Alpha 1-acid glycoprotein high-performance liquid chromatography column (EnantioPAC) as a screening tool for protein binding

An attempt was made to correlate retention behavior on a high-performance liquid chromatographic (HPLC) system employing an immobilized alpha 1-acid glycoprotein (AAG) column with AAG binding behavior for various compounds. Protein binding was assessed by propranolol displacement studies in an equilibrium dialysis system using isolated AAG. HPLC retention behavior poorly correlated with propranolol displacement from AAG. This system is not suitable for use as a screening tool for AAG affinity.     

A new method for the high performance liquid chromatographic determination of TA-870, a dopamine prodrug (catechol ester compound)

A new method for the high performance liquid chromatographic (HPLC) determination of N-(N-acetyl-L-methionyl)-O,O-bis(ethoxycarbonyl)dopamine (TA-870), a dopamine prodrug, in biological fluid has been developed. In order to measure with an electrochemical detector (ECD), TA-870 was passed first through an immobilized carboxylesterase column to be converted to the electrochemically active deethoxycarbonylated TA-870 (DEC-TA-870). The properties of this carboxylesterase immobilized on Sepharose 4B were examined by this flow injection system. Hydrolysis of TA-870 with this immobilized carboxylesterase was a maximum at pH 7-8 and 50 degrees C, and the activity decreased in the presence of organic solvent such as acetonitrile. For the determination of TA-870 in biological fluids, an HPLC-immobilized enzyme-ECD system using a column-switching technique was developed. The blood was deproteinized with ethanol, and TA-870 in the ethanol extracts was adsorbed in Bond Elut C18. The dichloromethane eluate from Bond Elut C18 was injected into the HPLC system. The HPLC apparatus was composed of three pumps, two separation columns (LiChrosorb Si 60 and mu Bondasphere), a trap column (Bond Elut), an enzyme column, ECD and the column-switching system. The calibration curve for TA-870 in blood was linear in the range from 2 to 200 ng/mL. This new assay method might be useful also for the determination of other catechol ester compounds.     

Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins

We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.     

Biointeraction analysis of carbamazepine binding to α1‐acid glycoprotein by high‐performance affinity chromatography

Interactions of the drug carbamazepine with the serum protein α₁‐acid glycoprotein (AGP) were examined by high‐performance affinity chromatography. Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low‐affinity interactions with the support and high‐affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (±0.1)×10⁵ M^?1, with values that ranged from 5.1 to 0.58×10⁵ M^?1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how high‐performance affinity chromatography can be used to examine biological interactions and drug–protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.     

Chromatographic study of the adsorption kinetics of albumin on monoclonal and polyclonal immunoadsorbents

Summary High-performance liquid chromatography (HPLC) has been used to study the adsorption kinetics of proteins on immunoadsorbents. The adsorption rate constant of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a silica HPLC support was determined by saturating the column with repeated pulse injections. Studies on polyclonal immunodsorbents of different capacities enable evaluation of the contribution of transport to the binding sites. The adsorption properties of two different monoclonal anti-HSA antibodies immobilized on a chromatographic support were characterized by different approaches. The location of the epitope on the HSA molecule was determined by enzyme-linked immunoassay (ELISA) with albumin fragments. The chromatographic method was used to determine the column capacity and the adsorption rate constant of HSA on the immunoadsorbent. To compare the affinity of the antibodies for the antigen, an indirect ELISA method was used to determine the equilibrium constant of antigen-antibody association in solution Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th september, 1996.     

Quantitative screening and matrix effect studies of drug discovery compounds in monkey plasma using fast-gradient liquid chromatography/tandem mass spectrometry.

