Metabolism Profile of Mequindox in Sea Cucumbers In Vivo Using LC-HRMS

In this work, the metabolism behavior of mequindox (MEQ) in sea cucumber in vivo was investigated using LC-HRMS. In total, nine metabolites were detected and identified as well as the precursor in sea cucumber tissues. The metabolic pathways of MEQ in sea cucumber mainly include hydrogenation reduction, deoxidation, carboxylation, deacetylation, and combinations thereof. The most predominant metabolites of MEQ in sea cucumber are 2-iso-BDMEQ and 2-iso-1-DMEQ, with deoxidation and carbonyl reduction as major metabolic pathways. In particular, this work first reported 3-methyl-2-quinoxalinecarboxylic acid (MQCA) as a metabolite of MEQ, and carboxylation is a major metabolic pathway of MEQ in sea cucumber. This work revealed that the metabolism of MEQ in marine animals is different from that in land animals. The metabolism results in this work could facilitate the accurate risk assessment of MEQ in sea cucumber and related marine foods.


Introduction
Quinoxalines, a class of synthetic quinoxaline veterinary drugs with a Quinoxaline 1,4-di-N-oxides structure (QdNOs), were reported with broad-spectrum antimicrobial activity [1]. QdNOs have been applied as food additives in farm animal husbandry. As a major QdNO, Mequindox (3-methyl-2-quinoaxlinacetyl-1,4-dioxide, MEQ) was developed in the 1980s. Due to its high antibacterial activity, MEQ was used as an additive in swine and chicken feed [2]. Recently, QdNOs were reported with potential carcinogenicity and mutagenicity [3,4]. In particular, carbadox (CBX) and olaquindox (OLA), two members of QdNOs, were banned by the European Commission (EC) [5]. Deriving from the same family of QdNOs with a corresponding basic structure, MEQ has attracted increasing attention due to corresponding toxic risk, including induced apoptosis, carcinogenicity, DNA damage, etc. [6,7].
It was reported that MEQ could be easily metabolized after ingestion by animals, and some of the metabolites were detected and their structure identified in previous metabolic investigations in chicken and swine [8,9]. N → O group reduction and carboxylation were the major metabolic pathways in farm animals [10]. In farm animals, the major N → O group reduction metabolites were 1-desoxymequindox (1-DMEQ) and 1,4-bisdesoxymequindox (BDMEQ). Moreover, carboxylation metabolites and hydroxylation metabolites were also detected and identified. However, these metabolic investigations and the metabolites identified were mainly in land farm animal tissues and related products. There are few reports regarding metabolism investigations of QdNOs in aquatic animals. The analytical methods for QdNOs and metabolites detection in aquatic animals and related products were mainly based on metabolic results in land farm animals and the metabolites identified in chicken and swine [11,12]. The potential metabolic enzyme between land farm animals and aquatic animals might lead to different metabolic mechanisms [13,14]. To the best of our knowledge, there have been no related comprehensive metabolic investigations of MEQ in sea cucumber.
As a high-nutrition and high-value marine food, sea cucumbers have been farmed and exploited for commercial use as food and functional food [15,16]. Recently, sea cucumbers have been investigated and processed for different functional foods, and the sea cucumber industry is becoming one of the most important food commodities around the globe for health purposes [17,18]. Bioactive compounds in sea cucumbers were detected and reported with antioxidant, antihypertensive, anti-inflammatory, anticancer, antimicrobial activities [19]. Mequindox has been widely applied and used as a feed additive and antibacterial medicine in the sea cucumber farming industry [20]. Considering the extensive application of sea cucumber in food and health-care food industry, the identification of potentially hazardous compounds in sea cucumber appears to be necessary.
In this metabolism investigation, the major metabolites and metabolic pathways of MEQ in sea cucumber were examined. This work could provide the basis and target compound for further control of sea-cucumber-related food safety.

