Journal of Chromatography B (v.968, #C)
Editorial Board (i).
Preface by John Lough; Ruin Moaddel (vii).
Classification of LC chiral stationary phases: Wainer Types I–V revisited by W. John Lough (1-7).
The method of classifying LC chiral stationary phases (CSPs) introduced by Irving Wainer in 1987 became widely adopted largely because it was based on the mechanisms by which the different CSP Types (I–V) achieved chiral recognition and was an integral part of a ‘how to do chiral LC’ package. The classification became less used perhaps because it was thought that it was not clear to which Type some of the newer CSPs should be assigned. Its only modern day significance seemed to be in single enantiomer drug patent litigation cases in defining the ‘state-of-the-art’ of chiral LC in the late 1980s. However, on closer inspection, it is clear that even with the introduction of many new commercially available CSPs, the Wainer classification, perhaps with minor modifications, remains a useful suitable vehicle for distinguishing between different groups (Types) of CSPs as an aid to chiral LC method development.
Keywords: Chiral LC; Wainer Types I–V; Chiral stationary phases (CSPs); Retention mechanisms;
Enantioseparation of chiral pharmaceuticals in biomedical and environmental analyses by liquid chromatography: An overview by Ana R. Ribeiro; Alexandra S. Maia; Quezia B. Cass; Maria E. Tiritan (8-21).
This review aims to present the issues associated to enantioseparation of chiral pharmaceuticals in biological and environmental matrices using chiral stationary phases (CSP). Thus, it related some enantioselective methods in liquid chromatography (LC) and compares the importance given to chiral separation in biomedical and environmental fields. For that the most used CSP, the enantioselective chromatographic methods, their advantages and drawbacks were swiftly revised and compared. The recent advances and the limitations of chiral analytical methods in LC were also discussed.
Keywords: Chiral pharmaceuticals; Biological matrices; Environmental matrices; Chiral stationary phases; Enantioselective chromatography;
Green chiral HPLC study of the stability of Chiralcel OD under high temperature liquid chromatography and subcritical water conditions by S. Droux; M. Roy; G. Félix (22-25).
We report here the study of the stability under subcritical water conditions of one of the most popular polysaccharide chiral stationary phase (CSP): Chiralcel OD. This CSP was used under high temperature and reversed phase conditions with acetonitrile and 2-propanol as modifier, respectively. The evolution of selectivity and resolution was investigated both in normal and reversed mode conditions with five racemates after packing, heating at 150 °C and separations of some racemic compounds under different high temperatures and mobile phase conditions. The results show that after using at high temperature and subcritical water conditions the selectivity was only moderately affected while the resolution fell dramatically especially in reversed mode due to the creation of a void at the head of the columns which reflects the dissolution of the silica matrix.
Keywords: Subcritical water chromatography; Super heated water; Reversed phase and normal chromatography; Chiral separations; Substituted polysaccharide stationary phase;
Reprint of: fRMSDchiral: A novel algorithm to represent differences between positions of stereoisomers in complex with dissymmetric binding site by Rafal D. Urniaz; Ewelina E. Rutkowska; Anita Plazinska; Krzysztof Jozwiak (26-31).
The ability of molecules to distinguish between optical isomers is crucial for living systems. The change of position of one enantiomer in respect to the position of the second enantiomer within an asymmetric binding site may be analyzed on different levels. Root Mean Square Deviation (RMSD) may be used for such analyses with low precision. Additional fragment level variants of RMSD allow for more precise definition of differences in location of the main molecular features responsible for recognition of stereoisomers by a selector. Three fRMSDchiral parameters appear to be very useful to precisely quantify the change in orientations of stereoisomers. Proposed calculation emerges as interesting assistance in interpretation of consequences of formation differential interaction(s) responsible for a chiral recognition process.
Keywords: Chiral recognition; Molecular modeling; Docking; Molecular dynamics; Fragment RMSD;
Simultaneous quantification of mefloquine (+)- and (−)-enantiomers and the carboxy metabolite in dried blood spots by liquid chromatography/tandem mass spectrometry by Mirjam C.K. Geditz; Wolfgang Lindner; Michael Lämmerhofer; Georg Heinkele; Reinhold Kerb; Michael Ramharter; Matthias Schwab; Ute Hofmann (32-39).
