豆制品及蛋制品中农兽药残留检测方案

SIGMA-ALDRICH°sigma-aldrich.comTel：(800) 247-6628 (814) 359-3441Fax： (800) 447-3044 (814) 359-3044Technical Report 2 595 North Harrison Road， Bellefonte， PA 16823-0048 USA Extraction of Nitroimidazoles from Milk and Egg Samples usingSupelMIP SPE Nitroimidazoles and LC-MS-MS Olga Shimelis1， Anna-Karin Wihlborg?， Marcus Rudolfsson， Brian Boyd?， and An Trinh1， 1Supelco， Bellefonte， PA， USA，2MIP Technologies AB， Lund，Sweden In this report， a new SPE phase was evaluated for extraction of thenitroimidazoles dimetridazole (DMZ)， ipronidazole (IPZ)， metronidazole(MNZ)， and ronidazole (RNZ) and their hydroxylated metabolites(DMZOH and MNZOH). Using this molecularly imprinted polymer(SupelMIP) and associated method， nitroimidazoles were extractedfrom milk and fresh egg samples with high and reproducible recoverieswith low RSD. Background Nitroimidazoles are anti-bacteriallandt anticoccidialdrugs used for treatmentof cattle， poultry and pigs.These compounds and theirmetabolites are suspected tobe carcinogens and mutagens.Consequently， their useinveterinary practice isthusstrictly regulated within thecountries inthe EuropeanUnion (Council Regulation2377/90， Annex IV BannedCompounds). Dimetridazole (DMZ)， metro-nidazole (MNZ) and ronidazole(RNZ)areprohibited and belongto the list of pharmacologicallyactive substances for which no maximum residue limit (MRL) can be fixed， meaning that theiruse is forbidden in food-producing animals and that any residueof these compounds found in food producing animals or productsintended for human consumption has to be considered as a violationof the regulations. MNZ and ipronidazole (IPZ) are not licensed asveterinary drugs and are considered forbidden compounds. Thesurveillance of nitroimidazole drug residues requires the detection ofthese compounds as well as their main metabolites at trace levels inanimal products Molecularly Imprinted PolymersMolecularly imprinted polymers(MIPs) are a class of highly crosslinked polymer-based moailnecularrecognition elements engineeredtobind.onespecific targetcompounddor a class ofstructurally related compoundswith high selectivity. The MIPmaterial is designed with cavitiesthat are sterically and chemicallycomplementaryto the targetanalyte(s) As a result， multipleinteractions (e.g.， hydrogenbonding， ionic， Van der Waals，hydrophobic)can take placebetween the MIP cavity and theanalyte. Method Sample Pre-treatment One egg was homogenized. 10 g of the mix was spiked at 1，2 and5 ug/kg with nitroimidazoles， dimetridazole (DMZ)， ipronidazole (IPZ)，metronidazole (MNZ)， and ronidazole (RNZ) and their hydroxylatedmetabolites (DMZOH and MNZOH)， and 2 pg/kg with deuteratedstandards for each compound， respectively. For MNZ and MNZOH，DMZOH-ds was used as internal standard. The spiked sample was extracted by combining the sample with 10mL acetonitrile followed by centrifugation for 5 minutes. 2 g sodiumchloride was mixed with the supernatant， centrifuged， and the extractwas evaporated to dryness in a silanized glass tube. The residue wasreconstituted in 2 mL water and sonicated for 3 min. prior to SPEprocessing. Similar pre-treatment protocol was followed for milk samples exceptliquid-liquid extraction using acetonitrile and sodium chloride wasdone as one step. SupelMIP SPE Nitroimidazole Procedure The SPE material used was SupelMIP Nitroimidazoles SPE tubes 50mg/3 mL (Cat. No. 52734-U). The column was conditioned with1 mL toluene followed by 1ml acetonitrile and 1 mL buffer， pH 6.5.2 mL sample was loaded using gravity and the columns were thenwashed to remove interfering contaminants. Initially， 1 mL water wasapplied followed by the application of strong vacuum for 5 minutesto dry the column. 