Reversed-phase HPLC Retention Research Articles

Inclusion Reaction of Some Fluorophores with β-Cyclodextrin and Its Effect on Their Reversed-Phase HPLC Retention

The thermodynamics of the inclusion reaction of flavonol, 7-methoxyflavonol and 4-phenyl-7-hydroxycoumarin (L) with /8-cyclodextrin (CD) has been studied fluorometrically with special reference to the effect of organic solvents (S). The equilibrium constants for this reaction were determined in aqueous methanol, ethanol, acetonitrile, acetone, dimethyl sulfoxide, and A’.A-dimethylformamide. The dependence of the constants on the content of the respective S was attributable to the substitution of S included in CD with L as CD ·nS+L ⇌ CD · L+nS. Also, the inclusion constants of S with CD, βn=[CD · nS][CD]-1[S]-n, were found to be linearly correlated with the solubility parameter of S for both n=1 and 2. When aqueous methanol was employed as a mobile phase, the retention behavior of coumarins on a reversed-phase HPLC column was studied in the presence of j8-CD as a mobile-phase component. The change in the capacity factors could be reasonably explained by the same inclusion scheme as that mentioned above.

Monitoring of α-helical secondary structures in peptides by reversed-phase HPLC of replacement sets

HPLC studies of systematic double d-amino acid, methionine and methionine sulfoxide replacement sets were applied towards the structural characterization of α-helical peptides. Double d-amino acid replacements disturb the local structure of the helical binding domain, leading to significant reversed-phase HPLC retention time decreases. On the other hand, methionine and methionine sulfoxide replacements locally alter the hydrophobicity along helical peptides, and cause position-dependent retention time differences between methionine and methionine sulfoxide analogs with a 3–4 repeat pattern in amphipathic α-helices. The two approaches yield complementary information on α-helical secondary structure and the hydrophobic interaction domain of peptides with the stationary phase. These HPLC-based methods were successfully applied to determine the potential of α-helix formation in neuropeptide Y and corticotropin releasing hormone. The results clearly demonstrate that hydrophobically induced α-helical structures can be accurately characterized in biologically active peptides by this approach.

Relationship Study Between Reversed-Phase HPLC Retention Data and the PKa Values in a Series of 2-Amino-2-Oxazolines

In reversed-phase liquid chromatography, the retention behaviour of ionogenic compounds is strongly related to the pH mobile phase. An application is presented for three basic 5-substituted 2-amino-2-oxazolines. The pH dependence of their capacity factor values, expressed as log k'w, was investigated in a RP-HPLC procedure using a column packed with poly methacrylate. The plot of capacity factor vs pH was described by a sigmoidal curve, leading us to approach the ionization constants of the molecules. Finally, the pKa values have been discussed in regard to chromatographic parameters and to experimental conditions.

Interactions of renal cytochrome P450 (CYP), angiotensin (AII) and tumor necrosis factor-alpha (TNF): implications for ion transport.

In cells from the rabbit mTALH, arachidonic acid (AA) is metabolized primarily by the CYP pathway1, 2. Rabbit mTALH cells, when incubated with 14C-AA for 30 min, form products that segregate into two peaks, designated P1 and P2 based on their reverse phase HPLC retention times. Structural analysis by gas chromatography/mass spectrometry (GC/MS) disclosed three AA products generated by the CYP pathway in the mTALH: 20-hydroxyeicosatetraenoic acid (20-HETE), 20-eicosatetraenedioic acid (20-COOH-AA), and 19-HETE2. P2, the more polar peak, was comprised of 20-COOH-AA, an inhibitor of Na+-K+-ATPase, that was devoid of vasoactivity. P1 contained vasoactive material that was also capable of affecting Na+-K+-ATPase and yielded two products, 20-HETE and 19-HETE (Fig. 1).

Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: characterization of post-translational modifications and zinc binding

MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12972 +/- 2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys79 and Cys90, and 1-methylation of His106; the calculated mass was then 12971.8 Da. Methylation of His106 was further characterized after incubation of spleen cells with L-[methyl-3H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His106, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more 85Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100 beta. Ca2+ decreased Zn2+ binding in S100 beta but it did not influence binding to MRP14, suggesting that the Zn2+ binding site was distinct from and independent of the two Ca2+ binding domains.

