Leuco Crystal Violet Research Articles

Portable electrophoresis titration chip model for sensing of uric acid in urine and blood by moving reaction boundary

Assays of uric acid (UA) in human urine and blood are of great significance to clinic diagnoses of many diseases, such as gout, renal failure, and hyperuricemia. Portable and visual quantification method, however, has been rarely developed for POCT detection of UA in urine and blood samples. In this work, we developed the electrophoresis titration (ET) model, the relevant sensing method and the portable chip device for portable and visual detection of UA in both urine and blood based on moving reaction boundary (MRB). In the model, uricase catalyzes UA in the anode well to allantoin, producing hydrogen peroxide, which oxidizes leucocrystal violet dye without charge to crystal violet (CV+) with one positive charge. Under an electric field, a MRB was created between the violet CV+ moving from the anode well into the channel and the alkaline sodium acetate in channel, resulting in an ET sensing. The model indicated that the distance of MRB under given conditions was as a function of UA content, implying an extraordinary simple sensing for UA. Based on the MRB-ET model, a series of experiments were conducted. The results evidently validated the model and method of ET-MRB. The experiments not only demonstrated the high facility and portability of MRB-ET model, but also showed the visuality, selectivity and rapidity. In addition, the experiments revealed the fair sensitivity (<0.1 mM), linearity (0.1–4.0 mM, r2 = 0.9948), recovery (85%–106%) and stability (RSD 3.8–7.1%). Finally, the developed method was successfully used for the determination of UA both in urine and blood. All these results manifested the simple, portable and visual sensing of UA in human body fluid samples, and implied the potential of MRB-ET method to real POCT assay of UA in urine and blood samples.

Analysis of Triphenylmethane Dye Residues and their Leuco-Forms in Frozen Fish by LC-MS/MS, Fish Microbial Quality, and Effect of Immersion in Whole Milk on Dye Removal.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and used to quantify crystal violet (CV), leucocrystal violet (LCV), malachite green (MG), leucomalachite green (LMG), and brilliant green (BG) residues in frozen fish (121 samples) from various countries, in order to detect the use of prohibited antibiotic dyes in fish for human consumption. The microbial quality of the fish was also assessed along with the effectiveness of a simple treatment with whole fat milk to reduce the levels of CV and LCV contamination. CV and LCV were the only two residues detected. They were found in farmed Pangasius (0.362 to 41.34 μg/kg and 0.178 to 10.58 μg/kg, respectively) and Tilapia (1.24 to 9.48 μg/kg and 1.29 to 2.81 μg/kg). Based on aerobic plate count (APC), 74%, 59%, and 55% of the samples of Tilapia fillets (from China) and Pangasius fillets (United Arab Emirates and Vietnam), and 100% and 50% of the skin samples of Hake (Argentina and U.S.A.) were of unacceptable microbial quality (APC > 107 cfu/g). Human pathogens, namely Escherichia coli, Staphylococcus aureus, and Vibrio spp., were detected in most fish. A significant reduction in CV and LCV concentrations by more than a third was achieved after immersing Pangasius and Tilapia fillets in whole fat milk for 120 minutes. These findings support the necessity of regular inspections and monitoring of CV and other antibiotic dye residues in fish, along with routine assessments of fish microbial quality, in order to protect public health. PRACTICAL APPLICATION: The described LC-MS/MS method can be used to rapidly and simultaneously quantify antibiotic dye residues in frozen fish. CV and LCV were detected in farmed Pangasius and Tilapia fillets and their concentrations was reduced by more than one third after immersing the fillets in whole milk for 120 min, a treatment which is not intended to replace safe fish farming practices upstream to artificially lower the level of banned dyes in fish. The findings support the necessity of regular inspections and monitoring of CV and other antibiotic dye residues in fish, along with assessments of fish microbial quality, to protect public health.

