High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Measurement of urinary vanilmandelic acid and homovanillic acid by high-performance liquid chromatography with electrochemical detection following extraction by ion-exchange and ion-moderated partition

An improved protocol has been developed to isolate homovanillic acid (HVA) and vanilmandelic acid (VMA) from urine with strong anion-exchange resin. The sample is diluted with acetate buffer and passed through a disposable column. HVA, uric acid, and many hydrophobic organic acids are removed with 1.0 M acetic acid—ethanol, Then VMA is eluted with 0.5 M phosphoric acid. Two isocratic mobile phases allow rapid high-performance liquid chromatographic measurement of VMA (5 min) and HVA (8 mins) on a 5-μm ODS column. Selective conditions were developed with dual-electrode coulometric detection to permit specific measurement of VMA, HVA, and internal standards, with less than 5% between-run variation.     

High-performance liquid chromatographic analysis of a new β-lactam antibiotic, 6059-s (moxalactam)

Reversed-phase high-performance liquid chromatography was applied to the quantitative determination of a new β-lactam antibiotic, 6059-S, and its R- and S-epimers were resolved. The procedure was also applied to pharmaceuticals and human urine samples. Chromatographic separation was effected on a bonded hydrophobic stationary phase with two mobile phases: methanol-phosphate buffer for the resolution of the epimers and methanol-tetra-n-butylammonium phosphate for the quantitation of 6059-S. For the determination of 6059-S in human urine, the latter mobile phase was used successfully without interference by the other urine components. An in vivo experiment was conducted by administering intravenously 1 g of 6059-S to seven volunteers and analysing their urine by chromatographic and microbiological assays, and a comparison of the results gave a correlation coefficient of 0.9954. One-compartment model analysis of the time-course data revealed that 6059-S was excreted in urine intact with a rate constant of 0.433 h^-1.     

Solution nonideality related to solute molecular characteristics of amino acids.

By measuring the freezing-point depression for dilute, aqueous solutions of all water-soluble amino acids, we test the hypothesis that nonideality in aqueous solutions is due to solute-induced water structuring near hydrophobic surfaces and solute-induced water destructuring in the dipolar electric fields generated by the solute. Nonideality is expressed with a single solute/solvent interaction parameter I, calculated from experimental measure of delta T. A related parameter, I(n), gives a method of directly relating solute characteristics to solute-induced water structuring or destructuring. I(n)-values correlate directly with hydrophobic surface area and inversely with dipolar strength. By comparing the nonideality of amino acids with progressively larger hydrophobic side chains, structuring is shown to increase with hydrophobic surface area at a rate of one perturbed water molecule per 8.8 square angstroms, implying monolayer coverage. Destructuring is attributed to dielectric realignment as described by the Debye-Hückel theory, but with a constant separation of charges in the amino-carboxyl dipole. By using dimers and trimers of glycine and alanine, this destructuring is shown to increase with increasing dipole strength using increased separation of fixed dipolar charges. The capacity to predict nonideal solution behavior on the basis of amino acid characteristics will permit prediction of free energy of transfer to water, which may help predict the energetics of folding and unfolding of proteins based on the characteristics of constituent amino acids.     

Fimbriation ofEscherichia coli in urinary tract infections. Comparisons between bacteria in the urine and subcultured bacterial isolates

Midstream urine samples from 37 patients with urinary tract infections were studied by electron microscopy, hemagglutination, and the salt aggregation test (SAT) to measure the hydrophobicity of the bacterial surface.Escherichia coli subcultured from these urine samples were tested in the same way. Fimbriae were visualized onE. coli in the urine of 31 specimens, and all these urines containedE. coli that expressed pronounced surface hydrophobicity and aggregated in ammonium sulfate of 0.1–1.6 M final concentration. Hemagglutination of human and/or guinea pig erythrocytes was expressed by 21E. coli in the urine. TheE. coli strains subcultured from these 31 urine samples were also fimbriated, but the number of fimbriae per bacterium as well as the percentage of fimbriated bacteria varied compared with the directly collected strains. The surface hydrophobicity and hemagglutination were similar to the results with the directly collected bacteria. However, after serial transfer in CFA-broth under static conditions, all non-hemagglutinating strains expressed mannose-sensitive hemagglutination of guinea pig erythrocytes, and three strains also expressed weak mannose-resistant hemagglutination of human erythrocytes. Following serial transfer, fimbriae were also visualized on the sixE. coli strains that appeared non-fimbriate in the urine. It is thus concluded thatE. coli causing urinary tract infection are often fimbriated and express surface hydrophobicity in the urine. Based on these findings, a rapid method to isolate hydrophobic, possibly fimbriated bacteria was tried in which the urine was mixed with a hydrophobic gel. Hydrophobic bacteria bound to the gel and could be eluted from the sedimented gel.     

Relation between the damaging effect of surface-active compounds on Escherichia coli cells and the phase of culture growth

The techniques of cell electrophoresis and electro-orientation spectroscopy were used to study the effect of sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) on Escherichia coli K-12 cells from the culture at the exponential and stationary growth phases. SDS (2 x 10(-4) M) considerably damaged cells at the exponential phase, particularly at pH less than 6.0, whereas cells at the stationary phase were damaged to a less degree and only at pH less than 5.3 or after their treatment with Trilon B. The damaging effect of SDS decreased in an isotonic medium (0.25 M sucrose) as compared to a hypotonic medium (distilled water). CTAB also damaged cells at the exponential phase more than those at the stationary phase, and its damaging action decreased with pH. Mg2+, Ca2+, and Sr2+ cations diminished the degree of cell damage with CTAB, but did not exert any noticeable protection in the case of SDS. The different sensitivity of cells at the exponential and stationary growth phases may be associated with changes in their surface electric charge and with the existence of hydrophobic regions on the cell surface. The higher electric charge of cells at the stationary growth phase is presumed to stem from a rise in the amount of surface lipopolysaccharides which bear a negative electric charge.     

Polymeric nanoparticles with encapsulated superparamagnetic iron oxide and conjugated Cisplatin for potential bladder cancer therapy

Amphiphilic poly(ε-caprolactone)-b-poly(propargyl methacrylate-click-mercaptosuccinic acid-co-poly(ethylene glycol) methyl ether methacrylate) (PCL-b-P(PMA-click-MSA-co-PEGMA)) were synthesized by a combination of ring-opening polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization, and thiol-yne "click" reaction. The hydrophobic PCL core can be used to load superparamagnetic iron oxide nanoparticles (SPIONs), while the pendant dicarboxylic groups in the hydrophilic shell are used to coordinate cisplatin. These SPIONs-loaded, cisplatin-conjugated polymeric nanoparticles (Pt-Fe-PNs) are superparamagnetic at room temperature and are mucoadhesive. Release of cisplatin from Pt-Fe-PNs in artificial urine at 37 °C was characterized by an initial burst release with a release of ～30% of the cisplatin in the first 4 h followed by a slow sustained release over 4 days. The cisplatin release can be further enhanced by increasing the temperature. These Pt-Fe-PNs can effectively induce cytotoxicity against UMUC3 bladder cancer cells with IC(50) of 32.3 μM. These results indicate that Pt-Fe-PNs is potentially a promising cisplatin delivery vehicle which can be combined with SPIONs-induced hyperthermia for bladder cancer therapy.     

Evaluation of the stereoselective metabolism of the chiral analgesic drug etodolac by high-performance liquid chromatography

The enantiomers of the racemic analgesic drug etodolac have been resolved by fractional crystallization of the diastereomeric salts with optically active 1-phenylethylamine. A high-performance liquid chromatographic method to determine racemic etodolac (assay I) and its major metabolites (assay II) in urine using a conventional reversed-phase column is described. The determination of the enantiomeric ratios of etodolac and the two metabolites 7-hydroxyetodolac and 8-(1′-hydroxyethyl)etodolac was achieved using different protein-bonded chiral stationary phases. The urinary data for five volunteers are presented and show a marked stereoselectivity of the metabolism of etodolac in humans.     

Computational and QSAR study of the alkylnaphthyl ketones adsorption on silver-ion stationary phase

The chromatographic behaviour of α- and β- alkylnaphthyl ketones at different temperatures on the silver-loaded stationary phase is described based on the QSRR model. Complexation via an oxygen atom is favoured over the interaction through the aromatic fragment. The QSRR model and DFT/MP2 studies suggest that retention times of alkylnaphthyl ketones on silver-containing stationary phases are determined primarily by the dipole moment, length of the alkyl substituent and concentration of modifier in the mobile phase.     

