Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis

More efficient and faster separation conditions for qualitative as well as quantitative analysis of neuropeptides in human plasma using capillary zone electrophoresis (CZE) have been developed. The analysis method for neuropeptides has been improved specifically to study thyroid hormone related neuropetides for the regulation of thyroid disease. In this study, we investigated the pretreatment methods, composition of the running buffer and rinsing procedures between runs in order to obtain more sensitive and faster separation of trace neuropeptides in plasma by CZE. The tested neuropeptides were somatostatin (SOMA), vasopressin (VP), neurotensin (NT), and thyrotropin-releasing hormone (TRH). Plasma samples were pretreated by deproteinization and solid-phase extraction method. The fraction of neuropeptides was reconstituted in 40% acetonitrile followed by ultrafiltration, and then analyzed by CZE. Resolution and sensitivity was improved using the separation buffer composition with 100 mM Tris-phosphate buffer (pH 2.0) while the sensitivity was further improved via a stacking method using the sample buffer of 40% acetonitrile. These sample pretreatment methods and buffer condition permit quantitative analysis on tested neuropeptides at the 20 ng/mL level. The rinsing procedures between runs using 90% ethanol dramatically shortened the rinsing time to 30 min.     

Development of miniaturized sample preparation with fibrous extraction media

Introducing fine polymeric filaments as the extraction medium, a miniaturized sample preparation technique for micro-column liquid chromatography (micro-LC) has been developed along with the investigation of a reproducible preparation scheme of the extraction capillary. The polymeric filaments were packed longitudinally into either a fused-silica capillary or a polyether ether ketone (PEEK) capillary of appropriate dimensions, and the extraction capillary was installed to the injection valve in micro-LC system. The number of packed filaments should be precisely counted before the packing process to make sure the reproducible preparation of the extraction capillary. With conventional stationary phase materials for open-tubular gas chromatography, polymeric coating to the surface of the filaments was also studied in order to further enhance the extraction performance and selectivity. Coated with the polymeric material suitable for the extraction of particular analyte, a dramatic improvement on the extraction power was obtained. The results suggest that the future possibility of novel tailored fibrous extraction medium with an appropriate coating on it, especially for the analysis of complex sample matrices.     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.     

In-Valve Sample Preparation Cartridge Designed for Microcolumn Liquid Chromatography

Miniaturized sample preparation technique for complex sample matrices has been developed with a polymer-coated fibrous extraction medium. By using extraction cartridge packed longitudinally with polymer-coated fiber into a short PEEK capillary, the extraction efficiency was significantly improved. The extraction cartridge was installed in an injection valve as a sample loop and the on-line coupling with microcolumn liquid chromatography (micro-LC) was demonstrated. This on-line coupled sample preparation/separation system showed a good validity for the analysis of phthalates and tricyclic antidepressant drugs in typical water samples. The lowest limits of quantification for several phthalates and tricyclic antidepressants (TCA) drugs were less than 1 ng mL^?1. The effect of polymeric coating on the filament surface on the extraction power was also investigated.     

Short metal capillary columns packed with polymer-coated fibrous materials in high-temperature gas chromatography

The high-temperature gas chromatographic (GC) separation of several semivolatile compounds is studied with a short metal capillary column packed with fibrous material, having a polydimethylsiloxane coating thereon. Taking advantage of the excellent heat-resistance of the fiber and also the combination of the surface-deactivated metal capillary, a temperature-programmed separation up to 450 degrees C is successfully demonstrated for the separation of polymer standard samples. The average molecular weight of the commercially-available polymer standard samples for size exclusion chromatography (SEC) is estimated by high-temperature GC analysis and compared with the nominal value determined by a conventional SEC method. Although a slight deviation for the number-average molecular weight is observed between the GC and SEC analysis, the data for the weight-average molecular weight shows a good agreement in these methods. The results also suggest the future possibility of the fiber-packed metal capillary as a miniaturized GC column with an increased sample loading capacity.     

Decidability of Irreducible Tree Shifts of Finite Type

Journal of Statistical Physics - We consider N particles in the plane, influenced by a general external potential, that are subject to the Coulomb interaction in two dimensions at inverse...     

Highly enantioselective Michael addition of cyclic ketones to chalcones catalyzed by pyrrolidine-based imides

An efficient procedure for asymmetric Michael addition reaction of cyclic ketones with low activated chalcones catalyzed by pyrrolidine-based phthalimide and 1,8-Naphthalimide catalysts was developed. The corresponding products were obtained in high yields with high diastereoselectivities (up to 99:1 dr) and high enantioselectivities (up to 96% ee) under mild conditions.     

Synthesis and in vitro Fungicidal Activity of a Series of Novel N-(Heterocycly)phthalimides

A series of novel phthalimides was prepared and tested for antifungal activity in vitro against seven phytopathogenic fungi:Rhizoctonia solani, Fusarium oxysporum f.sp. cucumerinum Owen., Botrytis cinerea, Fulvia fulva, Sclerotinia sclerotiorum de Bary, Phytophthora capsici, Botryosphaeria berengeriama. The results showed that those compounds holding the structure of 6‐methylpyridin‐2‐yl exhibited good activity against Phytophthora capsici and Botrytis cinerea.