Akimasa Shibukawa

High-performance frontal analysis for drug–protein binding study ☆

High-performance frontal analysis (HPFA) is a novel analytical method which enables simultaneous determination of total and unbound drug concentrations under drug-plasma protein binding condition. HPFA can be achieved using separation systems such as HPLC and CE. This paper deals with the principle and feature of HPFA method and its application to the stereoselective protein binding study. HPFA allows a simple analysis following direct sample injection, and does not suffer from undesirable drug adsorption on membrane nor leakage of bound drug through the membrane which are often encountered in conventional ultrafiltration and dialysis methods. HPFA can be easily incorporated into on-line HPLC system. By coupling HPFA with a chiral HPLC column, the unbound concentration of a racemic drug can be determined enantioselectively. The detection limit can be improved by coupling of HPFA with a preconcentration column. High-performance capillary electrophoresis/frontal analysis (HPCE/FA) enables to determine unbound concentrations enantioselectively with ultramicro injection volume, and is hence useful especially for the binding study of proteins which are scarce and difficult to obtain.

High-performance capillary electrophoresis of SDS-proteins using pullulan solution as separation matrix

Sodium dodecyl sulphate (SDS) capillary electrophoresis using pullulan solution as a separation matrix was developed for the separation and molecular mass determination of proteins. The silanol functions on the inner surface of a fused-silica capillary were deactivated by coating with linear polyacrylamide through Si-C linkages, into which the pullulan solution was filled. The stability of the coating was examined by exposure to an alkaline buffer solution (pH 9.2) for up to 30 days. Compared with conventional coatings with linear polyacrylamide through siloxane linkages, the present capillary was more stable even under alkaline conditions and markedly reduced electroosmotic flow. Thus, polymer solutions of low viscosity such as pullulan solution could be stabilized in the capillary, resulting in a prolonged life-span of the capillary and improved reproducibility of separations. An excellent linear relationship was obtained between the mobility and the logarithm of the molecular mass of SDS-proteins. The relative standard deviation of migration times was below 0.5% when the pullulan solution was refilled in each analysis (n = 10). The calibration plots of the integrated peak areas at 214 nm vs. concentration of standard proteins were linear in the range 5 micrograms/ml-0.1 mg/ml.

Liquid chromatography with crown ether-containing mobile phases : II. Retention behaviour of β-lactam antibiotics in reversed-phase high-performance liquid chromatography

Abstract The retention behaviour of several β-lactam antibiotics in reversed-phase liquid chromatography with mobile phases containing crown ether(18-crown-6 or dicyclohexyl-18-crown-6) has been investigated. The capacity factors were determined with various concentrations of crown ether and methanol in the aqueous mobile phase at different pH values. The observed profiles of capacity factor, k, vs. crown ether concentration and k vs. pH are discussed with reference to an equation derived from a chromatographic model involving association of β-lactam antibiotics with crown ethers and bindings of free and associated species to the hydrophobic stationary phase. The effect of ionic salts on the capacity factor is also discussed. The applicability of the method is demonstrated by specific separations of ampicillin in urine and cephalexin in plasma.

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 the 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 nM 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 microM 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 microM in the HSA solution and 250 nM-10 microM in human plasma.

Enantioselective binding analysis of verapamil to plasma lipoproteins by capillary electrophoresis–frontal analysis

Capillary electrophoresis coupled with frontal analysis was applied to the study of enantioselective binding of verapamil (VER) to plasma lipoproteins. The drug-lipoprotein mixed solution, which had been in the binding equilibrium, was hydrodynamically introduced into a non-coated fused-silica capillary. Since VER is positively charged in the neutral run buffer (pH 7.4), the unbound VER enantiomers migrated toward the cathodic end much faster than negatively charged lipoproteins and their bound forms. Once unbound VER migrated apart from lipoprotein, the bound VER was quickly released from the protein to maintain the binding equilibrium. Thus, VER migrated as a zone through the capillary and gave a trapezoidal peak with a plateau region on the electropherogram. The VER concentration in this plateau region was equal to the unbound VER concentration in the initial sample solution. It was found that the bindings of VER to high-density lipoprotein (HDL), low-density lipoprotein (LDL) and oxidized LDL were not site-specific and not enantioselective. Partition-like binding to lipid part of these lipoproteins seemed to be dominant. The total binding affinities of LDL to VER were about seven-times stronger than those of HDL, and the oxidation of LDL by copper ion enhanced the binding affinities significantly.

