Attomole protein analysis by CIEF with LIF detection

We have coupled CIEF with an LIF detector that is based on a post‐column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the ε‐amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503‐labeled β‐lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.     

Minimal surfaces over stars

A JS surface is a minimal graph over a polygonal domain that becomes infinite in magnitude at the domain boundary. Jenkins and Serrin characterized the existence of these minimal graphs in terms of the signs of the boundary values and the side-lengths of the polygon. For a convex polygon, there can be essentially only one JS surface, but a non-convex domain may admit several distinct JS surfaces. We consider two families of JS surfaces corresponding to different boundary values, namely JS₀ and JS₁, over domains in the form of regular stars. We give parameterizations for these surfaces as lifts of harmonic maps, and observe that all previously constructed JS surfaces have been of type JS₀. We give an example of a JS₁ surface that is a new complete embedded minimal surface generalizing Scherk's doubly periodic surface, and show also that the JS₀ surface over a regular convex 2n-gon is the limit of JS₁ surfaces over non-convex stars. Finally we consider the construction of other JS surfaces over stars that belong neither to JS₀ nor to JS₁.     

Rapid determination of aristolochic acids I and II in herbal products and biological samples by ultra-high-pressure liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs) are a mixture of structural-related compounds, in which aristolochic acid I (AA I) and aristolochic acid II (AA II) are reported to be correlated with Aristolochic acid nephropathy (AAN). In this work, a rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine AA I and AA II in herbal products and biological fluids. By using gradient elution with a mobile phase composed of a mixture of 10 mM ammonium formate buffer (pH 3.0) and acetonitrile, AAs could be determined within 10 min. Under optimum UHPLC-MS/MS conditions, the limit of detections was 0.14 and 0.26 ng mL⁻¹ for AA I and AA II, respectively. Run-to-run repeatability and intermediate precision of peak area for AA I and AA II were less than 5.74% relative standard deviation (RSD). Accuracy was tested by spiking 10, 100 and 1000 ng mL⁻¹ in rat serum and the recoveries were within 76.5-92.9%. Matrix effects were within 78.8-127.7%. The developed method was successfully applied to determine AA I and AA II in several herbal products and to investigate their pharmacokinetic behavior in female Wister rats. The result shows that the developed UHPLC-MS/MS method is efficient, sensitive, and accurate for the determination of AA I and AA II in herbal products and biological samples.     

C-H bond functionalization in the synthesis of fused 1,2,3-triazoles

A highly modular approach to fused 1,2,3-triazoles has been developed featuring a one-pot procedure combining copper(I) catalyzed azide-alkyne cycloaddition and palladium-catalyzed C-H bond functionalization. A class of structurally unique heterocycles was synthesized in good yields.     

Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system

We report a multiplexed capillary electrophoresis system employing an array of 32 capillaries with a micromachined sheath-flow cuvette as the detection chamber. The sample streams were simultaneously excited with a 473-nm laser beam, and the fluorescence emission was imaged on a CCD camera with a pair of doublet achromat lens. The instrument produced mass detection limits of 380 ± 120 yoctomoles for fluorescein in zone electrophoresis. Capillary isoelectric focusing of fluorescent standards produced peaks with an average width of 0.0029 ± 0.0008 pH. Capillary coating stability limits the reproducibility of the analysis.     

The unique role of the nitro group in intramolecular interactions: chloronitromethanes

We have extended an earlier crystallographic/computational study which revealed an exceptionally short C–Cl bond in chlorotrinitromethane, Cl–C(NO₂)₃. We show that the C–Cl bond length progressively decreases when NO₂ groups are introduced into chloromethane. This is attributed to intramolecular attractive interactions between the chlorine and the closest NO₂ oxygens. Computed electrostatic potentials on molecular surfaces support this interpretation, as well as the N–O interactions between neighboring NO₂ groups that help to determine the molecular conformations. The calculated C–Cl bond energies decrease as the NO₂ groups are added, which is expected, but means that the usual inverse relationship between bond energy and bond length is not being obeyed. For purposes of comparison, the computational analyses, which were primarily at the B3PW91/6-311G(3d,2p) level, were also carried out for the corresponding chlorocyanomethanes and chlorofluoromethanes. These do not show anomalously short C–Cl bond distances.     

Global QSAR models of skin sensitisers for regulatory purposes

Background

The new European Regulation on chemical safety, REACH, (Registration, Evaluation, Authorisation and Restriction of CHemical substances), is in the process of being implemented. Many chemicals used in industry require additional testing to comply with the REACH regulations. At the same time EU member states are attempting to reduce the number of animals used in experiments under the 3 Rs policy, (refining, reducing, and replacing the use of animals in laboratory procedures). Computational techniques such as QSAR have the potential to offer an alternative for generating REACH data. The FP6 project CAESAR was aimed at developing QSAR models for 5 key toxicological endpoints of which skin sensitisation was one.

Results

This paper reports the development of two global QSAR models using two different computational approaches, which contribute to the hybrid model freely available online.

Conclusions

The QSAR models for assessing skin sensitisation have been developed and tested under stringent quality criteria to fulfil the principles laid down by the OECD. The final models, accessible from CAESAR website, offer a robust and reliable method of assessing skin sensitisation for regulatory use.