Buffer Concentration Research Articles

Region-specific mouse brain ganglioside distribution revealed by an improved isobaric aminoxyTMT labeling strategy with automated data processing.

Gangliosides are specialized glycosphingolipids most abundant in the central nervous system. Their complex amphiphilic structure is essential to the formation of membrane lipid rafts and for molecular recognition. Dysfunction of lipid rafts and ganglioside metabolism has been linked to cancer, metabolic disorders, and neurodegenerative disorders. Changes in ganglioside concentration and diversity during the progression of disease have made them potential biomarkers for early detection and shed light on disease mechanisms. Chemical derivatization facilitates whole ion analysis of gangliosides while improving ionization, providing rich fragmentation spectra, and enabling multiplexed analysis schemes such as stable isotope labeling. In this work, we report improvement to our previously reported isobaric labeling methodology for ganglioside analysis by increasing buffer concentration and removing solid-phase extraction desalting for a more complete and quantitative reaction. Identification and quantification of gangliosides are automated through MS-DIAL with an in-house ganglioside derivatives library. We have applied the updated methodology to relative quantification of gangliosides in six mouse brain regions (cerebellum, pons/medulla, midbrain, thalamus/hypothalamus, cortex, and basal ganglia) with 2 mg tissue per sample, and region-specific distributions of 88 ganglioside molecular species are described with ceramide isomers resolved. This method is promising for application to comparative analysis of gangliosides in biological samples.

A novel electrochemical method determining pKa and transport parameters of buffers using acid-base transport limitation response

Dissociation constant (logarithmic form, pKa) and diffusion coefficient (D) are two fundamental physical parameters for weak acids and bases (buffers). Their effortless determinations from one method are not available yet. Herein, we propose an innovative method to determine pKa and D values based on a brand-new principle of electrochemical acid-base transport limitation (eABTL). The significance of eABTL lies in its ability to relate inert buffers to electrode reactions, enabling the acquisition of electrochemical responses for inert species. Acid-base transport limiting current (jl) is the only variable that needs to be measured. The pKa and transport coefficient (m) are determined from the relationship of jl vs. solution pH and jl vs. buffer concentration, respectively. Three ways are available for D determinations by introducing the expressions of diffusion layer thickness. The method is demonstrated using rotating disc electrodes to implement hydrogen evolution reaction. The pKa, m and D values exhibit good repeatability in multiple determinations. A well-defined electrode geometry is necessary for accurate D determination. This method can be developed to a family of methods with the advantages for fast estimating practical parameter values of buffers.

A comprehensive evaluation of a polymeric zwitterionic hydrophilic monolith for nucleotide separation.

Rapid and effective separation of nucleotides (NTs) and their derivatives is crucial for studying their physiological functions. In this work, we comprehensively evaluated the separation ability of a zwitterionic hydrophilic monolith, i.e., poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine-co-N,N'-methylenebisacrylamide) (poly(SPP-co-MBA)) for NTs analysis, including its selectivity, chemical stability under extremely basic condition and compatibility with hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (HILIC-MS). The poly(SPP-co-MBA) monolith exhibited excellent chemical stability, as evidenced by the low relative standard deviation of retention time (0.16-1.05%) after 4000 consecutive injections over one month under strong alkaline elution condition (pH 10). After optimizing the separation conditions, including buffer pH and concentration, organic solvent content and column temperature, four nucleoside triphosphates, five nucleoside diphosphates and five nucleoside monophosphates were baseline separated within 7min. Additionally, the mixtures containing one nucleoside and its corresponding mono-, di-, and triphosphates were baseline separated within only 3min, respectively. It is good HILIC-MS compatibility was also confirmed by the satisfactory peak shape and high response of nine NTs. Overall, the proposed poly(SPP-co-MBA) monolith exhibited good mechanical stability and compatibility of HILIC-MS, making it a promising technique for NTs analysis.

