Use of thermospray liquid chromatography-mass spectrometry to aid in the identification of urinary metabolites of a novel antiepileptic drug, Lamotrigine.

The use of thermospray liquid chromatography-mass spectrometry allowed the structural elucidation of a number of urinary metabolites of Lamotrigine, 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine, formed after administering the drug to man, Cynomolgus monkey and rabbit. This data when combined with the data obtained from high-performance liquid chromatography with radiochemical detection enabled us to determine the types and amounts of unchanged drug and metabolites excreted in urine by man and a number of laboratory animal species. This technique was particularly useful as it highlighted a previously unknown fact that Lamotrigine is metabolised to form two different N-glucuronides, one of which is resistant to cleavage in vitro by a crude beta-glucuronidase preparation from Helix pomatia.     

A novel ATR-FTIR approach for characterisation and identification of ex situ immobilised species.

We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR-FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMAC) chips containing about 10 mum thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR-FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)-tagged synthetic peptides of 25 (25-His6) and 48 (48-His6) amino acids to the NTA-groups and 3) attachment of a ligand, mesyl amide, to the surface-bound 48-His6. Despite interference from H(2)O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of approximately 100 mg mL(-1) and a dissociation constant of 116+/-11 muM, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR-FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm(-1). Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand-alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set-ups.     

Structural elucidation of N‐oxidized clemastine metabolites by liquid chromatography/tandem mass spectrometry and the use of Cunninghamella elegans to facilitate drug metabolite identification

Cunninghamella elegans is a filamentous fungus that has been shown to biotransform drugs into the same metabolites as mammals. In this paper we describe the use of C. elegans to aid the identification of clemastine metabolites since high concentrations of the metabolites were produced and MSⁿ experiments were facilitated. The combination of liquid chromatography and tandem mass spectrometry with two different ionization techniques and hydrogen/deuterium exchange were used for structural elucidation of the clemastine metabolites. Norclemastine, four isomers of hydroxylated clemastine, and two N‐oxide metabolites were described for the first time in C. elegans incubations. The N‐oxidations were confirmed by hydrogen/deuterium exchange and deoxygenation (?16 Da) upon atmospheric pressure chemical ionization mass spectrometry. By MSⁿ fragmentation it was concluded that two of the hydroxylated metabolites were oxidized on the methylpyrridyl moiety, one on the aromatic ring with the chloro substituent, and one on the aromatic ring without the chlorine. Copyright © 2010 John Wiley & Sons, Ltd.     

A new interpretation of chlorine leaving group kinetic isotope effects; a theoretical approach

The chlorine leaving group kinetic isotope effects (KIEs) for the S(N)2 reactions between methyl chloride and a wide range of anionic, neutral, and radical anion nucleophiles were calculated in the gas phase and, in several cases, using a continuum solvent model. In contrast to the expected linear dependence of the chlorine KIEs on the C(alpha)-Cl bond order in the transition state, the KIEs fell in a very small range (1.0056-1.0091), even though the C(alpha)-Cl transition state bond orders varied widely from approximately 0.32 to 0.78, a range from reactant-like to very product-like. This renders chlorine KIEs, and possibly other leaving-group KIEs, less useful for studies of reaction mechanisms than commonly assumed. A partial explanation for this unexpected relationship between the C(alpha)-Cl transition state bond order and the magnitude of the chlorine KIE is presented.     

A high-resolution accurate mass approach to identification of graveoline metabolites using ultra-high-performance liquid chromatography combined with a photo diode array detector and quadrupole/time-of-flight tandem mass spectrometry

Graveoline is a biologically active ingredient extracted from Ruta graveolens. Current work aimed at investigating in vitro metabolism of graveoline using rat or human liver microsomes and hepatocytes. Graveoline (20 μM) was incubated with nicotinamide adenine dinucleotide phosphate–supplemented rat and human liver microsomes as well as hepatocytes. LC coupled to a photo diode array detector and quadrupole/time-of-flight tandem mass spectrometry was used to detect and identify the metabolites. The structures of the metabolites were identified by accurate mass, elemental composition, and indicative fragment ions. A total of 12 metabolites, comprising 6 phase I and 6 phase II metabolites, were obtained. The metabolic pathways included demethylenation, demethylation, hydroxylation, glucuronidation, and glutathion conjugation. The metabolite (M10) produced by opening the ring of the methylenedioxyphenyl moiety was detected as the most abundant in both liver microsomes and hepatocytes, mainly catalyzed by CYP1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. This study provides valuable information on the in vitro metabolism of graveoline, which is indispensable for further development and safety evaluation of this compound.     

