Chiral separation of tryptophan enantiomers by liquid chromatography with BSA-silica stationary phase

The separation of tryptophan enantiomers was carried out with medium-pressure liquid chromatography using BSA (bovine serum albumin)-bonded silica as a chiral stationary phase. The influence of various experimental factors such as pH and ionic strength of mobile phase, separation temperature, and the presence of organic additives on the resolution was studied. In order to expand this system to preparative scale, the loadability of sample and the stability of stationary phase for repeated use were also examined. The separation of tryptophan enantiomers was successful with this system. The data indicated that a higher separation factor (α) was obtained at a higher pH and lower temperature and ionic strength in mobile phase. Addition of organic additives (acetonitrile and 2-propanol) in mobile phase contributed to reduce the retention time of L-tryptophan. About 30% of the separation factor was reduced after 80 days of repeated use.     

Novel liquid chromatographic–tandem mass spectrometric methods using silica columns and aqueous–organic mobile phases for quantitative analysis of polar ionic analytes in biological fluids

Use of silica stationary phase and aqueous–organic mobile phases could significantly enhance LC–MS–MS method sensitivity. The LC conditions were compatible with MS detection. Analytes with basic functional groups were eluted with acidic mobile phases and detected by MS in the positive ion mode. Analytes with acid functional groups were eluted with mobile phases at neutral pH and detected by MS in the negative ion mode. Analytes poorly retained on reversed-phase columns showed good retention on silica columns. Compared with reversed-phase LC–MS–MS, 5–8-fold sensitivity increases were observed for basic polar ionic compounds when using silica columns and aqueous–organic mobile phase. Up to a 20-fold sensitivity increase was observed for acidic polar ionic compounds. Silica columns and aqueous–organic mobile phases were used for assaying nicotine, cotinine, and albuterol in biological fluids.     

Chromatographic analysis of carbamazepine binding to human serum albumin

In this study, high-performance affinity chromatography was used to characterize the binding of carbamazepine to an immobilized human serum albumin (HSA) column. Frontal analysis was first used to determine the association equilibrium constant and binding capacity for carbamazepine on this column at various temperatures. The non-specific binding of carbamazepine within the column was also considered. The results indicated that carbamazepine had a single binding site on HSA with an association equilibrium constant of 5.3 x 10(3)M(-1) at pH 7.4 and 37 degrees C. This was confirmed through zonal elution self-competition studies. The value of DeltaG for this reaction was -5.35 kcal/mol at 37 degrees C, with an associated change in enthalpy (DeltaH) of -6.45 kcal/mol and a change in entropy (DeltaS) of -3.56 cal/molK. The location of this binding region was examined by competitive zonal elution experiments using probe compounds with known sites on HSA. It was found that carbamazepine had direct competition with l-tryptophan, a probe for the indole-benzodiazepine site of HSA, but allosteric interactions with probes for the warfarin, tamoxifen and digitoxin sites. Changes in the pH, ionic strength, and organic modifier content of the mobile phase were used to identify the predominant forces in the carbamazepine-HSA interaction.     

Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human α1-acid glycoprotein variants

The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained with proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated α₁-acid glycoprotein.     

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.     

Comparison of the binding behavior of several histidine-containing proteins with immobilized copper(II) complexes of 1,4,7-triazacyclononane and 1,4-bis(1,4,7-triazacyclononan-1-yl)butane

The protein binding characteristics of the immobilized binucleating chelate system, 1,4-bis(1,4,7-triazacyclononan-1-yl)butane (tacn(2)butane), complexed with Cu(2+) ions have been investigated with hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome C, as well as three histidine-rich proteins, serum albumin, transferrin, and α(2)-macroglobulin, present in partially fractionated human serum. The effects of pH, ionic strength and elution buffers on protein binding have been examined and compared with those of the analogous immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). The Cu(2+)-tacn(2)butane system was generally found to exhibit higher protein binding affinities than the Cu(2+)-tacn system, suggesting that the presence of immobilized binuclear copper(II) species leads to enhanced coordinative interaction with surface-exposed amino acid residues of the studied proteins. However, under some buffer conditions the dependencies of protein binding and elution on pH and ionic strength with these immobilized metal ion affinity chromatographic (IMAC) systems were consistent with electrostatic, hydrophobic and π-bonding interactions playing a significant secondary role in addition to the dominant coordinative interactions. As such, the results indicated that the selectivities were not solely dependent on the histidine content of the protein. In accord with this conclusion, differences in the selectivities of the Cu(2+)-tacn and Cu(2+)-tacn(2)butane adsorbents for serum albumin, transferrin, and α(2)-macroglobulin were observed depending on the choice of elution buffer. This attribute suggests that additional selectivity features can be realised for the separation of specific proteins with this new class of adsorbent.     

Comparative studies of the binding of some ligands to human serum albumin non-covalently attached to immobilized Cibacron Blue, or covalently immobilized on Sepharose, by column affinity chromatography.

