Quality assurance in immunoassay performance—comparison of different enzyme immunoassays for the determination of caffeine in consumer products

Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3′,5,5′-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.

Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.     

Novel combination of non-aqueous capillary electrophoresis and multivariate curve resolution-alternating least squares to determine phenolic acids in virgin olive oil

This paper presents the development of a non-aqueous capillary electrophoresis method coupled to UV detection combined with multivariate curve resolution-alternating least-squares (MCR-ALS) to carry out the resolution and quantitation of a mixture of six phenolic acids in virgin olive oil samples. p-Coumaric, caffeic, ferulic, 3,4-dihydroxyphenylacetic, vanillic and 4-hydroxyphenilacetic acids have been the analytes under study. All of them present different absorption spectra and overlapped time profiles with the olive oil matrix interferences and between them. The modeling strategy involves the building of a single MCR-ALS model composed of matrices augmented in the temporal mode, namely spectra remain invariant while time profiles may change from sample to sample. So MCR-ALS was used to cope with the coeluting interferences, on accounting the second order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the data herein analyzed. The method was firstly applied to resolve standard mixtures of the analytes randomly prepared in 1-propanol and, secondly, in real virgin olive oil samples, getting recovery values near to 100% in all cases. The importance and novelty of this methodology relies on the combination of non-aqueous capillary electrophoresis second-order data and MCR-ALS algorithm which allows performing the resolution of these compounds simplifying the previous sample pretreatment stages.     

Alkylation of 3‐Phenyl‐1H‐pyrazolo[4,3‐c] quinoline: Theoretical Analysis of Regioselectivity

The interaction of 3‐phenyl‐1H‐pyrazolo[4.3‐c]quinoline with alkylating agents in the presence of NaH leads mainly to N1‐substituted derivatives. The regioselectivity of the reaction has been studied using AM1 semi‐empirical calculations in the isolated molecule approximation.     

The 3-Methyl-1,3-butadienyl Phenyl Sulfoxide I. Preparation and Some Properties

The title sulfoxide 5 is worthy of interest because of its isoprenoid structure and its synthetic potentialities¹     

Reactions of 1,2-Epoxy-2-Methyl-3-butene with Lithio Derivatives of Allylic Sulfoxides. Syntheses of 2,6-Dimethylocta-3,7-dien-2,6-diol and of 5,8-Dehydro-4-0xo-nerolidol

Our preceding studies have shown that lithio carbanions of protected cyanohydrins can undergo Michael additions to an isoprenic sulfoxide to yield lithio derivatives of allylic sulfoxides¹. These results have led us to investigate the utilization of these lithio sulfoxides for new simple syntheses of aliphatic terpenes by head-to-tail linking of functionalized isoprenoid units. For this purpose, 1,2-epoxy-2-methyl-3-butene 2 was one especially appropriate electro-phile, but at the outset of our work, there had been very few investigations of the reactivity of lithio carbanions of allylic sulfoxides with epoxides²     

Production of Antibodies and Development of Specific Polarization Fluoroimmunoassay for Acetochlor

Specific polyclonal antibodies towards acetochlor (2-chloro- N -(ethoxymethyl)- N -(2-ethyl-6-methylphenyl)acetamide) were obtained from rabbits immunized against a 3-mercaptopropionic acid derivative of acetochlor, covalently attached to bovine serum albumin. A polarization fluoroimmuoassay (PFIA) based on these antibodies was developed and optimized to detect acetochlor in water samples. The optimized PFIA had a detection limit of 9 µg/L, linear working range from 50 to 5500 µg/L and within-assay coefficient of variation less than 4%. Cross-reactivity studies demonstrated that these antibodies are capable of specific detection of acetochlor amongst structurally related chloroacetanilide herbicides. Assay cross-reactivity values were: alachlor 0%, metolachlor 2.4%, propachlor 0%, butachlor 0.2% and dimethachlor 0.5%. Five organic solvents commonly used in sample extraction were evaluated for their effect on acetochlor PFIA performance, and methanol and ethanol were found to be compatible with the assay up to 10% v/v.     