A novel approach for the homogenization of cellulose to use micro‐amounts for stable isotope analyses

Climate reconstructions using stable isotopes from tree‐rings are steadily increasing. The investigations concentrate mostly on cellulose due to its high stability. In recent years the available amount of cellulose has steadily decreased, mainly because micro‐structures of plant material have had to be analyzed. Today, the amounts of cellulose being studied are frequently in the milligram and often in the microgram range. Consequently, homogeneity problems with regard to the stable isotopes of carbon and oxygen from cellulose have occurred and these have called for new methods in the preparation of cellulose for reliable isotope analyses. Three different methods were tested for preparing isotopically homogenous cellulose, namely mechanical grinding, freezing by liquid nitrogen with subsequent milling and ultrasonic breaking of cellulose fibres. The best precision of isotope data was achieved by freeze‐milling and ultrasonic breaking. However, equipment for freeze‐milling is expensive and the procedure is labour‐intensive. Mechanical grinding resulted in a rather high loss of material and it is also labour‐intensive. The use of ultrasound for breaking cellulose fibres proved to be the best method in terms of rapidity of sample throughput, avoidance of sample loss, precision of isotope results, ease of handling, and cost. Copyright © 2009 John Wiley & Sons, Ltd.     

A new approach to the chiral separation of novel diazenes

Two new types of potential liquid‐crystalline azo compounds were synthesized in the form of racemic mixtures and as the individual S enantiomers. Both materials consisting of two substituted aromatic rings in the molecular core and one chiral center at the aliphatic moiety showed mesomorphic behavior. The separation of the R and S enantiomers by chiral high‐performance liquid chromatography was unsuccessful when the azo compounds were in their natural E state. However, the irradiation of the compounds in the solution by UV light led to an almost quantitative transition to their Z forms. The chromatographic behavior of the compounds in their Z forms was significantly different, and partial separation of the individual enantiomers on chiral polysaccharide‐based stationary phases was obtained. Thus, the proposed procedure represents a novel approach to the enantioseparation of chiral diazenes.     

Semi‐supervised kernel partial least squares fault detection and identification approach with application to HGPWLTP

In this paper, fault detection and identification methods based on semi‐supervised Laplacian regularization kernel partial least squares (LRKPLS) are proposed. In Laplacian regularization learning framework, unlabeled and labeled samples are used to improve estimate of data manifold so that one can establish a more robust data model. We show that LRKPLS can avoid the over‐fitting problem which may be caused by sample insufficient and outliers present. Moreover, the proposed LRKPLS approach has no special restriction on data distribution, in other words, it can be used in the case of nonlinear or non‐Gaussian data. On the basis of LRKPLS, corresponding fault detection and identification methods are proposed. Those methods are used to monitor a numerical example and Hot Galvanizing Pickling Waste Liquor Treatment Process (HGPWLTP), and the cases study show effeteness of the proposed approaches. Copyright © 2016 John Wiley & Sons, Ltd.     

Microstructure and melting in fractions of polyethylene characterized with the aid of a novel folded-chain-lamella model

Folded-chain-lamellae are considered as “cooperative units” with frozen matter flow and with a locally heterogeneous atomistic order the degree of which is reversibly regulated by surface-melting or surface crystallization. A consequent thermodynamic treatment of restricted micro-phase equilibrium in semicrystalline fractions of polyethylene of different molecular weights can be shown to deliver a characterization of such nonequilibrium states by relating additional internal variables to parameters of the microstructure (DSC-measurements, electron-micrographs, SAXS). What appears to be very satisfactory is the conclusion that the excess situation produced by noncrystallizable defects in the semicrystalline samples is clearly related to the original state of order in the melt: Approximative invariance of penetration is directly elucidating that any matter flow in the regime of the chains of sufficient length is frozen during folded-chain crystallization. Molecular-weight effects in semicrystalline fractions of polyethylene can be interpreted on these lines.     

