High-performance Frontal Analysis Column Research Articles

Protein binding study of catechin hydrate and genistein by high-performance frontal analysis

High-performance frontal analysis (HPFA) was used for the protein binding study of catechin hydrate and genistein to human serum albumin (HSA). The experiment was performed on a Develosil 100Diol-5 column, and sodium phosphate buffer (pH 7.4 and ionic strength of 0.17) was used as the mobile phase. The mixtures of the drug-HSA solution were directly injected into the HPFA column, the HSA was eluted first and the unbound drugs were eluted out as a trapezoidal peak with a plateau region. The unbound drug concentration was determined from a plateau height of the plateau region and the experimental data were fitted by Scatchard equation. The binding constants (K) and binding affinities (nK) of the drug to HAS were K=1.32×104 (L mol−1), nK=0.47×104 (L mol−1) for catechin hydrate, and K=5.17×104 (L mol−1), nK=2.14×104 (L mol−1) for genistein.

High-Performance Frontal Analysis of the Binding of Thyroxine Enantiomers to Human Serum Albumin Binding of Thyroxine Enantiomers to Human Serum Albumin Kimura

The aim of this study was to characterize the binding property between thyroxine and human serum albumin (HSA) qualitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system consisting of an HPFA column, an extraction column, and an analytical HPLC column was developed to be used to determine the unbound concentrations of thyroxine enantiomers. Both enantiomers were bound to human serum albumin at two high-affinity sites with similar affinities. The binding constant (K) and the number of binding sites on an HSA molecule (n) evaluated from Scatchard plot analysis were K = 1.01 x 10(6)m(-1) and n = 1.90 for L: -thyroxine, and K = 9.71 x 10(5) m(-1) and n = 1.97 for D: -thyroxine. The binding sites were identified using phenylbutazone and diazepam as site-specific probes for sites I and II, respectively, and each enantiomer was found to bind to both sites. Incorporation of a chiral HPLC column into the on-line system permitted the investigation of enantiomer-enantiomer interactions, which revealed that both enantiomers competitively bind to the same binding sites without significant allosteric effects.

Simultaneous determination of unbound thyroid hormones in human plasma using high performance frontal analysis with electrochemical detection

A direct injection HPLC method in combination with high-performance frontal analysis (HPFA) and electrochemical detection (ECD) was developed for the simultaneous and sensitive determination of unbound thyroid hormones (thyroxine, triiodothyronine, and reverse triiodothyronine) in human plasma. The present on-line HPLC/HPFA system consists of an HPFA column, an extraction column and an analytical HPLC column connected through a column-switching device, and the eluent from the analytical column was monitored by ECD. The calibration lines showed good linearity (rsq. > 0.999) within 7.4–148.2 pM for T4 and 1.5–74.1 pM for T3 and rT3. Unbound T4 and T3 concentrations determined by the present system were 16.4 ± 2.4 pM ( n = 15) and 7.14 ± 1.04 pM ( n = 15), which were in agreement with those determined by the EIA method. The unbound rT3 concentration was 2.30 ± 0.27 pM ( n = 15). The CV% values of intra-day and inter-day assays ( n = 15) were less than 14.9% for T4, 14.5% for T3 and 13.2% for rT3. The present system was also applied to a competitive binding study of these thyroid hormones in human plasma.

Protein Binding Study of S-Ibuprofen Using High-Performance Frontal Analysis

High-performance frontal analysis (HPFA) was used for the determination of the binding constant of Sibuprofen to human serum albumin (HSA). This experiment was based on an Inertsil 100 Diol 5 column and sodium phosphate buffer (pH 7.4 and ionic strength of 0.17) as the mobile phase. The mixture of S-ibuprofen and HSA (70 <TEX>$\mu$</TEX>M) solution were directly injected into the HPFA column. An injection volume of 200 <TEX>$\mu$</TEX>L and a “estricted injection”method were applied to ensure the drug to be eluted as a zonal peak with a plateau. The unbound drug concentration was calculated from the peak height of the zonal peak. Scatchard analysis was used for evaluation of the binding constant (K) and binding affinity (nK) of S-ibuprofen to HSA, and the results were K = 2.833 <TEX>${\times}$</TEX> 10<TEX>$^4$</TEX> [L mol<TEX>$^{-1}$</TEX>], nK = 4.935 <TEX>${\times}$</TEX> 10<TEX>$^4$</TEX> [L mol<TEX>$^{-1}$</TEX>], respectively.

