High-performance Frontal Analysis Research Articles

Determination of plasma protein binding of positron emission tomography radioligands by high-performance frontal analysis

Positron emission tomography (PET) is an imaging technique based on the use of radioligands labeled with short lived radionuclides, such as 11C (t½=20.4min) and 18F (t½=109.8min), which as a consequence often requires rapid plasma protein binding analysis methods. In addition, PET radioligands can suffer from non-specific binding to the membrane when ultrafiltraion, which is the most commonly used method for measuring protein binding in PET, is employed. In this study a high-performance frontal analysis (HPFA) method based on incorporation of a gel filtration column (discovery® BIO GFC 100, 50mm×4.6mm, 5μm, 100Å) into a radio-LC system with phosphate buffered saline (PBS, pH 7.4) at a flow rate of 3ml/min as mobile phase was developed and investigated for four PET radioligands. The minimum injection volume (MIV) of plasma, which is a crucial factor in HPFA, was determined to be 200μl (human), 500μl (monkey), 700μl (human) and 1000μl (monkey) for these four radioligands. The MIV values increased as a higher fraction of the radioligand was present in the protein-free form. The protein binding results obtained were in good agreement with ultrafiltration and the method did not suffer from non-specific binding. The short analysis time (<12min) allowed multiple protein binding measurements during time course of a human [11C]PBR28 PET study.

Simultaneous Determination of Protein-Free and Total Positron Emission Tomography Radioligand Concentrations in Plasma Using High-Performance Frontal Analysis Followed by Mixed Micellar Liquid Chromatography: Application to [11C]PBR28 in Human Plasma

A two-dimensional liquid chromatographic (LC) system was developed for the determination of protein-free and total (free + bound forms) positron emission tomography (PET) radioligand concentrations in plasma by direct plasma injection. The unbound PET radioligand was first analyzed by high-performance frontal analysis using a short gel-filtration column and phosphate buffered saline solution as the mobile phase. All the collected effluent from the gel-filtration column was then transferred to the second dimension consisting of a monolithic C-18 column and mixed (anionic and nonionic surfactants) micellar eluent for determination of the total PET radioligand concentration. The simultaneous analysis of protein binding and radiometabolism of [(11)C]PBR28 was completed within 11 min without any pretreatment of plasma and employing a single analytical system. This system allowed highly sensitive analysis of total [(11)C]PBR28 with a limit of detection (LOD) of 1.6 becquerel (Bq). The LOD for the determination of unbound [(11)C]PBR28 was 21 Bq. Finally, simultaneous measurements of protein binding and radiometabolism of [(11)C]PBR28 in human plasma were achieved for up to 50 min after radioligand administration.

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.

Biantennary Glycans as Well as Genetic Variants of α1-Acid Glycoprotein Control the Enantioselectivity and Binding Affinity of Oxybutynin

The purpose of this study was to investigate the role of biantennary branching glycans of alpha1-acid glycoprotein (AGP) and its genetic variants in the enantioselective binding of oxybutynin (OXY). Human native AGP was separated using imminodiacetate-copper (II) affinity chromatography into two fractions, the A variant and a mixture of the F1 and S variants (F1-S). These fractionated AGPs were further separated by concanavalin A affinity chromatography into two fractions, with and without biantenarry glycans. An on-line high-performance liquid chromatography (HPLC) system consisting of a high-performance frontal analysis column, an extraction column, and an analytical HPLC column was developed to determine the binding affinities of OXY enantiomers for respective AGP species. The total binding affinity as well as the enantiomeric selectivity of OXY in the F1-S mixed variant was significantly higher than that for the A variant, indicating that the chiral recognition ability of native AGP for the OXY enantiomers highly depends on the F1-S mixed variant. Furthermore, not only the genetic variants but also bianntenary glycans of AGP affect the binding affinity of OXY and are also responsible for the enantioselectivity. Both genetic variants and glycan structures significantly contribute to the enantioselectivity and the binding affinity of OXY.

Single run measurements of drug‐protein binding by high‐performance frontal analysis capillary electrophoresis and mass spectrometry

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.

Protein binding study of isoflavone, perillyl alcohol and S-ibuprofen by high-performance frontal analysis

High-performance frontal analysis (HPFA) was used for a protein binding study of isoflavones (daidzein, genistin, and genistein), enantiomers of perillyl alcohol and S-ibuprofen to human serum albumin (HSA). The analyses were performed on a Develosil and Inertsil 100-Diol-5 column (10 cm×4.6mm). Sodium phosphate solution (pH 7.4, ionic strength 0.17) was used as the mobile phase at a flow rate of 1 ml/min. To ensure the drug to be eluted as a trapezoidal peak with a plateau, injection volumes were each fixed up the zonal profile with an evident plateau appears. The unbound drug concentration was determined from a plateau height of the plateau region after that experimental data were fitted by Scatchard equation. The binding constants (K) and total binding affinities (nK) of drugs to HSA were calculated, respectively.

