Fast and sensitive detection of nitroxynil using a chalcone-based supramolecular fluorescent sensor.

Nitroxynil as a veterinary drug has been widely used for treatment of parasitic worms in food-producing sheep and cattle. However, the residual nitroxynil in edible animal products can lead to severe adverse effects on human health. Thus, development of an effective analytical tool for nitroxynil is of great significance. In the present study, we designed and synthesized a novel albumin-based fluorescent sensor, which was capable of detecting nitroxynil with the fast response (<10 s), high sensitivity (limit of detection ∼8.7 ppb), high selectivity, and excellent anti-interference property. The sensing mechanism was clarified by using the molecular docking technique and mass spectra. Moreover, this sensor showed the detection accuracy comparable to standard HPLC method, and meanwhile exhibited much shorter response time and higher sensitivity. All the results demonstrated that this novel fluorescent senor could serve as a practical analytical tool for determination of nitroxynil in real food samples.

A portable paper-based testing device for fast and on-site determination of nitroxynil in food.

Nitroxynil (NTX) is a common anthelmintic veterinary drug for the management of fascioliasis in food-producing sheep and cattle. Since excessive NTX residue in food can lead to several adverse side effects, such as allergic skin reaction and respiratory irritation, it is of great importance to develop an efficient analytical method for NTX determination. Herein, we report a simple fluorescent detection method based on a novel supramolecular probe capable of detecting NTX with a fast response (5 s), high sensitivity (107 nM), high selectivity, and acceptable anti-interference property. Moreover, the portable paper-based test strips were facilely prepared and successfully realized on-site determination of NTX in real edible animal products simply with the aid of a smartphone. To the best of our knowledge, this is the very first report on the portable detection of NTX. This study also provides a promising strategy for the fast and portable detection of analyte based on the host-guest system, which will lead to improved fluorescent probe design for food analysis.

Spectrofluorometric determination of residual amounts of nitroxynil in food samples.

Because of the frequent treatment of dairy cows with nitroxynil (NTX), the presence of the later residues in animal tissues and milk has a significant concern. The quantum yield of the reaction product was calculated. A highly sensitive and rapid spectrofluorometric method for determining the anthelmintic drug (NTX) residual amounts is developed. The proposed approach is based on using Zn/HCl to reduce NTX nitro group to an amino group, resulting in a highly fluorescent derivative that was detected at each of λem 302 nm and 364 nm after excitation at λex = 277 nm. The experimental conditions were carefully studied and optimized. The proposed approach showed good linearity (r2 ≥ 0.9998) with linearity range of 10.0-100.0 ng/mL with a detection limit of 1.89 ng/mL, 1.27 ng/mL and quantification limit of 5.73 ng/mL, 3.86 ng/mL at λem 302 nm and 364 nm, respectively; that is far below the Minimum Regulatory Limits (MRLs) of NTX in animal tissues and milk. The proposed approach was successfully applied for the analysis of the drug in its veterinary formulations, and the obtained results agreed well with those of the official British Pharmacopeia method. Moreover, the proposed method's application was extended to efficiently determine the residues of nitroxynil in meat, liver, kidney and milk samples.

A rapid colloidal gold-based immunochromatographic strip assay for monitoring nitroxynil in milk.

BACKGROUND: Nitroxynil (NIT) is a veterinary drug against hepatic fluke disease for food-producing cattle and sheep. NIT has a long half-life time in animals since it is highly bound to plasma protein. Therefore NIT possibly remains in animal edible tissues or milk due to drug abuse. In this study, a specific murine monoclonal antibody (mAb) against NIT was prepared and an immunochromatographic strip assay based on the mAb was developed for screening NIT in milk. RESULTS: The affinity constant of the anti-NIT mAb was 2.93 × 1010 and the anti-NIT mAb had almost no cross-reactivity with other analogs, so that it showed good specificity. The cutoff value of this test strip was considered to be 50 ng mL-1 by the naked eye. When detected by the strip reader, the half maximum inhibition concentration (IC50 ) of the immunoassay strip was calculated to be 5.716 ng mL-1 and the limit of detection (LOD) was 1.146 ng mL-1 . Intra-assay recoveries from 88.80 to 97.13% were obtained, with the highest coefficient of variation (CV) at 9.01%; inter-assay recoveries ranged from 84.60 to 106.87%, with the highest CV at 9.93%. CONCLUSION: The operative procedure of the proposed method can be completed within 10 min. The strip developed in this study was a practical tool for rapid semiquantitative and quantitative detection of NIT in milk. This study suggested great potential for analytically monitoring NIT in other food samples. © 2019 Society of Chemical Industry.

