Development and implementation of (Q)SAR modeling within the CHARMMing web‐user interface

Recent availability of large publicly accessible databases of chemical compounds and their biological activities (PubChem, ChEMBL) has inspired us to develop a web‐based tool for structure activity relationship and quantitative structure activity relationship modeling to add to the services provided by CHARMMing ( www.charmming.org ). This new module implements some of the most recent advances in modern machine learning algorithms—Random Forest, Support Vector Machine, Stochastic Gradient Descent, Gradient Tree Boosting, so forth. A user can import training data from Pubchem Bioassay data collections directly from our interface or upload his or her own SD files which contain structures and activity information to create new models (either categorical or numerical). A user can then track the model generation process and run models on new data to predict activity. © 2014 Wiley Periodicals, Inc.     

Predictive modeling and computational machine learning simulation of adsorption separation using advanced nanocomposite materials

Adsorption process was simulated in this study for removal of Hg and Ni from water using nanocomposite materials. The used nanostructured material for the adsorption study was a combined MOF and layered double hydroxide, which is considered as MOF-LDH in this work. The data were obtained from resources and different machine learning models were trained. We selected three different regression models, including elastic net, decision tree, and Gradient boosting, to make regression on the small data set with two inputs and two outputs. Inputs are Ion type (Hg or Ni) and initial ion concentration in the feed solution (C₀), and outputs are equilibrium concentration (Ce) and equilibrium capacity of the adsorbent (Qe) in this dataset. After tuning their hyper-parameters, final models were implemented and assessed using different metrics. In terms of the R2-score metric, all models have more than 0.97 for Ce and more than 0.88 for Qe. The Gradient Boosting has an R2-score of 0.994 for Qe. Also, considering RMSE and MAE, Gradient Boosting shows acceptable errors and best models. Finally, the optimal values with the GB model are identical to dataset optimal: (Ion = Ni, C₀ = 250, Ce = 206.0). However, for Qe, it is different and is equal to (Ion = Hg, C0 = 121.12, Ce = 606.15). The results revealed that the developed methods of simulation are of high capacity in prediction of adsorption for removal of heavy metals using nanostructure materials.     

Effects of the gradient profile, sample volume and solvent on the separation in very fast gradients, with special attention to the second-dimension gradient in comprehensive two-dimensional liquid chromatography

Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions and therefore should be used in comprehensive two-dimensional LC×LC, both in the first and in the second dimension, where, however, gradients are limited to a short time period available for separation, usually 1 min or less. Gradient conditions spanning over a broad mobile phase composition range in each second-dimension fraction analysis are used with generic "full in fraction" (FIF) gradients. "Segment in fraction" (SIF) gradients cover a limited gradient range adjusted independently to suit changing lipophilicity range of compounds transferred to the second dimension during the first-dimension gradient run and to provide regular coverage of the two-dimensional retention space. Optimization of the gradient profiles is important tool for achieving high two-dimensional peak capacity and savings of the separation time in comprehensive LC×LC. Calculations based on the well-established gradient-elution theory can be used to predict the elution times and bandwidths in fast gradients, taking into account increased probability of pre-gradient or post-gradient elution. The fraction volumes transferred into the second dimension may significantly affect the second-dimension bandwidths, especially at high elution strength of the fraction solvent, which may cause even band distortion or splitting in combined normal-phase (HILIC)-RP systems, but also in some two-dimensional RP-RP systems. In the present work, the effects of the fast gradient profile, of the sample volume and solvent on the elution time and bandwidths were investigated on a short column packed with fused-core porous-shell particles, providing narrow bandwidths and fast separations at moderate operating pressure.     

Effect of buffer concentration on gradient chromatofocusing performance separating protiens on a high-performance DEAE column

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta-lactoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chromatofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, beta-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of beta-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.     