Binding study of desethyloxybutynin using high-performance frontal analysis method

Plasma protein binding of N-desethyloxybytynin (DEOXY), a major active metabolite of oxybutynin (OXY), was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of DEOXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human α 1-acid glycoprotein (AGP) solutions. DEOXY is bound in human plasma strongly and enantioselectively. The unbound drug fraction in human plasma samples containing 5 μ M ( R)- or ( S)-DEOXY was 1.19±0.001 and 2.33±0.044%, respectively. AGP plays the dominant role in this strong and enantioselective plasma protein binding of DEOXY. The total binding affinity ( nK) of ( R)-DEOXY and ( S)-DEOXY to AGP was 2.97×10 7 and 1.31×10 7 M −1, respectively, while the nK values of ( R)-DEOXY and ( S)-DEOXY to HSA were 7.77×10 3 and 8.44×10 3 M −1, respectively. While the nK value of ( S)-DEOXY is weaker than that of ( S)-OXY (1.53×10 7 M −1), the nK value of ( R)-DEOXY is 4.33 times stronger than that of ( R)-OXY (6.86×10 6 M −1). This suggests that the elimination of an ethyl group weakens the binding affinity of the ( S)-isomer because of the decrease in hydrophobicity, while the binding affinity of the ( R)-isomer is enhanced by the decrease in steric hindrance. The total binding affinity of DEOXY to HSA is much lower than that of DEOXY-AGP binding as well as OXY-HSA binding (2.64×10 4 and 2.19×10 4 M −1 for ( R)-OXY and ( S)-OXY, respectively). The study on competitive binding between OXY and DEOXY indicated that DEOXY enantiomers and OXY enantiomers are all bound competitively at the same binding site of AGP molecule.

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human α 1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2–10 μ M ( R)- or ( S)-OXY was higher than 99%, and the unbound fraction of ( R)-OXY was 1.56 times higher than that of ( S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities ( nK) of ( R)- and ( S)-OXY to AGP were 6.86×10 6 and 1.53×10 7 M −1, respectively, while the nK values of ( R)- and ( S)-OXY to HSA were 2.64×10 4 and 2.19×10 4 M −1, respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity ( S/ R ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.

Binding Study of Semotiadil and Levosemotiadil with α1-Acid Glycoprotein Using High-Performance Frontal Analysis

High-performance frontal analysis (HPFA) was used to investigate the binding properties of human α1-acid glycoprotein (AGP) with semotiadil ((R)-isomer, Ca-channel blocker) and its antipode levosemotiadil ((S)-isomer, Ca- and Na-channel blockers). An on-line HPLC system consisting of a HPFA column, an extraction column, and an analytical HPLC column was used to determine the unbound concentrations of these enantiomers, and the experimental data were subsequently subjected to the Scatchard analyses to estimate their binding parameters. The binding affinity of the (R)-isomer (K = 3.17 × 107 M, n = 0.74) is approximately 1.2 times stronger than that of (S)-isomer (K = 2.59 × 107 M, n = 0.74). An enantioselective competitive binding study indicated that both enantiomers are bound at the same site on AGP molecules.

Binding Study of Semotiadil and Levosemotiadil with Human Serum Albumin Using High-Performance Frontal Analysis

High-performance frontal analysis (HPFA) wasapplied to investigate the binding properties of semotiadil(R-isomer, Ca-channel blocker) and levosemotiadil (S-isomer,Ca- and Na-channel blocker), an enantiomeric pair of drugs under devel-opment, to human serum albumin (HSA). An on-line HPLC systemconsisting of a HPFA column, an extraction column and an analytical HPLC column was used to determine the unbound concentrations of theseenantiomers. The experimental data were subjected to Scatchard analyses to estimate the binding parameters. The binding affinity of levosemo-tiadil(K=6.59×105 M-1, n=0.97) is aboutthree-times stronger than that of semotiadil (K=2.15×105M-1, n=0.99). These results did not change in thepresence of warfarin, but were significantly decreased by the addition ofdiazepam, indicating that both enantiomers are bound at the diazepam siteon HSA molecule.

