Fragment-imprinted microspheres for the extraction of sulfonamides

Fragment-imprinted microspheres (FIMs) were synthesized by suspension polymerization of 4-sulfa-6-chloropyrimidine (the template), methacrylic acid and styrene (the mixed functional monomers), divinylbenzene (the crosslinker), and azobisisobutyronitrile (the initiator). The optimum conditions were obtained by an orthogonal experiment. After removal of the templates, the microspheres serve as a material to fairly specifically bind sulfonamides, the absorption capacity being 6.32 mg g^–1, whereas the absorption by non-imprinted microspheres is 1.68 mg g^?1. A method was worked out for the simultaneous determination of five sulfonamides by solid phase extraction (using the FIMs) coupled to HPLC, and applied to analyze milk samples. The recoveries are within 87.6 and 96.5 %, with relative standard deviations between 1.25 and 2.89 %.

A portable electrochemical magnetoimmunosensor for detection of sulfonamide antimicrobials in honey

A new electrochemical magnetoimmunosensor (EMIS) has been developed for the screening of residues of sulfonamide antimicrobials in honey samples. The immunosensor is able to detect up to ten different sulfonamide congeners at levels below the action points established in some European countries (25 μg kg^?1) after a hydrolysis step in which the sulfonamides are released from the corresponding conjugates formed in samples of this type. In spite of the complexity of the sample after the hydrolysis procedure, the EMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. For example, sulfapyridine, used as a reference, can be detected in hydrolyzed honey with a limit of detection (IC₉₀) of 0.1?±?0.03 μg kg^?1. Considering that the use of antibiotics for bee treatment is prohibited in the European Union, the immunosensor presented here could be an excellent screening tool. Moreover, several samples can be processed in parallel, which facilitates the analysis, reducing the necessity to use more costly confirmatory methods for just screening. As a proof of concept, a set of blind honey samples (spiked and incurred) were analyzed and the results were compared with those obtained by high-performance liquid chromatography–tandem mass spectrometry, demonstrating the potential of the EMIS as a screening tool.

Assessing hazelnut allergens by protein- and DNA-based approaches: LC-MS/MS,ELISA and real-time PCR

Hazelnut (Corylus avellana L.) is responsible for a significant part of the allergies related to nuts. Still, it is a very much appreciated nut and as consequence is widely used in all types of processed foods, such as chocolates. Correct food labelling is currently the most effective means of preventing the consumption of allergenic ingredients, namely hazelnut, by the sensitised/allergic individuals. Thus, to verify labelling compliance and to ensure allergic patient protection, the development of highly sensitive methodologies is of extreme importance. In this study, three major methodologies, namely enzyme-linked immunosorbent assays (ELISA), liquid chromatography coupled with mass spectrometry and real-time polymerase chain reaction, were evaluated for their performance regarding the detection of hazelnut allergens in model chocolates. The sandwich ELISA and respective antibodies were in-house developed and produced. With sensitivity levels of approximately 1 mg kg^?1 and limits of quantification of 50–100 mg kg^?1, all the performed methods were considered appropriate for the identification of hazelnut in complex foods such as chocolates. To our knowledge, this was the first successful attempt to develop and compare three independent approaches for the detection of allergens in foods.

Gas chromatography–tandem mass spectrometry analysis of 52 monohydroxylated metabolites of polycyclic aromatic hydrocarbons in hairs of rats after controlled exposure

