Rapid and sensitive HPLC-MS/MS method for the quantification of dopamine, GABA, serotonin, glutamine and glutamate in rat brain regions after exposure to tobacco cigarettes.

Tobacco smoking is a preventable main cause of fatal diseases. Accurate measurements of the effects it has on neurotransmitters are essential in developing new strategies for smoking cessation. Moreover, measurements of neurotransmitter levels can aid in developing drugs that counteract the effects of smoking. The aim of this study is to develop and validate a fast, simultaneous and sensitive method for measuring the levels of neurotransmitters in rat brain after the exposure of tobacco cigarettes. The selected neurotransmitters include dopamine, GABA, serotonin, glutamine and glutamate. The method is based on high-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved within 3 min using a Zorbax SB C18 column (3.0 × 100 mm, 1.8 µm particle size). The mobile phase consisted of HPLC-grade water and acetonitrile each containing 0.3% heptafluorobutyric acid and 0.5% formic acid at gradient conditions. The linear range was 0.015-0.07, 825-7,218, 140-520, 63.42-160.75 and 38.25 × 103 to 110.35 × 103  ng/ml for dopamine, GABA, serotonin, glutamine and glutamate, respectively. Inter- and intra-run accuracy were in the range 97.82-103.37% with a precision (CV%) of ≤0.90%. The results revealed that 4 weeks of cigarette exposure significantly increased neurotransmitter levels after exposure to tobacco cigarettes in various brain regions, including the hippocampus and the amygdala. This increase in neurotransmitters levels may in turn activate the nicotine dependence pathway.

Highly sensitive spectrofluorimetric method for the determination of the genotoxic methylglyoxal in glycerol-containing pharmaceuticals and dietary supplements.

Methylglyoxal (MGO) is a genotoxic α-dicarbonyl compound. Recently, it was found to be formed in glycerol preparations during storage through auto-oxidation. A simple fluorimetric determination of the carcinogenic degradation product of glycerol, MGO, was developed and validated. The proposed method is based on the derivatization of MGO with 4-carbomethoxybenzaldehyde (CMBA) and ammonium acetate to yield a fluorescent imidazole derivative that can be measured at 415 nm after excitation at 322 nm. The optimized conditions were determined to be 0.2 M CMBA, 1.0 M ammonium acetate and a reaction time of 40 min at 90°C using ethanol as diluting solvent. The linear range was 10.0-200.0 ng/ml. Detection and quantification limits were 2.22 and 6.72 ng/ml, respectively. The proposed method was validated according to International Council for Harmonisation (ICH) guidelines and compared with the reported method and no significant difference was found. It was successfully applied for the determination of MGO in six different glycerol-containing pharmaceutical preparations and dietary supplements.

Lactate and pyruvate levels correlation with lactate dehydrogenase gene expression and glucose consumption in Tamoxifen-resistant MCF-7 cells using capillary electrophoresis with contactless conductivity detection (CE-C4 D).

Breast cancer is the second leading cause of cancer death in women after lung cancer. The first-line treatment of metastatic breast cancer in premenopausal women relies on tamoxifen. The development of tamoxifen resistance is not fully understood. In this study, capillary electrophoresis with capacitively coupled contactless conductivity detector was developed to monitor the changes in lactate and pyruvate levels in supernatant media of three models of developed MCF-7 tamoxifen-resistant cells and correlate these metabolites changes with lactate dehydrogenase genes expression and glucose consumption. The electrophoretic separation was achieved under reversed electroosmotic flow conditions. The linear ranges were 0.15-5 and 0.01-1 mM with a correlation coefficient of 0.9966 and 0.9971 and the limits of detection were 0.01 and 0.02 µM for lactate and pyruvate, respectively. Inter- and intrarun accuracy were in the range of 96.88-105.94% with precision (CV, %) of ≤7.35%. The method was completely validated and the results were in agreement with those obtained using the lactate and glucose assay kits. The results revealed a significant increase in both lactate and pyruvate production in the three tamoxifen-resistant MCF-7 cells models compared to control cells. This increase was correlated with the increase of lactate dehydrogenase genes expression and the increase of glucose consumption.

High Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes.

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography-tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid-ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11-2.25, 0.012-0.227, and 0.02-0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.

An Open Microfluidic Chip for Continuous Sampling of Solute from a Turbulent Particle Suspension.

High solids content complicates in situ analysis of chemical processing, biological suspensions, and environmental streams. In most cases, analytical methods require at least one pre-treatment step of a small volume of sample before a particle-free fluid can be analyzed. We have developed a continuous in situ sampler that can "sip" particle-free solution from a turbulent high solids content stream (a slurry). An open microfluidic chip with an extended slit opening shields the internal laminar flow from the turbulence outside. Unlike other open chips, our chip does not require close proximity to a solid surface and operates in turbulent environments for hours without maintenance. Two applications are demonstrated: monitoring FeIII in a stirred slurry of mixed ore particles at high solids loading (4 %wt) and paracetamol tablet dissolution profiles for two different formulations.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016-2018).

One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2014-2016).

One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biennial reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods in capillaries and microchips, covering the period July 2014-June 2016. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2012-2014).

One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods, covering the period July 2012-July 2014. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to ITP, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.

Electrokinetic Size and Mobility Traps for On-site Therapeutic Drug Monitoring.

