Determination of tuberculosis-related volatile organic biomarker methyl nicotinate in vapor using fluorescent assay based on quantum dots and cobalt-containing porphyrin nanosheets.

Methyl nicotinate (MN) is a representative and typical volatile organic marker of Mycobacterium tuberculosis, and the specific detection of MN in human breath facilitates non-invasive, rapid, and accurate epidemic screening of tuberculosis infection. Herein, we constructed a fluorescent assay consisted of CdTe quantum dots (QD) and cobalt-metalized tetrakis(4-carboxyphenyl) porphyrin (CoTCPP) nanosheets to determine methyl nicotinate (MN) in vapor samples. Red-emission QD (λex=370 nm, λem=658 nm) acts as signal switches whose fluorescence signals can be effectively quenched by CoTCPP nanosheets but restored in the presence of MN. The strategy relied on the distinct binding affinity of cobalt ion and MN. MN restored the fluorescence of QD quenched by CoTCPP in a concentration-dependent manner, which exhibited a well-linear relationship in the range 1-100 µM, and a limit of detection of 0.59 µM. The proposed platform showed sensitivity and selectivity to detect MN in vapor samples with satisfactory RSD below 3.33%. The method is cheap, simple, and relatively rapid (detected within 4 min), which suggests a potential in tuberculosis diagnosis in resource- and professional-lacked areas.

Simultaneous determination of tazarotene, clindamycin phosphate and their active metabolites in Bama mini-pig skin by LC-MS/MS: Application to the development of a tazarotene/clindamycin phosphate cream.

Topical tazarotene combined with clindamycin phosphate can significantly improve the adherence and outcomes for the treatment of acne vulgaris than monotherapy, a novel tazarotene (0.05%)/clindamycin phosphate (1.2%) cream is thus developed. However, the pharmacokinetics and potential interaction of tazarotene and clindamycin phosphate in skin when formulated together remain unknown, which should be investigated to assess this novel cream. In the present work, a sensitive and rapid LC-MS/MS method for simultaneous determination of tazarotene, clindamycin phosphate and their active metabolites tazarotenic acid, clindamycin in Bama mini-pig skin was developed and reported for the first time. After pretreatment of the skin samples, the analytes were well separated on a Hypersil BDS C8 column (4.6 × 100 mm, 2.4 µm) using 0.2% (v/v) formic acid-0.1% (w/v) ammonium acetate water solution and acetonitrile as mobile phase in linear gradient elution. Quantification of tazarotene, clindamycin phosphate and their active metabolites tazarotenic acid, clindamycin was conducted under positive electrospray ionization mode using multiple reactions monitoring detection. The LC-MS/MS method was fully validated and then applied to the dermal pharmacokinetic study of the tazarotene/clindamycin phosphate cream. According to the obtained results, tazarotene and clindamycin phosphate did not have any drug-drug interaction when they were formulated together in the cream for topical application. Their absorption and metabolism features in the skin were also characterized, which can support the clinical medication regimen of tazarotene/clindamycin phosphate cream.

Development of selective preconcentration/clean-up method for imidazolinone herbicides determination in natural water and rice samples by HPLC-PAD using an imazethapyr imprinted poly(vinylimidazole-TRIM).

The development of a novel molecularly imprinted solid-phase extraction (MISPE) method for simultaneous preconcentration of imazapyr (IMP), imazapic (IMZ) and imazethapyr (IMT) with determination by HPLC-PAD (High performance liquid chromatography - photodiode-array detector) is proposed. The polymer synthesis was performed using imazethapyr as template molecule and 1-vinylimidazole as functional monomer. The method is based on preconcentration of 100.0 mL of sample through 200.0 mg of molecularly imprinted poly(vinylimidazole-TRIM) (MIP-1VN) at pH 4.0, followed by elution with 2.0 mL of MeOH:CH2Cl2:HAc (34:62:4, v/v). The range of analytical curve (0.29-200.0, 0.21-200.0 and 0.15-200.0 µg L-1), limits of detection (0.09, 0.06 and 0.04 µg L-1) and preconcentration factors (92, 96 and 98) determined for the herbicides, IMP, IMZ and IMT, respectively, were greatly superior when compared with those ones obtained with commercial adsorbents. The analytical method was successfully applied to spiked surface water and rice samples with good results of recovery values (86-107%).