A novel “Turn-on” fluorometric assays triggered reaction for β-glucosidase activity based on quercetin derived silicon nanoparticles and its potential use for cell imaging

β-Glucosidase (β-Gluco) is an enzyme that is crucial to numerous diseases, including cancer, and in sector of industries, it is used in the manufacturing of food. Measuring its enzymatic activity is critical for biomedical studies and other activities. Herein, we have developed a novel and precise fluorescent sensing method for measuring β-Gluco activity based on the production of yellow-green fluorescent quercetin-silicon nanoparticles (Q-SiNPs) produced from quercetin (QN) as a reducing agent and 3-[2-(2-aminoethyl amino) ethylamino] propyl-trimethoxy silane (AEEA) as a silane molecule. β-Gluco hydrolyzed quercetin-3-O-β-d-glucopyranoside (QO-β-DG) to produce QN, which was then used to produce Q-SiNPs. Reaction parameters, including temperature, time, buffer, pH, and probe concentration, were carefully tuned in this study. Subsequently, the fluorescence intensity was performed, showing good linearity (R2 = 0.989), a broad linear dynamic range between 0.5 and 12 U L−1, and a limit of detection (LOD) as low as 0.428 U L−1, which was proven by fluorescence measurements. Most importantly, various parameters were detected and characterized with or without β-Gluco. The designed probe was successively used to assess β-Gluco activity in human serum and moldy bread. However, the mathematical findings revealed recoveries for human serum ranging from 99.3 to 101.66% and for moldy bread from 100.11 to 102.5%. Additionally, Q-SiNPs were well suited to being incubated in vitro with L929 and SiHa living cells, and after using an Olympus microscope, imaging showed good fluorescence cell images, and their viability evinced minimal cytotoxicity of 77% for L929 and 88% for SiHa. The developed fluorescence biosensor showed promise for general use in diagnostic tests. Therefore, due to this outstanding sensing modality, we anticipate that this research can provide a novel schematic project for creating simple nanostructures with a suitable plan and a green synthetic option for enzyme activity and cell imaging.

Development, validation and greenness assessment of a new electro-driven separation method for simultaneous analysis of cefixime trihydrate and linezolid in their fixed dose combination

The establishment and validation of a straightforward, accurate, and eco-friendly capillary zone electrophoretic-diode array detection (CZE-DAD) procedure has been presented for concurrent measurement of two common antibiotics, namely, linezolid (LIN) and cefixime trihydrate (CEF), in their binary mixture or combined dosage form. The selected fused silica capillary has total and effective lengths equal to 58.5 cm and 50 cm, respectively, with a 50 µm internal diameter. Injections were performed utilizing 100 mM borate buffer at pH 10.2 as the background electrolyte (BGE) with a 15.0 s injection time. The finally utilized voltage was 30 kV. DAD was programmed to measure LIN at 250 nm and CEF at 285 nm. In less than 6 min, the two cited drugs were resolved at 2.51 and 5.47 min for LIN and CEF respectively. The introduced procedure had a linear response in the concentration range of 5–50 μg/mL for both analytes with correlation coefficients > 0.9999. Detection and quantification limits were 1.213 and 4.042 μg/mL, respectively, for LIN and 0.301 and 1.004 μg/mL, respectively, for CEF. Validation was conducted according to the International Council for Harmonization (ICH), concerning linearity, detection and quantitation limits, range, accuracy, precision, selectivity, and robustness. Precision was found acceptable due to the low relative standard deviation (RSD%) values that did not exceed 1.86% either for repeatability or for intermediate precision. Additionally, the adequately recovered concentrations and the low values of percentage relative error (Er%) provide evidence of the accuracy of the proposed method. On the other hand, the robustness of the introduced method was affirmed by the acceptable RSD% values that did not exceed 0.6% after deliberate changes in the following procedure parameters: buffer concentration, buffer pH, and wavelength. Finally, the ability of the presented method to quantify the two tested drugs in laboratory-prepared tablets was confirmed by the adequate recoveries (≥ 99%) utilizing the standard-addition procedure, along with the absence of any significant difference between the proposed method and the reference method as proven by the student’s t-test and the variance-ratio F-test values that did not exceed the theoretical ones. The analytical Eco-Scale and the analytical GREEness metric (AGREE) were the tools utilized for greenness assessment. This CZE procedure is the first electro-driven separation method that was utilized for the analysis of both antibiotics in their combined laboratory-prepared tablets with no interference from the co-formulated adjuvants.

