,sample pretreatment methods based on functionalized NFsMs as novel SPE adsorbents coupled with ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)for the determination of veterinary drug residues in various animal-derived foods were established.The research content is divided into the following two parts:(1)A SPE method based on Polyaniline nanofiber mat(PANI NFsM)as adsorbent was established for sample pretreatment,combining with UPLC-MS/MS,for the rapid detection of the seven NSAIDs residues in meat and eggs.The factors affecting the extraction efficiency of SPE were optimized.After the sample being extracted by acetonitrile,the extract was diluted with ultrapure water,which was no need to adjust the pH value and ionic strength,and then the sample solution was purified by only 5mg PANI NFsM and concentrated by 500μL 1%acetic acid/methanol as eluent,which was collected and directly analyzed by UPLC-MS/MS.Compared with China’s national standard method and other literature methods,the whole sample pretreatment process only needs 4 steps,including extraction,dilution,purification and concentration,which is far less than other methods(8～12 steps).Moreover,this method is more efficiency,24 samples can be pre-treatment at the same time and the average time of each sample is less than 1 minute,and the time consumption is reduced by 5-10 times.This method is also economical and environment friendly,the adsorbents usage is 1/20-1/100 of other methods,and the consumption of organic solvent is only 1/4-1/55 of existing methods.The proposed method was evaluated by real meat and egg samples.The limits of detection(LODs)and limits of quantification(LOQs)of 7 targets were 0.6-12.2μg/kg and 2.0-37.1μg/kg,respectively.In the linear ranges of each targets’LOQ to 1500μg/kg,good correlation coefficients were obtained(R~2>0.99).At the low,middle and high spiked levels,the recoveries were 85.18%-107.31%and the relative standard deviations(RSDs)were 2.74%-16.01%,which proved good accuracy and precision.In addition,the matrix purification ability of PANI NFsM for meat and eggs was investigated.The matrix effects of the 7 targets were-17.44%～-55.91%without SPE,showing strong matrix inhibition.However,after SPE purification,the matrix effect was significantly reduced,ranging from-4.37%to-13.88%,indicating that PANI NFsM had ideal matrix purification ability.The method was applied to the determination of 7 NSAIDs residues in 40 meat samples and 40 egg samples to evaluate its practical application feasibility.The results showed that 4 meat samples and 3 egg samples were detected with target residues.(2)Polydopamine polystyrene nanofibers mat(PDA-PS NFsM)was prepared,and based on which a novel and efficient sample pretreatment method was established,combined with UPLC/MS/MS,for the simultaneous determination of 18 VDs belonging to 8 classes(β-lactams,macrolides,lincoamides,chloramphenicol,tetracycline,nitroimidazoles,fluoroquinolones and sulfonamides)in various animal-derived foods(meat,fish,egg and honey).Polystyrene(PS)nanofiber mats were prepared by electrospinning as templates.PDA-PS NFsM was obtained by polydopamine(PDA)being spontaneously synthesized by oxidative self-polymerization of dopamine-hydrochloride in alkaline solutions(pH>7.5).Fourier Transform infrared Spectroscopy(FT-IR)and field emission scanning electron microscopy(FE-SEM)were applied to characterize the synthesized PS NFsM and PDA-PS NFsM.It showed that there are infrared absorption peaks of characteristic functional groups appears,such as N-H amino groups,aromatic ring and-OH on catechol,proving PDA was successfully modified on the PS NFsM.After modification,the structure of PDA-PS NFsM showed a rough outer core shell structure and a honeycomb structure inside in SEM figures.The adsorption and desorption properties of PDA-PS NFsM were investigated by adsorption/desorption kinetics experiment,adsorption thermodynamics experiment and adsorption isotherm experiment.The results show that PDA-PS NFsM is a kind of outstanding adsorbent with fast mass transfer rate,high specific surface area and strong specific selectivity.The adsorption process of PDA-PS NFsM was consistent with Langmuir adsorption model.At298K,318K and 338K,PDA-PS NFsM can simultaneously adsorb 18 targets,and the maximum static adsorption capacity was 97.28 mg/g 101.01 mg/g 123.76 mg/g,respectively.The adsorption and desorption of PDA-PS NFsM for 18 targets reached equilibrium within 5-10minutes,and the desorption rate was 70%-90%.In addition,the matrix effects before and after SPE purification were compared to prove the matrix purification ability of PDA-PS NFsM.After PDA-PS NFsM based SPE,the matrix inhibition effect decreased from-12.49%～-60.96%to-2.07%～-13.41%,indicating that PDA-PS NFsM can effectively eliminate the matrix interference in meat,egg,fish and honey samples.Then,the factors affecting the efficiency of SPE were optimized,including adsorbent usage,the flow rate,pH and ionic strength of the sample solution,the composition of eluent.The sample was extracted by the extraction solution,and then being diluted with ultra-pure water.The sample solution was purified by 20 mg PDA-PS NFsM without adjusting its pH value and ionic strength.The targets were eluted with 1 mL 1%formic acid and ethyl acetate(containing 20%methanol),and then was redissolved with 0.1 mL 10%methanol aqueous solution(containing 0.2%formic acid)after nitrogen drying.Based on the excellent adsorption/desorption performance of PDA-PS NFsM,this method can extract 18 drug residues from meat,fish,eggs and honey simultaneously,and PDA-PS NFsM can be reused for 4 times after regeneration.Compared with China’s national standard method and the existing methods,this method can detect more targets and applied in more matrix types.Furthermore,this method needs less steps(5 steps),less time consumption(SPE process only 5 minutes),less adsorbent usage(1/10-1/30 of other methods)and less organic solvent(1/10-1/50 of other methods).The quality control samples of meat,fish,eggs and honey were prepared to evaluate the proposed method.The results showed that the LODs and LOQs were 0.1-3.2μg/kg and 1.0-10.0μg/kg,respectively.In the linear range of each targets’LOQ to 1000.0μg/kg,good correlation coefficients were obtained(R~2>0.996).At low,medium and high levels,the recoveries were 92.59%-103.30%,and the RSDs of intra-and inter-day were 2.11%-10.66%in meat,fish,eggs and honey samples.The application of the method was evaluated by analyzing 10 meat samples,10 fish samples,10 egg samples and 10 honey samples.There are1 fish sample and 2 honey samples being detected with targets,while no target was detected in other samples(lower than LODs).The results are consistent with those of national standard and industry standard,which verifies the reliability of this method.In this research,based on the outstanding adsorption/desorption performance of functional nanofibers mats,novel sample pretreatment methods were established,achieving the detection of multiple representative drug residues in animal-derived foods.The developed functional nanofibers mat can efficiently extract a variety of targets with different properties,and has good purification ability to complex sample matrix.After rapid extraction and simple dilution,the sample can be directly processed for SPE without any treatment.The pretreatment time of a single sample is less than 2 minutes,requiring only a few milligrams of adsorbent and a few milliliters of organic solvent,and the adsorbent can be reused multiple times through regeneration,which indicating great potential in the routine monitoring of multiple drug residues in animal-derived foods.