TY - JOUR AU1 - Gao,, Xun AU2 - Si,, Xinxin AU3 - Yuan,, Yunxia AU4 - Chen,, Kexin AU5 - Qin,, Kunming AB - Abstract Background A simple, rapid and sensitive method coupling ultrasound-assisted dispersive liquid-liquid microextraction (DLLME) with ultra-high performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of malachite green (MG) and crystal violet (CV) in different water samples. Objective In ultrasound-assisted DLLME procedure, several parameters affecting the extraction efficiency, including pH, type and volume of the extraction and dispersive solvents, extraction time, ionic strength, were optimized to improve the accuracy and precision of this method. Methods MG and CV were extracted and preconcentrated using dichloromethane and acetonitrile as the extraction and dispersive solvents, respectively. Results Under the optimum conditions, the proposed method affords good linearity in the range of 0.40–20.0 ng/L, and the limit of detections were 0.21 and 0.32 ng/L for MG and CV, respectively. The recoveries of the method at three spiked levels were in the range of 83.4–94.2% with relative standard deviations lower than 4.7% (n = 3). Conclusions Satisfactorily, no significant matrix effect has been found as the data ranged between 68% and 102%. Introduction Both malachite green (MG) and crystal violet (CV), triphenylmethane dyes, were once extensively used throughout the world as antifungal and antiparasitic agents in aquaculture and as dyes in textile and dyeing industries (1, 2). Unfortunately, previous researches have shown that MG and CV appeard some adverse characteristics such as carcinogenesis, mutagenesis and other toxic effects (3–5), for which, in the USA and countries in European Union (EU), the use of MG and CV were banned in aquaculture and fisheries a few decades ago (6–8). In 2002, many countries in the EU formulated laws and regulations to list MG drugs as prohibited drugs in fishery and aquaculture, not to be used in the production of aquatic products, and made strict requirements in terms of technical methods, and set very low detection limits for export. The US Food and Drug Administration explicitly banned the use of MG in fish farming in 1991 due to its suspected carcinogenic properties (9, 10). The competent department of agriculture in China is aware of the harm and influence of the drug residues in aquatic products, and in 2002, through technical demonstration, MG and CV were included in the list of prohibited fishery drugs and their compounds for food animals (announcement No. 193 of the Ministry of Agriculture). Nevertheless, they are still illegally used in aquaculture due to their low cost and ready availability, especially high efficiency against fungus, bacteria and parasite. MG and CV are stable, soluble in water and not easy to be decomposed by metabolism. Studies (11, 12) have confirmed that MG or CV can be biotransformed and reduced into fat-soluble recessive malachite green (LMG) or recessive crystal violet (LCV), LMG after entering human or animal bodies, which can regenerate MG under the action of oxidase in vivo. LMG and LCV have high toxicity, high residue, carcinogenic, mutagenic and other side effects. They have been classified to be recalcitrant dye which remains in the environment for a long period, which, eventually, would cause serious consequences when the two compounds are discharged into environmental water in terms of industrial wastewater. In this sense, there is an increasing demand for simple and reliable determination of MG and CV in water. Various detection technologies have been applied for the simultaneous detection of MG and CV in water samples and aquatic products, including spectrophotometric method (13), enzyme-linked immunosorbent assay (14, 15), capillary electrophoresis (16, 17), chemometric-assisted method (18) and chromatographic techniques (19, 20). ELISA often provides false-positive result. Although capillary electrophoresis has good sensitiveness, it suffered from the disadvantages of poor reproducibility. On top of this, spectrophotometric method and chemometric-assisted method are not capable of measuring below nanomolar concentration levels. On the contrary, chromatographic