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Xiuhui Tian Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Dianfeng Han Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Yanmei Cui Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Lihua Ren Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Fang Jiang Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Hui Huang Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Xianghong Gong Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Jinglin Xue Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Jiawei Li Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Huihui Liu Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Yingjiang Xu Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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Xiaojun Luo Guangdong Provincial Key Laboratory of Environmental Protection and Resources Utilization, Guangzhou Institute of Geochemistry, Chinese Academy of Science, Guangzhou, 510640, China

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Xiaojing Liu Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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https://orcid.org/0000-0002-5522-244X
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Xiuzhen Zhang Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai, 264006, China

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https://orcid.org/0000-0003-3972-8077
Open access

Abstract

A sensitive and validated method for determining quinocetone and its main metabolites (3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone) was established in aquatic products using ultra-high-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS). Samples were extracted with 2.0 mol L−1 hydrochloric acid, then purified on MAX columns. After extraction and purification, the supernatant was evaporated to dry nearly under a gentle stream of nitrogen at 40 °C. Formic acid-acetonitrile-water (0.1/30/70, v/v/v) was adjusted to 1.00 mL final volume. An aliquot (10 μL) was injected into the C18 column for separation with the mobile phase of acetonitrile and 0.5% formic acid in water at 0.25 mL min−1. Calibration curves were linear ranged from 10.00 ng mL−1 to 200.0 ng mL−1 for quinocetone and 3-methylquinoxaline-2-carboxylic acid, and 20.00 ng mL−1 to 400.0 ng mL−1 for dedioxoquinenone. Mean recoveries were 70%–89%, 73%–83% and 72%–84%, respectively. The limit of detection (LOD) was 1.00 μg kg−1, 1.00 μg kg−1 and 2.00 μg kg−1, and quantification (LOQ) were 2.00 μg kg−1, 2.00 μg kg−1 and 4.00 μg kg−1 for quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone. Based on the method above, the analytes were determined in Apostichopus japonicus, three fishes (including Ctenopharyngodon idellus, Crucian carp and Oreochromis mossambicus), Penaeus vannamei, Penaeus chinensis, and Chlamys farreri. The method shows good sensitivity, linearity, precision, and accuracy. In short, the proposed method was reliable for the determination of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in aquatic products.

Abstract

A sensitive and validated method for determining quinocetone and its main metabolites (3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone) was established in aquatic products using ultra-high-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS). Samples were extracted with 2.0 mol L−1 hydrochloric acid, then purified on MAX columns. After extraction and purification, the supernatant was evaporated to dry nearly under a gentle stream of nitrogen at 40 °C. Formic acid-acetonitrile-water (0.1/30/70, v/v/v) was adjusted to 1.00 mL final volume. An aliquot (10 μL) was injected into the C18 column for separation with the mobile phase of acetonitrile and 0.5% formic acid in water at 0.25 mL min−1. Calibration curves were linear ranged from 10.00 ng mL−1 to 200.0 ng mL−1 for quinocetone and 3-methylquinoxaline-2-carboxylic acid, and 20.00 ng mL−1 to 400.0 ng mL−1 for dedioxoquinenone. Mean recoveries were 70%–89%, 73%–83% and 72%–84%, respectively. The limit of detection (LOD) was 1.00 μg kg−1, 1.00 μg kg−1 and 2.00 μg kg−1, and quantification (LOQ) were 2.00 μg kg−1, 2.00 μg kg−1 and 4.00 μg kg−1 for quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone. Based on the method above, the analytes were determined in Apostichopus japonicus, three fishes (including Ctenopharyngodon idellus, Crucian carp and Oreochromis mossambicus), Penaeus vannamei, Penaeus chinensis, and Chlamys farreri. The method shows good sensitivity, linearity, precision, and accuracy. In short, the proposed method was reliable for the determination of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in aquatic products.

Introduction

Quinocetone (3-methyl-2-quinoxalinbenzenevinylketo-1,4-dioxide, CAS No.: 81810-66-4, C18Hl4N2O3, Fig. 1A) is one of the quinoxaline 1,4-dioxides family, developed by Lanzhou Institute of Animal Husbandry and Veterinary Drugs, Chinese Academy of Agricultural Sciences (Lanzhou, PR China) [1, 2]. Quinocetone has been reported to obtain better growth enhancing activity and promote animal growth broadly now, including chicken, pig, fish, and goats, as a replacement for olaquindox and carbadox in China since 2003 [3]. It is also used as an antimicrobial agent against Salmonella, Escherichia coli, Brachyspira hyodysenteriae and other Gram-negative bacterial infections [4, 5].

Fig. 1.
Fig. 1.

Schematic formula of quinocetone, 3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2022.01001

Available data have illustrated that quinocetone could induce genotoxic and cytoxic effects in several in vitro. High doses of quinocetone (at least 4-10 folds) can induce pathological (such as liver and renal tissues) or behavioral alterations in rats or mice [6–8]. Quinocetone can induce marked cytoxicity and genotoxicity in African green monkey cell lines (Vero cells) and human hepatoma cells (HepG2) [9, 10]. It was revealed that the viability of HepG2 cells was inhibited significantly by quinocetone in a dose- and time-dependent manner, which was characterized by cell and nuclei morphology change, cell membrane phosphatidylserine translocation, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and a cascade activation of caspase-8, caspase-9 and caspase-3 [11]. Meanwhile, quinocetone has been demonstrated reproductive toxicity and teratogenic potential in Wistar rats [12].