Exact mass measurement of product ions for the structural elucidation of drug metabolites with a tandem quadrupole orthogonal-acceleration time-of-flight mass spectrometer

We herein report upon an approach whereby the interpretation of tandem mass spectrometry spectra can be both expedited and simplified via the accurate mass assignment of product ions utilizing a tandem quadrupole time-of-flight mass spectrometer (QqTOF). The applicability of the QqTOF in the drug metabolism laboratory is illustrated by the elucidation and differentiation of the dissociative pathways for Bosentan and its hydroxylated and demethylated metabolites. Target analyte fragmentation mechanisms were readily achieved by the measurement of product ions with a mass accuracy <5 ppm, possible by single-point internal recalibration using the residual precursor ion as calibrant. Differentiation of both precursor and product ions from nominally isobaric matrix species derived from biological extracts is demonstrated by operation of the QqTOF at resolutions of 8000 (m/Δm_FWHM).     

Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC‐CI‐MS/MS: a methandienone case study

Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC‐CI‐MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long‐term metabolite (18‐nor‐17β‐hydroxymethyl,17α‐methyl‐androst‐1,4,13‐trien‐3‐one) could be detected up to 26 days by using GC‐CI‐MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long‐term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright © 2016 John Wiley & Sons, Ltd.     

Speciation analysis of bromine-containing drug metabolites in feces samples from a human in vivo study by means of HPLC/ICP-MS combined with on-line isotope dilution

The aim of this work was speciation analysis of metabolites in feces samples collected within a clinical study during which a bromine-containing anti-tuberculosis drug (TMC207) was administered to patients with multi-drug resistant tuberculosis infection. Owing to slow elimination of the drug, no ¹⁴C label was used within this study. Quantification of the bromine species was accomplished using high performance liquid chromatography coupled to inductively coupled plasma–mass spectrometry (HPLC/ICP-MS) in combination with on-line isotope dilution (on-line ID), while structural elucidation of the species was performed using HPLC coupled to electrospray ionization–mass spectrometry. The ICP-MS-based method developed shows a good intra- and inter-day reproducibility (relative standard deviation = 3.5%, N = 9); the limit of detection (1.5 mg TMC207 L⁻¹) is of the same order of magnitude as that for HPLC/radiodetection; the dynamic range of the method covers more than two orders of magnitude. Furthermore, the column recovery was demonstrated to be quantitative (recoveries between 90.6% and 99.5%). Based on the excellent figures of merit, the “cold” HPLC/ICP-MS approach could be deployed for the actual human in vivo metabolism study, such that exposure of the human volunteers to the ¹⁴C radiolabel was avoided.     

Preparation and characterization of a novel magnetized nanosphere as a carrier system for drug delivery using Plantago ovata Forssk. hydrogel combined with mefenamic acid as the drug model

The aim of the present study was to magnetize Plantago ovata Forssk. hydrogel and produce a nanosphere system to carrier mefenamic acid as the drug model. For this propose, P. ovata seeds hydrogel (POSH) was extracted and magnetized by Fe₃O₄ being functionalized using tetraethyl orthosilicate and trimethoxyvinysilane. Thereafter, mefenamic acid (MFA) was loaded on the carrier system. The final product, as the magnetic drug loaded nanosphere (Fe/POSH/MFA), was fully characterized through different techniques involving X-ray diffraction (XRD), scanning electron microscopy (SEM), vibrating-sample magnetometer (VSM), thermal gravimetric analysis (TGA), dynamic light scattering (DLS), and FT-IR spectroscopy. The results confirmed the successful production of the drug loaded nanosphere system with particles magnetization of 25 emu/g over a range size of 40–50 nm. However, the size distribution less than 100 nm was measured through DLS analysis. The hydrogel showed a pH sensitivity swelling behavior representing the best efficacy at pH 7.4. The efficiency of the drug encapsulation was found to be 64.35%. The drug releasing was studied using a dialysis bag at pH = 7.4. The highest in vitro drug releasing was found to be 57.3 ± 0.6% after 72 h, as well. The findings of the current report account for the potential use of P. ovata hydrogel as an effective delivery system for encapsulation of water insoluble basic drugs, e.g., MFA in a magnetized carrier system.     