techniques, especially liquid chromatography coupled with mass spectrometry, can offer high separation efficiency, low limits of detection and enhanced selectivity (21, 22). Figure 1 Open in new tabDownload slide Structure of malachite green. Figure 1 Open in new tabDownload slide Structure of malachite green. Figure 2 Open in new tabDownload slide Structure of crystal violet. Figure 2 Open in new tabDownload slide Structure of crystal violet. Sample pretreatment step prior to analysis is considered to be essential, which could not only preconcentrate analytes of interest but also decrease the interference of endogenous components in sample matrices. Likewise, plenty of pretreatment methods have been proposed to determine MG and CV in the different sample matrix. Besides conventional technique such as liquid-liquid extraction (LLE) (23) and solid-phase extraction (SPE) (21), there are also many new approaches including monolithic fiber-based solid-phase microextraction (MF/SPME-HPLC) (24), molecularly imprinted solid-phase extraction (MISPE) (25), cloud point extraction (CPE) (13), accelerated solvent extraction, ionic liquid-microwave-assisted extraction (IL-MAE) (26) and salt-assisted graphene oxide dispersive solid-phase microextraction (SA-GO-DSPME) (27). MF/SPME-HPLC has the advantages of high extraction speed, satisfactory enrichment, easy-to-operate, low-cost and environmental friendliness (24). MISPE offers excellent recovery of the template and its analogues, and it is suitable to routine use for the multi-residue extraction of MG, GV and their metabolites in aquatic products (25). The preseparation method using CPE is simple and convenient (13). IL-MAE is a simple, environmentally friendly, high-performance and powerful preconcentration method (26). Compared with the existing fiber-based GO-SPME, SA-GO-DSPME showed the merits of fast mass transfer, short extraction time and ease of operation (27). Nevertheless, the approaches mentioned above often require complicated operations, large consuming of time and high cost of organic solvent. Recently, a new approach called dispersive liquid-liquid microextraction (DLLME) was developed by Rezaee et al. (28), in which sample extraction and preconcentration could be achieved by a single step. A cloudy mixture, full of fine droplets of the extraction solvent dispersed in the aqueous phase, is formed by rapidly injecting an appropriate mixture of extraction solvent and dispersive solvent into an aqueous solution, which markedly enlarges the contact surface between the extraction solvent and aqueous phase. Based on a ternary solvent system, DLLME only needs a little amount of extraction solvent, contributing to the less use of extraction solvent and the reduction of extraction time compared with LLE. DLLME shows numerous advantages: simplicity of operation, low-cost of poisonous organic solvents, rapidity and high recovery. Zhang et al. (29) has proposed a method named temperature-controlled ionic liquid dispersive liquid-liquid microextraction (TC-IL-DLLME) to determinate MG and CV in environmental water. However, this means high cost and long time-consuming. Thus, in this study, an improved ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) method was developed to simultaneously analyze MG and CV in different water samples. Table I Mass Spectrometric Parameters of Malachite Green and Crystal Violet Antibiotics . Precursor ion . Transitions (m/z) . Cone voltage (V) . Collision energy (eV) . Malachite green [M − Cl]+ 329 → 312 46 32 329 → 208 46 32 Crystal violet [M − Cl]+ 372 → 356 11 52 372 → 340 11 32 Antibiotics . Precursor ion . Transitions (m/z) . Cone voltage (V) . Collision energy (eV) . Malachite green [M − Cl]+ 329 → 312 46 32 329 → 208 46 32 Crystal violet [M − Cl]+ 372 → 356 11 52 372 → 340 11 32 Open in new tab Table I Mass Spectrometric Parameters of Malachite Green and Crystal Violet Antibiotics . Precursor ion . Transitions (m/z) . Cone voltage (V) . Collision energy (eV) . Malachite green [M − Cl]+ 329 → 312 46 32 329 → 208 46 32 Crystal violet [M − Cl]+ 372 → 356 11 52 372 → 340 11 32 Antibiotics . Precursor ion . Transitions (m/z) . Cone voltage (V) . Collision energy (eV) . Malachite green [M − Cl]+ 329 → 312 46 32 329 → 208 46 32 Crystal violet [M − Cl]+ 372 → 356 11 52 372 → 340 11 32 Open in new tab Figure 3 Open in new tabDownload slide Chromatograms of real sample. Figure 3 Open in new tabDownload slide Chromatograms of real sample. Materials and methods Chemicals and reagents The purity of the standards of MG and CV is >98%, obtained from the National Institute for Food and Drug Control (Beijing, China) and the structures are described in Figures 1 and 2. Acetonitrile and formic acid were of chromatographic grade and purchased from Fisher Scientific (Pittsburgh, PA, USA). Methanol of chromatographic grade was obtained from Yuwang Industrial Co., Ltd. (Shandong, China). Stock solutions of the interested analytes at a concentration of 1 mg/mL were prepared with methanol and stored at 4°C under dark conditions before use. The working solutions were obtained by diluting the stock solution with methanol to the required concentration. Ultrapure water purified by a Milli-Q Reagent Water system (Millipore, Bedford, MA) was used throughout the experiment. The solvents involved in the experiment such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane and chlorobenzene were all analytical grade and purchased from Shandong Yuwang Industrial Co., Ltd. Instrumentation An ACQUITY™ UPLC system (Waters Corp., Milford, MA, USA), consisting of an autosampler, a quaternary pump, micro degasser, and thermostatic column oven, was used for separation. MG and CV were separated on a Waters ACQUITY UPLC® BEH Phenyl (50 mm × 2.1 mm, 1.7 μm). The optimum separation was achieved isocratically using aqueous formic acid (0.1%, v/v) (A) and acetonitrile (B) as the mobile phase in an 80% (v/v) ratio. The column temperature was maintained at 35°C and the flow rate was set at 0.2 mL/min. The injection volume was 5 μL. A Micromass Quattro micro™ API mass spectrometer (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization (ESI), was used for the determination of MG and CV, in which multiple reaction monitoring was used for the quantification (external calibration) of the trace amount of MG and CV. The mixed standard reserve solution of the different volume was added to the water sample to form a standard sample containing different concentrations of target analytes. The water sample without adding standard was used as a blank, extracted and detected according to the optimized conditions, and the peak area was read. The working curve was obtained and quantified. Positive ESI was selected in which mode the higher response values could be reached. The parameters of MS analysis are presented in Table I. And other instrument conditions were set as follows: capillary voltage, 3.0 kV; desolvation temperature, 350°C and source temperature, 150°C; desolvation gas flow, 700 L/h. Argon was served as the collision gas in all cases and nitrogen as the auxiliary and shield gas in the ESI source. The centrifuge adopts TGL-16G high-speed desktop centrifuge purchased from Shanghai Anting Scientific instrument Factory. The centrifuge participates in the phase separation step. The speed was 4000 rpm. UA-DLLME procedure Five milliliters of water sample (previously acidified by 0.1 mol/L hydrochloric acid to pH 4) was placed in a 10 mL glass conical centrifuge tube. A mixture of 400 μL of dichloromethane (as extraction solvent) and 700 μL of acetonitrile (as dispersive solvent) was rapidly injected into the sample solution with a micropipette. And very fine droplets were formed throughout the water samples. The dispersion then was ultrasonicated for 2 min in an ultrasonic bath to form a homogeneous cloudy solution. The phase separation was performed by centrifugation for 10 min at 4000 rpm. The upper aqueous phase was removed with a syringe and discarded, and then the sedimented phase was evaporated to dryness under a gentle stream of nitrogen at 35°C. The residue obtained was reconstituted with 100 μL mobile phase. Finally, 5 μL of the above solution filtered with 0.22 μm filter membrane was injected into the chromatographic system. The chromatograms of the real sample are shown in Figure 3 and the chromatograms of the two target analytes for quantitative