Search

BACKGROUND Densitometric quantification of myeloma paraproteins (M-proteins) is used to monitor secretory myeloma related disorders in humans and dogs. The previous work in dogs used agarose gel electrophoresis (AGE) but did not establish if other methods of serum protein electrophoresis, such as capillary zone electrophoresis (CZE), were comparable. OBJECTIVES We aimed to determine if the densitometric quantification of M-proteins using CZE would yield results comparable to AGE methods. METHODS Fifty-one serum samples from 22 dogs and 18 cats with confirmed monoclonal gammopathies and previously performed AGE were evaluated using CZE on a Sebia Minicap system. Samples were run in duplicate, and their M-proteins were densitometrically measured using the corrected perpendicular drop method previously described. Human-based quality control samples were used to determine the inter-run coefficient of variation (CV). Patient samples were used to calculate the intra-run CV. Method comparison was performed using simple linear regression, Passing-Bablok regression, and Bland-Altman analyses, and Medx evaluations. RESULTS Inter-run and intra-run CVs for CZE were 3.71%-7.65% and 2.89%-4.74%, respectively. Simple linear regression demonstrated an excellent correlation (r > 0.98). Passing-Bablok regression was compatible with the presence of proportional bias in the entire population, and Bland-Altman plots revealed a proportional bias in the feline cases. The Medx evaluation suggested that the two methods did not perform similarly in clinical samples with poor performance at a decision limit of 0.5 gm/dL. CONCLUSIONS Capillary zone electrophoresis is an acceptable method for M-protein densitometric quantification in canine and feline sera but cannot be used interchangeably with AGE-based evaluations.

Bee venom from honey bees (Apis Mellifera L.) is known to have many pharmacological and biological properties. Melittin, a peptide consisting of 26 amino acids, is known as the main component of bee venom. The study aims to develop a rapid capillary electrophoresis method for separating and quantifying melittin in honeybee venom. Since melittin is a basic peptide, it will adhere to the capillary wall during separation. Two different methods were developed in this study for the capillary electrophoretic separation of melittin. As a first approach, a low pH buffer system was used. For the second approach, the capillary column was coated with a positively charged polymer (PEI). With both methods developed, the migration of melittin in the capillary was achieved by preventing wall adsorption. Melittin migrated in 6 min when the low-pH buffer system was applied, whereas its migration time is longer than 10 min in the PEI-coated capillary column. Thus, a low-pH buffer system was preferred for the analysis of the actual bee-venom sample. 100 mmol L-1 phosphoric acid/sodium dihydrogen phosphate system at pH 1.55 was chosen as separation buffer. As a conclusion, a fast and reliable method was developed for the determination of melittin in honeybee venom. The method was applied to an Anatolian bee venom sample to highlight the melittin amount. The melittin amount was found as 24.5 ± 3.4 g 100 g-1 in the bee venom sample.

Nitrate and nitrite ions taken from food are the sources of bioavailable nitric oxide (NO) in the nitrogen cycle. Some beneficial effects of honey on health are attributed to the ability of honey to increase NO production. The variation of nitrate and nitrite levels of honey samples collected from different Anatolia regions were clarified using capillary electrophoresis technique. The sensitivities of both anions were improved with the application of the sample stacking method. Separation buffer consisted of 30 mmol L−1 formic acid and 30 mmol L−1 sodium sulfate at a pH of 4.0. The CE technique revealed that 18 honey samples contained nitrate anion ranged between 2.53 and 31.8 mg kg−1. Nitrite amounts were found in lower amounts in the honey samples as between non-detected and 0.533 mg kg−1. The observed differences in nitrate levels between honey varieties may be a way to determine honey's origin.

Recently, there has been a revival of interest in the use of natural pigments in various industries. It is of great interest to obtain melanins from plant raw materials. An effective method has been developed for cleaning grape squeezes from impurities before the alkaline extraction of melanin. A schematic flow diagram of the isolation and purification of water-soluble melanin from grape squeezes is presented. The obtained pigment was identified by spectroscopic methods. It has been established by thermal treatment that the amorphous precipitate of melanin is stable at temperatures up to 120 °C, and the acid resistance of the obtained melanin solution is in the pH range of > 4.0. Gel filtration, electrophoresis and HPLC showed that the pigment preparation contained four fractions that differed by their molecular weights. The quantitative distribution and the molecular weights of the obtained melanin fractions were determined. Qualitative reactions and spectroscopic methods have shown that these fractions are typical melanins. The elemental composition of the obtained melanin and its antioxidant properties, the amino acid composition of the melanin protein, and its solubility were determined. The yield of melanin from the feedstock was 17.3%. Based on the physicochemical properties of the resulting melanin, it can be used as an effective natural food coloring and powerful antioxidant substance.

