This study developed an ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection of three major metabolites of mequindox, including 3-methyl-quinoxaline-2-carboxylic acid, 1-desoxymequindox, and 1,4-bisdesoxymequindox (MQCA, 1-DMEQ, and BDMEQ), in holothurian. Target analytes were simplified with ultrasound-assisted acidolysis extracted without complicated enzymolysis steps. After that, each sample was centrifuged and purified by an Oasis MAX cartridge. Then, the processed samples were separated and monitored by UPLC-MS/MS. This developed method has been validated according to FDA criteria. At fortified levels of 2, 10, and 20
Mequindox (MEQ, 3-methyl-2-quinoxaline acetyl-1,4-dioxide) is a newly synthetic quinoxaline-1,4-dioxides (QdNOs) antibiotic. The QdNOs have been widely applied as growth-promoting agents in animal husbandry in last decades [
Many metabolites of MEQ were detected, and structure was identified in previous metabolic investigations [
For the control of these chemical compounds in foods, there were many literatures reported for the detection of QdNOs and the metabolites in the previous studies, such as high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detector [
However, these current methods mainly focused on the land animal-derived foods. To the best of our knowledge, there were no related methods for the detection of MEQ and metabolites in aquatic animal-derived foods, especially in holothurian. In this work, for the control of MEQ residues in holothurian, we presented a novel, sensitive, and rapid UPLC-MS/MS (ultraperformance liquid chromatography coupled with triple-quadrupole mass spectrometry) protocol for simultaneous detection of the three major metabolites of MEQ (MQCA, 1-DMEQ, and BDMEQ structures are shown in Figure
Chemical structures of 1-DMEQ, BDMEQ, MEQ, and MQCA.
MQCA (98.0%) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). 1-desoxymequindox (1-DMEQ, 98.0%) and bisdesoxymequindox (BDMEQ, 98.0%) were synthesized by Green Bayer Biotechnology Company (Wuhan, China).
HPLC grade acetonitrile was purchased from Dima Technology Inc. (Muskegon, MI, USA). HPLC grade formic acid was obtained from Fisher Scientific Inc. (Pittsburgh, PA, USA). The Milli-Q synthesis system was adopted for the production of deionized water (Millipore, MA, USA). Oasis MAX SPE cartridges (3 cc, 60 mg) were purchased from Agilent Technologies (Santa Rosa, CA, USA). Other reagents and chemicals used in this work were of analytical grade which were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).
Stock standard solutions of MQCA (1.0 mg/mL), 1-DMEQ (1.0 mg/mL), and BDMEQ (1.0 mg/mL) were prepared by dissolving 5 mg of each standard in 5 mL of methanol. These stock standard solutions were stored at −20°C in brown amber bottles until use, and they were stable for at least 3 months. Working mixed standard solutions were prepared by diluting the stock standard solutions with methanol. These working solutions were stored at −20°C in the brown amber bottle and stable for at most 1 week.
A model HQ-60 vortex mixer was purchased from North TZ-Biotech Development Co. (Beijing, China). Nitrogen evaporation equipped with an Evap 111 evaporator was purchased from Organomation Associates Inc. (Berlin, MA, USA). Tissue homogenizer was purchased from MeiDi (Foshan, China). Syringe filters (0.22
The mixed targets of the three metabolites were were separated via an Acquity BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size) on a Waters AcquityTM UPLC system (Waters, Milford, MA, USA). The column oven temperature was maintained at 40°C. The mobile phase was composed of solvent A (acetonitrile containing 0.5% formic acid) and solvent B (water containing 0.5% formic acid). Gradient elution was adopted in this research, and the program was performed as follows: 0–0.25 min, 5% A; 0.25–7.75 min, 5–95% A; 7.75–8.50 min, maintain 95% A; 8.50–8.51 min, 95–5% A; and 8.51–10.0 min, maintain 5% A. The flow rate was settled at 0.25 mL/min with an injection volume of 10
The Mass Quattro Premier XE triple quadrupole mass spectrometer (Waters, Manchester, UK) was fitted to UPLC system with an electrospray ionization (ESI) source. Typical source conditions were optimized for maximum intensity as follows: desolvation gas at 700 L/h with the temperature of 350°C, capillary voltage at 3.0 kV, source temperature at 80°C, cone gas flow rate at 50 L·h−1, and collision at 0.11 mL/min. For all compounds, MS instrument was operated in ESI positive (ESI+) multiple reaction monitoring (MRM) mode. Optimized MS/MS parameters are summarized in Table
MS/MS parameters for MQCA, 1-DMEQ, and BDMEQ in the positive electrospray ionization mode.