Comparison of ZIC-HILIC Columns for the Simultaneous Analysis of Antiviral Drugs in Dosage Forms by Hydrophilic Interaction Liquid Chromatography

Background:: Antiviral drugs are vital since many viruses can produce fatal infections, as we have recently seen with the COVID-19 pandemic. Antiviral drugs can battle viruses at multiple stages of their life cycles, including neuraminidase, nucleic acid synthesis, protease, and virion fusion or entry [1]. Objective:: Antiviral drugs have poor retention in reversed-phase liquid chromatography, which makes it difficult to analyze a mixture of antiviral medications using high-performance liquid chromatography. Using zwitterionic hydrophilic interaction liquid chromatography (ZICHILIC), the paper highlights the simultaneous quantification of three antiviral drugs as active constituents in pharmaceutical formulations. Moreover, the influence of the length of the spacer between the two charges in two stationary phases on the retention behavior of antiviral drugs has been discussed. Methods:: Two homemade stationary phases (ZIC1-HILIC and ZIC5-HILIC) were utilized for the qualitative and quantitative analysis of three antiviral drugs, and UV was used as the detector. Several chromatographic conditions were examined, such as the organic modifier concentration, buffer concentration, and pH value. Results:: After optimizing the parameters, the devised method was applied to analyse three antiviral medications quantitatively. The initial results demonstrated the current procedure for separating and determining these three antiviral drugs to be sensitive, robust, and effective. Consequently, the present method has shown excellent repeatability, a broad linear range (0.1- 16.5 μgml-1), and excellent sensitivity (LOD 0.04-0.072 μgml-1). The RSD value of the method was less than 1. Conclusion:: A mixed mode of hydrophilic and ion exchange interactions was the predominant mode of antiviral medications with two ZIC-HILIC stationary phases. The ZIC5-HILIC stationary phase had a lower detection and limit of quantitation for three antiviral drugs and a prolonged retention time compared to the ZIC1-HILIC stationary phase with a shorter chain length.

Preparation of porous organic cage and its use as chiral stationary phase for capillary electrochromatography

Porous organic cages (POCs) are a unique type of microporous materials composed of discrete molecules with internal cavities that are accessible to various compounds. In this study, a prismatic chiral POC with good thermochemical stability was synthesized by condensing (1R,2R)-diaminocyclohexane and 3,3',5,5'-teturonic-4,4'-biphenediol via the Schiff base reaction and characterized by proton nuclear magnetic resonance spectroscopy, infrared (IR) spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy. The IR spectrum of the POC revealed a strong characteristic absorption peak at 1635 cm-1, indicating that it formed imine bonds (C=N). The absorption peak at 3425 cm-1 was attributed to the stretching vibrations of -OH, the absorption peaks at 2925 and 2858 cm-1 were attributed to the stretching vibrations of N=C-H and C-H, and the absorption peaks at 1446 and 1383 cm-1 were attributed to the stretching vibrations of C=C-H and C=C in the benzene ring. High-resolution mass spectral analysis of the POC showed a molecular ion peak at m/z 1363.7228, indicating its successful synthesis. TGA was performed from 25 to 800 ℃ at a rate of 10 ℃/min, and the results of this analysis showed that the POC was stable up to approximately 300 ℃. The POC was dissolved in dichloromethane and uniformly coated on the inner wall of a quartz capillary via the dynamic coating method to prepare a capillary electrochromatographic column. The experimental results revealed that the chiral electrochromatographic column could not only resolve ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol but also separate the isomers of o-, m-, and p-toluidine and o-, m-, and p-chloroaniline, indicating its good chiral separation ability. Investigation of the optimal separation conditions for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol revealed that the voltage, buffer solution concentration, and pH significantly affected their separation degree. In particular, the optimal separation voltage for ofloxacin, Troger's base, and 2-amino-1-butanol was 15 kV, while that for 1-phenyl-1-amyl alcohol was 17 kV. The optimal buffer concentration and pH for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol were 0.100 mol/L and 7.5. Under optimal chromatographic conditions, the resolution values for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-pentanol were 1.80, 3.33, 1.69, and 1.18, respectively. The results collectively demonstrate that the prepared POC may serve as a good chiral stationary phase for capillary electrochromatography with a certain chiral resolution ability and has good application prospects in chromatographic separation.

Urea-Self Powered Biosensors: A Predictive Evolutionary Model for Human Energy Harvesting.