Mefloquine (MQ), a racemic mixture of (+)-(11S,12R)- and (−)-(11R,12S)-MQ, has been used for treatment and prophylaxis of malaria for almost 30 years. MQ is metabolized by the cytochrome P450 3A subfamily to 4-carboxymefloquine (CMQ), which shows no antimalarial activity in vitro. Highly stereospecific pharmacokinetics of MQ have been reported, although with contradictory results. This might be due to incorrect assignment of the absolute configuration as shown only recently. Gastrointestinal as well as neuropsychiatric adverse events were described after prophylaxis and treatment with MQ. Data are indicating that the tolerability of the enantiomers may vary considerably. An involvement of the main metabolite CMQ in the development of neuropsychiatric adverse events has also been supposed. Due to these inconsistent results we established a novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of MQ enantiomers and the metabolite CMQ to investigate the attribution of efficacy and adverse effects to the single enantiomers as well as the main metabolite. Separation of the MQ enantiomers was achieved on a quinidine-based zwitterionic chiral stationary phase column, CHIRALPAK® ZWIX(−) (3.0 × 150 mm, 3 μm) in an isocratic run using a pre-mixed eluent consisting of methanol/acetonitrile/water (49:49:2 v/v) with 25 mM formic acid and 12.5 mM ammonium formate. We used stable isotope-labelled analogues as internal standards. The method was validated according to the FDA guidelines. With a linear calibration range from 5 to 2000 nM for the MQ enantiomers and from 13 to 2600 nM for CMQ respectively, the method was successfully applied to dried blood spot (DBS) samples from patients under prophylactic MQ treatment. The method was also applicable for plasma samples.
Keywords: Antimalarial drug; Mefloquineenantiomers; Carboxymefloquine; LC–MS/MS; Dried blood spots; Chiral separation;
Reprint of: Enantiomeric separation of functionalized ethano-bridged Tröger bases using macrocyclic cyclofructan and cyclodextrin chiral selectors in high-performance liquid chromatography and capillary electrophoresis with application of principal component analysis by Choyce A. Weatherly; Yun-Cheol Na; Yasith S. Nanayakkara; Ross M. Woods; Ankit Sharma; Jérôme Lacour; Daniel W. Armstrong (40-48).
The enantiomeric separation of a series of racemic functionalized ethano-bridged Tröger base compounds was examined by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Using HPLC and CE the entire set of 14 derivatives was separated by chiral stationary phases (CSPs) and chiral additives composed of cyclodextrin (native and derivatized) and cyclofructan (derivatized). Baseline separations (R s ≥ 1.5) in HPLC were achieved for 13 of the 14 compounds with resolution values as high as 5.0. CE produced 2 baseline separations. The separations on the cyclodextrin CSPs showed optimum results in the reversed phase mode, and the LARIHC™ cyclofructan CSPs separations showed optimum results in the normal phase mode. HPLC separation data of the compounds was analyzed using principal component analysis (PCA). The PCA biplot analysis showed that retention is governed by the size of the R1 substituent in the case of derivatized cyclofructan and cyclodextrin CSPs, and enantiomeric resolution closely correlated with the size of the R2 group in the case of non-derivatized γ-cyclodextrin CSP. It is clearly shown that chromatographic retention is necessary but not sufficient for the enantiomeric separations of these compounds.
Keywords: Cyclofructan-based stationary phase; Cyclodextrin-based stationary phase; Enantiomer; Functionalized ethano-Tröger base; Principal component analysis;
Analysis of biomolecular interactions using affinity microcolumns: A review by Xiwei Zheng; Zhao Li; Sandya Beeram; Maria Podariu; Ryan Matsuda; Erika L. Pfaunmiller; Christopher J. White II; NaTasha Carter; David S. Hage (49-63).
Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods.
Keywords: Affinity microcolumns; High-performance affinity chromatography; Biointeraction analysis; Zonal elution; Frontal affinity chromatography; Ultrafast affinity extraction;
Multiple protein stationary phases: A review by N.S. Singh; K-L. Habicht; K.S.S. Dossou; R. Shimmo; I.W. Wainer; R. Moaddel (64-68).
