Simultaneous Determination Of Residues Of Six Kinds Of Zeranols In Foodstuffs Of Animal Origin

Zeranols are a nonsteroidal mycotoxin, belong to the group ofresorcyclic acid lactones, Which contains α-zeranol, β-zeranol, α-zearalenol,β-zearalenol, zearalanone and zearalenone. Because of its anabolic effect,zeranol is used in animal husbandry with effects such as growth promotionand improvements in feed conversion efficiency. However, during the pastdecade, the residue of anabolic growth promoters in foods has beenconcerned issues for their impact of endocrine disruptors on human healthand wildlife. They are recognized to cause direct contamination in animaltissues, presenting a health concern at the parts per billion levels, and thepossible toxic effects on public health, which are associated with manydiseases such as breast cancer in humans and hermaphroditism in wildlife.So their use in food-producing animals has been prohibited by theEuropean Union by Council Directive96/22/EC. Therefore, monitoring forillicit use of hormones is carried out under the terms of EU Directive96/23/EC, which is implemented through surveillance according to theNationals Plans of the individual Member States.The current standard used liquid chromatography-tandem massspectrometry to detect zeranols in foodstuffs of animal origin which cleanup by HLB column. For controls at retail level, as well as for productsimported into the EU, efficient methods applicable to foodstuffs of animal origin are necessary. To establish the presence of anabolic agents and theirmetabolites in tissue samples, analytical techniques for detecting traceamounts of these compounds are required. Because of the low levels in thetissues of animal origin and the complexity of biological matrices, theanalysis of hormones and hormone-like substances is a challenging task.This implies that an effective sample preparation process and sensitiveanalytical instruments are necessary to achieve the optimal sensitivity,selectivity and specificity. This study establishes a simple, fast, safe andaccurate determination method to simultaneously detect six kinds ofzeranols in foodstuffs of animal origin. The full text is composed of the twoparts.Part I Simultaneous determination of residues of six kinds of zeranolsin foodstuffs of animal origin by high performance liquidchromatography with immunoaffinity clean up columnObjective: To develop an immunoaffinity cleanup-high performanceliquid chromatography (IAC-HPLC) method for the simultaneousdetermination of residues of six kinds of zeranols (α-zeranol, β-zeranol, α-zearalenol, β-zearalenol, zearalanone and zearalenone) in foodstuffs ofanimal origin. Methods: Enzymolyzed samples were extracted with ethylether, and subsequently reextracted with a sodium hydroxide solution. Afteradjust pH to7, the extract was cleaned up on an immunoaffinity column.The chromatographic separation was performed on an Agilent EclipseXDB-C18column (150mm×4.6mm i.d.,3.5μm) usingmethanol-acetonitrile-water (50：15：35, v/v/v) as mobile phase at a flow rate of1.0mL/min and UV detection at270nm. Results: Six kinds ofzeranols had good linear relationships in the range of0.01～0.2μg/mL, thecorrelation coefficient was higher than0.9997; average recoveries of sixtarget compounds at different concentrations ranged were71.5%～102.3%and relative standard deviation range was less than11.0%. Conclusions:The method could be used for determination of trace residues of zeranoland its analogs in foodstuffs of animal origin, and stable, reliable andaccurate.Part II Simultaneous determination of residues of six kinds of zeranolsin foodstuffs of animal origin by HPLC-MS/MS with immunoaffinityclean up columnObjective: To develop an immunoaffinity cleanup-high performanceliquid chromatography-tandem mass spectrometry method(IAC-HPLC-MS/MS) for the simultaneous determination of residues of sixkinds of zeranols (α-zeranol, β-zeranol, α-zearalenol, β-zearalenol,zearalanone and zearalenone) in foodstuffs of animal origin. Methods:Enzymolyzed samples were extracted with ethyl ether, and subsequentlyre-extracted with a sodium hydroxide solution. After cleaned up byimmunoaffinity column, the analysis was carried on HPLC-MS/MS. Theanalytes were separated on an Shimadzu VP-ODS C18clounm（150mm2.0mm，5m）, eluted by gradient with mobile phase of acetonitrile(A)and2mmol/L ammonium acetic contained0.2%formic acid(B) as0~4.0 min,25%A;4.0~7.0min,25%~70%A;7.0~7.5min,70%~25%A;7.5~8.0min,25%A. The flow rate was0.3mL/min, the column temperature was40℃and the injection volume was10mL. Results: The LOQs of sixanalogs were between0.05~0.63μg/kg; the average recoveries werebetween71.3%～99.6%at different concentrations, and the RSDs wereless than11.1%. Conclusions: The method was high sensitivity, highaccuracy, good repeatability, and could be used for determination of traceresidues of zeranols in foodstuffs of animal origin.