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.

Protein Binding Study of Isoflavones by High-Performance Frontal Analysis

High-performance frontal analysis was used for protein binding study of isoflavones (daidzein, genistin, and genistein) to human serum albumin. The analysis was performed on a Develosil 100-Diol-5 column (10 cm × 4.6 mm). Sodium phosphate solution (pH 7.4, ionic strength 0.17) was used as the mobile phase at a flow rate of 1 mL min−1. UV wavelength was set at 260 nm. To ensure the drug to be eluted as a trapezoidal peak with a plateau, injection volumes of 700 μL for daidzein and genistin, 900 μL for genistein were chosen, respectively. Experimental data were fitted by Scatchard equation. The binding constants (K) and binding affinities (nK) of isoflavones to HSA were: K=1.581 × 105 (L mol−1), nK=0.77 × 104 (L mol−1) for daidzein, K=1.082 × 105 (L mol−1), nK=0.32 × 104 (L mol−1) for genistein, and K=3.533 × 105 (L mol−1), nK=0.70 × 104 (L mol−1) for genistin, respectively.

Protein Binding Study of Perillyl Alcohol Enantiomers by High Performance Frontal Analysis

High‐performance frontal analysis (HPFA) was used for protein binding study of perillyl alcohol (POH) enantiomers to human serum albumin (HSA). The analysis was performed on a Develosil 100‐Diol‐5 (10 cm × 4.6 mm I.D.) column. Sodium phosphate solution (pH 7.4, ionic strength 0.17) was used as the mobile phase at a flow rate of 1 mL/min. UV wavelength was set at 205 nm. An injection volume of 600 µL was chosen to ensure the drug to be eluted as a trapezoidal peak with a plateau. Experimental data were fitted by Scatchard analysis. The binding constant (K) and binding affinity (nK) of POH enantiomers to HSA were: K = 5.20 × 106 (1/mol), nK = 1.74 × 104 (1/mol) for (S)‐POH and K = 3.71 × 106 (1/mol), nK = 1.51 × 104 (1/mol) for (R)‐POH, respectively.

Novel sulfamethazine ligand used for one-step purification of immunoglobulin G from human plasma

To replace conventional affinity ligand like protein A or protein G, a pseudobioaffinity ligand seems to be an alternative for the purification of immunoglobulin G (IgG). In this study, sulfamethazine (SMZ) was chosen as novel affinity ligand for investigating its affinity to human IgG. Monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) (PGMA) beads were employed as the support for high-performance affinity chromatography. SMZ was immobilized on PGMA beads using bisoxirane (ethanediol diglycigyl ether) as spacer. The resultant affinity media presented minimal non-specific interaction with other proteins. Results of high-performance frontal analysis indicated that the media showed specific affinity to human IgG with a dissociation constant on the order of 10 −6 M. The SMZ affinity column proved useful for a very convenient one-step purification of IgG from human plasma. Antibody purity after a one-step purification was higher than 90%, as determined by densitometric scanninng of sodium dodecyl sulfate–polyacrylamide gel electrophoresis of purified fraction under reducing condition. The results obtained indicate that SMZ is a valuable affinity ligand for purification of human IgG.

Study on the protein binding of ketoprofen using capillary electrophoresis frontal analysis compared with liquid chromatography frontal analysis.

A method of capillary electrophoresis frontal analysis (CEFA) is developed for the first time to study the binding of ketoprofen to human serum albumin (HSA) and compared with high-performance liquid chromatography frontal analysis (LCFA). The separation is performed in an uncoated fused-silica capillary (60-cm x 75- micro m i.d., 50-cm effective length) with a phosphate buffer (pH 7.4, ionic strength of 0.17M) as the running buffer. The applied voltage is 13 kV and the detection is set at 254 nm. A trapezoidal peak of the unbound ketoprofen appears after HSA elution in the electropherogram. The plateau height of the peak is employed to determine the unbound concentration of ketoprofen in the HSA equilibrated sample solution. The CEFA method provides the advantages of small sample injection volume and rapidity and the disadvantage of low sensitivity compared with LCFA. CEFA is applicable to the binding parameter estimation of ketoprofen to the secondary binding site; an association constant (K(2)) of 0.24 x 10(6)M(-1) and the number for the binding site per molecule HSA of 2.54 is estimated. In contrast, LCFA measures parameters for both primary and secondary sites, which are 1.05 x 10(6)M(-1) and 0.94 for K(1) and n(1), respectively, and 0.12 x 10(6)M(-1) and 3.16 for K(2) and n(2), respectively. It is found that ketoprofen binds mainly at the primary site at a molecular ratio of ketoprofen versus HSA lower than 0.75, and the binding at the secondary site occurs at a higher ratio.

Specific determination of unbound oxacillin in protein solution with cefoperazone by high-performance frontal analysis with chemiluminescence detection.