The extraction of target analytes from biological samples is a bottleneck in analysis. A microfluidic device featuring an electrokinetic size and mobility trap was formed by two nanojunctions of different pore size to extract and concentrate analytical targets from complex samples. The trap was seamlessly coupled with electrophoretic separation for quantitative analysis. The device was applied to the analysis of ampicillin levels in blood within 5 min and a linear response over the range of 2.5-20 µg mL(-1). This covers the recommended levels for treating sepsis, a critical condition with 30 to 50% mortality and unpredicted drug levels. The device provides a new opportunity for on-site therapeutic drug monitoring, which should enable quick and accurate dosing and may save lives in such critical conditions.

Cost-effective three-dimensional printing of visibly transparent microchips within minutes.

One-step fabrication of transparent three-dimensional (3D) microfluidic to millifluidic devices was demonstrated using a commercial 3D printer costing $2300 with 500 mL of clear resin for $138. It employs dynamic mask projection stereolithography, allowing fast concept-to-chip time. The fully automated system allows fabrication of models of up to 43 mm × 27 mm × 180 mm (x × y × z) at printing speeds of 20 mm/h in height regardless of the design complexity. The minimal cross sectional area of 250 µm was achieved for monolithic microchannels and 200 µm for positive structures (templates for soft lithography). The colorless resin's good light transmittance (>60% transmission at wavelengths of >430 nm) allows for on-chip optical detection, while the electrically insulating material allows electrophoretic separations. To demonstrate its applicability in microfluidics, the printer was used for the fabrication of a micromixer, a gradient generator, a droplet extractor, and a device for isotachophoresis. The mixing and gradient formation units were incorporated into a device for analysis of nitrate in tap water with standard addition as a single run and multiple depth detection cells to provide an extended linear range.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2010-2012).

CE has been alive for over two decades now, yet its sensitivity is still regarded as being inferior to that of more traditional methods of separation such as HPLC. As such, it is unsurprising that overcoming this issue still generates much scientific interest. This review continues to update this series of reviews, first published in Electrophoresis in 2007, with updates published in 2009 and 2011 and covers material published through to June 2012. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction and sweeping. Attention is also given to online or inline extraction methods that have been used for electrophoresis.

Spectrofluorimetric determination of selected genotoxic impurities in pharmaceutical raw materials and final products.

A green spectrofluorimetric method was introduced for the determination of selected genotoxic impurities; 2-aminopyridine and 3-aminopyridine in different pharmaceutical raw materials and dosage forms. The method relied on the native fluorescence of these impurities in acidic medium. The experimental conditions were carefully studied and optimized, and the method was validated according to International Council on Harmonisation (ICH) guidelines. The linear range for both analytes was 2.50-100 ng/mL with good determination coefficients of 0.9995 and 0.9992 and detection limits of 0.62 ng/mL and 0.74 ng/mL for 2-aminopyridine and 3-aminopyridine, respectively. The method was successfully applied for determination of 2-aminopyridine and 3-aminopyridine in four active pharmaceutical ingredients and nine dosage forms with satisfactory percentage recoveries and without interference from co-formulated excipients. Analytical performance of the proposed method was comparable to that of the reported methods; hence, the proposed method can be used as a simple and low-cost alternative in quality control laboratories.

Development and validation of a novel Spectrofluorimetric method of oral anticoagulant Edoxaban via derivatization with 9-fluorenyl methyl chloroformate: green assessment of the method by Eco-Scale and ComplexGAPI.

A precise, sensitive eco-friendly, simple, rapid, and derivative spectrofluorimetric method was developed to quantify edoxaban tosylate monohydrate in pure form and pharmaceutical dosage form. Sudden death due to pulmonary embolism as a consequence of coronavirus infection (covid-19) is an emerging problem. As a result, the world health organization introduced new guidelines to treat patients with COVID-19 with oral anticoagulants. Edoxaban tosylate monohydrate is an oral anticoagulant that doesn't require hospitalization after dose adjustment. This spectrofluorimetric method relies on the derivatization by 9-fluorenyl methyl chloroformate at room temperature in borate buffer pH 9.0. After excitation at 265 nm, the product is highly fluorescent at 309 nm. Many experimental factors influencing the reaction's stability and development were thoroughly investigated and optimized. The method validation was evaluated by using ICH guidelines and showed high precision and accuracy with an average percent recovery of 101.46% ± 1.02. The linear range was 5.0-50.0 ng/mL with a correlation coefficient of 0.9999, the LOD was 1.5 ng/mL, and the LOQ was 4.5 ng/mL. The green assessment of the method was achieved utilizing the eco-scale and the Green Analytical Procedure Index. There was no significant difference between the results of the suggested method and those of the reported method according to Statistical analysis.

Electrokinetics for sample preparation of biological molecules in biological samples using microfluidic systems.

Sample preparation is the first part of every analytical method, but is often considered only after the optimization of the method. It is traditionally performed using a range of techniques requiring extensive manual handling, with solid-phase extraction, liquid-liquid extraction, protein precipitation and ultracentrfiguation, among others, being used depending on the targets and the application. In this article, we will focus on alternatives based on electrokinetics for applications including sample clean-up, concentration and derivatization of large biological molecules (DNA, peptides and proteins) of diagnostic importance, as well as small molecules as a tool for therapeutic drug monitoring. This article describes these approaches in terms of mechanisms, applicability and potential to be integrated into a lab-on-a-chip device for directly processing biological samples. Examples dealing with treated or clean samples have been excluded except where they show exceptionally high value.

Tuneable nanochannel formation for sample-in/answer-out devices.

Control of the dielectric breakdown of PDMS was achieved by limiting the current during the breakdown process. This enabled tuning of the nanochannel pore size and hence their permeability for molecules of different molecular weights. This method enabled the analysis of the drug quinine from whole blood in 3 min using a simple, disposable microfluidic device.