The objective of this study is to create a reliable predictive model for the electrochemical performance of self-powered biosensors that rely on urea-based biological energy sources. Specifically, this model focuses on the development of a human energy harvesting model based on the utilization of urea found in sweat, which will enable the development of self-powered biosensors. In the process, the potential of urea hydrolysis in the presence of a urease enzyme is employed as a bioreaction for self-powered biosensors. The enzymatic reaction yields a positive potential difference that can be harnessed to power biofuel cells (BFCs) and act as an energy source for biosensors. This process provides the energy required for self-powered biosensors as biofuel cells (BFCs). To this end, initially, the platinum electrodes are modified by multi-walled carbon nanotubes to increase their conductivity. After stabilizing the urease enzyme on the surface of the platinum electrode, the amount of electrical current produced in the process is measured. The optimal design of the experiments is performed based on the Taguchi method to investigate the effect of urea concentration, buffer concentration, and pH on the generated electrical current. A general equation is employed as a prediction model and its coefficients calculated using an evolutionary strategy. Also, the evaluation of effective parameters is performed based on error rates. The obtained results show that the established model predicts the electrical current in terms of urea concentration, buffer concentration, and pH with high accuracy.

Co-reactant confined and vertically ordered silica nanochannel regulated electrochemiluminescence for homogeneous detection of miRNA

Electrochemiluminescence (ECL) arises from the collisional annihilation between the electro-excited luminophore and co-reactant. Most ECL-based bioassays require extensive free co-reactants that collide with luminophores randomly. Herein, the N,N-dimethylaniline (DMA) group was electro-grafted on the ITO electrode surface as the bottom co-reactant layer, and the vertically ordered mesoporous silica film (VMSF) was prepared as the upper layer to regulate the accessibility of Ru(bpy)32+. Thus, the dual-layer functionalized VMSF/N-ITO was newly built for the controllable ECL study. Relevant conditions, including the DMA electro-grafting, VMSF thickness, buffer concentration and pH value, were discussed to improve the ECL efficiency. Based on the VMSF/N-ITO platform, a homogeneous ECL bioassay was developed for miRNA-21 detection. The target nucleotide sequence induced the hybridization chain reaction (HCR) of probes H1 and H2 to absorb free Ru(bpy)32+ as much as possible. Residual Ru(bpy)32+ permeated the VMSF nanochannels to collide with the DMA co-reactant in producing an ECL response. There was a linear relationship between ∆ECL intensity and logarithmic concentration of miRNA-21 in the 1 fM ∼ 100 pM range, and the detection limit was 0.25 fM. The developed approach was probes immobilization-free, highly sensitive, and anti-interference, opening a new avenue for ECL-based bioassays.

Deep eutectic solvents combined with beta-cyclodextrin derivatives for chiral separation of typical adrenergic receptor agonists by capillary electrophoresis with amperometric detection

Enantioseparation has always been one of the research hotspots and difficulties in the field of modern separation science. In this work, a binary chiral electrophoretic separation system was constructed using deep eutectic solvents (DESs) coupled with beta-cyclodextrin derivatives based on capillary electrophoresis with amperometric detection system, and five groups of typical adrenergic receptor agonists (adrenaline, salbutamol, isoproterenol, norepinephrine and terbutaline) were selected as the model enantiomers. The effects of additive types and contents of DESs and cyclodextrins, and the pH value and concentration of the running buffer on the resolution of the selected chiral compounds were investigated in detail. The mechanism of DESs improving separation was explored preliminarily by means of UV spectrophotometry, which was further verified based on the comparison of single and mixed components of choline chlorine-urea DES. Under the optimum conditions, the relative standard deviations for inter-day and intra-day repeatability of the migration time, peak area and resolution for adrenaline and salbutamol were within 8.7%, and the limits of detection reached 0.030 μg mL−1 (S/N = 3). The recovery data were in the range of 96.3–118.7%. The developed methods have been applied for the analyses of (+)-adrenaline hydrochloride injection and (±)-salbutamol aerosol. This binary chiral electrophoretic separation system by CE-AD has high detection sensitivity and low analytical cost, providing an alternative for the separation and analysis of chiral drugs.

Potential application of a chromatographic method in the accurate simultaneous determination of amikacin sulfate and its preservation additives in different pharmaceutical dosage forms