Background Serum protein electrophoresis is one of the core investigations for screening for monoclonal proteins. Among the available capillary systems, the Helena V8 system has been evaluated in a limited number of studies. Methods In total, 310 sera samples were assessed on the Helena V8 system and compared with the Sebia Capillarys instrument. Abnormalities suggestive of monoclonal proteins were confirmed by immunofixation. Imprecision studies and reference intervals were determined. Results The imprecision of the Helena V8 was inferior or equal to 5.8%. The mean bias of Helena V8 vs Sebia Capillarys was about −0.9 g/L for albumin; −0.2 g/L for alpha-1; 1.1 g/L for alpha-2; −0.2 g/L for beta; 0.3 g/L for gamma; −0.5 g/L for monoclonal protein in beta; and 0.3 g/L for monoclonal protein in gamma. Among the 56 samples with monoclonal proteins confirmed by immunofixation, all were seen on both methods, with only 1 discordant result at a cutoff of 5.0 g/L. Reference intervals were statistically different between the 2 analyzers, except for the beta fraction. Conclusions Our evaluation confirms the good analytical performance of the Helena V8 analyzer as a suitable alternative to the Sebia Capillarys instrument.

This article reviews the various analytical methods reported so far in the literature for the determination of stability and impurity profile the lenalidomide and palbociclib anti cancer drugs in single or combination with other drugs in bulk, pharmaceutical dosage forms, biological fluids, stability indicating and impurity profiling methods. The analytical methods used for the estimation of lenalidomide and palbociclib anticancer drugs reviewed in this paper includes ultraviolet spectrophotometry,high performance liquid chromatography (HPLC) ,ultra performance liquid chromatography (UPLC) ,liquid chromatography-mass spectrometry (LC-MS) and electrophoresis. This review focus on the effect of all chromatographic parameters so as to provide as fast, reliable and cost effective methodology of testing. Method development is the process of proving that analytical method is acceptable for use to measure the concentration of active pharmaceutical ingredient in a specific compound dosage form which must be validated to provide reliable data for regulatory submissions. This reviewed is mainly on analytical method development and validation, stability indicating methods, simultaneous estimation methods and bioanalytical methods. The review covers the time period from 2007 to 2019 during which analytical methods including all types of spectrophotometric and chromatographic techniques were reported. The Review covers lenalidomide and palbociclib API and formulation analytical and bioanalytical methods.

The main objective of this project was to formulate novel amphiphilic PLGA nanoparticles having better physicochemical properties for the delivery of the novel peptide (CK-10) to be used for targeting the cancerous/tumour tissue. Double emulsion/Solvent evaporation and novel microfluidic techniques were used to formulate the nanoparticles. Loading efficiency and in-vitro release were measured by a modified Lowry assay. Size and zeta potential were characterized by dynamic light scattering, tuneable pore resistive sensing, and laser obscuration time. Images were scanned by scanning, transmission electron microscopes, and laser obscuration time. Stability was checked by high-performance liquid chromatography and capillary zone electrophoresis. Water absorption and its associated changes in the physicochemical properties were measured by various titration techniques. PLGA/Poloxomer nanoparticles had the highest peptide loading efficiency by 56.13 % for the novel microfluidic technique as well as the highest in-vitro release and water absorption values. It also had the smallest size with the lowest PDI (208.90 nm, 0.11) which are vital parameters for targeting cancer/tumour tissue. The successful development of better physicochemical properties for the CK-10 loaded PLGA nanoparticles could improve the RAN blocking by CK-10.

The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA. Detection of topoisomers was achieved by incorporating ethidium bromide in the separation medium. The detector response was improved by 3 orders of magnitude by utilizing a 605-nm optical filter with a 15-nm bandwidth. Separations of linear, open circle, supercoiled, and multimer DNA plasmids ranging from 4.2 to 10.5 kbp were accomplished in under 6 min using an unmodified fused silica capillary (50-μm internal diameter). The background electrolyte was comprised of 0.5% gel, which was hydroxypropylmethyl cellulose, 1 mM ethylenediaminetetraacetic acid, and 50 mM N-(2-acetamido)-2-aminoethanesulfonic acid (pH of 6.25). The separations, which balanced the bulk electroosmotic flow, the electrophoretic mobility of the DNA, and gel sieving were dependent upon the pH of the electrolyte and the gel concentration. Reproducibility was dependent upon the procedure used to prepare the gel as well as other factors including the ethidium bromide concentration and capillary conditioning. A single unmodified capillary operated for more than 150 runs had an across-day migration time precision of 1% relative standard deviation and percent area precision of 10% relative standard deviation.

Aims: In this study, a simple, green, and rapid capillary zone electrophoresis (CZE) method coupled with a diode array detector (DAD) was applied for the analysis of avanafil (AVA) and dapoxetine hydrochloride (DAP) as a binary mixture using vardenafil (VAR) as an internal standard (IS) in pure form and pharmaceutical formulation. Methodology: The separation was done using fused silica capillary (58.5 cm total length, 50 cm effective length, and 50 μm internal diameter) and the running background electrolyte (BGE) was 100 mM acetate buffer at pH 3.6. During the separation process, the applied voltage was 30 KV, while the temperature was 25 °C. The sample injection was applied at a pressure of 50 mbar for 10 s, and detection was carried out at 210 nm for DAP and 248 nm for AVA and VAR. Results: Analysis of the tested drugs and the internal standard was carried out in less than 6.5 min, where the migration times were 4.29, 4.90, and 6.02 min for IS, DAP and AVA respectively. The proposed method showed linearity in the concentration range 5-80 and 5-70 μg/mL with correlation coefficients 0.9996 and 0.9999 for AVA and DAP respectively. The limit of detection (LOD) was 0.523 and 0.531 for AVA and DAP respectively, while the limit of quantification (LOQ) was 1.585 and 1.608 in respective order. The Peak purity and identity in the proposed method were validated by DAD. Conclusion: The proposed CZE method was validated according to ICH guidelines and applied successfully for the estimation of AVA and DAP in their combined pharmaceutical preparation.

Nucleic acids dye Goldview is widely used in agarose gel electrophoresis (AGE). However, in this study, a sample of multiplasmid DNA (multi-pDNA) stained with Goldview analyzed by AGE showed its instability at low temperature. Three types of DNA samples were analyzed, including linear DNA (ladder), single-plasmid DNA (single-pDNA), and multi-pDNA, electrophoretic conditions were optimized by adjusting the dye, the buffer, and the temperature (1-50°C). The results showed that the light intensity of Gelred is 2.2-times higher than that of Goldview in staining multi-pDNA. Compared with the single-pDNA and the linear DNA, the multi-pDNA stained with Goldview was greatly affected by temperature. This short communication indicated that Gelred is a highly applicable dye for analyzing multiplasmid samples. The degree and the way of binding of Goldview to multi-pDNA are greatly affected by temperature.

Sodium dodecyl sulfate (SDS) provides numerous benefits for proteome sample preparation. However, the surfactant can be detrimental to downstream mass spectrometry analysis. Although strategies are available to deplete SDS from proteins, each is plagued by unique deficiencies that challenge their utility for high-throughput proteomics. An optimal approach would rapidly and reproducibly achieve less than 10 ppm residual SDS while simultaneously maximizing analyte recovery. Here, we describe improvements to a simple electrokinetic device termed transmembrane electrophoresis, which we previously reported for automated, rapid SDS depletion of proteome samples. Voltage-driven transport of SDS across a molecular weight cutoff membrane is enhanced at higher electric fields, which is herein achieved by integrating an active cooling mechanism to mitigate the impacts of Joule heating. We report 99.9% reduction of SDS (final concentration5 ppm) in 5 min. The device is employed in a detergent-based proteomic workflow for analysis of an enriched yeast membrane proteome extract, demonstrating quantitative protein recovery (98%) and increasing the number of identifications by liquid chromatography-tandem mass spectrometry.

Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the diethylaminoethyl-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than CE, and resolution approaching CE.

Capillary electrophoresis coupled online with mass detection is a modern tool for analyzing wide ranges of compounds in complex samples, including urine. Capillary electrophoresis with mass spectrometry allows the separation and identification of various analytes spanning from small ions to high molecular weight protein complexes. Similarly to the much more common liquid chromatography-mass spectrometry techniques, the capillary electrophoresis separation reduces the complexity of the mixture of analytes entering the mass spectrometer resulting in reduced ion suppression and a more straightforward interpretation of the mass spectrometry data. This review summarizes capillary electrophoresis with mass spectrometry studies published between the years 2017 and 2021, aiming at the determination of various compounds excreted in urine. The properties of the urine, including its diagnostical and analytical features and chemical composition, are also discussed including general protocols for the urine sample preparation. The mechanism of the electrophoretic separation and the instrumentation for capillary electrophoresis with mass spectrometry coupling is also included. This review shows the potential of the capillary electrophoresis with mass spectrometry technique for the analyses of different kinds of analytes in a complex biological matrix. The discussed applications are divided into two main groups (capillary electrophoresis with mass spectrometry for the determination of drugs and drugs of abuse in urine and capillary electrophoresis with mass spectrometry for the studies of urinary metabolome).

A study of the degradation profiles of drugs based on recombinant monoclonal antibodies under stress conditions made it possible to determine the dynamics and mechanisms of degradation processes, to identify drug degradation products, and to reveal differences in the stability of biosimilar drugs for relatively short times. A strategy for accelerated (35 d) stability assessment under stress conditions of the original product Soliris® (Switzerland) and its biosimilar PRK-001 (Russia) was proposed. Their degradation profiles after exposure to extreme pH, oxidative stress, and UV radiation were studied. Antibody fragments were analyzed using size-exclusion chromatography, capillary isoelectric focusing, electrophoresis, and mass spectrometry. Both products showed similar degradation profiles and similar changes in isoform contents.

After entering human blood circulation, small-molecule drugs interact extensively with various plasma proteins, such as human serum albumin and α1-acid glycoprotein. These interactions profoundly affect the distribution of drugs in vivo and the binding of drugs to targets, thus affecting the efficacy of drugs. In-depth investigation of drug-plasma protein interactions is of great significance for the optimization of drug properties, the development of new drugs, risk assessment, and combination therapy of drugs. Therefore, it is essential to develop highly efficient, sensitive, and accurate methods for elucidating drug-plasma protein interactions. Chromatography is a powerful tool with high throughput, high separation performance, and high sensitivity in the characterization of drug-protein interactions. High-performance affinity chromatography (HPAC) and capillary electrophoresis (CE) have been widely utilized in this field. These methods include the determination of the effects of the posttranslational modification of proteins on binding and the competitive binding of multiple drugs. In addition, various chromatographic methods are used to obtain interaction information such as the binding constant, binding-site number, and dissociation rate constant. In this review, the common strategies and recent advances in HPAC and CE in the study of drug-plasma protein interactions are briefly reviewed. The immobilization methods of proteins, the principles and applications of frontal analysis, zonal elution, ultrafast affinity extraction, peak profiling, and peak decay analysis are discussed for HPAC and affinity capillary electrophoresis (ACE) and capillary electrophoresis frontal analysis (CE-FA) for CE. HPAC relies on the fixation of proteins on the surfaces of chromatographic stationary phases by covalent linking or physical adsorption, followed by obtaining the drug-protein interaction information through a variety of chromatographic methods. In the frontal chromatography analysis, mobile phases with different concentrations of drugs are passed through the HPAC column to obtain different breakthrough times. The process can determine the number of drug binding sites and the binding constant of each site in the affinity protein with high accuracy. The zonal elution method can detect the drug binding sites on proteins using site-specific probes to determine whether there is competition between drugs and probes. The sample consumption and analysis time of the zonal elution method are much less than those in frontal chromatography analysis. The ultrafast affinity extraction method can inject complex samples, such as serum, into affinity columns to determine the free drug components. It can measure the combination and dissociation constants of drug-protein interactions by changing the chromatography flow rate. Peak profiling and peak decay analyses are both effective methods for investigating the dissociation of drugs and proteins. In CE analysis, the drug and protein samples are dissolved in an electrophoresis buffer, and their interactions are measured during electrophoresis with high accuracy and low sample consumption. However, the adsorption of proteins on the capillary wall can compromise CE performance. Common CE methods in drug-protein interaction analysis are ACE and CE-FA. ACE is usually performed by changing the effective mobility of drugs via the addition of different concentrations of proteins. This method has been widely used, and several variant techniques have been developed recently. CE-FA involves the sampling of a drug premixed at a known concentration with a target protein. Compared with other CE methods, CE-FA exhibits the unique advantages of high throughput, automatic online analysis, and the ability to determine high-order drug-protein interactions. Finally, the shortcomings of current chromatography methods are summarized, and the application prospects and development direction of chromatography technology in the field of drug-plasma protein interaction research are discussed.

