Reversed-phase HPLC Retention Research Articles

Polymyxins with Potent Antibacterial Activity against Colistin-Resistant Pathogens: Fine-Tuning Hydrophobicity with Unnatural Amino Acids.

In view of the increased prevalence of antimicrobial resistance among human pathogens, antibiotics against multidrug-resistant (MDR) bacteria are in urgent demand. In particular, the rapidly emerging resistance to last-resort antibiotic colistin, used for severe Gram-negative MDR infections, is critical. Here, a series of polymyxins containing unnatural amino acids were explored, and some analogues exhibited excellent antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Hydrophobicity of the compounds within this series (as measured by retention in reversed-phase analytical HPLC) exhibited a discernible correlation with their antimicrobial activity. This trend was particularly pronounced for colistin-resistant pathogens. The most active compounds demonstrated competitive activity against a panel of Gram-negative pathogens, while exhibiting low in vitro cytotoxicity. Importantly, most of these hits also retained (or even had increased) potency against colistin-susceptible strains. These findings infer that fine-tuning hydrophobicity may enable the design of polymyxin analogues with favorable activity profiles.

Chromatographic behaviour of peptides modified with amine-reacting tags for relative protein quantitation in proteomic applications

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs.

Development of a Reverse Phase HPLC Retention Index Model for Nontargeted Metabolomics Using Synthetic Compounds.

The MolFind application has been developed as a nontargeted metabolomics chemometric tool to facilitate structure identification when HPLC biofluids analysis reveals a feature of interest. Here synthetic compounds are selected and measured to form the basis of a new, more accurate, HPLC retention index model for use with MolFind. We show that relatively inexpensive synthetic screening compounds with simple structures can be used to develop an artificial neural network model that is successful in making quality predictions for human metabolites. A total of 1955 compounds were obtained and measured for the model. A separate set of 202 human metabolites was used for independent validation. The new ANN model showed improved accuracy over previous models. The model, based on relatively simple compounds, was able to make quality predictions for complex compounds not similar to training data. Independent validation metabolites with feature combinations found in three or more training compounds were predicted with 97% sensitivity while metabolites with feature combinations found in less than three training compounds were predicted with >90% sensitivity. The study describes the method used to select synthetic compounds and new descriptors developed to encode the relationship between lipophilic molecular subgraphs and HPLC retention. Finally, we introduce the QRI (qualitative range of interest) modification of neural network backpropagation learning to generate models simultaneously based on quantitative and qualitative data.

Systematization of the results of the chromatography–mass spectrometry identification of the products of quercetin oxidation by atmospheric oxygen in aqueous solutions

The coordination of the results of different works for the systematization of the structures of products previously detected in the complex samples is an important stage in the interpretation of the results of the identification of the components of complex samples of natural origin by chromatography–mass spectrometry with the low reproducibility of their mass spectra under the conditions of electrospray ionization and limited reference information. The data processing of this kind was carried out for the products of the oxidation of the most common natural flavonoid quercetin (3,5,7,3',4'-pentahydroxyflavone) by atmospheric oxygen in weakly alkaline aqueous alcohol solutions. For the correlation of peaks in chromatograms with the structures of oxidation products, their reversed-phase HPLC retention indices in the scale of reference n-alkyl phenyl ketones were determined for the first time. It was confirmed that not all of the oxidation products are stable in solution; some of them can accumulate or disappear during the storage of the samples.

A float mechanism of retention in reversed-phase chromatography

A float mechanism of retention in reversed-phase HPLC is proposed as an alternative to the known mechanisms of the distribution and hydrophobic expulsion of sorbate to the surface of a sorbent. Experimental data that the sorption of a flavylium structure is poorly sensitive to the position of OH groups, and that the retention of anthocyanins depends on the length of bonded alkyl radicals of reversed phase, form the basis of the proposed hypothesis. It is noted that the retention of anthocyanins depends on the orientation of hydroxyl groups in carbohydrate radicals, due to which the chromatographic behavior of anthocyanins is different for glucosides and galactosides, for arabinosides and xylosides, and so on. In other words, retention is a reliable indicator of the composition of a carbohydrate fragment. It is concluded that carbohydrate radicals serve as unique floats, while flat flavilic ions penetrate into the bonded phase. The existence of floats is the main reason for the lower efficiency (of the number of theoretical plates) of the peaks of anthocyanins. It is shown that if two carbohydrate radicals are present at different sites of aglycone (a two-float sorbate), the peaks of the substance are characterized by substantial additional broadening.

Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

Reversed-phase HPLC retention data in correlation studies with in lipophilicity molecular descriptors of carotenoids

Influence of chemical structure on the lipophilicity of isolated free carotenoids from paprika oleoresin has been studied by QSRR approach (Quantitative structure-retention relationship). The chromatographic behavior of these compounds was investigated by using reversed phase high-pressure liquid chromatography (RP HPLC). Retention mechanism has been determined using the following mobile phase: acetone -water, on reversed - phase column (SB-C18). A variety of lipophilicity parameters (logP) were calculated by use of different software products. On the basis of correlations, the nonlinear structure-activity models were derived between the retention constants, tr (retention time of investigation compounds), and logP values. Five high quality QSRR models were found to have a good predictive ability and close agreement between experimental and predicted values. The study showed that the retention constants can be used as a measure of lipophilicity of investigated compounds at a high significant level.

Reversed-Phase TLC and HPLC Retention Data in Correlation Studies with in Silico Molecular Descriptors and Druglikeness Properties of Newly Synthesized Anticonvulsant Succinimide Derivatives

The properties relevant to pharmacokinetics of two series of newly synthesized succinimide derivatives have been studied. The properties under consideration have been either determined empirically, by reversed-phase liquid chromatography (TLC and HPLC technique), or calculated with the use of established theoretical medicinal chemistry/drug design software. Chromatographic techniques allowed determination of the retention constants R(M)⁰ and log k(w), which characterize lipophilicity of compounds. Considering potential pharmaceutical importance of succinimide derivatives, we (i) examined the retention behavior in the reversed-phase liquid chromatographic (RP LC) systems, in both planar and column LC, and (ii) determined the relationships between chromatographic data and selected structural features of analytes that are believed to markedly affect their processes of absorption, distribution, metabolism, excretion and toxicity (ADMETox). Significant relationships were found between the retention constants, R(M)⁰ and log k(w), and the in silico calculated bioactivity descriptors, in particular HIA (human intestinal absorption) and PPB (plasma protein binding) parameters. The R(M)⁰ and log k(w) values of the investigated compounds have been recommended for description of their lipophilicity and evaluating pharmacokinetic properties. In view of results of this study the newly synthesized succinimide agents meet pharmacokinetic criteria of preselection of drug candidates and hence qualify for pharmacodynamic phase of antiepileptic drug development. Best compromising human intestinal absorption and plasma protein binding features appear to be compounds A4, A5, A10 and A11.

On the Presence and Role of Polar Lipids in Meibum

On the Presence and Role of Polar Lipids in Meibum

Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of δ and ω‐conotoxins

The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.

Stereospecific Enzymatic Transformation of α-Ketoglutarate to (2S,3R)-3-Methyl Glutamate during Acidic Lipopeptide Biosynthesis

The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues DptI from the daptomycin producer Streptomyces roseosporus and LptI from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, DptI, and LptI are S-adenosyl methionine (SAM)-dependent alpha-ketoglutarate methyltransferases that catalyze the stereospecific methylation of alpha-ketoglutarate (alphaKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase IlvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of alphaKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of beta-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes.