Determination of nitroxynil residues in tissues and bovine milk by immunobiosensor.

Nitroxynil is an anthelmintic drug mainly used for the control of liver fluke in sheep and cattle. The European Commission has established maximum residue limits in bovine and ovine muscle (400 µg kg(-1)), fat (200 µg kg(-1)), liver (20 µg kg(-1)) and kidney (400 µg kg(-1)), and more recently in bovine and ovine milk (20 µg kg(-1)). To ensure that these limits are not exceeded through incorrect use of the drug, it is necessary to monitor samples using robust and reliable methods capable of low-level detection. An inexpensive and rapid immunobiosensor-based screening procedure, capable of high sample throughput, was developed that is capable of detecting nitroxynil at <10 µg kg(-1) in bovine milk, at <10 µg kg(-1) in bovine liver, and at <200 µg kg(-1) in bovine and ovine muscle. The methods were fully validated and the milk assay was utilised in a comparison study of nitroxynil-incurred samples.

Investigation of the persistence of nitroxynil residues in milk from lactating dairy cows by ultra performance liquid chromatography tandem mass spectrometry.

Nitroxynil is an anthelmintic used in the treatment of liver fluke. In this study, six dairy cows were treated during lactation with Trodax, a 34% solution containing nitroxynil as its N-ethylglucamine salt, indicated for the treatment of fascioliasis in cattle and sheep. Samples were collected twice daily for 16 days and later at weekly intervals up to 58 days post-treatment. Nitroxynil residues were extracted from milk samples using acetonitrile; magnesium sulfate and sodium chloride were added to induce liquid-liquid partitioning and purified by dispersive solid phase extraction for clean-up. Nitroxynil was determined by ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in negative ionization mode. The limit of detection (CCα) of the method is 0.24 µg/kg. Maximum concentration of nitroxynil in the samples was in the range of 688-1358 µg/kg, with levels persisting for 58 days in four of the six lactating cows. Incurred nitroxynil samples were treated with sulfatase and ß-glucuronidase from Helix pomatia ; the results indicated the presence of glucuronide conjugates in samples at early withdrawal times. At later withdrawal times the concentration of free nitroxynil was lower than the concentration in the control samples, indicating potential degradation during enzymatic treatment.

Determination of bithionol, bromophen, nitroxynil, oxyclozanide, and tribromsalan in milk with liquid chromatography coupled with tandem mass spectrometry.

LC/MS/MS was developed to determine the residues of bithionol (BTN), bromofen (BMF), nitroxynil (NTX), oxyclozanide (OCZ), and tribromsalan (TBS) in milk. Samples were extracted with ethyl acetate and cleaned up by liquid-liquid separation with acetonitrile and n-hexane. The compounds were determined by RP-LC using a C18 column with 0.1% formic acid-methanol. Mass spectral acquisition was performed in the negative mode by applying selected-reaction monitoring. The method was validated in milk spiked with these compounds at 5-600 microg/kg; average recoveries were in the range 83.8-97.1%, with RSD values of 1.4-8.0%. The interassay RSDs were less than 11%. The LODs of these compounds in milk were 0.1 microg/kg. The method was applied to 24 raw milk samples. The concentration of these compounds in all samples was lower than the Japanese maximum residue limits. The method is rapid, sensitive, and specific for monitoring residues of BTN, BMF, NTX, OCZ, and TBS in milk.

Voltammetric assay of the anthelmintic veterinary drug nitroxynil in bulk form and formulation at a mercury electrode.

The electrochemical behavior of the anthelmintic veterinary drug nitroxynil at the mercury electrode was studied in a series of Britton-Robinson universal buffer of pH 1.9-11 containing 20% (v/v) ethanol using dc-polarography cyclic voltammetry and controlled-potential coulometry. The voltammograms exhibited two irreversible cathodic steps over the pH range 1.9-10.2; the height of the first step is double that of the second one. Controlled-potential coulometry in the B-R universal buffer of pH 1.9-10 at a mercury pool working electrode revealed the consumption of four and two electrons via the first and second reduction steps, respectively, which attributed to reduction of the NO2 group to the hydroxylamine stage (first step), and then to the amine stage (second step). Three voltammetric analytical procedures including dc-polarography, differential-pulse adsorptive stripping voltammetry and square-wave adsorptive stripping voltammetry were optimized for the direct determination of bulk nitroxynil. The three proposed procedures were applied for analysis of bulk nitroxynil with limits of detection of 3 x 10(-5), 1.31 x 10(-8) and 8.4 x 10(-10)M and limits of quantification of 1 x 10(-5), 4.36 x 10(-8) and 2.80 x 10(-9)M, respectively. The three procedures were successfully applied to the determination of nitroxynil in formulation (Dovenix, 25% nitroxynil injection solution) without the necessity for sample pretreatment and/or time-consuming extraction steps prior to the analysis.