Antioxidant properties of citric acid interfere with the uricase-based measurement of circulating uric acid

BackgroundCirculating uric acid (UA) is an important biomarker, not only in the detection and management of gout, but also in assessing the risk of related comorbidity. The impact of collection methods on clinical UA measurements has been the subject of limited study. After observing significant differences between UA concentrations of blood samples obtained by different collection tubes, we began examining the effects of exogenous tube components on measured UA concentrations. We aimed to: (1) demonstrate the variability in uricase-based UA measurements attributable to different collection methods and (2) identify factors influencing this variability. MethodsBlood samples from human subjects were collected using Serum Separator Tubes (SST tubes), Acid Citrate Dextrose (ACD) tubes, and Sodium Citrate (SC) tubes. Circulating UA concentrations were measured by chemistry analyzers utilizing the uricase method. Absorbance assays were run in order to determine the effects of citric acid, sodium citrate, and dextrose on measured absorbance in the presence of leuco crystal violet dye, hydrogen peroxide, and peroxidase. Statistical analyses—including Student’s T tests and ANOVA—were used to compare results. ResultsUA concentrations of blood samples collected in ACD tubes were significantly lower than those collected in SST tubes (P < 0.01). Samples collected in SC tubes trended towards lower UA measurements than samples collected in SST tubes, although this difference did not reach statistical significance (P = 0.06). Blood samples spiked with separate concentrations of sodium citrate (3.2 and 22.0 g/L), citric acid (8.0 g/L), and dextrose (24.5 g/L) demonstrated significantly lower UA measurements compared to controls (P < 0.01). Absorbance assays demonstrated that increasing concentrations of citric acid and sodium citrate—in the presence of leuco crystal violet, hydrogen peroxide, and peroxidase—decreased the amount of oxidized dye in the uricase method of UA measurement in a dose-dependent manner (P < 0.01). In contrast, dextrose did not significantly alter the amount of oxidized dye available. DiscussionOur results indicate that citric acid obstructs accurate uricase-based UA measurement, providing falsely low values. Citric acid, a known antioxidant, scavenges hydrogen peroxide, a key intermediate using the uricase method. By scavenging hydrogen peroxide, citric acid decreases the amount of oxidized leuco dye leading to falsely low UA measurements. Therefore, collection tubes, like ACD and SC tubes, which contain concentrations of citric acid or its conjugate base sodium citrate should not be used to measure circulating UA levels when utilizing uricase-based measurement methods.

Dye Residue Analysis in Raw and Processed Aquaculture Products: Matrix Extension of AOAC INTERNATIONAL Official Method 2012.25.

Background: Triphenylmethane dyes and metabolites are known or suspected mutagens and are prohibited in animals intended for human consumption. Despite toxicity, triphenylmethane dyes are used illegally as inexpensive treatments for fungal and parasite infections in aquatic animals. Objective: AOAC INTERNTIONAL Official Method 2012.25 for the LC-MS/MS determination of malachite green, crystal violet, brilliant green, and metabolites leucomalachite green and leucocrystal violet in seafood products was previously validated for finfish (trout, salmon, catfish, and tilapia) and shrimp, but had not been fully validated for other types of aquacultured products such as eel, molluscan shellfish, or frog or for processed seafoods. Methods: Method 2012.25 was applied to a wide scope of raw and processed aquaculture products including Arctic char, barramundi, eel, frog legs, hybrid striped bass, pompano, scallops, seabream, smoked trout, dried shrimp, and highly processed canned eel and dace products. The canned products contained oil, salt, sugar, flavorings, spices, sauces, and/or preservatives. Results: Dyes and metabolites were recovered with >85% accuracy and precision generally <20% relative standard deviation. The method detection limit was ≤0.60 μg/kg and LOQ was <1.0 μg/kg. Compounds were identified in 99% of 330 fortified and incurred samples. Conclusions: This study supports the use of Method 2012.25 for triphenylmethane dye residue analysis in a wide variety of aquacultured and seafood products. Highlights: Method 2012.25 performed well with results consistent with previous validation studies, regardless of presence of additional food ingredients or the type of processing.

