Core-shell column
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Recently, we confirmed that the well-established theory of gradient elution can be employed for prediction of retention in gradient elution from the isocratic data, method development and optimization in fast gradient chromatography employing short packed fully porous and monolithic columns and gradient times in between 1 and 2min, or even less. In the present work, we extended this study to short core-shell reversed-phase columns. We investigated the effects of the specification of the stationary phase in the core-shell structure on the prediction of gradient retention data. Two simple retention models describing the effects of the mobile phase on the retention by two-parameter equations yield comparable accuracy and can be used for prediction of elution times. The log-log model provides improved prediction of gradient bandwidths, especially for less retained compounds. A more sophisticated three-parameter model did not offer significant improvement of prediction. We compared the efficiency, selectivity and peak capacity of fast gradient separations of alkylbenzenes, phenolic acids and flavones on seven core shell columns with different lengths and chemistry of bonded shell stationary phase. Within the limits dictated by a fixed short separation time, appropriate adjustment of the range of the composition of mobile phase during gradient elution is the most efficient means to optimize the gradient separation. The gradient range affects sample bandwidths equally or even more significantly than the column length. Both 5-cm and 3-cm core-shell columns may provide comparable peak capacity in a fixed short gradient time.
Indole-3-carbinol is a natural glucosinolate known for prevention of human breast, prostate and other types of cancer and it started to be used in commercial preparations, as food supplements. However no analytical method has been proposed for quality control of nutraceuticals with this substance yet. In this paper a new high-performance liquid chromatography (HPLC) method using core-shell column for separation of indole-3-carbinol and its condensation/degradation products was developed and used for the quantitative determination of indole-3-carbinol in nutraceuticals. Separation of indole-3-carbinol, its condensation/degradation products and internal standard ethylparaben was performed on the core-shell column Kinetex 5μ XB-C18 100A (100×4.6mm), particle size 5.0μm, with mobile phase acetonitrile/water according to the gradient program at a flow rate of 1.25mLmin(-1) and at temperature 50°C. The detection wavelength was set at 270nm. Under the optimal chromatographic conditions good linearity of determination was achieved. Available commercial samples of nutraceuticals were extracted with 100% methanol using ultrasound bath. A 5-μL sample volume of the supernatant was directly injected into the HPLC system. The developed method provided rapid and accurate tool for quality control of nutraceuticals based on cruciferous vegetable extracts with indole-3-carbinol content. The presented study showed that the declared content of indole-3-carbinol significantly varied in the different nutraceuticals available on the market. Two analyzed preparations showed the presence of condensation/degradation products of indole-3-carbinol which were not officially declared by the manufacturer. Moreover, further two analyzed nutraceutical preparations showed absolutely no content of declared amount of indole-3-carbinol.
In this paper, a new ultra-high performance liquid chromatography (UHPLC) method using a core-shell column with a pentafluorophenyl stationary phase for separation of seven active compounds of a Silybum marianum extract was developed and validated. Silymarin, an extract of Silybum marianum, is known for its abilities to protect the liver from toxic substances, hepatitis therapy, and anti-tumour activity. Silymarin is currently being widely used in commercial preparations and herbal teas. Separation of seven compounds contained in the Silybum marianum extract (taxifolin, silychristin, silydianin, silybin A, silybin B, isosilybin A, isosilybin B) and other substances occurring in real samples was performed on the Kinetex 1.7μ F5 100A (150×2.1mm), 1.7μm particle size core-shell column, with a mobile phase methanol/100mM phosphate buffer pH 2.0 according to the gradient program. A mobile phase 0.35mLmin(-1) flow rate and 50°C temperature was used for the separation. The detection wavelength was set at 288nm. Under optimal chromatographic conditions, good linearity with a correlation coefficient of R(2) >0.999 for all compounds was achieved. The available commercial samples of herbal teas and food supplements were extracted with methanol using an ultrasonic bath. After dilution with water and centrifugation, a 2μL sample of the filtered supernatant was directly injected into the UHPLC system. The use of a pentafluorophenyl stationary phase with methanol as the organic component of the mobile phase showed new ways to effectively separate isomeric compounds in herbal extracts, which could not be done with the conventional C18 stationary phase.