A higher-throughput bioanalytical method based on fast-gradient (1 min run time) high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) was developed for screen-type analyses of plasma samples from early drug discovery studies in support of exploratory pharmacodynamic studies. The HPLC system equipped with minibore column was interfaced with either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) ionization techniques. The matrix ion suppression effect of both quantitative HPLC/MS/MS analyses was compared using the post-column infusion system. The use of the described methods provided advantages such as a shorter chromatographic region of ion suppression, less solvent consumption and shorter run times in comparison with standard analytical column HPLC/MS/MS methods. The analytical results obtained by both HPLC/MS/MS methods were in good agreement (within 15% of error) and displayed a good correlation with the pharmacodynamic outcome.     

Direct zonal liquid chromatographic method for the kinetic study of actinomycin-DNA binding

The binding of an anticancer drug (actinomycin D or ACTD) to double-stranded DNA (dsDNA) was studied by means of high-performance liquid chromatography (HPLC). ACTD is an antitumor antibiotic containing one chromophore group and two pentapeptidic lactone cycles that binds dsDNA. Incubations of ACTD with DNA were performed at physiological pH. The complexed and free ligand concentrations of the mixture were quantified at 440 nm from their separation on a size-exclusion chromatographic (SEC) column using the same buffer for the elution and the sample incubation. The DNA and the ACTD-DNA complexes were eluted at the column exclusion volume while the ligand was retained on the support. An apparent binding curve was obtained by plotting the amount emerging at the exclusion column volume against that eluted at free ACTD retention volume. A dissociating effect was evidenced and the binding parameters were significantly different from those obtained at equilibrium by visible absorbance titration. The equilibrium binding parameters determined by absorption spectroscopy were used as starting data in the numerical simulations of the chromatographic process. The results showed a strong dependency of the apparent binding parameters on the reaction kinetics. Finally the comparison of the apparent binding curve obtained from the HPLC experiments and from the numerical simulations permitted an evaluation of the dissociation rate constant (kd = 0.004 s(-1)).     

Microscopic modes and free energies of 3-phosphoinositide-dependent kinase-1 (PDK1) binding with celecoxib and other inhibitors

Celecoxib, also known as Celebrex (approved by FDA in 1998) and remembered as the fastest-selling drug in history, was used as a cyclooxygenase-2 (COX-2) selective inhibitor having both anti-inflammatory and anticancer activities. Most recent studies have revealed that the apoptotic activity of celecoxib (and its derivatives) is actually independent of the COX-2 inhibitory activity and that celecoxib also inhibits the kinase activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1), suggesting that the well-known anticancer activity of celecoxib is not due to the inhibition of COX-2, but possibly is due to the inhibition of PDK1. It is highly desirable to develop new celecoxib derivatives as PDK1-specifc inhibitors to avoid the side effects of COX-2 inhibitors. To understand how PDK1 binds with celecoxib and its derivatives, we have performed extensive molecular docking and combined molecular dynamics (MD) simulations and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on eight representative PDK1 inhibitors, leading to the finding of a new, more favorable binding mode which is remarkably different from the previously proposed binding mode. Based on the determined most stable binding structures, the calculated binding free energies are all in good agreement with the corresponding experimental data, and the biological activity data available for celecoxib and its derivatives can be better interpreted. The obtained new insights, concerning both the binding mode and computational protocol, will be valuable not only for future rational design of novel, more potent PDK1-specific inhibitors as promising anticancer therapeutics, but also for rational design of drugs targeting other proteins.     

Characterization of drug interactions with soluble beta-cyclodextrin by high-performance affinity chromatography