Modelling the long-term feed-to-fillet transfer of leuco crystal violet and leuco malachite green in Atlantic salmon (Salmo salar)

ABSTRACTLeuco crystal violet (LCV) and leuco malachite green (LMG) are the main metabolites of two dyes that are forbidden for use in food production, but can be present at low background concentration in novel Atlantic salmon feed ingredients such as processed animal proteins (animal by-product [ABP]). In this study, the potential transfer of dietary LCV or LMG to the fillet of farmed Atlantic salmon was investigated. The uptake and elimination rate kinetics were determined in seawater-adapted Atlantic salmon (initial weight 587 ± 148 g) fed two levels of either LCV- or LMG-enriched diets (~500 and 4000 µg kg−1, respectively) for 40 days, followed by a 90-day depuration period with feeding on control diets (<0.15 μg kg−1 LCV and LMG). A three-compartmental model was developed, based on a fillet fat, fillet muscle and a central body compartment comprising all other tissues. Model calibrations showed a good fit with measured values during overall uptake and elimination period; however, the model poorly predicted the short-term (days) peak measured values at the end of the exposure period. The model was used to simulate the long-term (>16 months) LCV and LMG feed-to-fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth and fillet fat deposition. The model predictions gave highest expected LCV and LMG fillet concentrations of approximately 0.12 and 0.45 μg kg−1, depending on the dietary levels of ABP and background level of LCV and LMG contamination. These levels are under the reference point for action of 2 µg kg−1 for the sum of MG and LMG that EFSA assessed as adequate to protect public health. However, for LCV, the predicted highest levels exceed the analytical decision limit (CCα) of 0.15 µg kg−1 for the method used in this paper.

Effect of Bleaching Gel Viscosity on Tooth Whitening Efficacy and Pulp Chamber Penetration: An In Vitro Study.

Whitening efficacy has been related to hydrogen peroxide (HP) diffusion into tooth structure. However, little information is available relating rheological properties to whitening efficacy. The purpose was to evaluate the whitening efficacy and HP penetration level of a 10% HP gel at three different viscosities and to compare them to a strip delivery system. Extracted molars (n=120) were randomly assigned into five groups (n=24/ group): NC_MED (negative control; median): medium viscosity gel without HP; LOW: 10% HP gel (low viscosity experimental gel, Ultradent Products Inc); MED: 10% HP gel (medium viscosity experimental gel, Ultradent); HIGH: 10% HP gel (high viscosity gel, Ultradent); and CWS: Crest 3D Whitestrips 1-Hour Express (Procter & Gamble). All teeth were subjected to five 60-minute whitening sessions. Instrumental color measurements were performed at baseline (T0), and 1-day after each application (T1-T5), and 1-month after whitening (T6). HP penetration was estimated with leucocrystal violet and horseradish peroxidase. A Kruskal-Wallis test and post hoc Bonferroni test were performed to assess the difference in tooth color change and HP penetration among the groups (α=0.05). Hydrogen peroxide penetration levels and overall color changes at T6 were 0.24 μg/mL / 2.80; 0.48 μg/mL / 8.48; 0.44 μg/mL / 7.72; 0.35 μg/mL / 8.49; 0.36 μg/mL / 7.30 for groups NC, LOW, MED, HIGH, and CWS, respectively. There was a significant difference for HP penetration, while there was no significant difference among the four experimental groups for tooth color change. Rheological properties should be considered when developing new whitening formulations.

Theoretical Calculation and Experimental Verification Demonstrated the Impossibility of Finding Haptens Identifying Triphenylmethane Dyes and Their Leuco Metabolites Simultaneously.

Detection of triphenylmethane dyes (TDs), especially the widely used malachite green (MG) and crystal violet (CV), plays an important role in safety control of aquatic products. There are two chromatic forms of TDs: oxidized or reduced. Usually, only one form can be detected by reported ELISA antibodies. In this article, molecular shape superimposing and quantum mechanics calculation were employed to elucidate the differences between MG, CV, and their reduced chromatic forms (leucomalachite green, LMG and leucocrystal violet, LCV). A potential hapten was rationally designed and synthesized. Polyclonal antibodies were raised through immunizing New Zealand white rabbits and BALB/C mice. We tested the cross-reactivity ratios between the hapten and TDs. The cross-reactivity ratios were correlated with the difference in surface electrostatic potential. The determination coefficients (r2) of the correlations are 0.901 and 0.813 for the rabbit and mouse antibody, respectively. According to this linear model, the significant difference in the atomic charge seemed to make it impossible to find a hapten that can produce antibodies with good cross-reactivities with both reduced and oxidized TDs.

