Advanced chemometric methods for simultaneous quantitation of caffeine, codeine, paracetamol, and p-aminophenol in their quaternary mixture.

Two different multivariate techniques have been applied for the quantitative analysis of caffeine, codeine, paracetamol and p-aminophenol (PAP) in quaternary mixture, namely, Partial Least Squares (PLS-1) and Artificial Neural Networks (ANN). For suitable analysis, a calibration set of 25 mixtures with various ratios of the drugs and PAP impurity were established using a 4-factor 5-level experimental design. The most meaningful wavelengths for the chemometric models were chosen using Genetic Algorithm (GA) as a variable selection technique. By using an independent validation set, the validity of the proposed methods was evaluated. A comparative study was established between the three multivariate models (PLS-1, GA-PLS and GA-ANN). The comparison between the various models revealed that the GA-ANN model was superior at resolving the highly overlapped spectra of this quaternary combination. The drugs were successfully quantified in their pharmaceutical dosage form utilizing the GA-ANN models.

A solvent-free HPLC method for the simultaneous determination of Favipiravir and its hydrolytic degradation product.

During COVID-19 pandemic, Favipiravir (FPV) showed a great efficacy against COVID-19 virus, it produced noticeable improvements in recovery of the patients. The aim of this study was to develop a new, green and simple method for the simultaneous determination of FPV and its acid-induced degradation product (ADP) in its pure and pharmaceutical dosage forms. This method will be key for the inevitable development of FPV solution and inhaler formulations. A green micellar RP-HPLC method was developed using an RP-VDSPHERE PUR 100 column (5 µm, 250 × 4.6 mm) and an isocratic mixed micellar mobile phase composed of 0.02 M Brij-35, 0.1 M SDS and 0.01 M potassium dihydrogen orthophosphate anhydrous and adjusted to pH 3.0 with 1.0 mL min-1 flow rate. The detection was performed at 280 nm with a run time of less than six min. Under the optimized chromatographic conditions, linear relationship has been established between peak area and concentration of FPV and its ADP in the range of 5-100 and 10-100 µg mL-1 with elution time of 3.8 and 5.7 min, respectively. The developed method was validated according to the ICH guidelines and applied successfully for determination of FPV in its pharmaceutical dosage form.

Continuous wavelet transform for solving the problem of minor components in quantitation of pharmaceuticals: a case study on the mixture of ibuprofen and phenylephrine with its degradation products.

The presence of minor components represents a challenging problem in spectrophotometric analysis of pharmaceuticals. If one component has a low absorptivity or present in a low concentration compared to the other components, this will hinder its quantitation by spectrophotometric methods. Continuous Wavelet Transform (CWT) as a signal processing technique was utilized to figure out a solution to such a problem. A comparative study was established between traditional derivative spectrophotometry (Numerical Differentiation, ND) and CWT to indicate the advantages and limitations of each technique and possibility of solving the problem of minor components. A mixture of ibuprofen (IBU) and phenylephrine (PHE) with its degradation products forming a ternary mixture was used for comparing the two techniques. The two techniques were applied on raw spectral data and on ratio spectra data resulting in four methods, namely ND, CWT, Derivative Ratio-Zero Crossing (DRZC) and Continuous Wavelet Transform Ratio-Zero Crossing (CWTR-ZC) methods. By comparing the results in laboratory prepared mixtures, CWT technique showed advantages in analysis of mixtures with minor components than ND. The proposed methods were validated according to the ICH guideline Q2(R1), where their linearity was established with correlation coefficient ranging from 0.9995 to 0.9999. The linearity was in the range 3-40 µg/mL for PHE in all methods, while for IBU it was 20-180 and 30-180 µg/mL in CWT and ND methods, respectively. The CWT methods were applied for quantitative determination of the drugs in their dosage form showing the ability of the methods to quantitate minor components in pharmaceutical formulations.

Development of an eco-friendly HPLC method for the stability indicating assay of binary mixture of ibuprofen and phenylephrine.

