Analysis of free analyte fractions by rapid affinity chromatography

The invention is generally directed toward an analytical method to determine the concentration of the free analyte fraction in a sample. More particularly, the method encompasses applying a sample comprising a free and bound analyte fraction to an affinity column capable of selectively extracting the free fraction in the millisecond time domain. The signal generated by the free fraction is then quantified by standard analytical detection techniques. The concentration of the free fraction may then be determined by comparison of its signal with that of a calibration curve depicting the signal of known concentration of the same analyte

Bioanalysis Young Investigator Award – sponsored by Waters- Michelle J. Yoo

Supervisor’s supporting comments have always been impressed with Michelle’s ability to conduct research in an independent and yet highly effective manner. Part of her research in my group has examined the use of affinity columns to examine drug–protein binding with serum proteins, such as human serum albumin. This work is extremely important to the fields of pharmaceutical chemistry and clinical chemistry in providing the data needed for the development of new drugs or in the optimization of treatments for patients with new, or existing, drugs. Another topic that Michelle has examined in her research is the use of new supports based on monolithic materials and ultrafast-extraction methods for affinity-based separations of biological samples and high-throughput screening of drug–protein binding. She was the lead author on a review written on this topic and also has several research publications related to this area of work. During her graduate studies, Michelle has emerged as a real leader in my group. She has excellent people and communication skills and is highly motivated in her pursuit of an advanced degree in analytical chemistry and bioanalysis. I have extremely high expectations for her in the future as she continues her career

Bioanalysis Young Investigator Award – sponsored by Waters- Michelle J. Yoo

Supervisor’s supporting comments have always been impressed with Michelle’s ability to conduct research in an independent and yet highly effective manner. Part of her research in my group has examined the use of affinity columns to examine drug–protein binding with serum proteins, such as human serum albumin. This work is extremely important to the fields of pharmaceutical chemistry and clinical chemistry in providing the data needed for the development of new drugs or in the optimization of treatments for patients with new, or existing, drugs. Another topic that Michelle has examined in her research is the use of new supports based on monolithic materials and ultrafast-extraction methods for affinity-based separations of biological samples and high-throughput screening of drug–protein binding. She was the lead author on a review written on this topic and also has several research publications related to this area of work. During her graduate studies, Michelle has emerged as a real leader in my group. She has excellent people and communication skills and is highly motivated in her pursuit of an advanced degree in analytical chemistry and bioanalysis. I have extremely high expectations for her in the future as she continues her career

An Overview of Capillary Electrophoresis (CE) in Clinical Analysis

The development and general applications of capillary electrophoresis (CE) in the field of clinical chemistry are discussed. It is shown how the early development of electrophoresis was closely linked to clinical testing. The rise of gel electrophoresis in clinical chemistry is described, as well as the eventual developments that lead to the creation and the use of modern CE. The general principles of CE are reviewed and the potential advantages of this method in clinical testing are examined. Finally, an overview is presented of several areas in which CE has been developed and is currently being explored for use with clinical samples

Characterization of the Binding Of Sulfonylurea Drugs to HSA by High-Performance Affinity Chromatography

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions

Development and Evaluation of Silica-Based Lectin Microcolumns for Glycoform Analysis of Alpha1-Acid Glycoprotein

Silica-based lectin microcolumns were developed and optimized for the separation and analysis of glycoform fractions in alpha1-acid glycoprotein (AGP) based on both the degree of branching and level of fucosylation. Concanavalin A (Con A) and Aleuria Aurantia lectin (AAL) were immobilized onto HPLC-grade silica by reductive amination and packed into 2.1 mm i.d. × 5.0 cm microcolumns. Factors examined for these microcolumns include their protein content, binding capacity, binding strength and band-broadening under isocratic conditions (Con A) or step elution conditions (AAL) and in the presence of various flow rates or temperatures. These factors were examined by using experiments based on frontal analysis, zonal elution, peak profiling and peak decay analysis. Up to 200 μg AGP could be loaded onto a Con A microcolumn and provide linear elution conditions, and 100 μg AGP could be applied to an AAL microcolumn. The final conditions for separating retained and non-retained AGP glycoform fractions on a Con A microcolumn used a flow rate of 50 μL min−1 and a temperature of 50 °C, which gave a separation of these fractions within 20 min or less. The final conditions for an AAL microcolumn included a flow rate of 0.75 mL min−1, a temperature of 50 °C, and the use of 2.0 mM L-fucose as a competing agent for elution, giving a separation of non-retained and retained AGP glycoforms in 6 min or less. The inter-day precisions were ± 0.7–4.0% or less for the retention times of the AGP glycoforms and ± 2.2–3.0% or less for their peak areas

Analysis of free analyte fractions by rapid affinity chromatography

The invention is generally directed toward an analytical method to determine the concentration of the free analyte fraction in a sample. More particularly, the method encompasses applying a sample comprising a free and bound analyte fraction to an affinity column capable of selectively extracting the free fraction in the millisecond time domain. The signal generated by the free fraction is then quantified by standard analytical detection techniques. The concentration of the free fraction may then be determined by comparison of its signal with that of a calibration curve depicting the signal of known concentration of the same analyte

Evaluation of the Overall Binding of Acetohexamide and Tolbutamide with Methyl Glyoxal-Modified HSA by High-Performance Affinity Chromatography

High performance affinity chromatography (HPAC) was used in a zonal competition format to investigate the overall binding of two sulfonylurea drugs with modified human serum albumin (HSA). The in vitro modifications of this protein that were examined included glycation, as occurs in the presence of elevated glucose levels during diabetes, and the formation of advanced glycation end-products (AGEs) due to the reaction of HSA with methylglyoxal (MGo). The sulfonylurea drugs acetohexamide and tolbutamide, which are used to treat type II diabetes, were each injected onto 10 mm x 2.1 mm i.d. affinity microcolumns that contained normal HSA and HSA modified with MGo. Competition studies based on a zonal elution format were carried out by using R-warfarin as a site –specific probe for Sudlow site I of HSA and L-tryptophan as a site-specific probe for Sudlow site II. The studies were performed with eight mobile phase concentrations of the drugs, ranging from 0-25 µM, and at a flowrate of 0.5 mL/min and a temperature of 37°C. The resulting information made it possible to determine the site-specific association equilibrium constants for each drug with normal and modified HSA. A decrease of approximately 0.4-fold to 0.8-fold in binding strength was found when comparing normal HSA vs control MGo-modified HSA and normal HSA vs diabetic MGo-modified HSA. These experiments illustrated how affinity microcolumns and zonal competition could be used in detailed studies of interactions by sulfonylurea drugs with normal HSA and modified forms of this protein. Such information may be useful in future applications within the field of personalized medicine for the development of customized treatments for patients with type II diabetes

Affinity Chromatography: A Historical Perspective

Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods