Lactoferrin is a multifunctional protein with antimicrobial activity and others tohealth beneficial properties. The main aim of this work was to propose easy to usetechnique for lactoferrin isolation from cow colostrum samples. Primarily we utilizedsodium dodecyl sulphate - polyacrylamide gel electrophoresis for isolation of lactoferrinfrom the real samples. Moreover we tested automated microfluidic Experionelectrophoresis system to isolate lactoferrin from the collostrum sample. The welldeveloped signal of lactoferrin was determined with detection limit (3 S/N) of 20 ng/ml. Inspite of the fact that Experion is faster than SDS-PAGE both separation techniques cannotbe used in routine analysis. Therefore we have tested third separation technique, ionexchange chromatography, using monolithic column coupled with UV-VIS detector (LCUV-VIS). We optimized wave length (280 nm), ionic strength of the elution solution (1.5M NaCl) and flow rate of the retention and elution solutions (0.25 ml/min and 0.75 ml/min.respectively). Under the optimal conditions the detection limit was estimated as 0.1 μg/mlof lactoferrin measured. Using LC-UV-VIS we determined that lactoferrin concentrationvaried from 0.5 g/l to 1.1 g/l in cow colostrums collected in the certain time interval up to 72 hours after birth. Further we focused on miniaturization of detection device. We testedamperometric detection at carbon electrode. The results encouraged us to attempt tominiaturise whole detection system and to test it on analysis of real samples of humanfaeces, because lactoferrin level in faeces is closely associated with the inflammations ofintestine mucous membrane. For the purpose of miniaturization we employed thetechnology of printed electrodes. The detection limit of lactoferrin was estimated as 10μg/ml measured by the screen-printed electrodes fabricated by us. The fabricatedelectrodes were compared with commercially available ones. It follows from the obtainedresults that the responses measured by commercial electrodes are app. ten times highercompared with those measured by the electrodes fabricated by us. This phenomenonrelates with smaller working electrode surface area of the electrodes fabricated by us(about 50 %) compared to the commercial ones. The screen-printed electrodes fabricatedby us were utilized for determination of lactoferrin faeces. Regarding to fact that sample offaeces was obtained from young and healthy man the amount of lactoferrin in sample wasunder the limit of detection of this method.
Application of the poly-ɛ-caprolactone composite sorbent consisting of the micro- and nanometer fibers for the on-line extraction of non-steroidal anti-inflammatory drugs from a biological matrix has been introduced. A 100 μL human serum sample spiked with ketoprofen, naproxen, sodium diclofenac, and indomethacin was directly injected in the extraction cartridge filled with the poly-ɛ-caprolactone composite sorbent. This cartridge was coupled with a chromatographic instrument via a six-port switching valve allowing the analyte extraction and separation within a single analytical run. The 1.5 min long extraction step isolated the analytes from the proteinaceous matrix was followed by their 13 min HPLC separation using Ascentis Express RP-Amide (100 × 4.6 mm, 5 µm) column. The recovery of all analytes from human serum tested at three concentration levels ranged from 70.1% to 118.7%. The matrix calibrations were carried out in the range 50 to 20,000 ng mL-1 with correlation coefficients exceeding 0.996. The detection limit was 15 ng mL-1, and the limit of quantification corresponded to 50 ng mL-1. The developed method was validated and successfully applied for the sodium diclofenac determination in real patient serum. Our study confirmed the ability of the poly-ɛ-caprolactone composite sorbent to remove the proteins from the biological matrix, thus serving as an alternative to the application of restricted-access media.
- Keywords
- biological samples, column-switching chromatography, microfibers, nanofibers, on-line extraction, restricted access media,
- Publication type
- Journal Article MeSH
The vertebral anatomy of snakes has attracted the attention of researchers for decades and numerous studies have been made for extinct and extant species. The present study investigated the morphological variations in vertebral structure among different vertebral regions in the dice snake Natrix tessellata, and provides a detailed anatomical and microstructural description of the vertebral column. Vertebrae were analyzed and compared using x-ray imaging, scanning electron microscopy, micro-computed tomography, and histological techniques. The vertebral column of N. tessellata is divided into three regions: precloacal, cloacal, and caudal. Unlike in many other tetrapods and snakes, the atlas of N. tessellata does not form a complete ring. It has a flat and roughly trilobate shape with a prominent middle lobe. The axis has two hypapophyses. The anterior precloacal region of the vertebral column has longer and more paddle-shaped hypapophyses, distinguishing it from the posterior and mid-trunk vertebrae. The anterior cloacal vertebrae have a short hypapophysis rather than a hemal keel, and the lymphapophysis extends outward, curving slightly. The cotyle and condyle of the caudal vertebrae exhibited a closer resemblance to a rounded shape, while the pleurapophysis extended ventrolaterally and curved ventrally near its distal end. Paired hemapophyses were present at the posterior-most point of the centrum instead of a hypapophysis. In light of previous fossil findings, our anatomical comparison of the vertebral and transverse processes indicates that the extant Natrix has a more flexible and less rigid spine than its ancestors. Overall, the vertebral differences among snake anatomical regions or taxa are a testament to the remarkable diversity and adaptability of these fascinating reptiles.
