Alpha 1-acid Glycoprotein Column Research Articles

Stepwise frontal affinity chromatography model for drug and protein interaction.

Frontal affinity chromatography is an efficient technique that combines affinity interaction and high-performance liquid chromatography, and frontal analysis has been used in studying the interaction between drugs and proteins. Based on frontal analysis, stepwise frontal analysis has been established. The present study aimed to use the Lineweaver-Burk plot in stepwise frontal analysis by taking the weighted average of time data. Commercial human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP) columns were used as an affinity column. Warfarin and digitoxin were chosen as model drugs for the HSA column, whereas verapamil and tamsulosin were selected as model drugs for the AGP column. The time data obtained by frontal analysis and stepwise frontal analysis were compared, and the results revealed good correlation (r2 = 0.9946-0.9998). Frontal analysis and stepwise frontal analysis were also used to analyze the equilibrium dissociation constants (Kd) of model drugs on the HSA and AGP columns. The Kd values were compared with literature values, which revealed the same order of magnitude. These results illustrate that conversion of the time data is reasonable and feasible. The Lineweaver-Burk plot can be used in the stepwise frontal analysis model to study the characteristics of the interaction between drugs and proteins. Graphical abstract ᅟ.

Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns

In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110–830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10–20min when using only 3–10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

Investigation of the retention behavior of structurally diverse drugs on alpha1 acid glycoprotein column: Insight on the molecular factors involved and correlation with protein binding data

Retention of 49 structurally diverse drugs on alpha1 acid glycoprotein column was investigated under different chromatographic conditions. Acetonitrile and 2-propanol were used as organic modifiers at different percentages and the pH was adjusted at 7.0 using PBS. Analysis of extrapolated and isocratic retention in terms of lipophilicity and electrostatic interactions revealed significant effect of the nature and percentage of organic modifier, which was attributed to the different shielding degree of the charged sites on the stationary phase by the buffer constituents. AGP retention factors were compared to HSA retention factors analyzed previously. Application of LSER analysis, extended to incorporate fractions ionized, demonstrated hydrogen bond acidity, dipolarity/polarizability and excess molar refraction as the most significant parameters for all AGP chromatographic indices, elucidating the differentiation of AGP retention from octanol–water partitioning and HSA retention. An attempt to correlate AGP chromatographic indices to AGP association constants, available in literature, supported the importance of stationary shielding in retention mechanism. Thus, isocratic retention factors logk10(ACN)AGP show a moderate but still better performance than lipophilicity in the case of A variant and may be a useful tool for the estimation of relevant association constants. For F1/S binding simulation lower stationary phase shielding is needed to obtain a significant two term regression equation, where logk20(ACN)AGP exerts a secondary contribution next to the most important bulk effect expressed by molecular weight.

Evaluation of a hydrazide‐linked α1‐acid glycoprotein chiral stationary phase: Separation of R‐ and S‐propranolol

The binding and chiral separation of R- and S-propranolol was investigated on a new type of alpha1-acid glycoprotein (AGP) column. This column was prepared through the controlled and mild oxidation of AGP, followed by the immobilization of this protein to hydrazide-activated silica. The effects of temperature, pH, ionic strength, and organic modifiers on the retention and separation of R- and S-propranolol were investigated on this column. Both the association equilibrium constants and number of binding sites for R/S-propranolol on the AGP column were found to increase with temperature and affect the measured retention factors for these compounds. Regarding the other factors, a change in the organic modifier concentration was found to give the largest change in retention and separation. It was found through these studies that both coulombic and hydrophobic interactions played important roles in determining the retention of R- and S-propranolol on the AGP column. The efficiency and separation impedance of this system were also considered. Under the final optimum conditions identified in this study, it was possible to separate R- and S-propranolol with a resolution of greater than 1.38 in less than 5 min on a 4.1 mm I.D. x 5 cm column.

Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and microdialysis samples using liquid chromatography/tandem mass spectrometry

Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective separation was achieved on an alpha1-acid glycoprotein column within 14 min for all methods. A cetirizine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were detected by multiple reaction monitoring using transitions m/z 389.1 --> 200.9 and 396.1 --> 276.1, respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea pig plasma, the assay was linear over the range 0.25-5000 ng/mL for both S-CZE and R-CZE, with a lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples, the assays were linear over the range 2.5-250 ng/g and 0.25-50 ng/mL, respectively, and the LLOQ values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were < or =7.1% and < or =12.6%, respectively, and the intra- and inter-day accuracy varied by less than +/-8.0% and +/-6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.

Enantioselective analysis of ritalinic acids in biological samples by using a protein-based chiral stationary phase.