The efficient n-doping of [6,6]-phenyl C61-butyric acid methyl ester by leuco-crystal violet to enhance the performance of inverted organic solar cells

Inverted organic solar cells (OSCs) have been fabricated using the photoactive blend thin films based on regioregular poly(3-hexylthiophene) (P3HT), [6,6]-phenyl C61-butyric acid methyl ester (PCBM), and leuco-crystal violet (LCV). It was found that the LCV as an efficient n-dopant could significantly increase intrinsic electron concentration of PCBM zone. The electron mobility of P3HT:PCBM:LCV blend thin film was measured 1.75 times as high as that of P3HT:PCBM blend thin film, as a result of LCV-induced trap filling in the bandgap of PCBM. The power conversion efficiency for the inverted device using the photoactive layer of P3HT:PCBM:LCV could be 1.22 times as high as that for the inverted device using the conventional photoactive layer of P3HT:PCBM, mostly because (1) the higher electron mobility could enhance the exciton dissociation and thereby short-circuit current density in the former relative to the latter; (2) the increase in the electron concentration of PCBM zone in P3HT:PCBM:LCV blend thin film may help blocking holes diffusion towards cathode, improving the hole collection efficiency and thereby fill factor of device. We provide a new insight on optimizing the electron-conducting property of bulk-heterojunction photoactive thin film, useful for pushing forward inverted OSCs towards the cost-effective commercialization.

Response Surface and Kinetic Modeling of Dimethyl Sulfide Oxidation – On the Origin of Dimethyl Sulfoxide in Malt

ABSTRACTDimethyl sulfide (DMS) contributes significantly to beer aroma. Malt contains considerable amounts of its oxidized form dimethyl sulfoxide (DMSO), the precursor of DMS during fermentation. Yet, detailed information regarding mechanisms of DMSO formation in malt is limited. Recently, hydrogen peroxide was suggested as a potential DMS oxidant in malt. The aim of this study was to characterize the reaction of hydrogen peroxide and DMS in buffered model solutions. Initially, hydrogen peroxide was identified in green malt using leuco crystal violet and horseradish peroxidase. Based on these findings the reaction of DMS with H2O2 was investigated in buffered model solutions at varying temperatures and pH using response surface modeling (RSM). Significant amounts of DMSO were formed by H2O2 mediated oxidation of DMS (∼0–41%), which was determined by temperature and hydrogen peroxide concentration (P ≤ 0.003). The effect of the pH was insignificant (P ≥ 0.4). Kinetics of DMS oxidation by H2O2 (DMS:H2O2 = 1:3) at 95°C followed a pseudo first order mechanism (k = 0.0041 min−1). Molecular oxygen was not capable of oxidizing DMS, whereas hydroxyl radical mediated DMS oxidation was faster than DMS oxidation aroused by H2O2. Potential reaction mechanisms and their relevance in DMS oxidation are discussed as a first approach to minimize DMSO levels in malt.

A paper-based visible light-responsive imaging material

In this paper, we have developed a paper-based optical imaging material responding to visible light. This material consists of a thioxanthone (TX) derivative with the additional phenyl carbazole chromophore, namely 1-phenyl-11,12a-dihydrothiochromeno (3,2-b) carbazol-13 (5H)-one (TX-PC), and leuco crystal violet (LCV). TX-PC is newly synthesized and characterized by 1HNMR, FTIR and high-resolution mass spectrometer. As a visible light absorbing Type II photoinitiator, TX-PC has an excellent absorption property in the visible region in the absence of an added co-initiator. In other words, TX-PC degrades into lively free radicals when irradiating under visible light which resulting in a color reaction on LCV. The degree of reaction is characterized by UV–vis spectrophotometer. In this study, this material is used for imaging within visible light because of the high efficiency color reaction. We coat a paper substrate with this TX-PC/LCV solution and cover with a pattern photomask. Under the irradiation of the visible light, color reaction occurs to irradiated area while the rest remains colorless. Thus the two areas are distinguished to obtain a clear image as same as the original version.