This study examined the use of an immobilized human serum albumin (HSA) column to study solution-phase reactions between drugs and beta-cyclodextrin (beta-CD). Chromatographic equations were developed to characterize the binding of chemicals to a soluble ligand (beta-CD) in the presence of an independent immobilized ligand (HSA). Situations considered included the presence of both a homogeneous and heterogeneous immobilized ligand, as well as complex interactions between the chemical of interest and soluble ligand. Three drugs (warfarin, tamoxifen, and phenytoin) were examined by this approach. This method involved injecting a small amount of each drug onto an HSA column in the presence of various concentrations of beta-CD in the mobile phase. By measuring the change in the drug's retention factor as the concentration of beta-CD was varied, it was possible to determine the stability constant between the injected drug and beta-CD. With this approach, warfarin and beta-CD were found to have 1:1 interactions with a stability constant of 5.2 x 10(2) M(-1) at 37 degrees C and pH 7.4, a result in close agreement with previous literature values. Tamoxifen and phenytoin were also found to have 1:1 interactions with beta-CD and had stability constants of 0.9-1.2 x 10(4) and 6-9 x 10(2) M(-1) respectively. With these latter solutes, the effects of secondary binding to the chromatographic support had to be considered. The theory and methods described in this report are not limited to these drugs and beta-CD but can be applied to other analytes and soluble ligands.     

键合型聚丙烯酰胺衍生物手性固定相的制备与手性识别性能

高效液相色谱(HPLC)被广泛认为是分离制备光学纯单一对映体的最有效方法。在高效液相色谱手性拆分中,手性固定相(CSP)的性能直接影响到色谱柱的手性分离能力。在众多手性固定相中,键合型手性固定相具有溶剂耐受性好,分离模式灵活等优点,已经发展成为一类重要的手性固定相。本文通过两步化学反应合成了新型的光学活性丙烯酰胺衍生物--(S)-1-丙烯酰-2-(N-苯基甲酰胺基)吡咯烷((S)-APACP),采用核磁共振氢谱表征了(S)-APACP的化学结构;通过3步化学反应制备了键合型聚丙烯酰胺衍生物手性固定相,采用热重分析法表征了聚合物的键合量,采用HPLC评价了键合型手性固定相的识别能力,分析了影响其手性识别能力的因素。研究结果表明,APACP聚合物成功地键合到硅胶表面制备了具有良好溶剂耐受性的键合型手性固定相,其聚合物键合量为10.2%~11.8%,该键合型手性固定相对若干种对映体显示了较好的手性识别能力。     

Separation and evaluation of soybean protein hydrolysates prepared by immobilized metal ion affinity chromatography with different metal ions

Metal ion affinity chromatography is widely used to purify peptides on the basis of the dissimilarities of their amino acids. However, researchers are interested in the separation differences between different metal ions in this method. In our study, four kinds of commonly used metal ions are compared by the amount of immobilized metal ion on iminodiacetic acid-Sepharose and binding amount of soybean peptide to immobilized iminodiacetic acid-Mn(+) adsorbents and evaluated by high-performance liquid chromatography (HPLC) profiles. The results show that due to the different adsorption behaviors of metal ions, the binding ability order of soybean protein peptide on the column should be Fe(3+) > Cu(2+) > Zn(2+) > Ca(2+). The HPLC profiles show that peptides adsorbed by four kinds of metal ions display similar strong hydrophobic characteristics.     

高效迎头分析法测定药物-人血清白蛋白混合液中游离药物浓度

 用高效迎头分析法 (HPFA)测定了药物 人血清白蛋白 (HSA)混合液中游离药物的浓度。样品溶液不经任何处理直接进样到装有内表面反相固定相的色谱柱中 ,用 67mmol/L磷酸盐缓冲液 ( pH 7 4 ,I =0 17mol/L)作流动相。当进样体积足够大时 ,游离药物以平顶峰的形式被洗脱出来 ,平顶峰区域洗脱液中的药物浓度等于样品溶液中游离药物的浓度。收集平顶峰区域的洗脱液 ,然后将一定体积的洗脱液注入到反相色谱柱中 ,测定游离药物的浓度。用该法测定酮基布洛芬 HSA和头孢哌酮 HSA两种混合液中游离药物的浓度。     

