[Confirmation of unknown additives in enrofloxacin powder]

In order to ensure the safety of animal food and regulate the application of veterinary drugs, it is necessary to strictly monitor their content, and to constantly improve the methods used to detect non-specific, illegally added substances in veterinary drugs. A study about the screening, analysis, and confirmation of illegal additives in enrofloxacin powder (used for aquaculture) using non-targeted analysis technology was introduced. First, an enrofloxacin powder test solution under acidic conditions was prepared by adding formic acid, and an enrofloxacin powder test solution under alkaline conditions was prepared by adding sodium carbonate. An ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) was used to assay the test solutions for the presence of unknown additives. Results revealed two high-response unknown peaks in the acidified test solution, with retention times of 1.870 min and 5.122 min respectively. In the alkalized test solution, only one high-response unknown peak was found, with a retention time of 5.122 min. The ultraviolet spectrum characteristic peaks at 5.122 min in acidified and alkalized test solutions were similar, but the peak area in the alkalized test solution was almost ten times that in the acidified solution. Two potential unknown substances were detected. Unknown substance 1 (1.870 min) and unknown substance 2 (5.122 min) may transform under acidic or alkaline conditions. Ultra-performance liquid chromatography-time of flight high resolution mass spectrometry (UPLC-TOF-HRMS) was used to analyze the unknown compounds in more detail. The acidified and alkalized test solutions were detected in the positive and negative ion modes of mass spectrometry, respectively. Accurate mass of the precursor ion, characteristics of secondary ion fragments, and isotopic intensity ratio of the two unknown substances were collected. This information was imported into SCIEX OS software. The molecular formula of the parent ion of unknown substance 2 was found to fit to C₁₁H₈O₂, and its secondary fragment structure may contain a benzene ring and two carbonyl groups, with a propylene structure connected to them through ring formation. From this, unknown substance 2 was presumed to be a menadione. The molecular ion peak of unknown substance 1 was found to fit to C₁₁H₉O₅S^-, only HSO₃^- was collected in the secondary fragments, and the missing part was consistent with unknown substance 2. Considering the most common derivatives of menadione, unknown substance 1 can be proposed to be menadione sodium bisulfite. Finally, we used menadione and menadione sodium bisulfite as reference substances in a comparative study. The same treatment method was used to prepare menadione, menadione sodium bisulfite reference solution, and enrofloxacin powder test solution. After UPLC-PDA detection, unknown substance 1 and menadione sodium bisulfite, unknown substance 2 and menadione, were found to have similar retention times and UV spectra. When the reference solution was added to the enrofloxacin powder test solution, the peak purity of the unknown substance did not change, and were all single peaks. UPLC-TOF-HRMS analysis revealed that the retention time of unknown substance 1 was consistent with that of sodium menadione bisulfite: compared to its accurate mass number in theory, the mass accuracy error was 1.0×10^-6, and the matching degree of fragmentation information in the library was 100%. The retention time of unknown substance 2 was same as the menadione: compared to its accurate mass number in theory, the mass accuracy error was 0.6×10^-6, and the matching degree of fragmentation information in the library was 99.7%. The structures of unknown substances 1 and 2 were confirmed. Menadione sodium bisulfite is known to participate in the synthesis of thrombin in the liver, and also promotes the formation of prothrombin, and accelerates coagulation. The indication of enrofloxacin powder (used for aquaculture) is the treatment of hemorrhage and sepsis in aquaculture animals such as fish and eel. The pharmacological effects of the two drugs correspond to each other, which can cause producers to take risks and add them illegally. With the strict supervision and severe restrictions on the addition of veterinary drugs, illegal additives are becoming more and more subtle. Conventional targeted analysis does not always meet the monitoring requirements. In this paper, the non-targeted analysis of unknown substances using UPLC-PDA combined with UPLC-TOF-HRMS is described in detail. The results may provide a technical reference for screening and identifying illegal additives in drugs, food, health care products, cosmetics, and pesticides.