Method for Determining Chloramphenicol Residues in Milk Using Anyeep TQ9120

Chloramphenicol is a broad-spectrum antibiotic produced by *Streptomyces venezuelae* that inhibits bacterial growth. However, it poses significant toxicity risks to humans, including bone marrow suppression leading to allergic reactions and aplastic anemia. Long-term consumption of animal products containing chloramphenicol residues may disrupt gut flora and inhibit antibody formation.

This document follows the "GB29688-2013 National Food Safety StandardsChlorine in milkrequirements for the determination of macrolide residues by liquid chromatography-tandem mass spectrometry, developedChlorine in milkLiquid chromatography-tandem mass spectrometry method for determination of macrolide residues.This method exhibits high sensitivity, good linearity, and precision.Recovery RateAll methodological parameters meet the standard's requirements.
Instruments and Reagents

Anyeep TQ9120 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)

Ethyl acetate, n-hexane (HPLC grade, CNW Technologies GmbH, Germany)

Sodium Chloride (Analytical Grade, Anpel, Shanghai)

Chloramphenicol Standard and Internal Standard (Shanghai ANPEL)

C18 Solid-Phase Extraction Cartridge (SBEQ-CA0853, ANPEL, Shanghai)

Liquid Chromatography Conditions

Mobile PhaseA: Water

Mobile Phase B: Acetonitrile

Chromatography column: CNW Athena UHPLC C18, 2.1 mm × 100 mm, 1.8μm 

Column Temperature：40 ℃

Flow rate: 0.3 mL/min

Injection volume：10 μL

Mass Spectrometry Conditions

Ionization Source： Electrospray Ionization Source (ESI)-）

Source Temperature： 550 ℃

Spray Voltage： -4500 V

air curtain： 35 psi

Mist gas： 50 psi

Auxiliary Gas： 70 psi

Crash： 8

Scan Mode：MRM

Sample Processing

Sample Processing:Weigh 10.0 g of the sample to a precision of 0.05 g into a 50 mL centrifuge tube and add 250 μL of chloramphenicol internal standard working solution at 20 ng/mL.μAdd 20 mL ethyl acetate to L, shake for 15 min, and centrifuge at 6000 rpm for 10 min. Transfer the ethyl acetate layer to a pear-shaped flask. Extract the residue again with 20 mL of ethyl acetate, then combine both extracts. 45℃Rotary evaporate the water bath to dryness. Dissolve the residue with 4% sodium chloride solution (5 mL), add 5 mL of n-hexane, shake for 1 min, and allow to stand until layers separate. Discard the n-hexane layer, then add another 5 mL of n-hexane to repeat the extraction. Collect the lower aqueous layer for use.

Condition the C18 solid-phase extraction cartridge sequentially with 5 mL methanol and 5 mL water. Pass the sample through the cartridge at a flow rate of one drop every 3-4 seconds. Rinse with 5 mL of water, dry under vacuum, and elute with 5 mL of methanol. Collect the eluate in 50.℃Dry under nitrogen, reconstitute with 50% acetonitrile (1.0 mL), and pass through 0.22.μm filter, ready for injection.

Standard Curve:Dilute the chloramphenicol standard and internal standard with 50% acetonitrile solution to prepare a series of standard solutions with an internal standard concentration of 10 µg/mL and chloramphenicol concentrations of 0.02, 0.05, 0.10, 0.25, 0.50, 1.0, 2.0, and 5.0 ng/mL.

Recovery Rate Sample:Add a certain amount of chloramphenicol standard to the sample according to "Sample ProcessingPerform the operation to configure the final content as 0.01, 0.02, 0.05, and 0.10.μg/kgChloramphenicol recovery samples. Prepare 6 replicates at each concentration level.

Summary

This article establishes Anyeep TQ9120 Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry SystemMethod for analyzing chloramphenicol residues in milkAs the results show，The method exhibits excellent sensitivity, stability, and a wide linear range.All relevant metrics meet the required standards.Suitable forChloramphenicol Residue in MilkofRoutine analysis and testing.