Removal of enrofloxacin using Eichhornia crassipes in microcosm wetlands.

The global consumption of antibiotics leads to their possible occurrence in the environment. In this context, nature-based solutions (NBS) can be used to sustainably manage and restore natural and modified ecosystems. In this work, we studied the efficiency of the NBS free-water surface wetlands (FWSWs) using Eichhornia crassipes in microcosm for enrofloxacin removal. We also explored the behavior of enrofloxacin in the system, its accumulation and distribution in plant tissues, the detoxification mechanisms, and the possible effects on plant growth. Enrofloxacin was initially taken up by E. crassipes (first 100 h). Notably, it accumulated in the sediment at the end of the experimental time. Removal rates above 94% were obtained in systems with sediment and sediment + E. crassipes. In addition, enrofloxacin was found in leaves, petioles, and roots (8.8-23.6 µg, 11-78.3 µg, and 10.2-70.7 µg, respectively). Furthermore, enrofloxacin, the main degradation product (ciprofloxacin), and other degradation products were quantified in the tissues and chlorosis was observed on days 5 and 9. Finally, the degradation products of enrofloxacin were analyzed, and four possible metabolic pathways of enrofloxacin in E. crassipes were described.

Binding the gap between experiments, statistics, and method comparison: A tutorial for computing limits of detection and quantification in univariate calibration for complex samples.

The present tutorial aims to review the most frequently reported criteria for the calculation of the limits of detection (LOD) and quantification (LOQ) in univariate calibration, summarizing their fundamentals, advantages, and limitations. The current criteria for estimating LOD and LOQ are based on diverse theoretical and/or empirical assumptions and require different amounts of experimental data, making the calculation rather complex in some cases. Moreover, alternative forms for calculating LOD/LOQ frequently lead to dissimilar results. This scenario might worsen in the case of complex analytical systems. Throughout this tutorial, different forms of calculating LOD/LOQ are illustrated using previously reported experimental datasets in the environmental chemistry field as examples. The influence of the sample matrix during the estimation of LOD/LOQ parameters is investigated through one calibration approache. The discrepancies in the obtained results with different criteria for the calculation of LOD/LOQ are highlighted. Finally, general guidelines and recommendations regarding experimental and data processing issues are proposed, aiming to promote fair criteria for the comparison of different analytical methodologies in terms of prediction ability and detection capability.

Natural deep eutectic solvent: A novelty alternative as multi-walled carbon nanotubes dispersing agent for the determination of paracetamol in urine.

This work reports for the first time the analytical performance of glassy carbon electrodes (GCE) modified with a dispersion of multi-wall carboxylated carbon nanotubes (MWCNTs-COOH) using a mixture of a natural deep eutectic solvent (NADES - LGH/lactic acid-glucose-water), ethylene glycol (EG) and water (GCE/MWCNT-LGH-EG) for the determination and N-(4-hydroxyphenyl) acetamide (paracetamol) (APAP) in urine samples. The optimization of both dispersion and measurement conditions was carried out using experimental design. The modified electrode exhibited enhanced current responses, demonstrating excellent electrochemical response towards APAP oxidation compared to MWCNTs-LGH, MWCNTs-EG, MWCNTs-H2O-EtOH and MWCNTs-H2O. The linear dependence between the anodic peak currents and the square root of scan rates over the range of 0.010-0.300 Vs-1 demonstrates that the electro oxidation of APAP occurs under diffusional control. The MWCNT-LGH-EG modified GCE displayed an analytical sensitivity of 10.72 mL µg-1 (r = 0.9994) and a detection limit of 100 ng mL-1 for the selective determination of APAP in urine samples. The proposed electrochemical sensor was successfully applied for quantifying APAP in urine samples in the presence of uric acid. In addition, the accuracy and precision of the method was contrasted against a HPLC reference method.

Determination of residual enrofloxacin in eggs due to long term administration to laying hens. Analysis of the consumer exposure assessment to egg derivatives.