- Keywords
- Natrix, SEM, micro‐CT, snake, vertebra,
- MeSH
- Colubridae * MeSH
- Histological Techniques MeSH
- Spine diagnostic imaging MeSH
- X-Ray Microtomography MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Turkey MeSH
Spinal deformities such as scoliosis and kyphosis are incurable, and can lead to decreased physical function, pain, and reduced quality of life. Despite much effort, no clear therapies for the treatment of these conditions have been found. Therefore, the development of an animal model for spinal deformity would be extremely valuable to our understanding of vertebral diseases. In this study, we demonstrate that mice deficient in the mitochondrial enzyme isocitrate dehydrogenase 2 (IDH2) develop spinal deformities with aging. We use morphological analysis as well as radiographic and micro-CT imaging of IDH2-deficient mice to characterize these deformities. Histological analysis showed increased abnormalities in IDH2-deficient mice compared to wild type mice. Taken together, the results suggest that IDH2 plays a critical role in maintaining the spinal structure by affecting the homeostatic balance between osteoclasts and osteoblasts. This indicates that IDH2 might be a potent target for the development of therapies for spinal deformities. Our findings also provide a novel animal model for vertebral disease research.
- MeSH
- Isocitrate Dehydrogenase genetics MeSH
- Disease Models, Animal * MeSH
- Mice, Knockout MeSH
- Spinal Diseases diagnostic imaging etiology pathology MeSH
- Spine pathology MeSH
- X-Ray Microtomography MeSH
- Aging pathology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Isocitrate Dehydrogenase MeSH
- isocitrate dehydrogenase 2, mouse MeSH Browser
Knowing the adsorption isotherms of the components of a mixture on the chromatographic system used to separate them is necessary for a better understanding of the separation process and for the optimization of the production rate and costs in preparative high-performance liquid chromatography (HPLC). Currently, adsorption isotherms are usually measured by frontal analysis, using conventional analytical columns. Unfortunately, this approach requires relatively large quantities of pure compounds, and hence is expensive, especially in the case of pure enantiomers. In this work, we investigated the possible use of packed micro-bore and capillary HPLC columns for the determination of adsorption isotherms of benzophenone, o-cresol and phenol in reversed-phase systems and of the enantiomers of mandelic acid on a Teicoplanin chiral stationary phase. We found a reasonable agreement between the isotherm coefficients of the model compounds determined on micro-columns and on conventional analytical columns packed with the same material. Both frontal analysis and perturbation techniques could be used for this determination. The consumption of pure compounds needed to determine the isotherms decreases proportionally to the second power of the decrease in the column inner diameter, i.e. 10 times for a micro-bore column (1 mm I.D.) and 100 times for capillary columns (0.32 mm I.D.) with respect to 3.3 mm I.D. conventional columns.
Polycaprolactone composite nanofibers coated with a polydopamine layer are introduced as a new type of absorption material for on-line solid phase extraction (SPE) in chromatographic system. A hybrid technology combining the electrospinning and melt blowing was used for the preparation of 3D-structured microfiber/nanofibrous polycaprolactone composite. The dopamine coating was then applied to functionalize the micro/nanofibers. Polydopamine-coated polycaprolactone fibers were tested as an extraction phase in on-line SPE prior to HPLC separation and UV detection. Four groups of biologically active substances including bisphenols (Bisphenol S, Bisphenol AF, Bisphenol A, Bisphenol C, Bisphenol AP, Bisphenol Z, Bisphenol BP, and Bisphenol M), betablockers (Timolol, Metoprolol, Labetalol, and Propranolol), nonsteroidal antiphlogistic drugs (Salicylic acid, Ketoprofen, Naproxen, Indomethacin, Diclofenac, Ibuprophen, and Meclofenamic acid), and phenolic acids (Chlorogenic acid, Caffeic acid, Sinapic acid, m-Coumaric acid, Benzoic acid, and Cinnamic acid) were used as the model analytes. Neat and coated fibers were compared and applied as sorbents for the on-line extraction set-up. Both materials produced good extraction potential for the determination of bisphenols and nonsteroidal drugs in model biological and environmental samples including river water, human urine, and blood serum. However, the polydopamine layer significantly increased the extraction efficiency of polar drugs. Typical repeatability of on-line extraction procedure on polydopamine coated fibers was in the range 0.12-4.11% for bisphenols, 0.55-1.41% for antiphlogistic drugs, 0.59-2.52% for phenolic acids, and 1.01-1.65% for betablockers. Graphical abstract Schematic representation of polycaprolactone composite nanofibers coated with a polydopamine layer as an advanced absorption material for on-line solid phase extraction in chromatography.