Enantioselective protein binding of semotiadil and levosemotiadil determined by high-performance frontal analysis

An on-line frontal analysis HPLC system was developed for the determination of the unbound concentrations of semotiadil, a new calcium antagonist with non-dihydropyridine structure, and its antipode (levosemotiadil), and was applied to the enantioselective investigation of their plasma protein binding properties. This system consists of a high-performance frontal analysis (HPFA) column, an extraction column, and an analytical column, which are connected via two switching valves. After the direct injection of the sample solution into the HPFA column, the drug was eluted as a zonal peak with a plateau region. The unbound drug concentration was determined as the drug concentration in the plateau. As low as 1.04 nM of the unbound drug was determined with good reproducibility. Semotiadil ( R-isomer) and levosemotiadil ( S-isomer) are bound strongly and enantioselectively to human serum albumin (HSA) and human α 1-acid glycoprotein (AGP), and the enantioselectivity was reversed between these plasma proteins. While HSA binds S-isomer more strongly than the antipode, human AGP binds R-isomer more strongly. In human plasma, the unbound drug fraction was less than 1%, and the enantioselectivity was similar to that observed in AGP solution.

Protein-Binding High-Performance Frontal Analysis of (R)- and (S)-warfarin on HSA with and without Phenylbutazone

Applicability of high-performance frontal analysis (HPFA) to the stereoselective study of drug–drug interaction upon plasma protein binding has been investigated. Racemic warfarin and phenylbutazone were used as model drugs. An on-line HPFA/HPLC system consisting of a HPFA column (diol-silica column), an extraction column, and a chiral separation column was developed, and human serum albumin solution containing racemic warfarin and/or phenylbutazone was injected directly to the HPFA column. When the injection volume was large enough, the binding equilibrium in the sample solution was reproduced in the column, and consequently a plateau region appeared on the chromatogram. This plateau region contains unbound drug(S). A given volume of eluent in the plateau part was transferred into the extraction column by column-switching. The concentrated drug(S) was then transferred to the chiral separation column to determine the unbound concentrations of the enantiomers and/or the competitor. The results agreed with those obtained by a conventional ultrafiltration–HPLC method. The influence of phenylbutazone upon the protein binding of warfarin is enantioselective. In warfarin and human serum albumin mixed solution, the unbound concentration of (R)-warfarin was 1.22 times higher than that of the S-isomer. By addition of phenylbutazone, the unbound concentration of (S)-warfarin increased more than that of (R)-warfarin, resulting in the reversed enantioselectivity, i.e., the unbound concentration of (S)-warfarin became 1.19 times larger than that of (R)-warfarin. The present method was also applicable to human plasma samples.

Theoretical Study of High-Performance Frontal Analysis: A Chromatographic Method for Determination of Drug−Protein Interaction

High-performance frontal analysis (HPFA), a chromatographic method to determine unbound drug concentration in drug-protein binding equilibrium, has been considered on the basis of a theoretical plate model, where a rapid equilibrium of drug-protein binding in the mobile phase in the interstices of packing materials and a chromatographic partition equilibrium of the drug were taken into account simultaneously. When a certain excess volume of drug-protein mixed solution is injected directly into a HPFA column packed with a restricted-access type phase that excludes protein but retains drug in the micropores, the drug is eluted as a zonal peak with a plateau region. The elution profile can be well simulated by the mass balance equation derived according to a relatively simple plate theory concept, which confirms that the drug concentration in the plateau range agrees with the unbound drug concentration in the sample solution. The model was applied to the theoretical and systematic investigation of the dependence of the HPFA profile on several chromatographic conditions and the properties of the sample solution, such as injection volume of sample solution, drug and protein concentrations in sample solution, capacity factor of the drug, theoretical plate number, and binding parameters. The smaller capacity factor and the higher column efficiency lead to the larger plateau volume. The lower drug concentration, the higher protein concentration, and the stronger binding constant, which give the lower unbound drug fraction, lead to the larger plateau volume and allow frontal analysis with a smaller sample size.

