biocommaIntroduction
For the LC-MS detection method of oxytetracycline, tetracycline,chlortetracycline, and doxycycline residues in seafood such as fish,shrimp, crab, and sea cucumber, Biocomma conducted experiments andoptimized some parameters to establish an SPE-HPLC-MS/MS methodwith good recovery and stability for your reference.
Experiment
Preparation
Prepare Na2EDTA-Mcllvaine buffer (0.1 mol/L): weigh 12.9 g of citricacid, 10.9 g of disodium hydrogen phosphate, and 37.2 g of disodiumethylenediaminetetraacetate, and dissolve by water. Dilute with water to1000 mL, and adjust the pH to 4.0±0.05 with 0.1 mol/L HCl or 0.1 mol/LNaOH.
Prepare lead acetate solution (20.0 g/L): dissolve 20.0 g of lead acetatein water and dilute to 1000 mL.
Extraction
Weigh 2 g (accurate to 0.02 g) of ground aquatic samples such asfish and shrimp into a clean centrifuge tube. Add 6 mL of Na2EDTAMcllvaine (pH=4±0.05) and 2 mL lead acetate. Vortex for 1 min, andextract by ultrasonic treatment for 10 min. After centrifuge at 8000 r/min, 4 ℃ for 5 min, transfer the supernatant to another clean centrifugetube. Add 6 mL of Na2EDTA-Mcllvaine (pH=4±0.05) and repeat theextraction procedures twice. Combine all the extracted solutions. Add 10ml of n-hexane. Vortex for 1 min, and then centrifuge at 8000 r/min for5 min. Discard the n-hexane layer and take 10 mL of the lower layer forpurification.
Purification (Copure® HLB Cartridge, 60 mg/ 3 mL)
Activation: activate the Copure® HLB Cartridge by 5 mL of methanol,then 5 mL of water.
Loading: add the prepared solution to the activated cartridge.Washing: wash the cartridge by 5 mL of water, then 5 mL of 5%methanol-water solution. Drain the cartridge.
Elution : add 5 mL of methanol . Collectall the eluate .Evaporate sample to 100 μL at 45 ℃ . Dilute to 1 mL by 0.1% formicacid. After filter by nylon membrane, the sample is ready for analysis.
Preparation of Standard Curve Solution
Prepare blank sample in the same procedures. Add internal standard toprepare the concentrations of 5 μg/L, 10 μg/L, 50 μg/L, 100 μg/L, and200 μg/L for standard curve.
Instrumental Conditions
1.Chromatographic conditions
Instrument: UPLC-MS/MS (Thermo Fisher TSQ Endura)
Chromatographic column: Hypersil GOLD C18 (2.1 mm×100 mm, 1.9μm)
Mobile phase: A: water (containing 0.1% formic acid) B: methanol(containing 0.1% formic acid)
Flow rates: 0.3 mL/min
Column temperature: 30 ℃
Injection volume: 5 μL
Table 1. Gradient Elution Program
2.Mass spectrometry conditions
Ion source: HESI
Electrospray voltage: 3500 V
Sheath gas pressure: 40 arb
Auxiliary gas pressure: 2 arb
Ion transfer tube: 380 ℃
Auxiliary air temperature: 350 ℃
Table 2. Targets, Retention Times and Characteristic Ions (QuantitativeIon)
Results
Table 3. Results of Chloramphenicol Spiking Recovery
Ordering Information
