Simultaneous Determination of Amphenicols and Metabolites in Animal-Derived Foods Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry

Amphenicols are widely used to prevent and treat animal diseases. However, amphenicol residues accumulate in livestock and poultry and harm consumers. We hypothesized that one can combine solid-phase extraction (SPE) with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to simultaneously determine amphenicols and metabolites in pork, beef, lamb, chicken, and their products and meet government regulations for maximum residue limits. We extracted crude samples with ethyl acetate and ammonia water (98:2, v/v), purified the samples with a CNW Si SPE column, defatted the samples with acetonitrile-saturated n-hexane, and then determined the resulting analytes by UHPLC-MS/MS. The limit of detection of the analytes in livestock and poultry meat was 0.03–1.50 μg/kg, and the limit of quantification was 0.05–5.00 μg/kg. Measured chloramphenicol, thiamphenicol, and florfenicol concentrations were linear over the range 0.50–50 μg/kg; and the florfenicol amine concentration was linear over the range 5.00–200 μg/kg (all with correlation coefficients >0.9990). The recovery of the spiked samples was between 72% and 120%. The intraday relative standard deviation (RSD) ranged from 1% to 9%, and the interday RSD ranged from 1% to 12%. Based on the above results, the current method is sensitive, accurate, and reproducible with the detection limits being well below the maximum residue limits as per Chinese standard GB 31650-2019, and thus, our research hypothesis could be confirmed.


Introduction
China produces and consumes a large quantity of livestock and poultry meat; corresponding quality and safety problems harm consumer health and create barriers to the import and export trade [1]. Among these quality and safety problems, veterinary drug residues are especially pertinent. Chloramphenicol (CAP), thiamphenicol (TAP), and florfenicol (FF) are amphenicols, substances that are widely used in livestock and poultry breeding and production. ese substances have broad-spectrum antibacterial properties, are low cost, and have high potency [2][3][4]. However, CAP is highly toxic to the human hematopoietic system and can cause side effects such as aplastic anemia [5,6]. As a derivative of CAP, TAP replaces the para-nitro group of CAP with a methanesulfonyl group and is less toxic than CAP but can inhibit the production of red blood cells, white blood cells, and platelets [7,8]. FF is synthesised from TAP by replacing the 3-hydroxy group with fluorine; it is an animalspecific amphenicol and has higher antibacterial activity than CAP and TAP but has embryo toxicity [9,10]. FF is metabolized to florfenicol amine (FFA) via bioconversion pathways in treated animals ( Figure 1) [11]. erefore, to regulate the use of toxic amphenicols and reduce their prevalence in livestock and poultry products, the use of amphenicols is strictly restricted by laws and regulations.
Currently, China, the United States, the European Union, Japan, and other countries have banned the use of CAP in animal feeds. As shown in Table 1, regulations in China, the United States (US), and the European Union (EU) have stipulated that the maximum residue limits (MRLs) of TAP in animal-derived foods such as the muscle, fat, liver, kidney, and milk are 50 μg/kg; and the MRLs of FF (calculated as the sum of both FF and FFA) in the muscle, liver, kidney, skin, and fat are 100-3700 μg/kg [12][13][14][15][16][17]. TAP is not recommended for animals from which eggs are produced for human consumption, and FF is not recommended for animals from which milk and eggs are produced for human consumption in China and the EU Various approaches for the determination of amphenicols in animal-derived foods are available in the literature and apply detection systems such as biosensors [18][19][20][21], enzyme-linked immunosorbent assays [22,23], high-performance liquid chromatography (HPLC) [24][25][26], liquid chromatography-tandem mass spectrometry (LC-MS/MS) [27][28][29][30], gas chromatography (GC) [31], and GC-MS/MS [32,33]. Among them, LC-MS/MS is the main detection and positive confirmation method for amphenicols. Ultrahighperformance liquid chromatography (UHPLC)-MS/MS is an LC-MS/MS technology that is more suitable than conventional LC-MS/MS for the analysis and detection of amphenicols because of a short analysis time, higher separation efficiency, and higher sensitivity. Currently, several analytical methods have been reported for the analysis of some of amphenicols and metabolites [28,30,34,35]. In addition, these methods are usually developed for one or two matrices [7,36,37]. To our knowledge, there are few reports on simultaneously detecting the entire family of amphenicols and metabolites in animal-derived foods. In this study, we combined solid-phase extraction (SPE) with UHPLC-MS/MS in both negative and positive electrospray ionization (ESI) mode for simultaneous determination of CAP, TAP, FF, and FFA in pork, beef, lamb, chicken, and their products under the optimal experimental conditions. Our goal is to promote the development of international trade in animalderived foods, provide references for quality and safety monitoring, and standardize supervision and testing methods of animal-derived foods.

