Determination of β-Agonist Residues in Pork by Using Wayeal LCMS-TQ9200 Liquid Chromatography-Tandem Mass Spectrometry System

This experiment refers to "GB 31658.22-2022 National Food Safety Standard — Determination of β-Agonist Residues in Animal-Derived Foods by Liquid Chromatography-Tandem Mass Spectrometry" and utilizes the Wayeal LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system to determine the content of β-agonists in pork. The experimental results indicate that the system suitability test demonstrated well-shaped peaks and good linearity, meeting the experimental requirements. The retention time repeatability for the β-agonist standard working solution was below 1%, and the peak area repeatability was less than 3%, indicating good repeatability. For the sensitivity test solution of β-agonists, the signal-to-noise ratios of the main peaks at concentrations of 0.2ng/mL and 0.5ng/mL were significantly greater than 3 and 10, respectively, meeting the experimental requirements.

No β-agonists were detected in the pork sample test. All the above data meet the instrument requirements specified by the standard method.

Keywords: Liquid Chromatography-Tandem Mass Spectrometry; β-Agonists; Clenbuterol; Food Safety.

1. Instruments and Reagents

1.1 Configuration List of LCMS

Table 1 Instrument Configuration List

1.2 Reagents and Standards

Table 2 List of Reagents and Standards

1.3 Experiment Material and Auxiliary Equipment

Ultrasonic Cleaner

Vortex Mixer

Water Bath Constant Temperature Shaker

High-Speed Centrifuge

2. Experiment Method

2.1 Sample Pretreatment

2.1.1 Enzymatic Hydrolysis and Extraction

Weigh 2g of the sample (accurate to ±0.05g) into a 50mL centrifuge tube. Add 6mL of 0.2mol/L ammonium acetate buffer solution and 40μL of β-glucuronidase/arylsulfatase. Vortex to mix thoroughly, and incubate at 37°C in the dark with shaking in a water bath for 16 hours. Allow to cool to room temperature for subsequent use.

2.1.2 Extraction, Purification, and Concentration

Take the prepared solution, add 100μL of the 100ng/mL internal standard working solution, vortex to mix thoroughly, and centrifuge at 8000r/min for 8 minutes. Collect the supernatant, add 5mL of 0.1mol/L perchloric acid solution, vortex to mix, and adjust the pH to 1.0 ± 0.2 using perchloric acid. After centrifugation at 8000r/min for 8 minutes, adjust the pH of the supernatant to 10 ± 0.5 using 10mol/L sodium hydroxide solution. Add 15mL of ethyl acetate, shake at medium speed for 5 minutes, and centrifuge at 5000r/min for 5 minutes. Collect the upper organic layer. To the remaining aqueous layer, add 10mL of tert-butyl methyl ether, shake at medium speed for 5 minutes, centrifuge at 5000r/min for 5 minutes, and collect the upper organic layer. Combine all organic layers and evaporate to dryness under a nitrogen stream at 50°C. Dissolve the residue in 5mL of 2% formic acid solution and set aside. Prepare a mixed-mode cation exchange solid-phase extraction column by sequentially activating it with 3mL of methanol and 3mL of 2% formic acid. Load the prepared solution onto the column, sequentially rinse with 3mL of 2% formic acid and 3mL of methanol, and dry under vacuum. Elute the column with 3mL of 5% ammoniated methanol solution. Evaporate the eluate to dryness under a nitrogen stream at 50°C. Redissolve the residue in 0.5mL of methanol–0.1% formic acid solution (10:90, v/v), mix thoroughly, and filter through a 0.22μm microporous membrane for analysis by liquid chromatography-tandem mass spectrometry.

2.2 Experiment Conditions

2.2.1 Liquid Chromatographic Conditions

Chromatography column” C18, 1.8μm 2.1*100mm

Mobile phase : A : 0.1% Formic Acid in Water; B: Methanol

Flow rate: 0.3 mL/min

Colum temperature: 40°C

Injection volume: 3μL

3.System Suitability Test

The system suitability test shows that the target peaks are well-shaped with no interference from other impurity peaks, meeting the experimental requirements.

Fig 1 Chromatogram of Seven β-Agonists Test (1ng/mL)

3.2 Linear Range

Taking β-agonist standard solutions and using intermediate concentration working solutions, a standard curve was prepared stepwise. The linear range for the seven β-agonists was 0.5–50ng/mL, with R² > 0.99, indicating a good linear relationship.

Table 3 Table of Linear Ranges for β-Agonists

Fig 2 Standard Curve Chromatogram of Seven β-Agonist

3.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

In this method, the limit of detection (LOD) and limit of quantitation (LOQ) for β-agonists are set at 0.2ng/mL and 0.5ng/mL, respectively. For each compound in this method, the signal-to-noise ratio at the specified concentrations for LOD and LOQ is greater than 3 and 10, respectively, meeting the experimental sensitivity requirements.

Table 4 Corresponding Signal-to-Noise Ratios for LOD and LOQ of Each Compound

Fig 3 Ion Chromatograms of Seven β-Agonists at LOD and LOQ Concentration

3.4 Precision Test

A mixed solution of β-agonist standard substances at low, medium, and high concentrations was taken and injected consecutively six times to compare retention time and peak area deviations. The results are shown in the table below. All β-agonists showed retention time deviations of less than 1% and peak area deviations of less than 3%, meeting the experimental requirements.

Table 5 Precision Test for Each Compound

Fig 4Precision Testing Chromatograms of Seven β-Agonists (6 Injections)

3.5 Sample Test

The pork samples purchased from the market were tested according to the mentioned pretreatment method, and no β-agonists were detected. No chromatographic peaks were observed at the retention times corresponding to the seven β-agonists, while the other peaks represent the matrix signals after enzymatic hydrolysis.

Fig 5 Pork Sample Test Chromatogram

4. Conclusion

This method utilizes the Wayeal LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system for the determination of β-agonists in pork. The data demonstrate that all chromatographic peaks exhibit good shape without tailing, sensitivity meets experimental requirements, linear correlation coefficients exceed 0.99, and precision is satisfactory with retention time deviations below 1% and peak area deviations within 3% for six consecutive injections of each compound. No β-agonists were detected in the pork sample tests. These results indicate that the method, equipped with the Wayeal LC-MS/MS system, fulfills the routine qualitative and quantitative detection requirements for the target analytes.