CLOVER TC-M160 Pro Fungal Toxins Multi functional Purification Column

CLOVER TC-M160 Pro Fungal Toxins Multi functional Purification Column

Aspergillus flavus and zearalenone in feedT-2Determination of toxins

--－LC-MSlaw

1.Target audience

This method refers toNY/T2071-2011Aflatoxin in the sampleCorn gibberellin ketone andT-2 toxin was extracted with acetonitrile solution, defatted with n-hexane, purified with a multifunctional purification column, dried with nitrogen, dissolved in formic acid acetonitrile solution, and determined by liquid chromatography.

This method uses aflatoxinandThe detection limit of T-2 toxin is 1.0ug/kg, and the quantification limit is 2.0ug/kg; The detection limit of zearalenone is 5.0ug/kg, and the quantification limit is 10.0ug/kg.

GB13078-2017The hygiene standards for feed stipulate that:

2.Experimental preparation

2.1solution preparation

Formic acid solution（0.1%）Accurately measure formic acid1mlAdd water until1000mlShake well to obtain.

Acetic acid solution（0.02%）Accurately measure glacial acetic acid0.2mlAdd water until1000mlShake well to obtain.

Formic acid acetonitrile solution: Take formic acid solution50mlAdd acetonitrile to100mlShake well to obtain.

2.2Mixed standard reserve solution:

Extract a certain amount separately6The fungal toxin standard was prepared in a brown volumetric flask using methanol at a concentration of1ug/mlThe mixed reserve solution is stored in4In the refrigerator at ℃.

2.3Matrix standard working fluid:

Take a certain amount of fungal toxin standard reserve solution separately, add blank sample extraction solution, dry with nitrogen, and dilute with mobile phase to a concentration of1.0ug/L,5.0ug/L,10.0ug/L,50ug/L,100ug/L,200ug/LThe mixed standard series working fluid is newly prepared for immediate use.

3.Sample pretreatment

（1）Weighing5g±0.02gFeed samples50mlAccurately add to the centrifuge tube25mlExtraction solution (acetonitrile: water)=84：16）Vortex mixing2min, placed in an ultrasonic cleaner for ultrasonic extraction20minIntermediate oscillation2-3Next time.

（2）Take it out, in8000r/mincentrifugation5minPour the supernatant into a separating funnel and add15mlN-hexane, fully oscillate. After static stratification, accurately measure the lower layer liquid5ml, to be used.

（3）Put the above5mlLiquid, multifunctional purification column, controlled flow rate2ml/minCollect the effluent and60Blow dry under nitrogen at ℃.

（4）Use1.0mlDissolve residue in formic acid acetonitrile solution, vortex30s, through0.22umAfter filter membrane filtration, perform machine testing.

4 liquid chromatography-Tandem mass spectrometry

4.1Liquid chromatography conditions

Chromatographic column:C18pillar,3.0×150mm（3.0um）； Or other equivalent chromatographic columns.

Column temperature:33℃

Injection volume:20ul

The mobile phase and flow rate are shown in the table below.

a.ESI+Source gradient elution conditions

a. ESI source gradient elution conditions

4.2 Mass spectrometry conditions

aIon source: electric spray ion source.

bScanning modes: positive ion scanning mode and negative ion scanning mode.

cDetection method: Multi reaction monitoring.

dThe solvent removal gas and cone hole gas are both high-purity nitrogen gas, and the collision gas is high-purity argon gas. Before use, the flow rates of each gas should be adjusted to ensure that the mass spectrometry sensitivity meets the detection requirements.

eThe voltage values of capillary tube voltage, cone hole voltage, collision energy, etc. should be optimized toSensitivity.

fQualitative ion pairs, quantitative ion pairs, retention time, and corresponding cone hole voltage and collision energy reference values are shown in the table below

a.ESI +Monitoring Mode

b.ESI-Monitoring Mode

5.Qualitative determination

Under the same experimental conditions, the deviation between the retention time of the test substance in the sample and the retention time of the standard solution shall not exceed ± 1% of the retention time of the standard solution2.5%If the relative abundance of qualitative ions in each ancestral tomb in the sample is compared with the relative abundance of corresponding qualitative ions in a standard solution with similar concentration, and the deviation does not exceed the range in the following table, it can be determined that there is a corresponding analyte in the sample.

Large allowable deviation in relative ion abundance during qualitative confirmation（%）

6.quantitative determination

Under the working conditions of the instrument, mixed standard working solution and sample are alternately injected, calibrated using matrix matching standard solution, and quantified using external standard method.

CLOVER TC-M160 Pro Fungal Toxins Multi functional Purification Column

Brand: Clover