The development and validation of the stability indicating HPLC technique has contributed to the understanding of the stability profile of ibuprofen (IBU) and phenylephrine (PHE). Stability profile was achieved for PHE; the drug was found to be liable to be influenced by stress oxidative conditions; two oxidative degradants (Deg1 & Deg2) were formed and their structures were confirmed using IR and mass spectrometry. The drugs and degradation products were successfully separated using a gradient elution method on YMC-C8 column with 0.1% hexanesulfonic acid and acetonitrile as a mobile phase at pH 6.6. The flow rate was 1.0 mL/min, and a diode array detector operating at 220 nm was used for UV detection. The retention times of degradants Deg1, Deg2, ibuprofen (IBU), and phenylephrine hydrochloride (PHE) were 2.0, 2.2, 3.2 and 7.0 min, respectively. The proposed method was validated with respect to linearity, accuracy, precision, specificity, and robustness using ICH guidelines. The linearities of ibuprofen and phenylephrine hydrochloride were in the range of 10-100 µg/mL and 0.3-10 µg/mL, respectively. The % recoveries of the two drugs were found to be 100.75 ± 1.44%, 99.67% ± 1.67, and the LOD was found to be 2.75/mL and 0.09/mL for IBU, and PHE, respectively. The method was successfully applied to the estimation of ibuprofen and phenylephrine hydrochloride combination in pharmaceutical dosage form. The proposed technique was validated using ICH guidelines and its greenness was assessed according to Analytical Eco Scale metric (AES). Molecular docking was used to assess the two drugs and PHE oxidative degradants interaction with the stationary phase and to confirm the outcomes of the proposed method with regard to the order of elution of the two drugs and PHE degradation products. Eco-friendly and environmental safety were assessed through the application of one of the most applicable greenness assessment tool; Analytical Eco Scale metric (AES).

Fluorescence imaging approaches for eco-friendly determination of perampanel in human plasma and application for therapeutic drug monitoring.

Antiepileptic drugs are among the most common medications that require therapeutic drug monitoring (TDM). Indeed, TDM provides a realistic approach to adjust drug doses for epilepsy based on plasma concentrations to optimize its clinical outcome. The most common technique for TDM is high-performance liquid chromatography, which has a very low green profile among analytical techniques. Perampanel (PER) is an inherently fluorescent compound that its fluorophore readily allows sensitive and quantitative measurements. This paper describes the development and validation of a sensitive, specific, and eco-friendly spectrofluorimetric method for the determination of PER. Experimental parameters affecting fluorescence intensity of the compound, including solvent dilution, temperature, and excitation wavelength, were studied and optimized. The developed spectrofluorimetric method was established in acetonitrile at λex  = 295 nm and λem  = 431 nm over a concentration range of 5-60 ng/ml. The adopted method was applied for the determination of PER in human plasma; it was effective in the range of 15-50 ng/ml. The proposed method was found to be sensitive and specific for PER and can be applied successfully in TDM of PER and in quality control laboratories.

Dietary supplement mislabelling: case study on selected slimming products by developing a green isocratic HPLC method for their quality control.

Nowadays, a huge population consumes Dietary supplements for losing weight. Products are often claimed as botanical blends, yet they aren't necessarily safe. Misleading labels are also very common. Thus, validated analytical methods for a wide range of slimming compounds are highly needed. Herein, we present a simple HPLC/PDA method for the quantitation of seven popular slimming ingredients. Studied compounds were Caffeine, Raspberry Ketone, trans-Resveratrol, p-Synephrine, p-Octopamine, p-Hordenine and 2-phenethylamine. After optimization, separation was carried out on a C18 column and mobile phase was a mixture of Acetonitrile:Water containing 0.1% phosphoric acid (50:50, %v/v). The last compound was eluted at 9.76 min. Separation was efficient showing baseline- separated symmetric peaks, without using any gradient programs, organic mobile phase modifiers or modified stationary phases. Method validation was done following ICH guidelines. Calibration curves were linear over wide concentration ranges and calculated LOD values were in the range 0.02-0.09 µg/mL. Method greenness was assessed using Analytical Eco-scale, GAPI and AGREE metric tools. Further, four random sample products purchased from online supplement stores were assayed. Results proved some mislabeling actions. To support our findings, standard addition was carried out and average % recoveries were 96.67 - 101.44% with standard deviation ≤ 2.83 between measurements.

Advanced chemometric methods as powerful tools for impurity profiling of drug substances and drug products: Application on bisoprolol and perindopril binary mixture.

Impurity profiling has a rising importance nowadays due to the increased health problems associated with impurities and degradation products found in several drug substances and formulations. Three advanced, accurate and precise chemometric methods were developed as impurity profiling methods for a mixture of bisoprolol fumarate (BIS) and perindopril arginine (PER) with their degradation products which represent drug impurity or a precursor to such impurity. The methods applied were Partial Least Squares (PLS-1), Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Artificial Neural Networks (ANN). Genetic Algorithm (GA) was used as a variable selection tool to select the most significant wavelengths for the three chemometric models. For proper analysis, a 5-factor 5-level experimental design was used to establish a calibration set of 25 mixtures containing different ratios of the drugs and their degradation products (impurities). The validity of the proposed methods was assessed using an independent validation set. The designed models were able to predict the concentrations of the drugs and the degradation products/impurities in the validation set and pharmaceutical formulation. The proposed methods presented a powerful alternative to traditional and expensive chromatographic methods as impurity profiling tools.