A method based on gas chromatography–tandem mass spectrometry after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide was developed for the analysis of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in hair. The method focused on 52 target compounds corresponding to two- to six-ring monohydroxylated metabolites of polycyclic aromatic hydrocarbons (PAHs). The limits of quantification ranged from 0.2 to 50 pg mg^?1. The method was then applied to the analysis of hair samples collected from rats exposed to 12 PAHs at 0.01, 0.1, and 1 mg kg^?1, by intraperitoneal injection, for 28 days. The results of this study confirm that these metabolites can be incorporated in hair after intraperitoneal administration of the corresponding parent compound. Only 20 of the 52 metabolites were actually detected in hair samples and corresponded to nine parent PAHs. The mean concentrations of OH-PAHs in rat hair samples exposed to PAHs at 1 mg kg^?1 ranged from 0.6?±?0.2 pg mg^?1 for 8-hydroxybenzo[b]fluoranthene to 6.7?±?1.0 pg mg^?1 for 1-hydroxypyrene. The results also demonstrated that hair pigmentation has no influence on the concentration of most OH-PAHs. This animal experiment confirmed the incorporation of PAH metabolites in hair and demonstrated that the method was sufficiently sensitive to detect low levels of exposure to PAHs. These results confirmed the usefulness of hair analysis in the biomonitoring of human exposure to PAHs.

Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity

Immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have suffered from high selectivity for individual sulfonamides and a wide range of selectivities for different sulfonamides. In this study, five synthesized haptens, HS, BS, CS, SA10, and TS and two sulfonamides, SG and SMX were used as haptens, which may or may not contain a ring structure at the N1 position of the sulfonamides, were selected to evaluate the effectiveness for producing group-specific monoclonal antibodies (MAbs). Mice immunized with three different two-ring haptens were used for hybridoma production, which resulted in three unique MAbs recognizing 10, 13, and 15 sulfonamides showing 50 % inhibition (IC₅₀) at concentrations below 100 ng?mL^–1. MAb 4D11 derived from one novel immunizing hapten could recognize 12 sulfonamides with IC₅₀ values ranging from 1.2 to 12.4 ng?mL^–1, almost within 1 order of magnitude. These produced MAbs show lower IC₅₀ values in addition to significantly improved group specificity compared with previously generated MAbs. This study clearly indicates that the careful selection of the immunizing hapten has an important effect on the specificity of the generated antibodies.

Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg^?1 and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg^?1. Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg^?1 (15 %). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n?=?6), mean intra-day precision 3.2 % (n?=?3 within a plate; n?=?2 repeated within a day), mean inter-day precision 3.7 % (n?=?3), mean reproducibility over the whole procedure including sample preparation 4.1 % (n?=?2), and mean recovery 106.9 % (n?=?5 different concentrations; n?=?4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2 %. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8 %. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.

A sensitive and specific ELISA for determining a residue marker of three quinoxaline antibiotics in swine liver

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is a possible residue marker for three quinoxaline veterinary medicines (olaquindox, mequindox, and quinocetone). The wide application of mequindox/quinocetone or the illegal use of olaquindox leads to MQCA residue in animal’s original food, thereby threatening the safety of human food. The indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) with a specific coating antigen and monoclonal antibody (MAB) was established and optimized for detecting MQCA in swine liver. Samples were acidified with 2 mol?l^?1 hydrochloric acid, extracted with ethyl acetate–hexane–isopropanol (8?+?1?+?1, v/v/v) and then detected by IC-ELISA. The logarithm correlation of standards to OD values ranged from 0.2 to 200 μg?l^?1, with IC₅₀ of 6.46 μg?l^?1. Negligible cross-reactivity happened to five quinoxaline antibiotics (olaquindox, mequindox, quinocetone, carbadox, and cyadox) and the metabolite of carbadox and cyadox (quinoxaline-2-carboxylic acid). When spiked with 1 to 100 μg?kg^?1 of MQCA, the recoveries ranged from 85.44 to 100.02 %, with the intra-assay coefficient of variation (CV) of 6.64–10.57 % and inter-assay CV of 7.29–10.88 %. The limit of detection for MQCA was 1.0 μg?kg^?1 in swine liver. Furthermore, incurred samples were detected by the IC-ELISA and then conformed by a reported LC/MS/MS method, it shown that there was good correlation between the two methods. All these results indicated that the IC-ELISA method is appropriate for surveillance MQCA residue in animal tissues.

