MSE with mass defect filtering for in vitro and in vivo metabolite identification

Metabolite identification studies involve the detection and structural characterization of the biotransformation products of drug candidates. These experiments are necessary throughout the drug discovery and development process. The use of high-resolution chromatography and high-resolution mass spectrometry together with data processing using mass defect filtering is described for in vitro and in vivo metabolite identification studies. Data collection was done using UPLC coupled with an orthogonal hybrid quadrupole time-of-flight mass spectrometer. This experimental approach enabled the use of MS(E) data collection (where E represents collision energy) which has previously been shown to be a powerful approach for metabolite identification studies. Post-acquisition processing with a prototype mass defect filtering program was used to eliminate endogenous interferences in the study samples, greatly enhancing the discovery of metabolites. The ease of this approach is illustrated by results showing the detection and structural characterization of metabolites in plasma from a preclinical rat pharmacokinetic study.     

Liquid chromatography/tandem mass spectrometric quantification with metabolite screening as a strategy to enhance the early drug discovery process

Throughput for early discovery drug metabolism studies can be increased with the concomitant acquisition of metabolite screening information and quantitative analysis using ultra-fast gradient chromatographic methods. Typical ultra-fast high-performance liquid chromatography (HPLC) parameters used during early discovery pharmacokinetic (PK) studies, for example, employ full-linear gradients over 1-2 min at very high flow rates (1.5-2 mL/min) on very short HPLC columns (2 x 20 mm). These conditions increase sample throughput by reducing analytical run time without sacrificing chromatographic integrity and may be used to analyze samples generated from a variety of in vitro and in vivo studies. This approach allows acquisition of more information about a lead candidate while maintaining rapid analytical turn-around time. Some examples of this approach are discussed in further detail.     

Synthesis of stereodefined polysubstituted olefins. 1. Sequential intermolecular reactions involving selective, stepwise insertion of Pd(0) into allylic and vinylic halide bonds. The stereoselective synthesis of disubstituted olefins

Palladium-catalyzed allylic substitution and cross-coupling reactions have been combined into a sequential procedure to provide a range of disubstituted olefin products starting from two-, three-, and four-carbon common olefin templates. Diverse application of this template strategy is demonstrated in a variety of model studies and in a parallel synthesis (combinatorial) approach to prepare an allylic amine molecular library. An approach toward the preparation of astaxanthin beta-D-diglucoside, an interesting antioxidant whose total synthesis has yet to be reported, using the olefin-template approach is also discussed.     

Using a charging coordinate in studies of ionization induced partial unfolding

The ionization of groups in proteins may sometimes involve a partial unfolding and/or water penetration. Unfortunately the corresponding structural changes might not be captured by microscopic free energy perturbation (FEP) approaches due to activation barriers that are not surmounted in nanosecond FEP simulations. This problem is apparent, for example, from mutation experiments that introduced ionizable groups in protein interiors and from the difficulties to reproduce the corresponding pKa changes by microscopic approaches. Here we develop a new general approach for treating such challenging cases. Our approach drives the protein structural change by increasing the charge of the ionized group beyond its physical value and thus overcoming the barriers for the partial unfolding by a physically consistent process. The potential of our approach is illustrated by the evaluation of the pKa of the Val66Glu mutant of staphylococcal nuclease. In this case it is first demonstrated that standard FEP approaches give extremely disappointing results for this pKa. On the other hand, our "overcharging" approach gives a much more realistic result. We believe that the present approach represents a breakthrough in FEP studies of ionizable residues in proteins, and expect this strategy to be useful in studies of a wide range of challenging problems including simulations of hydrogen exchange processes.     

Global approach for calculation of minimum miscibility pressure

An algorithm has been developed for calculation of minimum miscibility pressure (MMP) for the displacement of oil by multicomponent gas injection. The algorithm is based on the key tie line identification approach initially addressed by Wang and Orr [Y. Wang and F.M. Orr Jr., Analytical calculation of minimum miscibility pressure, Fluid Phase Equilibria, 139 (1997) 101–124]. In this work a new global approach is introduced. A number of deficiencies of the sequential approach have been eliminated resulting in a robust and highly efficient algorithm. The time consumption for calculation of the MMP in multicomponent displacement processes has been reduced significantly and can now be performed within a few seconds on a PC for a 15-component gas mixture. The algorithm is hence particularly suitable for gas enrichment studies or other case studies where a large number of MMP calculations is required. Predicted results from the key tie line identification approach are shown to be in excellent agreement with slimtube data and with other multicell/slimtube simulators presented in the literature.     