This study was to develop and validate a new chiral HPLC-UV method for the quantitative analysis of enantiomeric ritalinic acid (RA) in human plasma. An alpha1-acid glycoprotein column was used with the mobile phase containing 0.4% acetic acid and 0.1% dimethyloctylamine, pH 3.4. The detection of enantiomeric RAs was at 220 nm. A baseline separation for d- and l-RA was achieved by a separation factor of 2.08. Methylphenidate, the precursor of RA, was eluted with the front solvent, and thus does not interfere in the analysis of RA in our method. The assay was successfully applied for the in vitro analysis of enantiomeric ritalinic acids produced by human and dog plasma and dog liver. Data demonstrated that the esterase(s) in human plasma metabolize d-methylphenidate faster than its l-isomer. The yielded intrinsic clearances (Clint) are 1.02 and 2.17 microl/min/mg protein, respectively, for d and l-methylphenidate.

Analysis of bioactive components in traditional Chinese medicines by molecular biochromatography with alpha1-acid glycoprotein stationary phase.

Molecular biochromatography with alpha1-acid glycoprotein (AGP) stationary phase was proposed to screen and analyse the biologically active components in traditional Chinese medicines (TCMs) with extracts from Radix Salviae miltiorrhizae as a tested sample. More than ten peaks were resolved based on their affinity to AGP. The effects of concentrations of acetonitrile, pH, concentration of inorganic salt and temperature on the retention behaviors of several major active components were also investigated, and it was found that the hydrophobic effect is the major contributor to retention. Tanshinone IIA was identified as one of the principal bioactive components, which is the marker for the quality control of Radix Salviae miltiorrhizae and a complicated remedy named YiXiTongMai. The amount of tanshinone IIA in Radix Salviae miltiorrhizae and YiXiTongMai determined by this method was 2.9 mg/g (net weight, RSD 4.9%, n=5) and 0.078 mg/g (net weight, RSD 2.5%, n=3), respectively. The possibility for fast differentiation of the TCM sources was also studied by the comparison of the fingerprint of chromatograms for eight typical TCMs on the AGP column. It was observed that different TCMs showed different fingerprint characteristics. Even for the same plant, Rhizoma cimicifugae from three different geographical sources, although there were common characteristics, distinct differences in types and concentrations of biologically active components were clearly observed. It was shown that molecular biochromatography was an effective and fast way for the analysis and screening of biologically active compounds in traditional Chinese medicines.

Enantioselective Separation of Methadone and Its Main Metabolite in Human Hair by Liquid Chromatography/Ion Spray-Mass Spectrometry

Optical isomers exhibit significant differences in their affinities for receptor sites, biotransformation and binding to serum and tissue proteins. Methadone has been used for the substitution of heroin addicts since 1964. The racemic form is used, i.e., a mixture of the biologically active R-form and the practically inactive S-form. To investigate methadone distribution, a chiral separation of the isomers was developed in human hair samples. The method involves decontamination of hair with water and acetone, pulverization in a ball mill, enzymatic hydrolysis in presence of deuterated internal standards, solid-phase extraction, and liquid chromatography/ion spray-mass spectrometry. Enantioselective separation of methadone and its main metabolite, EDDP, was obtained using an alpha1-acid glycoprotein column (100 by 4 mm ID). In all nine specimens obtained from subjects under racemic methadone treatment in a detoxification center, R- and S-enantiomers of methadone and EDDP were identified with the following concentrations: 2.58-10.22, 1.89-9.53, 0.42-1.73, and 0.40-2.10 ng/mg for R-methadone, S-methadone, R-EDDP, and S-EDDP, respectively. Results are suggestive of a predominance of the Renantiomer of methadone in human hair.

Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester.

A stereospecific HPLC method for the quantitation of CGP 49309 in samples of its corresponding enantiomer valsartan has been developed and validated. The enantiomeric separation was achieved on a 5 micron silica-bonded, alpha 1-acid glycoprotein column (Chiral AGP) with a phosphate buffer, pH 7, containing 2% (v/v) 2-propanol as a mobile phase. The linearity was established in the range 0.1-4% (r > 0.999). The limit of quantitation was 0.1% and the limit of detection was 0.04%. The accuracy of the method was found to be 96.7% (average). For the precision (repeatability), a relative standard deviation value of 2.4% was found. Similarly, a stereoselective HPLC method was also developed and validated for the quantitation of the enantiomer of the starting material used for the synthesis of valsartan, namely (R)-valinebenzyl ester tosylate. Baseline resolution of the enantiomers of valinebenzyl ester tosylate could be achieved on the chiral crown ether column Crownpak CR (Daicel) at 50 degrees C using water-methanol-trifluoroacetic acid (850:150:1, v/v) as a mobile phase. The linearity was established in the range 0.5-5% (r > 0.999). The accuracy of the method was found to be 100.5% (average). For the precision (repeatability), a relative standard deviation value of 3.4% was found. Both methods were found to be suitable for the analysis of the respective analytes.

