Journal of Pharmaceutical and Biomedical Analysis, Apr 1, 2008
An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was dev... more An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or a monolith based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of HSA in the silica monolith was similar to values obtained with silica particles and a GMA/EDMA monolith. However, the higher surface area of the silica monolith gave a material that contained 1.3-to 2.2-times more immobilized HSA per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the HSA silica monolith were evaluated using two chiral analytes: D/L-tryptophan and R/S-warfarin. The separation of R-and S-ibuprofen was also considered. The HSA silica monolith gave higher retention and higher or comparable resolution and efficiency when compared with HSA columns that contained silica particles or a GMA/EDMA monolith. The silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with immobilized HSA as a chiral stationary phase.
High‐performance affinity microcolumns were used to characterize binding by the anti‐diabetic dru... more High‐performance affinity microcolumns were used to characterize binding by the anti‐diabetic drugs repaglinide and nateglinide with normal and glycated forms of human serum albumin. The microcolumns contained only nmol amounts of protein and provided a detailed analysis of these drug interactions with good precision and in a matter of minutes per experiment. The overall binding by repaglinide to normal and glycated albumin fits a model with two types of binding sites: a set of one or two moderate‐to‐high affinity regions and a larger set of weaker regions with association equilibrium constants of ∼105 and 103 M−1, respectively, at pH 7.4 and 37°C. Competition studies gave site‐specific association constants for repaglinide and nateglinide at Sudlow site I of 4.2 × 104 and 5.0 × 104 M−1 for normal albumin, with a decrease of 26%–30% being seen for nateglinide with glycated albumin and no significant change being noted for repaglinide. At Sudlow site II, repaglinide and nateglinide h...
Journal of Pharmaceutical and Biomedical Analysis, 2021
During diabetes human serum albumin (HSA), an important drug transport protein, can be modified b... more During diabetes human serum albumin (HSA), an important drug transport protein, can be modified by agents such as glyoxal (Go) and methylglyoxal (MGo) to form advanced glycation end-products. High-performance affinity microcolumns and zonal elution competition studies were used to compare interactions by the anti-diabetic drugs repaglinide and nateglinide with normal and Go- or MGo-modified HSA at Sudlow sites I and II of this protein. Both drugs had their strongest binding at Sudlow site II for the normal and modified forms of HSA. The association equilibrium constants at this site for repaglinide and nateglinide with normal HSA were 6.1 (± 0.2) × 104 M-1 and 7.1 (± 0.8) × 105 M-1, respectively, at pH 7.4 and 37⁰C; these values increased by up to 3.6-fold for repaglinide and decreased by up to 45-55 % for nateglinide when HSA was modified by Go or MGo at levels seen in prediabetes or diabetes. Both drugs were also found to bind at Sudlow site I, with association equilibrium constants at this site on normal HSA of 4.2 (± 0.3) × 104 M-1 for repaglinide and 5.0 (± 0.1) × 104 M-1 for nateglinide. The binding strength for repaglinide at Sudlow site I increased by 1.3- to 1.7-fold with the Go-modified HSA and decreased slightly (i.e., up to 19 %) for the MGo-modified HSA, while nateglinide showed only a small or insignificant change in binding with the same modified HSA samples. These results indicated that binding by repaglinide and nateglinide with HSA can be altered significantly by modification of this protein with Go or MGo, making these modifications of potential interest in the treatment of patients with these drugs during diabetes.