Approaches to the Synthesis of Candelabrone Synthesis of (±)-Margocilin O-Methyl Ether

Model studies to elaborate a synthetic approach to candelabrone have provided an intermediate from which (±)-margocilin O-methyl ether has been synthesised.     

Temperature-controlled 'breathing' of carbon dioxide bubbles

We report a microfluidic (MF) approach to studies of temperature mediated carbon dioxide (CO(2)) transfer between the gas and the liquid phases. Micrometre-diameter CO(2) bubbles with a narrow size distribution were generated in an aqueous or organic liquid and subsequently were subjected to temperature changes in the downstream channel. In response to the cooling-heating-cooling cycle the bubbles underwent corresponding contraction-expansion-contraction transitions, which we term 'bubble breathing'. We examined temperature-controlled dissolution of CO(2) in four exemplary liquid systems: deionized water, a 0.7 M aqueous solution of NaCl, ocean water extracted from Bermuda coastal waters, and dimethyl ether of poly(ethylene glycol), a solvent used in industry for absorption of CO(2). The MF approach can be extended to studies of other gases with a distinct, temperature-dependent solubility in liquids.     

Through the channel and around the channel: Validating and comparing microscopic approaches for the evaluation of free energy profiles for ion penetration through ion channels

Microscopic calculations of free energy profiles for ion transport through biological ion channels present a very serious challenge to modern simulation approaches. The main problem is due to the major convergence problems associated with the heterogeneous landscape of the electrostatic environment in ion channels and with the need to evaluate the profile associated with the transfer of the ion from bulk water to the channel environment. This problem is compounded by the lack of reliable and relevant benchmarks that can discriminate between alternative approaches. The present study is aimed at reducing the above problems by defining benchmarks that are directly relevant to ion channels and can also give converging results. This is done by constructing a series of models of a truncated gramicidin channel with different numbers of water molecules and by comparing the profiles for going around the channel and through the channel. These discriminating models are then used to validate and compare the adiabatic charging free energy perturbation (FEP) approach combined with an umbrella sampling approach (Warshel, A. J. Phys. Chem. 1982, 86, 2218) and the potential of mean force (PMF) approach used frequently in studies of ion channels. It is found that both approaches work quite well until one moves to the case of the fully solvated channel. In this limit, the PMF approach may give different results for the overall work of going through the channel and around the channel, while the FEP approach gives physically consistent results. The present benchmark also indicates that the weighted histogram analysis method (WHAM) approach does not offer a significant advantage over earlier approaches at least as much as studies of ion channels are concerned. Finally, it is concluded that the FEP approach may be more useful in evaluating the overall barrier for moving ions from water to ion channels and that in some cases it might be beneficial to use the FEP approach for selective points along the channel and then to connect these points by PMF calculations.     

Improvements in the determination of eight polycyclic aromatic hydrocarbons through a stepwise interlaboratory study approach

A summarising account of a systematic stepwise approach based on interlaboratory studies carried out by a number of laboratories from European Union and EFTA countries is given. This approach has been designed to improve the analytical state of the art in the determination of selected polycyclic aromatic hydrocarbons in different environmental matrices. The approach resulted in a certification exercise to produce a sewage sludge as Certified Reference Material (CRM). The results of the programme showed that HPLC and GC are equally reliable for PAH analysis at submicrogram to microgram per gram levels in various environmental matrices. Major improvements were achieved during the programme, resulting in reduced coefficients of variation and between-laboratory differences. Several recommendations emerging from the programme experience are presented.     

Synthesis and characterization of chiral enantiopure bis-chromanones: a Baylis-Hillman approach

Enantiopure bis-chromanones were prepared from (S)-Binol and bromo esters via a Baylis-Hillman approach. Chiroptical studies indicate that the two-naphthyl units of the chromanone system are non-coplanar.     

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.     

Fourier transform infrared imaging for high-throughput analysis of pharmaceutical formulations

Fourier transform infrared (FTIR) spectroscopic imaging with infrared array detectors has recently emerged as a powerful materials characterization tool. We report a novel application of FTIR imaging for high-throughput analysis of materials under controlled environment. This approach combines the use of spectroscopic imaging with an attenuated total reflection (ATR)-IR cell, microdroplet sample deposition system, and a device that controls humidity inside the cell. By this approach, it was possible to obtain "chemical snapshots" from a spatially defined array of many different polymer/drug formulations (more than 100) under identical conditions. This method provides direct measurement of materials properties for high-throughput formulation design and optimization. Simultaneous response (water sorption, crystallization, etc.) of the array of formulations to the environmental parameters was studied. Implications of the presented approach range from studies of smart polymeric materials and sensors to screening of pharmaceuticals and biomaterials.     

