Simultaneous Determination of Amphenicols in Animal-Derived Foods by Solvent and Solid Phase Extraction With Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry.

Background: The consumption of foods containing amphenicols, a type of antibiotic, is a major concern for human health. A stable and accurate detection method can provide technical support for food-safety monitoring.

Objective: An effective and efficient method was established for determining amphenicols in animal-derived foods through the simultaneous use of solid-phase extraction (SPE) cleanup and ultrahigh-performance liquid chromatography/mass spectrometry (UPLC-MS/MS).

Method: Samples were extracted using 1.0% ammoniated ethyl acetate solution, degreased with n-hexane, and then concentrated and cleaned using a C18 SPE column. Next, gradient elution was performed using methanol and 0.05% aqueous ammonia as the mobile phase, followed by separation using a C18 column. The target compound was detected using electrospray ionization, both in positive and negative modes, through multiple reaction monitoring, and quantified using an internal-standard method.

Results: The content of chloramphenicol (CAP), florfenicol (FF), and florfenicol amine (FFA) (content range: 0.2-8.0 µg/kg) as well as that of thiamphenicol (TAP; content range: 1.0-40.0 µg/kg) show a good linear relationship, with a correlation coefficient of r > 0.999. Furthermore, recoveries of 86.7-111.9% and relative standard deviations of <9.0% were achieved. The limits of detection and quantification are obtained as 0.03-0.33 and 0.1-1.0 μg/kg, respectively.

Conclusions: The proposed method has excellent stability and accuracy, and can be successfully used for the qualitative and quantitative determination of amphenicols, i.e., CAP, TAP, FF, and FFA residues in 210 animal-derived food samples, of which FF and FFA were detected in four samples.

Highlights: A stable and accurate method was successfully established for the simultaneous determination of CAP, TAP, FF, and FFA in animal-derived foods using UPLC-MS/MS. Effective sample pretreatment was established, lipids were removed using n-hexane, concentration and cleanup were achieved with the C18 SPE column, and matrix effects were effectively reduced, thus improving the method's accuracy and stability. The method was validated for eight common animal-source foods, including beef, lamb, pork, chicken, egg, milk, fish, and honey. This method has good applicability for CAP, TAP, FF, and FFA in animal-derived foods.