Micro-HPLC 1: Linear “breakthrough-gradients” using the discontinuous interface between eluents in a packed gradient-generator column

A new approach is described for micro-HPLC for generating nearly linear gradients in the 40 to 550 μl volume range. These “linear” gradients have a straight line middle portion with a gentle curved onset of the gradient andd rapid attainment of the final 100% composition. Such “breakthrough-gradients” are produced by the interface between weak and strong eluent when a small (3 × 0.46 cm) packed “gradient-generator” column is switched abruptly from weak to strong eluent. The model system described here, without an analytical column, shows gradients from weak eluent (water) to strong eluent (modele with water plus acetone) using a 12-port high pressure valve to 1) vent the “flush” eluent, and simultaneously 2) begin the gradient and 3) make the sample injection. The volume of the gradient can be changed in two ways. If the breakthrough-generator is packed with very large (300 to 1600 μm nonporous particles, the gradient volume is nearly independent of the flush flow rate. The flush flow is used to quickly move the breakthrough-interface to the head of the micro-HPLC column. However, the gradient volume can be increased by using larger solid particles in the gradient-generator column because of increased eddy diffusion. If the gradient-generator is packed with porous particles (75–150 μm), the gradient volume can be changed over a broad range by simply changing the flush flow rate. This simple control over the gradient volume is due to the increased mass transfer of weak eluent out of the porous particles into the strong eluent at higher flush flows. The breakthrough-interface gradient-generator column approach provides the following advantages for micro-HPLC:

• – Gradient volumes can be readily varied over the micro-HPLC range 40–550 μl.
• – Gradient are linear with smooth and rapid onset of intial and final concentrations.
• – Gradient are produced inexpensively with a single high-pressure precision pump.
• – Gradient can be automated with a timer and single 12-port valve that provides both column regeneration and sample injection.

     

Quasi-linear gradients for capillary liquid chromatography with mass and tandem mass spectrometry

Gradient elution, capillary liquid chromatography mass spectrometry was performed with linear, static gradients constructed by laminar flowing ten, 1.5 microL volume steps of decreasing organic concentration into tubing of small internal diameter. Sample loading, gradient formation, and sample elution were accomplished entirely by means of a commercially available micro-autosampler and single-syringe drive pump. The procedure was simple, fast, stable, and reproducible. Essentially linear gradients were produced without the use of additional valves, mixers, pumps or software. It took less than 10 minutes to form a gradient and less than 30 minutes to construct the set of individual buffer vials. The gradients were shown to be stable to storage. One hour after forming, peak retention times were reproduced to +/-0.5%. Long-term retention time reproducibility was found to vary by +/-2%. Chromatographic resolution was comparable or superior to that obtained by gradient elution with conventional dynamic mixing and split flow. The procedure was adapted with a 'peak parking' method which extended the time for generating peptide fragmentation data up to 10 minutes per peptide with the triple quadruple mass spectrometer. Using this technique, collision data were collected at the 25 femtomole level on nine of ten tryptic peptides in a single run.     

PASS: identification of probable targets and mechanisms of toxicity

Toxicity of chemical compound is a complex phenomenon that may be caused by its interaction with different targets in the organism. Two distinct types of toxicity can be broadly specified: the first one is caused by the strong compound's interaction with a single target (e.g. AChE inhibition); while the second one is caused by the moderate compound's interaction with many various targets. Computer program PASS predicts about 2500 kinds of biological activities based on the structural formula of chemical compounds. Prediction is based on the robust analysis of structure-activity relationships for about 60,000 biologically active compounds. Mean accuracy exceeds 90% in leave-one-out cross-validation. In addition to some kinds of adverse effects and specific toxicity (e.g. carcinogenicity, mutagenicity, etc.), PASS predicts approximately 2000 kinds of biological activities at the molecular level, that providing an estimated profile of compound's action in biological space. Such profiles can be used to recognize the most probable targets, interaction with which might be a reason of compound's toxicity. Applications of PASS predictions for analysis of probable targets and mechanisms of toxicity are discussed.     