Global proteomic analysis distinguishes biologic differences in head and neck squamous carcinoma

The goal of this study was to establish a method for detecting biologically significant differences in protein expression of head and neck squamous cell carcinoma (HNSCC) obtained from the same samples utilized in gene expression analyses. Proteins from two head and neck tumor cell lines, SCC-25 and FaDu, were isolated from the denatured protein solution remaining from the TRIzol extraction procedure used for isolation of total RNA for microarray analysis. Peptides resulting from chemical and enzymatic digestion of the proteins were first separated by strong cation-exchange chromatography, followed by liquid chromatography-mass spectrometry (LC-MS) analysis on a QqTOF mass spectrometer. Stable isotope-labeled synthetic peptides were added to each ion-exchange fraction as internal standards, for reversed-phase HPLC retention time alignment. Protein extraction and digestion were repeated three times for each cell line and each extract was analyzed three times by LC-MS. To discriminate between technical vs biological variation, the ion-exchange fraction, retention time, normalized mass and signal intensity of these nine data sets were constructed into numerical arrays for statistical analysis. Of the ∼50 000 signals, 90 peptide ions were found to discriminate the two cell lines with high stringency. Of those, six peptides were derived from vimentin and four peptides were derived from annexin II; both expressed more in SCC-25. Follow-up analysis of some of these signals by LC-MS/MS and RNA expression profiling revealed both concordance and discordance of RNA and protein expression. This study demonstrates that this procedure is highly reliable for identifying peptides that distinguish biological variability among samples, indicating that this method can be applied to study clinical samples, to identify potential prognostic biomarkers for HNSCC.

QSRR of Flavones: Evaluation of Substituent Contributions to RP HPLC Retention

A non‐parameter method based on the assumption of additive substituent increments on retention is employed for estimation of the effects of the substituents in the flavone ring on the retention in the reversed phase HPLC. The influence of OH‐ and OCH3‐groups in the positions: 3, 5, 6, 7, 8, 3′, 4′, and 5′ on reversed phase HPLC retention is studied in a group of 21 flavones. The multiple linear regressions performed using 15 substituent codes as independent variables and the logarithm of the retention factor (log k) of each solute obtained with isocratic elution as dependent variables, gave a good correlation with R 2 from 0.9884 to 0.9984, and R¯2 from 0.9535 to 0.9937. The regression coefficients confirm that incorporation of an OH‐group does not necessarily reduce retention, and OCH3‐groups, depending on the position, can either cause a decrease or increase in retention. The proposed approach was used for prediction of the structure of an unknown flavone. The predicted substituent pattern of an unknown flavone found in an extract of Teucrium polium is then experimentally confirmed by mass spectrometry and nuclear magnetic resonance of the isolated flavone–cirsiliol.

Prediction of Microclimate pH in Poly(lactic-co-glycolic Acid) Films

An equilibrium mathematical model that accurately predicts microclimate pH (mupH) in thin biodegradable polymer films of poly(lactic-co-glycolic acid) (PLGA) is described. mupH kinetics was shown to be primarily a function of: (i) kinetics of water-soluble acid content and composition in the polymer matrix and (ii) polymer/water partition coefficient of water-soluble degradation products (P(i)). Polymers were coated on standard pH glass electrodes, and mupH was measured potentiometrically. Water-soluble acid distribution and content in PLGA films were determined by pre-derivatization HPLC. Polymer degradation products partitioned favorably in the polymer phase relative to water (P(i) range: approximately 6-100), and P(i) increased with increasing hydrophobicity of the acidic species according to a linear free energy law related to reversed phase HPLC retention time for the corresponding derivatized bromophenacyl esters. The mupH predicted by the model was in excellent agreement with experimental mupH for several PLGAs as a function of time and PLGA lactic/glycolic acid ratio. These data may be useful to slowly release pH-sensitive PLGA-encapsulated bioactive substances and provide a general framework for predicting partitioning behavior of degradation products in biodegradable polymers.

Effect of the counter-anion type and concentration on the liquid chromatography retention of β-blockers

Analysis of β-blockers, basic pharmaceutical compounds with p K a values greater than 8.5, in reversed-phase HPLC can sometimes be challenging in terms of selection of the mobile phase pH, buffer concentration, and acidic modifier. The effect of the type and concentration of various mobile phase additives on the reversed-phase HPLC retention of these compounds was studied. HPLC analysis was performed at a mobile phase pH of 3 ensuring the protonation of the β-blockers. It was found that at increasing perchlorate anion concentration at a constant mobile phase pH the retention factor for all β-blocker compounds studied increased to varying degrees. The relative increase in the retention was attributed to ion interaction with the anionic mobile phase additive. Similar trends were observed when other types of inorganic salts such as NaH 2PO 4, NaPF 6, NaBF 4, and CF 3CO 2Na were employed. Differences in selectivity of the β-blockers were obtained at a constant pH and an equimolar concentration of the different additives throughout the whole concentration range studied.