Compound | Parent ion ( |
Daughter ion ( |
Cone voltage (V) | Collision energy (eV) |
---|---|---|---|---|
1-DMEQ | 203 | 143 |
30 | 25 |
185 | 17 | |||
BDMEQ | 187.1 | 145.1 | 33 | 25 |
159.15 |
20 | |||
MQCA | 189 | 92 | 19 | 24 |
143.1 |
17 |
Blank holothurian samples were obtained from the breeding base of Shandong Marine Resource and Environment Research Institute (Yantai, Shandong, China) which were previously characterized using UPLC-MS/MS. Samples were homogenized by a domestic food blender and then stored at −20°C till use.
An amount of 5.00 g (±0.025 g) of holothurian samples was weighed and transferred into a 50 mL polypropylene centrifuge tube. Samples were divided into four groups. Three fortified groups were prepared by adding mixed working standard solution to yield final concentrations of 2, 10, and 20
Oasis MAX SPE cartridges (3 cc, 60 mg, Waters, USA), which were mainly consisted of anionite, were adopted for the purification process. The cartridges were conditioned with 3 mL of methanol and 3 mL of hydrochloric acid (2 mol/L). Then, the supernatant was loaded onto the prepared cartridge by gravity. Each was rinsed with 3 mL of sodium acetate-methanol solution (0.05 mol/L, 6.12 g of sodium acetate was dissolved in 900 mL of water and adjusted to pH 7.0 with sodium hydroxide, and then, the volume was kept constant at 1000 mL). Before elution, each cartridge was dried with a vacuum pump, and then target analytes were eluted with 3 mL of methanol/ethyl acetate (2/98,
The linearity, limit of detection (LOD), limit of quantification (LOQ), and accuracy and precision were validated for this method according to the FDA criteria [
The linearity was validated by matrix-matched calibration curves by preparing external standard calibrations. Matrix-matched standards were prepared at 8 point concentrations of 1, 2, 5, 10, 20, 50, 100, and 200 ng/mL by adding working mixed standard solutions to negative control samples.
LOD is determined by a signal-to-noise ratio (S/N) ≥ 3, and it stands for the lowest concentration for the detection of each analyte. LOQ is determined by S/N ≥ 10, and it stands for the lowest measured concentration of each analyte.
Each analyte in spiked samples was detected in this developed procedure with six replicates on three separate days with the spiked levels at 2, 10, and 20
In order to obtain a satisfactory recovery, the first and most crucial step is the extraction procedure. In the previous literature, ethyl acetate was evaluated for the extraction of these target analytes in animal tissues [
In order to simplify the extraction process, in this work, ethyl acetate and hydrochloric acid (2 mol/L) were evaluated as the extraction solution without acidolysis, alkaline hydrolysis, or enzymolysis procedure. The results showed that ethyl acetate led to a low recovery for MQCA, which was in accordance with the previous studies [
The ultrasound-assisted extraction time was also evaluated to obtain higher recovery in this research at 15, 30, 45, 60, 90, and 120 min. The results showed that the recoveries for each metabolite increased with the ultrasound time during 60 min. Specially, when the extraction reached 60 min, the recovery for each metabolite was satisfactory, and 90 and 120 min extraction time did not lead to significant higher recoveries. Therefore, 60 min ultrasound-assistedextraction time was adopted in this research.
For trace amount analysis, it is important to get rid of the matrix inhibition in order to enhance the sensitivity in UPLC-MS/MS analysis process. Based on the previous literatures, the most common SPE cartridges for the purification of MEQ metabolites were Bond Elut C18, HLB, MAX, and MCX to eliminate possible interferences from crude sample extract [
In this research, MAX, MCX, and HLB cartridges were evaluated for the purification of target analytes. The result is shown in Table
SPE purification efficiency of MCX, MAX, and HLB cartridges for target analytes.
SPE cartridge | Recovery (%) | ||
---|---|---|---|
1-DMEQ | BDMEQ | MQCA | |
MCX | 3.21% | 4.23% | 2.38% |
MAX | 88.31% | 92.58% | 99.63% |
HLB | 42.38% | 36.54% | 38.82% |
UPLC was performed in this research via an Acquity BEH C18 column. The separation condition was optimized with different mobile phases, percentage of formic acid, and gradient elution program. These target analytes could be separated within 4 min using a micromass Quattro Premier XE triple quadrupole mass spectrometer coupled to UPLC system by fitting with electrospray ionization (ESI). All the analytes exhibited high response in the positive mode (ESI+). Acetonitrile (containing 0.5% formic acid) and water (containing 0.5% formic acid) were adopted as the mobile phase in order to improve the ionization efficiency of mass spectrometer detection.