Literature about quinocetone metabolites have been reported in animals, such as rats, pigs, broilers, chickens, and carp [13–17]. Also, 3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone were illustrated as the primary metabolites in aquatic products (CAS No.: 74003-63-7, C10H8N2O2, Figs 1B and C), Apostichopus japonicus as representative and pig [18, 19]. The residues of quinocetone and its metabolites in edible tissues of aquatic animals may endanger human health. Although a withdrawal interval for quinocetone has been established to ensure food safety, it cannot prevent excessive residues caused by off-label use [20]. Therefore, it is necessary to establish a method to make up the residue monitoring methods for quinocetone and its metabolites in aquatic animals.

Recently, many analytical techniques have been used to determine the primary metabolites of quinocetone, such as high-performance liquid chromatography coupled with ultraviolet light (HPLC-UV) or mass spectrometry (HPLC-MS/MS) [21–23]. Samples are concentrated in edible tissues of swine and chickens mainly [24, 25]. However, few methods have been developed to analyze the metabolites in aquatic products. There is no detection method capable of analyzing quinocetone along with their metabolites currently.

The objectives of this research were to (1) confirm the main metabolites of quinocetone in aquatic products, A. japonicus as representative; (2) establish the UHPLC-MS/MS method for determining quinocetone and the main metabolites, including 3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone; (3) evaluate the method established in the aspects of matrix effect, linearity, stability, limit of detection and quantification, accuracy, reproductivity and precision. The UHPLC-MS/MS method was developed and validated in aquatic products, with the solid phase extraction (SPE) column to better recovery. The conditions of pretreatment, chromatography, and spectrometry were optimized, which had the virtues of efficient separation and accurate characterization, addressing the requirement for analysis of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone. The result was satisfactory after optimization of pretreatment and instrumental conditions. The present study has contributed to improving the safety of aquatic products and the residue of the metabolite after disposition of quinocetone.

Experimental

Materials and reagents

Powdered quinocetone was purchased from Anpel Laboratory Technologies Inc. (Shanghai, China), and purity more than 98%. 3-methylquinoxaline-2-carboxylic acid were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and purity more than 98%. Dedioxoquinenone was synthesized by Jinan Weiran Biotechnology Co., Ltd. (Jinan, China), and its purity was more than 98%. Methanol, acetonitrile, ethyl acetate, n-hexane, dichloromethane, and formic acid were liquid chromatography grades obtained from Merck (Darmstadt, Germany). Hydrochloric acid, anhydrous sodium sulfate, and sodium acetate were of guaranteed reagent grade. Ultrapure water was obtained in a Milli-Q system from Millipore (Boston, USA). MAX (mixed anion exchange column), MCX (mixed cation exchange column), and HLB column (hydrophilic-lipophilic balance column) columns (3 cc, 60 mg) were obtained from Waters (Milford, USA).

Preparation of aquatic samples

Samples were obtained from a local supermarket. Sea cucumber were prepared as follows: viscera and gonad removed and the edible part taken. Fish were prepared as follows: scale and skin removed, muscle collected along back, and scale and skin collected separately. Shrimp were prepared as follows: head shell and intestinal gland removed, and muscle taken. Shellfish were cleaned, peeled off, then all soft tissues and body fluids were collected. The sample above was crus, mixed, well-homogenized fully in a blender, and stored at −18 °C. Laboratory samples were analyzed, and those found to contain no analytes were used as negative controls.

To confirm the main metabolites of quinocetone, A. japonicus as representative, quinocetone powder, and commercial fodder were mixed evenly to prepare a feed containing 20 mg kg−1 quinocetone, fed for 24 h. Then the edible tissue was taken and well-homogenized. In the pre-experiment, too much fat and protein were extracted with methanol, time-consuming in the concentration step.

Preparation of the stock and working solutions

Because quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone were soluble in organic solvents but water poorly. The stock solution (100 μg mL−1) was prepared, dissolving 0.0100 g of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in acetonitrile (liquid chromatography grades) using a 100 mL volumetric flask, respectively. Quinocetone and 3-methylquinoxaline-2-carboxylic acid were prepared using 100 μL of each stock standard solution (100 ng mL−1, 200 ng mL−1, 500 ng mL−1, 1,000 ng mL−1 and 2,000 ng mL−1) and 900 μL formic acid-acetonitrile-water (0.1/30/70, v/v/v) to establish the curves. The final concentrations were 10 ng mL−1, 20 ng mL−1, 50 ng mL−1, 100 ng mL−1, and 200 ng mL−1. Dedioxoquinenone were prepared using 100 μL of each stock standard solution (200 ng mL−1, 400 ng mL−1, 1,000 ng mL−1, 2,000 ng mL−1, and 4,000 ng mL−1) and 900 μL formic acid-acetonitrile-water (0.1/30/70, v/v/v) to establish the curves. The final concentrations were 10 ng mL−1, 20 ng mL−1, 50 ng mL−1, 100 ng mL−1, 200 ng mL−1, and 400 ng mL−1. Working solutions were diluted on the day of analysis. Each solution was ultrasonicated over 1.0 min and filtered through a 0.22 μm Millipore cellulose filter (Boston, USA) before injection.

