Detection of β2-agonists in pork by Agilent SampliQ SCX solid phase extraction column coupled with liquid chromatography/tandem mass spectrometry
Application Report / Food Safety
Author: Chenhao Zhai Agilent Technologies Co., Ltd. China Shanghai Waigaoqiao Free Trade Zone, 412 England Road Shanghai, 200131 China
Jianzhong Li and Yue Song Agilent Technologies Co., Ltd. 318 Fuzhou Road, 11th Floor, Gaoteng Building, Shanghai 200001 China
Abstract <br>This application report develops and validates a method for the simultaneous detection of four β2-agonist residues, including terbutaline, salbutamol, clenbuterol and formoterol. The analytes were purified by liquid-liquid extraction (LLE) and solid phase extraction (SPE) and quantified by liquid chromatography coupled electrospray ionization mass spectrometry (LC-ESI-MS/MS) in positive ion multiple reaction monitoring mode.
This method provides a detection limit (LOD) for sub-ng/g levels and a dynamic calibration range of 0.25 to 5 ng/g for the detection of four β2-agonists in pork. The overall recovery was 78% to 101% and the relative standard deviation (RSD) was 1.8% to 7.2%.
Introduction β2-agonists are an illegal growth promoter widely used in pork production. The recent poisoning incident is due to the high level of beta-agonist (clenbuterol) in pork. This application report uses Agilent's new solid phase extraction (SPE) product to extract and concentrate beta-agonists in pork and analyze them using LC-MS/MS. Table 1 gives the names and structures of the four β-agonist compounds.
Table 1. β2-agonist compounds used in this study
EXPERIMENTAL <br> Reagents and Pharmaceuticals All reagents are pure-spectrum, HPLC-grade or analytical grade.
Acetonitrile and water are supplied by Scharlau. Ethyl acetate and isopropanol were supplied by Fisher. Standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP). Pork is sourced from the local market. A single standard solution (1.0 mg/mL) was prepared in methanol and refrigerated at 4 °C.
The combined working solutions (10 μg/mL) were prepared in methanol-water (10:90) and refrigerated at 4 °C. The addition solution should be prepared weekly, prepared by appropriately diluting the combined working solution.
Equipment and materials
Agilent 1200 High Performance Liquid Chromatography System Agilent 6460 Triple Quadrupole LC-MS/MS System Agilent SamliQ SCX Polymer Column, 50 x 3 mL Tube, 60 mg (Part Number: 5982-3236)
Agilent ZORBAX Eclipse Plus C18 column, 50 × 2.1 mm, 1.8 μm (part number: 959741-906)
Agilent Vacuum Manifold Process Kit (Part No.: 5982-9120)
Sample Preparation <br> Liquid-Liquid Extraction 2 g pork (accurate to ±0.01 g) was weighed into a 15 mL polypropylene tube with a lid. Add 8 mL of 0.2 M sodium acetate (pH 5.2) solution and vortex to mix well. Then 100 μL of β-glucuronidase (1000 U/mL) was added and vortexed for 2 minutes. The sample was hydrolyzed at 37 °C for 16 hours.
The hydrolyzate was shaken for 15 minutes and centrifuged in a centrifuge at 4000 rpm for 10 minutes. Take 4 mL of the supernatant to another centrifuge and add 5 mL.
0.1 M perchloric acid solution and adjust the pH to 1 ± 0.3, then centrifuge in a centrifuge at 4000 rpm for 10 minutes. The supernatant was transferred to another tube and the pH was adjusted to 11 with 10 M sodium hydroxide solution.
Add 10 mL of saturated sodium chloride solution and isopropanol-ethyl acetate (60:40) mixture to the sample tube and shake for 5 minutes. After centrifugation at 4000 rpm for 5 minutes, the organic layer was carefully transferred to another centrifuge tube. The addition, shaking treatment, centrifugation, and transfer of the organic layer of the isopropyl alcohol-ethyl acetate mixture were repeated twice, and all the supernatants were combined.
The sample was dried with nitrogen at 40 ° C and the residue was re-dissolved in 5 mL of 0.2 M sodium acetate (pH 5.2) solution. This sample will be used for solid phase extraction purification.