Proteins can be separated according to their size by gel electrophoresis and further analyzed by Western blotting. The proteins can be transferred to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF), which results in a replica of the proteins' separation patterns. The proteins on the membrane can be detected by specific antibodies followed by visualization either on the membrane itself, on film or by CCD cameras. Western blotting is a sensitive technique to verify data obtained from difference gel electrophoresis (DIGE)-based proteomics.

Two-dimensional comparative fluorescence gel electrophoresis (CoFGE) uses an internal standard to increase the reproducibility of coordinate assignment for protein spots visualized on 2D polyacrylamide gels. This is particularly important for samples, which need to be compared without the availability of replicates and thus cannot be studied using differential gel electrophoresis (DIGE). CoFGE corrects for gel-to-gel variability by co-running with the sample proteome a standardized marker grid of 80-100 nodes, which is formed by a set of purified proteins. Differentiation of reference and analyte is possible by the use of two fluorescent dyes. Variations in the y-dimension (molecular weight) are corrected by the marker grid. For the optional control of the x-dimension (pI), azo dyes can be used. Experiments are possible in both vertical and horizontal (h) electrophoresis devices, but hCoFGE is much easier to perform. For data analysis, commercial software capable of warping can be adapted.

Biosurfactants are biologically secreted amphiphilic molecules which resemble with the chemical surfactant in function. Compared to the chemical surfactants, they are biodegradable, biocompatible and less toxic. However, commercialization of biosurfactant production is still challenging due to high production and recovery cost, less efficient strain availability, and less knowledge about biosynthetic or metabolic pathways of biosurfactant production. Therefore, the attempt was made to optimize the production parameter for improving the biosurfactant production using Central Composite Design (CCD) from Sphingobacterium thalpophilum DP9. In addition, application of biosurfactant as shampoo ingredient was also studied. Results showed that the biosurfactant production was increased by 4.94 fold when 1% bacterial inoculum was inoculated in Bushnell-Hass medium (pH–11) supplemented with 8 g% sucrose. Biosurfactant was effectively purified by acid precipitation from cell free supernatant. Chromatographic techniques (TLC and HPTLC) and Native–PAGE electrophoresis confirmed glycoprotein nature of the biosurfactant with 85.75 ± 0.05% carbohydrate and 4.58 ± 0.03% protein part. In addition, two fragments of protein (37 kD and 44 kD) were also observed in electrophoretic analysis. According to the ‘blind touch test’ data of volunteer it was noted that the biosurfactant based shampoo showed satisfactory (conditioning) activity compared to commercially available shampoo. Therefore, it was concluded that glycoprotein produced from S. thalpophilum DP9 can be used in cosmetic or personal health products.

Ionic liquids (ILs) are widely used in separation methods such as chromatography and capillary electrophoresis due to their unique properties. This review discusses the prospects for using imidazole-based ILs as electrophoretic system modifiers in capillary zone electrophoresis and micellar and microemulsion electrokinetic chromatography for the determination of biologically active substances (amino acids, catecholamines, steroid hormones, and polyphenolic antioxidants). New approaches to selective electrophoretic determination and efficient online preconcentration of analytes with the participation of imidazolium ILs are considered; the application of developed approaches to the analysis of natural objects (urine, blood plasma, and plant extracts) is exemplified.