Journal of Pharmaceutical and Biomedical Analysis, 2021
2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension ... more 2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension and opioid withdrawal. It is known these drugs bind to serum proteins and have significant variations within this class of compounds in the overall level of this binding. However, little specific information is available on the interactions of these compounds with the two major transport proteins for many drugs, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP). This study examined binding by 2-imidazolines to these proteins by using 25 mm × 2.1 mm i.d. high-performance affinity microcolumns that contained HSA or AGP. The drugs that were examined were antazoline, clonidine, dexmedetomidine, lofexidine, moxonidine, phentolamine, and tizanidine, which represented a wide range of structures and pharmaceutical applications. The major metabolite of lofexidine, N-(2-aminoethyl)-2-(2,6-dichlorophenoxy) propenamide (LADP), was also examined. All these 2-imidazolines were found to have weak-to-moderate binding to HSA, with global affinities that ranged from 1.62 × 102 to 1.07 × 104 M-1 at pH 7.4 and 37 °C. These compounds had stronger binding with AGP, with global affinities constants ranging from 3.80 × 102 to 1.85 × 104 M-1. No stereoselectivity was observed by HSA for the enantiomers of dexmedetomidine, lofexidine, or LADP. However, AGP did show some stereoselectivity for lofexidine and LADP but not for dexmedetomidine. These results provide a better understanding of interactions of 2-imidazoline with HSA vs AGP in the circulation and of how this binding can change between drugs within this class of compounds.
The overall goal of this study is to develop a non-covalent immobilization process for quantitati... more The overall goal of this study is to develop a non-covalent immobilization process for quantitative studies of drug-protein binding, or related biointeractions, by high-performance affinity chromatography (HPAC). A series of polymeric monolithic supports based on glycidyl methacrylate-co-ethylene dimethacrylate (GMA-co-EDMA) with different porogen compositions were prepared. On-column entrapment was employed to immobilize human serum albumin (HSA) on monolith supports which had been converted into a hydrazide-activated and capped with oxidized glycogen. Warfarin and L-tryptophan were used as site-specific probes to evaluate preparation of these stationary phases through their binding with these solutes. Zonal elution studies with these materials also showed good agreement with the binding properties that have been reported for soluble HSA. This combined approach of using protein entrapment and HPAC gave a fast means for screening and studying drug-protein interactions. On-column entrapment of proteins using monolithic support can be a suitable mean of investigating drug-protein interactions in HPAC. The effectiveness of entrapment in immobilizing HSA within various monoliths, and the consequent change in drug binding, was seen to vary with the porogen composition that was used to make the monolith. Lowering the percentage of 1-dodecanol vs. cyclohexanol in the polymerization mixture gave entrapped HSA supports with higher retention for drugs. Since this immobilization method should result in little or no loss of protein activity, the data obtained in zonal elution studies such as used in this report should be useful in directly determining binding constants for drug-protein interactions in solution. The future work will involve estimation of the moles of binding sites for each drug on HSA by also analyzing the support’s protein content. This analysis will make it possible to directly measure the association equilibrium constant for interactions of the drug with HSA. Frontal analysis of these columns will also be performed to evaluate the overall binding of drugs with proteins in these supports
Atrazine is the most widely used herbicide in the U.S. and has been detected in surface water and... more Atrazine is the most widely used herbicide in the U.S. and has been detected in surface water and groundwater. Technologies are needed for onsite and in situ remediation of water and soil containing atrazine. We investigated the potential of using fine-grained, zero-valent iron (Fe0) to remove atrazine and promote its degradation in contaminated water and soil. Atrazine loss from aqueous solution increased with increasing Fe0concentration (w/v). Agitating 20 μg14C-ring-labeled atrazine L−1with 10% Fe0(w/v) removed 92% of the14C from solution within 48 h. Only about 4% of the14C lost from solution was extractable from the iron with 3 mM CaCl2(readily available pool), 81% was extractable with CH3CN (potentially available pool), and 11% was unextractable residues. A companion experiment indicated that most of the14C extracted from the iron with 3 mM CaCl2after the 48-h Fe0treatment was unaltered atrazine, while the CH3CN extract contained approximately 33% atrazine and 48% was unidenti...