The use of the antibiotic enrofloxacin (ENR) in poultry is controversial. A high-performance liquid chromatography coupled to fast-scanning fluorescence detection (HPLC-FSFD) method for the determination of ENR in egg white, egg yolk, and lyophilized samples was developed. In a first analysis, the long-term administration of ENR (100 days) to laying hens was carried out to determine its presence in egg white, yolk, or both. The predominance of ENR was observed in egg white and variations in the weight of egg white and eggshell was evidenced, showing a potential problem in the industry. Eventually, the presence of ENR was confirmed in commercial lyophilized egg white samples in concentration values around 350 µg kg-1. The consumer exposure assessment was estimated for children, adolescents, and adults. The result displayed that, in an intake of lyophilized egg white with food-producing animals, the %ADI exceeds 100%, showing toxicological levels.

Multivariate optimization of a dispersive liquid-liquid microextraction method for the determination of six antiparasite drugs in kennel effluent waters by using second-order chromatographic data.

Six veterinary active ingredients (imidacloprid, albendazole, fenbendazole, praziquantel, fipronil and permethrin) were extracted and quantified by liquid chromatography with diode array detection in water samples from a wetland system used for the treatment of waste from a dog breeding plant. Response surface methodology, based on least-squares and artificial neural networks modelling, was applied for the optimization of a dispersive liquid-liquid microextraction (DLLME) procedure. Firstly, two experimental designs were built for screening and optimization, respectively. Then, the desirability function was implemented for the simultaneous optimization of the six recoveries (chromatographic areas of the six compounds). The optimum conditions were: 600 µL of acetone (dispersive solvent), 670 µL of dichloromethane (extractant solvent) and 0.6 min of vortex mixing. The preconcentration factor was 37.5. Then, in order to identify and quantify the six drugs, second-order calibration with MCR-ALS modeling of HPLC-DAD data was implemented attaining successful results. The limits of quantification were 4 ng mL-1 for imidaclopril, albendazole and fenbendazole; 8 ng mL-1 for praziquantel and fipronil; and 26 ng mL-1 for permethrin. The developed method allowed the quantitation of the target analytes, even in the presence of unexpected compounds from dirty water samples. The following maximum levels of veterinary drugs were found (in ng mL-1): imidaclopril, 7; albendazole, 46; fenbendazole, 21; praziquantel, 29; fipronil, 29 and permethrin, 217.

Modelling of bioprocess non-linear fluorescence data for at-line prediction of etanercept based on artificial neural networks optimized by response surface methodology.

In the last years, regulatory agencies in biopharmaceutical industry have promoted the design and implementation of Process Analytical Technology (PAT), which aims to develop rapid and high-throughput strategies for real-time monitoring of bioprocesses key variables, in order to improve their quality control lines. In this context, spectroscopic techniques for data generation in combination with chemometrics represent alternative analytical methods for on-line critical process variables prediction. In this work, a novel multivariate calibration strategy for the at-line prediction of etanercept, a recombinant protein produced in a mammalian cells-based perfusion process, is presented. For data generation, samples from etanercept processes were daily obtained, from which fluorescence excitation-emission matrices were generated in the spectral ranges of 225.0 and 495.0 nm and 250.0 and 599.5 nm for excitation and emission modes, respectively. These data were correlated with etanercept concentration in supernatant (measured by an off-line HPLC-based reference univariate technique) by implementing different chemometric strategies, in order to build predictive models. Partial least squares (PLS) regression evidenced a non-linear relation between signal and concentration when observing actual vs. predicted concentrations. Hence, a non-parametric approach was implemented, based on a multilayer perceptron artificial neural network (MLP). The MLP topology was optimized by means of the response surface methodology. The prediction performance of MLP model was superior to PLS, since the first is able to cope with non-linearity in calibration models, reaching percentage mean relative error in predictions of about 7.0% (against 12.6% for PLS). This strategy represents a fast and inexpensive approach for etanercept monitoring, which conforms the principles of PAT.

Enhancement of multianalyte mass spectrometry detection through response surface optimization by least squares and artificial neural network modelling.

In this work, the use of design of experiments and posterior data modelling by artificial neural network (ANN) and least squares (LS) is presented as a suitable analytical tool for the performance optimization of a tandem mass spectrometric detector coupled to ultra-high performance liquid chromatography for the analysis of seventeen veterinary drugs. Firstly, a central composite design was built considering as factors the cone, capillary, extractor and radio frequency voltages of the mass spectrometer in order to obtain a proper combination to improve the sensitivity of the method. Secondly, a one factor design considering the collision voltage was built to define the adequate voltage for each daughter ion. The response surface methodology (RSM) was then applied, and the prediction capability of ANN and LS were compared. As conclusion, the ANN modelling provided better results than LS, both in terms of the ANOVA and predicted areas results. The accuracy of the model prediction was between 85 and 125%, confirming that the estimates of the model were correct, and endorsing the optimization procedure as a suitable way to gather excellent results. The suitability of the new approach and its implications on the simultaneous analysis of seventeen veterinary drugs by ultra-high liquid chromatography coupled to tandem mass spectrometry detection are discussed.