Optimization of azeotropic protein separations in gradient and isocratic ion-exchange simulated moving bed chromatography

The separation of dilute binary mixtures of proteins by salt aided ion-exchange simulated moving bed (SMB) chromatography is optimized with respect to throughput, desorbent consumption and salt consumption. The optimal flow-rate ratios are analytically determined via an adopted "triangle theory". Azeotropic phenomena are included in this procedure. The salt concentrations in the feed and recycled liquid are subsequently determined by numerical optimization. The azeotropic separation of bovine serum albumin and a yeast protein is used to illustrate the procedure. Gradient operation of the SMB is generally preferred over isocratic operation. A feed of azeotropic salt concentration can only be separated in a gradient SMB. Desorbent and salt consumption are always lower in gradient than in isocratic SMB chromatography.     

Quantitative analysis of tea using ytterbium‐based internal standard near‐infrared spectroscopy coupled with boosting least‐squares support vector regression

The present study demonstrated the possibility of utilizing the ytterbium (Yb)‐based internal standard near‐infrared (NIR) spectroscopic measurement technique coupled with multivariate calibration for quantitative analysis of tea, including total free amino acids and total polyphenols in tea. Yb is a rare earth element aimed to compensate for the spectral variation induced by the alteration of sample quantity during the spectral measurement of the powdered samples. Boosting was invoked to be combined with least‐squares support vector regression (LS‐SVR), forming boosting least‐squares support vector regression (BLS‐SVR) for the multivariate calibration task. The results showed that the tea quality could be accurately and rapidly determined via the Yb‐based internal standard NIR spectroscopy combined with BLS‐SVR method. Moreover, the introduction of boosting drastically enhanced the performance of individual LS‐SVR, and BLS‐SVR compared favorably with partial least‐squares regression. Copyright © 2013 John Wiley & Sons, Ltd.     

The gradient volume principle for fast evaluation of optimum conditions for isocratic analysis

Summary The paper describes by simple experiments in a pragmatical way by easy rules of thumbs gradient optimization. Besides selection of the stationary phase and initial and final conditions the two other important variables are program time and eluent flow rate. It is demonstrated, that when the product of both, the gradient volume, is kept constant, the solutes are always eluted with the same eluent composition at column outlet. At constant gradient volume, peak broadening depends on flow rate and on the eluent properties (viscosity) at which the solutes elute, and on the time the solutes spend in the column. Because peak broadening increases with increasing gradient volume, the peak capacity in gradient elution shows an optimum at gradient volumes around 15 empty column volumes (program times 45 to 60 min at flow rates of 1 ml/min with standard columns).Gradient elution can also be used for fast evaluation of optimum eluent composition for isocratic analysis. This procedure requires a calibration of the equipment for determination of eluent composition at column outlet. The sample is chromatographed in a standard gradient run of 10 to 15 empty column volumes. The eluent composition at which the solute of interest elutes during the gradient is used for isocratic analysis, where the k' value of this solute will then be around 2.Part of Ph. D. Thesis H. Elgass, Saarbrücken, 1978, present address Hewlett-Packard, Waldbronn, FRG. In part presented at Eastern Analytical Symposium, New York, 1982.     

Guanidine improves DEAE anion exchange-based analytical separation of plasmid DNA

Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the diethylaminoethyl-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than CE, and resolution approaching CE.     

Gradient extremals and valley floor bifurcations on potential energy surfaces

Gradient extremals are curves in configuration space denned by the condition that the gradient of the potential energy is an eigenvector of the Hessian matrix. Solutions of a corresponding equation go along a valley floor or along a crest of a ridge, if the norm of the gradient is a minimum, and along a cirque or a cliff or a flank of one of the two if the gradient norm is a maximum. Properties of gradient extremals are discussed for simple 2D model surfaces including the problem of valley bifurcations.     

A method of finding an informative set of molecular graph indices for the prediction of chemical compound properties

The method and algorithms for predicting properties of chemical compounds by their quantitative structural characteristics, in particular, molecular graph indices, are presented. The prediction procedure consists of establishing the priority of indices for training sample compounds, classifying control sample compounds in the Euclidean space of indices, and finding a locally optimum (informative) index set giving a least prediction error. The algorithms have been successfully tested using the BACC system (analysis and classification of biologically active compounds), created at the S. V. Sobolev Institute of Mathematics of the Siberian Branch of the Russian Academy of Sciences. Translated fromZhumal Strukturnoi Khimii, Vol. 38, No. 4, pp. 795–802, July–August, 1997.     

