| Cheese is made from milk by fermentation, it is a kind of high nutritional value of food. Inrecent years,because people’s consumption level improve, the per capita consumption of cheesealso increased year by year. Such as raw material, process parameter control in the process ofproduction and mature of cheese can produce biological toxin,that can endanger human bodyhealth.Biogenic amine and aflatoxin M1is most common biological toxins in cheese.The harm forhuman body is deep. For both two toxins many researchers at home and abroad have established avariety of detection methods, but these methods have different degrees of low detection sensitivity,time consuming, high cost, testing instruments and equipment Difficult to achieve.Therefore, thisstudy from the cheese sample pretreatment, pre-column derivatization, liquid chromatographydetection conditions of several aspects, to establish detection method for the biological toxins incheese, in order to solve these problems. This study got the following results:（1）The conclusion of the research of HPLC test for Biogenic amines in cheese:1Respectively using trichloroacetic acid, perchloric acid, hydrochloric acid, anhydrousethanol, anhydrous methanol extract biogenic amines in cheese sample and calculate the6kinds ofbiogenic amines’ addition recoveries and relative standard deviation. Experimental results showthat using hydrochloric acid to extract the biogenic amines in cheese sample, the extractionefficiency for six kinds of biogenic amines is the best one. The recovery rate is highest, relativestandard deviation less than6.7%,have the best extraction efficiency.2Choose dansyl chloride （Dns-Cl） as derivative agent, through the test to determine the mostsuitable volume of the5mg/ml dansyl chloride solution is1ml.When the derivative reaction is completed, The solution will contains impurities such asinorganic salts, in order to avoid damage the chromatographic column, and safeguard biogenicamine’s detection accuracy, the enrichment and purification for biogenic amine derivatives isnecessary. By comparing the ODS C18solid phase extraction method and ethyl acetate, ethyl ether,chloroform, three kinds of liquid-liquid extraction methods,and calculate six kinds of biogenicamine’s recoveries and relative standard deviation. Determine the ODS-C18solid phase extractionmethod is most suitable, and simpler operation method than the other. Determined by test, using15%aqueous solution of acetone as wash solvent of solid phase extraction small column issuitable,the volume of elution solvent for biogenic amine derivatives is5ml of acetonitrile. Time consumption of the solid phase extraction process of biogenic amine derivatives is10min.3Established the optimum mobile phase for the determination of six kinds of biogenic aminesin cheese Take advantage of the HPLC-FD technology, Mobile phase is acetonitrile and water,Gradient elution,the concentration of acetonitrile rose from30%to100%within25min; Theseparation result for Histamine, tyramine and tryptamine, putrescine, cadaverine,β-phenethylamine is good within25min.When the linear range of six kinds of biogenic amines’ concentration is0.110mg·L-1, theaverage recovery rate of histamine was85.1%90.3%; the average recovery rateofβ-phenethylamine was78.2%82.6%; The rest of the four kinds of biogenic amines’ recoverymore than92%on average. Relative standard deviation （RSD） was1.7%6.7%.The limit ofdetection of tryptamine was0.0052mg·L-1；histamine was0.0037mg·L-1；Tyramine was0.0158mg·L-1；β-phenethylamine was0.0063mg·L-1；Cadaverine was0.0072mg·L-1；Putrescine was0.0182mg·L-1。The detection accuracy meet the daily needs. Time consuming of this method fromsample preparation to final completion is8090min，however, current national standard time is150200min, detection speed is doubled. To sum up, this detection method for6kinds of biogenicamines in cheese sample is both quick and accurate.（2）The conclusion of the research of HPLC test for aflatoxin M1in cheese:1Respectively using acetonitrile, Methanol, acetone extract aflatoxin M1（AFM1） in cheesesample and calculate the addition recoveries and relative standard deviation of AFM1.The resultsshow that using acetonitrile as extracting solution of cheese samples, the recovery rate of AFM1ishighest, and have best extraction efficiency.2Because the cheese is complex food substrate,so the extracting solution must be have muchimpurities. In order to get a higher precision, AFM1purification step is must be done.Thisexperiment by comparing the purification effect of OASISHLB and AFM1immune affinitycolumn to determine the best method. The test results show that the purification effect of the twomethods for AFM1are equal. Take into account of the price of OASISHLB is more cheaper thanimmune affinity column.So OASISHLB is more suitable than immune affinity column.Andthrough the test to determine the25%acetonitrile-water solution as wash solvent,5ml ofacetonitrile as elution solvent.3When the linear range of AFM1concentration is0.14ng/mL, the average recovery rate ofAFM1was87.1%92.2%; relative standard deviation （RSD） is1.4%5.7%; The limit of detectionof AFM1was0.037μg·kg-1. The accuracy of this method Compare with the current nationalstandard of the second method—immune affinity chromatography purification high performanceliquid chromatography （HPLC） method had no significant difference. Time consuming of thismethod from sample preparation to final completion is5060min，however, the time consuming ofcurrent national standard is80100min, detection speed is increased3040min. And the cost ofOASISHLB is about one-third of immune affinity column. To sum up, this detection method for AFM1in cheese sample is both quick and accurate. |
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