Simultaneous determination of sulfonamides and metabolites in manure samples by one-step ultrasound/microwave-assisted solid–liquid–solid dispersive extraction and liquid chromatography–mass spectrometry

An in-line matrix cleanup method was used for the simultaneous extraction of 15 sulfonamides and two metabolites from manure samples. The ultrasound/microwave-assisted extraction (UMAE) combined with solid–liquid–solid dispersive extraction (SLSDE) procedure provides a simple sample preparation approach for the processing of manure samples, in which the extraction and cleanup are integrated into one step. Ultrasonic irradiation power, extraction temperature, extraction time, and extraction solvent, which could influence the UMAE efficiency, were investigated. C₁₈ was used as the adsorbent to reduce the effects of interfering components during the extraction procedure. The extracts were concentrated, and the analytes were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) without any further cleanup. The isotopically labeled compounds sulfamethoxazole-d ₄, sulfamethazine-d ₄, sulfamonomethoxine-d ₄, and sulfadimethoxine-d ₆ were selected as internal standards to minimize the matrix effect in this method. The recoveries of the antibiotics tested ranged from 71 to 118 % at the three spiking levels examined (20, 200, and 500 μg?·?kg^-1). The limits of detections were 1.2–3.6 μg?·?kg^-1 and the limits of quantification were 4.0–12.3 μg?·?kg^-1 for the sulfonamides and their metabolites. The applicability of the method was demonstrated by analyzing 30 commercial manure samples. The results indicated that UMAE–SLSDE combined with LC–MS/MS is a simple, rapid, and environmentally friendly method for the analysis of sulfonamides and their metabolites in manure, and it could provide the basis for a risk assessment of the antibiotics in agricultural environments.     

Study on the PK profiles of magnoflorine and its potential interaction in Cortex phellodendri decoction by LC-MS/MS

Magnoflorine, an aporphine alkaloid in Cortex phellodendri, is increasingly attracting research attention because of its antidiabetic effects. However, at present, little information on its pharmacokinetics (PK) in vivo is available. In this study, a sensitive, rapid, and selective method was developed to determine the magnoflorine content in rat plasma using liquid chromatography–tandem mass spectrometry. Following liquid–liquid extraction, the calibration curve showed good linearity within the concentration range of 2.93 to 1,500 ng ml^?1. The intra- and inter-day precisions were all below 7.8 %, and the accuracy ranged from 94.9 to 103.4 %. The method was successfully applied in investigating the PK of magnoflorine in rats. The compound had low bioavailability, a high absorption rate, and a high elimination rate. However, area under the curve, T _1/2, and MRT increased approximately twofold when the same dosage of the compound was administered in a C. phellodendri decoction (20.8 g kg^?1). Moreover, T _max was prolonged from 0.3 to 3.33 h. Furthermore, a comparison of coadministration of the mixture group, magnoflorine (40 mg kg^?1) and berberine (696.4 mg kg^?1), with the C. phellodendri decoction group, revealed that no statistical difference (P?>?0.05) was found in the parameter AUC, and certain similar changes in the PK trend to the herbal medicine group were also observed. These results suggested that oral administration of the herbal medicine decreased the absorption and elimination rates of magnoflorine and increased its bioavailability. Berberine played a significant role in interacting with magnoflorine and in affecting the PK profiles of magnoflorine in the C. phellodendri decoction group.

