A-sensitive-and-validated-method-for-determination-of-melamine-residue-in-liquid-milk-by-reversed-ph.pdf

A sensitive and validated method for determination of melamine residuein liquid milk by reversed phase high-performance liquid chromatographywith solid-phase extractionHanwen Suna,*,Lixin Wanga,Lianfeng Aib,Shuxuan Lianga,Hong WuaaCollege of Chemistry and Environmental Science,Hebei University,Key Laboratory of Analytical Science and Technology of Hebei Province,Baoding 071002,ChinabHebei Entry-Exit Inspection and Quarantine Bureau,Shijiazhuang 050051,Chinaa r t i c l ei n f oArticle history:Received 5 May 2009Received in revised form 27 September2009Accepted 2 October 2009Available online xxxxKeywords:MelamineLiquid milkSolid-phase extractionLiquid chromatographya b s t r a c tA sensitive and validated method for the determination of melamine residue in liquid milk is developedusing reversed phase high-performance liquid chromatography-diode array detection(RP-HPLC-DAD)with solid-phase extraction(SPE).The conditions of the extraction,SPE and HPLC were investigatedand optimized.The linearity is satisfactory in the range of 0.150lg/mL with a correlation coefficientof 0.9998.Under the optimal conditions,the method limit of detection(LOD)and method limit of quan-tification(LOQ)were 18lg/kg and 60lg/kg,respectively.The recovery of melamine for milk samplesspiked with 0.103 mg/kg was in the range of 85.599.3%with the RSDs(n=3)of 2.33.7%.The intra-day assay precision(RSD)was 5.6%for five replicates of quality control milk sample at 2 mg/kg level.Con-firmation of the identities of melamine was achieved by monitoring the two transitions in multiple-reac-tion monitoring(MRM)mode,and has been applied successfully for the determination of melamineresidue in liquid milk samples.The confirmatory method can permit the detection of melamine residuesat levels as low as 60lg/kg in different liquid milks.?2009 Elsevier Ltd.All rights reserved.1.IntroductionMelamine has been found in animal food because these animalswere fed melamine-contaminated feeds.In 2008,melamine hascaused the death of certain infants in China,who had drunk milkcontained this compound.Certain liquid milk and milk powderwere adulterated with melamine to increase their total nitrogenconcentration.Melamine as the environmental contaminants hascaused the attention in the world.It is important to monitor mel-amine in raw milk and milk products.A series of analytical techniques are available to determine mel-amineina varietyof petfoodsandanimalfeeds.Recentlymelaminein pet food was determined by Kim et al.(2008)using enzymeimmunoassay,high-performance liquid chromatography(HPLC)andultra-performanceliquidchromatographytandemmassspectrometry(UPLCMS/MS).Analysis of melamine and relatedcompounds in human foods is an important work for health safety.HPLC is more attractive than GC because no preliminary derivatiza-tionproceduresarerequired.Muiz-Valenciaetal.(2008)developed a simple HPLC method for the determination ofmelamine and its degradation products in rice protein concentratewith the decision limit of 65lg/g and detection capability of75lg/g for melamine.A method for the determination of melamineresidueinplantoriginproteinpowderswasdevelopedbyDingetal.(2008)using HPLC-diode array detection(HPLC-DAD)for prelimin-ary screening of the samples for melamine with an LOQ of 10 mg/kgand HPLCMS/MS was used in the confirmatory of melamine withLOQ of 0.5 mg/kg.An isotope dilution LCMS method was reportedfor determine melamine and cyanuric acid in catfish,pork,chicken,and pet food with LOD of 10lg/kg(Varelis&Jeskelis,2008).HPLCMS/MS method has been used for the quantification and con-firmatory of melamine in catfish,trout,tilapia,salmon and shrimpwith LOD of 3.2lg/kg(Andersen et al.,2008),porcine muscle tissuewith LOD of 1.7lg/kg(Filigenzi,Tor,Poppenga,Aston,&Puschner,2007),chard with LOD of 10lg/kg(Sancho,Ibaez,Grimalt,Pozo,&Hernandez,2005),and kidney tissue(Filigenzi,Puschner,Aston,&Poppenga,2008).Recently,Feng et al.