β-agonists, chemically belonging to the phenylethanolamine group, are chemical analogs of catecholamines, epinephrine and norepinephrine. Source: Food Safety Monitoring Technology (Chemical Industry Press)
Operation method
Gas Chromatography-Mass Spectrometry
Materials and Instruments
Liver, kidney, meat. Move 1. Extraction For more product details, please visit Aladdin Scientific website.
Sodium acetate buffer solution Metoprolol Perchloric acid Concentrated ammonium hydroxide solution
Centrifuge bottle Thermostat Triangular funnel Horizontal oscillator Solid phase columns
Weigh (10 ± 0.1) g of mashed sample in a 100 mL centrifuge bottle, add 15 mL of sodium acetate buffer solution (0.2 mol/L, pH 5.2), and mix for 2 min. Add 120 ng of metoprolol as an internal standard to all the samples of liver, kidney, and meat. Add 50 uL of β-glucosidase-arylsulfatase, cover and ultrasonicate for 15 min, and incubate at 37 ℃ in a thermostat for 16 h. Add 10 mL of 0.1 mol/L perchloric acid, mix well, and adjust the pH with 11.7 mol/L perchloric acid (concentrated) to 1 ± 0.3. Centrifuge the samples at 4400 r/mL for 10 min, then pass the supernatant through a triangular chamber stuffed with a little desiccated cotton. After centrifugation at 4400 r/mL for 10 min, the supernatant was filtered through a triangular funnel stuffed with a little desiccated cotton into another 100 mL centrifuge tube. The pH was adjusted to 9.5 ± 0.3 with concentrated ammonium hydroxide solution (~17% concentration) and the aqueous phase was saturated with sodium chloride. Extract with 20 mL and 15 mL of isopropanol-ethyl acetate (6 + 4) mixture, respectively, for 2 min each, shaking on a horizontal shaker for 15 min and centrifuging at 3000 r/min for 10 min. Transfer the organic phase to a 50 mL rotary evaporation flask and evaporate the organic phase to dryness at (45 ± 5) °C. Add 12 mL of phosphate buffer to a 100 mL tube. Add 12 mL of phosphate buffer solution to make the evaporation residue into a suspension, and then centrifuge at 2000 r/min for 5 min.
2. Purification
The C18 column (500 mg, 3 mL) and SCX column (500 mg, 3 mL) were loaded in order from top to bottom. Sequentially activate the column with 5 mL of water, 5 mL of methanol and 5 mL of 0.03 mol/mL hydrochloric acid solution at a certain flow rate. Transfer the solution to the top of the solid phase column and pump the column carefully and slowly for at least 10 min. Wash the column with 2 mL of 1 mol/L acetic acid for at least 15 min to dryness, and 12 mL of methanol under strong negative pressure for at least 10 min to dryness. remove the C18 column. The components to be analyzed were eluted from the column with 12 mL of well-mixed ethyl acetate-ammonium hydroxide (97 + 3, v/v) solution. Evaporate the eluate on a heating block at (40 ± 5) °C by venting nitrogen. Add 200 uL of methanol and shake with vigorous vortexing to dissolve the evaporation residue. Transfer the solution to a reaction glass vial. Another 200 uL of methanol was used to dissolve the residue that was not completely dissolved in the previous step and the solution was combined in a reaction glass vial. Evaporate the methanol to dryness on a heating block at temperature (40 ± 5) °C.
3. Derivatization reaction
100 uL of N,O-bis(trimethylsilyl)-ammonium trifluoroacetate (BSTFA) was added and vigorously vortexed and shaken. The reaction vial was placed in a thermostat at (65 ± 5) °C and heated for (30 ± 5) min. After allowing the vial and the heating block to cool to room temperature, a small stream of nitrogen was passed through to evaporate to dryness. 500 L of toluene was added to dissolve the evaporation residue with vigorous vortex shaking. The solution was analyzed by GC-MS.