Aminoglycosides are antibiotics with high clinical importance that face a great challenge of detection in the UV range. This study aimed to develop an accurate, simple, and sensitive validated high-performance liquid chromatographic (HPLC) method for the determination of amikacin sulfate, one of the widely used aminoglycoside antibiotics in the pharmaceutical formulations, as the target analyte without interference from its preservation additive sodium metabisulfite in the pharmaceutical dosage forms without the usage of any derivatization reactions. The developed method is based on direct UV detection at 205 nm using a gradient mobile phase consisting of acetonitrile as an organic solvent and buffered solution; pH 3.0. This method was developed on a reversed-phase ZORBAX ECLIPSE XDB-C8 (4.6 × 250 mm, 5-μm) analytical column with a flow rate of 1.5 mL min−1 at a column temperature of 35 °C. The separation of amikacin sulfate and sodium metabisulfite was achieved at 3.7, and 1.5 min, respectively. The developed method validation was performed as prescribed by the International Conference on Harmonization (ICH) guidelines to confirm its linearity, precision, and accuracy. Finally, using simple, commonly available analytical tools, safe buffer concentration on the HPLC system, and without any complicated equipment or challenging derivatization steps, our validated HPLC method was applied for the simultaneous determination of amikacin and sodium metabisulfite in commercially available pharmaceutical formulations in a single method of analysis.

Amherstia Nobilis: an Approach to in Vitro Anti-inflammatory Activity

Herbal plant compounds were used in the inhibition of inflammation from traditional period and its main benefit is the less side effects. The aim of this study is to determine the anti-inflammatory potential of Amherstia nobilis by albumin denaturation method. The leaves of the plant were collected, dried and powdered. The egg albumin, phosphate buffer and different concentrations of the plant extracts were incubated at 37°C for 15 minutes and heated at 70°C for 5 minutes. The rate of inhibition of protein denaturation is calculated. The result verified the anti-inflammatory potential of Amherstia nobilis and its prospective use in alleviating inflammation as a natural remedy.

Effect of phosphate buffer concentration on the isomerization of galactose to rare sugars under subcritical water conditions

Galactose was treated in sodium phosphate buffer at various concentrations (0.1–500 mmol/L) under subcritical water conditions (140 °C), and the effects of the buffer concentration and reaction time (0–300 s) on the reaction behavior were evaluated. The reaction proceeded rapidly at higher buffer concentrations. Rare sugars (tagatose, talose, and sorbose) were formed from galactose by isomerization. The highest yield of the main product, tagatose, was approximately 14 % in 50 mmol/L buffer. However, the tagatose yield did not increase further with increasing buffer concentration. On the other hand, the formation of talose and sorbose was accelerated at higher buffer concentrations, with the highest yields of approximately 5 % and 12 %, respectively, in 500 mmol/L buffer. At the same time, the formation of byproducts (organic acids and colored substances) was also accelerated in high-concentration buffers. These results suggest that phosphate buffer promoted all reactions occurring under subcritical water conditions.

The effects of pH, temperature, and buffer concentration on the self-assembling behavior, secondary structure, and surface hydrophobicity of donkey and bovine β-casein

The self-assembling behavior, secondary structure, and surface hydrophobicity of purified donkey β-casein in terms of pH, temperature, and buffer concentration were investigated in comparison with commercial bovine β-casein. Critical micelle concentration of both β-caseins decreased with the lowering of pH (pH 8.0–6.0) and the increasing temperatures (25–50 °C). Critical micelle temperature of both β-caseins increased moving from pH 6.0 to 8.0 and aggregates larger than micelles formed at pH 6.0 that is close to their isoelectric point. Fluorescence spectroscopy analysis demonstrated that the maximum surface hydrophobicity was achieved at pH 6.0. The secondary structure was examined using circular dichroism spectroscopy, highlighting an increase of α-helix content and a decrease of unordered structures with the decrease of pH and increase of temperature. This work provides insights on parameters promoting molecular interactions involved in donkey β-CN self-association, useful to develop nanocarriers for encapsulating bioactive compounds in pharmaceutical and nutraceutical applications.

Application of a Quality by Design Approach to Develop a Simple, Fast, and Sensitive UPLC-MS/MS Method for Quantification of Safinamide, an Antiparkinson’s Drug, in Plasma

Safinamide is an orally active, selective monoamine oxidase-B inhibitor with dopaminergic and non-dopaminergic properties approved by the European Medicine Agency and US Food and Drug Administration for the treatment of mid- to late-stage fluctuating Parkinson’s disease (PD) used in combination with other PD medications such as levodopa. In this study, an analytical quality by design (AQbD) approach was applied to optimize an LC-MS/MS bioanalytical method to determine safinamide in human plasma. A full 33 factorial design was used to optimize safinamide separation conditions, with a method first screened and optimized using chromatographic responses, including peak area and retention time. The results showed that temperature had a significant indirect effect on retention time and peak area (p < 0.05), while ammonium acetate concentration had an insignificant indirect impact on peak area or retention time. However, the temperature was significantly agonistic to the effect of buffer concentration (p < 0.05). The resultant optimized chromatography conditions utilized 9.0 mM ammonium acetate buffer and acetonitrile (22.0:78.0) as mobile phases at a column temperature of 23.2 °C. The assay was linear from 0.1–1000 ng/mL, met acceptance criteria for inter- and intra-assay precision and accuracies across three quality controls, and was successfully applied to in vitro microsomal metabolic stability. The UPLC/MS/MS method was found to be adequately sensitive and suitable for routine safinamide pharmacokinetic studies.