Reverse phase liquid chromatography of beta-endorphins and related peptides

Anomalous band broadening of beta-endorphin related polypeptides chromatographed on hydrophobic high performance stationary phases can be attributed to ligand induced conformational changes associated with polypeptide folding. In the presence of anionic lipids the size exclusion chromatographic behaviour of members of the beta-endorphin family also exhibits similar behaviour. These structure-retention and band broadening behaviour of these polypeptides were in accord with predictions made by hydropathy algorithms and amphipathic helix representations. These observations on surface accessibility of key amino acid residues and their interaction as a conformationally induced domains with stationary phase ligands are equally relevant to other peptidic solutes and neurotransmitters.     

Immobilized receptor- and transporter-based liquid chromatographic phases for on-line pharmacological and biochemical studies: a mini-review

This review addresses the synthesis and characterization of two different types of receptor-based liquid chromatographic supports, one based upon a trans-membrane ligand gated ion channel receptor (the nicotinic acetylcholine receptor) and the other a soluble nuclear receptor (the estrogen receptor). In addition, studies with the P-glycoprotein transporter are also reported. The nicotinic receptor was immobilized via hydrophobic insertion into the interstitial spaces of an immobilized artificial membrane (IAM) stationary phase. the estrogen receptor was tethered to a hydrophilic stationary phase and the membranes containing the Pgp transporter were coated on the surface of the IAM stationary phase. The stationary phases were characterized using known ligands and substrates for the respective non-immobilized proteins. The results from zonal and frontal chromatographic experiments demonstrated that the stationary phases could be used to determine binding affinities (expressed as dissociation constants, Kd,'s) and to resolve mixtures of ligands according to their relative affinities. In addition. competitive ligand binding studies on the P-glycoprotein-based stationary phase have established that this phase can be used to identify and characterize competitive displacement and allosteric interactions. These studies demonstrate that immobilized-receptor phases can be used for on-line pharmacological studies and as rapid screens for the isolation and identification of lead drug candidates from complex biological or chemical mixtures.     

Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols

Problems inherent in the isolation of thiols from natural sources, such as oxidation, undesirable addition reactions, and low concentration of thiol species in cell-free extracts, can be circumvented by reversible derivatization to a less labile form which can be concentrated selectively. These objectives are realized by converting thiols to heterodisulfides in which the thiol partner is an apolar thiol with strong affinity for hydrophobic stationary phases. When reacted with 2-S-(2(')-thiopyridyl)-6-hydroxynaphthyldisulfide at pH<5, where most thiol species are relatively stable to atmospheric oxidation, mixed disulfides with 2-mercapto-6-hydroxynaphthalene as the apolar partner are obtained in good yield and can be concentrated onto a hydrophobic stationary phase. Such heterodisulfides exhibit excellent chromatographic properties when separated on reversed-phase media and the derivatization reaction can, therefore, be conveniently monitored. Following their isolation as the heterodisulfides the thiol species of interest are recovered by reduction and facile separation from the apolar 2-mercapto-6-hydroxynaphthalene partner.     

Screening and determination of β-blockers, narcotic analgesics and stimulants in urine by high-performance liquid chromatography with column switching

A fast method is described for the screening of eleven β-blockers, two narcotic analgesics and two stimulants in urine by HPLC with column switching. The urine sample (100 μl), buffered tto pH 9–9.5, is injected onto a short extraction column packed with CN stationary phase. The extraction is flushed with water for 2.5 min to elute polar matrix components to waste. The retained components are then backflushed by means of a six-port valve onto the ODS analytical column where they are separated. Phosphate buffer pH 3.0 and acetonitrile were used as mobile phase. Gradient elution was applied in the screening method to improve separation. Detection was performed with diode-array detector at 220, 235 and 300 nm. Recoveries were near 100%, precision was excellent and sensitivity about 0.25 μg/1. The speed up the quantitative analysis, the same method but with isocratic elution was successfully applied to the determination of acebutolol and metoprolol in urine samples collected 4 h after administration of the compounds as single doses.     