Development of monolith-based stir bar sorptive extraction and liquid chromatography tandem mass spectrometry method for sensitive determination of ten sulfonamides in pork and chicken samples

A highly sensitive method was developed for the simultaneous determination of ten sulfonamides in pork and chicken samples by monolith-based stir bar sorptive extraction (SBSE) coupled to high-performance liquid chromatography tandem mass spectrometry. The samples were freeze-dried and extracted by acetonitrile, then enriched and further extracted by SBSE which was based on poly(vinylphthalimide-co-N,N-methylenebisacrylamide) monolith (SBSE-VPMB) as coating. To achieve optimum extraction performance of SBSE for sulfonamides, several parameters, including pH value and ionic strength in the sample matrix and extraction and desorption time, were investigated in detail. Under the optimal conditions, the limits of detection (S/N?=?3) for target sulfonamides were 1.2–6.1 ng/kg in pork and 2.0–14.6 ng/kg in chicken, respectively. Real samples spiked at the concentration of 0.5 and 5.0 μg/kg showed recoveries above 55 % and relative standard deviations below 12 %. At the same time, the extraction performances of target sulfonamides on SBSE-VPMB were compared with other SBSE based on porous monolith and commercial SBSE.

Preconcentration of ultra-trace amounts of iron and antimony using ion pair solid phase extraction with modified multi-walled carbon nanotubes

Ion pair solid phase extraction was applied to the simultaneous preconcentration of iron and antimony. The ion pairs consisting of FeCl₄ ^? or SbCl₄ ^? anions and the benzyldimethyltetradecyl ammonium cation were formed on the surface of multi-walled carbon nanotubes, then eluted with nitric acid, and the elements finally quantified by ETAAS. The adsorption capacities of the impregnated MWCNTs are 9.2 mg g^?1 for iron and 27.5 mg g^?1 for antimony. The following analytical figures of merit were determined for iron and antimony, respectively: Enrichment factors of 210 and 230, assay precisions of ±5.3 % and ±4.8 %, linear calibration plots from 0.7 to 9.4 and 13.0 to 190 ng L^?1, and detection limits of 0.17 and 3.5 ng L^?1. The method was applied to the determination of iron and antimony in human hair, synthetic sample, and to the certified reference materials gold ore (MA-1b) and trace elements in water (SRM 1643d).

Simultaneous Analysis of Selected Typical Antibiotics in Manure by Microwave-Assisted Extraction and LC–MS n

Analysis of antibiotics in the environment has become an urgent issue. A novel method entailing microwave-assisted extraction (MAE) and high-performance liquid chromatography–electrospray mass spectrometry (LC–MS ⁿ ) has been developed for determination of selected typical antibiotics, including quinolones, sulfonamides, and tetracyclines, in manure. Compared with ultrasonic extraction, MAE significantly increased recovery of fluoroquinolones (63–106%), sulfonamides (64–133%), and tetracyclines (64–109%) from manure. Acetonitrile acidified with formic acid buffer solution (pH 4.0) was used for extraction of swine manure whereas chicken manure was extracted with 0.1 M EDTA–McIlvaine buffer solution. Limits of quantification (LOQ) for all compounds were in the range 5.12–168.4 μg kg^?1 dry matter, which were satisfactory for analysis of all samples. The suitability of the method was assessed by analysis of manure from six different sites.     

Determination of soy proteins in food samples by dispersive solid-phase immunoextraction and dynamic long-wavelength fluorometry

We report on a method for the determination of soy proteins in food samples via dispersive solid-phase immunoextraction using gold-coated magnetic nanoparticles (NPs) as a support. Soy proteins were first extracted using anti-soy protein antibodies immobilized on the NPs, and then quantified by measuring the increase in fluorescence of the long-wavelength fluorophore cresyl violet in the presence of the anionic surfactant sodium dodecyl sulfate at neutral pH in a flow system. The method involves the use of two standard or sample aliquots. The fluorescence intensity of one aliquot is directly measured whereas that of the other aliquot is measured after immunoextraction. The difference between the peak heights of both aliquots serves as the analytical information that is directly proportional to the protein concentration. The limit of detection is 0.35 mg L^?1, the linear range is from 1 to 15 mg L^?1, and the relative standard deviation is <?5 %. Proteins such as bovine serum albumin and globulins do not interfere at the same concentration level. The method was applied to the analysis of soy-based beverages and gave recoveries in the range between 80.0 and 107.3 %.

