**1. Introduction**

Enrofloxacin (ENR) is a broad-spectrum antibiotic found in animals as a secondgeneration fluorinated quinolone. Because of its efficaciousness against common bacterial pathogens, ENR is applied in treating and preventing various bacterial diseases [1], including furunculosis, vibriosis, and bacterial kidney diseases in aquaculture [2–4]. At present, ENR is licensed for use at levels below maximum residue limits (MRL), set at 100 μg/kg for ENR and ciprofloxacin (CIP) in fish farming in China, the European Union, and Vietnam [5]. In the United States, no fluorinated quinolone has been approved for

**Citation:** Tang, Y.; Yang, G.; Fodjo, E.K.; Wang, S.; Zhai, W.; Si, W.; Xia, L.; Kong, C. Improved LC/MS/MS Quantification Using Dual Deuterated Isomers as the Surrogates: A Case Analysis of Enrofloxacin Residue in Aquatic Products. *Foods* **2023**, *12*, 224. https://doi.org/10.3390/ foods12010224

Academic Editors: Dapeng Peng and Yongzhong Qian

Received: 31 October 2022 Revised: 23 December 2022 Accepted: 26 December 2022 Published: 3 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

use in food-producing animals (except in poultry) since 1997 [6]. Despite this constraint, ENR is extensively applied beyond the set limit value to control diseases in aquaculture, cattle, pigs, and poultry farms [7]. Recent reports have shown high detection rates of 11.4% and 50.4% of ENR residues in aquatic products in South Korea [8] and China [9], respectively. Residues range between N.D. and 785 μg/kg. This excessive use of ENR is the result of over-exploited domestic fisheries, with intensive and high-density culture being adopted to obtain high yields and profits. These activities lead to high antibiotic residues in treated aquatic animals, aquaculture-related sediments and soils, and natural water environments [10–12]. This, in turn, leads to potential exposure of human health to risks induced by the ultimate accumulation of ENR in humans and the alarmingly high issues related to antibiotic bacterial resistance [13]. Growing concerns about quality and safety necessitate the monitoring of ENR residues in aquatic products for the safety of human consumption.

The methods for quantitative determination of ENR in aquaculture have been extensively reported. Rapid detection with competitive indirect enzyme-linked immunosorbent assay (ELISA) was performed in field tests due to its easy operation and rapid analysis [14,15]. Furthermore, several studies reported the quantitation of ENR using high-performance liquid chromatography (HPLC) with fluorescence detectors [16,17]. On the other hand, high-performance liquid chromatography–tandem mass spectrometry (HPLC−MS/MS) was also extensively used for the determination of ENR in recent years [18,19]. Because of its high specificity and sensitivity, LC–MS/MS attracted much attention in various fields when applied to the analysis of antibiotics [20–23]. Generally, the pretreatment of ENR includes solvent extraction, followed by purification with liquid–liquid extraction or solid-phase extraction, concentration, and redissolution before analysis using LC–MS/MS assays. In most cases, the purification and concentration processes can cause the loss of the target analyte, resulting in decreased sensitivity in the detection method.

In order to solve this issue, and to obtain more accurate results, an isotope surrogate was introduced as the control of stochastic and/or systematic variation in analyte extraction and analysis [24,25]. Furthermore, the inaccuracy resulting from matrix effects and sample preparation can be effectively compensated by the addition of a fixed amount of isotope surrogate to each sample at the beginning of the process [26]. Therefore, ENR was extensively estimated using the isotope standard method with HPLC–MS/MS in most cases to obtain an accurate measurement. In previous reports, samples containing ENR with concentrations significantly above MRL, such as 785 μg/kg in loach [8], 148.4 μg/kg in carp [27], and 2200 μg/kg in grass carp [28], were found. These high residue values can be attributed to the overapplication of this antibiotic. However, the MS detector tends to produce unstable and imprecise results with the regular detection method, which might be caused by the range of the calibration curve, a high discrepancy between the analyte and the isotope surrogate, and/or the saturated response on the instrument [29]. According to our experience, the linear response range of ENR in the MS detector was not over 500 ng/mL in an actual sample test (Figure 1), as can be found in the recent literature [30,31]. Obviously, the response values of ENR at concentrations of more than 500 ng/mL do not follow the linear calibration curve and tend to be saturated (Figure 1).

Thus, ENR determination requies a repeated sample preparation to obtain more accurate results. It is noteworthy that the dilution of the original sample can reduce the response of MS to resolve the difficulty in MS saturation of the instrument, but the measured value of the diluted sample is still beyond the range of the calibration curve using the quantitative method with the isotope standard [32]. Moreover, the dilution of injection samples cannot resolve the enormous discrepancy in concentration between the analyte and the isotope surrogate to obtain stable and precise results. Therefore, the samples need to be further analyzed by changing the amount of added isotope surrogate and establishing a new procedure, as follows: (i) a new calibration curve is prepared to match the sample concentration; (ii) a repeated sample preparation should be performed with a reasonable amount of added isotope surrogate [33]. However, the high amount of ENR may saturate the MS system detector, resulting in no suitable calibration curve. Consequently, the sample requires laborious re-preparation to resolve the linearity range of the calibration curve, saturation of the MS detector, and the vast discrepancy in concentration between the analyte and the isotope surrogate; this, therefore, increases the time spent on analysis and generates more hazards for the environment. Moreover, the repeatability and reproducibility of the determined results could be poor if a lower amount of isotope surrogate is used than the target ENR in the final solution to be analyzed [34]. This could occur even though the detection values fall within the range of the new calibration curve and the linear response range of the instrument. Therefore, it is necessary to develop a simple, fast, and reliable quantitative method for the analysis of ENR residue levels, which vary from low to high amount in aquatic animals. The use of two different levels of isotope surrogates can provide two ranges of calibration curves, which effectively extends the upper limit of the curve and leads to accurately quantitate the analyte at low and high concentrations, respectively. Moreover, the high addition level of the isotope surrogate can be diluted directly with solutions to adapt the instrument's response.

**Figure 1.** Linear range profile of ENR in the MS detector.

This detection strategy for use with an HPLC–MS instrument has not been proposed and validated in the previous literature. In this study, therefore, a new isotope standard, ENR-*d3*, for ENR was synthesized and characterized by 1HNMR and MS. We firstly established and validated a novel quantitative method for ENR in aquatic animals using two isotope surrogates with HPLC–MS/MS, achieving a wide range in the calibration curve, single-time sample preparation, direct dilution for the instrument limit, and accurate quantification from low to high residue levels. Moreover, the method involves easy sample preparation, high sensitivity, and a low amount of reagent. Finally, the new method was applied to the determination of ENR in actual positive samples for various aquatic species.