Pretreatment of samples

Samples (5 g ± 0.05 g) were weighed into the 50 mL polypropylene tubes. 15 mL of 2.0 mol L−1 hydrochloric acid was added, and the samples were homogenized for 30 s, then ultrasonicated for one hour at room temperature. The homogenates were centrifuged at 10,000 r/m for 10 min at 4 °C. Then the supernatant was transferred to a clean 50 mL polypropylene tube for purification furtherly.

The solid-phase extraction was applied, including MAX, MCX, and HLB columns. The purification process was developed at 100 μg L−1, 200 μg L−1 and 500 μg L−1 of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone. 1.00 mL of the standard solution was to dryness under a gentle stream of nitrogen in a 40 °C water bath, and dissolved in 1.00 mL 2.0 mol L−1 hydrochloric acid, and passed through the activated MAX, MCX, and HLB columns directly. Then the column was eluted with 6.0 mL formic acid-ethyl acetate (2/98, v/v) and to dryness under a gentle stream of nitrogen in a 40 °C, and the formic acid-acetonitrile-water (0.1/30/70, v/v/v) was added to give 1.00 mL final volume and filtered using a 0.22 μm cellulose filter into an autosampler vial.

SPE process with MAX column can be summarized as follows: (1) activated with 3 mL of methanol, 3 mL of 2.0 mol L−1 hydrochloric acid (both steps at 3.0 mL min−1); (2) loaded at less than 2.0 mL min−1; (3) rinsed with 3 mL of 0.05 mol L−1 sodium acetate methanol solution for three times, and drawn to dryness nearly; (4) eluted with 12 mL dichloromethane and 3 mL formic acid-ethyl acetate (2/98, v/v) at less than 2.0 mL min; (5) the eluate was collected in a 10 mL stoppered glass centrifuge tube, dehydrated over 10.0 g of anhydrous sodium sulfate and to dryness under a gentle stream of nitrogen in a 40 °C water bath; (5) formic acid-acetonitrile-water (0.1/30/70, v/v/v) was added to give a 1.0 mL final volume and filtered using a 0.22 μm cellulose filter into an autosampler vial. An aliquot (10 μL) was injected onto the C18 column for analysis.

UHPLC–MS/MS conditions

UHPLC-MS/MS system comprised an Acquity UHPLC system with a Xevo TQ-S Micro tandem mass spectrometer (Waters, USA). The column performed was ACQUITYTM BEH C18 reversed-phase column (2.1 × 100 mm, 1.7 μm particle size, Waters, USA) maintained at 40 °C. The mobile phase was acetonitrile (A) and 0.5% formic acid in water (B). Table 1 showed the linear gradient programmed after sample injection (10 μL).

Table 1.

Program of gradient elution

Time (min) Flowrate (mL min−1) A% (acetonitrile) B% (0.5% formic acid in water) Entry curve
0.00 0.25 5 95
0.25 0.25 5 95 6
4.00 0.25 95 5 6
6.00 0.25 95 5 6
6.10 0.25 5 95 6
7.00 0.25 5 95 1

The entire eluate was electrosprayed, ionized, and monitored by MS/MS in multiple reaction monitoring (MRM) using positive electrospray ionization. Table 2 showed the parameters optimized to provide the highest sensitivity. The drying gas (N2) flow rate and temperature were 750 L h−1 and 350 °C, respectively. The flow of the cone gas (N2) was 50 L h−1. The collision gas (Ar) flow was 0.35 mL min−1, and the capillary voltage was 2,500 V. The dwell time was set at 100 ms for each transition. The extractor voltage was 4 V and RF lens voltage 0.4 V. The source temperature was 140 °C. LM1, LM2, HM1 and HM2 resolution were 15.0, 13.0, 15.0, and 13.0. The entrance and exit voltages were −1 and 2. Ion energy and multiplier were set at 1.0 and 650. The smoothing method was mean, and window size (scan) was ±1, and the number of smooths was set at 2. The software version was Masslynx V4.2.

Table 2.

The optimum settings for the analysis of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone

Compound name Precursor ion (m/z) Daughter ion (m/z) Cone voltage/V Collision energy/V
Quinocetone 307 103 34 42
143* 34 34
3-methylquinoxaline-2-carboxylic acid 189 143 19 17
145* 19 14
Dedioxoquinenone 275 143 30 26
247* 30 16

*stood for ions of quantification.