New prodrugs derived from 6-aminodopamine and 4-aminophenol as candidates for melanocyte-directed enzyme prodrug therapy (MDEPT)

Two novel tyrosinase mediated drug delivery pathways have been investigated for the selective delivery of cytotoxic units to melanocytes from urea and thiourea prodrugs. The synthesis of these prodrugs is reported, as well as oximetry data that illustrate that the targets are substrates for tyrosinase. The stability of each of the prodrugs in (i) phosphate buffer and (ii) bovine serum is discussed, and the urea prodrugs are identified as lead candidates for further studies. Finally, HPLC studies and preliminary cytotoxicity studies in a melanotic and an amelanotic cell line, that illustrate the feasibility of the approach, are presented.     

Wastewater-based epidemiology to assess human exposure to personal care and household products – A review of biomarkers,analytical methods,and applications

Humans are nowadays exposed to numerous chemicals in our day-to-day life, including parabens, UV filters, phosphorous flame retardants/plasticizers, bisphenols, phthalates and alternative plasticizers, which can have different adverse effects to human health. Estimating human’s exposure to these potentially harmful substances is, therefore, of paramount importance. Human biomonitoring (HBM) is the existing approach to assess exposure to environmental contaminants, which relies on the analysis of specific human biomarkers (parent compounds and/or their metabolic products) in biological matrices from individuals. The main drawback is its implementation, which involves complex cohort studies. A novel approach, wastewater-based epidemiology (WBE), involves estimating exposure from the analysis of biomarkers in sewage (a pooled urine and feces sample of an entire population). One of the key challenges of WBE is the selection of biomarkers which are specific to human metabolism, excreted in sufficient amounts, and stable in sewage. So far, literature data on potential biomarkers for estimating exposure to these chemicals are scattered over numerous pharmacokinetic and HBM studies. Hence, this review provides a list of potential biomarkers of exposure to more than 30 widely used chemicals and report on their urinary excretion rates. Furthermore, the potential and challenges of WBE in this particular field is discussed through the review of pioneer WBE studies, which for the first time explored applicability of this novel approach to assess human exposure to environmental contaminants. In the future, WBE could be potentially applied as an “early warning system”, which could promptly identify communities with the highest exposure to environmental contaminants.     

Visualizing zeptomole (electro)catalysis at single nanoparticles within an ensemble

The relationship between the structural properties, such as the size and the shape, of a catalytic nanoparticle and its reactivity is a key concept in (electro)catalysis. Current understanding of this relationship is mainly derived from studies involving large ensembles of nanoparticles (NPs). However, the results necessarily reflect the average catalytic behavior of an ensemble, even though the properties of individual particles may vary widely. Here, we demonstrate a novel approach using scanning electrochemical cell microscopy (SECCM) to locate and map the reactivity of individual NPs within an electrocatalytic ensemble, consisting of platinum NPs supported on a single carbon nanotube. Significantly, our studies show that subtle variations in the morphology of NPs lead to dramatic changes in (potential-dependent) reactivity, which has important implications for the design and assessment of NP catalysts. The instrumental approach described is general and opens up new avenues of research in functional imaging, nanoscale electron transfer, and catalysis.     

Acid-catalyzed intramolecular cyclization of N-(4,4-diethoxybutyl)sulfonamides as a novel approach to the 1-sulfonyl-2-arylpyrrolidines

Herein we report our studies on the acid-catalyzed cyclisation of N-(4,4-diethoxybutyl)sulfonamides at the presence of polyatomic phenols as an efficient one-pot approach to the synthesis of 1-sulfonyl-2-arylpyrrolidines from the acyclic precursors.     

Engineering a beta-sheet protein toward the folding speed limit

Recent experimental studies have shown that alpha-helical proteins can approach the folding "speed limit", where folding switches from an activated to a downhill process in free energy. beta-sheet proteins are generally thought to fold more slowly than helix bundles. However, based on studies of hairpins, folding should still be able to approach the microsecond time scale. Here we demonstrate how the hPin1 WW domain, a triple-stranded beta-sheet protein with a sharp thermodynamic melting transition, can be engineered toward the folding "speed limit" without a significant loss in thermal denaturation cooperativity.     