2 aliquots of 1 mL hexane were added followedby 1 mL heptane：toluene (3：1 V/V). Between each wash step， a briefvacuum was applied and after the last heptane：toluene wash step， 3minutes of strong vacuum was applied to dry the column. To elute thenitroimidazoles， 2 aliquots of 1 mL 60：40 acetonitrile：water containing0.5% acetic acid was applied. The flow during the elution was controlledto 0.2 mL/minutes. The eluted fractions were evaporated to 50 pL insilanized glassware and reconstituted to 500 uL with 0.1% formic acidin water. See also Figure 1. Performance Evaluation Relative recovery was determined against internal standards for eachof the spike levels tested (n=5). The data was also used to determine reproducibility and to estimateLLOD. The influence of matrix (ionization effects) was tested by spikingnitroimidazoles into blank post-SPE extracts prior to LC-MS analysis.The results were quantified against an external calibration curve inbuffer. LC-MS conditions are described in Table 4. Figure 1. SupelMIP SPE Nitroimidazole Procedure Condition column with： 1 mL toluene Eluted fraction evaporated to 50 uL at 45℃ underN， and reconstitute in 500 pL LC-MS mobile phase，filter prior to analysis if necessary. Results High recoveries， reproducibility and clean extracts allowlow detection levels The reported recoveries are high and reproducible， allowing highquality analysis. Relative recoveries are presented in Table 1. Therecovery and relative standard deviation were 95% resp. 7% inaverage. Representative ion-chromatograms of egg samples (1 ng/gspike) are presented in Figure 2. These chromatograms illustrate lowbackground. Samples spiked into post-SPE extracts revealed good sample cleanupand low ion-suppression. The matrix ionization effects were minimalas evident from 90-120% recovery values in the post-spiked samples(Table 2). The estimated detection levels for nitromidazoles ranged from 0.010-0.16 pg/kg in milk and 0.016-0.12 ug/kg in egg. The LODs for thedifferent nitroimidazoles for each sample matrix are shown in Table 3. TheLODs were estimated based on the analyte and background responselevels of the lowest spike concentration. Using this information theLOD concentration was estimated based on signal-to-noise calculationof 3：1. Table 1. Recoveries of Nitroimidazoles from Spiked Eggand Milk Samples (n=5) Sample DMZ1 DMZOH1 IPZ1 MNZ2 MNZOH2 RNZ1 Egg 1 pg/kg 111(7) 106(5) 105(6) 109(3) 83(6) 105(7) Egg 2 pig/kg 84(5) 90(11) 94(5) 91(9) 58(8) 113(20) Egg 5 pg/kg 88(5) 92(8) 90(1) 92(8) 57(13) 134(3) Milk 1 pg/kg 100(8) 1076) 108(6) 105(2) 76(4) 104(3) Milk 2 pg/kg 88(7) 94(3) 97(10) 92(14) 59(7) 111(10) Milk 5 pg/kg 96(10) 101(10) 95(5) 107(9) 62(8) 123(4) 1Deuterated standard is available， 2Used DMZOH-d； as an internal standard Sample DMZ DMZOH IPZ MNZ MNZOH RNZ Egg 91 92 103 96 99 89 Milk 118 110 123 116 98 109 Figure 2. MS-MS Chromatograms of the NitroimidazoleExtractfromEggat1ng/g，Dimetridazole(DMZ)，Ipronidazole(IPZ)， Metronidazole (MNZ)， and Ronidazole (RNZ) and theirHydroxylated Metabolites (DMZOH and MNZOH) Recommended Analytical Technique： LC-MS-MS or LC-MS Ordering Information Description Oty. Cat. No. SupelMIP SPE-Nitroimidazoles， 50 mg/3 mL 50 52734-U Ascentis C18 Column， 15 cmx 2.1 mm l.D.， 3 pm particle size 1 581302-U World Headquarters3050 Spruce St.， St. Louis， MO 63103(314)771-5765sigma-aldrich.com ( Order/Customer Service ( 800) 325-3010· Fax(800) 325-5052Technical Service ( 800) 325-5832·sig m a-aldrich.com/techservice Development/Custom Ma n ufacturing Inquiries SAFC (800) 2 4 4-1173 ) ( ◎ 2009 Si g ma-Aldri c h Co . Al l ri g hts reserved. S IGMA， S ， S A FC， SAFC'， SI G M A-AL D RICH， A LDRIC H ， A， FLUKA， A ， and S U PELCO， 4 a re trad e ma r ks belong i n g t o S i gma- A l d ri c h Co . a nd i t s aff il iat e Sig m a-Aldrich Biotechnology， L . P . Sigma brand products are s ol d t h r ou g h Sigma- A ldrich ， I n c. S igma-Aldrich， I n c. w arrants tha t i t s p r oducts c onform t o t h e i n f o rmat i or c ont ai ned i n t his an d o ther S i g m a - Aldric h pu b li c a t ions. 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