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.

Retention properties of internal-surface reversed-phase silica parking and recovery of drugs from human plasma

The properties of internal-surface reversed-phase silica packings developed for use in high-performance liquid chromatography, were investigated with regard to the effects of mobile phase conditions (pH, ionic strength, nature and concentration of organic modifiers, ion-pairing agent and surfactant) on the retention of some selected compounds having different polarities. The results indicated that pi-electron interactions play the main role in solute retention, although it is weaker than that on phenylsilica supports, and that weak cation-exchange properties exert a secondary effect on the retention of ionic solutes. The ion-pairing effect was found to be markedly weakened in mobile phases containing an anionic counter ion. In contrast, the addition of a surfactant (SDS) caused a marked increase in the retention of certain drugs at low pH and expanded the pH range of the mobile phase applicable to plasma samples. Based on these findings, the separation and recovery of several drugs (probenecid, lidocaine, cefpiramide and cefaclor) from human plasma were investigated, with emphasis on their protein bindings. The contour chromatogram of human plasma is also demonstrated in relation to the selection of the detection wavelength.

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 alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (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 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR 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.

EFFECT OF SIALIC ACID RESIDUES OF HUMAN ALPHA 1-ACID GLYCOPROTEIN ON STEREOSELECTIVITY IN BASIC DRUG-PROTEIN BINDING

The function of sialic acid groups at the terminal of sugar chains of human alpha 1-acid glycoprotein (AGP) was investigated with respect to chiral discrimination between optical isomers of basic drugs, using high-performance capillary electrophoresis/frontal analysis (HPCE/FA), a novel analytical method developed for the determination of unbound drug concentration with ultramicrosample volume (100-200 nl). Native human AGP and desialylated AGP were used as test proteins, and propranolol (PRO) and verapamil (VER) were used as model drugs. The unbound concentration of (S)-VER was 1.31 times higher than that of (R)-VER in native AGP solution. This selectivity was not affected by desialylation. Further, enzymatic elimination of galactose residues, which neighbored sialic acid groups, did not change the binding of either isomer of VER. On the other hand, the unbound concentration of (R)-PRO was 1.27 times higher than that of (S)-PRO in native AGP solution. Desialylation caused the unbound concentration of (S)-PRO to rise to the same level of (R)-PRO, resulting in loss of enantioselectivity. Thus, it follows that sialic acid groups of AGP, as a whole, are not responsible for chiral recognition between enantiomers of VER but are involved in enantioselectivity toward the isomers of PRO.

Frontal analysis of drug-plasma lipoprotein binding using capillary electrophoresis.

High performance frontal analysis coupled with capillary electrophoresis (HPFA/CE) was applied to the ultramicroanalysis of enantioselective binding of drug to plasma lipoproteins. A small volume (ca. 80 nl) of (R)- or (S)-propranolol (PRO, 25-150 microM) and human high-density lipoprotein (HDL, 2.63 g/l) or human low-density lipoprotein (LDL, 4.37 g/l) mixed solution, which was in the state of binding equilibrium, was introduced hydrodynamically into a non-coated fused silica capillary. Positively charged unbound PRO enantiomers migrated toward cathodic end much faster than negatively charged lipoproteins and the bound form. Once unbound PRO migrated apart from lipoprotein, the bound PRO was quickly released from the lipoprotein to maintain the binding equilibrium. Thus, PRO migrated as a zone in the capillary, giving a peak with a plateau region, where the concentration is the same as the unbound PRO concentration in the original sample solution. The unbound PRO concentration calculated form the plateau height agreed with that determined by a conventional ultrafiltration method used as a reference method. It was found that the bindings of PRO to HDL and PRO to LDL were not enantioselective, while the total binding affinity of PRO to LDL (4.01 x 10(5) per M) was 17 times higher than that of PRO-HDL binding (2.38 x 10(4) per M).