Study on the LC-MS/MS Determination Method for Residues of Nine β-Agonists in Animal-Derived Foods
Introduction and Background Overview
β-Agonists are a class of compounds with a phenethylamine structure, primarily used clinically to treat respiratory diseases such as bronchial asthma. However, when these substances are illegally added to animal feed, they can promote animal growth and increase lean meat yield. Long-term consumption of animal-derived foods containing residues of β-agonists can pose serious health risks to humans, including palpitations, dizziness, muscle tremors, and in severe cases may even lead to death.
The Ministry of Agriculture's Announcement No. 1025-18-2008 clearly stipulates the maximum residue limits (MRLs) for nine types of β-agonists in animal-derived foods. This study is based on that standard method and employs solid-phase extraction combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology to establish detection methods for residues of nine β-agonists in common animal-derived foods such as pork, pig liver, beef, and lamb. The method features high sensitivity, good selectivity, and high accuracy to meet food safety regulatory requirements.
Experimental Materials and Methods
Experimental Reagents and Instruments
This experiment utilized cleanert® pcx solid-phase extraction columns (60mg/3ml) produced by Tianjin Bona Aijier Technology Co., Ltd., for sample pretreatment. These solid-phase extraction columns exhibit excellent adsorption performance and stable reproducibility; they are particularly suitable for extracting polar compounds like β-agonists. The β-glucuronidase/arylsulfatase enzymes used were purchased from Sigma; other reagents were chromatographic or analytical grade.
Main instruments include: ultra-high-performance liquid chromatography-tandem mass spectrometer (equipped with an electrospray ion source), high-speed centrifuge (maximum speed 15000rpm), nitrogen blow-down apparatus, pH meter, vortex mixer, constant temperature water bath oscillator etc. All glassware was thoroughly cleaned to avoid cross-contamination.
Target Compound Information
The nine types of β-agents detected in this study include: terbutaline, cimaterol , albuterol , fenoterol , chlorpromazine , ractopamine , clenbuterol , tulobuterol , and mabuterol . These compounds share a similar phenethylamine backbone but differ structurally due to their substituents which affects their polarity and mass spectral behavior differently. By optimizing chromatographic conditions and mass spectrometric parameters effective separation can be achieved along with accurate quantification.
Sample Pretreatment Process
Sample Enzymatic Hydrolysis Process Accurately weigh 2.00g homogenized animal tissue samples (pork,pig liver,cattle or sheep meat) into a 50ml brown centrifuge tube.Add 8 ml 0 .2 mol/L ammonium acetate buffer solution(pH=5 .2).This buffer system maintains optimal pH environment during enzymatic hydrolysis.Subsequently add40μLβ-glucuronidase/arylsulfatase mixed enzyme solution,mix well using vortexing,and incubate at37℃in dark under constant temperature water bath shakerfor16 hours.This step effectively releases metabolites boundto glucuronic acid or sulfate increasing recovery rateof target analytes. n n Extraction And Purification Steps n After enzymatic hydrolysis coolthe sampleto roomtemperaturecentrifuge at10000 rpmfor10 minutes separatingsolid-liquid phases.Takethe supernatant adding5 ml0 .1 mol/L perchloric acid adjustingpHto1±0 .2.The acidic condition favors protein precipitation andreleaseoftargetanalytes.Centrifugationagain adjustsupernatant pHt9 .5±0 .2using10mol/L NaOHsolution.Atthispointβ-receptor agonistsexistmolecularly facilitatingorganic solventextraction.Use two-stepliquid-liquid extractionmethod:firstlyadd15 mL ethyl acetate,vortex mix then shakefor10minutes.Centrifugeseparationafterwardsadding10mL tert-butyl methyl ether forthe secondextraction.Combine organic phase after evaporatingnitrogen at50℃until dry.Reconstitutewith5 mL 2% formic acid aqueous solutionpreparingfor subsequentsolid-phase extractionprocess.Solid-phasextraciton processusescleanert®pcx column sequentiallyactivatingcolumnwithmethanolwaterand2%formic acidsolution each3mL.After loading use2%formicacid aqueoussolutionand methanol each3mLas wash remove impurities finally elutingtargetcompoundswith3%ammonia-methanol solution(25ml).After evaporationunder nitrogenreconstituteusingmethanol -0 .1 % formicsolution(10 :90,v/v )thencentrifuge at15000rpmfor10 minutescollectsupernatant performLC-MS/MS analysis.Instrumental Analysis ConditionsThisexperiment strictlyfollowsagricultural ministry announcementNo1025182008specifiedmethods.AnalyticalchromatographywasperformedontheC18reversephasecolumnflow phaseconsistedof0 .1 % formicaqueoussolutionmethanol employinggradientelutionprogramachievingbaseline separationnineβ-receptor agonist.Massspectrometrydetectedbyelectrospraypositiveionmode(ESI+),multi reaction monitoring(MRM)scanning mode selectingtwo pairscharacteristicionsqualitativequantitativeanalysis.Instrumentparameterswereoptimizedsystematicallyincludingion sourcetemperature nebulizing gas flow collision energy ensuringbest signal-to-noise ratio sensitivity.Duringanalysis blanksamples spiked samplesalternated injectionmonitorinstrumentperformanceavoidingcross contamination.Data processingutilizesprofessionalmassspectrometry workstationsoftwareestablishingstandardcurve quantitative calculation.Method Validation Results DiscussionMethodological ValidationByaddingstandard solutionsnine types beta receptor agonistin different matrices(pork,liver,cattle,sheepmeat )at levelsof0 ..ng/g assessmethod recovery precision.Results showedall targetcompoundsaveraged75%-110%,relative standard deviation(RSD)<15%,fully meetingresidue analysisrequirements.Limitsof Detection(LOD)&Quantitation(LOQ)determinedbysignal-to-noiseratio approach.Under optimizedconditions LODs9typesbeta receptoragonistarebetween0..05 ng/g& LOQsrangefromO.lng/g farbelowChina’smaximummicrobiological limit standards.Matrixeffectassessmentindicatedisotopeinternalstandardscouldeffectivelycorrectmatrix suppressionor enhancementeffects fromdifferentanimal tissues.SampleAnalysisResultsUsingthismethoddetectmarketedanimalderivedfoodsresults indicatedcleanert®pcxsolidphaseextractioncolumns exhibitedexcellentpurificationefficiency effectivelyremovinginterferingfatty proteins obtainingclearchromatograms.Effectstudyvariousanimal tissues matrixondetectionresultsshowedliverorganshigh fat content requireparticularattentionduringpretreatmenthoweverthroughoptimizingextractionconditions satisfactoryrecoveriescanstillbeobtained.Longtermvalidationdemonstratestabilitygoodreproducibilityapplicablelarge-scale routine testing.Comparedtraditionalimmunoassay techniques LC-MSMSapproacheshigher specificityaccuracy effectivelyavoidingfalse positive results.Conclusion Application ProspectsThe establishedanalytical methodbasedcleanert®pcxsolidsorbent &LCMSMStechnology accuratelydeterminesresidualamountnine beta receptor agentsinanimalsourcedfoodsthis procedurehasreasonablepretreatmentsignificant purification effecthighsensitivitycompletely meetsfood safetyregulatoryrequirements.Aspeople’sdemandforsafetycontinuesincrease detectionwillmove towards higher throughputlowerlimitsfutureconsider expandingthis methodology simultaneousdetectionmoretypesbeta receptor agentsdevelopsimplifiedrapidsampletreatmenttechniques.Appropriatelyappliedbothroutine regulatorytestingprovidetechnicalsupportrelatedfood safetystandard revisions.