biocommaExperiment
Extraction
Weigh 2 g of sample into a 50 mL centrifuge tube. Add6 mL of 0.2 mol/L ammonium acetate buffer solutionand 40 μL of β-glucuronidase/aryl sulfatase. Vortex tomix well. Oscillate in a water bath at 37 ℃ , and keepaway from light exposure for 16 h. Leave it reach roomtemperature. Add 125 μL of 20 ng/mL internal standardsolution. Vortex to mix. Centrifuged at 9500 r/min for5min. Transfer the supernatant into another 50 mLcentrifuge tube. Add 5 mL of 0.1 mol/L perchloric acidvortex to mix well. Adjust the pH with perchloric acid to1.0±0.2. Centrifuged at 9500 r/min for 5min. Transfer thesupernatant a clean 50 mL centrifuge tube, and adjustthe pH to 10±0.5 with 10 mol/L NaOH solution. Add 15mL of ethyl acetate, shaking for 5min. Centrifuged at9500 r/min for 5min. Pipette the upper organic phase toa new tube. Add 10 mL of tert-butyl methyl to residues,shaking for 5min. Centrifuged at 9500 r/min for 5min.Pipette the upper organic phase to the same tube.Evaporate to nearly dryness at 40℃ . Dissolve with 5 mLof 2% formic acid. The sample is ready for purification.
Purification (Copure® MCX Cartridge, 60 mg/ 3 mL)
Activation: activate the Copure® MCX Cartridge by 3 mLof methanol, then 3 mL of 2% formic acid.
Loading: add prepared sample.
Washing: add 3 mL of 2% formic acid, then 3 mLmethanol. Drain the cartridge.
Elution: add 5 mL of 5% ammoniated methanol andcollect the eluate. Evaporate to near dryness at 40 °C.Add to 0.5 mL of methanol with 0.1% formic acid (10+90,V/V). After filter by 0.22 μm membrane, the sample isready for analysis.
Preparation of Standard Curve Solution
Accurately measure the standard solution and internalstandard solution. Dilute with methanol with 0.1%formic acid (10+90, V/V) to prepare series of standardconcentrations of 0.500 ng/mL, 1.00ng/mL, 2.00 ng/mL,5.00 ng/mL, 10.0 ng/mL, and 20.0 ng/mL. The internalstandard is 5.00 ng/mL.
Instrumental Conditions
1.Chromatographic conditions
Instrument: LC-MS/MS (Triple Quad 5500+)Chromatographic column: ZORBAX RRHD Eclipse Plus95Å C18, 2.1 x 100 mm, 1.8 µm
Mobile phase: A: 0.1% formic acid in water, B:acetonitrile
Mobile phase gradient: initial 98% A, 98% A (0 min~0.3min), 10% A (0.3 min~3.0 min), 10% A (3.0 min~4.0min), 98% A (4.0 min~4.2 min), 98% A (4.2 min~6.0 min)Flow rate: 0.3 mL/min
Column temperature: room temperature
Injection volume: 4.0 μL
2.Mass Spectrometry Conditions
Detection method: multi-reactive ion monitoring (MRM)
Table 1. Ion Source Control Conditions
Table 2. Targes Characteristic Ions (*Quantifier Ion)
Results
Table 3. Spiked β-Agonist in Beef Recovery at 1.50 μg/kg
Ordering Information
