Stability-indicating UHPLC and TLC-densitometric Methods for the Determination of Tiamulin Fumarate

Two novel stability-indicating methods were developed for the determination of tiamulin fumarate. in the presence of its degradation products. The first was UHPLC-UV method. Efficient separation was achieved by isocratic elution with a mobile phase of 0.1% aqueous ortho-phosphoric acid (pH 3.5 ± 0.5) and methanol (20:80, v/v) with UV detection at 210 nm. Linearity was obtained in the range of 0.5-10.0 µg mL-1 with mean accuracy of 100.40 ± 0.71. The second method was a TLC-densitometric evaluation of a thin-layer chromatogram of the intact drug using a mobile phase of methanol: pentanol: ethyl acetate: 16.5% ammonia (5:4:2:4, by volume). The TLC-plates were scanned densitometrically at 220 nm where Rf values were 0.58, 0.48 and 0.74 for tiamulin F, its acidic and oxidative degradants, respectively. Moreover, the plates were sprayed with 16% sulfuric acid, heated at 105°C for 10 min. where a yellow-coloured band appeared corresponding to the intact drug was scanned densitometrically at 450 nm. While the bands of the two degradants were no longer observed anymore. Tiamulin F was determined in the range of 1.0-10.0 μg/band with mean accuracy of 100.27% ± 1.47 at 220 nm and 99.93% ± 1.38 at 450 nm. The proposed methods were successfully applied for the determination of drug in marketed oral solution. The obtained results were statistically analyzed and found to be in accordance with those obtained by a reported method.

OPTIMASI SUHU DAN LAMA PEMANASAN TERHADAP HASIL SINTESIS SENYAWA BENZOILTIOUREA DENGAN METODE ASILASI

To find new compounds acting on central nervous system (CNS), this research has been done the synthesis of benzoylthiourea by acylating the thiourea with benzoyl chloride, using tetrahydrofuran as solvent. Reaction temperature were respectively done at 90oC, 100oC, 110oC and 120oC, and heating duration was respectively accomplished at 0.5 hours, 1.0 hours, 1.5 hours, 2.0 hours and 2.5 hours. This research was aimed at finding out optimal heating duration and temperature to obtain maximal percentage yield of benzoylthiourea synthesis. The highest percentage yield of 59.89% obtained in optimum temperature 100oC and 2.0 hours heating duration. The purity test of the synthesis product is shown by the single spot on the thin layer chromatogram (TLC) and narrow range of melting point. Based on the structure identification with UV and IR spectrophotometries, H-NMR spectrometries and Gas Chromatography- Mass Spectrometries (GC-MS), it was concluded that the structure of the synthesis product was accordance to the prediction.

Using UPLC-QTOF-MS Method to Analyse the Spot Constituents of the Thin Layer Chromatograms for Chuzhou Stemona sessilifolia (Miq.) Miq.

A simple, rapid, and highly sensitive analytical method was established for identification of constituents in the spot of the thin layer chromatogram of Chuzhou Stemona sessilifolia (Miq.) Miq. (Chuzhou S. sessilifolia) with ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The technology was applied to systematically analyze and detect the targeted spots. Compared with the fragmentation behaviors of more than thirty reference constituents, the possibly existing compounds of the target spots were identified or tentatively identified by their exact masses and diagnostic fragment ions. Finally, the four clear spots of the thin layer chromatograms of Chuzhou S. sessilifolia were screened and identified of possible molecular formula and structures.

Enteropathogenic EscherichiacoliVirulence Factor Bundle-Forming Pilus Has a Binding Specificity for Phosphatidylethanolamine

ABSTRACT The bundle-forming pilus (BFP) of enteropathogenicEscherichia coli (EPEC), an established virulence factor encoded on the EPEC adherence factor (EAF) plasmid, has been implicated in the formation of bacterial autoaggregates and in the localized adherence of EPEC to cultured epithelial cells. While understanding of the pathogenic mechanism of this organism is rapidly improving, a receptor ligand for BFP has not yet been identified. We now report, using both solid-phase and liposome binding assays, that BFP expression correlates with phosphatidylethanolamine (PE) binding. In a thin-layer chromatogram overlay assay, specific recognition of PE was documented for BFP-expressing strains, including E2348/69, a wild-type EPEC clinical isolate, as well as a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP did not bind PE, including a bfpAdisruptional mutant of E2348/69, EAF plasmid-cured E2348/69, and HB101. E2348/69 also aggregated PE-containing liposomes but not phosphatidylcholine- or phosphatidylserine-containing liposomes, while BFP-negative strains did not produce aggregates with any tested liposomes. Purified BFP preparations bound commercial PE standards as well as a PE-containing band within lipid extracts from human epithelial cells and from E2348/69. Our results therefore indicate a specific interaction between BFP and PE and suggest that PE may serve as a BFP receptor for bacterial autoaggregation and may promote localized adherence to host cells, both of which contribute to bacterial pathogenesis.

Glycosphingolipid Binding Specificities of Rotavirus: Identification of a Sialic Acid-Binding Epitope

ABSTRACT The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcα3-Galβ) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcα3Galβ is preferred to NeuAcα3Galβ. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.

Binding of Actinobacillus pleuropneumoniae Lipopolysaccharides to Glycosphingolipids Evaluated by Thin-Layer Chromatography

ABSTRACT The binding profile of Actinobacillus pleuropneumoniaeserotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay. A. pleuropneumoniae whole cells recognized glucosylceramide (Glcβ1Cer), galactosylceramide (Galβ1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO3-3Galβ1Cer), lactosylceramide (Galβ1-4Glcβ1Cer), gangliotriaosylceramide GgO3(GalNAcβ1-4Galβ1-4Glcβ1Cer), and gangliotetraosylceramide GgO4 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb3, globoside Gb4, or Forssman Gb5 glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b. The A. pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine or ceramide. Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO3 and GgO4glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO3 and GgO4, where GalNacβ1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that A. pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.