analysis are shown in Figure 4, with 10 μg·L−1 added to each analyte. Figure 4 Open in new tabDownload slide Chromatograms of the two analytes of spiked sample (10 μg·L−1). Figure 4 Open in new tabDownload slide Chromatograms of the two analytes of spiked sample (10 μg·L−1). Samples collection Tap water samples were collected from the tap in the laboratory. River water (RW) samples were taken from the South Canal of Shenyang. Drinking water samples were taken from barreled drinking water produced in Shenyang. The Influent (IWW) and effluent (EWW) wastewaters were collected at the urban wastewater treatment plant of Shenyang. All samples were immediately filtered through 0.45 μm nylon membranes syringe filter membrane after collection and then stored at −20°C in brown glass bottles prior to analysis. Results and discussion Optimization of UA-DLLME conditions In order to obtain high extraction efficiency, the effect of different factors, such as the pH of samples, the type and the volume of extraction and dispersive solvent, extraction time and ionic strength were investigated. In this experiment, 2 mL of Milli-Q water spiked at 10 ng/L of each compound was used to optimize the extraction conditions. All experiments were carried out in triplicate. The optimization of sample pH In DLLME, sample pH is an important factor that affects the extraction efficiency of target analytes to a great extent as it affects the hydrolysis status and the solubility of the analytes in the aqueous phase (25). The pKa of MG and CV are 6.9 and 0.8, respectively, so the water samples should be adjusted to acidity in which case target analytes exist in the terms of a molecule and their solubility in water will be decreased (29). In order to investigate the effect of pH on the extraction effect, the pH of the sample solution was adjusted to 2, 3, 4, 5, 6, 7 using 0.1 mol/L of hydrochloric acid. As shown in Figure 5, with the increase of pH, the recovery rate increases until the highest recovery rate is obtained when pH is 4.0; when pH is >4, the recovery rate of pH decreases, because when pH 4.0, most antibiotics are in a molecular state, which is more favorable for extraction, while under other pH conditions, antibiotics are in ionic state and tend to be dissolved in the aqueous phase. Therefore, pH 4.0 was chosen for further investigation. Figure 5 Open in new tabDownload slide Effect of pH of DLLME on the recovery of targets. Figure 5 Open in new tabDownload slide Effect of pH of DLLME on the recovery of targets. The optimization of the type and volume of extraction solvent The type of extraction solvent is the major factor for DLLME process. It was previously reported that the extraction solvent must meet the following requirements: low solubility in water, high extraction capability of interested compounds (30, 31). Dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane and chlorobenzene was investigated in this experiment based on the above demands (Figure 6). Nearly the same higher recoveries were obtained when dichloromethane and chloroform were separately used as extraction solvent. At last, dichloromethane was selected as a dispersive solvent while take it into consideration that the toxicity of dichloromethane is much lower than that of chloroform. Figure 6 Open in new tabDownload slide Effect of extraction solvent of DLLME on the recovery of targets. Figure 6 Open in new tabDownload slide Effect of extraction solvent of DLLME on the recovery of targets. Then, different volumes of dichloromethane (200, 300, 400, 500, 600 μL) were further investigated. Among 100–400 μL the recoveries of MG and CV were significantly improved as the volume of dichloromethane was increased, which remained constant when the volume of dichloromethane was further increased. The experimental results showed that when the volume of dichloromethane was 400 μL, the adsorption reaches saturation and the best extraction effect was obtained. Thus, the volume of extraction solvent was determined as 400 μL. And the results of the kind and volume of extraction solvent were presented in Figures 6 and 7, respectively. Figure 7 Open in new tabDownload slide Effect of extraction solvent volume of DLLME