Insights into the degradation chemistry of tazarotene, a third generation acetylenic retinoid: LC-HRMS (Orbitrap), LC-MSn and NMR characterization of its degradation products, and prediction of their physicochemical and ADMET properties.

Tazarotene is a prodrug that belongs to the acetylenic class of retinoids. The drug was subjected to hydrolytic, oxidative and photolytic stress testing to establish its comprehensive degradation chemistry. The drug proved to be unstable under acidic and basic hydrolytic conditions, yielding tazarotenic acid, which is a known major degradation product (DP) and an active metabolite. Additionally, two DPs each were generated upon interaction of drug and tazarotenic acid with HCl, used as an acid stressor. These were experimentally proven as pseudo DPs, as they did not originate when H2SO4 was employed as the stressor. The drug was also unstable under oxidative and photolytic conditions, yielding six DPs. All the products were separated on reversed phase (C18) column, using mobile phase composed of 10 mM ammonium formate (pH 3.5) and acetonitrile, which was run in a gradient mode. The separated DPs were subjected to LC-HRMS and LC-MSn studies for their initial characterization. Seven hydrolytic and oxidative DPs that could be isolated using semi-preparative column were subjected to extensive 1D (1H, 13C and DEPT-135) and 2D (COSY, HSQC and HMBC) NMR studies to confirm their structures. In total, five novel DPs were characterized, apart from two previously reported DPs, viz., tazarotenic acid and tazarotene sulfoxide, and four additional pseudo DPs. The complete degradation pathway of the drug was established. In silico ADMET properties of the drug and its DPs were evaluated using ADMET Predictor™.

UV spectrophotometric method for simultaneous estimation of betamethasone valerate and tazarotene with absorption factor method: Application for in-vitro and ex-vivo characterization of lipidic nanocarriers for topical delivery.

The present study elucidates the development of an accurate, precise and simple simultaneous estimation method for the routine analysis of Betamethasone Valerate (BV) and Tazarotene (TZ). This combination is widely used in the treatment of psoriasis. No method has been reported so far for the simultaneous estimation of BV and TZ in topical dosage forms. The method proposed by this study for the quantification of BV and TZ is the Absorption factor method. The developed method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline. The validated method was found to be linear in a concentration range of 10-38 µg/mL and 4-14 µg/mL for BV and TZ respectively with a regression coefficient >0.990. The method was validated for accuracy and precision which revealed the recovery of >99.80% with RSD <2.0. The method was found to be precise with RSD <2% for inter and intraday. The developed method was employed for quantification of BV and TZ in lipid based nanocarriers formulation and their in-vitro drug release samples. Further, the developed method was successfully applied for the estimation of BV and TZ in the ex-vivo skin matrix. This showed that the method can sensitively determine the drugs in aqueous and biological samples.

Review on Characteristics and Analytical Methods of Tazarotene: An Update.

Tazarotene (TZR) is the first topical receptor-selective retinoid prodrug derived from vitamin A used for management of plaque psoriasis and efficacious in dealing of acne vulgaris, and photo aging. As per US food and drug administration (FDA), 0.1% strength of TZR is permitted for the treatment of acne. This article draws attention to various advanced and conventional analytical methods. The hyphenated and conventional chromatographic techniques such as LC-MS/MS and HPTLC, HPLC respectively. Moreover, spectrophotometric methods like UV/visible spectroscopy also used to quantify TZR as active pharmaceutical ingredient and its formulations, especially in topical preparations. Moreover, the TZR is required alternative methods for routine quality control and to estimate TZR in pharmaceutical dosage form especially in pharmacokinetic studies of topical preparation. This write up focus on critical review of characteristics, uses and the information about the physicochemical, pharmacokinetics properties, mechanisms of action and more emphasis on different analytical methods for estimation of TZR in pharmaceutical formulations.

Adsorption-Desorption Behavior of Polar Imidazolinone Herbicides in Tropical Paddy Fields Soils.