Occurrence, Distribution, and Ecological Risk of Fluoroquinolones in Rivers and Wastewaters.

The use of fluoroquinolones for the treatment of infections in humans and animals has increased in Argentina, and they can be found in large amounts in water bodies. The present study investigated the occurrence and associated ecological risk of 5 fluoroquinolones in rivers and farm wastewaters of San Luis, Santa Fe, Córdoba, Entre Ríos, and Buenos Aires provinces of Argentina by high-performance liquid chromatography coupled to fast-scanning fluorescence detection and ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry detection. The maximum concentrations of ciprofloxacin, enrofloxacin, ofloxacin, enoxacin, and difloxacin found in wastewater were 1.14, 11.9, 1.78, 22.1, and 14.2 µg L-1 , respectively. In the case of river samples, only enrofloxacin was found, at a concentration of 0.97 µg L-1 . The individual risk of aquatic organisms associated with water pollution due to fluoroquinolones was higher in bacteria, cyanobacteria, algae, plants, and anurans than in crustaceae and fish, with, in some cases, risk quotients >1. The proportion of samples classified as high risk was 87.5% for ofloxacin, 63.5% for enrofloxacin, 57.1% for ciprofloxacin, and 25% for enoxacin. Our results suggest that the prevalence of fluoroquinolones in water could be potentially risky for the aquatic ecosystem, and harmful to biodiversity. Environ Toxicol Chem 2019;38:2305-2313. © 2019 SETAC.

Determination of six veterinary pharmaceuticals in egg by liquid chromatography: Chemometric optimization of a novel air assisted-dispersive liquid-liquid microextraction by solid floating organic drop.

Two extraction strategies for albendazole, chloramphenicol, trimethoprim, enrofloxacin, oxitetracycline and nicarbazin in egg were optimized for its quantitation by liquid chromatography. First, two designs were built to find out the optimized condition for the air assisted-dispersive liquid-liquid microextraction based on solidification of organic drop: a fractional factorial and a central composite design. The optimum conditions were 1140µL of water, 125mg of ZnSO4, 1175µL of acetonitrile, 1200µL of methyl alcohol and 740µL of propanone, using 1.00g of homogenized egg and 50µL of 1-dodecanol. Second, two designs were also built to optimize the dispersive liquid-liquid microextraction: a central composite design and a mixture design to set the combination of the re-suspended solvents. The optimum conditions were 1840µL of acetonitrile and 160µL of dichloromethane and the re-suspended mixture consisting in acetonitrile and sodium phosphate buffer 10mmolL-1 pH = 3.50 (30:70v/v).

Exploiting the synergistic effect of concurrent data signals: Low-level fusion of liquid chromatographic with dual detection data.

A low-level data fusion strategy was developed and implemented for data processing of second-order liquid chromatographic data with dual detection, i.e. absorbance and fluorescence monitoring. The synergistic effect of coupling individual information provided by two different detectors was evaluated by analyzing the results gathered after the application of a series of data preprocessing steps and chemometric resolution. The chemometric modeling involved data analysis by MCR-ALS, PARAFAC and N-PLS. Their ability to handle the new data block was assessed through the estimation of the analytical figures of merits achieved in the prediction of a validation set containing fifteen fluorescent and non-fluorescent veterinary active ingredients that can be found in poultry litter. Eventually, the feasibility of the application of the fusion strategy to real poultry litter samples containing the studied compounds was verified.

Simultaneous multi-residue determination of twenty one veterinary drugs in poultry litter by modeling three-way liquid chromatography with fluorescence and absorption detection data.