Improved Method for Converting an Unmodified Peptide to an Energy-Transfer Substrate for a Proteinase

This paper describes refinements to a method for generating energy-transfer-based fluorogenic substrates for proteinases [Geoghegan, K. F., et al. (1993) Bioconjugate Chem. 4, 537-544]. An unprotected peptide is taken as starting material and coupled by three steps of chemistry to two dyes that form an energy donor-energy acceptor pair. The donor group is Lucifer Yellow CH (lambda cx 420-430 nm, lambda em 530 nm), and the acceptor is 5-carboxytetramethylrhodamine, a strong chromophore that effectively quenches Lucifer Yellow fluorescence in intact substrates. Periodate oxidation of N-terminal Ser gives the peptide an alpha-N-glyoxylyl moiety, OHC-CO-peptide, which is allowed to react with Lucifer Yellow CH (a carbohydrazide derivative) to attach the dye through an adequately stable hydrazone bond. Derivatization is completed by allowing the succinimidyl ester of 5-carboxytetramethylrhodamine to react with the epsilon-amino group of a single Lys placed near the C terminus. In the revised method, the first two steps of chemistry are completed sequentially in a single vessel to eliminate the labor and losses associated with isolating the alpha-N-glyoxylyl intermediate. In addition, peptides taken as starting material for this method are now designed according to the scheme (Ser)-(cleavable sequence)-(Lys-Arg). The addition of Arg at the C terminus promotes aqueous solubility of the final substrate without complicating the chemistry; multiple Arg might also be used, through this has not been tried here. It has also been found that relative reversed-phase HPLC retention of Lucifer Yellow/5-carboxytetramethylrhodamine substrates is predictable on the basis of the hydrophobicity of the original peptide. Substrates prepared from peptides with Bull and Breese indices of > 200 cal/ mol are readily separated from residual dye in the second and final chromatographic step of the synthesis, simplifying the only moderately difficult step in the preparation. As with all fluorogenic substrates, assays using these substrates are subject to internal filtering effects that can lead to serious error. This problem becomes significant at a range of substrate concentration that is predictable when the size and geometry of the fluorescence cell are taken into account. The reasoning applied in determining this range is applicable to substrates constructed with any donor-acceptor pair.

Reversed-Phase HPLC Retention of Deoxyribonucleosides as a Function of Mobile Phase Composition

Abstract Comparison of approaches describing retention of deoxynucleosides (dCyd, dUrd, dGuo, dThd, and dAdo) as a function of the mobile phase composition has been performed. The volume fraction (F) of organic modifier was changed from 0.05 to 0.30, and to 0.12 for methanol and acetonitrile, respectively. Linear regression has been carried out for the following equations: log k'=log k'w—S X F (1), log k'=H + Klog(1/F) (2), k'=A + B(1/F) (3), where k' is capacity factor, and log k'w, S, H, K, A and B are experimental coefficients. The best fits were obtained for the eqns. (1) and (3), and the poorest correlations were obtained for eqn. (2). The slope ratios Bmethanol/Bacetonitrile and Sacetonitrile/Smethanol are changed in comparatively narrow ranges with average values of 2.86 and 2.30, respectively. Unlike the slope S of eqn. (1), the slope B of eqn. (3) can characterize the properties of solute and organic modifier simultaneously. For each solute, the intercepts A calculated for acetonitrile and methan...

Direct synthesis and characterization of site-specific adenosyl adducts derived from the binding of a 3,4-dihydroxy-1,2-epoxybenzo[c]phenanthrene stereoisomer to an 11-mer oligodeoxyribonucleotide.

