Method for Determination of Sulfonamides and Antibacterial Synergists Residues in Bee Products Using the Anyeep TQ9120 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry System

Sulfonamides (SAs) are a class of chemotherapeutic agents characterized by the p-aminobenzenesulfonamide structure, used to prevent and treat bacterial infections. Thousands of sulfonamide variants exist, with dozens widely applied and clinically effective. As a common category of antibacterial and anti-inflammatory drugs in modern medicine, sulfonamides encompass a vast "family" of diverse compounds.

Routine testing covers animal-derived foods, honey products, milk, and dairy products. Applicable standards include GB/T 22966-2008, GB/T 22951-2008, GB/T 22947-2008, GB/T 21316-2007, GB/T 20759-2006, GB/T 18932.17-2003, GB/T 31657.10-2025, SC/T 9436-2020, SN/T 5140-2019, NY/T 3167-2017, SN/T 4816-2017, GB 31658.17-2021, and SN/T 4057-2014. This method is based on GB 31657.10-2025 and uses external standard quantification.

Instruments and Reagents

Anyeep TQ9120 Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry System

Acetonitrile, Methanol (MS grade, ANPEL Shanghai)

Formic acid (chromatographic grade, Anpel Shanghai)

Liquid Chromatography Conditions

Mobile Phase A: Water with 0.1% formic acid

Mobile Phase B: Methanol

Column: Waters ACQUITY UPLC BEH C18, 1.7 µm, 2.1×100 mm

Column Temperature: 35 °C

Flow rate: 0.3 mL/min

Injection volume: 5 μL

Mass Spectrometry Conditions

Ionization Source: Electrospray Ionization (ESI+)

Source Temperature: 550°C

Spray voltage: 5000V

Air Curtain Air: 30 psi

Atomizing gas: 50 psi

Auxiliary Air: 7 psi

Collision Gas: 10

Scan Mode: DMRM

Sample Processing

Transfer a honey sample weighing (5 ± 0.05) g into a 50 mL centrifuge tube. Add 5 mL of 0.1% formic acid solution and vortex-mix. Then add 10 mL of acetonitrile, shake for 5 min, and add 4 g of anhydrous sodium sulfate and 1 g of sodium chloride. Vortex for 1 min, then centrifuge at 8000 r/min for 5 min. Collect the supernatant and set aside.

Purification: Transfer the supernatant into a 15 mL centrifuge tube. Add 20 mg PSA, 150 mg C18, and 900 mg anhydrous sodium sulfate. Vortex for 1 min and centrifuge at 8000 r/min for 5 min. Collect 2 mL of the supernatant and evaporate to dryness under a nitrogen stream in a water bath at 40 °C. Reconstitute the residue with 1 mL of 10% methanol solution, vortex thoroughly, filter through a microporous membrane, and analyze by LC-MS/MS.

Preparation of Matrix-Matched Calibration Curves Accurately pipette an appropriate volume of the mixed standard working solution and add it to honey blank sample residue that has been extracted and purified. Dry under a nitrogen stream in a water bath at 40 °C. Add 1 mL of 10% methanol solution to the honey sample, vortex to dissolve the residue, and prepare matrix-matched standard working solutions with concentrations of 0.1 μg/L, 0.2 μg/L, 0.5 μg/L, 1 μg/L, 5 μg/L, 10 μg/L, 50 μg/L, and 100 μg/L. Filter through a microporous membrane and analyze using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Summary

This study developed a detection method for sulfonamides and antibacterial synergists residues in bee products using the Anyeep TQ9120 ultra-high-performance liquid chromatography-tandem mass spectrometry system. Results demonstrate that the method exhibits excellent precision and accuracy, making it suitable for routine analysis and testing of these residues in bee products.