Analysis of herbicides sorption behavior in soil is critical in predicting their fate and possible harmful side effects in the environment. Application of polar imidazolinone herbicides is growing in tropical agricultural fields. Imidazolinones have high leaching potential and are persistent. In this study, adsorption-desorption of imazapic and imazapyr herbicides were evaluated in different types of Malaysian agricultural soils. Effects of soil parameters were also investigated on the soils' sorption capacities. The adsorption data fitted best to Freundlich isotherm (R2 > 0.991). The herbicides adsorptions were physical and spontaneous processes as ΔG values were negative and below 40 kJ/mol. The adsorption correlated positively with clay content, total organic carbon (TOC) content, and cation exchange capacity (CEC). There were strong negative correlations between hysteresis index and these factors indicating their importance in imidazolinones immobilization and, thus, their pollution reduction in the environment.

Stability indicating liquid chromatographic method for simultaneous quantification of betamethasone valerate and tazarotene in in vitro and ex vivo studies of complex nanoformulation.

Low-potency corticosteroid betamethasone valerate and vitamin-A tazarotene are used in combination for effective treatment of psoriasis. There is no robust high-performance liquid chromatography analytical technique available for simultaneous estimation of betamethasone valerate and tazarotene in conventional and nanocarriers based formulations. A simple, accurate, robust isocratic high-performance liquid chromatography method was developed for simultaneous estimation of betamethasone valerate and tazarotene in topical pharmaceutical formulations. The developed method was validated as per the regulatory guidelines. The validated method was linear over the concentration range of 150-6000 ng/mL (r2  > 0.999) at 239 nm wavelength. Limits of detection and quantification of two analytes were 50 and 150 ng/mL, respectively. The %relative standard deviation for intraday and interday precision was less than 2%. The method was also evaluated in the presence of forced degradation conditions. The developed method was successfully applied for in vitro and ex vivo drug release studies of in-house designed nanoformulations.

Quantification and In Silico Toxicity Assessment of Tazarotene and its Impurities for a Quality and Safe Drug Product Development.

Tazarotene is internationally accepted common name for ethyl 6-[(4,4-dimethylthiochroman-6-yl)ethynyl]nicotinate. It is a synthetic retinoid used for the topical treatment of mild to moderate plaque psoriasis, acne vulgaris and photo aging. To ensure the quality of drug product and drug substance, a LC-MS compatible UHPLC method was developed for quantification of drug and its related substances. Stationary phase with fused core particle technology is used for the separation of impurities. Limit of quantification and limit of detection of the method are 0.1 and 0.03%, respectively. Precision of the method for Tazarotene and all its related substances is less than 2.2% RSD. The correlation coefficient is >0.999. Accuracy of method is ranged from 95.3% to 107.0%. Application of this method in stability analysis has been demonstrated by analyzing stressed samples. Experimental design is used for the verification of robustness of the method. To ensure the safety, an in silico toxicity of the drug and its related substances were determined using TOPKAT and DEREK toxicity predictions Both UHPLC and in silico methods were validated as per the ICH Q2 and ICH M7 guidelines, which will enable a rapid product development of Tazarotene topical formulations while ensuring the safety and quality of product.

Development of a simple method for simultaneous determination of tazarotene and betamethasone dipropionate and their metabolites using LC-MS method and its application to dermatopharmacokinetic study.

In our study, a method for the determination for tazarotene and betamethasone dipropionate in human tissue-engineered skin was established. Tazarotene gel, betamethasone dipropionate cream or a combination cream was administered to the skin. Then the skin was taken off at 0.25, 0.75, 1.75, 3, 5, 8, 12, 24, 36, 48 h time points after the residual drug was removed. The concentrations of tazarotene, betamethasone dipropionate and their major metabolites in skin were determined by LC-MS. Tazarotene and tazarotenic acid were detected in the concentration range of 2-200 µg/mL with an LLOQ of 2 µg/mL. Betamethasone dipropionate was detected in the concentration range 0.5-300 µg/mL with an LLOQ of 0.5 µg/mL, and betamethasone was detected at 2-200 µg/mL with an LLOQ of 2 µg/mL. The intra- and inter-day precisions of the four analytes in the skin homogenate were all <15% (RSD, %). The results showed that tazarotene could be metabolized to tazarotenic acid and betamethasone dipropionate could be metabolized to betamethasone in tissue-engineered skin. The results also revealed that this method was suitable for the simultaneous determination of tazarotene, betamethasone dipropionate and their metabolites in tissue-engineered skin.