A relatively fast and reproducible CE separation was developed for the glycoform analysis of α 1a... more A relatively fast and reproducible CE separation was developed for the glycoform analysis of α 1acid glycoprotein (AGP). Factors that were considered included the pH for this separation and various techniques for coating the capillary and/or to minimize electroosmotic flow and protein adsorption. Optimum resolution of the AGP glycoforms was obtained at pH 4.2 with a running buffer containing 0.1% Brij 35 and by using static and dynamic coatings of PEO on the capillary. These conditions made it possible to separate nine AGP glycoform bands in about 20 min. The limit of detection (based on absorbance measurements) ranged from 0.09 to 0.38 μM for these AGP glycoform bands, and the linear range extended up to a total AGP concentration of at least 240 μM. The migration times for the glycoform bands had typical within-day and day-today precisions of ± 0.16-0.23% or less, respectively, on a single treated capillary and the variation between capillaries was ± 0.56% or less. A charge ladder approach was employed to examine the mass or charge differences in the glycoforms that made up these bands, giving a good fit to a model in which the neighboring bands differed by one charge (e.g., from a sialic acid residue) and had an average mass difference of approximately 0.7-0.9 kDa. The approaches used to develop this separation method are not limited to AGP but could be extended to the analysis of other glycoproteins by CE.
Journal of pharmaceutical and biomedical analysis, Jan 11, 2017
The interactions of drugs with serum proteins are often stereoselective and can affect the distri... more The interactions of drugs with serum proteins are often stereoselective and can affect the distribution, activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on affinity chromatography, and particularly high-performance affinity chromatography (HPAC), have been used as tools to study these interactions. This review describes the general principles of affinity chromatography and HPAC as related to their use in drug binding studies. The types of serum agents that have been examined with these methods are also discussed, including human serum albumin, α1-acid glycoprotein, and lipoproteins. This is followed by a description of the various formats based on affinity chromatography and HPAC that have been used to investigate drug interactions with serum proteins and the historical development for each of these formats. Specific techniques that are discussed include zonal elution, frontal analysis, and kinetic methods such as those that make use...
The present invention generally relates to a microcolumn capable of separating an analyte from a ... more The present invention generally relates to a microcolumn capable of separating an analyte from a sample in the millisecond time domain. The microcolumn is capable of such rapid separation by employing small column volumes that can tolerate medium to high flow rates. The invention also relates to a method of loading a microcolumn capable of separating an analyte from a sample in the millisecond time domain using plural injections of the packing material
Contaminants in Drinking and Wastewater Sources, 2020
The presence of emerging man-made contaminants in wastewater and water sources in the environment... more The presence of emerging man-made contaminants in wastewater and water sources in the environment is a growing problem worldwide. Affinity chromatography is one approach that has been explored for the detection and analysis of such compounds. This approach is a chromatographic technique in which target analytes are captured based on their selective interactions with a biologically related binding agent that has been immobilized onto a support and placed within a column. Various forms of affinity chromatography have been utilized for the analysis of emerging contaminants in wastewater and the environment. Binding agents such as antibodies, molecularly imprinted polymers, and chiral stationary phases have been employed in this work, with the latter including macrocyclic antibiotics, proteins, and polysaccharide derivatives. Each of these binding agents is considered in this chapter with regards to their applications for emerging contaminants in water. Formats in which these binding agents have been used will also be described, such as off-line or online extraction, chromatographic immunoassays, and methods in which affinity chromatography has been combined with liquid chromatography, gas chromatography, and liquid chromatography-mass spectrometry. Other binding agents, such as aptamers and boronates, will also be briefly described that may be employed in future work with affinity-based separations for the analysis of emerging contaminants in water.
Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the... more Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the extraction of environmental contaminants from water. Computer simulations based on a countercurrent distribution model were also used to study the behavior of these sorbents. Several model drugs, pesticides and hormones of interest as emerging contaminants were considered in this work, with carbamazepine being used as a representative analyte when coupling the albumin column on-line with liquid chromatography and tandem mass spectrometry. The albumin column was found to be capable of extracting carbamazepine from aqueous solutions that contained trace levels of this analyte. Further studies of the bovine serum albumin sorbent indicated that it had higher retention under aqueous conditions This article is protected by copyright. All rights reserved. 2 than a traditional C 18 support for most of the tested emerging contaminants. Potential advantages of using these protein-based sorbents included the low cost of bovine serum albumin and its ability to bind to a relatively wide range of drugs and related compounds. It was also shown how simulations could be used to describe the elution behavior of the model compounds on the bovine serum albumin sorbents as an aid in optimizing the retention and selectivity of these supports for use with liquid chromatography or methods such as liquid chromatography with tandem mass spectrometry. This article is protected by copyright. All rights reserved. 4 2 Materials and Methods 2.1 Reagents BSA was obtained from Sigma-Aldrich (St. Louis, MO) and had a purity of 98%. The Nucleosil 300-5 silica (300 Å pore size, 5 µm diameter) was from Macherey-Nagel (Düren, Germany). The carbamazepine, 17β-estradiol, estrone, ibuprofen and testosterone were purchased from Sigma-Aldrich. Atrazine and 2,4-dichlorophenoxyacetic acid (2,4-D) were obtained from Riedel-de Haën/Sigma-Aldrich (Seelze, Germany), and caffeine was obtained from Alfa Aesar (Ward Hill, MA). All other chemicals were of the purest grades available. All solutions were prepared using water from a Nanopure purification system (Barnstead, Dubuque, IA). 2.2 Apparatus The chromatographic system used for the determination of binding capacities and retention consisted of an LC-10AD solvent delivery system from Shimadzu (Kyoto, Japan), a 515 HPLC pump from Waters (Milford, MA), a LabPro injection valve from Rheodyne (Oak
Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a mono... more Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target-specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, and dye-ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.
A one-site immunometric assay based on affinity microcolumns was developed for the analysis of al... more A one-site immunometric assay based on affinity microcolumns was developed for the analysis of alpha 1-acid glycoprotein (AGP) as a model protein biomarker. In this assay, a sample containing AGP was incubated with an excess amount of a labeled binding agent, such as fluorescein-labeled anti-AGP antibodies or F ab fragments. The excess binding agent was removed by passing this mixture through a microcolumn that contained an immobilized form of AGP, while the signal was measured for the binding agent-AGP complex in the non-retained fraction. Theoretical and practical factors were both considered in selecting the concentration of labeled binding agent, the incubation time of this agent with the sample, and the application conditions for this mixture onto the microcolumn. The effects of using various labeling methods and intact antibodies vs F ab fragments were also considered. The final assay was performed with fluorescein-labeled anti-AGP antibodies and a 2.1 mm i.d. × 1.0 cm AGP microcolumn operated at 0.30 mL min −1. This method required only 1 μL of serum, had a detection limit of 0.63 nM AGP, and gave a potential throughput of 2 min per sample. This assay was used to measure AGP in normal serum and serum from patients with systemic lupus erythematosus, giving good agreement with the literature and a reference method. The same approach and guidelines can be used to create assays for other protein biomarkers by changing the labeled binding agent and immobilized protein within the microcolumn.
The first albumin structure in complex with testosterone and the hormone's binding affinity m... more The first albumin structure in complex with testosterone and the hormone's binding affinity measured with two methods.
The Journal of nutritional biochemistry, Jan 25, 2018
Persistent activation of the mechanistic target of rapamycin complex 1 (mTORC1) is linked to sust... more Persistent activation of the mechanistic target of rapamycin complex 1 (mTORC1) is linked to sustained inflammation and progression of colorectal cancer. Widely available dietary phenolics, curcumin and piperine are purported to have antiinflammatory and anticarcinogenic activities through yet-to-be-delineated multitarget mechanisms. Piperine is also known to increase the bioavailability of dietary components, including curcumin. The objective of the study was to determine whether curcumin and piperine have individual and combined effects in the setting of gut inflammation by regulating mTORC1 in human intestinal epithelial cells. Results show that curcumin repressed (a) mTORC1 activity (measured as changes in the phosphorylation state of p70 ribosomal protein S6 kinase B1 and 40S ribosomal protein S6) in a dose-dependent manner (2.5-20 μM, P<.007) and (b) synthesis of nascent proteins. Piperine inhibited mTORC1 activity albeit at comparatively higher concentrations than curcumin...