A DNA aptamer as a new target-specific chiral selector for HPLC

In this paper, a DNA aptamer, known to bind stereospecifically the D-enantiomer of an oligopeptide, i.e., arginine-vasopressin, was immobilized on a chromatographic support. The influence of various parameters (such as column temperature, eluent pH, and salt concentration) on the L- and D-peptide retention was investigated in order to provide information about the binding mechanism and then to define the utilization conditions of the aptamer column. The results suggest that dehydration at the binding interface, charge-charge interactions, and adaptive conformational transitions contribute to the specific D-peptide-aptamer complex formation. A very significant enantioselectivity was obtained in the optimal binding conditions, the D-peptide being strongly retained by the column while the L-peptide eluted in the void volume. A rapid baseline separation of peptide enantiomers was also achieved by modulating the elution conditions. Furthermore, it was established that the aptamer column was stable during an extended period of time. This work indicates that DNA aptamers, specifically selected against an enantiomer, could soon become very attractive as new target-specific chiral selectors for HPLC.     

Characterization of the Penetration of the Blood–Brain Barrier by High-Performance Liquid Chromatography (HPLC) Using a Stationary Phase with an Immobilized Artificial Membrane

The biological activity of drugs on organisms is associated with the pharmacokinetic properties, such as the ability to penetrate through environments of varying polarity such as cellular organelles. In this area, particular attention is turned to the physicochemical properties that determine the potential of drugs to pass across the blood–brain barrier and thus to act on the central nervous system. In this study, special effort has been devoted to the simulation of passive diffusion of seven drugs (propranolol, ibuprofen, atenolol, promazine, chlorpromazine, imipramine, and desipramine) through the blood–brain barrier by high-performance liquid chromatography (HPLC) using a column with an immobilized artificial membrane. Gradient reverse elution was used to develop a linear correlation model for the capacity factors k_IAM and the in vivo logarithmic values of brain-to-blood drug concentration ratios (log BB) with R of 0.9851. Eleven additional pharmaceuticals were determined by the same method to predict their potential to penetrate the blood–brain barrier. The reported analytical method represents an alternative tool for rapid and noninvasive assessment of the absorption properties of chemicals, especially for the development of novel drugs. The retention of the studied compounds on the immobilized artificial membrane column was also compared with three other C18-based stationary phases. Herein, the results of the HPLC determination of drugs using an immobilized artificial membrane are briefly discussed with respect to a general application of the method for evaluating a broader spectrum of pharmaceutical compounds.     

Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

Summary A new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.     

A highly sensitive determination of individual serum bile acids using high-performance liquid chromatography with immobilized enzyme

We have developed a highly sensitive method for the simultaneous determination of individual bile acids in serum using high-performance liquid chromatography (HPLC) combined with immobilized 3 alpha-hydroxysteroid dehydrogenase. Both the HPLC column and the immobilized-enzyme column are suitable for use with alkaline solutions necessary in working with this enzyme system. With this method we were able to determine simultaneously fifteen different serum bile acids.     

Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry

An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.     

New liquid chromatography columns for highlighting the interaction of ligand candidates with humic acid

Humic acid was the main compound in soil and reduced the availability of some organic compounds in soils. In this work, humic acid was immobilized for the first time on a homemade neutravidin poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary column with a 20 μm i.d. for the screening of potential ligands to humic acid and the evaluation of their molecular recognition mechanism. This homemade humic acid column enabling it to work at very low backpressure (0.60 MPa at 20 nl/min flow rate), had a long lifetime, excellent repeatability, and negligible non-specific binding sites. The performance of this affinity humic acid column was demonstrated by the evaluation of recognition assay for a series of known ligands of humic acid (a series of rodenticide molecules) which is the heart of the fragment-based drug design. In addition, this column was used successfully for highlighting the binding mechanism to humic acid of the severe acute respiratory syndrome coronavirus-2-spike protein. As well this new humic acid miniaturized liquid chromatography column developed in this work could be used in the feature for another solute molecule-humic acid binding studies or for a separative mode.     

Study on the degeneracy of antisense peptides using affinity chromatography

The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. Similar result was obtained from another experiment that Arg-substituted antisense peptide of fusion peptide (1-11) of influenza virus A was also shown the highest affinity binding to immobilized fusion peptide.     

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions

In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).