Validation and application of a QuEChERS based method for estimation of half-life of imidazolinone herbicides in soils by LC-ESI-MS/MS.

A modified QuEChERS (quick, easy, cheap, effective, rugged and safe) method was validated and applied for the estimation of half-lives of two imidazolinone group herbicides, imazethapyr and imazapic, in the Dystric Plinthosol (FX) and Ferralsol (LVA) agricultural soils using liquid chromatography tandem mass spectrometry. The solutions were prepared in a matrix extract to avoid matrix effects. The analytical method showed satisfactory specificity, selectivity, linearity (R2 > 0.99), recoveries (range 85.0-117.0%), with RSD between 6.7% and 16.6%, and precision (range 94.7-108.5%), with RSD between 6.0% and 11.9%. The limit of detections for imazethapyr and imazapic in the soils were 2.2 µg kg-1 and 2.0 µg kg-1, and the limit of quantifications were 6.6 µg kg-1 and 6.1 µg kg-1. The half-lives of imazethapyr (35.7 and 97.9 days) and imazapic (40.4 and 64.4 days), in the FX and LVA soils, respectively, indicate that they are medium-persistence herbicides with possibility of leaching into groundwater. In addition, high concentrations of imazethapyr and imazapic were found in the soil samples after the time of application, meaning that there is a potential for prolonged soil residual activity due to carryover.

Behavior of pre-mix formulation of imazethapyr and imazamox herbicides in two different soils.

Imidazolinone group herbicides are known for longer persistence in soil. Therefore, a laboratory study was performed to evaluate the persistence of pre-mix formulation of two imidazolinone herbicides-imazethapyr and imazamox in clay and sandy loam soils. Herbicide formulation was applied at 70 and 140 g a.i. ha-1 equivalent to recommended doses in legumes. For achieving efficient sample preparation, three methods namely ultrasonic-assisted extraction (UAE), matrix solid phase dispersion (MSPD), and solid phase extraction (SPE) were optimized. MSPD gave better recoveries (85.22 to 96.00%) over SPE (80.10 to 84.78%) and UAE (56.44 to 66.20%). Residues were estimated using gas chromatography tandem mass spectrometry (GC-MS/MS) which is previously not reported in open literature. Dissipation followed first-order kinetics and half-life period of 23.5 to 43.3 days in clay loam and 19.6 to 39.8 days in sandy loam soil. The results revealed the persistent nature of pre-mix formulation of both herbicides as only 64.2 to 86.6% residues dissipated after 90 days of application in both soils.

Determination of nitenpyram and 6-chloronicotinic acid in environmental samples by ion chromatography coupled with online photochemically induced fluorescence detector.

A simple, cost-effective, sensitive, and quick method for the determination of nitenpyram and its metabolite 6-chloronicotinic acid in environmental samples was developed by coupling an ion chromatograph with a fluorescence detector and a post-column photochemical reactor. This developed analytical method involved a rapid sample extraction by modified and miniaturized quick, easy, cheap, effective, rugged, and safe method followed by isocratic ion chromatographic separation of nitenpyram and 6-chloronicotinic acid into an IonPac™ AS11-HC column protected by IonPac™ AG11A guard column by running 30 mM NaOH + 10% acetonitrile mobile phase. A homemade post-column photochemical reactor was also integrated with the ion chromatographic system for online transformation of both analytes into their respective highly fluorescent photoproduct in basic media without using an extra pump. The developed method was validated by following SANTE/11945/2015 guidelines on analytical quality control and validation procedures. The method showed a good linear response (r > 0.999), improved limit of detection (0.101-0.132 µg/L), minimum or no matrix effect, excellent recoveries (90.2-100.10%) and relative standard deviations were found to be ≤6.50%.

Mechanism of the effect of pH and biochar on the phytotoxicity of the weak acid herbicides imazethapyr and 2,4-D in soil to rice (Oryza sativa) and estimation by chemical methods.