A method for the simultaneous investigation of twenty one veterinary active ingredients in poultry litter based on MCR-ALS modeling of three-way liquid chromatography with fluorescence and UV detection data is presented. The chromatographic procedure was optimized in terms of both the nature of the organic solvent and the pH of the mobile phase to maximize the resolution of the analytes. In order to improve the simultaneous extraction efficiency of the twenty one veterinary drugs, a simplex-centroid design with combinations of the three components of the extracting mixture, i.e. MeOH, ACN and sodium phosphate buffer 10mmolL-1 pH =3.50, was carried out. The second-order advantage was exploited in the analysis of highly complex samples containing unmodeled components. The qualitative and quantitative results showed that the application of MCR-ALS was appropriate to resolve highly overlapped peaks in the presence of unknown matrix compounds. Limits of quantification, relative errors of prediction (REP) and average recoveries ranging from 0.02 to 0.61µgg-1, 3.09-9.35% and 91.0-105.6%, respectively, were obtained. Eventually, the method was successfully applied to the determination of active ingredients in five poultry litter samples collected from different poultry livestock in Argentina.

Dispersive liquid-liquid microextraction of quinolones in porcine blood: Validation of a CE method using univariate calibration or multivariate curve resolution-alternating least squares for overlapped peaks.

In the previously published part of this study, we detailed a novel strategy based on dispersive liquid-liquid microextraction to extract and preconcentrate nine fluoroquinolones in porcine blood. Moreover, we presented the optimized experimental conditions to obtain complete CE separation between target analytes. Consequently, this second part reports the validation of the developed method to determine flumenique, difloxacin, enrofloxacin, marbofloxacin, ofloxacin, ciprofloxacin, through univariate calibration, and enoxacin, danofloxacin, and gatifloxacin through multivariate curve resolution analysis. The validation was performed according to FDA guidelines for bioanalytical assay procedures and the European Directive 2002/657 to demonstrate that the results are reliable. The method was applied for the determination of fluoroquinolones in real samples. Results indicated a high selectivity and excellent precision characteristics, with RSD less than 11.9% in the concentrations, in intra- and interassay precision studies. Linearity was proved for a range from 4.00 to 30.00 mg/L and the recovery has been investigated at four different fortification levels, from 89 to 113%. Several approaches found in the literature were used to determinate the LODs and LOQs. Though all strategies used were appropriate, we obtained different values when using different methods. Estimating the S/N ratio with the mean noise level in the migration time of each fluoroquinolones turned out as the best studied method for evaluating the LODs and LOQs, and the values were in a range of 1.55 to 4.55 mg/L and 5.17 to 9.62 mg/L, respectively.

Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae.

The ecological risks posed by two ß-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1-1000µgL-1) under standard laboratory conditions for 96h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10µgL-1 of CPX were lower compared to controls (Dunnett post hoc p<0.05) and presented signs of emaciation. Concentrations of 1000µgL-1of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10µgL-1 of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.

Dispersive liquid-liquid microextraction of quinolones in porcine blood: Optimization of extraction procedure and CE separation using experimental design.

A dispersive liquid-liquid microextraction procedure was developed to extract nine fluoroquinolones in porcine blood, six of which were quantified using a univariate calibration method. Extraction parameters including type and volume of extraction and dispersive solvent and pH, were optimized using a full factorial and a central composite designs. The optimum extraction parameters were a mixture of 250 µL dichloromethane (extract solvent) and 1250 µL ACN (dispersive solvent) in 500 µL of porcine blood reached to pH 6.80. After shaking and centrifugation, the upper phase was transferred in a glass tube and evaporated under N2 steam. The residue was resuspended into 50 µL of water-ACN (70:30, v/v) and determined by CE method with DAD, under optimum separation conditions. Consequently, a tenfold enrichment factor can potentially be reached with the pretreatment, taking into account the relationship between initial sample volume and final extract volume. Optimum separation conditions were as follows: BGE solution containing equal amounts of sodium borate (Na2 B4 O7 ) and di-sodium hydrogen phosphate (Na2 HPO4 ) with a final concentration of 23 mmol/L containing 0.2% of poly (diallyldimethylammonium chloride) and adjusted to pH 7.80. Separation was performed applying a negative potential of 25 kV, the cartridge was maintained at 25.0°C and the electropherograms were recorded at 275 nm during 4 min. The hydrodynamic injection was performed in the cathode by applying a pressure of 50 mbar for 10 s.

Plasma retinoids concentration in Leptodactylus chaquensis (Amphibia: Leptodactylidae) from rice agroecosystems, Santa Fe province, Argentina.

Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis of vertebrates: Alteration in endogenous retinoids concentration is linked with teratogenic effects. Retinol (ROH), retinoid acid (RA), and isoform 13-Cis-retinoic acid (13-Cis-RA), in plasma of a native adults frog, Leptodactylus chaquensis from a rice field (RF) and a forest (reference site; RS) were measured. ROH did not vary between treatment sites. RA and 13-Cis-RA activities were higher (93.7±8.6 µg mL(-1) and 131.7±11.4 µg mL(-1), respectively) in individuals collected from RF than in those from RS (65.5±8.6 µg mL(-1) and 92.2±10.2 µg mL(-1), respectively). The ratios retinoic acid-retinol (RA/ROH) and 13-Cis-RA/ROH revealed significantly higher values in RF than in RS. RA and 13-Cis-RA concentrations in plasma on wild amphibian's species such as L. chaquensis would be suitable biomarkers of pesticide exposure in field monitoring. Finally, the mechanism of alteration in retinoid metabolites alteration should be further explored both in larvae and adult, considering that the potential exposition and uptake contaminants vary between the double lives of these vertebrates.

Multiple responses optimization in the development of a headspace gas chromatography method for the determination of residual solvents in pharmaceuticals.

An efficient generic static headspace gas chromatography (HSGC) method was developed, optimized and validated for the routine determination of several residual solvents (RS) in drug substance, using a strategy with two sets of calibration. Dimethylsulfoxide (DMSO) was selected as the sample diluent and internal standards were used to minimize signal variations due to the preparative step. A gas chromatograph from Agilent Model 6890 equipped with flame ionization detector (FID) and a DB-624 (30 m×0.53 mm i.d., 3.00 µm film thickness) column was used. The inlet split ratio was 5:1. The influencing factors in the chromatographic separation of the analytes were determined through a fractional factorial experimental design. Significant variables: the initial temperature (IT), the final temperature (FT) of the oven and the carrier gas flow rate (F) were optimized using a central composite design. Response transformation and desirability function were applied to find out the optimal combination of the chromatographic variables to achieve an adequate resolution of the analytes and short analysis time. These conditions were 30 °C for IT, 158 °C for FT and 1.90 mL/min for F. The method was proven to be accurate, linear in a wide range and very sensitive for the analyzed solvents through a comprehensive validation according to the ICH guidelines.

Rapid determination of retinoic acid and its main isomers in plasma by second-order high-performance liquid chromatography data modeling.

This paper reports the development of a method based on high-performance liquid chromatography (HPLC) coupled to second-order data modeling with multivariate curve resolution-alternating least-squares (MCR-ALS) for quantification of retinoic acid and its main isomers in plasma in only 5.5 min. The compounds retinoic acid (RA), 13-cis-retinoic acid, 9-cis-retinoic acid, and 9,13-di-cis-retinoic acid were partially separated by use of a Poroshell 120 EC-C18 (3.0 mm × 30 mm, 2.7 µm particle size) column. Overlapping not only among the target analytes but also with the plasma interferents was resolved by exploiting the second-order advantage of the multi-way calibration. A validation study led to the following results: trueness with recoveries of 98.5-105.9 % for RA, 95.7-110.1 % for 13-cis-RA, 97.1-110.8 % for 9-cis-RA, and 99.5-110.9 % for 9,13-di-cis-RA; repeatability with RSD of 3.5-3.1 % for RA, 3.5-1.5 % for 13-cis-RA, 4.6-2.7 % for 9-cis-RA, and 5.2-2.7 % for 9,13-di-cis-RA (low and high levels); and intermediate precision (inter-day precision) with RSD of 3.8-3.0 % for RA, 2.9-2.4 % for 13-cis-RA, 3.6-3.2 % for 9,13-di-cis-RA, and 3.2-2.9 % for 9-cis-RA (low and high levels). In addition, a robustness study revealed the method was suitable for monitoring patients with dermatological diseases treated with pharmaceutical products containing RA and 13-cis-RA.

Enhanced high-performance liquid chromatography method for the determination of retinoic acid in plasma. Development, optimization and validation.