on the recovery of targets. Figure 7 Open in new tabDownload slide Effect of extraction solvent volume of DLLME on the recovery of targets. The optimization of the type and volume of dispersive solvent Dispersive solvent is another crucial parameter that affects extraction efficiency. The role of the dispersive solvent in DLLME is to promote the formation of emulsion and increase the contact area between water and extraction solvent. For this reason, it is necessary for dispersive solvent to be miscible well with both water and dichloromethane (32). Methanol, ethanol, acetonitrile and acetone were investigated due to their widely use as a dispersive solvent. As shown in Figure 8, the extraction effect of acetonitrile was the best, which may be due to the fact that each target analyte was more soluble in acetonitrile, so acetonitrile was chosen as the dispersant in this experiment. Therefore, acetonitrile was chosen as the dispersion solvent. Figure 8 Open in new tabDownload slide Effect of dispersive solvent of DLLME on the recovery of targets. Figure 8 Open in new tabDownload slide Effect of dispersive solvent of DLLME on the recovery of targets. At the same time, the effect of dispersant volume on the extraction effect was also investigated. The extraction rates were investigated when the volume of dispersant was 200, 300, 400, 500 and 600 μL, respectively. The results were shown in Figure 9. In the range of 200–400 μL, the recovery of target analyte increases with the increase of acetonitrile volume. When the recovery was above 400 μL, the recovery decreases slightly, which may be due to the fact that the dispersed solvent also increases the solubility of the extractant in water. The recovery rate was the highest when the volume of dispersant was 400 μL, so the volume of the dispersing solvent was 400 μL. Figure 9 Open in new tabDownload slide Effect of dispersive solvent volume of DLLME on the recovery of targets. Figure 9 Open in new tabDownload slide Effect of dispersive solvent volume of DLLME on the recovery of targets. Effect of ionic strength As we all known, adding salt is the most commonly means to change the ionic strength of the solution. Whereas, it should be noted that the effect of adding salt is different in the various system. Therefore, 0–10% of sodium chloride was separately added into the water samples to evaluate the effect of ionic strength. The data obtained showed that the salinity of samples had no significant effect on the recoveries of target analytes. Thus, no salt was added in the subsequent experiments. Table II The Performance Characteristics of UPLC-MS/MS Compound . Linear range (ng/L) . r . LOD (ng/L) . LOQ (ng/L) . Intra-day precision RSD % (n = 6) . Inter-day precision RSD % (n = 6) . Repeatability RSD % (n = 6) . Malachite green 0.40–20.0 0.9969 0.21 0.70 2.1 2.8 2.4 Crystal violet 0.40–20.0 0.9982 0.32 1.06 1.8 2.3 1.6 Compound . Linear range (ng/L) . r . LOD (ng/L) . LOQ (ng/L) . Intra-day precision RSD % (n = 6) . Inter-day precision RSD % (n = 6) . Repeatability RSD % (n = 6) . Malachite green 0.40–20.0 0.9969 0.21 0.70 2.1 2.8 2.4 Crystal violet 0.40–20.0 0.9982 0.32 1.06 1.8 2.3 1.6 Open in new tab Table II The Performance Characteristics of UPLC-MS/MS Compound . Linear range (ng/L) . r . LOD (ng/L) . LOQ (ng/L) . Intra-day precision RSD % (n = 6) . Inter-day precision RSD % (n = 6) . Repeatability RSD % (n = 6) . Malachite green 0.40–20.0 0.9969 0.21 0.70 2.1 2.8 2.4 Crystal violet 0.40–20.0 0.9982 0.32 1.06 1.8 2.3 1.6 Compound . Linear range (ng/L) . r . LOD (ng/L) . LOQ (ng/L) . Intra-day precision RSD % (n = 6) . Inter-day precision RSD % (n = 6) . Repeatability RSD % (n = 6) . Malachite green 0.40–20.0 0.9969 0.21 0.70 2.1 2.8 2.4 Crystal violet 0.40–20.0 0.9982 0.32 1.06 1.8 2.3 1.6 Open in new tab Table III Spiked Recoveries of the Water Sample Antibiotics . Spiked (ng/L) . Found (ng/L) . Recovery (%) . RSD (%) (n = 3) . Malachite green 1 3.70 90.0 3.1 5 6.97 83.4 4.7 10 11.92 91.2 3.4 Crystal violet 1 3.02 92.0 2.5 5 6.81 94.2 2.4 10 11.38 92.8 1.7 Antibiotics . Spiked (ng/L) . Found (ng/L) . Recovery (%) . RSD (%) (n = 3) . Malachite green 1 3.70 90.0 3.1 5 6.97 83.4 4.7 10 11.92 91.2 3.4 Crystal