The existing form of an ionizable organic compound can simultaneously affect its soil adsorption and plant bioactivity. In this experiment, the adsorption and bioactivity of two weak acid herbicides (WAHs), imazethapyr and 2,4-D, were studied to explore the predominant mechanism by which the soil pH and the addition of biochar can influence the phytotoxicity of WAHs in soil. Then, the WAH concentration extracted by hollow fiber-based liquid-phase microextraction (CHF-LPME), the in situ pore water concentration (CIPW) and the added concentration (CAC) were employed to estimate the phytotoxicity. The results showed that with increased pH from 5.5 to 8.5, the phytotoxicity of the WAHs to rice increased about 1-fold in the soil, but decreased in aqueous solutions, the IC50 values for imazethapyr and 2,4-D at pH 5.0 were 3- and 2-fold higher than that at pH 8.0. In addition, the soil adsorption decreased, indicating that the adsorption process was the dominant factor for the variation of the phytotoxicity of the WAHs in the tested soil instead of the decreasing bioactivity. The concentration that inhibits plant growth by 50% (IC50) calculated by the CAC in different pH and biochar soils ranged from 0.619 to 3.826 mg/kg for imazethapyr and 1.871-72.83 mg/kg for 2,4-D. The coefficient of variation (CV) of the IC50 values reached 65.61% for imazethapyr and 130.0% for 2,4-D. However, when IC50 was calculated by CIPW and CHF-LPME, the CVs of the IC50 values decreased to 23.51% and 36.23% for imazethapyr and 40.21% and 50.93% for 2,4-D, respectively. These results suggested that CIPW and CHF-LPME may be more appropriate than CAC for estimating the phytotoxicity of WAHs.

Imazethapyr persistence in sandy loam detected using white mustard bioassay.

Field experiments were conducted during two years at Srem region to investigate the influence of meteorological conditions, time and rate of application on soil persistence of imazethapyr in sandy loam type of soil. Imazethapyr was applied PRE- and POST-EM and in both cases in three application rates: 80, 120 and 160 g a.i./ha. Soil samples were collected from the day of herbicide application in predetermined intervals up to one year after application and residual concentrations were determined with a white mustard root bioassay. Imazetapyr persistence was significantly influenced by meteorological conditions with average half-life being 6 days longer in season with lower precipitation level. Time of application induced slower imazethapyr dissipation resulting in higher average t1/2 (seven and nine days in first and second year of examination, respectively). Application rates had no consistent effect on imazethapyr persistence. Imazethapyr residue level one year after application caused no visible injuries on white mustard shoots, while root growth reduction ranged from 4.6 to 27.7%. Obtained residue levels were further compared with known data on crop sensitivity in order to assess possibility of crop injuries one year after imazethapyr application.

Integrated Analytical and Statistical Two-Dimensional Spectroscopy Strategy for Metabolite Identification: Application to Dietary Biomarkers.

A major purpose of exploratory metabolic profiling is for the identification of molecular species that are statistically associated with specific biological or medical outcomes; unfortunately, the structure elucidation process of unknowns is often a major bottleneck in this process. We present here new holistic strategies that combine different statistical spectroscopic and analytical techniques to improve and simplify the process of metabolite identification. We exemplify these strategies using study data collected as part of a dietary intervention to improve health and which elicits a relatively subtle suite of changes from complex molecular profiles. We identify three new dietary biomarkers related to the consumption of peas (N-methyl nicotinic acid), apples (rhamnitol), and onions (N-acetyl-S-(1Z)-propenyl-cysteine-sulfoxide) that can be used to enhance dietary assessment and assess adherence to diet. As part of the strategy, we introduce a new probabilistic statistical spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference Matching), that uses 2D J-resolved 1H NMR spectra for enhanced information recovery using the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a reference. RED-STORM provided new information for subsequent experiments (e.g., 2D-NMR spectroscopy, solid-phase extraction, liquid chromatography prefaced mass spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate the benefit of acquiring J-resolved experiments alongside conventional 1D 1H NMR as part of routine metabolic profiling in large data sets and show that application of complementary statistical and analytical techniques for the identification of unknown metabolites can be used to save valuable time and resources.

Improvement of Nicotinic Acid and Nicotinamide Analysis in Meats and Meat Products by HPLC and LC-MS/MS with Solid-Phase Extraction.

