Enhanced albumin protein separations and protein-drug binding constant measurements using anti-inflammatory drugs as run buffer additives in affinity capillary electrophoresis.

An affinity capillary electrophoresis (ACE) method for improving albumin protein separations has been developed. Separation efficiencies for bovine serum albumin (BSA) and human serum albumin (HSA) are dramatically improved by using anti-inflammatory compounds as run buffer additives. The anti-inflammatory drugs used as biospecific ligands to improve the protein separation include ibuprofen (IB), flurbiprofen (FL), and ketoprofen (KE). The binding constants of proteins (BSA and HSA) for the anti-inflammatory ligands (FL and IB) are estimated by ACE and compared to literature values.

Enhanced chiral separation of dansylated amino acids with cyclodextrin-dextran polymer network by capillary electrophoresis.

A method for the chiral separation of dansyl-DL-amino acid mixtures using a dextran (M(r) 2,000,000) polymer network containing beta-cyclodextrin (beta-CD) in HPCE was developed. Mixtures of up to seven amino acids could be baseline resolved by this method under neutral pH conditions. Resolution was dependent on the dextran concentration in the polymer network. Temperature effects on the chiral separation were studied. Optimal efficiency and resolution of DL-enantiomeric pairs of amino acid samples were obtained at 25 degrees C. The resolution of different amino acids used in this work decreased with an increase in temperature.

Quantitative relationships between the structure of beta-adrenolytic and antihistamine drugs and their retention on an alpha 1-acid glycoprotein HPLC column.

Chromatographic retention parameters of a series of 7 beta-adrenolytics and of 12 antihistamine drugs were determined employing an alpha 1-acid glycoprotein (AGP) high-performance liquid chromatographic (HPLC) column. For the group of antihistamines capillary electrophoretic (CE) retention was additionally measured in the presence of either AGP or human serum albumin (HSA). Two series of solutes hydrophobicity parameters were obtained by reversed-phase HPLC on an immobilized artificial membrane (IAM) column. The solutes studied were subjected to molecular modelling and the structural descriptors obtained were applied in studies of quantitative structure-retention (protein binding) relationships (QSRR). It was found that retention on AGP correlates well with the literature on physiological protein binding data. This retention was demonstrated to depend on hydrophobicity: to a lesser extent in the case of beta-adrenolytics and strongly in the case of antihistamines. Hydrophobicity, along with molecular width and electron excess charge on aliphatic nitrogen was demonstrated to describe retention of antihistamines on AGP. The AGP column is recommended as a convenient reactor for studies of drug-protein interactions. Preliminary CE data do not correlate with the HPLC data.

Chiral separation of leucovorin with bovine serum albumin using affinity capillary electrophoresis.

A method for the determination of the (6R)- and (6S)-stereoisomers of leucovorin using electrokinetic chromatography (EKC) in the affinity mode has been developed. Bovine serum albumin (BSA) is used as a run buffer additive to incorporate enantiomeric selectivity into the system. Protein-wall interactions are minimized by using a poly(ethylene glycol) (PEG) coated capillary. Chiral resolution is obtained in 12.5 min with efficiencies greater than 200,000 theoretical plates using BSA as an additive, while no resolution is obtained in the absence of BSA. A general equation is derived to calculate the free energy of interaction between the leucovorin isomers and the BSA molecule. This method represents a new means of obtaining thermodynamic data for substrate binding interactions and for the general study of drug cross-reactions and interactions of drugs with serum and other proteins.

Separation of serum bilirubin species by micellar electrokinetic chromatography with direct sample injection.

Four major bilirubin species in serum were separated by micellar electrokinetic chromatography with 25 mM sodium dodecyl sulfate (SDS) and 20 mM sodium tetraborate-boric acid buffer at pH 8.5. Due to the solubilization of the serum proteins by the SDS micelles, serum samples were injected directly into a 50 cm x 75 microns I.D. fused-silica capillary and complete separation of the four bilirubin species was accomplished within ca. 10 min without extensive sample pretreatment. Detection was performed by absorbance at 450 nm and average limit of detection was in the 6.0 microM concentration range. The usefulness of this method was demonstrated for the separation and detection of a number of bilirubin species present in pathological human serum samples.