Quality control of dietary supplements: An economic green spectrofluorimetric assay of Raspberry ketone and its application to weight variation testing.

Although Dietary supplements are readily accessible and extensively used worldwide, they are inadequately regulated and consumers are victims of manufacturers' fraud. Thus, quality regulations are required to ensure safety of products available to the public. We propose the first native spectrofluorimetric quality control assay of raspberry ketone, a popular dietary supplement ingredient for weight loss. This work relies on the constant wavelength synchronous scan of the Raspberry Ketone native fluorescence, overcoming the demerits of conventional excitation/ emission spectra. For the best measurement conditions, several parameters were optimized including Δλ value, diluting solvent, medium pH and the effect of surfactants/ macromolecules. In aqueous medium (Δλ = 110 nm), a linear relationship exists between synchronous fluorescence intensity at peak maximum 405.6 nm and solution concentration in the range 300-1500 ng/mL. Method sensitivity was recorded with LOD and LOQ values 60.63 and 183.72 ng/mL; respectively. Validation was done in accordance to International Conference on Harmonization (ICH) guidelines. This simple procedure was successfully applied to the analysis of Raspberry Ketone in commercially available dietary supplement capsules with average recovery 98.67% ± 1.74 and further extended to weight variation testing following the official United States Pharmacopeial (USP) guidelines. Finally, green assessment was done using the ''Analytical Eco-scale'' tool. The total score was 89/100 points revealing excellent greenness of our proposal. Our proposal is simple, eco-friendly and cheap. It can be conveniently adopted for routine quality control practices especially in developing countries.

Microfabricated potentiometric sensor for personalized methacholine challenge tests during the COVID-19 pandemic.

The methacholine challenge test is considered to be the gold standard bronchoprovocation test used to diagnose asthma, and this test is always performed in pulmonary function labs or doctors' offices. Methacholine (MCH) acts by inducing airway tightening/bronchoconstriction, and more importantly, MCH is hydrolyzed by cholinesterase enzyme (ChE). Recently, the American Thoracic Society raised concerns about pulmonary function testing during the COVID-19 pandemic due to recently reported correlation between cholinesterase and COVID-19 pneumonia severity/mortality, and it was shown that cholinesterase levels are reduced in the acute phase of severe COVID-19 pneumonia. This work describes the microfabrication of potentiometric sensors using copper as the substrate and chemically polymerized graphene nanocomposites as the transducing layer for tracking the kinetics of MCH enzymatic degradation in real blood samples. The in-vitro estimation of the characteristic parameters of the MCH metabolism [Michaelis-Menten constant (Km) and reaction velocity (Vmax)] were found to be 241.041 µM and 56.8 µM/min, respectively. The proposed sensor is designed to be used as a companion diagnostic device that can (i) answer questions about patient eligibility to perform methacholine challenge tests, (ii) individualize/personalize medical dosing of methacholine, (iii) provide portable and inexpensive devices allowing automated readouts without the need for operator intervention (iv) recommend therapeutic interventions including intensive care during early stages and reflecting the disease state of COVID-19 pneumonia. We hope that this methacholine electrochemical sensor will help in assaying ChE activity in a "timely" manner and predict the severity and prognosis of COVID-19 to improve treatment outcomes and decrease mortality.

Optimization of localized surface plasmon resonance hot spots in surface-enhanced infrared absorption spectroscopy aluminum substrate as an optical sensor coupled to chemometric tools for the purity assay of quinary mixtures.