Preparation of Standard Stock and Working Solutions.
Standard stock solutions of CAP, TAP, FF, FFA, CAP-D5, and FFA-D3 were prepared at a concentration of 1000 μg/ mL by dissolving each analyte in methanol. All the aforementioned standard stock solutions were stored at −20°C for up to 6 mo. Mixed standard working solutions of CAP, TAP, FF, and FFA at a concentration of 10 μg/mL were prepared by diluting the standard stock solutions with methanol. An internal standard working solution at a concentration of 1.0 μg/mL was prepared by diluting the internal standard stock solutions with methanol. e aforementioned standard working solutions were stored at −20°C for up to 3 mo.

UHPLC-MS/MS Instrumentation and Operating Conditions.
A ermo Scientific Vanquish ultrahigh-performance liquid chromatograph coupled to a ermo Scientific TSQ Quantis mass spectrometer ( ermo Fisher Scientific, Waltham, MA, USA) was used. Separation was performed with a Waters Acquity UPLC high-strength silica (HSS) C18 column (50 mm × 2.1 mm, 1.8 μm; Waters, Milford, MA, USA), with the front end connected to a Waters Acquity UPLC HSS C18 VanGuard precolumn (5 mm × 2.1 mm, 1.8 μm; Waters, Milford, MA, USA). e column temperature was 40°C, and the sample injection volume was 2.0 μL. Gradient elution was performed with water and ACN as the mobile phases, at a flow rate of 0.3 mL/ min: 0-4 min, 4% (v/v) ACN increased to 96% (v/v); 4-6 min, 96% (v/v) ACN; and 6-10 min, 96% (v/v) ACN decreased to 4% (v/v). e MS/MS was equipped with an ESI source operating in the positive ionization (PI) mode, negative ionization (NI) mode, and selected reaction monitoring mode. CAP, TAP, FF, and CAP-D5 were analyzed in the NI mode, whereas FFA and FFA-D3 were analyzed in the PI mode. e ESI source was operated with the following capillary voltage: 3.5 kV in PI mode and 2.5 kV in NI mode; sheath gas: 50 Arb; auxiliary gas: 10 Arb; ion transfer tube temperature: 325°C; and vaporizer temperature: 350°C.