A comparative study of the ligand-binding properties of human serum albumin was performed by the technique of affinity chromatography with the protein attached to immobilized Cibacron Blue F3GA (Blue Sepharose), or covalently immobilized on Sepharose. The binding strength of octanoate, decanoate and dodecanoate is much weaker when human serum albumin is attached to immobilized Cibacron Blue, indicating that the binding sites for fatty acids are involved in the attachment of human serum albumin to immobilized Cibacron Blue. The results revealed additional alterations of the ligand binding when human serum albumin was attached to immobilized Cibacron Blue, involving sites outside of the binding domains of fatty acids. Thus the stereoselective binding of L-tryptophan was abolished, and the resolution of the warfarin enantiomers was impaired. However, the binding strength of warfarin and salicylic acid was rather close to the values observed with human serum albumin covalently immobilized on Sepharose. It is suggested that the availability of the binding sites for L-tryptophan, warfarin and salicylic acid is partially blocked by the complex between albumin and the dye without direct participation in the complex-formation. An alternative interpretation involves an allosteric mechanism brought about by complex-formation between serum albumin and the immobilized Cibacron Blue.     

The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase

The DNA unwinding element binding protein (DUE-B) plays a key role in DNA replication. The DUE-B protein has been immobilized on a liquid chromatography support and the resulting immobilized protein column was used for the on-line screening of a series of steroids. The DUE-B protein was expressed with an added C-terminal sequence of six adjacent histidine residues, a His6-tag and immobilized on a chiral ligand exchange support, the CLC-L column, using Ni2+ as the coordinating metal ion. The chromatographic retentions of 12 steroids were determined on the DUE-B/CLC-L column. The magnitudes of the steroid-immobilized DUE-B interactions, reflected by the observed retention times, correlated to the effect of the steroids in the cell-free replication system, i.e. the longer the retention, the greater the increase in DNA replication. The coefficient of determination for the %DNA activities linear relation to retention time was 0.9694. The data suggest that the DUE-B/CLC-L phase can be used for on-line pharmacological studies. The results also indicated that His-tagged proteins can be directly immobilized on the CLC-L stationary phase and the resulting columns used as rapid screens for the isolation and identification of small molecule or protein ligands from complex biological or chemical mixtures.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Characterisation of the binding of digitoxin and acetyldigitoxin to human serum albumin by high-performance affinity chromatography

Zonal elution and high-performance affinity chromatography were used to examine interactions of the drugs digitoxin and acetyldigitoxin with the protein human serum albumin (HSA). This was done by injecting small amounts of digitoxin and acetyldigitoxin onto an immobilized HSA column in the presence of mobile phases that contained various concentrations of digitoxin, acetyldigitoxin or other solutes as competing agents. A fixed concentration of β-cyclodextrin was also present in the mobile phase as a solubilising agent. It was found that digitoxin and acetyldigitoxin each had strong interactions at a single common binding site on HSA, but with slightly different equilibrium constants for this region. Neither compound showed any competition with warfarin or L-tryptophan, which were used as probes for binding at the warfarin-azapropazone and indole-benzodiazepine sites of HSA. These results confirmed the presence of a separate binding region on HSA for digitoxin-related compounds.     

Optical resolution of some biologically active compounds by chiral liquid chromatography on BSA-silica (resolvosil) columns

Optical resolution on the analytical scale of a number of racemic pharmaceuticals and some other biologically active compounds has been studied using immobilized bovine serum albumin (BSA) as the stationary phase. For some of the compounds the elution order was determined by the use of optically enriched fractions obtained from a preceding passage of a sample through a preparative column containing microcrystalline triacetylcellulose (MCTA). The reversal in the sign of optical rotation shown in the polarimetric elution profile from the latter, combined with the integrated peak area ratio obtained on resolution on the analytical column, gave directly the order of elution. For one of the benzothiadiazines studied (bendroflumethiazide), increasing the pH of the mobile phase produced opposite effects on the retention of the two enantiomers, leading to a large effect on the separation factor. For many of the compounds studied, high separation factors (α > 2) could be achieved.     

Chiral resolution of histidine using an anti-D-histidine L-RNA aptamer microbore column

In this paper, we report a new anti-amino acid aptamer chiral stationary phase (CSP). The enantiomers of histidine were separated using an immobilized histidine-specific L-RNA aptamer (40-mer) and an aqueous buffer as mobile phase. The effects of the variation of different operating parameters, including the mobile phase pH and the MgCl2 concentration as well as the column temperature, on the solute retention were assessed. The results suggested that (i) the protonated form of histidine was involved in the stereospecific RNA binding and (ii) Mg2+ was essential for the target enantiomer binding to the specific aptamer sites. From a practical point of view, it appeared that the baseline resolution in a minimum analysis time can be achieved at a column temperature of 35 degrees C for an eluent containing 10 mM of MgCl2, pH 5.5.     