Cellular membrane affinity chromatography stationary phases have been extensively used to characterize immobilized proteins and provide a direct measurement of multiple binding sites, including orthosteric and allosteric sites. This review will address the utilization of immobilized cellular and tissue fragments to characterize multiple transmembrane proteins co-immobilized onto a stationary phase. This approach will be illustrated by demonstrating that multiple transmembrane proteins were immobilized from cell lines and tissue fragments. In addition, the immobilization of individual compartments/organelles within a cell will be discussed and the changes in the proteins binding/kinetics based on their location.
Keywords: Bioaffinity chromatography; G-protein coupled receptors; Ligand-gated ion channels; ATP-binding cassette transporters;
Species-dependent binding of tocainide analogues to albumin: Affinity chromatography and circular dichroism study by Marco Pistolozzi; Cecilia Fortugno; Carlo Franchini; Filomena Corbo; Marilena Muraglia; Myriam Roy; Guy Félix; Carlo Bertucci (69-78).
A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts.
Keywords: High performance liquid affinity chromatography; Protein binding; HSA; RSA; Tocainide analogues; Circular dichroism;
Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications by Enrica Calleri; Daniela Ubiali; Immacolata Serra; Caterina Temporini; Giulia Cattaneo; Giovanna Speranza; Carlo F. Morelli; Gabriella Massolini (79-86).
We described the development of a biochromatographic system which uses a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) for the evaluation of the substrate specificity on nucleoside libraries. AhPNP has been covalently immobilized on a fused silica Open Tubular Capillary (OTC) via Schiff base chemistry. The resulting bioreactor has been characterized by the determination of kinetic constants (K m and V max) for a natural substrate (inosine) and then assayed versus all natural purine (deoxy)ribonucleosides and a small library of 6-substituted purine ribosides. Characterization of the bioreactor has been carried out through a bidimensional chromatographic system with the sample on-line transfer from the bioreactor to the analytical column for the separation and quantification of substrate and product. Comparison with the soluble enzyme showed that the AhPNP-based bioreactor is reliable as the same ranking order, with respect to the standard activity assay, was obtained. The stability of the IMER was also assessed and the system was found to be stable up to 60 reactions.
Keywords: Purine nucleoside phosphorylase from Aeromonas hydrophila; On-line enzyme reactor; Phosphorolysis reaction; Nucleosides screening;
Immobilized cholinesterases capillary reactors on-flow screening of selective inhibitors by Adriana Ferreira Lopes Vilela; Joyce Izidoro da Silva; Lucas Campos Curcino Vieira; Gilberto C.R. Bernasconi; Arlene Gonçalves Corrêa; Quezia Bezerra Cass; Carmen Lúcia Cardoso (87-93).
The discovery of selective inhibitors for acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) is extremely important for the development of drugs that can be used in the treatment of patients diagnosed with the Alzheimer's disease (AD). For this reason, there is a growing interest in developing rapid and effective assays techniques for cholinesterases (ChE) enzymes ligand screening. Herein is presented the results of selective screening assays of a coumarin derivatives library using BChE and AChE covalently immobilized onto silica fused capillaries (ICERs, 15 cm × 0.1 mm ID). The statistical comparison of the ICERs screening assay with that of the free enzymes is reported and highlights the advantages of the on-flow ICERs assay. Two out of 20 coumarin derivatives could be highlighted: compound 17 is more active toward BChE (IC50 = 109 ± 21 μM) and 19 showed activity against both enzymes (BChE IC50 = 128 ± 28 μM and hu-AChE IC50 = 144 ± 40 μM). The statistical evaluation of the results of the ICERs and free enzyme assays showed no difference between them, further validating the ICERs assay model. The ICERs ability to recognize selective ligands and its use for characterization of the inhibition mechanisms of the hits consolidates the approach here reported.
Keywords: Selective inhibitor; Screening; Statistical comparation; Butyrylcholinesterase; Acetylcholinesterase; Enzymatic inhibition assays;
Reprint of: Liquid chromatographic enzymatic studies with on-line Beta-secretase immobilized enzyme reactor and 4-(4-dimethylaminophenylazo) benzoic acid/5-[(2-aminoethyl) amino] naphthalene-1-sulfonic acid peptide as fluorogenic substrate by Angela De Simone; Claudia Seidl; Cid Aimbiré M. Santos; Vincenza Andrisano (94-100).