Determination of nitroxynil in cow milk by reversed-phase high-performance liquid chromatography with dual-electrode coulometric detection.

A sensitive and specific method is described for the determination of nitroxynil (fasciolicide) residues in cow milk. The milk samples were extracted with acetone and acetonitrile, following clean-up using a simple liquid-liquid extraction step. Nitroxynil was separated from the matrix peaks by reversed-phase high-performance liquid chromatography and detected using dual-electrode coulometric detection. The mobile phase was a mixture (20:80, v/v) of acetonitrile and 0.05 M potassium dihydrogenphosphate with the pH adjusted to 4.0. The flow-rate was 1 ml/min at 40 degrees C. The applied potentials of detectors 1 and 2 were maintained at -0.7 and +0.2 V, respectively. Average recoveries (n = 5) of nitroxynil from milk samples spiked at concentrations of 0.01 and 0.1 micrograms/ml were found to be 92.0% and 97.0% with coefficients of variation of 6.2% and 2.2%, respectively. The detection limit of nitroxynil in milk was 0.7 ng/ml. During the analysis of 30 raw cow milk and 140 market milk samples, nitroxynil was detected at a level of 4 ng/ml in one raw cow milk sample.

[Investigation of the residue of the fasciolicide nitroxynil in milk and dairy products].

A sensitive gas chromatographic method with electron capture detection of the fasciolicide, nitroxynil, in milk and dairy products was developed and was applied to assess nitroxynil concentration in cow's milk after subcutaneous injection of three lactating cows. The level of nitroxynil in cow's milk reached a maximum (0.25-0.26 micrograms/ml) in 6-30 hours, and was undetectable within 8 weeks. Analysis of nitroxynil concentrations in cream, skimmed milk, curd and whey prepared from nitroxynil excreting cow's milk showed that the chemical became concentrated in cream and curd. Nitroxynil appeared to be stable at temperature used in LTLT and HTST pasteurization with the rate of degradation in milk being less than 10%. Investigation of nitroxynil residues in milk (raw, liquid whole, processed) and dairy products (processed cheese, natural cheese, butter, sweetened condensed milk, evaporated skimmed milk, skimmed milk powder, formulated milk powder) was performed during 1976-1979. Nitroxynil was detected in 20% of milk samples at a maximum level of 39 ng/ml, with one formulated milk powder at level of 0.34 ng/g. Confirmation of nitroxynil was performed by GCMS. The results of this investigation were promptly reported to the government. Since then administrative guidance was provided leading to appropriated use of nitroxynil thereafter. The ordinance controlling dairy production amended in 1979 revised the withdrawal time for medicines administrated to cows from "three days" to "the period when medicine remains in the milk". There was no mutagenicity of nitroxynil evident by the Ames test.

Determination of nitroxynil residues in tissues using high-performance liquid chromatography-thermospray mass spectrometry.

A method is described for the determination of nitroxynil residues in muscle, liver and kidney. The samples were extracted into diethyl ether and cleaned-up using a simple liquid-liquid extraction step. Any nitroxynil present was separated from interfering compounds by high-performance liquid chromatography and detected using thermospray mass spectrometry. The assay is specific and sensitive, with a detection limit of 2 ng g-1 in tissues.

[Residues of the antifluke preparations, dovenix and acredist, in sheep milk and their effect on the biochemical blood indices of sheep]. / Ostatuchni kolichestva ot protivometilnite preparati doveniks i atsedist v ovche mliako i vliianieto im vurkhu niakoi biokhimichni pokazateli na kruvta na ovtse.

The excretion of residual amounts of acedist and dovenix with the milk of sheep that had been treated with these preparations was established on the 24th, resp., up to the 27th day following treatment, though their presence in the milk was found to drop sharply after the third day. The treatment with acedist led to a dependable decrease (P less than 0.05) in the hemoglobin amount, the total protein, and the erythrocyte count, and a dependable increase (P less than 0.05) in the leukocyte count. The intensity of these changes subsided parallel to the decrease in the residual amounts of the preparation in the milk excreted. The treatment with dovenix did not result in any deviations of the indices refered to.