Role of helix formation for the retention of peptides in reversed-phase high-performance liquid chromatography

In order to get insight into the role of helix formation for retention in reversed-phase HPLC, we have studied the isocratic retention behavior of amphipathic and non-amphipathic potentially helical model peptides. Plots of the logarithmic capacity factor in absence of organic solvent (ln k 0) versus 1/ T were used to derive the enthalpy, Δ H 0, the free energy, Δ G 0, the entropy of interaction, Δ S 0, and the heat capacity change, Δ C p. Retention of all peptides was accompanied by negative Δ C p revealing that hydrophobic interactions play a large role independent of peptide sequence and secondary structure. Δ H 0 was negative for the amphipathic analogs and was attributed mainly to helix formation of these peptides upon interaction with the stationary phase. In contrast, Δ H 0 was considerably less exothermic or even endothermic for the non-amphipathic analogs. The differences in helix formation between the individual analogs were quantified on the basis of thermodynamic data of helix formation previously derived for peptides in a hydrophobic environment. Correlation of the helicity with the free energy of stationary phase interaction revealed that helix formation accounts for ∼40–70% of Δ G 0, and is hence in addition to the hydrophobic effect a major driving force of retention.

Practical implications of some recent studies in electrospray ionization fundamentals.

In accomplishing successful electrospray ionization analyses, it is imperative to have an understanding of the effects of variables such as analyte structure, instrumental parameters, and solution composition. Here, we review some fundamental studies of the ESI process that are relevant to these issues. We discuss how analyte chargeability and surface activity are related to ESI response, and how accessible parameters such as nonpolar surface area and reversed phase HPLC retention time can be used to predict relative ESI response. Also presented is a description of how derivitizing agents can be used to maximize or enable ESI response by improving the chargeability or hydrophobicity of ESI analytes. Limiting factors in the ESI calibration curve are discussed. At high concentrations, these factors include droplet surface area and excess charge concentration, whereas at low concentrations ion transmission becomes an issue, and chemical interference can also be limiting. Stable and reproducible non-pneumatic ESI operation depends on the ability to balance a number of parameters, including applied voltage and solution surface tension, flow rate, and conductivity. We discuss how changing these parameters can shift the mode of ESI operation from stable to unstable, and how current-voltage curves can be used to characterize the mode of ESI operation. Finally, the characteristics of the ideal ESI solvent, including surface tension and conductivity requirements, are discussed. Analysis in the positive ion mode can be accomplished with acidified methanol/water solutions, but negative ion mode analysis necessitates special constituents that suppress corona discharge and facilitate the production of stable negative ions.

Predicting electrospray response from chromatographic retention time.

The relationship between electrospray ionization response and HPLC retention time was explored. For the series of small peptides studied, higher ESI response was observed for analytes with longer reversed-phase HPLC retention times. This correlation existed for both experimentally measured retention times and those calculated from amino acid retention coefficients. This study is useful t

Low Dietary Salt Regulates Renal Vascular 20-Hydroxyeicosatetraenoic Acid Levels.