High throughput screening (HTS) techniques are required for the fast hit inhibitors selection in the early discovery process. However, in Beta-secretase (BACE1) inhibitors screening campaign, the most frequently used methoxycoumarin based peptide substrate (M-2420) is not widely applicable when aromatic or heterocycle compounds of natural source show auto-fluorescence interferences. Here, in order to overcome these drawbacks, we propose the use of a highly selective 4-(4-dimethylaminophenylazo)benzoic acid/5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (DABCYL/1,5-EDANS) based peptide substrate (Substrate IV), whose cleavage product is devoid of spectroscopic interference. HrBACE1-IMER was prepared and characterized in terms of units of immobilised hrBACE1. BACE1 catalyzed Substrate IV cleavage was on-line kinetically characterized in terms of K M and v max, in a classical Michaelis and Menten study. The on-line kinetic constants were found consistent with those obtained with the in solution fluorescence resonance energy transfer (FRET) standard method. In order to further validate the use of Substrate IV for inhibition studies, the inhibitory potency of the well-known BACE1 peptide InhibitorIV (IC50: 0.19 ± 0.02 μM) and of the natural compound Uleine (IC50: 0.57 ± 0.05) were determined in the optimized on-line hrBACE1-IMER. The IC50 values on the hrBACE1-IMER system were found in agreement with that obtained by the conventional methods confirming the applicability of Substrate IV for on-line BACE1 kinetic and inhibition studies.
Keywords: Liquid chromatography with fluorescence detection; Immobilized enzyme reactor; BACE1; Inhibition studies; Uleine;
Preliminary studies on trigonelline as potential anti-Alzheimer disease agent: Determination by hydrophilic interaction liquid chromatography and modeling of interactions with beta-amyloid by Joanna Makowska; Damian Szczesny; Agnieszka Lichucka; Artur Giełdoń; Lech Chmurzyński; Roman Kaliszan (101-104).
For trigonelline, a quaternary-base pyridine alkaloid of presumed Alzheimer's disease-preventing activity, a method of determination has been proposed, based on the hydrophilic interaction chromatography (HILIC). That method might be applied to study the agent's bioavailability, in particular its permeation through blood–brain barrier, which is an inevitable property for the potential central nervous system affecting drugs. Providing that trigonelline possesses the requested pharmacokinetic properties, once attaining pharmacodynamic phase it must interact effectively with the relevant molecular site in the brain, which is characteristic to neurodegenerative diseases, namely the beta-amyloid peptide. Here it was demonstrated by molecular modeling that affinity of trigonelline to the Aβ(1–42) peptide is high and similar to that of an anti-Alzheimer's disease drug candidate – cotinine.
Keywords: Trigonelline; HILIC; Beta-amyloid; Aβ(1–42); Docking; Alzheimer disease;
The identification of a novel SIRT6 modulator from Trigonella foenum-graecum using ligand fishing with protein coated magnetic beads by N. Singh; S. Ravichandran; K. Spelman; S.D. Fugmann; R. Moaddel (105-111).
SIRT6 is a histone deacetylase that has been proposed as a potential therapeutic target for metabolic disorders and the prevention of age-associated diseases. Thus the identification of compounds that modulate SIRT6 activity could be of great therapeutic importance. We have previously developed an H3K9 deacetylation guided assay with SIRT6 coated magnetic beads (SIRT6-MB). With the developed assay, we identified quercetin, naringenin and vitexin as SIRT6 inhibitors from T. foenum-graecum seed extract using a candidate approach. Currently, the predominant method for the identification of active compounds from a plant extract is carried out through a dereplication process. A novel targeted approach for the direct identification of active compounds from a complex matrix could save time and resources. Herein, we report the application of the SIRT6-MB for ‘fishing’ experiments utilizing T. foenum-graecum seed extract. In which orientin, and seventeen other compounds were identified as SIRT6 binders. This is the first use of this method for ‘fishing’ out active ligands from a botanical matrix, and sets the basis for the identification of active compounds from a complex matrix.
Keywords: SIRT6; Fenugreek seed extract; Orientin; Ligand fishing; Magnetic beads;