(2008)established anLCMS/MS method for determination of melamine and cyromazineresidues in milk and dairy products with the LOD of 10lg/kg.Solid-phase extraction(SPE)with OASIS MCX column was used forclean-up of pet food prior to HPLC(Wang et al.,2008),and forclean-up of the foods prior to HPLCMS/MS detection(Liu et al.,2008).Recently,CleanertPCXSPEcolumn is popular,andfrequentlyused for separation andpre-concentrationpurposestodetect0956-7135/$-see front matter?2009 Elsevier Ltd.All rights reserved.doi:10.1016/j.foodcont.2009.10.008*Corresponding author.Tel.:[phone];fax:[phone].E-mail address:(H.Sun).Food Control xxx(2009)xxxxxxContents lists available at ScienceDirectFood Controljournal homepage: IN PRESSPlease cite this article in press as:Sun,H.,et al.A sensitive and validated method for determination of melamine residue in liquid milk by reversed phasehigh-performance liquid chromatography with solid-phase extraction.Food Control(2009),doi:10.1016/j.foodcont.2009.10.008melamineinfeetandmilkpowderbyliquidchromatographyquad-rupole mass spectrometry(Ming&Wang,2008),and in raw milkanddairyproducts byHPLC(China NationalStandardizingCommit-tee,2008a,2008b).Agela Technologies has developed an effective,improved and reliable method for determining melamine in rawmilk,milk powder and dairy products by using the solution kit con-taining Cleanert PCX SPE column and Venusil ASB C8 chromato-graphic column(Beijing Agela Sci-Tech.Company Limited,2008).An interim method for the determination of melamine residue infoods using LCMS/MS was proposed by the Food and Drug Admin-istration(FDA)(Smoker&Krynitsky,2008),and the method limit ofquantification(LOQ)for melamine was 25lg/kg for tissue and li-quid formula and 200lg/kg for dry infant formula products.Thismethod is the principal analytical method for detection and quanti-fication of melamine in foods.To our knowledge,there are few re-ports for the determination of melamine in liquid milk and milkproducts.Recently,a national standard method(GB/T 224002008)for rapid determination of melamine in raw milk using LChas been issued in China with the limit of detection of 50lg/kg(China National Standardizing Committee,2008a,2008b).A HPLCmethod presented by Yan et al.(2008)and a hydrophilic interactionchromatographyelectrospray ionizationMS/MS method reportedby He,Liu,Huang,Yang,and Liao(2008)were applied for the deter-mination of melamine in milk powder and dairy milk with LOQ of1 mg/kgand50lg/kg,respectively.It isofcriticalimportancetode-velop simple and sensitive methods for melamine detection in foodsystems information on toxicity of melamine and on the levels ofmelamine compounds in edible tissues would be useful for futureassessments.To meet detection need for melamine contaminated,a reliableand simple SPEHPLC method was developed in our work for thedetermination of melamine in liquid milk with method LOD of18lg/kg and LOQ of 60lg/kg.The proposed method was validatedby LCMS/MS,and has been applied successfully for routine deter-mination of melamine of different milk samples.2.Materials and methods2.1.Chemicals and reagentsCleanert PCX-SPE cartridges(3 mL/60 mg)were obtained fromBeijing Agela Technologies Company(Beijing,China).Sodiumn-heptanesulfonate(chromatographic grade)was used as an ionpair reagent.Melamine was purchased from Kermel ChemicalReagents Development Center(Tianjin,China).An individual stockstandard solution,1000lg/mL,was prepared by dissolving themelamine in a mixture of methanol and water(1:4,v/v),and