Combined approach for high-throughput preparation and analysis of plasma samples from exposure studies

In drug discovery today, drug exposure is determined in preclinical efficacy and safety studies and drug effects are related to measured concentrations rather than to the administered dose. This leads to a strong increase in the number of bioanalytical samples, demanding the development of higher throughput methods to cope with the increased workload. Here, a combined approach is described for the high-throughput preparation and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of drug levels in plasma samples from the preclinical efficacy and safety studies, i.e. exposure studies. Appropriate pharmacokinetic (PK) compartmental models were fitted to data from PK screening studies in the rat, which were subsequently used to simulate the expected plasma concentrations of the respective exposure studies. Information on the estimated drug concentrations was used to dilute the samples to appropriate concentration levels. A Tecan Genesis RSP liquid handling system was utilized to perform automated plasma sample preparation including serial dilution of standard solutions, dilution of plasma samples, addition of internal standard solution and precipitation with acetonitrile. This robotic sample preparation process permitted two studies of 1-96 samples each to be run simultaneously. To ensure the performance of this method the accuracy and precision for diazepam were examined. Two novel drugs were used to illustrate the suggested approach. In conclusion, our method for sample preparation of exposure samples, based on the combined use of PK simulations, a liquid handling system and a fast LC/MS/MS method, increased the throughput more than three times and minimized the errors, while maintaining the required accuracy and precision.     

Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information-dependent acquisition on a QTrap instrument

Bioanalytical support of plasma pharmacokinetic (PK) studies for drug discovery programs primarily involves the quantitative analysis of dosed compounds using liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/MS/MS) operated in selected reaction monitoring (SRM) mode. However, there is a growing need for information on the metabolism of new chemical entities (NCEs), in addition to the time-concentration profiles from these studies. In this paper, we present a novel approach to not only quantify parent drugs with SRM, but also simultaneously screen for metabolites using a hybrid triple quadrupole/linear ion trap (QqQ(LIT)) instrument. This was achieved by incorporating both the conventional SRM-only acquisition of parent compounds and the SRM-triggered information-dependent acquisition (IDA) of potential metabolites within the same scan cycle during the same LC/MS/MS run. Two test compounds were used to demonstrate the applicability of this approach. Plasma samples from PK studies were processed by simple protein precipitation and the supernatant was diluted with water before injection. The fast scanning capability of the linear ion trap allowed for the information-dependent acquisition of metabolite MS/MS spectra (<1 s/scan), in addition to the collection of adequate data points for SRM-only channels. The MS/MS spectra obtained from potential metabolites in post-dose samples correlated well with the spectra of the parent compounds studied, therefore providing additional confirmatory structure information without the need for repetitive analyses. Relative quantitative time-concentration profiles of identified metabolites were also obtained. Furthermore, this articulated SRM+SRM-IDA approach generated equivalent quantitative results for parent compounds to those obtained by conventional SRM-only analysis. This approach has been successfully used to support discovery PK screening programs.     

Non‐covalent Versus Covalent Control of Self‐Assembly and Chirality of Nile Red‐modified Nucleoside and DNA

A DNA‐based covalent versus a non‐covalent approach is demonstrated to control the optical, chirooptical and higher order structures of Nile red ( Nr ) aggregation. Dynamic light scattering and TEM studies revealed that in aqueous media Nr ‐modified 2′‐deoxyuridine aggregates through the co‐operative effect of various non‐covalent interactions including the hydrogen bonding ability of the nucleoside and sugar moieties and the π‐stacking tendency of the highly hydrophobic dye. This results in the formation of optically active nanovesicles. A left‐handed helically twisted H‐type packing of the dye is observed in the bilayer of the vesicle as evidenced from the optical and chirooptical studies. On the other hand, a left‐handed helically twisted J‐type packing in vesicles was obtained from a non‐polar solvent (toluene). Even though the primary stacking interaction of the dye aggregates transformed from H→J while going from aqueous to non‐polar media, the induced supramolecular chirality of the aggregates remained the same (left‐handed). Circular dichroism studies of DNA that contained several synthetically incorporated and covalently attached Nr ‐modified nucleosides revealed the formation of helically stacked H‐aggregates of Nr but—in comparison to the noncovalent aggregates—an inversed chirality (right‐handed). This self‐assembly propensity difference can, in principle, be applied to other hydrophobic dyes and chromophores and thus open a DNA‐based approach to modulate the primary stacking interactions and supramolecular chirality of dye aggregates.