Site-specifically modified oligonucleotides were obtained in milligram quantities by reacting racemic 3t,4r-dihydroxy-1,2t-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE-2, or anti-B[c]PhDE) with the single deoxyadenosine (dA) residue in the oligodeoxynucleotide d(CTCTCACTTCC). Enzyme digestion of the covalently modified oligonucleotides with the exonuclease spleen phosphodiesterase yielded covalently linked B[ca]PhDE-N6-deoxyadenosyl monophosphate (dAMP) adducts. Comparisons of the reverse phase HPLC retention times and CD spectra of these B[c]PhDE-3'-dAMP mononucleotide adducts, with those of standards derived from the reaction of the enantiomers (+)- and (-)-anti-B[c]PhDE with 3'-dAMP, show that two major oligonucleotide adducts (I and II) were obtained upon reacting racemic anti-B[c]PhDE with d(CTCTCACTTCC). In oligonucleotide adduct I, the lesion is a (+)-trans-anti-B[c]PhDE-N6-dA residue, and in oligonucleotide adduct II it is a (-)-trans-anti-B[c]PhDE-N6-dA residue. These assignments were further confirmed using a standard 32P postlabeling assay of B[c]PhDE-3'-dAMP mononucleotide adducts obtained from the digestion of oligonucleotides I and II by spleen phosphodiesterase. The melting points (Tm) of duplexes of modified oligonucleotides I and II and their natural complementary strands are not affected significantly by the presence of the covalently bound benzo[c]phenanthrenyl residues. Opposite stereoselective resistance to enzyme digestion by the exonucleases snake venom phosphodiesterase and spleen phosphodiesterase is exhibited by the stereoisomeric (+)-trans- and (-)-trans-anti-B[c]PhDE-modified oligonucleotide adducts I and II; these results are consistent with the intercalative insertion of the benzo[c]phenanthrenyl residues on the 5'-side of the modified dA residue in adduct I, and its insertion on the 3'-side of the dA residue in adduct II, as observed in the duplexes by high resolution NMR techniques [Cosman et al. (1993) Biochemistry 32, 12488-12497, and Cosman et all, Biochemistry, in press.

Location of an amphipathic alpha-helix in peptides using reversed-phase HPLC retention behavior of D-amino acid analogs.

The reversed-phase HPLC retention behavior of D-amino acid replacement sets of an amphipathic model peptide, neuropeptide Y, and corticotropin releasing factor has been studied. The results demonstrate that D-amino acid substitutions destabilized the amphipathic alpha-helix, leading to a decrease of fractional helicity as determined by circular dichroism. The effect is enhanced by substitution of two adjacent D-amino acids and correlates well with a decrease of hydrophobic interaction during reversed-phase HPLC, caused by disturbance of the preferred binding domain of the stationary phase-bound peptide. In contrast, D-amino acid substitutions in nonamphipathic or disordered regions of peptides do not influence the retention time to the same extent. Thus, the "retention profile" that results from plotting the retention time vs the position of the double D-amino acid replacements provides an indication of the presence and location of an amphipathic alpha-helical secondary structure in peptides.

Reversed-Phase HPLC Selectivity and Retention Characteristics of Conformationally Different Bonded Alkyl Stationary Phases

Reversed-Phase HPLC Selectivity and Retention Characteristics of Conformationally Different Bonded Alkyl Stationary Phases

Quantitative structure chromatography relationships in reversed-phase high performance liquid chromatography: Prediction of retention behaviour using theoretically derived molecular properties

The use of theoretically calculated molecular properties as predictors for retention in reversed-phase HPLC has been explored. HPLC retention times have been measured for a series of 47 substituted aromatic molecules in three solvent mixtures and steric and electronic properties of these compounds have been derived using semi-empirical molecular orbital and empirical theoretical methods. A subset of the experimental data (a training set) was used to derive property-retention time relationships and the remaining data were then used to test the predictive capability of the methods. Good retention time prediction was possible using derived regression equations for individual solvents and after including solvent parameters it was possible to predict retention for all solvents using a single equation. This method showed that the most useful properties were calculated log P and the calculated dipole moment of the solutes, and the calculated solvent polarisability. In addition, 90% of the data were used to train an artificial neural network and the remaining 10% of the data used to test the network; excellent prediction was obtained, the neural network approach being as successful as the regression analysis.

A comparative study of the reversed-phase HPLC retention behaviour of S-adenosyl- l-methionine and its related metabolites on Hypersil ODS and Supelcosil™ LC-ABZ stationary phases

S-Adenosyl- l-methionine (SAM) and its metabolites S-adenosyl- l-homocysteine (SAH) and methylthioadenosine (MTA) are endogenous compounds that are heavily involved in a variety of biochemical processes, and have therefore been the target for several assays in body fluids and tissues. Reversed-phase chromatographic behaviour of SAM and its metabolites has been studied by using Supelcosil™ LC-ABZ column, specially designed for analysis of acidic, basic, zwitterionic and neutral compounds, and on a Hypersil ODS column as a function of mobile phase pH. The retentions of the compounds, expressed by the capacity ratio ( k′), are measured on both column with mobile phases comprised of 10% acetonitrile and 10 mM ammonium formate buffer with pH values ranging from 2 to 9. Higher selectivity is observed on Supelcosil LC-ABZ within pH range 4–6. Different retention properties are observed at very low pH and seemed as if the Supelcosil LC-ABZ column reduced the effect of the mobile phase pH by about 1 pH unit. Whilst the Supelcosil column can be recommended for the routine analysis of SAM and its related metabolises in biological fluids by using mobile phase pH 5, the Hypersil ODS column may be suggested for use with mobile phase pH values of 3–4.