Validation procedures

Identification was confirmed by two pairs of MRM fragments from the precursor ions at the defined retention time windows, which was set ±5% concerning that of the closest matching concentration of the standard solution used, and matching of the specific tolerance of the relative abundance of major ions as stated in the Commission Decision 2002/657/EC [26–29]. Linearity based on quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone area as a function of the nominal concentration was assessed by least square regression (1/x 2). Line equations, linearity range, and correlation coefficient were also calculated. Standard calibration and quality control (QC) samples were analyzed in the three consecutive analyses.

Results and discussions

Optimization of the extraction reagent of 3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone

Trichloroacetic acid had little influence on the ion response of positive ions but reduced negative ions significantly, such as chloramphenicol, when detection for other compounds. Acetonitrile could cause denaturation of protein, as preliminary purification, but too much fat extracted at once. According to the structure of the targets and previous literature [18], two reagents, 2.0 mol L−1 HCl and ethyl acetate, were compared. When extracted with 2.0 mol L−1 HCl solution, the bound metabolites, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone, can be released through hydrolysis. The content of 3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone was 2–3 times that of ethyl acetate. Therefore, 2.0 mol L−1 HCl was selected as the extraction reagent. The effects of extraction time (30 min, 60 min, 90 min, and 120 min) were investigated. It was found that if the extraction time was more than 90 min, the efficiency was not increased. Therefore, 90 min was selected for extraction.

Optimization of purification of SPE process

In view of fat, protein, and other interfered aquatic products, liquid-liquid extraction could not achieve the satisfying result in the purification process. The recoveries were calculated with calibration curves of matrix-matched shown in Fig. 2, illustrating that quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dioxoquinenone could achieve better retention in the MAX column, chosen for the concentration purification.

Fig. 2.
Fig. 2.

Comparison of recoveries of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone using different columns of Apostichopus japonicus (n = 3)

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2022.01001

Optimization of the elution and reconstituted solution

MAX column was applied for the concentration and enrichment of acidic compounds generally, and better effect can be achieved if acidic solution for elution. Dichloromethane and formic acid-ethyl acetate (2/98, v/v) were chosen for the eluent. The dichloromethane volume (6 mL, 12 mL, 18 mL, and 24 mL) was discussed, and 12 mL was selected. Meanwhile, the volume of formic acid-ethyl acetate (2/98, v/v) was discussed, and 3 mL was chosen. The results were indicated that the target compound was eluted completely using 12 mL dichloromethane and 3 mL formic acid-ethyl acetate (2/98, v/v). Acetonitrile-water was used for the reconstituted solution, and the proportion was also tested. Formic acid could achieve better ion response and peak shape. Formic acid-acetonitrile-water (0.1/30/70, v/v/v) was used for reconstituted solution for better ion response.

Optimization of MS/MS conditions

Tuning solution (1.0 μg mL−1) was introduced into the electrospray source by direct infusion (20 μL min−1) during the tuning process. Figure 3 showed the mass spectrum infused into the triple quadruple mass spectrometer. The main diagnostic ions produced in MS/MS, 77, 90, 103, 131, and 143 of quinocetone, 77, 92, 102, 143 and 145 of 3-methylquinoxaline-2-carboxylic acid, and 119, 143, 171, 184 and 247 of dioxoquinenone, were identified in positive ionization mode. Two diagnostic ions were selected with the higher response and better ratio of signal to noise, 103 and 143 for quinocetone, 143, and 145 for 3-methylquinoxaline-2-carboxylic acid, 143, and 247 dioxoquinenone, respectively.

Fig. 3.
Fig. 3.

The primary diagnostic ions produced of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dioxoquinenone in MS/MS mode

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2022.01001

All parameters were optimized to increase sensitivity, which fulfilled the recommendations of the European Union concerning identification, since two transitions from the ionized molecule of the targets providing four points in the scale-a value regarded as sufficient for unequivocal identification. Figs 4 and 5 illustrated the possible bond breaking and remapping modes for the main ion fragments of quinocetone (m/z 307) and 3-methylquinoxaline-2-carboxylic acid (m/z 189).

Fig. 4.
Fig. 4.

The possible bond breaking and remapping modes for the ion fragments of quinocetone (m/z 307)

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2022.01001

Fig. 5.
Fig. 5.

The possible bond breaking and remapping modes for the ion fragments of 3-methylquinoxaline-2-carboxylic acid (m/z 189)

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2022.01001

Matrix effect

Matrix effect was evaluated by the peak area dissolved in blank solution with the reconstituted solution. If the ratio was to be <85% or >115%, a standard solution diluted by blank solution was adopted to calculate the concentration. If 85%–115%, the matrix effect was absent. In the experiment, no interfering peak was observed in the aquatic products at the retention time of quinocetone, 3-methylquinoxaline-2-carboxylic acid, or dedioxoquinenone. Meanwhile, the ratio, 0.81–1.06, was shown in Table 3, so the matrix effect could be absent.

Table 3.