When determining endogenous compounds in biological samples, the lack of blank or analyte-free matrix samples involves the use of alternative strategies for calibration and quantitation. This article deals with the development, optimization and validation of a high performance liquid chromatography method for the determination of retinoic acid in plasma, obtaining at the same time information about its isomers, taking into account the basal concentration of these endobiotica. An experimental design was used for the optimization of three variables: mobile phase composition, flow rate and column temperature through a central composite design. Four responses were selected for optimization purposes (area under the peaks, quantity of peaks, analysis time and resolution between the first principal peak and the following one). The optimum conditions resulted in a mobile phase consisting of methanol 83.4% (v/v), acetonitrile 0.6% (v/v) and acid aqueous solution 16.0% (v/v); flow rate of 0.68 mL min(-1) and an column temperature of 37.10 °C. Detection was performed at 350 nm by a diode array detector. The method was validated following a holistic approach that included not only the classical parameters related to method performance but also the robustness and the expected proportion of acceptable results lying inside predefined acceptability intervals, i.e., the uncertainty of measurements. The method validation results indicated a high selectivity and good precision characteristics that were studied at four concentration levels, with RSD less than 5.0% for retinoic acid (less than 7.5% for the LOQ concentration level), in intra and inter-assay precision studies. Linearity was proved for a range from 0.00489 to 15.109 ng mL(-1) of retinoic acid and the recovery, which was studied at four different fortification levels in phuman plasma samples, varied from 99.5% to 106.5% for retinoic acid. The applicability of the method was demonstrated by determining retinoic acid and obtaining information about its isomers in human and frog plasma samples from different origins.

Hybrid hard- and soft-modeling of spectrophotometric data for monitoring of ciprofloxacin and its main photodegradation products at different pH values.

A simple and fast on line spectrophotometric method combined with a hybrid hard-soft modeling multivariate curve resolution (HS-MCR) was proposed for the monitoring of photodegradation reaction of ciprofloxacin under UV radiation. The studied conditions attempt to emulate the effect of sunlight on these antibiotics that could be eventually present in the environment. The continuous flow system made it possible to study the ciprofloxacin degradation at different pH values almost at real time, avoiding errors that could arise from typical batch monitoring of the reaction. On the base of a concentration profiles obtained by previous pure soft-modeling approach, reaction pathways have been proposed for the parent compound and its photoproducts at different pH values. These kinetic models were used as a constraint in the HS-MCR analysis. The kinetic profiles and the corresponding pure response profile (UV-Vis spectra) of ciprofloxacin and its main degradation products were recovered after the application of HS-MCR analysis to the spectra recorded throughout the reaction. The observed behavior showed a good agreement with the photodegradation studies reported in the bibliography. Accordingly, the photodegradation reaction was studied by high performance liquid chromatography coupled with UV-Vis diode array detector (HPLC-DAD). The spectra recorded during the chromatographic analysis present a good correlation with the ones recovered by UV-Vis/HS-MCR method.

A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: chemometric tools for improving the optimization and validation.

The development, optimization and validation of an ion-pairing high performance liquid chromatography method for the simultaneous determination of both nicarbazin (NIC) components: 4,4'-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in bulk materials and feed additives are described. An experimental design was used for the optimization of the chromatographic system. Four variables, including mobile phase composition and oven temperature, were analyzed through a central composite design exploring their contribution to analyte separation. Five responses: peak resolutions, HDP capacity factor, HDP tailing and analysis time, were modelled by using the response surface methodology and were optimized simultaneously by implementing the desirability function. The optimum conditions resulted in a mobile phase consisting of 10.0 mmol L(-1) of 1-heptanesulfonate, 20.0 mmol L(-1) of sodium acetate, pH=3.30 buffer and acetonitrile in a gradient system at a flow rate of 1.00 mL min(-1). Column was an INERSTIL ODS-3 (4.6 mm×150 mm, 5 µm particle size) at 40.0°C. Detection was performed at 300 nm by a diode array detector. The validation results of the method indicated a high selectivity and good precision characteristics, with RSD less than 1.0% for both components, both in intra and inter-assay precision studies. Linearity was proved for a range of 32.0-50.0 µg mL(-1) of NIC in sample solution. The recovery, studied at three different fortification levels, varied from 98.0 to 101.4 for HDP and from 99.1 to 100.2 for DNC. The applicability of the method was demonstrated by determining DNC and HDP content in raw materials and commercial formulations used for coccidiosis prevention. Assays results on real samples showed that considerable differences in molecular ratio DNC:HDP exist among them.