The MRM conditions of each analytes were optimized by injecting standard solutions. The most optimized parameters adopted in this research are summarized in Table
Negative holothurian samples were analyzed to verify the specificity of the proposed analytical method. MRM chromatograms of fortified samples spiked at 10.0
MRM chromatograms of blank holothurian samples fortified at 10
The linearity was evaluated, and matrix-matched linear regression calibration curves ranged from 1.0 to 200 ng/mL are shown in Table
Parameters (including the standard curve, LOD, and LOQ) of MQCA, 1-DMEQ, and BDMEQ in holothurian samples.
Matrix | Analyte | Linear range (ng/mL) | Regression equitation |
|
LOD ( |
LOQ ( |
---|---|---|---|---|---|---|
Chicken | 1-DMEQ | 1.0∼200 |
|
0.9982 | 0.48 | 1.59 |
BDMEQ | 1.0∼200 |
|
0.9996 | 0.21 | 0.69 | |
MQCA | 1.0∼200 |
|
0.9995 | 0.24 | 0.79 |
LODs determined by S/N ≥ 3 in this research ranged from 0.21 to 0.48
Accuracy and precision were determined by the recoveries of each analyte in fortified sample. Each analyte in the spiked levels was analyzed with six replicates each day (
The accuracy and precision for the analysis of MQCA, 1-DMEQ, and BDMEQ residues in holothurian.
Matrix | Analyte | Spiked level ( |
Mean recovery (%) | Intraday RSD% ( |
Interday RSD% ( |
---|---|---|---|---|---|
Holothurian | 1-DMEQ | 2 | 88.0 | 4.96 | 11.8 |
10 | 83.7 | 6.07 | 6.27 | ||
20 | 90.5 | 6.96 | 8.06 | ||
BDMEQ | 2 | 88.0 | 3.14 | 5.85 | |
10 | 82.5 | 5.90 | 6.51 | ||
20 | 88.5 | 7.27 | 7.95 | ||
MQCA | 2 | 93.5 | 3.82 | 5.37 | |
10 | 85.7 | 5.56 | 6.94 | ||
20 | 85.5 | 3.80 | 6.61 |
The developed analytical method was successfully applied for the determination of all the 3 metabolites in 20 samples obtained from the local supermarket. Totally, 14 positive samples were detected positive with concentrations of 1-DMEQ, BDMEQ, and MQCA ranged from 2.27 to 6.44
Concentrations of 1-DMEQ, BDMEQ, and MQCA contamination in commercial holothurian samples.
Commercial sample | Sample code | Concentration of 1-DMEQ ( |
Concentration of BDMEQ ( |
Concentration of MQCA ( |
---|---|---|---|---|
Holothurian | 1 | ND | ND | ND |
2 | 2.27 | 4.68 | 3.99 | |
3 | ND | 6.88 | 6.52 | |
4 | 3.96 | 6.24 | 7.96 | |
5 | 5.68 | 3.95 | ND | |
6 | 6.26 | 6.21 | ND | |
7 | 6.44 | 3.698 | ND | |
8 | ND | ND | ND | |
9 | ND | ND | ND | |
10 | 3.98 | 5.68 | 3.57 | |
11 | ND | ND | ND | |
12 | ND | ND | 9.22 | |
13 | 4.69 | 6.09 | 8.69 | |
14 | ND | ND | ND | |
15 | 6.10 | 4.33 | ND | |
16 | ND | ND | 5.39 | |
17 | ND | ND | ND | |
18 | 5.62 | 4.18 | ND | |
19 | 5.33 | 5.62 | 3.59 | |
20 | ND | 5.19 | ND |
ND: not detected.
This research describes a novel ultrasound-assisted acidolysis-based UPLC-MS/MS method for the determination of three major metabolites of mequindox (1-DMEQ, BDMEQ, and MQCA) in holothurian samples. Target analytes were simplified with ultrasound-assisted acidolysis extracted without complicated enzymolysis steps. After that, each sample was centrifuged and purified by an Oasis MAX cartridge. Then, the processed samples were separated and monitored by UPLC-MS/MS. At fortified levels, the developed method exhibited satisfactory performance with good linearity, lower LODs, and good accuracy and precision. On application to commercial samples, 14 of 20 samples were detected positive for the three target analytes, with positive rate at 70 percentage. The result indicated that this method was specific, sensitive, and suitable for the quantification and conformation of the three major metabolites of MEQ in holothurian.
The authors declare that they have no conflicts of interest.
This work was supported by the Development of Science and Technology Project of Shandong Province (2012GHY11517).