Columns and optimum gradient conditions for fast second-dimension separations in comprehensive two-dimensional liquid chromatography

Gradient elution provides significantly higher peak capacity in comparison to the isocratic elution mode, hence it is very useful in online comprehensive two-dimensional liquid chromatography (LC). We compared suitability of five commercial core-shell columns and one monolithic column for fast gradients in the second LC dimension, where the time of separation is strictly limited by the fraction cycle time. In two-dimensional reversed-phase systems with partially correlated retention, the resolution, the peak capacity, and the regularity of coverage of the second-dimension retention space can be improved by appropriate adjusting the gradient time and the gradient range to suit the sample properties. We developed a new strategy for adjusting the gradient mobile phase composition range in the second-dimension, employing the retention data of representative sample standards characterizing the sample properties, which can be calibrated using the reference alkylbenzene series. Optimized second-dimension gradients with single-step or segmented profiles covering two or more fraction ranges, employed for the separation of subsequent fractions from the first-dimension, improve significantly the resolution, the separation time, and the regularity of coverage of the two-dimensional retention plane. The approach was applied to the two-dimensional comprehensive separation of phenolic acids and flavonoid compounds occurring as natural antioxidants.     

A comparison of gradient capacity anion chromatography using macrocycles D-2.2.2 and D-2.2.1 in constant or variable temperature mode

The ability of macrocyclic ligands to complex alkali metal cations has been exploited to perform chromatographic separations of anions. Macrocycles adsorbed to reversed phase columns can complex eluent cations, thus generating anion exchange sites. Gradient separations of anions can be performed by changing the column capacity during the course of the separation, either by changing the eluent cation or by changing the column temperature. Gradient anion separations are performed by changing the eluent from sodium hydroxide to lithium hydroxide with the cryptand D-2.2.2, while similar anion separations are achieved with D-2.2.1 by a KOH-LiOH gradient. Since the complexation of cations by macrocycles is exothermic, increasing the column temperature decreases the anion column capacity, allowing temperature gradient separations. The experimentally measured DeltaH values for D-2.2.1 are higher than for D-2.2.2, leading to steeper gradients and thus better separations with D-2.2.1.     

DNA purification from crude samples for human identification using gradient elution isotachophoresis

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure‐driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On‐line DNA quantification using laser‐induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.     

Stable and sensitive method for the simultaneous determination of N5-methyltetrahydrofolate, leucovorin, methotrexate and 7-hydroxymethotrexate in biological fluids

A high-performance liquid chromatographic method for the determination of N5-methyltetrahydrofolic acid, leucovorin, methotrexate and 7-hydroxymethotrexate in plasma and liquor samples is presented. Gradient elution is used to increase the sensitivity. Four sample preparation methods were compared with respect to the stability of the injectable sample. Samples can be pretreated with a simple deproteinization method. For enhanced selectivity a solid-phase extraction procedure is described.     

Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices

Stability is an important pre-analytical variable for quantitative LC-MS/MS analysis of drug molecules and/or their metabolites in biological matrices. Instability of an analyte in any stage of the bioanalytical process, including sample collection, processing, storage, extraction and LC-MS/MS analysis, can result in under-/over-estimation if an adequate preventive procedure is not in place. In the current review on practical strategies in quantitative LC-MS/MS bioanalysis of unstable small molecules, the common causes of analyte instability were examined. The instability of some analytes is readily predictable because of the presence of certain chemically or biologically labile moieties in the molecules or because the compounds are in an inter-convertible form, e.g. lactone vs hydroxyl carboxylic acid. However, the instability of many other analytes is not readily predictable. Necessary evaluation needs to be conducted to identify the possible instability issues. The current review highlighted some general considerations and specific approaches for developing a robust LC-MS/MS method. In particular, incurred samples should be used as part of routine short-term stability assessment of any unstable analyte during the early stages of method development and validation. This can help unveil any 'hidden' instability issues that, if left unaddressed, could lead to the invalidation of a 'validated' method.