Analysis of free fractions for chiral drugs using ultrafast extraction and multi-dimensional high-performance affinity chromatography.
Ontology highlight
ABSTRACT:
SUBMITTER: Zheng X
PROVIDER: S-EPMC3812820 | biostudies-literature | 2013 Nov
REPOSITORIES: biostudies-literature
Publications
Analysis of free fractions for chiral drugs using ultrafast extraction and multi-dimensional high-performance affinity chromatography.
Zheng Xiwei X Yoo Michelle J MJ Hage David S DS
The Analyst 20131101 21
A multi-dimensional chromatographic approach was developed to measure the free fractions of drug enantiomers in samples that also contained a binding protein or serum. This method, which combined ultrafast affinity extraction with a chiral stationary phase, was demonstrated using the drug warfarin and the protein human serum albumin. ...[more]
Similar Datasets
Project description:In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum.
| S-EPMC4724389 | biostudies-literature
Project description:Ultrafast affinity extraction and a multi-dimensional affinity system were developed for measuring free drug fractions at therapeutic levels. This approach was used to compare the free fractions and global affinity constants of several sulfonylurea drugs in the presence of normal human serum albumin (HSA) or glycated forms of this protein, as are produced during diabetes. Affinity microcolumns containing immobilized HSA were first used to extract the free drug fractions in injected drug/protein mixtures. As the retained drug eluted from the HSA microcolumn, it was passed through a second HSA column for further separation and measurement. Items that were considered during the optimization of this approach included the column sizes and flow rates that were used, and the time at which the second column was placed on-line with the HSA microcolumn. This method required only 1.0 ?L of a sample per injection and was able to measure free drug fractions as small as 0.09-2.58% with an absolute precision of ±0.02-0.5%. The results that were obtained indicated that glycation can affect the free fractions of sulfonylurea drugs at typical therapeutic levels and that the size of this effect varies with the level of HSA glycation. Global affinity constants that were estimated from these free drug fractions gave good agreement with those predicted from previous binding studies or determined through a reference method. The same approach could be utilized with other drugs and proteins or modified binding agents of clinical or pharmaceutical interest.
| S-EPMC4254497 | biostudies-literature
Project description:Ultrafast affinity extraction (UAE) has recently been developed and employed for measuring non-bound (or free) fractions and binding or rate constants for drugs and other targets with soluble binding agents such as serum proteins. This study examined the long-term stability of 10 mm × 2.1 mm i.d. affinity microcolumns when used in UAE at both low and high flow rates (e.g., 0.5 and 3.5 mL/min) over an extended series of injections. This stability was investigated by using immobilized human serum albumin (HSA) and samples containing the drug warfarin with or without soluble HSA as a model system. The free warfarin fractions measured at 0.5 mL/min in the presence of soluble HSA were stable up to 150 injections and changed by <10% at 3.5 mL/min. The association equilibrium constant for warfarin with HSA that was estimated by UAE at 3.5 mL/min had no significant change over 50 injections and a change of only ∼18-22% over 100-150 injections. The dissociation rate constant for warfarin from HSA was found by combining UAE results at 0.5 and 3.5 mL/min and employing a new two-point approach, with no significant changes in this value being seen even after 200 injections. The effects of extended microcolumn use on the retention time, peak width, and peak asymmetry for warfarin, and on the backpressure of the microcolumn, were also considered. These results indicated that UAE and HSA microcolumns could be used to provide consistent values for free solute fractions, binding constants, and rate constants over a large series of injections. These results should be useful in future work by providing guidelines for the assessment, further development, and use of UAE in characterizing interactions involving other drugs and binding agents in solution-based samples.
| S-EPMC8639762 | biostudies-literature
Project description:Various organic-based monoliths were prepared and optimized for immobilization of the protein human serum albumin (HSA) as a binding agent for chiral separations and high-performance affinity chromatography. These monoliths contained co-polymers based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or GMA and trimethylolpropane trimethacrylate (TRIM). A mixture of cyclohexanol and 1-dodecanol was used as the porogen, with the ratio of these solvents being varied along with the polymerization temperature to generate a library of monoliths. These monoliths were used with both the Schiff base and epoxy immobilization methods and measured for their final content of HSA. Monoliths showing the highest protein content were further evaluated in chromatographic studies using R/S-warfarin and d/l-tryptophan as model chiral solutes. A 2.6-2.7-fold increase in HSA content was obtained in the final monoliths when compared to similar HSA monoliths prepared according to the literature. The increased protein content made it possible for the new monoliths to provide higher retention and/or two-fold faster separations for the tested solutes when using 4.6mm i.d.× 50 mm columns. These monoliths were also used to create 4.6mm i.d.× 10 mm HSA microcolumns that could separate the same chiral solutes in only 1.5-6.0 min. The approaches used in this study could be extended to the separation of other chiral solutes and to the optimization of organic monoliths for use with additional proteins as binding agents.