A Novel Approach to Monitor the Concentration of Phosphate Buffers in the Range of 1 M to 0.1 M Using a Silicon-Based Impedance Sensor.

We present a novel and easy approach using a silicon-based impedance chip to determine the concentration of the given aqueous buffer solution. An accurate determination of the post-dilution concentration of the buffers is necessary for ensuring optimal buffer capacity, pH stability, and to assess solution reproducibility. In this study, we focused on phosphate buffer as the test liquid to achieve precise post-dilution concentration determinations. The impedance chip consisting of a top gold ring electrode, where a test volume of 20 μL to 30 μL of phosphate buffer was introduced for impedance measurements within the frequency range of 40 Hz to 1 MHz. For impedance investigation, we used phosphate buffers with three different pH values, and the impedance was measured after diluting the phosphate buffers to a concentration of 1.00 M, 0.75 M, 0.50 M, 0.25 M, 0.10 M, 0.05 M, and 0.01 M. In order to analyze the distinctive changes in the measured impedance, an equivalent circuit was proposed and modeled. From the impedance modeling, we report that the circuit parameter RAu/Si showed exponential dependence on the concentration of phosphate buffer and no dependence on the pH values of the phosphate buffer and on the added volume inside the ring electrode. The proposed silicon-based impedance chip is quick and uses reduced liquid volume for post-dilution concentration measurements of buffers and has perspective applications in the pharmaceutical and biological domains for regulating, monitoring, and quality control of the buffers.

Optimization and in-house validation of the analytical procedure for official control of bentonites as aflatoxin inactivators

In Europe, bentonites are allowed as feed additives for aflatoxin mitigation (1m558) provided they have specific mineralogical characteristics and an aflatoxin-binding capacity (BCAfB1) above 90%. BCAfB1 is determined by an official adsorption assay using an aflatoxin solution (4 mg/L) in acetate buffer (pH 5.0) and a bentonite at 0.02% (w/v). To date, the robustness of this method has not been investigated.In this work, we addressed this challenge and performed a robustness study by analyzing six bentonite samples that met the mineralogical requirements for claim code 1 m558. Leading factors selected for robustness testing were (1) preparation mode of bentonite suspension, (2) residual amount of acetonitrile in the test trial, (3) acetate buffer concentration, (4) incubation time, and (5) centrifugation. It was statistically evinced that factors 2 and 5 affected the results. Due to its weakness, the method excluded 4 out of six bentonites to be marketed in EU, being BCAfB1 < 90%. A new protocol was developed by keeping the main experimental parameters of the official assay and was in-house validated. This protocol yielded BCAfB1 > 90% for all test bentonites and showed satisfactory precisions with a RSDI of 3.4% and HorRat < 2. Its validity was proven by the isotherm approach, comparing Langmuir adsorption parameters with BCAfB1 values. Application of the protocol to bentonites other than montmorillonite was demonstrated.

Tetraethylenepentamine-derived carbon dots and tetraethylenepentamine co-immobilized silica stationary phase for hydrophilic interaction chromatography