wasstablefor al least 1 monthifstored at 4?C.A freshworkingstandardsolution was prepared daily by diluting the stock solution with amixture of methanol and water(1:4,v/v)for different studies.The solution was filtered through 0.45lm microporous membraneof mixed cellulose ester.The acetonitrile was filtered through0.22lm microporous membrane of polyvinylidene fluoride beforeused.A 10 mM sodium n-heptanesulfate(pH adjusted with citricacid)acetonitrile(83:17,v/v)were used as mobile phase for HPLC.All the reagents were of analytical grade except for additional illus-tration.Doubly distilled water obtained from quartz distillationapparatus.2.2.InstrumentThe HPLC equipment was a Shimadzu HPLC system(Shimadzu,Kyoto,Japan)with a binary pump,a gradient controller(SCL-10Avp),an on-line-degasser(DGU-12A),a column thermostat(CTO-10Avp),and a diode array detector(SPD-M10Avp).CLASS-VPworkstation was used as the data acquisition system.The analyti-cal column was a ZY1104 C18 column(250?4.6 mm I.D.5lm).An ultrasonic cleaner(Ultrasonic Instrument Co.,Ltd.,Kunshan,China)and PHS-3C pH meter(Shanghai precision&scientificinstrument Co.,Ltd.,Shanghai,China)were used in sample treat-ment.An TGL-16 M centrifuge(Xiangyi Centrifuge Co.,Ltd.,Hunan,China)was used in sample treatment.Confirmation of melamine in milk sample was performed byLCMS/MS(Thermo Electron Corp.,San Jose,CA,USA)consistingof a Surveyor MS pump with an on-line-degasser,a Surveyor auto-sampler,and a TSQ Quantum triple stage quadrupole mass spec-trometer equipped with an electrospray ionization(ESI)sourceoperated in positive ion mode.LC separations were performed onan SeQuant ZIC HILIC column,2.1?150 mm PEEK,5lm(The NestGroup)a guard column(2.1 mm?12.5 mm)that was at 30?C.LCmobile phase was composed of 10 mM ammonium acetate andacetonitrile(90:10,v/v)at a flow rate of 0.2 mL/min.The injectionvolume was 10lL and between injections,the needle was rinsedwith methanol.TSQ Quantum mass spectrometer was calibratedwith a solution of polytyrosine-1,3,6 according to the manufac-turer.For method development,a standard solution containing1lg/mL of melamine was infused at 10lL/min with 200lL/minmobile phase into the ESI source.The optimized source parameterswere as follows:sheath gas pressure,20(arbitrary units);auxiliarygas flow,30(arbitrary units);spray voltage,4000 V;capillary tem-perature,350?C;tube lens offset,78 V;and source collision-in-duced decomposition(CID),10 V.For quantification,the massspectrometer was set to the data acquisition mode of multiple-reaction monitoring(MRM).The acquisition parameters were:scanwidth(m/z)0.01,scan time 0.1 s,peak width(FWHM)0.7 for bothQ1 and Q3,and collision gas pressure 1.5 mTorr,the primary tran-sition:m/z 127/85,Collision Energy 25 eV,Secondary transition:m/z 127/68;Collision Energy 25 eV.Data acquisition and analysiswere accomplished with LC quan software v.2.5(Thermo ElectronCorp.,San Jose,CA,USA).2.3.Sample extraction and clean-upA 50 mL of 1%trichloroacetic acid solution and 2 mL 2.2%leadacetate solution were added to 5 g of liquid milk sample in orderto eliminating protein and extracting analyte.A 30 mL of the mix-ture was placed in ultrasonic cleaner for 20 min to mix well,stand-ing for 2 min.Then mixed solution was centrifuged for 10 min at10,000 rpm.A 10 mL volume of the supernatant was applied to aPCX-SPE cartridge which had been previously conditioned with3 mL of methanol and 3 mL of water.SPE cartridge was washedin turn with 5 mL of water and 5 mL of methanol and the eluatewas discarded.Melamine was eluted with 6 mL of 25%ammoniasolutionmethanol(1:20,v/v).The eluate was evaporated to dry-ness at 50?C under a stream of nitrogen and residue was re-dis-solved in 0.5 mL of methanolwater(1:4,v/v).Then the solutionwas