| S-EPMC3578315 | biostudies-literature
Project description:A flow-based method employing a reverse displacement immunoassay was combined with ultrafast immunoextraction and near-infrared fluorescence detection for the analysis of free drug fractions, using phenytoin as a model analyte. Factors considered in the design of this method included the sample application conditions, the design of the immobilized drug analog column, the utilization of antibodies or F(ab) fragments as labeled binding agents, and the label application and column regeneration conditions. In the final method, sample injections led to the displacement of labeled binding agents from an immobilized phenytoin analog column. This displacement peak appeared within 20-30 s of sample injection and was proportional in size to the free phenytoin concentration in the sample. It was possible with this method to regenerate the column using only the application of additional label between sample injections. This method was used to measure clinically relevant concentrations of free phenytoin in serum and drug/protein mixtures and gave good correlation with ultrafiltration, while also being faster to perform and requiring significantly less sample. This technique was not limited to free phenytoin measurements but could be adapted for other drugs or analytes through the use of appropriate columns and binding agents.
| S-EPMC3237916 | biostudies-literature
Project description:Interactions with serum proteins such as alpha1-acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 104-106M-1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s-1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest.
| S-EPMC5479935 | biostudies-literature
Project description:Getting insights on tissue or cell specific EV composition in pathophysiological conditions for developing therapeutics, biomarkers and diagnostic tools in modern medicine still remains a major bottleneck. Similarly, lack of standards for EV comparison also hampers progression in the field. Here, we therefore generated a tagged CD81 fusion protein highly enriched in EVs, enabling affinity isolation of EVs from complex matrices in a non-destructive and pure form without disturbing the here tested EV characteristics.
2024-11-22 | GSE251842 | GEO
Project description:Ultrafast affinity extraction was evaluated and used with microcolumns containing human serum albumin (HSA) to measure the global affinity constants and dissociation rate constants for several second- and third-generation sulfonylurea drugs with solution-phase normal HSA or glycated HSA. Glibenclamide, glimepiride and glipizide were used as model drugs for this work. Both single- and two-column systems were considered for the analysis of global affinities for the model drugs. These methods were optimized with respect to the flow rates, column sizes and sample residence times that were employed with each drug for ultrafast affinity extraction. Data acquired with single-column systems were further utilized to estimate the dissociation rate constants for normal HSA and glycated HSA with the given drugs. The binding constants obtained by the single- and two-column systems showed good agreement with each other and with values obtained from the literature. Use of a single-column system indicated that levels of glycation found in controlled or advanced diabetes resulted in a 18-44% decrease in the overall binding strength of the model drugs with HSA. Although the two-column system allowed work with smaller free drug fractions and clinically-relevant drug/protein concentrations, the single-column system required less protein, provided better precision, and was easier to use in binding studies.
| S-EPMC6251404 | biostudies-literature
Project description:Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42-2.63 × 105 M-1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M-1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07-3.31 × 105 M-1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.
| S-EPMC10015259 | biostudies-literature
Project description:USArray, a pioneering project for the dense acquisition of earthquake data, provides a semi-uniform sampling of the seismic wavefield beneath its footprint and greatly advances the understanding of the structure and dynamics of Earth. Despite continuing efforts in improving the acquisition design, network irregularity still causes spatial sampling alias and incomplete, noisy data, which imposes major challenges in array-based data analysis and seismic imaging. Here we employ an iterative rank-reduction method to simultaneously reconstruct the missing traces and suppress noise, i.e., obtaining free USArray recordings as well as enhancing the existing data. This method exploits the spatial coherency of three-dimensional data and recovers the missing elements via the principal components of the incomplete data. We examine its merits using simulated and real teleseismic earthquake recordings. The reconstructed P wavefield enhances the spatial coherency and accuracy of tomographic travel time measurements, which demonstrates great potential to benefit seismic investigations based on array techniques.
| S-EPMC6768879 | biostudies-literature