Optimization of Sample Preparation
In order to obtain more comprehensive metabolites, the first and most important step is target compounds and metabolites extraction. In this work, three schemes were optimized according to the previous literature [21][22][23], with some modification. An experiment group sample and bland control of 2.00 ± 0.02 g were weighed into a 50 mL polypropylene centrifuge tube, respectively. In total, 10 compounds, including MEQ and 9 metabolites, were evaluated in different pretreatment schemes. By comparing the extraction effects of three different schemes, it was obvious that Scheme 1 led to the most optimized results with the highest abundance of all target compounds ( Figure 1). Additionally, Scheme 1 was used for MEQ and metabolites pretreatment of sea cucumber. metabolites were also detected and identified. However, these metabolic investigation and the metabolites identified were mainly in land farm animal tissues and related prod ucts. There are few reports regarding metabolism investigations of QdNOs in aquatic an imals. The analytical methods for QdNOs and metabolites detection in aquatic animal and related products were mainly based on metabolic results in land farm animals and the metabolites identified in chicken and swine [11,12]. The potential metabolic enzym between land farm animals and aquatic animals might lead to different metabolic mecha nisms [13,14]. To the best of our knowledge, there have been no related comprehensiv metabolic investigations of MEQ in sea cucumber.
As a high-nutrition and high-value marine food, sea cucumbers have been farmed and exploited for commercial use as food and functional food [15,16]. Recently, sea cu cumbers have been investigated and processed for different functional foods, and the se cucumber industry is becoming one of the most important food commodities around th globe for health purposes [17,18]. Bioactive compounds in sea cucumbers were detected and reported with antioxidant, antihypertensive, anti-inflammatory, anticancer, antimi crobial activities [19]. Mequindox has been widely applied and used as a feed additiv and antibacterial medicine in the sea cucumber farming industry [20]. Considering th extensive application of sea cucumber in food and health-care food industry, the identifi cation of potentially hazardous compounds in sea cucumber appears to be necessary.
In this metabolism investigation, the major metabolites and metabolic pathways o MEQ in sea cucumber were examined. This work could provide the basis and target com pound for further control of sea-cucumber-related food safety.

Optimization of Sample Preparation
In order to obtain more comprehensive metabolites, the first and most important step is target compounds and metabolites extraction. In this work, three schemes were opti mized according to the previous literature [21][22][23], with some modification. An experi ment group sample and bland control of 2.00 ± 0.02 g were weighed into a 50 mL poly propylene centrifuge tube, respectively. In total, 10 compounds, including MEQ and metabolites, were evaluated in different pretreatment schemes. By comparing the extrac tion effects of three different schemes, it was obvious that Scheme 1 led to the most opti mized results with the highest abundance of all target compounds ( Figure 1). Addition ally, Scheme 1 was used for MEQ and metabolites pretreatment of sea cucumber.

Identification of Metabolites in Sea Cucumber
After administration, all experimental and control group samples were simply pretreated according to Scheme 2 and determined by HRMS. According to the elemental composition of MEQ and the accuracy mass difference value between the metabolites and MEQ, the elemental composition of metabolites could be speculated. In total, nine metabolites were detected and identified in sea cucumber in vivo. The predicted elemental compositions ([M + H] + ), mass errors, retention times (RT), fragmentation ions, and relative percentages of peak area are presented in Table 1. The errors between the measured and predicted masses were within less than 6 ppm, which demonstrated the high mass accuracy of the instrument. The chromatograms of MEQ and metabolites are shown in Figure 2, and the fragmentation ions and potential neutral losses are shown in Figure 3.

Identification of Metabolites in Sea Cucumber
After administration, all experimental and control group samples were simply pretreated according to Scheme 2 and determined by HRMS. According to the elemental composition of MEQ and the accuracy mass difference value between the metabolites and MEQ, the elemental composition of metabolites could be speculated. In total, nine metabolites were detected and identified in sea cucumber in vivo. The predicted elemental compositions ([M+H] + ), mass errors, retention times (RT), fragmentation ions, and relative percentages of peak area are presented in Table 1. The errors between the measured and predicted masses were within less than 6 ppm, which demonstrated the high mass accuracy of the instrument. The chromatograms of MEQ and metabolites are shown in Figure 2, and the fragmentation ions and potential neutral losses are shown in Figure 3.

Metabolic Pathway of MEQ in Sea Cucumber In Vivo
The results show that mequindox can be metabolized in sea cucumber after administration. In total, nine metabolites of MEQ are detected and identified by HRMS in sea cucumber. The metabolic pathways of MEQ in sea cucumber mainly include hydrogenation reduction, deoxidation, carboxylation, deacetylation, and combinations of these metabolic pathways ( Figure 4). Table 1   In the previous literature on MEQ metabolism investigations, the predominant metabolite of MEQ in rat is 1-DMEQ, which takes almost half of total MEQ and metabolites, followed by BDMEQ and 3-hydroxymethyl-1-DMEQ. In chicken, the predominant metabolite is 1-DMEQ, as in rat, followed by BDMEQ, 2-isoethanol-1-DMEQ, and 2′-hydrox- In the previous literature on MEQ metabolism investigations, the predominant metabolite of MEQ in rat is 1-DMEQ, which takes almost half of total MEQ and metabolites, followed by BDMEQ and 3-hydroxymethyl-1-DMEQ. In chicken, the predominant metabolite is 1-DMEQ, as in rat, followed by BDMEQ, 2-isoethanol-1-DMEQ, and 2 -hydroxyacetyl-1-DMEQ. According to a MEQ metabolism study in pig, the predominant metabolite is 2-isoethanol-1-DMEQ. Additionally, 1-DMEQ, 2-isoethanol-MEQ, and BDMEQ are also reported as major metabolites. From the literature, N→O group reduction is the most predominant metabolic pathway of MEQ metabolism in land animals [21]. Different from land animals, in the metabolism investigation of MEQ in sea cucumber, the most predominant metabolic pathways are deoxidation and carbonyl reduction, with M6 (2-isoethanol-BDMEQ) and M4 (2-isoethanol-1-DMEQ) as major metabolites. In particular, carboxylation is also detected as a major metabolic pathway, with MQCA (M7) as one of the metabolites of MEQ. MQCA was reported as a residue marker of olaquindox in land farm animals in previous literature works [24,25]. This work first reported MQCA as one of the metabolites of MEQ in sea cucumber.