Sample Preparation.
Blank samples used in the validation procedure were obtained from the Supervision, Inspection, and Testing Center for Quality of Meat Products (Nanjing, China). Pork, beef, lamb, and chicken muscle were chopped and homogenized by an HM6300 intelligent homogenizer (Lab Precision Beijing Technology Co., LTD, Beijing, China) at 10,000 r/min, and aliquots of 5.0 g were weighed in a 50-mL polypropylene centrifuge tube. Samples were spiked with 10 μL of the internal standard working solution, corresponding to a concentration of 2 μg/kg of CAP-D5 and FFA-D3 in tissue. en 15 mL of ethyl acetate with 2% (v/v) ammonia were added. e mixture was thoroughly homogenized using an IKA Vortex 3 (IKA, Staufen, Germany). After mixing, the homogenate was centrifuged at 10,621 g for 5 min at 4°C, and the supernatant was transferred into a 50-mL polypropylene tube. e extraction step was repeated by the addition of 15 mL of ethyl acetate with 2% (v/v) ammonia, shaken, and centrifuged again under the same conditions. e resulted supernatant was merged with the previous extract in the 50 mL polypropylene tube, and the extracts were evaporated to dryness at 40°C for 2.5 h under a nitrogen stream with an N-EVAP-11 nitrogen evaporator (Organomation, Berlin, MA, USA). e residual solution was dissolved in acetone/n-hexane (1:9, v/v; 5 mL) and loaded onto a CNW Si SPE (200 mg) cartridge, which was preconditioned with water (5 mL) and acetone/n-hexane (1:9, v/v; 5 mL). e cartridge was eluted with acetone/n-hexane (6:4, v/v; 5 mL), and then the eluate was evaporated to dryness at 40°C for 10 min under a nitrogen stream. e residual solution was reconstituted by adding 1.0 mL of ACN-water (50:50; v/v) and then vortexed for 1 min. Next, 5 mL of hexane saturated with pure ACN was added to the tube, and the mixture was vortexed for 30 s. After centrifugation for 1 min at 10,621 g, the hexane layer was discarded. is defatting step was repeated twice. Finally, an aliquot of 1 mL of the bottom layer was poured through a 0.22 μm filter into an autosampler vial, and then 2 μL of the liquid in the aforementioned vial was analyzed using UHPLC-MS/MS. e standard curves were constructed using the mass concentration of amphenicols (μg/kg) as the abscissa (x) and the analyte/internal standard peak area ratio versus as the ordinate (y), and the squares of the correlation coefficients (R 2 ) were calculated.

Method Validation
Negative livestock and poultry meat were added to the internal standard working solution and the mixed standard working solution and then detected with reference to the method described above. e limit of detection (LOD) was the minimum added concentration when the signalto-noise ratio (S/N) ≥3, and the limit of quantification (LOQ) was the minimum added concentration when S/ N ≥ 10 [38].

Recovery and Precision
Test. Solutions were spiked into livestock and poultry meat as follows: standard working solutions of CAP, TAP, and FF at three concentration levels (1, 5, and 10 μg/kg) and the standard working solution of FFA at three concentration levels (5, 10, and 50 μg/kg). Pretreatment was performed in accordance with the method described above, and each concentration was set in six parallels. e recovery was calculated as follows: where C E is the experimental concentration determined from the calibration curve and C M is the spiked concentration. e precision was determined by calculating the relative standard deviation (RSD). Samples with three levels of spiked concentrations were measured in the same day and in six consecutive days, and then the intraday RSD and the interday RSD were calculated. e RSD was calculated as follows: where SD is the standard deviation and C A is the average of the experimental concentration determined from the calibration curve.

Optimization of UHPLC-MS/MS
Conditions. e use of formic acid improved the response and peak shape corresponding to FFA but generated ion suppression of CAP, TAP, and FF-which we monitored in the NI mode [28].
us, to better separate amphenicols and metabolite residues, we used ACN-0.1 v/v% formic acid, ACN-0.2 v/v% formic acid, and ACN-0.3 v/v% formic acid as the mobile phase for UHPLC-MS/MS analyses in this experiment. e separation effect of the four target compounds was excellent when ACN-water was a mobile phase ( Figure 2). e FFA peak became bimodal after adding formic acid to the mobile phase. erefore, we selected ACN-water as a mobile phase to separate the four analytes.
We directly injected 1 μg/mL of CAP, TAP, FF, FFA, CAP-D5, and FFA-D into the mass spectrometer at a flow rate of 20 μL/min, and the instrument scanned in Full Scan Q1 simultaneously in ESI (+) and ESI (−) to determine the precursor ion of each compound. en the instrument scanned in SIM Q3. For each compound, we optimized two pairs of characteristic ion pairs with the highest response as qualitative and quantitative ion pairs. We also optimized the declustering voltage and collision energy of each ion pair. In doing so, we obtained corresponding mass spectrometry parameters ( Table 2).