Effects of hypophysectomy on galaninergic neurons in the rat hypothalamus.

To understand better the relationship between hypothalamic galaninergic neurons and the pituitary gland, we studied the effects of hypophysectomy on hypothalamic galanin (GAL) content and distribution by radioimmunoassay and immunohistochemistry, and on GAL mRNA by Northern blot analysis. Three weeks after hypophysectomy, performed at 5 or 8 weeks of age, the hypothalamic concentrations of GAL and GAL mRNA were reduced by 30-50% in both male and female rats, compared to age- and sex-matched controls. Similar reverse-phase HPLC retention times of hypothalamic GAL were observed in intact and hypophysectomized rats. The reduction of hypothalamic GAL concentration following hypophysectomy was time-dependent, as peptide levels were unaffected one week after surgery. Immunohistochemistry showed regional differences in the effect of hypophysectomy on galaninergic neurons. In the hypophysiotropic hypothalamus, the scarce GAL immunoreactivity normally observed in the arcuate nuclei was no longer detectable in hypophysectomized rats, and the intense GAL immunoreactivity of the external zone of the median eminence progressively decreased and completely disappeared 3 and 6 weeks after hypophysectomy. In contrast, in the neurohypophyseal system, there was an increase of GAL labelling of the perikarya and emerging axons in the supraoptic and lateral-paraventricular nuclei, 1 and 3 weeks after hypophysectomy, that disappeared 6 weeks after hypophysectomy. An increase of GAL immunoreactivity was also observed in the internal zone of the median eminence 1 week but not 3 weeks after hypophysectomy. We conclude that hypophysectomy reduces the content of GAL and GAL mRNA in the rat hypothalamus. These changes are time-dependent and clearly detected after 3 weeks. The neurohypophyseal and hypophysiotropic galaninergic systems respond differently to hypophysectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Physico-chemical modelling of solute retention in reversed-phase HPLC with methanol-phosphate buffer mobile phase

In this paper the new retention model primarily established for RP-HPLC systems employing methanol-water mobile phases has been adapted to the description of solute retention in analogous systems using methanol-phosphate buffer mixtures. The basic physical relationship of this approach was very carefully tested upon experimental material, and excellent agreement found between theory and practice of the phenomenon considered. This relationship can be utilized in the future for the optimization of solute retention in RP-HPLC systems with methanol-phosphate buffer mobile phases.

Linear discriminant and multiple regression analyses of anticoccidial triazines

Quantitative structure-activity relationships among some anticoccidial 2-(substituted-phenyl)-1,2,4-triazine-3,5-(2H,4H)-diones were studied by multiple regression analysis (MRA, the Hansch approach) and by linear discriminant analysis (LDA). With MRA the potencies of these compounds are correlated with their reverse-phase HPLC retention times and their 1H NMR chemical shifts at the 6-position. While the coefficients of the variable terms are significant, the moderate R2 (0.56) of the correlating equation suggests that predictions made from this analysis are not likely to be accurate. LDA supports the idea that these descriptors are related to potency, but the discriminant function does not lead to good classification. However, when coupled with a graphic display of the results, LDA gives a more immediate sense of the synthetic direction to take when seeking highly potent analogues. It is apparent that other important but not yet identified factors also play a role in determining the potencies of these compounds.