Plasma levels of trimipramine and metabolites in four patients: determination of the enantiomer concentrations of the hydroxy metabolites.

A two-step high-performance liquid chromatography method is described, using a CN column and an alpha 1-acid glycoprotein column, which allows the measurement of the enantiomers of the hydroxy metabolites of trimipramine in plasma of trimipramine-treated patients. Of the four patients analyzed, three showed approximately equimolar concentrations of the (D)- and (L)-enantiomers of the hydroxy metabolites (2-hydroxy-trimipramine and 2-hydroxy desmethyltrimipramine), and one was found to have roughly twice as much of the (L)-form and of the (D)-form of 2-hydroxy trimipramine and 2-hydroxy desmethyltrimipramine. From the data available on the pharmacological effects of the enantiomers of trimipramine, it is postulated that this interindividual variability in its pharmacokinetics is another factor that could contribute to the interindividual variability in its pharmacodynamics.

Measurement of the (R)- and (S)-isomers of warfarin in patients undergoing anticoagulant therapy.

An achiral/chiral high-performance liquid chromatographic system for the analysis of total warfarin together with the (R)- and (S)-enantiomers in clinical samples has been developed. The achiral analysis is achieved using a C8 column, which is coupled to a chiral stationary phase, alpha 1-acid glycoprotein (AGP), thereby allowing for analysis of warfarin isomers without interfering serum peaks. A 0.015 M phosphate buffer mobile phase with 15% v/v propan-2-ol (pH 7.0) was used on the C8/AGP system. UV analysis at 308 nm was used for quantitation of total warfarin on the C8 column and fluorescence (excitation 300 nm, emission 390 nm) detection was employed for isomer quantitation on the AGP. Retention time of total warfarin on the C8 column was 5.95 min, while that of the (S)- and (R)-warfarin on the AGP column was 10.38 and 12.69 min, respectively. Peak resolution of the warfarin isomers was 1.64. All serum samples were subjected to solid-phase extraction. Data from two patients in a single dose study indicate that a two-compartmental model could represent the warfarin concentration-time data with enterohepatic circulation. In some patients studied during steady state therapy, concentrations of (S)-warfarin were greater than (R)-warfarin indicating that the clearance of the former is slower in these patients.

Computer-aided optimisation of drug enantiomer separation in chiral high-performance liquid chromatography

The advent of several new column materials for the resolution of chiral compounds in high-performance liquid chromatography has opened up new possibilities for the analysis of drug enantiomers both in the dosage form and in bioanalytical studies. The utility of simplex optimisation, modified simplex and response surface mapping are considered with reference to the antischistosomal drug, oxamniquine, separated on an alpha 1-acid glycoprotein column. The resolution of the enantiomers of three closely related benzodiazepines, temazepam, oxazepam and lorazepam, is attempted on three new column systems: cellulose triacetate, beta-cyclodextrin and the reversed-phase column porous graphitic carbon with beta-cyclodextrin as a mobile phase additive.

Direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using a chiral alpha 1-acid glycoprotein column.

A 10-cm long alpha 1-acid glycoprotein column is used for the enantiomeric resolution of the clinically used racemic aminoglutethimide (+/- AG) and its acetylated metabolite (+/- AAG). A direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite is accomplished without any derivatizations. Maximum resolutions of 1.37 and 0.73 are obtained for the enantiomers of aminoglutethimide and its acetylated metabolite, respectively. The effect of the 2-propanol content in mobile phase on retention and enantioselectivity of aminoglutethimide and its acetylated metabolite is demonstrated. The variation of the separation factors (alpha) with pH in enantiomeric separation of aminoglutethimide is also shown.

Direct HPLC resolution of beta-aminoalcohol (tazifylline, ranolazine, sotalol) enantiomers

A routine chiral analysis has been developed to control the optical purity of chiral drugs and to monitor their asymmetric synthesis. The recent advent of new chiral stationary phases for HPLC enabled us to achieve the direct resolution of chiral drugs without any derivatization. The factors affecting chiral resolution on a new alpha1-acid glycoprotein column (EnantioPac, LKB) were assessed with three beta-aminoalcohols.

Enantioselective analysis of chloroquine and desethylchloroquine after oral administration of racemic chloroquine.

An enantioselective high-performance liquid chromatographic method for chloroquine and desethylchloroquine was developed using a chiral alpha 1-acid glycoprotein column. This method was used to determine concentrations of chloroquine and desethylchloroquine enantiomers in plasma and urine from volunteers given single oral doses of racemic chloroquine. The disposition of the enantiomers was different. The renal clearance of the chloroquine enantiomers was indicative of stereoselective renal secretion of the drug, and evidence for stereoselective metabolism also was found.