Solid Phase Extraction <br> The solid phase extraction process (SPE) is shown in Figure 1. The Agilent SampliQ SCX column was first activated with 3 mL of methanol and then equilibrated with 3 mL of water. Load 5 ml of the sample solution into the column and pass the column by gravity (about 1 mL/min). Use 2 mL water and 2 mL
Rinse the SPE cartridge with 2% aqueous formic acid and discard all eluents. Vacuum the SPE cartridge. Finally, it was eluted with 5 mL of 5% ammonia in methanol at a flow rate of about 1 mL/min. The eluate was blown dry at 40 ° C under nitrogen and the residue was redissolved in 1 mL of 0.1% formic acid in water/acetonitrile (90:10). The sample was vortexed and sonicated to completely dissolve and transferred to a 1.5 mL centrifuge tube and treated at 3000 rpm for 5 minutes. Finally, transfer the sample to a 2 mL chromatography vial for analysis.
Figure 1. Cleaning and Concentration of Pork - Solid Phase Extraction Process
Instrument condition
HPLC column: Agilent ZORBAX Eclipse Plus C18 column,
50 × 2.1 mm, 1.8 μm (part number: 959741-906)
Flow rate: 0.4 mL/min
Column temperature: 40 °C
Injection volume: 5 μL
Mobile phase: water (0.1% formic acid solution + 2 mM ammonium acetate, A),
Acetonitrile (0.1% formic acid, B)
Gradient elution: 
Mass Spectrometry Conditions <br> Four compounds were monitored in positive mode. The ion source conditions are shown in Figure 2, and the multiple reaction monitoring channels are shown in Table 2.
Figure 2. Mass spectrometry source parameters for four compounds
Table 2. Quality monitoring of multiple reaction monitoring modes
Results and Discussion <br>Linear and Detection Limits The standard operating curves (0.25, 0.5, 1.0, 2.0, and 5.0 ng/g) were prepared by adding an appropriate amount of mixed working solution to a blank matrix. The blank matrix is ​​prepared by subjecting pork to hydrolysis, liquid-liquid extraction and solid phase extraction. The results of the calibration curve are shown in Table 3. The limit of detection (LOD) is defined as the concentration at which each compound gives a signal to noise ratio (S/N) greater than 3:1. The limits of detection for each compound are also listed in Table 3.
Table 3. Linearity and detection limits for β2-agonists
Recovery and reproducibility
The concentration of the standard was 0.5, 1.0, 2.0 ng/g in the pork, and the recovery and reproducibility were calculated. The analysis was repeated 6 times for each concentration. Recovery and reproducibility data are listed in Table 4. A chromatogram of the addition of pork extract (1.0 ng/g) is shown in Figure 3.
Table 4. Recovery and reproducibility of β2-agonists in solid-phase extracted pork using Agilent SampliQ SCX; (part number: 5982-3236), average recovery 90%, average relative standard deviation 4.4% 
Figure 3. Chromatogram of 1.0 ng/g added pork sample extract
Conclusions This study demonstrates that Agilent SampliQ SCX can be used as an effective method for the purification and concentration of various β2-agonists in complex matrices such as pork. The recovery and reproducibility measured by standard addition matrix are in line with Chinese national standards for the detection of β2-agonist residues in pork. Impurities and matrix effects do not interfere with the quantification of any target compound, and the minimum quantitation limit is significantly lower than the maximum residue limit [1, 2].
references
1. GB/T 21313-2007 "Analysis of β2-agonists in animal-derived foods by high performance liquid chromatography-tandem mass spectrometry"
2. SN/T 1924-2007 "Determination of Clenbuterol, Retopamine, Salbutamol and Terbutaline Residues in Animal-derived Foods by High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry"
For more information: For more information on our products and services, visit
Â
Yingmed Homecare Devices offers you different support products to help people who can not stand or walk to move. The wheelchair is ideal for transporting immobile patients in hospitals, practices and care facilities. The Folded Wheelchair provide a space-saving storage solution when it is not being used or when taking it along in a medical transport vehicle. The Commode wheelchair has the advantage of enabling the disabled and patients siting on the wheelchair to solve the problem of defecating and urinating without help. Compared with wheelchair, walker and crutch is more cheaper and portable. Collapsible is easy to taken along and save space to store.
Foldaway Strecher, Emergency bed, Spine board are designed for who suffered a serious emergency injury and can`t move. You will find them in the category of Emergency Aids Devices. Plaster Of Paris Bandage is the essential fixation dressing.
Homecare Devices
Homecare Devices,Homecare Electrotherapy Device,Homecare Air Cervical Neck,Portable Homecare Devices
NINGBO YINGMED MEDICAL INSTRUMENTS CO.,LTD , https://www.chinayingmed.com