Ligand interaction energies and molecular recognition by chloramphenicol acetyltransferase

The apparent binding energy for the interaction of the 3-hydroxyl group of chloramphenicol (CM) with the proposed general base (His-195) in chloramphenicol acetyltransferase (CAT) was determined by comparison of the dissociation constants of CM and 3-deoxyCM with CAT. The delta Gapp for this hydrogen bond to the N-3 of the imidazole ring is 1.5 kcal mol-1. Extending the use of modified ligands, in an approach which is complementary to that of site-directed mutagenesis, the binding affinity of each of a family of 3-halo-3-deoxychloramphenicol derivatives was observed to increase in the series F less than Cl less than Br less than I and is dominated by hydrophobic considerations. There is a linear free energy relationship between the dissociation constants for binding to CAT and an empirical hydrophobicity scale derived from reverse-phase HPLC retention times. The existence of such a relationship allows a true estimate of the total energetic contribution of interactions between the 3-hydroxyl group of CM and its contacts at the active site of CAT to be made on the basis of a regression analysis. The calculated value of delta Gbind (2.7 kcal mol-1) must include not only the hydrogen bond but also some favorable van der Waals interactions. The results demonstrate some of the advantages of an analysis of the energetics of ligand binding using modified ligands, in an approach that is formally analogous with and complementary to the use of site-directed mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Binding of Xanthine Derivatives to Guinea Pig Serum Albumin

Binding of the bronchodilators N3-alkylxanthine and N3-alkyl-N1-methylxanthine derivatives to guinea pig serum albumin was investigated in vitro using the ultrafiltration method. A marked difference in the binding parameters of xanthine derivatives was observed, and binding was shown to be concentration dependent. Significant relations were observed among their binding parameter, dissociation constant (Kd), and hydrophobicity (log PC). The extent of binding of xanthine derivatives was increased both when a N3-methyl group was replaced by a longer alkyl chain and when a N3-alkylxanthine molecule was additionally replaced by a methyl group. Reversed-phase HPLC retention, as an index of hydrophobicity of xanthine derivatives, was also determined. Significant relationships were found between the adjusted retention time data for each xanthine derivative and their hydrophobicity or biological activities, such as their abilities to cause muscle relaxation and cyclic AMP phosphodiesterase (PDE) inhibition. These findings indicate that the difference in the extent of binding among xanthine derivatives is related to hydrophobicity, which is an important determinant of their biological activities.

Structure-activity Relationships for Unsaturated Dialdehydes. V. Estimation of the Lipophilicity of Nineteen Sesquiterpenoid Unsaturated Dialdehydes, and Determination of their Chemical Stability in Three In Vitro Assay Media

The lipophilicity, expressed as log P, has been estimated for 19 sesquiterpenoid unsaturated dialdehydes by the determination of the 1-octanol/water partition coefficient for 12 of the compounds, and the reversed-phase HPLC retention data for all the compounds. The log P values obtained varied between 0.5 and 3.02. The stability of 17 unsaturated dialdehydes in three common in vitro assay media at 37°C was investigated by HPLC analysis of samples dissolved in the media. All compounds investigated were essentially stable for up to 24 hours in a phosphate buffered saline, while several of them disappeared rapidly in media containing amino acids and fetal calf serum. This chemical instability must be considered when results from biological assays of unsaturated dialdehydes are compared.

Prediction of the soil adsorption coefficient Koc for aromatic pollutants

The prediction of the adsorption factor Koc of eleven aromatic compounds has been made by using a multilinear relation with log Kow (Kow = octanol/water partition coefficient) and δ (Hildebrand's solubility parameter). Very significative relations could also be obtained using reverse phase HPLC retention factors instead of log Kow. The contribution of the δ term is not constant and decreases when the polarity of the stationary phase increases.

Physico-chemical modelling of solute retention in reversed-phase HPLC with temary mobile phases

In our previous publications we have established a new physico-chemically grounded retention model valid for the adsorption and partition mechanism of retention using multicomponent mobile phases. Moreover, we have demonstrated its practical usefulness for predicting solute retention in several different chromatographic modes (i.e. in TLC, normal-phase and reversed-phase HPLC) employing binary mobile phases. In this paper we give an ultimate check to the physicochemical correctness of our new retention model, using it for the description of solute retention in reversedphase HPLC systems with ternary mobile phases of the B+AB1+AB2 type. The complexity of these mobile phases and of the possible intermolecular interactions among the consitituent solvents was supposed to rigidly scrutinize the premises of the assumed new approach. We have observed a good agreement between the experimental retention parameter for a number of solutes chromatographed with the use of two different mobile phases of the B+AB1+AB2 type, and the retention parameter calculated according to the assumed model. Therefore, we believe to have sufficiently tested our new approach and confirmed its physico-chemical correctness.