Journal of Pharmaceutical and Biomedical Analysis, Apr 1, 2008
An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was dev... more An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or a monolith based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of HSA in the silica monolith was similar to values obtained with silica particles and a GMA/EDMA monolith. However, the higher surface area of the silica monolith gave a material that contained 1.3-to 2.2-times more immobilized HSA per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the HSA silica monolith were evaluated using two chiral analytes: D/L-tryptophan and R/S-warfarin. The separation of R-and S-ibuprofen was also considered. The HSA silica monolith gave higher retention and higher or comparable resolution and efficiency when compared with HSA columns that contained silica particles or a GMA/EDMA monolith. The silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with immobilized HSA as a chiral stationary phase.
High‐performance affinity microcolumns were used to characterize binding by the anti‐diabetic dru... more High‐performance affinity microcolumns were used to characterize binding by the anti‐diabetic drugs repaglinide and nateglinide with normal and glycated forms of human serum albumin. The microcolumns contained only nmol amounts of protein and provided a detailed analysis of these drug interactions with good precision and in a matter of minutes per experiment. The overall binding by repaglinide to normal and glycated albumin fits a model with two types of binding sites: a set of one or two moderate‐to‐high affinity regions and a larger set of weaker regions with association equilibrium constants of ∼105 and 103 M−1, respectively, at pH 7.4 and 37°C. Competition studies gave site‐specific association constants for repaglinide and nateglinide at Sudlow site I of 4.2 × 104 and 5.0 × 104 M−1 for normal albumin, with a decrease of 26%–30% being seen for nateglinide with glycated albumin and no significant change being noted for repaglinide. At Sudlow site II, repaglinide and nateglinide h...
Journal of Pharmaceutical and Biomedical Analysis, 2021
During diabetes human serum albumin (HSA), an important drug transport protein, can be modified b... more During diabetes human serum albumin (HSA), an important drug transport protein, can be modified by agents such as glyoxal (Go) and methylglyoxal (MGo) to form advanced glycation end-products. High-performance affinity microcolumns and zonal elution competition studies were used to compare interactions by the anti-diabetic drugs repaglinide and nateglinide with normal and Go- or MGo-modified HSA at Sudlow sites I and II of this protein. Both drugs had their strongest binding at Sudlow site II for the normal and modified forms of HSA. The association equilibrium constants at this site for repaglinide and nateglinide with normal HSA were 6.1 (± 0.2) × 104 M-1 and 7.1 (± 0.8) × 105 M-1, respectively, at pH 7.4 and 37⁰C; these values increased by up to 3.6-fold for repaglinide and decreased by up to 45-55 % for nateglinide when HSA was modified by Go or MGo at levels seen in prediabetes or diabetes. Both drugs were also found to bind at Sudlow site I, with association equilibrium constants at this site on normal HSA of 4.2 (± 0.3) × 104 M-1 for repaglinide and 5.0 (± 0.1) × 104 M-1 for nateglinide. The binding strength for repaglinide at Sudlow site I increased by 1.3- to 1.7-fold with the Go-modified HSA and decreased slightly (i.e., up to 19 %) for the MGo-modified HSA, while nateglinide showed only a small or insignificant change in binding with the same modified HSA samples. These results indicated that binding by repaglinide and nateglinide with HSA can be altered significantly by modification of this protein with Go or MGo, making these modifications of potential interest in the treatment of patients with these drugs during diabetes.
