biocommaIntroduction
For the detection method soft etracy clines ,sulfonamides, and quinolones in beef, lamb, Beef,and chickens, Biocomma conducted experiments andoptimized some parameters to establish an SPE-HPLCMS/MS method with good recovery and stability for yourreference.
Experiment
Preparation
Na2EDTA-Mcllvaine buffer: weigh 12.9 g of citric acid,10.9 g of disodium hydrogen phosphate, and 39.2 g ofdisodium ethylenediaminetetraacetate, and dissolve by900mL of water. Adjust the pH to 5.0±0.2 with 1 mol/LNaOH. Dilute to 1000 mL with water.
phosphate buffer: dilute 190 mL of 0.05 mol/L sodiumdihydrogen phosphate solution to 1000 mL with 0.05mol/L disodium hydrogen phosphate solution.
Extraction
Weigh 1.00 g of ground meat sample into a cleancentrifuge tube. Add 8 mL of Na2EDTA-Mcllvaine buffer,and vortex to mix well. Extract by ultrasonic treatmentfor 20 min. After centrifuge at 120,000 r/min, -5 ℃for 5 min, transfer the supernatant to another cleancentrifuge tube. Add 8 mL of phosphate buffer to thesample residue and repeat the extraction procedurestwice. Combine all the extracted solutions. Centrifuge at120,000 r/min, -5℃ for 5 min. The supernatant is readyfor purification.
Purification (Copure® HLB Cartridge, 200 mg/ 6 mL)
Activation: activate the Copure® HLB Cartridge by 5 mLof methanol, then 5 mL of water
Loading: add the prepared solution to the activatedcartridge.
Washing: wash the cartridge by 5 mL of water, then 5mL of 5% methanol-water solution. Drain the cartridge.Elution: use 8 mL of methanol: ethyl acetate: ammoniawater = 50:50:2 solution for elution.
Collect all the eluate, blow it with nitrogen to about100 μL at 45°C. Evaporate sample to 100 μL at 45 ℃ .Dilute to 1 mL by 0.1% formic acid. After filter by PTFEhydrophilic membrane, the sample is ready for analysis.
Preparation of Standard Curve Solution
Prepare 6 blank sample in the same procedures. Addinternal standard to prepare the concentrations of 2 μg/L,10 μg/L, 50 μg/L, 100 μg/L, 200 μg/L, and 500 μg/L forstandard curve.
Instrumental Conditions
1.Chromatographic conditions
Instrument: UPLC-MS/MS (Thermo Fisher TSQ Endura)Chromatographic column: Hypersil GOLD C18 (2.1mm×100 mm, 1.9 μm)
Mobile phase: A: water (0.1% formic acid), B: methanol:acetonitrile = 2:8 (0.1% formic acid)
Flow rate: 0.3 mL/min
Column temperature: 35 °C
Injection volume: 10 μL
Table 1. Gradient Elution Program
2.Mass spectrometry conditions
Ion source: HESI
Electrospray voltage: 3500 V
Sheath gas pressure: 40 arb
Auxiliary air pressure: 2 arb
Ion exchange tube: 380 ℃
Auxiliary air temperature: 350 ℃
Table 2. Targets, Retention Times and Characteristic Ions (*Quantitative Ion)
Results
Table 3. Results of Muti-Residue of Veterinary Drugs Recovery Experiments
Optimization Tips:
1.Lowering the centrifuge temperature to -5°C during extraction inhibit the dispersion of the fat in the sample on the surface of the aqueous solutionbetter, which is beneficial to the extraction and transfer of the sample.
2.After ultrasonic treatment, centrifuging the combined sample again improved removal of matrix effects and facilitates loading.
3.Eluting with 5 mL of 5% methanol-water solution reduces the loss of some target substances during elution.
4.Although the experiment requires to evaporate sample to about 100 μL, minimizing this process reduces the loss of some target substances.
5.Diluting the evaporated sample to 1 mL by 0.1% formic acid effectively improves the peak shape and recovery rate of some targets.
6.In order to improve the loading flow rate, employ Copure® HLB Cartridge, 200 mg/ 6 mL (Cat. No.: COHLB6200-M).
Ordering Information