On-line high-performance liquid chromatography-post-column reaction-capillary gas chromatography analysis of lipids in biological samples.

The versatility of on-line liquid chromatography and gas chromatography is further expanded by the addition of on-line derivatization. The on-line fractionation, derivatization and separation system is applied to the characterization of lipids in biological samples. Separation of the triglycerides from the phospholipids was accomplished on a narrowbore (2.0 mm I.D.) 5 microns silica column. The entire triglyceride fraction was transferred to a heated fixed bed reactor for esterification of the fatty acid constituents. Transfer of the derivatized zones to the gas chromatograph was accomplished by the use of a retention gap. Application of the system to the separation and characterization of Staphylococcus aureus is presented.

High-performance liquid chromatography analysis of the thiobarbituric acid adducts of malonaldehyde and trans,trans-muconaldehyde.

A reversed-phase HPLC method is described for the separation and analysis of the thiobarbituric acid (TBA) adducts of the reactive aldehydes muconaldehyde (MUC) and malonaldehyde (MDA). The TBA adduct of malonaldehyde was synthesized, purified, and its structure elucidated, for use as standard in quantitative HPLC studies. A detection limit of 1 X 10(-14) mol was achieved for the MUC:TBA and MDA:TBA adducts using the double monochromator fluorometric detector, 7 X 10(-13) mol was the detection limit using a variable wavelength uv-visible detector. Direct on-line identification of the eluting aldehyde:TBA adducts was achieved by the use of a diode-array uv-visible detector. The chromatographic behavior of the adducts under different mobile phase conditions was also examined. This HPLC methodology was used for the identification of muconaldehyde as a product of benzene oxidation in a hydroxyl radical generating system.

The synthesis and characterization of chemically bonded silica-based zwitterion-exchangers for HPLC.

The synthesis and characterization of a zwitterionic stationary phase bonded onto microparticulate silica is described. The bonded zwitterionic phase was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and quantitative analysis of the ligands by high performance liquid chromatography (HPLC) following chemical cleavage from the silica backbone. Chromatographic evaluation of this novel bonded phase indicates that it functions as a weak cation exchanger at pH values above 4.5, an anion exchanger at pH values below 7, and as a zwitterionic phase between these two values. The simultaneous separation of a mixture of cationic, anionic and zwitterionic solutes with this novel bonded phase is shown. Using nucleotides as model compounds, a correlation was developed between maximum solute retention and the pH values corresponding to maximum solute/stationary phase zwitterion overlap. The possibility for a quadrupolar retention mechanism of the bonded zwitterionic phase for zwitterionic solutes is explored.

Mixed-mode hydrophobic ion exchange for the separation of oligonucleotides and DNA fragments using HPLC.

Hydrophobic anion exchangers were formed by cobonding both ionic and hydrophobic ligands to silica gel. These phases were used to separate single-stranded oligonucleotides and double-stranded DNA restriction fragments. By varying the ratio of n-octyldimethylsilane and either 3-chloropropyldimethylsilane or 4-chlorobutyldimethylsilane added during silanization a series of mixed-ligand or mixed-mode stationary phases was created. Concentration and ratio of bonded ligands were determined using a new gas chromatography fluorination method. Total ligand coverage was found to approach 2.1 ligands nm-2 for n-octyldimethylsilane. Bonding reproducibility for mixed-mode phases was good. Nucleic acid separations were achieved under gentle mobile phase conditions by using the stationary phase as an easily modifiable variable.

Quantitative retention relationships as a function of mobile and C18 stationary phase composition for non-cogeneric solutes.

Twelve non-homologous solutes with a wide range of functional groups were chromatographed on a series of well-characterized stationary phases with a range of methanol-water mobile phases. Retention correlations were found for quantum mechanically calculated molecular parameters. These parameters, the total molecular energy, and the maximal excess electronic charge difference, were found to be better descriptors of retention than the dipole moment or fragmental constants. The implication of these results in terms of retention mechanisms on reversed-phase materials are discussed.

Current state of the art in the HPLC analyses of free nucleotides, nucleosides, and bases in biological fluids.