- Keywords
- Chromatography, Column switching, Degradation, Dopamine coating, Micro-column, Microfiber, Nanofiber, Polymerization, Solid phase extraction, Surface modification,
- MeSH
- Anti-Inflammatory Agents, Non-Steroidal analysis isolation & purification MeSH
- Adrenergic beta-Antagonists analysis isolation & purification MeSH
- Water Pollutants, Chemical analysis isolation & purification MeSH
- Cinnamates analysis isolation & purification MeSH
- Solid Phase Extraction methods MeSH
- Phenols analysis isolation & purification MeSH
- Indoles chemistry MeSH
- Nanofibers chemistry MeSH
- Polyesters chemistry MeSH
- Polymerization MeSH
- Polymers chemistry MeSH
- Rivers chemistry MeSH
- Reproducibility of Results MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Anti-Inflammatory Agents, Non-Steroidal MeSH
- Adrenergic beta-Antagonists MeSH
- Water Pollutants, Chemical MeSH
- Cinnamates MeSH
- Phenols MeSH
- Indoles MeSH
- polycaprolactone MeSH Browser
- polydopamine MeSH Browser
- Polyesters MeSH
- Polymers MeSH
A monolithic sulfobetaine polymethacrylate micro-column BIGDMA-MEDSA designed in our laboratory, shows dual retention mechanism: In acetonitrile-rich mobile phase, hydrophilic interactions control the retention (HILIC system), whereas in more aqueous mobile phases the column shows essentially reversed-phase behavior with major role of hydrophobic interactions. The zwitterionic polymethacrylate micro-column can be used in the first dimension of two-dimensional LC in alternating reversed-phase (RP) and HILIC modes, coupled with an alkyl-bonded core-shell or silica-based monolithic column in the second dimension, for HILIC×RP and RP×RP comprehensive two-dimensional separations. During the HILIC×RP period, a gradient of decreasing acetonitrile gradient is used for separation in the first dimension, so that at the end of the gradient the polymeric monolithic micro-column is equilibrated with a highly aqueous mobile phase and is ready for repeated sample injection, this time for separation under reversed-phase gradient conditions with increasing concentration of acetonitrile in the first dimension. The fast repeating reversed-phase gradients on a short silica-monolithic or core-shell column in the second dimension can be optimized independently of the actual running first-dimension gradient program. As the alternating HILIC and RP separations on the first-dimension zwitterionic methacrylate column are based on complementary retention mechanisms, the instrumental setup essentially represents two coupled two-dimensional systems. It is first time that such an automated dual LCxLC approach is reported. The novel system allows obtaining three-dimensional data in a relatively short time and can be applied not only to multidimensional gradient separations of flavones and related polyphenolic compounds.
- Keywords
- HILIC, Monolithic columns, Multidimensional chromatography, Polyphenolic compounds, Reversed-phase,
- MeSH
- Acetonitriles MeSH
- Betaine analogs & derivatives MeSH
- Chromatography, Liquid instrumentation methods MeSH
- Chromatography, Reverse-Phase instrumentation methods MeSH
- Flavones isolation & purification MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Hydroxybenzoates isolation & purification MeSH
- Polymethacrylic Acids * MeSH
- Silicon Dioxide * MeSH
- Solvents MeSH
- Water MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- acetonitrile MeSH Browser
- Acetonitriles MeSH
- Betaine MeSH
- Flavones MeSH
- Hydroxybenzoates MeSH
- Polymethacrylic Acids * MeSH
- Silicon Dioxide * MeSH
- phenolic acid MeSH Browser
- Solvents MeSH
- sulfobetaine MeSH Browser
- Water MeSH
The advantages of microbore columns for trace analysis by liquid chromatography are identified, with reference to on-column enrichment techniques performed on analytical micro-columns. The selectivity and high sensitivity of the amperometric detector are utilized in combination with a microbore column for a number of pharmaceutical and bioanalytical analyses, including phenothiazines, parabens, sulphonamides, catecholamines, tetracyclines, vitamins, amino acids and dipeptides.
- Publication type
- Journal Article MeSH
Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.
- Keywords
- comprehensive two-dimensional chromatography, monolithic columns, polymethacrylate monoliths, silica-based monolithic columns,
- Publication type
- Journal Article MeSH
Once a suitable stationary phase and column dimensions have been selected, the retention in liquid chromatography (LC) is traditionally adjusted by controlling the mobile phase composition. Solvent gradients enable achievement of good separation selectivity while decreasing the separation time as compared to isocratic elution. Capillary columns allow use of other programming parameters, i.e. temperature and applied electric fields, in addition to solvent gradient elution. This paper presents a review of programmed separation techniques in miniaturized LC, including retention modeling and method transfer from the conventional to micro- and capillary scales. The impact of miniaturized instrumentation on retention and the limitations of capillary LC are discussed. Special attention is focused on the gradient dwell volume effects, which are more important in micro-LC techniques than in conventional analytical LC and may cause significant increase in the time of analysis, unless special instrumentation and (or) pre-column flow-splitting is used. The influence of temperature upon retention is also discussed, and applications where the temperature has been actively used for retention control in capillary LC are included together with the instrumentation utilized. Finally the possibilities of additional selectivity control by applying an electric field over a packed capillary LC column are discussed.
- Publication type
- Journal Article MeSH