Surface-enhanced infrared absorption spectroscopy offers an alternative to conventional IR spectroscopy and utilizes the signal enhancement exerted by the plasmon resonance of nanostructured metal thin films. Citrate-capped silver nanoparticles were prepared in a single-step method, and their morphology was identified using transmission electron microscopy, scanning electron microscopy, ultraviolet/visible spectrophotometry, and Zetasizer. The nanoparticles generated were deposited on the surface of cheap aluminum slides for different durations aiming for the selection of the best time producing a thin film, suitable to act as a lab-on-a-chip SEIRA substrate. These substrates were coupled to partial least squares regression tools for simultaneous resolving of the quinary mixture in commercial dosage forms of bisoprolol, perindopril, bisoprolol acid degradation product, bisoprolol alkali degradation product, and perindoprilat in concentration ranges of 15-75, 60-300, 15-55, 12-60, and 20-80 µg/mL with limits of detection values of 0.69, 3.43, 0.97, 1.25, and 1.09 µg/mL, respectively. Overall, we could demostrate that the localized surface plasmon resonance sensor coupled to chemometrics provides cheap, simple, selective, multiplex, rapid, and molecular specific procedures for impurity detection, which would be beneficial in many applications for quality control and quality accuracy of active pharmaceutical ingredients.

A comparative study of two analytical techniques for the simultaneous determination of amprolium HCl and ethopabate from combined dosage form and in presence of their alkaline degradation.

Chemometric and separation-based techniques (HPLC) are the most applicable and versatile analytical techniques for the analysis of multicomponent mixtures, in the present contribution, a comparison was highlighted between the two analytical techniques of utmost importance as stability indicating assays: UV-spectrophotometry and HPLC-UV focusing on the greenness of each for the simultaneous determination of amprolium HCl (AMP) and ethopabate (ETHOP) in the presence of their alkaline degradation products. The first method was chemometric methods applied were PLS-1, GA-PLS and GA-ANN. To compare the prediction ability of the models, a 4-factor 5-level experimental design was used to establish a calibration set of 25 mixtures containing different ratios of the drugs and their degradation products. The validity of the proposed methods was assessed using an independent validation set of 5 mixtures. The comparison between the different models showed the superiority of ANN model in solving the highly overlapped spectra of the quaternary mixture, yet using inexpensive and easy to handle instruments like the UV-VIS spectrophotometer. The ANN method was used for the quantitative analysis of the drugs in pharmaceutical dosage form via handling the UV spectral data. The second method was based on liquid chromatographic HPLC determination of AMP and ETHOP using C18 column (250 × 4.6 mm2)-PRONTOSIL 5 µm, a mobile phase consisting of methanol: Hexane sulphonic acid sodium salt at (pH = 3.4 ± 0.2) adjusted by orthophosphoric acid (55: 45 v/v). Quantitation was achieved with UV detection at 270 nm at temperature 24 °C. Linearity, accuracy and precision were found to be acceptable over the concentration range of 10.0-70.0 and 1.0-25.0 µg·mL-1 for AMP and ETHOP, respectively. The proposed methods could be successfully applied for the routine analysis of the studied drugs either in their pure bulk powders or in their pharmaceutical preparations without any preliminary separation step. The results obtained were statistically compared with those obtained by applying the reported method.

Point-of-care diagnostics for drugs of abuse in biological fluids: application of a microfabricated disposable copper potentiometric sensor.

The major objective of this work was to develop a portable, disposable, cost-effective, and reliable POC solid-state electrochemical sensor based on potentiometric transduction to detect benzodiazepine abuse, mainly diazepam (DZP), in biological fluids. To achieve that, microfabricated Cu electrodes on a printed circuit board modified with the conducting polymer poly(3-octylthiophene) (POT) have been employed as a substrate. This polymer was introduced to enhance the stability of the potential drift (0.9 mV/h) and improve the limit of detection (0.126 nmol mL-1). Nernstian potentiometric response was achieved for DZP over the concentration range 1.0 × 10-2 to 5.0 × 10-7 mol L-1 with a slope of 55.0 ± 0.4 mV/decade and E0 ~ 478.9 ± 0.9. Intrinsic merits of the proposed sensor include rapid response time (11 ± 2 s) and long life time (3 months). In order to enhance the selectivity of the potentiometric sensor towards the target drug and minimize any false positive results, calix[4]arene (CX4) was impregnated as an ionophore within the PVC plastic ion-sensing membrane. The performance of the POC sensors was assessed using electrochemical methods of analysis and electrochemical impedance spectroscopy as a surface characterization tool. The studied sensors were applied to the potentiometric determination of DZP in different biological fluids (plasma, urine, saliva, and human milk) in the presence of its metabolite with an average recovery of 100.9 ± 1.3%, 99.4 ± 1.0%, 101.8 ± 1.2%, and 99.0 ± 2.0%, respectively. Graphical abstract.

Stability-Indicating RP-HPLC and CE Methods for Simultaneous Determination of Bisoprolol and Perindopril in Pharmaceutical Formulation: A Comparative Study.

