Silica hydride based phases for small molecule separations using automated liquid chromatography-mass spectrometry method development.

Silica hydride, or Type C silica, has been developed as an alternative chromatographic support material for liquid chromatography. There are various bonded phases available with this new support. For four such phases (Cholesterol, Bidentate C18, Diamond Hydride, and Diol), retention and selectivity behavior were investigated using liquid chromatography coupled with triple quadrupole mass spectrometry. A set of small molecules from several chemical classes of interest, and varying in their physicochemical properties, were chromatographed under both reversed-phase and aqueous normal phase modes. To screen the columns, column switching was performed using an automated platform controlled by associated software and an additional valve. A typical scouting gradient was implemented. The separation conditions were not further optimized since the goal was simply to evaluate the variable retention behavior of the phases and selectivity under generic conditions. Further, retention of the analytes were evaluated under isocratic conditions with varying percentages of organic phase to visualize the potential for dual retention modes on the same column for certain analytes. Four analytes (fentanyl, hydrocodone, hydromorphone, and matrine) showed dual mode retention behavior with all four phases. Especially, fentanyl exhibited dramatic "U-shaped" retention profiles on Cholesterol and Bidentate C18 phases. Overall, changes in the retention order between reversed phase and aqueous normal phases emphasized the potential for altered selectivity. Results showed that the Cholesterol phase provided the highest retention for most analytes compared to the other phases. The more polar Diol phase still provided good retention in reversed phase mode. Retention and selectivity were all highly reproducible.

Automated screening of reversed-phase stationary phases for small-molecule separations using liquid chromatography with mass spectrometry.

There are various reversed-phase stationary phases that offer significant differences in selectivity and retention. To investigate different reversed-phase stationary phases (aqueous stable C18 , biphenyl, pentafluorophenyl propyl, and polar-embedded alkyl) in an automated fashion, commercial software and associated hardware for mobile phase and column selection were used in conjunction with liquid chromatography and a triple quadrupole mass spectrometer detector. A model analyte mixture was prepared using a combination of standards from varying classes of analytes (including drugs, drugs of abuse, amino acids, nicotine, and nicotine-like compounds). Chromatographic results revealed diverse variations in selectivity and peak shape. Differences in the elution order of analytes on the polar-embedded alkyl phase for several analytes showed distinct selectivity differences compared to the aqueous C18 phase. The electron-rich pentafluorophenyl propyl phase showed unique selectivity toward protonated amines. The biphenyl phase provided further changes in selectivity relative to C18 with a methanolic phase, but it behaved very similarly to a C18 when an acetonitrile-based mobile phase was evaluated. This study shows the value of rapid column screening as an alternative to excessive mobile phase variation to obtain suitable chromatographic settings for analyte separation.

The entirely carbohydrate immunogen Tn-PS A1 induces a cancer cell selective immune response and cytokine IL-17.

The tumor-associated carbohydrate antigen/hapten Thomsen-nouveau (Tn; a-D-GalpNAc-ONH2) was conjugated to a zwitterionic capsular polysaccharide, PS A1, from commensal anaerobe Bacteroides fragilis ATCC 25285/NCTC 9343 for the development of an entirely carbohydrate cancer vaccine construct and probed for immunogenicity. This communication discloses that murine anti-Tn IgG3 antibodies both bind to and recognize human tumor cells that display the Tn hapten. Furthermore, the sera from immunization of mice with Tn-PS A1 contain cytokine interleukin 17 (IL-17A), which is known to possess anti-tumor function and represents a striking difference to an IL-2, and IL-6 profile obtained with anti-PS A1 sera.

Immunological response from an entirely carbohydrate antigen: design of synthetic vaccines based on Tn-PS A1 conjugates.

An entirely carbohydrate-based immunogen consisting of a zwitterionic polysaccharide (ZPS) PS A1 and the well-known tumor antigen Tn has been designed, synthesized, and studied for immunological effects. The PS A1 motif was included to act as an MHCII elicitor for a T-cell-dependent immune response with increased immunogenicity against tumor-associated carbohydrate antigens, providing an alternative to carrier proteins. Through the use of C57BL/6 mice, it has been shown that chemical modification of PS A1 does not alter the recognition sequence responsible for an MHCII-mediated, T-cell-dependent immune response. The Tn-PS A1 conjugate construct confers specificity toward the Tn antigen alone, and specific carbohydrate immunoglobulins, namely, IgG3, are generated from intraperitoneal immunizations with or without adjuvant. The properties of the vaccine candidate are attributed to a site-specific linking strategy that incurs significant incorporation of Tn antigen.