- MeSH
- bylinné čaje analýza MeSH
- farmaceutická chemie metody normy MeSH
- isomerie MeSH
- ostropestřec mariánský * MeSH
- potravní doplňky analýza MeSH
- rostlinné extrakty analýza chemie MeSH
- silymarin analogy a deriváty analýza chemie MeSH
- vysokoúčinná kapalinová chromatografie metody normy MeSH
- Publikační typ
- časopisecké články MeSH
Two chromatographic narrow-bore columns, a novel 2.6 μm particle-packed Kinetex™ C18 core-shell (50×2.1 mm id) and monolithic Chromolith(®) FastGradient RP-18e (50×2 mm id), were evaluated for the analysis of diastereoisomers of the flavonolignans silybin and 23-O-acetylsilybin under isocratic conditions. The main advantages of the core-shell column are markedly higher efficiency (hmin =2.8 versus 5.6 for silybin A) and better peak symmetry. The Kinetex column exhibits only a slight change in the height equivalent of the theoretical plate with a higher linear velocity of the mobile phase. The monolithic column shows notably higher selectivity in terms of selectivity factor (1.21 versus 1.12) in the analysis of critical-pair of diastereoisomers (silybin A and silybin B) and enables shorter run duration (approx. twofold) together with lower backpressure. The resolution power was found to be comparable, but the Kinetex column required a higher pressure of the mobile phase that, together with the higher chance of clogging, can be a disadvantage in the separation of biological samples. Successful baseline separation of silybin diastereoisomers in real pharmaceutical sample on monolithic column was accomplished.
The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high-throughput assay methods in bioanalysis. It can be achieved either by ultra-high-performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction--monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second-generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core-shell and fully porous sub-2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.
- MeSH
- alfa-tokoferol analýza krev MeSH
- lidé MeSH
- mateřské mléko chemie MeSH
- poréznost MeSH
- syntetické pryskyřice chemie MeSH
- velikost částic MeSH
- vitamin A analýza krev MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
For the purpose of in vivo pharmacokinetic studies, an HPLC method was developed and validated for the quantification of N-(ω)-hydroxy-nor-L-arginine, L-arginine and N-(ω)-ethyl-L-arginine (internal standard) in rat plasma. Sample processing involved a solid-phase extraction on the Waters MCX cartridges and on-line pre-column derivatization of the analytes with o-phthaldialdehyde and 3-mercaptopropionic acid. Separation of the derivatives was carried out on a core-shell Kinetex C18 column in a gradient elution mode with a mobile phase consisting of methanol and water (pH=3.00 adjusted with formic acid). Fluorimetric detection with the excitation/emission wavelengths of 235/450 nm was used. The method was validated according to the FDA guidelines and applied to pilot pharmacokinetic experiments. An unknown metabolite was extracted from the plasma of Wistar rats after a single bolus of N-(ω)-hydroxy-nor-L-arginine (i.v. 10 mg kg(-1)). The metabolite was identified as nor-L-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.
- MeSH
- arginasa antagonisté a inhibitory MeSH
- arginin aplikace a dávkování analogy a deriváty krev farmakokinetika MeSH
- extrakce na pevné fázi metody MeSH
- krysa rodu rattus MeSH
- lineární modely MeSH
- pilotní projekty MeSH
- potkani Wistar MeSH
- reprodukovatelnost výsledků MeSH
- stabilita léku MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Currently, for Sequential Injection Chromatography (SIC), only reversed phase C18 columns have been used for chromatographic separations. This article presents the first use of three different stationary phases: three core-shell particle-packed reversed phase columns in flow systems. The aim of this work was to extend the chromatographic capabilities of the SIC system. Despite the particle-packed columns reaching system pressures of ≤ 610 PSI, their conditions matched those of a commercially produced and optimised SIC system (SIChrom™ (FIAlab(®), USA)) with a 8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with a 4 mL reservoir and maximum system pressure of ≤ 1000 PSI. The selectivity of each of the tested columns, Ascentis(®) Express RP-Amide, Ascentis(®) Express Phenyl-Hexyl and Ascentis(®) Express C18 (30 mm × 4.6mm, core-shell particle size 2.7 μm), was compared by their ability to separate seven phenolic acids that are secondary metabolite substances widely distributed in plants. The separations of all of the components were performed by isocratic elution using binary mobile phases composed of acetonitrile and 0.065% phosphoric acid at pH 2.4 (a specific ratio was used for each column) at a flow-rate of 0.60 mL/min. The volume of the mobile phase was 3.8 mL for each separation. The injection volume of the sample was 10 μL for each separation. The UV detection wavelengths were set to 250, 280 and 325 nm. The RP-Amide column provided the highest chromatographic resolution and allowed for complete baseline separation of protocatechuic, syringic, vanillic, ferulic, sinapinic, p-coumaric and o-coumaric acids. The Phenyl-Hexyl and C18 columns were unable to completely separate the tested mixture, syringic and vanillic acid and ferulic and sinapinic acids could not be separated from one another. The analytical parameters were a LOD of 0.3 mg L(-1), a LOQ of 1.0 mg L(-1), a calibration range of 1.0-50.0 (100.0) mg L(-1) (r>0.997) and a system precision of 10 mg L(-1) with a RSD ≤ 1.65%. The high performance of the chromatography process with the RP-Amide column under optimised conditions was highlighted and well documented (HETP values ≤ 10 μm, peak symmetry ≤ 1.33, resolution ≥ 1.87 and time for one analysis <8.0 min). The results of these experiments confirmed the benefits of extending chromatographic selectivity using core-shell particle column technology in a SIC manifold.