Well‐Defined Molecular Magnesium Hydride Clusters: Relationship between Size and Hydrogen‐Elimination Temperature

A new tetranuclear magnesium hydride cluster, [{ NN ‐(MgH)₂}₂], which was based on a N? N‐coupled bis‐β‐diketiminate ligand ( NN ^2?), was obtained from the reaction of [{ NN ‐(MgnBu)₂}₂] with PhSiH₃. Its crystal structure reveals an almost‐tetrahedral arrangement of Mg atoms and two different sets of hydride ions, which give rise to a coupling in the NMR spectrum (J=8.5 Hz). To shed light on the relationship between the cluster size and H₂ release, the thermal decomposition of [{ NN ‐(MgH)₂}₂] and two closely related systems that were based on similar ligands, that is, an octanuclear magnesium hydride cluster and a dimeric magnesium hydride species, have been investigated in detail. A lowering of the H₂‐desorption temperature with decreasing cluster size is observed, in line with previously reported theoretical predictions on (MgH₂)_n model systems. Deuterium‐labeling studies further demonstrate that the released H₂ solely originates from the oxidative coupling of two hydride ligands and not from other hydrogen sources, such as the β‐diketiminate ligands. Analysis of the DFT‐computed electron density in [{ NN ‐(MgH)₂}₂] reveals a counterintuitive interaction between two formally closed‐shell H^? ligands that are separated by 3.106 Å. This weak interaction could play an important role in H₂ desorption. Although the molecular product after H₂ release could not be characterized experimentally, DFT calculations on the proposed decomposition product, that is, the low‐valence tetranuclear Mg(I) cluster [( NN ‐Mg₂)₂], predict a structure with two almost‐parallel, localized Mg? Mg bonds. As in a previously reported β‐diketiminate Mg^I dimer, the Mg? Mg bond is not characterized by a bond critical point, but instead displays a local maximum of electron density midway between the atoms, that is, a non‐nuclear attractor (NNA). Interestingly, both of the NNAs in [( NN ‐Mg₂)₂] are connected through a bond path that suggests that there is bonding between all four Mg^I atoms.     

Unzipping Nucleoside Channels by Means of Alcohol Disassembly

Gold nanoparticles capped with simple adenosine derivatives can form colloidal aggregates in nonpolar solvents. Theoretical calculations indicate the formation of organic channels by the supramolecular assembly of the nanoparticles by means of hydrogen bonds between the adenine moieties. The aggregates were only negligibly sensitive to nPrOH, iPrOH, and tBuOH, whereas some showed a similar response to MeOH and EtOH, and others showed high selectivity toward MeOH. DNA nucleoside derivatives (1‐(2‐deoxy‐β‐D ‐ribofuranosyl)‐5‐methyluracil and 2′,3′‐O‐isopropylideneadenosine) as well as thymine and other aromatic compounds such as pyrene derivatives (pyrene, 1‐chloropyrene, 1‐hydroxypyrene, (1‐pyrenyl)methanol, and 2‐hydroxynapthalene) did not induce disassembly of the nanoparticle aggregates. Data suggest that the nucleoside channels allow access to alcohols according to their size, and an efficient interaction between the alcohol and the adenine units destabilizes the hydrogen bonds, which eventually leads to nanoparticle disassembly.     

An improved capillary electrophoresis method for in vitro monitoring of the challenging early steps of Aβ1–42 peptide oligomerization: Application to anti‐Alzheimer's drug discovery

We report an improved CE method to monitor in vitro the self‐assembly of monomeric amyloid β‐peptide (42 amino acids amyloid β‐peptide, Aβ_1–42) and in particular the crucial early steps involved in the formation of the neurotoxic oligomers. In order to start the kinetics from the beginning, sample preparation was optimized to provide samples containing exclusively the monomeric form. The CE method was also improved using a dynamic coating and by reducing the separation distance. Using this method, the disappearance of the monomer as well as the progressive formation of four species during the self‐assembly process can now be monitored and quantified over time. The hydrodynamic radius of the species present at the initial kinetics step was estimated around 1.8 nm by Taylor dispersion analysis while SDS‐PAGE analyses showed the predominance of the monomer. These results confirmed that the Aβ_1–42 species present at this initial time was the monomer. Methylene blue, an anti‐Alzheimer disease candidate, was then evaluated. In spite of an oligomerization inhibition, the enhanced disappearance of the Aβ_1–42 monomer provoked by methylene blue was demonstrated for the first time. This method, allowing the monomeric and smallest oligomeric species to be monitored, represents a new accurate and precise way to evaluate compounds for drug discovery.