filtered through a 0.45lm microporous membrane of mixedcellulose ester for HPLC analysis.2.4.HPLC analysisAfter the C18 column was conditioned with a mobile phase of10 mM sodium n-heptanesulfonateacetonitrile(83:17,v/v,pH2.7)at 1 mL/min at 30?C,a 20lL volume of sample solutionwas injected in the column,and then eluted with the mobilephase.The linear equation describing the relationship betweenmelamine concentration and its peak area was determined byleast-squares weighted for UV detection at 235 nm.Quantifica-tionwascarriedoutbyusingmatrix-matchedstandardscalibration.2H.Sun et al./Food Control xxx(2009)xxxxxxARTICLE IN PRESSPlease cite this article in press as:Sun,H.,et al.A sensitive and validated method for determination of melamine residue in liquid milk by reversed phasehigh-performance liquid chromatography with solid-phase extraction.Food Control(2009),doi:10.1016/j.foodcont.2009.10.0083.Results and discussion3.1.Extraction and clean-upFor the extraction of melamine from matrix,several reagentswere used,such as water for vegetable materials with the recoveryof 70.280.2%(Ni,2008),Andersen,Turnipseed,Karbiwnyk,andMadson(2007),Andersen et al.(2008)used a 50:50 acetoni-trile:water and hydrochloric acid for catfish tissue with the recov-ery of 76.3%,and acidic acetonitrile for catfish,trout,tilapia,salmon and shrimp with the recovery of 54.974.8%.Filigenziet al.(2007)utilized 50%acetonitrile for porcine muscle tissue withthe recovery of 83%.Melamine could be associated via some notspecified interactions with proteins.In this work,trichloroaceticacid solution was used to precipitate proteins and to dissociatethe target analyte from the sample matrix.When using 50 mL of1%trichloroacetic acid plus 2 mL 2.2%lead acetate for the extrac-tion of the analyte and the removal of protein,higher extractionefficiencies(85.595.5%)of melamine were achieved.The mixedsolution was centrifuged and the supernatant was applied forclean-up.An Oasis MCX SPE column was used to clean-up pet food matrixafter extraction with 1%ice-acidic acid solution(Wang et al.,2008),and to clean-up food matrix after extraction with 1%trichloroace-tic acid solution(Liu et al.,2008).Cleanert PCX SPE column is pop-ular,and frequently used for separation and pre-concentrationpurposes to detect melamine in foods prior to liquid chromato-graphic separation(China National Standardizing Committee,2008a,2008b;Ming&Wang,2008).The surface of the PCX-SPEcartridges was modified with sulfonic group on the sorbent of highpolar organic copolymer which was composed of polystyrene andpolydivinylbenzene.The retention mechanism of the PCX-SPE car-tridges is based on cation exchange adsorption and reversed phaseretention,which could keep stabilization between pH 014.Thecartridges could extract polar and non-polar compounds.The neu-tral and acidic interferences from the serum of milk are removed,while the weakbasicmelaminepasses awayto theeffluent.This may result in improved analytical performance in the quanti-fication of melamine.In this work,the supernatant obtained afterextraction with trichloroacetic acid plus lead acetate solutionwas applied to a PCX-SPE cartridges column previously condi-tioned with 3 mL of methanol and 3 mL of water.SPE cartridgewas washed in turn with 5 mL of water and 5 mL of methanoland the eluate was discarded,then,it was eluted with 6 mL of25%ammonia solutionmethanol(1:20,v/v).The eluate wasevaporated to dryness at 50?C under a stream of nitrogen.The obtained residue was re-dissolved with a methanolwatersolution(1:4,v/v),the lower dosage of which would increase theconcentration factor of melamine.The dosage of 0.5 mL of metha-nolwater(1:4,v/v)for dissolving the residue could satisfy therequest for HPLC sample analysis with higher con