Breast milk is a main source of fat-soluble vitamins for newborns and it is needful to monitor the nutritional status prior to its application. In this work a novel, high-throughput and low-cost method for monitoring of retinol and alpha-tocopherol in breast milk was developed, validated and compared with reference method using monolithic column. For this purpose five various porous shell and monolithic columns were tested on the basis of relationship between HETP and linear mobile phase velocity, analysis time and consumption of solvents. Finally the core-shell analytical column Kinetex C18 (2.6 μm, 100 Å, 100×4.6 mm) was chosen as the best and optimal values of flow rate, injection volume and temperature of analysis were established. The detection of retinol and alpha-tocopherol was carried out at 325 and 295 nm, respectively by diode array detector. The LOD 0.004 μmol/L and 0.078 μmol/L, the LOQ 0.012 μmol/L and 0.182 μmol/L for retinol and alpha-tocopherol, respectively were calculated. The validation data showed good linearity, repeatability of retention time with RSD 0.22% and 0.12%, repeatability of peak area with RSD 6.94% and 1.75%, recovery 114.1-116.3% and 99.0-108.6% for retinol and alpha-tocopherol, respectively. Moreover, the newly developed method substantially decreased the solvent consumption by about 263 mL per 100 samples with the total time of analysis 1.75 min in comparison with analysis time 1.80 of the reference method.
- MeSH
- alfa-tokoferol analýza MeSH
- časové faktory MeSH
- lidé MeSH
- limita detekce MeSH
- mateřské mléko chemie MeSH
- reprodukovatelnost výsledků MeSH
- vitamin A analýza MeSH
- vitaminy analýza MeSH
- vysokoúčinná kapalinová chromatografie ekonomika přístrojové vybavení MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
The presence of biogenic amines is a hallmark of degraded food and its products. Herein, we focused on the utilization of magnetic nanoparticles off-line coupled with ion exchange chromatography with post-column ninhydrin derivatization and Vis detection for histamine (Him) separation and detection. Primarily, we described the synthesis of magnetic nanoparticles with nanomaghemite core (γ-Fe₂O₃) functionalized with titanium dioxide and, then, applied these particles to specific isolation of Him. To obtain further insight into interactions between paramagnetic particles' (PMP) surface and Him, a scanning electron microscope was employed. It was shown that binding of histamine causes an increase of relative current response of deprotonated PMPs, which confirmed formation of Him-PMPs clusters. The recovery of the isolation showed that titanium dioxide-based particles were able to bind and preconcentrate Him with recovery exceeding 90%. Finally, we successfully carried out the analyses of real samples obtained from silage. We can conclude that our modified particles are suitable for Him isolation, and thus may serve as the first isolation step of Him from biological samples, as it is demonstrated on alfalfa seed variety Tereza silage.
A high-throughput miniaturized liquid-liquid extraction procedure followed by a simple ultra-high performance liquid chromatography method coupled with fluorescence detection for bioanalytical analysis of all tocopherol isomers and retinol in human serum has been developed and validated. In the extraction procedure, a synthetic internal standard tocol was used, which does not occur in the human body. The separation of structurally related vitamins was achieved using a new generation of pentafluorophenyl propyl core-shell stationary phase with elution using methanol and an aqueous solution of ammonium acetate. The fluorescence of retinol and tocopherol isomers was detected at λex = 325, 295 nm and λem = 480, 325 nm, respectively. The rapid baseline separation of all analytes was accomplished within 4.0 min. The sensitivity of method was demonstrated with lower limits of quantification: retinol 0.01 μM, α-tocopherol 0.38 μM, β-tocopherol 0.18 μM, γ-tocopherol 0.14 μM, and δ-tocopherol 0.01 μM. Possible application of this method in clinical practice was confirmed by the analysis of human serum samples from healthy volunteers. Finally, the simultaneous determination of retinol and all tocopherol isomers in human serum can enable the clarification of their role in metabolism and in diseases such as cancer.