Optimization of Sample Preparation.
e extraction solvent was ACN, which showed satisfactory recoveries for CAP. However, when the other analytes were introduced in the method, it was necessary to choose the extraction solvent to provide better recoveries rates for all analytes, not just for CAP. e solvent of election was ethyl acetate, which can promote the extraction of all analytes. Moreover, the use of ammonium hydroxide as an additive can promote an alkaline environment, which is favorable for the extraction of FFA. Taking into account the extraction efficiency of all the amphenicols and metabolites, it was necessary to choose an extraction solvent that was able to provide better recoveries for all the analytes. erefore, we chose the following extraction methods for comparison: After the extraction by method A, the recovery of amphenicols and metabolites in the four livestock and poultry meat products was all in the range 60% to 120% (Table 3). erefore, we chose method A as the optimal extraction method.
Because livestock and poultry meat contain a large quantity of fat and protein and are complex matrices, the samples require further purification after extraction. erefore, we compared the purification effects of the following columns: HyperSep Retain PEP (60 mg/3 mL; ermo Fisher Scientific, Waltham, MA, USA), Oasis HLB (200 mg/6 mL; Waters, Milford, MA, USA), and CNW Si SPE (60 mg/3 mL; ANPEL Experimental Technology Co., Shanghai, China). After purification with the CNW Si SPE column, the recovery of amphenicols and metabolites in the four livestock and poultry meat products was in the range from 60% to 120% (Table 4). erefore, we chose the CNW Si SPE column as the optimal purification method.   International Journal of Analytical Chemistry 0.03-1.50 μg/kg and 0.11-5.0 μg/kg in four types of livestock and poultry meat (Table 5). Compared with other literature reports [27,28,38], this method is fast and has high sensitivity and meets the requirements of the National Food Safety Standard on Maximum Residue Limits for Veterinary Drugs in Foods (GB 31650-2019) in China.

Recovery and Precision of Amphenicols and Metabolites.
To verify the recovery and precision of this method, we spiked blank samples at three concentrations within the ranges 1.0-10.0 μg/kg for CAP, TAP, and FF and 5.0-50.0 μg/kg for FFA (n � 6 at each level). Table 6 summarizes the results of the recovery and precision experiments. e average recovery of the four analytes in livestock and poultry meat samples was 71.77% to 120.30%, with an intraday RSD <9.22% and interday RSD <12.30%. us, our method has high recovery and good precision and can achieve simultaneous detection of amphenicols and metabolites in livestock and poultry meat.

Real Sample Analyses.
We selected 28 commercial livestock meat, poultry meat, and their products to verify practical applications of our method in real food matrices with possible interferences. We used one positive meat sample as the quality control sample. We detected 1.38 μg/kg of the banned veterinary drug CAP in fresh chicken, 0.35 μg/ kg of FF in pork sausage, and 0.54 μg/kg of FF in fried pork jerky (Qingdao, Shandong), yet none of the samples exceeded the MRL of 300 μg/kg for porcine muscle (GB 31650-2019; Table 7).    International Journal of Analytical Chemistry 7

Conclusions
Amphenicols are widely used in livestock and poultry breeding and production, but illegal or noncompliant use may occur. is causes amphenicol residues to accumulate in the body of livestock and poultry animals. Such accumulation is a threat to livestock and poultry food safety, as well as human health. erefore, we developed a detection method for amphenicols and metabolites in livestock meat, poultry meat, and their products. By optimizing MS, LC, and sample pretreatment conditions, we established a UHPLC-MS/MS detection method that can simultaneously detect amphenicols and metabolites in livestock meat, poultry meat, and their products. e peak time of the analytes was within 5 min; the LOD was 0.03-1.50 μg/kg; the LOQ was 0.05-5.00 μg/kg; the recovery of spiked samples at low, medium, and high concentrations was between 71.77% and 120.30%; the intraday RSD was <9.22%, and the interday RSD was <12.30%. us, this method has a short analysis time, a high sensitivity, and a good repeatability. We have

Data Availability
e original data used to support the findings of this study can be obtained from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this study.