Self reporting RNA probes as an alternative to cleavable small molecule mass tags

The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker

Acylthioureas as anion transporters: the effect of intramolecular hydrogen bonding

Small molecule synthetic anion transporters may have potential application as therapeutic agents for the treatment of diseases including cystic fibrosis and cancer. Understanding the factors that can dictate the anion transport activity of such transporters is a crucial step towards their application in biological systems. In this study a series of acylthiourea anion transporters were synthesised and their anion binding and transport properties in POPC bilayers have been investigated. The transport activity of these receptors is dominated by their lipophilicity, which is in turn dependent on both substituent effects and the formation and strength of an intramolecular hydrogen bond as inferred from DFT calculations. This is in contrast to simpler thiourea systems, in which the lipophilicity depends predominantly on substituent effects alone

Applications and implementation of modern ultrahigh-performance supercritical fluid chromatography-mass spectrometry

Chromatography and mass spectrometry (MS) are powerful analytical techniques that have seen significant technological advancements over the last 20 years. These have facilitated and enhanced their use as routine tools in many scientific laboratories. Since their introduction, the techniques have been mainly used in isolation with experts specialising in one field only. Recent developments in hyphenating (or coupling) the techniques mean that analysts need to be experts on both sides of the hyphen. i.e., be competent in the fundamental properties of chromatographic separation and all aspects of mass spectrometry. Modern technological advances in the field of ultra-high performance supercritical fluid chromatography (UHPSFC) mean that modern instrumentation is robust enough to be coupled to atmospheric pressure ionisation mass spectrometers. There is increased control of the pressure of the supercritical fluid and since it exhibits the properties of both a liquid and a gas, the optimum separation properties of both are utilised for chromatography. Sometimes referred to as convergence chromatography, the technology converges between liquid and gas chromatography. UHPSFC-MS is often considered as a replacement for normal phase chromatography as the mobile phase is non-polar, but by combining this solvent with a more polar co-solvent a larger range of compounds can be explored. This also means that SFC has the advantage of using the conventional reversed phase (RP) stationary phases such as the C18 to expand the range of compounds analysed. UHPSFC-MS has proved to be a powerful technique in the following application areas: • As a robust tool to be used in an open access environment to fill the analytical gap between RP UHPLC-MS and GC-MS for those unretained, or solvent incompatible samples • To identify and quantify small organic acids and purines in sweat as early markers for the detection of bedsores • As a rapid quality control screening and quantitation protocol for the active ingredients in home-made facemask testing solutions. Additional application areas include the fuel industry where it has been used as an alternative approach to analyse Fatty Acid Methyl Esters (FAMEs) in Aviation turbine fuel and to detect and quantify a new fuel marker (ACCUTRACE™ S10) in diesel fuel to combat fuel laundering. A further quantitation method was developed to analyse elemental sulfur in mineral oil to help understand sulfur-related power transformer failures

Open Access UHPSFC/MS - an additional analytical resource for an academic mass spectrometry facility

RATIONALE: Many compounds submitted for analysis in Chemistry at the University of Southampton either do not retain, elute or ionise using open access RP-UHPLC/MS and required analysis via infusion. An ultra-high performance supercritical fluid chromatography mass spectrometry approach was implemented to afford high through-put analysis of these compounds with chromatographic separation.METHODS: A UPC2-TQD MS system has been incorporated into the open access MS provision within Chemistry at the University of Southampton, using an ESCi source (electrospray and atmospheric pressure chemical ionization) and an atmospheric pressure photoionization (APPI) source. Access to instrumentation is enabled via a web-based interface (RemoteAnalyzer™). RESULTS: Compounds such as fluorosugars, fullerenes, phosphoramidites, porphyrins, and rotaxanes exhibiting properties incompatible with RP-UHPLC/MS have been analysed using automated chromatography and mass spectrometry methods. The speedy return of data enables research in these areas to progress unhindered by sample type. The provision of an electronic web format enables easy incorporation of chromatograms and mass spectra into electronic files and reports. CONCLUSIONS: The implementation of UHPSFC/MS increases access to a wide range of chemistries incompatible with reversed-phase chromatography and polar solvents, enabling more than 90% of submitted samples to be analysed using an open access approach. Further, chromatographic separation is provided where previously flow injection or infusion analysis were the only options.<br/

Development of ultrahigh‐performance liquid chromatography/mass spectrometry and ultrahigh‐performance supercritical fluid chromatography/mass spectrometry assays to determine the concentration of Bitrex™ and sodium saccharin in homemade facemask fit testing solutions

Rationale: Fast and easily transferable chromatography/mass spectrometry assays were required to detect and quantify the amount of Bitrex™ and sodium saccharin in homemade facemask fit testing solutions. Methods: Bitrex™ solutions were analysed using reversed-phase ultrahigh-performance liquid chromatography coupled with positive ion electrospray ionisation mass spectrometry (UHPLC/ESI-MS). Separation was achieved using a mobile phase gradient with an Acquity BEH C18-packed column. Sodium saccharin solutions were analysed using ultrahigh-performance supercritical fluid chromatography coupled with negative ion electrospray ionisation (UHPSFC/ESI-MS). Separation was achieved using isocratic elution with an Acquity UPC 2 Torus Diol packed column and a methanol (25 mM ammonium acetate) co-solvent. Results: The calibration curves obtained using the ratio of the active compound to an internal standard generated linear regression values (R 2) &gt;0.99. Samples analysed prior to and after an autoclave sterilisation process and bottling gave repeatable measurements within 10% of the expected concentration. Conclusions: The two assays afford a fast robust and quantitative analytical method for the detection of the active components used to test the efficacy of the homemade facemask testing solutions. </p