A LC-HRMS After QuEChERS Cleanup Method for the Rapid Determination of Dye Residues in Fish Products

The reliability of a rapid LC-HRMS method was studied in order to find a sensitive and accurate, simple, cheap, and rapid method for screening and quantitative determination of malachite green (MG), leucomalachite green (LMG), brilliant green (BG), crystal violet (CV), and leucocrystal violet (LCV) in fish muscle. All the analytes were extracted using a mixture of acetonitrile and citrate buffer, while the cleanup phase was carried out by Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method. All the data analyzed were acquired using both full MS and dd-MS2 modes. Good specificity, precision (relative standard deviation <11% for each sample tested), recovery (10–60%), decision limit (CCα between 0.55 and 0.62 μg kg−1), detection capabilities (CCβ between 0.65 and 0.78 μg kg−1), and ruggedness were achieved for the reliability of the method. Satisfactory results were obtained during the linearity test in the range of 0.10–2 ng mL−1 (r 2 > 0.999). Best recoveries were obtained by the QuEChERS cleanup method for all the analytes examined, presenting values between 70 and 104%. The application of 70,000 FWHM mass resolution and narrow mass windows significantly improved the selectivity of the method, leading to simultaneous screening and quantification of dye residues in comparison to other methods proposed in literature. The optimized method proposed in this work enables a simple and fast preparation; it offers exceptional sensitivity and selectivity and maximizes efficiency and reproducibility with a low consumption of reagents. Finally, the present method was successfully employed to detect dye residues in 73 fish samples, as provided for the national residue control plan.

Dyes in aquaculture and reference points for action.

The European Commission requested EFSA to evaluate whether a series of dyes are covered by the ‘Guidance on methodological principles and scientific methods to be taken into account when establishing Reference Points for Action (RPAs) for non‐allowed pharmacologically active substances present in food of animal origin’ and to which group they should be attributed according to this guidance. Although these substances are not registered for use in food‐producing animals in the European Union, they may be used illegally in aquaculture for their antimicrobial properties. It was concluded that acriflavine, 3‐aminoacridine, aminoacridine, basic blue 7, brilliant green, leucobrilliant green, C.I. basic blue 26, chloranil, crystal violet, leucocrystal violet, dichlone, ethyl violet, methylene blue, new methylene blue, Nile blue, pararosaniline base, proflavine, proflavine hydrochloride, rhodamine 6G and trypan red are covered by the guidance document and belong to group I. A toxicological screening value of 0.0025 μg/kg body weight per day is applicable. Azure blue and potassium permanganate were excluded from the evaluation due to their inorganic nature.

Leuco-crystal-violet micelle gel dosimeters: Component effects on dose-rate dependence

Designed experiments were performed to produce empirical models for the dose sensitivity, initial absorbance, and dose-rate dependence respectively for leucocrystal violet (LCV) micelle gel dosimeters containing cetyltrimethylammonium bromide (CTAB) and 2,2,2-trichloroethanol (TCE). Previous gels of this type showed dose-rate dependent behaviour, producing an ∼18% increase in dose sensitivity between dose rates of 100 and 600 cGy min-1. Our models predict that the dose rate dependence can be reduced by increasing the concentration of TCE, CTAB and LCV. Increasing concentrations of LCV and CTAB produces a significant increase in dose sensitivity with a corresponding increase in initial absorbance. An optimization procedure was used to determine a nearly dose-rate independent gel which maintained high sensitivity and low initial absorbance. This gel which contains 33 mM CTAB, 1.25 mM LCV, and 96 mM TCE in 25 mM trichloroacetic acid and 4 wt% gelatin showed an increase in dose sensitivity of only 4% between dose rates of 100 and 600 cGy min-1, and provides an 80% greater dose sensitivity compared to Jordan’s standard gels with similar initial absorbance.