Estimation of the number of enantioselective sites of bovine serum albumin using frontal chromatography

On a column with bovine serum albumin (BSA) immobilized covalently to silica, the adsorption isotherms of the enantiomers of mandelic acid, tryptophan, 2-phenylbutyric acid, and N-benzoylalanine are measured using a buffered mobile phase. Knowing the amount of BSA immobilized on the column (36 mg), the ratio of the number of enantiomer molecules needed to saturate the enantioselective retention mechanism to the number of BSA molecules is determined. The mean of the set of eight enantiomers is 0.28. These data confirm that at most one enantioselective site exists for each BSA molecule for the kind of enantiomers studied. © 1993 Wiley-Liss, Inc.     

Liquid chromatographic retention behavior and enantiomeric separation of three chiral center beta-blocker drug (nadolol) using heptakis (6-azido-6-deoxy-2, 3-di-O-phenylcarbamolyted) beta-cyclodextrin bonded chiral stationary phase

Nadolol, a beta-blocker used in the management of hypertension and angina pectoris, has three chiral centers and is currently marketed as an equal mixture of its four stereoisomers. Enantiomeric separation of nadolol by high-performance liquid chromatography was studied on a column packed with novel heptakis (6-azido-6-deoxy-2, 3-di-O-phenylcarbamolyted) beta-cyclodextrin bonded chiral stationary phase. The retention behavior and resolution of nadolol enantiomers were investigated and discussed with respect to the mobile phase composition and flow rate, pH, ionic strength, and temperature. The optimal separation condition was found; the mobile phase contained 80% buffer solution (1% triethylamine acetate, pH 5.5) and 20% methanol with 0.3 ml/min mobile phase flow rate at a temperature of 20 degrees C. At the optimal conditions, resolution of three stereoisomers of nadolol was obtained with a complete separation of the most active enantiomer, (RSR)-nadolol. Thermodynamic properties including enthalpy and entropy change of binding to the CSP for the enantiomeric separation were also determined.     

Binding measurements of indolocarbazole derivatives to immobilised human serum albumin by high-performance liquid chromatography

The binding properties of six indolocarbazole derivatives have been measured using immobilised human serum albumin (HSA) in an HPLC column. The compounds showed very strong binding to HSA which necessitated the application of a 30 to 40% concentration of 2-propanol in the mobile phase. This represents a much higher concentration than is recommended by the column manufacturers. This HSA column had not changed its binding property when it was used again with 4% 2-propanol and 96% phosphate buffer. The binding parameters were estimated by extrapolation to 0% 2-propanol and were above 99% for each indolocarbazole derivative. The correlation analysis, including the calculated octanol/water partition coefficient (logP),pK_a values as well as measured reversed-phase retention data of the compounds, revealed that the extremely strong binding can be explained by the hydrophobic and acidic properties of the compounds.     

Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes,pH and ionic strength on drug–receptor interactions

Nicotinic acetylcholine receptor (nAChR) α3-subunits, β4-subunits, α3/β4-subunit combination and α4/β2-subunit combination were immobilized on chromatographic stationary phases and the binding affinities of the different nAChR subtypes were chromatographically evaluated. The observed relative binding affinities of epibatidine were α4/β2>α3/β4 and epibatidine did not bind at α3-subunits and β4-subunits. No significant difference in binding affinities was observed on the α4/β2 nAChRs immobilized in immobilized artificial membrane (IAM) particles and those sterically immobilized on Superdex 200 beads. The effects of mobile phase pH and ionic strength on the binding affinities of the α3/β4 nAChRs support were also investigated. The results are consistent with the proposed ligand–nAChR binding model in which a cationic center exists at the binding site.     

Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges

The extracellular polymeric substances (EPS) extracted from three granular and one flocculant anaerobic sludges were characterised by size exclusion chromatography (SEC) using two serially linked chromatographic columns in order to obtain more detailed chromatograms. A Superdex peptide 10/300 GL (0.1–7 kDa) and Superdex 20010/300GL (10–600 kDa) from Amersham Biosciences were used in series with a mobile phase at pH 7 with an ionic strength of 0.223 M (phosphate buffer 50 mM and NaCl 150 mM). A part of the EPS molecules displays hydrophobic and/or ionic interactions with the column packing. Interactions could be modified by changing the mobile phase ionic strength or polarity (addition of acetonitrile). The detection wavelength (210 or 280 nm) affects strongly the EPS chromatogram. For a sludge originating from the same type of biofilms (i.e., anaerobic granules), the differences in EPS fingerprints are mainly due to differences in the absorbance of the chromatographic peaks, linked to EPS molecules content and composition. The EPS fingerprint changes significantly when the EPS originate from another type of anaerobic sludges. In addition, EPS fingerprints were affected by the extraction method used (centrifugation only; heat and centrifugation or cationic exchange resin and centrifugation). This phenomenon was observed mainly for the largest and smallest molecules and molecules which display interactions with column packing.     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin—azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.