Sample Preparation
Live sea cucumbers (150 ± 10 g, 3 years old) were obtained from local aquaculture and raised for 7 days to ensure they were MEQ free. Sea cucumbers were divided into the experimental group and the control group with six replicates each. Sea cucumbers in the experimental group were raised in sea water with 4 mg/L MEQ, while the control group were raised in sea water free of MEQ for 24 h. Afterward, sea cucumbers in the experimental and the control group were collected. Each sea cucumber sample was homogenized with a blender machine for further treatment. For sea cucumber preparation, 2.00 g ± 0.02 g experiment samples and blank control samples were weighed into a 50 mL polypropylene centrifuge tube, respectively. Detailed preparation procedures were optimized with three different schemes based on previous literature works [21][22][23], and each scheme was run in six repetitions. In Scheme 1, each sample was extracted with 5 mL of methanol by vortexing for 5 min and supersonic extraction for 2 min. Each sample was centrifuged at 10,000× g for 15 min at 4 • C in a refrigerated centrifuge, and the supernatant was transferred into another 50 mL polypropylene centrifuge tube. The residues were reextracted with 5 mL of ethyl acetate containing 0.1% formic acid. The supernatant was Antibiotics 2022, 11, 1599 8 of 10 combined after centrifugation and taken to dry under a stream of nitrogen at 45 • C. Then, the residue was re-dissolved with 1 mL of methanol-water (50:50, v/v) and filtered through a 0.22 µm syringe filter into auto-sampler vials for HRMS analysis. In Scheme 2, 20% metaphosphoric acid was added to each sample and settled in a water bath at 55 • C for 30 min for acidolysis and extraction. After centrifugation at 10,000× g for 15 min at 4 • C in a refrigerated centrifuge, the supernatants were purified with an OASIS HLB column. In Scheme 3, 2 mol/L hydrochloride acid was added, and each sample was put in a water bath at 55 • C for 30 min for acidolysis and extraction. After the water bath and centrifugation at 10,000× g for 15 min at 4 • C in a refrigerated centrifuge, the supernatant was purified with an OASIS HLB column. Each sample was filtered through a 0.22 µm syringe filter into auto-sampler vials for HRMS analysis.
The mass spectrum parameters for HRMS were operated in a data-dependent acquisition (DDA) mode with negative heated electrospray ionization (HESI+). The parameters for full mass scan in DDA were settled at a resolution of 70,000 FWHM, scan range of m/z 120-900, automatic gain control (AGC) target of 3.0 × 10 6 , and maximum ion implantation time (Maximum IT) of 64 ms. The ddMS2 parameters in the DDA mode were settled at a resolution of 17,500 FWHM, TOP N = 5, maximum IT 64 ms, isolation window 1.2 m/z, and normalized collisional energy (NCE) at 20, 40, and 60 eV.

Data Processing
In this study, Thermo's in-house software Xcalibur was used to demonstrate the high mass accuracy of the instrument by comparing the measured and predicted masses of targets [26]. Simultaneously, the metabolites could be detected and identified by comparing the precursor, fragment ions, and neutral loss between the experimental and control samples. The mass tolerance window was set to 10.0 ppm. The smoothing setting was enabled. The smoother type Gaussian was selected with 7 smoothing points.

Conclusions
In general, the metabolism behavior of MEQ in sea cucumber in vivo was investigated in the current study. The result shows that, in total, nine metabolites were detected and identified, as well as the precursor. Different from the metabolism result in land farm animals, the most predominant metabolites of MEQ in sea cucumbers were 2-iso-BDMEQ and 2-iso-1-DMEQ, with deoxidation and carbonyl reduction as major metabolic pathways in sea cucumber. In particular, this work first reported MQCA as a metabolite of MEQ, and carboxylation is a major metabolic pathway of MEQ in sea cucumbers. This work reveals that the metabolism of MEQ in marine animals is different from that in land animals. The metabolism results in this work could facilitate the accurate risk assessment of MEQ in sea cucumber and related marine foods.  Institutional Review Board Statement: The study was conducted according to the guidelines of the technical specification for ethical review of laboratory animal welfare and approved by the Ethics Committee) of Beijing Municipal Commission of Science and Technology (protocol code DB11/T 1734-2020 and date of approval in 2020).