Because of the use of HPLC for the determination of free nucleotides, nucleosides, and bases in samples of biological origin, investigations that were very difficult or impossible to perform only a decade ago are now possible. Microparticulate, totally porous, chhemically bonded particles appear to be the column packings that will be used routinely in the future because analyses are achieved with high sensitivity, selectivity, efficiency, and speed. These packings are stable, the results are reproducible, and the data are quantitative. In addition, the reversed-phase mode of HPLC offers improvements over the ion-exchange mode for the analysis of nucleosides, bases, and other UV-absorbing compounds which are not ionic. However, the ion-exchange mode may be used as a complementary method for very polar or ionic compounds which are eluted too rapidly with reversed phase. A sample can be chromatographed with each mode and a profile--a complete picture of nucleotide, nucleoside, and base levels in the sample--obtained. Alternatively, ion pairing can be used with the reversed-phase mode; thus both the ionic and lipophilic compounds can be determined in one separation.

Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography. I. Chromatographic methodology.

A comprehensive investigation of the high-performance liquid chromatographic separation of nucleosides, their bases and other low-molecular-weight UV-absorbing compounds that might be found in serum is reported. A buffer-methanol gradient was used in conjunction with chemically bonded, microparticulate columns to separate many of the biologically important compounds under study in minimal time with maximal resolution. Retention data, absorbance ratios (280/254 nm) and fluorescence responses are reported for 86 nucleosides, bases, nucleotides and other UV-absorbing compounds commonly encountered in biological studies.

Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography. II. Evaluation of human sera.

The reversed-phase mode of high-performance liquid chromatography was used to investigate the profiles of low-molecular-weight, UV-absorbing compounds in human serum. Identification techniques are described which allow for the identification of picomole amounts of the nucleosides, bases and other compounds in several microliters of serum ultrafiltrate. The sera from 31 normal subjects (17 males, 14 females) showed very consistent profiles. A total of 12 compounds were identified and quantified in normal serum. The analysis of sera from over 150 patients with various types of neoplasia and other diseases showed serum profiles significantly different from normal profile.

High performance liquid chromatographic determination of serum UV profiles of normal subjects and patients with breast cancer and benign fibrocystic changes.

High performance liquid chromatography (HPLC) was used to determine the UV profiles of serum samples taken postoperatively from 22 patients with histologically documented breast cancer, 8 patients with benign breast fibrocystic changes and 10 normal subjects. The analyses were performed on coded serum samples and after they were completed, the code was broken and the results correlated with the clinical data. Only one ml of serum was required for the HPLC analysis and identification. Detection limits for the nucleosides and bases were in the 10--20 pmol range and the injection volume of the deproteinated serum was 75 mul. The UV profiles of the normal subjects were very reproducible and similar to those of the patients with benign fibrocystic changes. The profiles of some of the cancer patients were distinctly different from the two other groups, 1-methylinosine and N2-methylguanosine, which were not detected in sera from normal subjects and patients with benign fibrocystic changes, were found in 45.5% and 22.7% of the cancer patients, respectively. Patients with the metastatic disease also showed elevated levels of guanosine and uridine. Only one false positive was found in the normal population. At present, it is not clear whether this indicates a subclinical manifestation of the disease and it must await further follow-up.

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum.

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5'-monophosphoric acid (AMP) in a buffer of required pH, 5'-nucleotidase was inhibited with Ni2+ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultraviolet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.

Reversed-phase liquid chromatographic separation of 3',5'-cyclic ribonucleotides.

A rapid, reversed-phase "high-performance" liquid-chromatographic separation of the five naturally occurring cyclic ribonucleotides is described. The separation, optimized for the measurement of these compounds in biological samples, is short (25 min), sensitive (50-100 pmol), and requires no sample pre-concentration steps. We also report an alternative isocratic elution mode, optimized for a rapid and selective analysis for adenosine 3',5'-cyclic phosphate. The identity of chromatographic peaks in biological extracts is confirmed by several methods: retention times, co-chromatography with the reference compounds, absorbance ratios, enzymatic peak-shift with cyclic nucleotide phosphodiesterase, and stopped-flow ultraviolet-scanning techniques.