Evaluation of matrix effects comparing the ratio of concentration in matrix matched to solvent based standards in Apostichopus japonicus

Concentration Quinocetone 3-methylquinoxaline-2-carboxylic acid Dedioxoquinenone
Ratio value RSD (%) Ratio value RSD (%) Ratio value RSD (%)
10.00 ng mL−1 0.88/0.82/0.87a 3.8 0.86/0.87/0.92a 3.6 0.87/0.96/0.92 4.9a
20.00 ng mL−1 0.81/0.94/0.88 7.4 0.91/0.92/0.86 3.6 0.89/0.93/0.87 3.4
50.00 ng mL−1 0.83/0.92/0.80 7.4 0.88/0.93/0.90 2.8 0.95/1.05/1.03 5.2
100.0 ng mL−1 0.85/0.95/0.81 8.3 0.89/0.93/0.87 3.4 0.89/1.06/0.88 10.7
200.0 ng mL−1 0.87/0.81/0.95 8.0 0.97/0.98/0.92 3.4 0.93/1.02/0.98 4.6

a: Mean of three replications.

Linearity validation

Calibration functions were calculated by plotting the ratio of peak areas on the y-axis and the concentration of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone on the x-axis, which was not forced through the origin. The final concentrations were 10 ng mL−1, 20 ng mL−1, 50 ng mL−1, 100 ng mL−1, and 200 ng mL−1 for quinocetone and 3-methylquinoxaline-2-carboxylic acid; 20 ng mL−1, 40 ng mL−1, 100 ng mL−1, 200 ng mL−1, and 400 ng mL−1 for dedioxoquinenone. A typical linear regression function was accepted only r > 0.99. The calibration curves were performed on the day of analysis suitable for quantification during intra-day and inter-day validation and stability tests. The typical linear regression functions were y = 2312.83x−196.59 (r = 0.9998) for quinocetone, y = 5610.17x−1762.54 (r = 0.9994) for 3-methylquinoxaline-2-carboxylic acid and y = 1122.21x+160.02 (r = 0.9992) for dedioxoquinenone.

Stability

The stability was evaluated by repeated injections of the test solution. The stock solutions were stable at 4 °C for six months. Quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone were stable in the matrix for 5 days at room temperature. Standard solutions containing quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone were stored at 4 °C for one week. It was also found that evaporation to dryness completely at 40 °C could lead to a lower recovery, showing only 30%–65% of quinocetone, 3-methylquinoxaline-2-carboxylic acid, or dedioxoquinenone, so evaporated to dry nearly under a gentle stream after purification could make better recoveries.

Limit of detection and quantification

The LOD of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone (minimum 3:1 signal-to-noise) was determined using blank samples spiked at 50 μL 100.0 ng mL−1 of quinocetone and 3-methylquinoxaline-2-carboxylic acid, or 100 μL 100.0 ng mL−1 of dedioxoquinenone. LOQ, the lowest concentration that could be determined accurately, was also calculated (minimum 10:1 signal-to-noise) using blank samples spiked at 100 μL 100.0 ng mL−1 of quinocetone and 3-methylquinoxaline-2-carboxylic acid, or 200 μL 100.0 ng mL−1 of dedioxoquinenone. in A. japonicus. Based on the detection limit of the instrument, sample mass and diluted volume, the limit of detection (LOD) were 1.00 μg kg−1, 1.00 μg kg−1, and 2.00 μg kg−1, and quantification (LOQ) were 2.00 μg kg−1, 2.00 μg kg−1 and 4.00 μg kg−1 for quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone. satisfying the requirements for the analysis of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone.

Accuracy, reproductivity, and precision

The method validated was applied to determine quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in aquatic products, such as A. japonicus, three fishes (including Ctenopharyngodon idellus, Crucian carp, and Oreochromis mossambicus), Penaeus vannamei, Penaeus chinensis, and Chlamys farreri successfully, without any interference observed. Confirmation was performed by two ion transitions and retention time. Specifically, the relative intensity, the ratio of ion abundance of 143 to 103, 145 to 143, and 247 to 143 for the three analytes, was unique and not changed appreciably over the concentration range and applied for confirmation. Variation of the relative ion intensities within 20% was acceptable for confirmation usually.

Ruggedness can be used for the measurement of the reproductivity in normal experiment conditions. The conditions were: flow rate 0.25 ± 0.02 mL min−1, column temperature 40 ± 5 °C, mobile phase composition 0.5% ± 0.1% formic acid, cone voltage 34 ± 3 V, 19 ± 2 V, and 30 ± 3 V, collision energy 42 ± 4 V, 34 ± 3 V, 17 ± 2 V, 14 ± 2 V, 26 ± 3 V and 16 ± 2 V for quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone. The flow rate and column temperature could influence the retention time, so it should be stable, meanwhile getting a shorter retention time on the premise of satisfactory separation. Formic acid can strengthen ion abundance, but ±0.1% variation had little influence. It was found that cone voltage and collision energy could affect the sensitivity significantly, so it should be optimized according to different instruments and kept constant.

Intra-day accuracy and precision (RSD, relative standard deviation) were assessed from six consecutive analyses of QC (quality control) samples at three different contents of A. japonicus, three fishes (including C. idellus, C. carp and O. mossambicus), P. vannamei, P. chinensis, and C. farreri. Inter-day accuracy and precision were obtained from consecutive analyses of the same batch of QC samples on three separate occasions.