Two fast, accurate and selective stability-indicating methods were developed and validated for the simultaneous determination of bisoprolol, perindopril and three of their possible degradation products. The first proposed method was a gradient reversed phase-high-performance liquid chromatography (HPLC) method, whereas the second was a capillary electrophoresis method. The structures of the obtained degradation products were elucidated using infrared and mass spectrometry. They were also confirmed to be either a drug impurity in the British Pharmacopoeia or a precursor to such impurity. The linearity for bisoprolol and perindopril was achieved in the range of 1-20 µg mL-1 and 5-30 µg mL-1 for HPLC and capillary electrophoresis methods, respectively. The proposed methods were validated according to the International Conference on Harmonisation guidelines. The HPLC method proved to be more sensitive and succeeded in the quantitative determination of the obtained degradation products. Also, it was able to quantify perindopril impurity up to three times lower than the desired limit set by the British Pharmacopoeia. They were successfully employed in the determination of bisoprolol and perindopril in their combined pharmaceutical formulation.

Strategies for stabilizing formulation and QbD assisted development of robust stability indicating method of azilsartan medoxomil/chlorthalidone.

Reasons for formulation instability were investigated either encountered during production or analytical processes of azilsartan medoxomil (AZM)/chlorthalidone hydrochloride (CLT) tablets. Through the identification of the most feasible degradation pathways, several strategies were proposed to enhance the stability of AZM/CLT formulation. Furthermore, a robust HPLC-UV method was developed and validated for the determination of AZM, CLT in the presence of their possible degradation products. For chromatographic method development, typical quality by design (QbD) approach was implemented. In order to optimize fourteen chromatographic responses, we have used a central composite design with four factors (pH, temperature, flow rate, and acetonitrile %). However, the developed method provides a design space, but optimum parameters were Inertsil C8 column (150 x 4.6 mm, 5 µm), mobile phase composed of 0.025 M phosphate buffer pH 2.7 and acetonitrile (52.5: 47.5%), with flow rate of 1.5 mL.min-1 and detection wavelength 225 nm at 33 °C. The method was then validated according to ICH guidelines and applied to quantitate AZM and CLT in the pharmaceutical formulation. To the best of our knowledge, this manuscript is the first attempt to discuss such instability issues, to propose strategies that enhance the stability of AZM/CLT tablet formulation, to develop robust stability-indicating method taking into consideration the realistic degradation products in addition to minor ones.

Vesicles stage of camel brain development / Etapas del desarrollo de las vesículas cerebrales del camello

A successive embryonic developmental study was conducted on the brain of twenty eight embryos and fetuses of one humped camel (Camelus Dromedarius), whose crown vertebral rump lengths (CVRL) ranged from 9 to 80 mm, collected from the El-Basateen (Cairo) and Belbees (ElSharqya) Slaughterhouse. The current investigation revealed that camel brain was found to consist of fore, mid and hind brains. The fore brain is divided into telencephalon and diencephalon while the rhombencephalon divided into metencephalon and myelencephalon. Flexures appeared between the vesicles are cervical flexure between the rhomencephalon and the spinal cord, cephalic flexure in the mesencephalon and pontine flexure between the metencephalon, and the myelencephalon of the hind brain (rhombencephalon). The cavity of the rhombencephalon is the fourth ventricle, while that of the diencephalon is the third ventricle, and those of the telencephalon are the lateral ventricles but that of mid brain is the cerebral aqueduct. myelencephalon becomes medulla oblongata and metencephalon developed to pons and cerebellum while mesencephalon gives rise to the cerebral crura and anterior and a posterior colliculus. Diencephalon gives the thalamus, hypothalamus, mamillary body, infundibulum and pineal body while telencephalon becomes the cerebral hemispheres and corpus striatum.

Se llevó a cabo un estudio del desarrollo embrionario cerebral de veintiocho embriones y fetos de camello jorobado (Camelus dromedarius). Las muestras fueron recolectadas en los mataderos de El-Basateen (El Cairo) y Belbees (ElSharqya). La investigación reveló que el cerebro de camello posee un cerebro anterior, medio y posterior. El cerebro anterior se divide en telencéfalo y diencéfalo, mientras que el rombencéfalo se divide en metencéfalo y mielencéfalo. Las flexiones encontradas entre las vesículas son la flexión cervical entre el rombencéfalo y la médula espinal; la flexión cefálica en el mesencéfalo; y la flexión pontina entre el metencéfalo y el mielencéfalo del cerebro posterior (rombencéfalo). La cavidad del rombencéfalo conforma el cuarto ventrículo, la del diencéfalo forma el tercer ventrículo, y las del telencéfalo a los ventrículos laterales. En el cerebro medio, la cavidad corresponde al acueducto cerebral. El mielencéfalo se convierte en médula oblonga y el metencéfalo deriva en puente y cerebelo, mientras que el mesencéfalo da lugar a la crura cerebral y a los colículos anterior y posterior. El diencéfalo origina el tálamo, el hipotálamo, el cuerpo mamilar, el infundíbulo y la hipófisis, mientras que del telencéfalo se originan los hemisferios cerebrales y el cuerpo estriado.