A method for nicotinic acid (NA) and nicotinamide (NAA) analysis in meats was developed. NA and NAA were extracted from meats or meat products with metaphosphate aqueous solution. The extract was cleaned up with an Oasis MCX cartridge. The cartridge was washed with 2% acetic acid (v/v) and acetic acid-methanol solution. NA and NAA were eluted with ammonia-methanol solution. NA and NAA in the eluate were chromatographed on a Scherzo SM-C18 (3.0×150 mm, 3.0 µm) column with 20 mmol/L ammonium acetate containing 0.1% acetic acid-acetonitrile (97 : 3) as a mobile phase and were monitored at 261 nm. Quantification was performed by LC and LC-MS/MS. Calibration curves showed high linearity (correlation coefficient>0.998) between 1-25 µg/mL for LC and LC-MS/MS. Recoveries were 84-108% (CV≦5.8%) by HPLC and 79-105% (CV≦9.0%) by LC-MS/MS. The limit of quantitation for NA was 0.005-0.01 g/kg and that for NAA was 0.01-0.02 g/kg.

Determination of contents of four alkaloids in Pericarpium arecae by quantitative analysis of multi-components by single-marker.

By using a typical component in traditional Chinese medicine Pericarpium Arecae (PA), quantitative analysis of multi-components by single-marker (QAMS) was performed to determine the contents of four alkaloids. With a column packed with strong cation exchange bonded silica particles, the alkaloids were well separated, showing linear relationships within certain ranges. The limit of detection, limit of quantitation, precision, stability, repeatability and recovery all met requirements. By employing arecoline as internal standard, relative correction factors for arecaidine, guvacine and guvacoline at five concentrations were detected with three HPLC systems and three HPLC columns. The peaks of arecaidine, guvacine and guvacoline were positioned, during which the columns with the same packing materials from different manufacturers significantly affected relative retention values and retention time differences of the alkaloids. However, the columns, from different batches, managed to give relative retention values satisfying the requirements of HPLC peak positioning. The Thermo Fisher Scientific column packed with strong cation exchange bonded silica particles was finally selected by considering resolution and peak time. Compared with the external standard method, QAMS detected the alkaloid contents in 12 PA samples more accurately and reliably. The results provide valuable evidence for content determination and quality control of alkaloids in PA.

Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

Imazapyr+imazapic herbicide determines acute toxicity in silver catfish Rhamdia quelen.

Imazapyr (IMY) and imazapic (IMI) are imidazolinone herbicides which have been associated in a commercial formulation (Kifix(®)). To date, there are no studies on the toxicity of an IMY+IMI herbicide in fish. This work aimed to assess the acute toxicity (24 and 96 h) of IMY+IMI (0, 0.488 and 4.88 µg/L) towards Rhamdia quelen through hematological, biochemical, immunological, ionoregulatory and enzymatic indexes. Red blood cell count was lower at 4.88 than at 0.488 µg/L (24 and 96 h); mean corpuscular volume was lower than control at both concentrations (24 h) and at 0.488 µg/L (96 h); lymphocytes declined at 4.88 µg/L comparing to control (96 h); and monocytes increased at 4.88 µg/L (96 h) in comparison with the respective control and with 4.88 µg/L at 24h. Aspartate aminotransferase was higher at 0.488 µg/L (96 h) than the respective control and the respective concentration at 24 h; uric acid reduced at 4.88 µg/L comparing with 0.488 µg/L (96 h); and cortisol was lower at 4.88 µg/L compared to 0.488 µg/L and control (96 h). Herbicide exposure lowered plasma bactericidal activity at both concentrations (24 h) and at 0.488 µg/L (96 h); and plasma complement activity declined at 4.88 µg/L comparing with 0.488 µg/L and control (96 h), and was lower at all concentrations at 96 h than at 24 h. Plasma K(+) levels were higher at 4.88µg/L than in the remaining groups (24 and 96h); and Na(+) levels decreased at 4.88 µg/L compared to control (96 h). Na(+)/K(+)-ATPase and H(+)-ATPase activities in gills were lower at 4.88 µg/L comparing with control (24 h) and with the respective concentration at 96 h; and AChE activity in brain was higher at 0.488 and 4.88 µg/L than control (24 h) and the respective concentrations at 96 h, while in muscle it was higher at 0.488 and 4.88 µg/L than control (96 h) and the respective concentrations at 24 h. The present findings demonstrate that, despite IMY+IMI targets the animal-absent AHAS enzyme, such formulation displayed an acute toxic effect upon R. quelen homeostasis by impacting on vital functions such as immune defense, metabolism, ionoregulation and neurotransmission.