For the aquatic samples above, the mean recoveries were 70%–89%, intra-day precision between 3.6% and 9.2%, and inter-day between 5.0% and 10.3% quinocetone. Mean recoveries were 73%–83%, and intra-day between 5.0% and 8.3%, inter-day between 5.7% and 10.9% for 3-methylquinoxaline-2-carboxylic acid. Mean recoveries were 72%–84%, and intra-day between 6.0% and 10.5%, and inter-day between 7.0% and 10.3% for dedioxoquinenone. All data obtained were acceptable for method validation, indicating efficient and reproducible for analysis of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone, as shown in Table 4 for A. japonicus.

Table 4.

Results for repeatability and within-laboratory reproducibility of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in Apostichopus japonicus

Analyte Spiked level (μg kg−1) Accuracy
Assay 1 repeatability Assay 2 repeatability Assay 3 repeatability Within-laboratory reproducibility
Mean (%) RSD (%)a Mean (%) RSD (%)a Mean (%) RSD (%)a Mean (%) RSD (%)b
Quinocetone 10.0 73 6.2 77 7.0 77 6.4 76 8.2
20.0 75 7.4 80 6.6 82 5.6 79 9.3
40.0 76 8.3 76 6.6 82 7.0 78 8.7
3-methylquinoxaline-2-carboxylic acid 10.0 80 5.0 76 8.2 77 7.2 78 7.2
20.0 78 5.7 74 7.6 81 6.2 78 8.0
40.0 81 7.3 81 7.7 80 6.4 80 8.7
Dedioxoquinenone 20.0 75 6.2 77 6.2 75 7.2 76 9.2
40.0 77 6.6 75 7.2 74 7.6 75 9.0
80.0 80 7.7 76 6.4 72 8.7 76 10.1

a: RSD represented as repeatability(n = 6); b: RSD represented as within-laboratory reproducibility (n = 18).

Application

The method was applied for determining quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in aquatic products successfully. Chromatograms of standard solution, negative control A. japonicus, negative control A. japonicus spiked at 10.0 μg kg−1 for quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone were shown in Fig. 6. The results were calculated as a graph of peak area versus quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone, and the recoveries were 70%, 83%, and 74%.

Fig. 6
Fig. 6

Quantification ions chromatograms of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in Apostichopus japonicus. A-C represent: (A) standard solution of 10.0 ng mL−1; (B) negative control; (C) negative control spiked at 5.00 μg kg−1.

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2022.01001

We also detected quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in A. japonicus, three fishes (including C. idellus, C. carp, O. mossambicus), P. vannamei, P. chinensis, and C. farreri, which were obtained from the local market. Quinenone was not detected in the samples above due to the rapid metabolism. 3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone were detected only in A. japonicus, and the content was 105 μg kg−1 to 870 μg kg−1 of 3-methylquinoxaline-2-carboxylic acid and 152 μg kg−1 to 308 μg kg−1 of dedioxoquinenone, respectively. The contents were much higher than the LOQs of this method, and they might be accumulated and harmful to the health for human body. It was also found that the interfering peaks were observed at the retention time of 3-methylquinoxaline-2-carboxylic acid and dedioxoquinenone transitions, but upon quantification, were so low as to be of little significance.

Comparison of the proposed method with others

The methods, including HPLC-UV and mass spectrometry, for determining quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone, these were limited to tissues, urine, plasma, serum, sewage, and drug so far [21–25]. The target compounds were quinocetone and 3-methylquinoxaline-2-carboxylic acid only. No method has been established for the simultaneous determination of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in A. japonicus, or other aquatic products yet. Part of the difficulties was active substances such as fat, protein and the fatty acid rich in aquatic products, presenting complex matrix, which can interfere with pretreatment and purification. The proposed method shows good response linearity, low LODs, and LOQs relatively, and good precision, providing the first characterization of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in aquatic products comprehensively.

Conclusions

A method allowing identification and quantification of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone have been proposed in aquatic products using UHPLC-MS/MS method. Compared to the traditional LC method, UHPLC has a very short single run time of 7.0 min per sample, which makes it an attractive procedure in the residue analysis. There is no published method for the simultaneous determination of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in aquatic products before. In short, the proposed method for determination of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone is reliable (with good response linearity, high recovery and precision, and low detection and quantification levels) and no interference in the matrix and is successful for the determination of quinocetone, 3-methylquinoxaline-2-carboxylic acid, and dedioxoquinenone in aquatic products.

Statement

The manuscript has not been published elsewhere and that has not been submitted simultaneously for publication elsewhere. The authors declare no competing financial interest.

Acknowledgement

The work was supported by Science and Technology Development Project of Shandong Province of 2012 (Grant No. 2012GHY11517), Guangdong Foundation for Program of Science and Technology Research (Grant No. 2020B1212060053), Science Foundation of Shandong Province (Grant No. ZR2021MD046), Shandong Provincial Modern Agricultural Industry Technology System (Grant No. SDAIT-26-05 and SDAIT-14-08) and Major Applied Agricultural Technology Innovation Projects in Shandong Province (Grant No. SF1805301301).