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of “electrical anisotropy” of retinal chromophore in the membranes. At about pH 7, there was no difference in the “electric anisotropy” between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the “electric anisotropy” are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of ~0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of ~0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 ±2 debye/bR.     

Localized, Positive Charge Mediates Adhesion of Rhodosporidium toruloides to Barley Leaves and Polystyrene

The physicochemical forces that mediate attachment of yeasts to the phylloplane are unknown. Cell surface charge and hydrophobicity and adhesion to polystyrene, glass, and barley were assessed for wild-type Rhodosporidium toruloides and attachment-minus (Att⁻) mutants. Cells were grown under conditions promoting (excess carbon) or not promoting (excess nitrogen) capsule production. Hydrophobicity was measured by adhesion to xylenes, and surface charge characteristics were assessed by attachment to either DEAE (positive)- or carboxymethyl (CM) (negative)-Sephadex ion-exchange beads. Hydrophobicity and adhesiveness of nonencapsulated, wild-type R. toruloides decreased from mid-log to late stationary phase. Encapsulated wild-type R. toruloides cells were more hydrophobic and more adhesive than nonencapsulated cells. However, two encapsulated Att⁻ mutants were more hydrophobic than the wild type and levels of adhesion of R. toruloides were similar on polystyrene and less hydrophobic glass surfaces. Adhesion of wild-type yeast to barley and polystyrene was correlated with attachment to CM-Sephadex beads, indicating a positive cell surface charge. Sixteen Att⁻ mutants did not exhibit a positive cell surface charge, and wild-type yeast cells that did not attach to CM-Sephadex did not adhere to either polystyrene or barley. Wild-type R. toruloides attached to CM-Sephadex beads by the poles of the cells, indicating a localization of positive charge which was also visualized with India ink. We conclude that localized, positive charge, and not hydrophobic interactions, mediates attachment of R. toruloides to barley leaves.     

Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles.

Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics.     

Binding of substituted phenol and aniline derivatives to the corn protein zein studied by high-performance liquid chromatography

The interaction of 12 substituted phenol, three aminophenol and four substituted aniline derivatives with the corn protein zein was studied on zein-coated silica and alumina stationary phases by high-performance liquid chromatography using bidistilled water as mobile phase. Solutes were eluted from the zein-coated supports with different retention times indicating that they bind to the protein with different forces. They were more strongly retained on silica-based than on alumina-based support proving that the original adsorptive character of the support remains even after impregnation. The retention of solutes on both zein-coated stationary phases significantly depended on the steric and electronic parameters of solutes and was independent of the calculated and measured lipophilicity parameters, indicating that hydrophobic forces are not included in the interaction of zein with these class of solutes. It has been concluded that the interaction is governed by steric and electrostatic forces.     

Internal electrostatic potentials in bilayers: measuring and controlling dipole potentials in lipid vesicles.

The binding and translocation rates of hydrophobic cation and anion spin labels were measured in unilamellar vesicle systems formed from phosphatidylcholine. As a result of the membrane dipole potential, the binding and translocation rates for oppositely charged hydrophobic ions are dramatically different. These differences were analyzed using a simple electrostatic model and are consistent with the presence of a dipole potential of approximately 280 mV in phosphatidylcholine. Phloretin, a molecule that reduces the magnitude of the dipole potential, increases the translocation rate of hydrophobic cations, while decreasing the rate for anions. In addition, phloretin decreases the free energy of binding of the cation, while increasing the free energy of binding for the anion. The incorporation of 6-ketocholestanol also produces differential changes in the binding and translocation rates of hydrophobic ions, but in an opposite direction to those produced by phloretin. This is consistent with the view that 6-ketocholestanol increases the magnitude of the membrane dipole potential. A quantitative analysis of the binding and translocation rate changes produced by ketocholestanol and phloretin is well accounted for by a point dipole model that includes a dipole layer due to phloretin or 6-ketocholestanol in the membrane-solution interface. This approach allows dipole potentials to be estimated in membrane vesicle systems and permits predictable, quantitative changes in the magnitude of the internal electrostatic field in membranes. Using phloretin and 6-ketocholestanol, the dipole potential can be altered by over 200 mV in phosphatidylcholine vesicles.