violet 1 3.02 92.0 2.5 5 6.81 94.2 2.4 10 11.38 92.8 1.7 Open in new tab Table III Spiked Recoveries of the Water Sample Antibiotics . Spiked (ng/L) . Found (ng/L) . Recovery (%) . RSD (%) (n = 3) . Malachite green 1 3.70 90.0 3.1 5 6.97 83.4 4.7 10 11.92 91.2 3.4 Crystal violet 1 3.02 92.0 2.5 5 6.81 94.2 2.4 10 11.38 92.8 1.7 Antibiotics . Spiked (ng/L) . Found (ng/L) . Recovery (%) . RSD (%) (n = 3) . Malachite green 1 3.70 90.0 3.1 5 6.97 83.4 4.7 10 11.92 91.2 3.4 Crystal violet 1 3.02 92.0 2.5 5 6.81 94.2 2.4 10 11.38 92.8 1.7 Open in new tab Table IV ME and its Precision (RSD) of the Proposed Method in Different Environmental Water Matrices Spiked at the 100 ng/mL level Antibiotics . ME (RSD) . Drinking water . Running water . RW . Influent water . Efluent water . Malachite green 93 (1) 92 (3) 87 (5) 68 (2) 85 (2) Crystal violet 102 (2) 86 (2) 84 (3) 74 (3) 99 (3) Antibiotics . ME (RSD) . Drinking water . Running water . RW . Influent water . Efluent water . Malachite green 93 (1) 92 (3) 87 (5) 68 (2) 85 (2) Crystal violet 102 (2) 86 (2) 84 (3) 74 (3) 99 (3) Open in new tab Table IV ME and its Precision (RSD) of the Proposed Method in Different Environmental Water Matrices Spiked at the 100 ng/mL level Antibiotics . ME (RSD) . Drinking water . Running water . RW . Influent water . Efluent water . Malachite green 93 (1) 92 (3) 87 (5) 68 (2) 85 (2) Crystal violet 102 (2) 86 (2) 84 (3) 74 (3) 99 (3) Antibiotics . ME (RSD) . Drinking water . Running water . RW . Influent water . Efluent water . Malachite green 93 (1) 92 (3) 87 (5) 68 (2) 85 (2) Crystal violet 102 (2) 86 (2) 84 (3) 74 (3) 99 (3) Open in new tab The optimization of extraction time In DLLME, extraction time was defined as a time interval between the formation of homogeneous cloudy solution and phase separation by centrifugation. Herein, ultrasound was applied to accelerate the formation of a fine cloudy solution, which would shorten equilibrium time and further enhance the extraction efficiency. The influence of extraction time was investigated in the range from 0 to 4 min. The result in Figure 10 revealed that the recoveries increase with the increasing extraction time within 2 min, and the recoveries were decreased slightly beyond 2 min. It was clear that the transfer of analytes into the extraction solvent was accelerated greatly with the help of ultrasonic irradiation. This may be the credit of the assisting-dissolving and emulsifying effect of the ultrasound irradiation. The decreased recoveries were probably because volatilization loss of the analytes and extraction solvent under ultrasound increased with the extension of ultrasonic time. As a result, the extraction time for the proposed method was set at 2 min for further experiments. Figure 10 Open in new tabDownload slide Effect of time of DLLME on the recovery of targets. Figure 10 Open in new tabDownload slide Effect of time of DLLME on the recovery of targets. Table V Determination of the Six Antibiotics in Real Water Samples (ng/L) Antibiotics . Drinking water . Running water . RW . Influent water . Effluent water . Malachite green – – 1.9 2.8 0.99. Three replicate extractions were performed for each concentration level. LOD and LOQ were determined from the spiked blank water samples that the amount of the analytes with the signal to noise ratios of 3 and 10, respectively. Drinking water samples were taken from barreled drinking water produced in Shenyang. LOD (based on signal-to-noise ratios of 3) and LOQ (based on signal-to-noise ratios of 10) were employed to represent the sensitivity of the method. LODs were 0.21 ng/L and 0.32 ng/L for MG and CV, respectively. And LOQs were 0.7 ng/L and 1.06 ng/L for MG and CV, respectively. Intra- and inter-day precision were selected to measure the instrumental precision of the method, expressed as the relative standard deviation (RSD). Intra-day test was performed by six repeated analyses of the same mixed standards solution within one day. As for the inter-day precision test, the mixed standards solution was examined in six replicates per day for three consecutive days. The results, <10%, show a satisfactory instrumental precision. To evaluate the repeatability of the method, six