A colorimetric probe for ascorbic acid based on copper-gold nanoparticles in electrospun nylon

We report on a colorimetric probe based on copper-gold alloy nanoparticles (NPs). The probe is capable of selectively detecting ascorbic acid (AA) as a result of the distance-dependent colour change of the nanoparticles immobilized in an electrospun nylon-6 nanofiber. The resulting white nanofibres undergo a colour change to blue as a result of the aggregation of the NPs induced by AA in the pH range 2–7. The probe is selective for AA even in the presence of dopamine, uric acid, saccharides, amino acids and certain organic acids. It covers the 1.76 x10^?2 mg L^?1 to 1.76 x10⁵ mg L^?1 concentration range, and exhibits a limit of detection of 1.76 x10^?2 mg L^?1 based on visual detection. Its application was demonstrated by the determination of ascorbic acid in fruit juices, urine, serum, and vitamin C tablets.

Matrix Segregation as the Major Cause for Sample Inhomogeneity in MALDI Dried Droplet Spots

The segregation in dried droplet MALDI sample spots was analyzed with regard to the matrix-to-sample ratio using optical microscopy, MALDI imaging mass spectrometry (MALDI MSI) and IR imaging spectroscopy. In this context, different polymer/matrix/solvent systems usually applied in the analysis of synthetic polymers were investigated. The use of typical matrix concentrations (10 mg mL^?1) in almost every case resulted in ring patterns, whereas higher concentrated matrix solutions always led to homogeneous sample spot layers. The data revealed that segregation is predominantly caused by matrix transport in the drying droplet, whereas polymer segregation seems to be only secondary.

Miniaturized and green method for determination of chemical oxygen demand using UV-induced oxidation with hydrogen peroxide and single drop microextraction

We report on a green method for the determination of low levels of chemical oxygen demand. It is based on the combination of (a) UV-induced oxidation with hydrogen peroxide, (b) headspace single-drop microextraction with in-drop precipitation, and (c) micro-turbidimetry. The generation of CO₂ after photolytic oxidation followed by its sequestration onto a microdrop of barium hydroxide gives rise to a precipitate of barium carbonate which is quantified by turbidimetry. UV-light induced oxidation was studied in the absence and presence of H₂O₂, ultrasound, and ferrous ion. Determinations of chemical oxygen demand were performed using potassium hydrogen phthalate as a model compound. The optimized method gives a calibration curve that is linear between 3.4 and 20 mg L^?1 oxygen. The detection limit was 1.2 mg L^?1 of oxygen, and the repeatability (as relative standard deviation) was around 5 %. The method was successfully applied to the determination of chemical oxygen demand in different natural waters and a synthetic wastewater.

Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment

A chemiluminescent competitive indirect enzyme-linked immunosorbent assay, based on a mutant single-chain variable fragment (scFv), was developed to detect a broad range of fluoroquinolones (FQs) in fish and shrimp matrices. In this study, the best scFvC4A9H1_mut2 was adopted, which showed 10-fold improved affinity to sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO), while the affinity to other FQs was fully inherited from wild-type scFvC4A9H1. In the optimized generic test, scFvC4A9H1_mut2 in combination with norfloxacin–ovalbumin conjugate and horseradish peroxidase-labeled anti-c-myc 9E10 antibody showed 50 % binding inhibition (IC₅₀) at 0.12 μg kg^?1 for norfloxacin in buffer. Screening for the class of FQ antibiotics is accomplished using a simple, rapid extraction carried out with ethanol/acetic acid (99:1, v/v). This common extraction was able to detect 20 FQ residues such as s ciprofloxacin (CIP), danofloxacin, DIF, enoxacin, enrofloxacin (ENR), fleroxacin, amifloxacin, flumequine, levofloxacin, lomefloxacin hydrochloride, marbofloxacin, norfloxacin (NOR), ofloxacin, orbifloxacin, pazufloxacin, pefloxacin-d5 (PEF), prulifloxacin, SAR, sparfloxacin, and TRO in fish and shrimp. The limit of detection (LOD) for NOR was 0.2 μg kg^?1 and the LODs for CIP and ENR were all <0.2 μg kg^?1. Values of LODs inferred from the cross-reactivity data will range from approximately 0.23 μg kg^?1 for PEF to 2.1 μg kg^?1 for TRO. Field fish and shrimp samples were analyzed and compared to the results obtained from liquid chromatography tandem mass spectrometric method. All five instances (from 0.25 to 15.6 μg kg^?1) in which FQs were present at concentrations near or above the assay LOD were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool.

