An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution. In this study, we evaluated the Labcyte Echo 525 liquid handler for its ability to deliver small volumes of sample preparations in biological matrix (plasma and serum) and to assess the feasibility of integration of the Echo with three types of bioanalytical assay platforms: microplate enzyme-linked immunosorbent assay, Gyrolab immunoassay, and liquid chromatography with tandem mass spectrometry. The results demonstrated acceptable consistency of dispensed plasma samples from multiple lots and species by the Echo. Equivalent assay performance demonstrated between the Echo and manual liquid procedures indicated great potential for the integration of the Echo with the bioanalytical assay, which allows the successful implementation of microsampling strategies in drug discovery and development.
This chapter focuses on the ordinarily used sample preparation strategies in drug research and development, as these are more applicable and practical to be used in real studies. For small molecule analytes, protein precipitation (PPT), liquid-liquid extraction (LLE), and solid-phase extraction (SPE) are the most commonly used sample preparation methods. Improving sensitivity has become a critical factor for the wider application of liquid chromatography-mass spectrometry (LC-MS) in protein bioanalysis. Especially, protein biomarkers are often present at low levels, demanding superior sensitivity for quantitative biomarker assays. There are three major components for an LC-MS bioanalytical assay: sample preparation, LC separation, and MS detection. Hydrophilic interaction LC (HILIC) can also provide greatly improved sensitivity and has been widely used the analysis of polar compounds. Post-column addition of organic solvent or modifier is an useful approach to improve the MS response of the analyte.
Substitution reactions of arsenazo III [3,6-bis(o-arsonophenylazo)-4,5-dihydroxynaphthalene-2,7-disulfonic acid] complexes of EuIII with the polyaminecarboxylates dtpa (diethylenetriaminepentaacetate) and edta (ethylenediaminetetraacetate) were studied as a function of polyaminecarboxylate concentration, pH, temperature, pressure and ionic strength by stopped-flow spectrophotometry within the acidity range 3.61 < pH < 5.56 and between 17.5 and 40 °C. Under all experimental conditions two consecutive steps (k1 and k2) were observed over different time-scales. For the fast step (k1) the substitution rate of EuIIIL2(H2O)(L = arsenazo III) by dtpa and edta (L′) increases with increasing acidity of the medium, k1=ka+kb[H+], and depends on the L′ concentration, i.e. k1=kc+kd[L′]. For the slow step (k2) plots of k2versus[H+] are linear and exhibit no intercept and k2 is independent of L′ concentration. Ionic strength studies indicated no significant dependence of k1 and k2 on [NaClO4]. Rate constant k1 increases with increasing pressure, and there is a very good linear relationship between ln k1 and pressure at any temperature, ligand concentration, pH and ionic strength. In contrast, k2 exhibits almost no pressure dependence for any temperature, pH and ligand concentration. It is suggested that this latter step involves acid-catalysed dechelation of arsenazo III accompanied by chelation of L′. In terms of intrinsic volume changes, bond formation and breakage contributions seem to cancel in order to account for the zero ΔV2‡ values. The substitution of arsenazo III by dtpa on EuIII is much faster than the same process on GdIII.
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