Non-diffusing radiochromic leuco-crystal violet hydrogel dosimeter

A systematic study to prepare mixed-micelle, radiochromic hydrogels found that non-diffusing dosimeters can be prepared. Subsequent experiments determined that sodium dodecyl sulfate (SDS) a negatively charged surfactant, binds positively charged crystal violet dye to gelatin below the critical micelle concentration. A typical formulation includes: 4% gelatin by mass, 1 mM hydrogen peroxide, 1 mM SDS, 1 mM leuco crystal violet (LCV) and 25 mM trichloroacetic acid (TCAA). This transparent material has an initial attenuation coefficient of 0.08 cm-1 and dose sensitivity of 0.015 cm-1 Gy-1. Which is a doubling of the dose sensitivity from the initial formulation with uncharged Triton X100 micelles. Reconstructed beam profiles from 3D optical CT scans performed 1, 14 and 85 hours post irradiation demonstrate no diffusion of the recorded dose distribution.

Direct inclusion of triphenylmethane and triphenylsilane into the pores of zeolite Y

A mixture of triphenylmethane (CHPh3, 1) and zeolite Y was heated to 423 K. The treatment resulted in the inclusion of 1 in the supercages of zeolite Y, up to 18 wt% (1.2 molecules of 1 per supercage). Similar phenomena were observed not only for 1 but also for the derivatives of 1 and for triphenylsilane (7). The inclusion of 1 was completed in less than 0.5 h, while the inclusion of 7 proceeded slowly (∼12 h), probably due to the bulk of 7. The included amounts of 1 and 7 tended to increase with a decreasing concentration of Al in zeolite Y. The Fourier-transform infrared spectra of 7-Y reveal the presence of three types of guest molecules, based on the appearance of three Si–H peaks in the Si–H stretching region. Calculations using molecular dynamics revealed that each of the three phenyl groups of 1 was inserted into the 12-membered rings of zeolite Y to form inclusion compounds. The catalytic activity of leucomalachite green and leucocrystal violet for the Knoevenagel reaction was significantly enhanced by the inclusion of these molecules into the SCs.

Evaluation of Radiochromic Gel Dosimeter and Optical CT

Three-dimensional (3D) radiation dosimeter has received growing interest with the implementation of highly conformal radiotherapy treatments. Optical Computed tomography (CT) scanners have been developed for 3D quantitative measurement of optical attenuation. Malachite green and Leuco Crystal Violet (LCV) dye radiochromic gels were recently developed as radiation dosimeters in combination with optical CT scanners. Malachite green and LCV are colorless dyes and they become green or violet after irradiation, respectively. The dyes mixed with gelatin and surfactant became hardened and can be used as a 3D gel dosimeter. The radiochromic gels dosimeter with optical CT scanners provides many of desired features such as: low cost, easy to fabricate, low toxicity and fast readout. In this article, the fundamental characteristics of radiochromic gel dosimeter with optical CT scanners are reviewed.

Isolation and Characterization of Microorganisms that Degrade Dimethoate

Microorganisms have the ability to degrade a large number of pollutants found in the environment. Microorganisms such as bacteria can degrade toxic compounds and chemicals in an environment friendly way. Dimethoate is an organophosphate pesticide. It is a broad-spectrum insecticide. In the present study, dimethoate degrading microorganisms have been isolated from various soil samples. The different isolates that were obtained were characterized by various biochemical tests. Standardization of degradation was performed. Leuco crystal violet reagent was used as an indicator and colorimetric analysis was done at 595 nm. In total, 8 isolates were got. 3 belonged to Bacillus sp. and 3 were identified as Staphylococcus aureus. 2 isolates were identified as Aspergillus sp. HPLC analysis was carried out for the organism that showed the best degradation results.