parallel influent wastewaters at the spiked concentration level of 0.1 ng/mL were extracted and analyzed. The RSDs were 2.4 for MG and 1.6 for CV, which explains acceptable repeatability for this approach. The recovery tests were carried out by spiking three different levels (1, 5 and 10 ng/L) of MG and CV into real samples (influent wastewater) under the optimal conditions and three replicate extractions was carried out for each level. The recoveries for target analytes were in the range of 83.4–94.2% with RSDs <5, indicating the method was accurate. Matrix effect There often exists a severe matrix effect (ME) when LC–MS/MS was used, especially in case of using ESI source (33). ME may lead to a signal suppression or enhancement, caused by the fact that the ESI source is highly susceptible to endogenous components of the matrix, which can obviously affect the quantitation of a trace amount of analytes. Thereby, ME should be evaluated so that the reliability of the results obtained could be ensured. In this work, ME was calculated by the following formula: $$ \mathrm{ME}=\left(C-A\right)/B $$ A represents the response of each real sample resulting from the existence of target analytes, B stands for the response of standard solution and C was the response of the samples spiked reference standard prior to extraction with the same concentration as B. The results, listed in Table IV, indicated that the ME has little influence when this UA-DLLME method was applied in different water samples. Application to real samples In order to evaluate the practical use of the proposed approach, it was applied to the simultaneous determination of MG and CV in five different kinds of water samples. As can be seen in Table V, only small amounts of MG and CV were detected in RW and influent wastewaters, and the amount of the former (1.9 ng/L for MG, 1.5 ng/L for CV) was slightly less than the latter (2.8 ng/L for MG). Comparison of UA-DLLME with other methods The advantages of the proposed UA-DLLME method were highlighted in comparison with others developed for the determination of MG and CV in water samples (Table VI). Among these, MF/SPME-HPLC allows a wide calibration range using relatively low volumes of sample. However, the SPME procedure requires about 30 min instead of the 2 min needed in this UA-DLLME approach. In addition, TC-IL-DLLME-HPLC requires about 50 min for the sample extraction, and the recoveries of MG and CV are >100% in most cases (29). Furthermore, the present method not only achieved higher LODs, but also reached lower consumption of sample than those represented in the table. Conclusion In this study, an novel method, ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) combined with ultra-high performance liquid chromatography-tandem mass spectrometry, for detected and quantify MG and CV at trace levels in different water samples was developed. The extraction efficiency of target analytes in DLLME was improved significantly by applying an ultrasound-assisted process, which can facilitate the formation of a cloudy solution. Thereby, the extraction efficiency and effect of the target analytes were enhanced. The method required less time of sample preparation and less use of harmful solvent than the conventional methods. The results demonstrated that UA-DLLME coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) was an efficient, sensitive, and reliable method for the analysis of MG and CV. Acknowledgements A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References 1. Schnick , R.A. ; The impetus to register new Therapeutants for aquaculture ; The Progressive Fish-Culturist , ( 1988 ); 50 ( 4 ): 190 – 196 . Google Scholar Crossref Search ADS WorldCat 2. 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For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) TI - Ultra-trace Extraction of Two Bactericides Via Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction JF - Journal of Chromatographic Science DO - 10.1093/chromsci/bmaa083 DA - 2021-01-14 UR - https://www.deepdyve.com/lp/oxford-university-press/ultra-trace-extraction-of-two-bactericides-via-ultrasound-assisted-cbvOXYQxfs SP - 182 EP - 190 VL - 59 IS - 2 DP - DeepDyve ER -