Graphene oxide functionalized magnetic nanoparticles as adsorbents for removal of phthalate esters

We show that magnetic nanoparticles can be functionalized with graphene oxide (GO-MNPs) in two reaction steps, and that such nanoparticles can be used as adsorbents for the removal of phthalate esters (PAEs) from water samples. The GO-MNPs were characterized by scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, zeta potential, and vibrating sample magnetometer. The impacts of contact time, sample pH, ionic strength and sample volume on the adsorption process were investigated. The maximum adsorption capacity for diethyl phthalate was calculated to be 8.71 mg g^?1 according to the Langmuir adsorption isotherm. The adsorption efficiency was tested by removal of PAEs. More than 99 % of the total quantity of PAEs (0.12 mg L^?1) in 500 mL real water samples can be removed when GO-MNPs (275–330 mg) were used as an adsorbent. In addition, other species (estriol and fluorene) containing benzene rings were also almost completely removed with the PAEs using GO-MNPs, indicating that GO-MNPs are suitable for the removal of the species containing π-electron system through π-π interactions.

Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of β-lactam antibiotics in foods

β-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of β-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by β-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual β-lactam was known. The IC₅₀ values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 μg L^?1. Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected β-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 μg kg^?1, respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 β-lactams in edible tissues, among which 11 β-lactams controlled by European Union could be detected below maximum residue limits.

Determination and pharmacokinetic study of the diacid metabolite of norcantharidin in beagle plasma by use of liquid chromatography–tandem mass spectrometry

Because norcantharidin (NCTD) is unstable and subject to ring opening and hydrolysis, the diacid metabolite of norcantharidin (DM-NCTD) is the stable form of NCTD found in normal saline solution. Conversion of NCTD to DM-NCTD is almost 100 %, making it possible to determine and investigate the pharmacokinetics of DM-NCTD converted from NCTD. In this paper, a sensitive, simple and selective liquid chromatographic–tandem mass spectrometric method was developed and validated for determination of DM-NCTD in beagle plasma. DM-NCTD was detected in multiple-reaction monitoring (MRM) mode by using the dehydrated ion 169.3 as precursor ion and its product ion 123.1 as the detected ion. Ribavirin was used as internal standard and detected in MRM mode by use of precursor ions, resulting in a product ion transition of m/z 267.1?→?135.1. This method was successfully used for a pharmacokinetic study of DM-NCTD in beagles after intravenous administration of DM-NCTD in normal saline solution at doses of 0.39, 0.78, and 1.6 mg kg^?1. DM-NCTD had dose-dependent kinetics across the dosage range investigated, with enhanced T _1/2α and AUC_0-12 and apparently decreasing V _d and CL with increasing dosage. After single-dose administration, T _1/2α ranged from 0.20 to 0.55 h, AUC_0-12 from 1.81 to 43.6 μg mL^?1 h^?1, V _d from 228 to 55.9 mL kg^?1, and CL from 220 to 36.5 mL kg^?1 h^?1 (P?<?0.01). The results indicated nonlinear pharmacokinetic behavior of DM-NCTD in beagles, suggesting that the risk of DM-NCTD in normal saline solution intoxication may be non-proportionally increased at higher doses.