Trichoderma asperellum laccase mediated crystal violet degradation–Optimization of experimental conditions and characterization

Biodegradation of crystal violet (CV) by Trichoderma asperellum laccase was herein reported. The process parameters viz., enzyme concentration, dye concentration, pH and incubation time were experimentally optimized for the maximum dye degradation. The degradation of CV was physico-chemically characterized by UV–vis spectroscopy, FTIR, HPTLC, HPLC and GC–MS analyses. In UV–vis spectroscopy, a major reduction in characteristic peak from 588nm to 575nm has suggested the disappearance of triphenyl methane chromophore. From the FT-IR spectra, the formation of new peaks at 3707cm−1 (OH stretching) and 1726cm−1 (CO stretching) indicates the successful transformation of the crystal violet. Similarly, the existence of a prominent peak (Rf−0.39) in HPTLC and a significant reduction in peak intensity of crystal violet had suggested the laccase mediated dye degradation. GC–MS analysis of crystal violet degraded metabolites were identified as Leucocrystal violet, Olivetol dimethyl ether (1,3-Dimethoxy-5-pentylbenzene) and 1,3,5-trimethoxybenzene by a hydride ion (H-) transfer followed by oxidative methylation. The genotoxicity studies by Allium cepa assay has shown that the degraded intermediates of CV are minimally toxic than the original dye. Owing to the above-discussed significance, T.asperellum laccase may be considered for the development of more environmental friendly treatment processes.

Development and Validation of a Monoclonal Antibody-Based Indirect Competitive ELISA for the Detection of Sudan I in Duck Eggs and Crystal Violet in Carp

In this study, two specific monoclonal antibodies, one for Sudan I and the other for leucocrystal violet, were prepared and then used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of two illegal synthetic dyes: Sudan I in duck egg and crystal violet in fish muscle. The Sudan I-specific antibody 1H7 was of the IgG2b isotype and had an IC50 of 3.1 μg L−1. Additionally, the crystal violet-specific antibody 4B10 was of the IgG2a isotype and had an IC50 of 5.3 μg L−1. Based on these antibodies, the limit of detection (LOD), recoveries, and coefficient of variation of the ic-ELISA developed for the detection of Sudan I residues in duck egg samples were 0.6 μg kg−1, 83.6∼113.8 %, and less than 8.4 %, and the values of the ic-ELISA developed for the detection of leucocrystal violet residues in fish muscle samples were 1.2 μg kg−1, 90.8∼108.4 %, and less than 12.3 %, respectively. Both assays exhibited a positive correlation (r > 0.99) with the results of HPLC-MS run on the samples. These data indicate that the developed ic-ELISAs are reliable and will be very reliable screening methods for the detection of the illegal dyes Sudan I and crystal violet in duck egg and fish muscle, respectively.

Temperature sensitive molecularly imprinted microspheres for solid-phase dispersion extraction of malachite green, crystal violet and their leuko metabolites

This article demonstrates the feasibility of an alternative strategy for producing temperature sensitive molecularly imprinted microspheres (MIMs) for solid-phase dispersion extraction of malachite green, crystal violet and their leuko metabolites. Thermo-sensitive MIMs can change their structure following temperature stimulation. This allows capture and release of target molecules to be controlled by temperature. The fabrication technique provides surface molecular imprinting in acetonitrile using vinyl modified silica microspheres as solid supports, methacrylic acid and N-isopropyl acrylamide as the functional monomers, ethyleneglycol dimethacrylate as the cross-linker, and malachite green as the template. After elution of the template, the MIMs can be used for fairly group-selective solid phase dispersion extraction of malachite green, crystal violet, leucomalachite green, and leucocrystal violet from homogenized fish samples at a certain temperature. Following centrifugal separation of the microspheres, the analytes were eluted with a 95:5 mixture of acetonitrile and formic acid, and then quantified by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with isotope internal calibration. The detection limits for malachite green, crystal violet and their metabolites typically are 30 ng·kg−1. Positive samples were identified by UHPLC-MS/MS in the positive ionization mode with multiple reaction monitoring. The method was applied to the determination of the dyes and the respective leuko dyes in fish samples, and accuracy and precision were validated by comparative analysis of the samples by using aluminum neutral columns.