In this work, tetraethylenepentamine (TEPA) was used as precursor and reaction medium to prepare tetraethylenepentamine-functionalized carbon dots (TEPACDs), the resultant mixture was subsequently silanized and then grafted on the surface of bare silica. The obtained tetraethylenepentamine-functionalized carbon dots and tetraethylenepentamine co-modified silica stationary phase (Sil-TEPA/CDs) was characterized by multiple ways, such as Fourier transformed infrared spectroscopy (FTIR), elemental analysis and transmission electron microscope, which revealed the successful preparation of the mixed stationary phase and higher density of functional groups on co-modified stationary phase than precursor single-modified stationary phase. The synergistic effect of TEPACDs and TEPA was proved by comparing the separation performance of Sil-TEPA/CDs and Sil-TEPA toward amino acids, nucleosides, and nucleobases, which distinctly enhanced the selectivity of Sil-TEPA/CDs. Thus, 12 nucleosides and nucleobases and 11 amino acids was nicely separated on Sil-TEPA/CDs. By study the influences of the changes of mobile phase composition, mobile phase buffer concentration and buffer pH on the retention behaviors of Sil-TEPA and Sil-TEPA/CDs, it was found that both hydrophilic partitioning and adsorption of analytes on Sil-TEPA/CDs were enhanced benefit from the co-existence of TEPA and TEPACDs, which provided the analytes better separation performance. By comparing the column quality of Sil-TEPA/CDs with four commercially available columns, Sil-TEPA/CDs exhibited the best peak asymmetry of 0.98, and second best column efficiency of 43895 m−1 using guanosine as analyte. The RSD (n = 9) of the retention times of five selected analytes on Sil-TEPA/CDs were within 0.30–0.61% during 40 h of continuously elution, which implied excellent stability of prepared packing material.

QuEChERS-assisted ion pair chromatography/fluorescence detection method for determination of antimigraine combination therapy in rabbit plasma samples: Application to a pharmacokinetic study

Antimigraine combination therapy has shown significant effectiveness in relieving pain, as well as reducing the frequency, duration, and severity of migraine attacks if compared to a single migraine medication. This work represents the first analytical investigation for emphasizing the synergistic effect of combining ophthalmic beta blockers with triptans in migraine treatment. The presented study was conducted to investigate the pharmacokinetic profile of almotriptan (ALM), a serotonin (5-HT1B/1D) receptor agonist used to treat migraine, when coadministered with timolol (TIM) or verapamil (VER) which are considered as an adjuvant therapy in migraine prevention. Ion pair chromatography (IPC) with online fluorescence detection was applied to simultaneously detect and quantify the binary mixtures of ALM/TIM and ALM/VER in rabbit plasma samples. The separation was achieved using a Platinum C18 analytical column with a mobile phase composed of methanol: 35 mmol L-1 phosphate buffer solution containing 10 mmol L-1 SDS at pH = 6.8 (60:40 v/v). Several parameters were evaluated during the optimization of separation conditions including mobile phase composition, buffer concentration, buffer pH and concentration of ion pair reagent. A thorough investigation of the retention mechanism was performed, and the results showed that Coulomb forces were the main contributors to the overall retention mechanism, which may be hydrophobically assisted. QuEChERS extraction technique was utilized to extract the investigated drugs from plasma samples and a detailed study was carried out to optimize partition/extraction solvents, pH, extraction salts, sample volume and clean-up step. The method had a limit of detection and quantitation of 5.6 and 16.9 ng mL-1 for ALM in ALM/TIM mixture and 2.5 and 7.6 ng mL-1 for ALM in ALM/VER mixture, with an overall recovery not less than 95.22%. This newly proposed method offers a faster alternative to existing chromatographic methods for extraction and determination of ALM in binary mixtures with TIM or VER in rabbit plasma and provides a platform for studying pharmacokinetic parameters. The coadministration of either TIM or VER with ALM resulted in a notable rise in Cmax (maximum plasma concentration) and AUC (area under the plasma concentration-time curve) of ALM, implying possible alterations in the absorption and overall exposure of ALM.

Enhancing biotechnological applications of l-asparaginase: Immobilization on amino-epoxy-agarose for improved catalytic efficiency and stability

l-asparaginase has been shown to be effective in reducing acrylamide formation in food and in treating certain cancers, such as acute lymphoblastic leukemia. This study aimed to improve the application of l-asparaginase in these areas, through its immobilization in two distinct supports: epoxy-agarose and amino-epoxy-agarose. Different immobilization conditions were investigated, including immobilization pH and buffer concentration. The best activity results were obtained employing amino-epoxy-agarose, being the 0.1 M sodium carbonate buffer pH 10 the conditions that offered the highest immobilization yield (76%), immobilization efficiency (89.8%) and relative activity (68.2%). Immobilization of l-asparaginase in amino-epoxy-agarose improved its catalytic efficiency and stability, including at pH 4 and 60 °C. All immobilized biocatalysts preserved more than 70% of their activity after 7 cycles of reuse, and maintained about 82% of their activity after 60 days of storage at 4°C. These results show the efficacy of immobilized biocatalysts in the preservation of enzymatic activity and highlight their potential for biotechnological applications.