P110 20-hydroxyeicosatetraenoic (HETE), a prohypertensive cytochrome P450 (CYP)-derived arachidonic acid (AA) metabolite, is a principal eicosanoid of preglomerular microvessels. Vascular 20-HETE release is stimulated by angiotensin II (AII) and is subject to metabolism by cyclooxygenase (COX)-2. As dietary salt alters AII levels and COX-2 expression, we studied 20-HETE levels from microdissected arcuate and interlobular arteries and interlobar arteries obtained from male Sprague-Dawley rats fed a control (0.4% NaCl) or low salt diet (LS; 0.05% NaCl). In controls (n=6), metabolism of 14 C-AA (7μM) in the presence of NADPH (1mM) and indomethacin (INDO; 10μM) to 20-HETE was higher (P&lt;0.05) in arcuate and interlobular arteries compared to interlobar arteries (25.2 ± 3.6 ng vs. 15.3 ± 2.3 ng/mg protein/30 min, respectively) based on reverse-phase HPLC retention times and on-line radiodetection. No regional differences in epoxide formation were evident between arcuate and interlobular arteries compared to interlobar arteries (21.9 ± 3.6 ng vs. 19.6 ± 3.7 ng/mg protein/30 min, respectively). LS treatment, for 7 days, selectively increased 20-HETE levels in interlobar arteries (24.6 ± 3.9 ng/mg protein/30 min) and increased medullary CYP-4A expression. However, in the absence of INDO, 20-HETE levels in arcuate and interlobular arteries, but not interlobar arteries, were diminished by 90% with LS treatment; changes that corresponded with induced cortical COX-2 expression. Thus, 20-HETE levels vary segmentally within the renal microvasculature. LS intake induces medullary CYP-4A expression and interlobar artery 20-HETE formation. Presumably, the diminished 20-HETE levels in arcuate and interlobular arteries, in the absence of COX inhibition, were a result of increased cortical COX-2 expression serving as a metabolic pathway for 20-HETE; the vasoconstrictor 20-HETE being metabolized by COX-2 to vasodilator prostaglandin analogs. Increased 20-HETE levels may account for the adverse effects of COX inhibitors with salt depletion.

Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland.

The glycosylation of a number of constituents of human saliva is known to modify its biological roles, such as its lubricating properties and binding of microbial flora. Gillece-Castro et al. [Gillece-Castro, B. L., Prakobphol, A., Burlingame, A. L., Leffler, H. & Fisher, S. J. (1991) J. Biol. Chem. 266, 17358-17368] have proposed that the major glycan on the salivary proline-rich glycoproteins is a trifucosylated biantennary sugar with one difucosylated and one unfucosylated antenna. Furthermore, they proposed that the non-fucosylated antenna mediated adherence to a peridontal pathogen, Fusobacterium nucleatum. The detailed structures and roles of other highly fucosylated glycans that co-exist in the parotid gland are not fully known. In view of the influence of outer-arm fucosylation on carbohydrate recognition processes in general, this paper reports the use of a combination of HPLC (normal and reversed phase), matrix-assisted laser-desorption/ionisation (MALDI) mass spectrometry and exoglycosidase digestions to dissect the detailed structures of the most abundant of these polyfucosylated glycans. For measurement of reversed-phase HPLC retention times, new calibration units were used which paralleled the glucose units used for normal-phase HPLC. These differed in that the difference in retention times were compared with those derived from a ladder of 2-aminobenzamide-labelled arabinose oligomers instead of the corresponding oligomers from partially hydrolysed dextran. Over sixty neutral sugars were identified from the parotid gland and many of these were additionally found substituted with sialic acid (both alpha2-3-linked and alpha2-6-linked) and sulphate. These glycans were mainly bi- and tri-antennary sugars with up to five and seven fucose residues respectively, containing fucose alpha1-3-linked to the outer-arm GlcNAc residues and alpha1-2-linked to the galactose. All fucosylated structures contained a core (alpha1-6-linked) fucose. The detailed structure of the trifucosylated biantennary glycan was confirmed, together with the structures of another 12 fucosylated biantennary glycans. Smaller amounts of hybrid and tetraantennary structures were also found and bisected glycans were shown to be constituents of parotid glycoproteins for the first time. Acidic glycans were mainly substituted with sialic acid. Most were monosialylated as the presence of fucose on the antennae was found to suppress the addition of extra sialic acid moieties. The possible functional significance of highly fucosylated N-glycans is discussed in relation to their modification of the availability of other non-reducing terminal monosaccharides for recognition processes.