Application of wavelet and Fuorier transforms as powerful alternatives for derivative spectrophotometry in analysis of binary mixtures: A comparative study.

Two signal processing methods, namely, Continuous Wavelet Transform (CWT) and the second was Discrete Fourier Transform (DFT) were introduced as alternatives to the classical Derivative Spectrophotometry (DS) in analysis of binary mixtures. To show the advantages of these methods, a comparative study was performed on a binary mixture of Naltrexone (NTX) and Bupropion (BUP). The methods were compared by analyzing laboratory prepared mixtures of the two drugs. By comparing performance of the three methods, it was proved that CWT and DFT methods are more efficient and advantageous in analysis of mixtures with overlapped spectra than DS. The three signal processing methods were adopted for the quantification of NTX and BUP in pure and tablet forms. The adopted methods were validated according to the ICH guideline where accuracy, precision and specificity were found to be within appropriate limits.

Development and validation of LC-MS/MS assay for the simultaneous determination of methotrexate, 6-mercaptopurine and its active metabolite 6-thioguanine in plasma of children with acute lymphoblastic leukemia: Correlation with genetic polymorphism.

Individualized therapy is a recent approach aiming to specify dosage regimen for each patient according to its genetic state. Cancer chemotherapy requires continuous monitoring of the plasma concentration levels of active forms of cytotoxic drugs and subsequent dose adjustment. In order to attain optimum therapeutic efficacy, correlation to pharmacogenetics data is crucial. In this study, a specific, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed for determination of methotrexate (MTX), 6-mercaptopurine (MP) and its metabolite 6-thioguanine nucleotide (TG) in human plasma. Based on the basic character of the studied compounds, solid phase extraction using a strong cation exchanger was found the optimum approach to achieve good extraction recovery. Chromatographic separation was carried out using RP-HPLC and isocratic elution by acetonitrile: 0.1% aqueous formic acid (85:15v/v) with a flow rate of 0.8mL/min at 40°C. The detection was performed by tandem mass spectrometry in MRM mode via electrospray ionization source in positive ionization mode. Analysis was carried out within 1.0min over a concentration range of 6.25-200.00ng/mL for the studied analytes. Validation was carried out according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the monitoring of the MTX, MP and TG and subsequent application to personalized therapy was demonstrated in a clinical study on children with acute lymphoblastic leukemia (ALL). Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment.

Transmission electron microscopic evaluation of neuronal changes in methylmercury-exposed zebrafish embryos (Danio rerio).

Our work aimed to elucidate the ultrastructural changes associated with brain neurons in wild-type zebrafish embryos exposed to different concentrations of methylmercury. Zebrafish embryos were exposed to one of five concentrations of methylmercury (0 [negative control], 5, 10, 50, and 80 parts per billion) starting at six hours post fertilization (hpf). At 96 hpf, cells in the zebrafish embryo brains were examined using transmission electron microscopy. The developing neurons of the control embryos sowed normal cellular ultrastructure. Few alterations were observed among the neurons of zebrafish embryos exposed to 5 ppb methylmercury. The cells of the embryos exposed to 10 ppb methylmercury showed slight cellular degeneration as demonstrated by the accumulation of electron dens bodies which were presumably lysosomes in different stages of formation. In embryos exposed to 50 ppb methylmercury, the neuronal cytoplasm conained large electron dense lysosomes and the rough endoplasmic reticulum appeared to be reduced and irregular in shape. Furthermore, the embryonic brain neurons exposed to 80 ppb methylmercury showed the most severe ultrastructural changes, including some that were consistent with different stages of the cell death process. Obvious cellular changes were observed in this highest exposure group included: disrupted or degenerating nuclei; fragmentation or vacuolization of mitochondrial cristae; and loss of mitochondrial matrix density. Based on these observations, we conclude that these different morphological patterns of cellular changes may reflect either different stages of the cell death process or different types of cell death due to 24 hours of exposure to 80 ppb methylmercury.