High-performance frontal analysis for the study of protein binding of troglitazone (CS-045) in albumin solution and in human plasma

An on-line frontal analysis HPLC system was developed for the determination of teh unbound concentration of troglitazone (CS-045), a new oral antidiabetic agent, in human serum albumin (HSA) solution and in human plasma. This system consists of a high-performance frontal analysis (HPFA) column, an extraction column, and an analytical column, which are connected via two switching valves. After the direct injection of the sample solution into the HPFA column, the drug was eluted as a zonal peak with a plateau region. The unbound drug concentration was determined as the drug concentration in the plateau. As low as 0.3 n M unbound CS-045 was determined with good reproducibility. It was found that CS-045 strongly binds with HSA, and the bound fraction in the 550 μ M HSA solution was 99.93%, which was very close to that in human plasma (99.89%). The bound fractions were constant within the total drug concentration range of 1–10 μ M in the HSA solution and 250 n M–10 μ M in human plasma.

High-Performance Frontal Analysis/High-Performance Liquid Chromatographic System for the Enantioselective Determination of Unbound Fenoprofen Concentration in Protein Binding Equilibrium

Abstract High-performance frontal analysis (HPFA) and high-perfon-ance liquid chromatography (HPLC) were incorporated in an on-line coupled column system for the enantioselective determination of unbound fenoprofen (FP) concentrations in the state of a protein binding equilibrium following direct sample injection. This system consists of a “restricted access” type column for HPFA and al-acid glycoprotein-immobilized silica column for chiral separation. When a 80-μL portion of a sample solution containing 50 or 90μg/mL FP and 300 or 550,μM human serum albumin (HSA) was directly injected into the HPFA column, FP gave a trapezoidal peak exhibiting a plateau region. The concentration in the plateau range was equal to the unbound FP concentration in the initial sample solution. By the delivery of a 90-μL portion of the eluent in this plateau region into the chiral separation column, the unbound concentrations of FP enantiomers were determined. The results agreed with those obtained by the conventional ultrafil...

High‐Performance Frontal Analysis—High-Performance Liquid Chromatographic System for Stereoselective Determination of Unbound Ketoprofen Enantiomers in Plasma after Direct Sample Injection

AbstractAn on‐line, high‐performance frontal analysis (HPFA)—high‐performance liquid chromatographic system was developed for the enantioselective determination of a low level of unbound ketoprofen (KP) that is in equilibrium with KP that is bound to protein. The system consists of three subsystems (HPFA system, preconcentration system, and chiral separation system) connected in series in the stated order via column‐switching valves. When either a 300‐μL portion of buffer solution containing 300 or 550 μM human serum albumin and 100 or 300 μM racemic KP or a 300‐μL portion of human plasma containing 12.5–100 μM racemic KP was directly injected onto the HPFA column with the mobile phase at a low flow rate, KP was separated from proteins and eluted as a zonal peak with a plateau. The KP concentration in the eluant of the plateau region was the same as the unbound‐KP concentration that was in equilibrium with protein‐bound KP in the initial sample solution. A 1‐mL portion of the eluant of the plateau region was switched to the preconcentration system, where KP was adsorbed and condensed on an octadecylsilyl silica (ODS) column. The adsorbed KP was eluted out of the ODS column and transferred to the chiral separation system, via another switching valve, where the enantiomers of unbound KP were separated and determined. The results agree well with those obtained by the conventional ultrafiltration method. The advantages of the present method over the widely used ultrafiltration method are (1) a very low concentration of unbound drug, due to strong binding to protein, can be determined enantioselectively without sample pretreatment and (2) undesirable adsorption of drug and leakage of proteins, which often cause trouble in the ultrafiltration method, can be avoided. Consequently, the therapeutic concentrations of unbound KP in human plasma could be determined with good reproducibility by injecting a relatively small volume (300 μL) of untreated plasma. The limit of determination (signal‐to‐noise ratio of 10 with UV detection at 262 nm) was 1.0 nM for each KP enantiomer. The present system was used to determine the profile of the concentration of unbound KP enantiomers in rat plasma versus time.