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  • 1.

    Carta, A. ; Corona, P. ; Loriga, M. Curr. Med. Chem. 2005, 12, 22592272.

  • 2.

    Vieira, M. ; Pinheiro, C. ; Fernandes, R. ; Noronha, J. P. ; Prudêncio, C. Microbiol. Res. 2014, 169, 287293.

  • 3.

    Wang, D. ; Zhong, Y. ; Luo, X. ; Wu, S. ; Xiao, R. ; Bao, W. ; Yang, W. ; Yan, H. ; Yao, P. ; Liu, L. Food Chem. Toxicol. 2011, 49, 477484.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Yang, W. ; Fu, J. ; Xiao, X. ; Yan, H. ; Bao, W. J. Sci. Food Agric. 2012.

  • 5.

    Ihsan, A. ; Wang, X. ; Zhang, W. ; Tu, H. ; Wang, Y. ; Huang, L. ; Iqbal, Z. ; Cheng, G. ; Pan, Y. ; Liu, Z. ; Tan, Z. ; Zhang, Y. ; Yuan, Z. Food Chem. Toxicol. 2013, 59, 207214.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Chen, Q. ; Tang, S. ; Jin, X. ; Zou, J. ; Chen, K. ; Zhang, T. ; Xiao, X. Food Chem. Toxicol. 2009, 47, 328334.

  • 7.

    Jin, X. ; Chen, Q. ; Tang, S. S. ; Zou, J. J. ; Chen, K. P. ; Zhang, T. ; Xiao, X. L. Toxicol. Vitro 2009, 23, 12091214.

  • 8.

    Wang, X. ; Zhang, W. ; Wang, Y. ; Peng, D. ; Ihsan, A. ; Huang, X. ; Huang, L. ; Liu, Z. ; Dai, M. ; Zhou, W. ; Yuan, Z. H. Regul. Toxicol. Pharmacol. 2010, 58, 421427.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Zhang, K. ; Wang, X. ; Wang, C. ; Zheng, H. ; Li, T. ; Xiao, S. ; Wang, M. ; Fei, C. ; Zhang, L. ; Xue, F. Environ. Toxicol. Pharmacol. 2015, 39, 555567.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Dai ; Chongshan ; Zhou ; Yan ; Li ; Daowen ; Zhang ; Shen ; Hui ; Xiao Food Chem. Toxicol. Int. J. Published Br. Ind. Biol. Res. 2016.

  • 11.

    Zhang, C. ; Wang, C. ; Tang, S. ; Sun, Y. ; Zhao, D. ; Zhang, S. ; Deng, S. ; Zhou, Y. ; Xiao, X. Food Chem. Toxicol. 2013, 62, 825838.

  • 12.

    Wang, X. ; Zhang, W. ; Wang, Y. L. ; Ihsan, A. ; Dai, M. H. ; Huang, L. L. ; Chen, D. M. ; Tao, Y. F. ; Peng, D. P. ; Liu, Z. L. ; Yuan, Z. H. Food Chem. Toxicol. 2012, 50, 16001609.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Liu, Z. Y. ; Huang, L. L. ; Chen, D. M. ; Dai, M. H. ; Tao, Y. F. ; Wang, Y. L. ; Yuan, Z. H. Anal. Bioanal. Chem. 2010, 396, 12591271.

  • 14.

    Shen, J. ; Yang, C. ; Wu, C. ; Feng, P. ; Wang, Z. ; Li, Y. ; Li, Y. ; Zhang, S. Rapid Commun. Mass Spectrom. 2010, 24, 375383.

  • 15.

    Wu, H. ; Yang, C. ; Wang, Z. ; Shen, J. ; Zhang, S. ; Feng, P. ; Li, L. ; Cheng, L. Eur. J. Drug Metab. Pharmacokinet. 2012, 37, 141154.

  • 16.

    Zhong, J. L. ; Wang, L. ; Zhao, N. J. South China Agric. Univ. 2012, 33, 248252.

  • 17.

    Li, J. ; Huang, L. ; Wang, X. ; Pan, Y. ; Liu, Z. ; Chen, D. ; Tao, Y. ; Wu, Q. ; Yuan, Z. Food Chem. Toxicol. 2014, 69, 109119.

  • 18.

    Ren, C. B. ; Xue, J. L. ; L, G. ; Tian, X. H. ; Yan, S. ; Liu, H. H. ; Xu, Y. J. ; Ping, G.E. ; Fei, L. ; Gong, X. H. Mod. Food ence Technol. 2015.

    • Search Google Scholar
    • Export Citation
  • 19.

    Wang, X. ; Wan, D. ; Ihsan, A. ; Liu, Q. ; Cheng, G. ; Juan, L. ; Liu, Z. ; Yuan, Z. Food Chem. Toxicol. 2015, 84, 115124.

  • 20.

    Li, J. ; Huang, L. ; Pan, Y. ; Chen, D. ; Wang, X. ; Ahmad, I. ; Tao, Y. ; Liu, Z. ; Yuan, Z. J. Agric. Food Chem. 2014, 62, 1034810356.

  • 21.

    Huang, L. ; Wang, Y. ; Tao, Y. ; Chen, D. ; Yuan, Z. J. Chromatogr. B Analyt Technol. Biomed. Life Sci. 2008, 874, 714.

  • 22.

    Wu, C.-M. ; Li, Y. ; Shen, J.-Z. ; Cheng, L.-L. ; Li, Y.-S. ; Yang, C.-Y. ; Feng, P.-S. ; Zhang, S.-X. Chromatographia 2009, 70, 1605.

  • 23.

    Li, Y. ; Liu, K. ; Beier, R.C. ; Cao, X. ; Shen, J. ; Zhang, S. Food Chem. 2014, 160, 171179.

  • 24.

    Huang, L. L. ; Xiao, A. G. ; Fan, S. X. ; Yin, J. Y. ; Chen, P. J. AOAC Int. 2005, 88, 472478.

  • 25.

    Zhang, J. ; Gao, H. ; Peng, B. ; Li, Y. ; Li, S. ; Zhou, Z. Talanta 2012, 88, 330337.

  • 26.

    Commission, E. Commission Decision 2002/657/EC of 12 August 2002. Official J. Eur. Community 2002, 836.

  • 27.

    Commission, E. , Commission Decision 2002/657/EC of 12 August 2002. 2002.

  • 28.

    Xu, Y. ; Ren, C. ; Han, D. ; Gong, X. ; Zhang, X. ; Huang, H. ; Jiang, F. ; Cui, Y. ; Zheng, W. ; Tian, X. J. Chromatogr. B-Analytical Tech. Biomed. Life Sci. 2019, 1126.

    • Search Google Scholar
    • Export Citation
  • 29.

    Xu, Y. ; Tian, X. ; Ren, C. ; Huang, H. ; Zhang, X. ; Gong, X. ; Liu, H. ; Yu, Z. ; Zhang, L. J. Chromatogr. B Analyt Technol. Biomed. Life Sci. 2012, 899, 1420.

    • Crossref
    • Search Google Scholar
    • Export Citation
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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa (1946-2023)

Editor(s)-in-Chief: Sajewicz, Mieczyslaw, University of Silesia, Katowice, Poland

Editors(s)

  • Danica Agbaba, University of Belgrade, Belgrade, Serbia
  • Łukasz Komsta, Medical University of Lublin, Lublin, Poland
  • Ivana Stanimirova-Daszykowska, University of Silesia, Katowice, Poland
  • Monika Waksmundzka-Hajnos, Medical University of Lublin, Lublin, Poland

Editorial Board

  • Ravi Bhushan, The Indian Institute of Technology, Roorkee, India
  • Jacek Bojarski, Jagiellonian University, Kraków, Poland
  • Bezhan Chankvetadze, State University of Tbilisi, Tbilisi, Georgia
  • Michał Daszykowski, University of Silesia, Katowice, Poland
  • Tadeusz H. Dzido, Medical University of Lublin, Lublin, Poland
  • Attila Felinger, University of Pécs, Pécs, Hungary
  • Kazimierz Glowniak, Medical University of Lublin, Lublin, Poland
  • Bronisław Glód, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland
  • Anna Gumieniczek, Medical University of Lublin, Lublin, Poland
  • Urszula Hubicka, Jagiellonian University, Kraków, Poland
  • Krzysztof Kaczmarski, Rzeszow University of Technology, Rzeszów, Poland
  • Huba Kalász, Semmelweis University, Budapest, Hungary
  • Katarina Karljiković Rajić, University of Belgrade, Belgrade, Serbia
  • Imre Klebovich, Semmelweis University, Budapest, Hungary
  • Angelika Koch, Private Pharmacy, Hamburg, Germany
  • Piotr Kus, Univerity of Silesia, Katowice, Poland
  • Debby Mangelings, Free University of Brussels, Brussels, Belgium
  • Emil Mincsovics, Corvinus University of Budapest, Budapest, Hungary
  • Ágnes M. Móricz, Centre for Agricultural Research, Budapest, Hungary
  • Gertrud Morlock, Giessen University, Giessen, Germany
  • Anna Petruczynik, Medical University of Lublin, Lublin, Poland
  • Robert Skibiński, Medical University of Lublin, Lublin, Poland
  • Bernd Spangenberg, Offenburg University of Applied Sciences, Germany
  • Tomasz Tuzimski, Medical University of Lublin, Lublin, Poland
  • Yvan Vander Heyden, Free University of Brussels, Brussels, Belgium
  • Adam Voelkel, Poznań University of Technology, Poznań, Poland
  • Brata Walczak, University of Silesia, Katowice, Poland
  • Wiesław Wasiak, Adam Mickiewicz University, Poznań, Poland
  • Igor G. Zenkevich, St. Petersburg State University, St. Petersburg, Russian Federation

 

KOWALSKA, TERESA (1946-2023)
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

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Acta Chromatographica
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