Journal of Pharmaceutical and Biomedical Analysis, 2021
2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension ... more 2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension and opioid withdrawal. It is known these drugs bind to serum proteins and have significant variations within this class of compounds in the overall level of this binding. However, little specific information is available on the interactions of these compounds with the two major transport proteins for many drugs, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP). This study examined binding by 2-imidazolines to these proteins by using 25 mm × 2.1 mm i.d. high-performance affinity microcolumns that contained HSA or AGP. The drugs that were examined were antazoline, clonidine, dexmedetomidine, lofexidine, moxonidine, phentolamine, and tizanidine, which represented a wide range of structures and pharmaceutical applications. The major metabolite of lofexidine, N-(2-aminoethyl)-2-(2,6-dichlorophenoxy) propenamide (LADP), was also examined. All these 2-imidazolines were found to have weak-to-moderate binding to HSA, with global affinities that ranged from 1.62 × 102 to 1.07 × 104 M-1 at pH 7.4 and 37 °C. These compounds had stronger binding with AGP, with global affinities constants ranging from 3.80 × 102 to 1.85 × 104 M-1. No stereoselectivity was observed by HSA for the enantiomers of dexmedetomidine, lofexidine, or LADP. However, AGP did show some stereoselectivity for lofexidine and LADP but not for dexmedetomidine. These results provide a better understanding of interactions of 2-imidazoline with HSA vs AGP in the circulation and of how this binding can change between drugs within this class of compounds.
The overall goal of this study is to develop a non-covalent immobilization process for quantitati... more The overall goal of this study is to develop a non-covalent immobilization process for quantitative studies of drug-protein binding, or related biointeractions, by high-performance affinity chromatography (HPAC). A series of polymeric monolithic supports based on glycidyl methacrylate-co-ethylene dimethacrylate (GMA-co-EDMA) with different porogen compositions were prepared. On-column entrapment was employed to immobilize human serum albumin (HSA) on monolith supports which had been converted into a hydrazide-activated and capped with oxidized glycogen. Warfarin and L-tryptophan were used as site-specific probes to evaluate preparation of these stationary phases through their binding with these solutes. Zonal elution studies with these materials also showed good agreement with the binding properties that have been reported for soluble HSA. This combined approach of using protein entrapment and HPAC gave a fast means for screening and studying drug-protein interactions. On-column entrapment of proteins using monolithic support can be a suitable mean of investigating drug-protein interactions in HPAC. The effectiveness of entrapment in immobilizing HSA within various monoliths, and the consequent change in drug binding, was seen to vary with the porogen composition that was used to make the monolith. Lowering the percentage of 1-dodecanol vs. cyclohexanol in the polymerization mixture gave entrapped HSA supports with higher retention for drugs. Since this immobilization method should result in little or no loss of protein activity, the data obtained in zonal elution studies such as used in this report should be useful in directly determining binding constants for drug-protein interactions in solution. The future work will involve estimation of the moles of binding sites for each drug on HSA by also analyzing the support’s protein content. This analysis will make it possible to directly measure the association equilibrium constant for interactions of the drug with HSA. Frontal analysis of these columns will also be performed to evaluate the overall binding of drugs with proteins in these supports
Atrazine is the most widely used herbicide in the U.S. and has been detected in surface water and... more Atrazine is the most widely used herbicide in the U.S. and has been detected in surface water and groundwater. Technologies are needed for onsite and in situ remediation of water and soil containing atrazine. We investigated the potential of using fine-grained, zero-valent iron (Fe0) to remove atrazine and promote its degradation in contaminated water and soil. Atrazine loss from aqueous solution increased with increasing Fe0concentration (w/v). Agitating 20 μg14C-ring-labeled atrazine L−1with 10% Fe0(w/v) removed 92% of the14C from solution within 48 h. Only about 4% of the14C lost from solution was extractable from the iron with 3 mM CaCl2(readily available pool), 81% was extractable with CH3CN (potentially available pool), and 11% was unextractable residues. A companion experiment indicated that most of the14C extracted from the iron with 3 mM CaCl2after the 48-h Fe0treatment was unaltered atrazine, while the CH3CN extract contained approximately 33% atrazine and 48% was unidenti...
