*Article* **Improved LC/MS/MS Quantification Using Dual Deuterated Isomers as the Surrogates: A Case Analysis of Enrofloxacin Residue in Aquatic Products**

**Yunyu Tang 1,†, Guangxin Yang 1,†, Essy Kouadio Fodjo 2, Shouying Wang 3, Wenlei Zhai 4, Wenshuai Si 3, Lian Xia <sup>5</sup> and Cong Kong 1,\***


**Abstract:** Extensive and high residue variations in enrofloxacin (ENR) exist in different aquatic products. A novel quantitative method for measuring ENR using high-performance liquid chromatography– tandem mass spectrometry was developed employing enrofloxacin-*d5* (ENR-*d5*) and enrofloxacin-*d3* (ENR-*d3*) as isotope surrogates. This reduced the deviation of detected values, which results from the overpass of the linear range and/or the large difference in the residue between the isotope standard and ENR, from the actual content. Furthermore, high residue levels of ENR can be directly diluted and re-calibrated by the corresponding curve with the addition of high levels of another internal surrogate without repeated sample preparation, avoiding the overflow of the instrument response. The validation results demonstrated that the method can simultaneously determine ENR residues from MQL (2 μg/kg) to 5000 × MQL (method quantification limit) with recoveries between 97.1 and 106%, and intra-precision of no more than 2.14%. This method realized a wide linear calibration range with dual deuterated isomers, which has not been previously reported in the literature. The developed method was successfully applied to the analysis of ENR in different aquatic products, with ENR residue levels varying from 108 to 4340 μg/kg and an interval of precision in the range of 0.175~6.72%. These results demonstrate that batch samples with a high variation in ENR residues (over the linear range with a single isotope standard) can be detected by the dual isotope surrogates method in a single sample preparation process.

**Keywords:** enrofloxacin; dual isotope surrogates; enrofloxacin-*d3*; contaminant detection; accurate quantification; aquatic animals