A chemometric approach based on a novel similarity/diversity measure for the characterisation and selection of electronic nose sensors

Electronic nose sensor signals provide a digital fingerprint of the product in analysis, which can be subsequently investigated by means of chemometrics. In this paper, the fingerprint characterisation of electronic nose data has been studied by means of a novel chemometric approach based on the partial ordering technique and the Hasse matrix. This matrix can be associated to each data sequence and the similarity between two sequences can be evaluated with the definition of a distance between the corresponding Hasse matrices. Since all the signals achieved along time are intrinsically ordered, the data provided by electronic nose can be also considered as sequential data and consequently characterized by means of the proposed approach. The similarity/diversity measure has been here applied in order to characterize the class discrimination capability of each electronic nose sensor: extra virgin olive oil samples of different geographical origin have been considered and Hasse distances have been used to select the sensors which appear more able to discriminate the olive oil origins. The distance based on the Hasse matrix has showed some useful properties and proved to be able to link each electronic nose time profile to a meaningful mathematical term (the Hasse matrix), which can be consequently studied by multivariate analysis.     

A new approach to the coordination of anions,novel polycobalticinium macrocyclic receptor molecules

The syntheses and electrochemistry of four novel polycobalticinium macrocyclic receptor molecules are reported. Preliminary anion coordination studies in one case reveal the receptor to be redox responsive to the binding of a Br⁻ guest anion, shifting the respective cobalticinium reduction wave to more negative potentials.     

A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase

We report a new structure-based strategy for the identification of novel inhibitors. This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall. The enzyme catalyzes the racemization of l- and d-alanine using pyridoxal 5-phosphate (PLP) as a cofactor. The restriction of AlaR to bacteria and some fungi and the absolute requirement for d-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design. Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects.This article describes the development of a receptor-based pharmacophore model for AlaR, taking into account receptor flexibility (i.e. a `dynamic' pharmacophore model). In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1sft) with a d-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer. The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program. Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing.The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets.     

Quindolinocryptotackieine: the elucidation of a novel indoloquinoline alkaloid structure through the use of computer‐assisted structure elucidation and 2D NMR

Numerous indoloquinoline alkaloid structures have been identified from extracts of the West African plant Cryptolepis sanguinolenta. Recently, through the use of 2D NMR methods and cryogenic NMR probe technology in conjunction with computer‐assisted structure elucidation (CASE) methods, the structures of some chemical degradation products of this family of alkaloids have also been reported. We now report the characterization of a novel indoloquinoline dimeric alkaloid, quindolinocryptotackieine, through the extensive utilization of CASE methods. The NMR data presented here were collected over a decade earlier before the elucidation of the structure was possible, since manual analysis did not present a conclusive structure, whereas CASE produced a series of structures from which the structure could be verified. The original mass spectrometric (MS) data collected for the sample were problematic. Contemporary MS data were instead recollected from remaining small quantities of this alkaloid using modern instrumentation. The re‐collected data gave a usable molecular ion and several key fragment ions that were diagnostically useful. Copyright © 2003 John Wiley & Sons, Ltd.     

A chemometric approach to the historical and geographical characterisation of different terracotta finds

The development of modern analytical instrumental techniques has allowed to solve a lot of problems concerning all kinds of disciplines, but often we can obtain for the analysed samples very numerous data, obtained by means of analytical techniques. So the effort of the analysts is more and more paid on the elaboration of a so high number of data. We can find a typical example in the classification and study of archaeological finds. In the archaeometry, that is the application of scientific methods and analysis techniques to archaeological issues, one of the most important step is the statistical elaboration of multivariate data obtained by physical and chemical analysis of ancients artefacts. This study was carried out on 20 different pottery samples coming from different periods. The 20 analysed terracotta finds come from 4 different archaeological sites, three Italian and one Libyan. For the analysis we used ICP–AES, thermogravimetric (TG) and thermomechanical (TMA) techniques; the main technique used to elaborate the data was the Principal Component Analysis (PCA). We already knew, approximately, the “burning age” of the finds and we had to check if some eigenvector of Principal Components Analysis could fit with that age. Few milligrams of finds were used for ICP–AES, TGA and TMA analysis obtaining, after a reduction, a matrix of 11 variables. The results show a good correlation between age and PC1.