A relatively fast and reproducible CE separation was developed for the glycoform analysis of α 1a... more A relatively fast and reproducible CE separation was developed for the glycoform analysis of α 1acid glycoprotein (AGP). Factors that were considered included the pH for this separation and various techniques for coating the capillary and/or to minimize electroosmotic flow and protein adsorption. Optimum resolution of the AGP glycoforms was obtained at pH 4.2 with a running buffer containing 0.1% Brij 35 and by using static and dynamic coatings of PEO on the capillary. These conditions made it possible to separate nine AGP glycoform bands in about 20 min. The limit of detection (based on absorbance measurements) ranged from 0.09 to 0.38 μM for these AGP glycoform bands, and the linear range extended up to a total AGP concentration of at least 240 μM. The migration times for the glycoform bands had typical within-day and day-today precisions of ± 0.16-0.23% or less, respectively, on a single treated capillary and the variation between capillaries was ± 0.56% or less. A charge ladder approach was employed to examine the mass or charge differences in the glycoforms that made up these bands, giving a good fit to a model in which the neighboring bands differed by one charge (e.g., from a sialic acid residue) and had an average mass difference of approximately 0.7-0.9 kDa. The approaches used to develop this separation method are not limited to AGP but could be extended to the analysis of other glycoproteins by CE.
Journal of pharmaceutical and biomedical analysis, Jan 11, 2017
The interactions of drugs with serum proteins are often stereoselective and can affect the distri... more The interactions of drugs with serum proteins are often stereoselective and can affect the distribution, activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on affinity chromatography, and particularly high-performance affinity chromatography (HPAC), have been used as tools to study these interactions. This review describes the general principles of affinity chromatography and HPAC as related to their use in drug binding studies. The types of serum agents that have been examined with these methods are also discussed, including human serum albumin, α1-acid glycoprotein, and lipoproteins. This is followed by a description of the various formats based on affinity chromatography and HPAC that have been used to investigate drug interactions with serum proteins and the historical development for each of these formats. Specific techniques that are discussed include zonal elution, frontal analysis, and kinetic methods such as those that make use...
The present invention generally relates to a microcolumn capable of separating an analyte from a ... more The present invention generally relates to a microcolumn capable of separating an analyte from a sample in the millisecond time domain. The microcolumn is capable of such rapid separation by employing small column volumes that can tolerate medium to high flow rates. The invention also relates to a method of loading a microcolumn capable of separating an analyte from a sample in the millisecond time domain using plural injections of the packing material
Contaminants in Drinking and Wastewater Sources, 2020
The presence of emerging man-made contaminants in wastewater and water sources in the environment... more The presence of emerging man-made contaminants in wastewater and water sources in the environment is a growing problem worldwide. Affinity chromatography is one approach that has been explored for the detection and analysis of such compounds. This approach is a chromatographic technique in which target analytes are captured based on their selective interactions with a biologically related binding agent that has been immobilized onto a support and placed within a column. Various forms of affinity chromatography have been utilized for the analysis of emerging contaminants in wastewater and the environment. Binding agents such as antibodies, molecularly imprinted polymers, and chiral stationary phases have been employed in this work, with the latter including macrocyclic antibiotics, proteins, and polysaccharide derivatives. Each of these binding agents is considered in this chapter with regards to their applications for emerging contaminants in water. Formats in which these binding agents have been used will also be described, such as off-line or online extraction, chromatographic immunoassays, and methods in which affinity chromatography has been combined with liquid chromatography, gas chromatography, and liquid chromatography-mass spectrometry. Other binding agents, such as aptamers and boronates, will also be briefly described that may be employed in future work with affinity-based separations for the analysis of emerging contaminants in water.
Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the... more Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the extraction of environmental contaminants from water. Computer simulations based on a countercurrent distribution model were also used to study the behavior of these sorbents. Several model drugs, pesticides and hormones of interest as emerging contaminants were considered in this work, with carbamazepine being used as a representative analyte when coupling the albumin column on-line with liquid chromatography and tandem mass spectrometry. The albumin column was found to be capable of extracting carbamazepine from aqueous solutions that contained trace levels of this analyte. Further studies of the bovine serum albumin sorbent indicated that it had higher retention under aqueous conditions This article is protected by copyright. All rights reserved. 2 than a traditional C 18 support for most of the tested emerging contaminants. Potential advantages of using these protein-based sorbents included the low cost of bovine serum albumin and its ability to bind to a relatively wide range of drugs and related compounds. It was also shown how simulations could be used to describe the elution behavior of the model compounds on the bovine serum albumin sorbents as an aid in optimizing the retention and selectivity of these supports for use with liquid chromatography or methods such as liquid chromatography with tandem mass spectrometry. This article is protected by copyright. All rights reserved. 4 2 Materials and Methods 2.1 Reagents BSA was obtained from Sigma-Aldrich (St. Louis, MO) and had a purity of 98%. The Nucleosil 300-5 silica (300 Å pore size, 5 µm diameter) was from Macherey-Nagel (Düren, Germany). The carbamazepine, 17β-estradiol, estrone, ibuprofen and testosterone were purchased from Sigma-Aldrich. Atrazine and 2,4-dichlorophenoxyacetic acid (2,4-D) were obtained from Riedel-de Haën/Sigma-Aldrich (Seelze, Germany), and caffeine was obtained from Alfa Aesar (Ward Hill, MA). All other chemicals were of the purest grades available. All solutions were prepared using water from a Nanopure purification system (Barnstead, Dubuque, IA). 2.2 Apparatus The chromatographic system used for the determination of binding capacities and retention consisted of an LC-10AD solvent delivery system from Shimadzu (Kyoto, Japan), a 515 HPLC pump from Waters (Milford, MA), a LabPro injection valve from Rheodyne (Oak
Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a mono... more Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target-specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, and dye-ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.
A one-site immunometric assay based on affinity microcolumns was developed for the analysis of al... more A one-site immunometric assay based on affinity microcolumns was developed for the analysis of alpha 1-acid glycoprotein (AGP) as a model protein biomarker. In this assay, a sample containing AGP was incubated with an excess amount of a labeled binding agent, such as fluorescein-labeled anti-AGP antibodies or F ab fragments. The excess binding agent was removed by passing this mixture through a microcolumn that contained an immobilized form of AGP, while the signal was measured for the binding agent-AGP complex in the non-retained fraction. Theoretical and practical factors were both considered in selecting the concentration of labeled binding agent, the incubation time of this agent with the sample, and the application conditions for this mixture onto the microcolumn. The effects of using various labeling methods and intact antibodies vs F ab fragments were also considered. The final assay was performed with fluorescein-labeled anti-AGP antibodies and a 2.1 mm i.d. × 1.0 cm AGP microcolumn operated at 0.30 mL min −1. This method required only 1 μL of serum, had a detection limit of 0.63 nM AGP, and gave a potential throughput of 2 min per sample. This assay was used to measure AGP in normal serum and serum from patients with systemic lupus erythematosus, giving good agreement with the literature and a reference method. The same approach and guidelines can be used to create assays for other protein biomarkers by changing the labeled binding agent and immobilized protein within the microcolumn.
The first albumin structure in complex with testosterone and the hormone's binding affinity m... more The first albumin structure in complex with testosterone and the hormone's binding affinity measured with two methods.
The Journal of nutritional biochemistry, Jan 25, 2018
Persistent activation of the mechanistic target of rapamycin complex 1 (mTORC1) is linked to sust... more Persistent activation of the mechanistic target of rapamycin complex 1 (mTORC1) is linked to sustained inflammation and progression of colorectal cancer. Widely available dietary phenolics, curcumin and piperine are purported to have antiinflammatory and anticarcinogenic activities through yet-to-be-delineated multitarget mechanisms. Piperine is also known to increase the bioavailability of dietary components, including curcumin. The objective of the study was to determine whether curcumin and piperine have individual and combined effects in the setting of gut inflammation by regulating mTORC1 in human intestinal epithelial cells. Results show that curcumin repressed (a) mTORC1 activity (measured as changes in the phosphorylation state of p70 ribosomal protein S6 kinase B1 and 40S ribosomal protein S6) in a dose-dependent manner (2.5-20 μM, P<.007) and (b) synthesis of nascent proteins. Piperine inhibited mTORC1 activity albeit at comparatively higher concentrations than curcumin...
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