biocommaExperiment
Extraction
Weigh 2 g of egg into a 50 mL centrifuge tube. Add 8 mLof Na2EDTA-Mcllvaine buffer. Vortex for 10 min, centrifugeat 14000 r/min at 4 °C for 10 min. Transfer the supernatantto another clean centrifuge tube. Add 8 mL of Na2EDTAMcllvaine buffer and repeat the extraction procedures twice.Combine all the extracted solutions. Then, centrifuge at14000 r/min at 4 °C for 10 min. Filter the supernatant forpurification.
Purification (Copure® HLB Cartridge, 60 mg/ 3 mL)
Activation: activate the Copure® HLB Cartridge by 3mLmethanol, then 5 mL of water.
Loading: add the prepared solution to the activatedcartridge.
Washing: wash the cartridge by 3 mL of water, then 3 mL of5% methanol-water solution. Drain the cartridge.
Elution: add 5 mL of methanol. Collect all the eluate.Evaporate sample to near dryness (remain few drops) at 40℃ . Dilute to 1 mL by 20% acetonitrile solution. Vortex for 30s to dissolve the residue. After filter by 0.22 μm membrane,the sample is ready for analysis.
Preparation of Standard Curve Solution
Prepare blank sample in the same procedures. Aftercollecting the eluent, add an appropriate amount of standardsolution. Evaporate and dilute the concentration of 1.00 ng/mL, 2.00 ng/mL, 5.00 ng/mL, 10.0 ng/mL, 20.0 ng/mL.
Instrumental Conditions
1.Chromatographic conditions
Instrument: Liquid Chromatography-Tandem MassSpectrometry (Triple Quad 5500)
Chromatographic column: ACQUITY UPLC BEH C18 1.7m2.150mm Column
Mobile phase: A: 0.1% formic acid in water, B: AcetonitrileMobile phase gradient: initial 95%A, 60%A (0 min~2.50min),10%A (2.50 min~3.00min),10%A (3.00min~4.00min), 95%A (4.00min~5.00min)
Flow rate: 0.300 mL/min
Column temperature: 35 ℃
Injection volume: 4.0 μL
2.Mass spectrometry conditions
Detection method: multiple reaction ion monitoring (MRM)Table 1. Ion Source Control Conditions
Table 2. Targets, Retention Times, Characteristic Ions,Declustering Voltage and Collision Energy (Quantitative Ion)
Results
Table 3. Results of Doxycycline Spiking RecoveryExperiments at 5.00 μg/kg
Figure 2. Characteristic Ion Mass Chromatogram of EggMatrix Matched Standard Solution (2.00ng/mL)
Ordering Information
