Method for Determining 18 β-Agonists in Pork Using Anyeep TQ9120

"Lean meat powder" is a collective term for drugs that inhibit fat synthesis in animals and promote lean muscle growth. Substances capable of such functions primarily belong to a class known as beta-agonists. Once introduced into livestock, these drugs alter metabolic pathways to stimulate muscle development. In recent years, poisoning incidents linked to the consumption of food contaminated with "lean meat powder" have occurred frequently, resulting in severe consequences and drawing significant attention from governments worldwide. On 2021 3, 19, the General Office of the Ministry of Agriculture and Rural Affairs issued the "Notice on Launching a Special Campaign Against 'Lean Meat Powder'," deploying a three-month nationwide campaign to crack down on the illegal use of "lean meat powder." Routine testing covers animal-derived foods and feed, adhering to standards including GB/T 21313-2007, GB/T 22286-2008, GB/T 22147-2008, SN/T 1924-2011, NY/T 3144-2017, NY/T 3145-2017, NY/T 3146-2017, Ministry of Agriculture Announcement 1025-11/18/6-2008, and Ministry of Agriculture Announcement 1029-1-2011. This method references GB 31658.22-2022 and employs the internal standard quantification technique.

Instruments and Reagents

Anyeep TQ120 Ultra-High Performance Liquid Chromatography-Tandem Mass SpectrometrySystem

Acetonitrile, Methanol(MS-grade, from Thermo Fisher Scientific, USA)

Formic acid (mass spectrometry grade, CNW Technologies, Germany)

Perchloric acid (CNW Technologies, Germany)

Ammonia (CNW, Germany)

Ethyl acetate (chromatographic grade, CNW Technologies, Germany)

tert-Butyl methyl ether (chromatographic grade, CNW Technologies, Germany)

Liquid Chromatography Conditions

Mobile PhaseA: 0.1% formic acid, water

Mobile Phase B: 0.1% formic acid, acetonitrile

Chromatography column：Phenomenex Kinetex F5, 100 Å, 2.6 µm, 3×100 mm

Column Temperature：40 ℃

Flow rate: 0.4 mL/min

Injection volume：5 μL

Mass Spectrometry Conditions

Ionization Source： Electrospray Ionization Source (ESI)+）

Source Temperature： 550 ℃

Spray Voltage： 5000 V

air curtain： 30 psi

Mist gas： 50 psi

Auxiliary Gas： 70 psi

Crash： 8

Scan Mode：DMRM

Sample Processing

Sample ProcessingWeigh 2 g of the test sample into a 50 mL centrifuge tube, add 6 mL of 0.2 mol/L ammonium acetate buffer, and 40 β-glucuronidase/arylsulfatase. μL, vortex mix, 37℃Shake the water bath in the dark for 16 h, then let it cool to room temperature before use.

Take the backup solution and add 100 μL of internal standard working solution at 100 ng/mL. μVortex mix, centrifuge at 8000 r/min for 8 min. Collect the supernatant and add 5 mL of 0.1 mol/L perchloric acid solution. Vortex mix again. Adjust pH to 1.0 ± 0.2 using perchloric acid. Centrifuge at 8000 r/min for 8 min. Adjust the supernatant pH to 10 ± 0.5 using 10 mol/L NaOH solution. Add 15 mL ethyl acetate. Shake at medium speed for 5 min, then centrifuge at 5000 r/min for 5 min. Collect the upper organic phase. To the lower aqueous phase, add 10 mL tert-butyl methyl ether. Shake at medium speed for 5 min, then centrifuge at 5000 r/min for 5 min. Collect the upper organic phase and combine with the previous fraction. Dry under nitrogen at 50 °C. Dissolve in 5 mL of 2% formic acid solution. Set aside.

Elute the mixed-mode cation exchange SPE cartridge with 2% formic acid solution (3 mL each), followed by methanol. Pass the备用液 through the cartridge, then wash sequentially with 2% formic acid solution and methanol (3 mL each). Dry under vacuum, then elute with 5% ammoniated methanol (3 mL). Evaporate the eluate to dryness under a nitrogen stream at 50 °C.

Add 0.5 mL of methanol containing 0.1% formic acid to the residue and dissolve thoroughly. Filter through 0.22.μmicro-porous membrane for analysis by liquid chromatography-tandem mass spectrometry.

Standard Curve PreparationAccurately measure an appropriate amount of the mixed standard working solution and the mixed internal standard working solution. Dilute with a methanol-0.1% formic acid solution to prepare a series of standard working solutions with concentrations ranging from 0.05 to 50 ng/mL, each containing 20 ng/mL of internal standard. Analyze by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Summary

This page was createdThis study established an ultra-high-performance liquid chromatography-tandem mass spectrometry (Anyeep TQ9120) system to analyze 18 compounds.beta-Detection method for receptor agonist drugs. As shown in the results, this method offers excellent sensitivity, stability, and linear range, making it suitable forbeta-Routine analysis and testing of receptor agonist drugs.