Unbound oxacillin concentrations in human serum albumin (HSA) solutions in the presence or absence of cefoperazone were determined using high-performance frontal analysis coupled with chemiluminescence detection (HPFA-CL). The HPFA was performed on an ISRP column with 67 mM potassium phosphate buffer of pH 7.4 and ionic strength of 0.17 as the mobile phase. The luminol-H(2)O(2)-Co(2+) system was employed in the chemiluminescence detection. The detection was highly specific for oxacillin in the presence of cefoperazone. Although both drugs in HSA solutions co-eluted in the same region in HPFA, cefoperazone did not interfere with the determination of unbound concentration of oxacillin. In the solution of 100 microM HSA and 11.33 micro M oxacillin the bound percentage of oxacillin to HSA was estimated as 80.5%. Addition of 30.98 micro M cefoperazone into the HSA-equilibrated solution produced little effect on the protein binding of oxacillin. In the presence of 154.9 micro M cefoperazone, however, the bound percentage of oxacillin was significantly reduced. This specific method could be applied to the investigation of drug-drug interaction in protein binding.

Chemiluminescence detection coupled to high-performance frontal analysis for the determination of unbound concentrations of drugs in protein binding equilibrium

High-performance frontal analysis coupled with chemiluminescence detection (HPFA–CL) was developed for the determination of unbound oxacillin concentration in human serum albumin solution. The HPFA system consisted of an ISRP column and a mobile phase of 67 m M potassium phosphate buffer of pH 7.4 and ionic strength of 0.17. The luminol–H 2O 2–Co 2+ system was used in the chemiluminescence detection. An enhancement of luminol chemiluminescence by oxacillin was investigated and employed for determining the concentration of oxacillin in the HPFA eluate. Sample solutions were directly injected onto the column; the drug was eluted as a zonal peak with a plateau region. The unbound drug concentrations were determined by using the height of the plateau. The results agreed with those obtained with conventional ultrafiltration–HPLC method. Good reproducibility was confirmed by the within run and between run RSD≤7.4%. HPFA–CL provided a selective method for determination of unbound drug concentration in protein binding equilibrium.

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 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.

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 μM) 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 (4.01×10 5 per M) was 17 times higher than that of PRO–HDL binding (2.38×10 4 per M).

Effect of oxidation of low-density lipoprotein on drug binding affinity studied by high performance frontal analysis-capillary electrophoresis.

The effect of oxidation of low-density lipoprotein (LDL) on the enantioselective drug binding affinity was investigated using high performance frontal analysis--capillary electrophoresis (HPFA-CE). Verapamil and nilvadipine enantiomers were used as the chiral model drugs. LDL was oxidized with copper sulfate for 0, 0.5, 1, 2, and 12 h at 37 degrees C. The HPFA-CE method enabled microdetermination of unbound drug concentrations in native and oxidized LDL solutions. It was found that the bindings between LDL and the model drugs were not enantioselective at any oxidation stage. The total binding affinity (nK) between LDL and verapamil enantiomers was increased by 3.3-, 4.6-, 7.0-, and 19-fold after 0.5, 1, 2, and 12 h oxidation, respectively, whereas the nK value between nilvadipine and LDLwas increased by 1.3-, 1.4-, 1.4-, and 1.7-fold in the same reaction times, respectively. These results indicate that the LDL oxidation enhances the drug binding affinity, and the affinity of verapamil is increased more sensitively than that of nilvadipine. The nK value of each model drug increased steeply after the first 2 h oxidation, followed by the gradual increase after the next 10 h oxidation. It is considered that the net increase in the negative charges and/or the formation of hydroperoxides in the first 2 h oxidation enhances the drug-LDL binding more significantly than the formation of aldehydes or Schiff bases in the following 10 h oxidation.

On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis

A microreactor by immobilized trypsin on the activated glycidyl methacrylate-modified cellulose membrane packed column was constructed. Immobilized trypsin mirrored the properties of the free enzyme and showed high stability. A novel method to characterize the activity and reaction kinetics of the immobilized enzyme has been developed based on the frontal analysis of enzymatic reaction products, which was performed by the on-line monitoring of the absorption at 410 nm of p-nitroaniline from the hydrolysis of N-α-benzoyl- dl-arginine- p-nitroanilide (BAPNA). The hydrolytic activity of the immobilized enzyme was 55.6% of free trypsin. The apparent Michaelis–Menten kinetics constant ( K m) and V max values measured by the frontal analysis method were, respectively, 0.12 m M and 0.079 m M min −1 mg enzyme −1. The former is very close to that observed by the static and off-line detection methods, but the latter is about 15% higher than that of the static method. Inhibition of the immobilized trypsin by addition of benzamidine into substrate solution has been studied by the frontal analysis method. The apparent Michaelis–Menten constant of BAPNA ( K m), the inhibition constant of benzamidine ( K i) and V max were determined. It was indicated that the interaction of BAPNA and benzamidine with trypsin is competitive, the K m value was affected but the V max was unaffected by the benzamidine concentration.