Self-reporting hybridisation assay for miRNA analysis

Hybridisation assays, which are commonly used to analyse oligonucleotides such as siRNAs and miRNAs, often employ detection probes with fluorescent tags. The signal emitted by a fluorescent tag covers a broad range of wavelengths and this limits the multiplexing potential due to overlapping signals. A novel method of indirect oligonucleotide analysis has been developed which combines a hybridisation assay with cleavable small molecule mass tags using HPLC-ESI MS detection. A self-reporting detection probe has been designed which incorporates a DNA/RNA chimeric oligonucleotide sequence in the reporter region, which generates small nucleotide products upon RNase cleavage of the ribose-phosphate backbone. These small nucleotides can then serve as mass tags for the indirect detection of oligonucleotide analytes. The narrow mass range covered by a small molecule mass tag combined with the wide range of possible mass tags provides a high degree of multiplexing potential. This approach has been demonstrated for the analysis of a synthetic miRNA

Lipophilic balance – a new design principle for transmembrane anion carriers

Despite extensive interest in transmembrane anion carriers (anionophores), the factors that govern activity are still only partly understood. Herein we report a study which identifies a new principle for anionophore design, that of “lipophilic balance”. A series of simple thioureas with identical molecular formulae has been prepared and assayed for chloride/nitrate transport activity in synthetic vesicles. The molecules differ only in the positioning of the phenylthiourea binding unit within an 11-carbon linear chain. They are shown to possess very similar lipophilicities and anion affinities, while a test for leaching establishes that they locate almost exclusively in the vesicle membranes. Notwithstanding their close similarities, activities across the series show &gt;5-fold variation, peaking when the phenylthiourea group is centrally located. The results suggest that transport is favoured by a balanced array of lipophilic substituents, possibly because this arrangement facilitates transfer of the complexed anion into the apolar membrane interio

Structural diversity in divalent group 14 triflate complexes involving endocyclic thia-macrocyclic coordination

A highly unusual series of M(II) (M = Ge, Sn, Pb) complexes with endocyclic thioether macrocyclic coordination and with coordination numbers ranging from three to nine have been prepared by the reaction of [9]aneS3 (1,4,7-trithiacyclononane), [12]aneS4 (1,4,7,10-tetrathiacyclododecane), or [24]aneS8 (1,4,7,10,13,16,19,22-octathiacyclotetracosane) with M(OTf)2 (M = Sn and Pb; OTf = CF3SO3-) or with GeCl2·dioxane and 2 mol equiv of TMSOTf (Me3SiO3SCF3) in a mixture of anhydrous CH2Cl2 and MeCN. The isolated bulk products are characterized by 1H, 13C{1H}, 19F{1H}, and 119Sn{1H} NMR and IR spectroscopy, high-resolution ESI+ MS, and microanalytical data. Crystal structures are also reported for [M(L)][OTf]2 (M = Ge, Sn, Pb; L = [9]aneS3, [12]aneS4) and for [M([24]aneS8)][OTf]2 (M = Sn, Pb). In all cases, the ligand is bound in an endocyclic fashion, but the coordination environment and number are highly dependent on the group 14 ion, the macrocyclic ring size, and the number of S-donor atoms it presents. Solution NMR spectroscopic data suggest that the metal-macrocycle coordination is retained in solution but that the triflate anions are extensively dissociated on the NMR timescale. Density functional theory calculations on the [M([9]aneS3)]2+ and [M([12]aneS4)]2+ (M = Ge, Sn, Pb) dications reveal that the HOMO is centered on the group 14 atom as a directional “lone pair”; it also retains a significant amount of positive charge.</p

Different detectors used with SFC

In addition to the common quality attributes of an SFC separation, i.e., resolution, peak shape, repeatability, and transferability, there are other detector-related attributes that need to be recognized for method development or routine operation. For example, response (peak height) and sensitivity required for the analyte and associated impurities will ultimately affect the SFC-detection method choice. Understanding the details of how these detectors operate and why they are desirable for use with SFC is an important part of making the most appropriate choice when considering method development using SFC. Herein, we emphasize the use of SFC with several common detectors. These different detection approaches are a significant testament to the importance of SFC as an analytical tool for many pharmaceutical lab applications.</p

Dispersity determination of poly(ethylene glycol)s using supercritical fluid chromatography-mass spectrometry and different mass analysers

Rationale: Dispersity values are considered critical quality attributes for the quality control of poly(ethylene glycol) formulations due to the direct impact on drug performance. However, when these polymers are analysed using mass spectrometry, the design of the mass analyser can impact the oligomer response and affect the obtained dispersity values, so further understanding is needed. Methods: The deconvoluted electrospray ionisation mass spectra of poly(ethylene glycol)s obtained using supercritical fluid chromatography (SFC) hyphenated to different mass analysers were compared, and visualisation diagrams were used to understand the differences in the dispersity value calculations. Five calibration approaches based on a surrogate single oligomer that represents the whole distribution, or the whole distribution itself, for response selection, were used to evaluate ionisation efficiency prior to quantitation. The impact of using an internal standard (ISTD) on the expanded uncertainty was also assessed. Results: Although there were challenges related to the resolution of multiply charged species when low-resolution instruments were used, similar quantitation capabilities were obtained to those when high-resolution mass analysers were used. Evaluation of approaches using a surrogate oligomer or the whole distribution suggested the independence of both approaches and a constant ionisation efficiency across the oligomer chain length. The higher degree of chromatographic resolution of SFC allowed incorporating a monodispersed ISTD to improve the accuracy and precision of the method. Conclusions: The use of low resolution mass analysers was sufficient to provide accurate and precise dispersity values; however, higher resolution instruments were recommended for characterisation due to the improved mass resolution of ions. The introduction of a monodispersed ISTD improved precision without compromising the calculated dispersity value due to the lack of analyte suppression.</p