Surprising absence of heparin in the intestinal mucosa of baby pigs.

Heparin, a member of a family of molecules called glycosaminoglycans, is biosynthesized in mucosal mast cells. This important anticoagulant polysaccharide is primarily produced by extraction of the mast cell-rich intestinal mucosa of hogs. There is concern about our continued ability to supply sufficient heparin to support the worldwide growth of advanced medical procedures from the static population of adult hogs used as food animals. While the intestinal mucosa of adult pigs is rich in anticoagulant heparin (containing a few hundred milligrams per animal), little is known about how the content of heparin changes with animal age. Using sophisticated mass spectral analysis we discovered that heparin was largely absent from the intestinal mucosa of piglets. Moreover, while the related, nonanticoagulant heparan sulfate glycosaminoglycan was present in significant amounts we found little chondroitin sulfate E also associated with mast cells. Histological evaluation of piglet intestinal mucosa showed a very low mast cell content. Respiratory mast cells have been reported in baby pigs suggesting that there was something unique about the piglets used in the current study. These piglets were raised in the relatively clean environment of a university animal facility and treated with antibiotics over their lifetime resulting in a depleted microbiome that greatly reduced the number of mast cells and heparin content of the intestinal mucosal in these animals. Thus, from the current study it remains unclear whether the lack of intestinal mast cell-derived heparin results from the young age of these animals or their exposure to their depleted microbiome.

Development and validation of a new stability indicating reversed phase liquid chromatographic method for the determination of prednisolone acetate and impurities in an ophthalmic suspension.

A new stability indicating reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated under current International Conference of Harmonisation (ICH) guidance for the determination of prednisolone acetate (PAC) and impurities in an ophthalmic suspension. The developed method is presented as an alternative to a modified version of the current RP-HPLC method described in the USP monograph for the assay of PAC in an ophthalmic suspension. Along with the assay of PAC, the new method is also capable of identifying and quantifying eight selected PAC impurities and degradation products in an ophthalmic suspension. Using an Agilent Poroshell 120 EC-C18 100 mm × 4.6mm (dp: 2.7 µm) column set to 60°C with step gradient elution generated using mobile phase A: acetonitrile/water (10:90) (v/v) and mobile phase B: acetonitrile delivered at 1.2 mL min(-1), all peaks of interest are eluted in 33 min with resolution of 1.5 between the critical pairs. The developed method was validated for PAC and impurities to ICH recommendations for accuracy, linearity, precision (repeatability), limit of detection, limit of quantitation, robustness and specificity.

Characterization of surface confined ionic liquid stationary phases: impact of cation revisited.

Modification of the Linear Solvation Energy Relationship (LSER) equation to account for ionic interactions in the retention of ionizable compounds has enabled the elucidation in the effect of the imidazolium cation identity on retention. Three Surface Confined Ionic Liquid stationary phases were synthesized from an octylbromide phase on silica: 1-octyl-3-methylimidazolium bromide (MIM), 1-octyl-3-butylimidazolium bromide (BIM), and 1-octyl-3-benzylimidazolium bromide (BzIM). These phases were probed via a 35 analyte probe set, including 6 phenolic acids, 5 anilinic bases, and 2 pyridinic bases, and the resulting column parameters compared with previously reported interactions of ionic liquids or Surface Confined Ionic Liquids. The correlation between experimental and calculated retention for the conventional, 6-parameter LSER equation was very poor: r(2)=0.64 (MIM), 0.60 (BIM), and 0.62 (BzIM). By accounting for the ionic interactions between stationary phase and analytes, linearity for the modified, 8 parameter LSER equation was significantly improved to r(2)=0.997 (MIM), 0.996 (BIM), and 0.997 (BzIM). The primary difference between cation identities is within the retention of acids where BIM>BzIM>MIM. We conjecture that the accessibility of bulky, acidic analytes to the on-top interaction of the imidazolium ring is the major contributor to increased anion retention.

Heparin-induced circular dichroism of an achiral, bicyclic species.

Antimalarial drugs have shown potential in suppressing the role of glycosaminoglycans (GAGs) in the pathology of prion protein conformational disorders (e.g. "Mad Cow" disease) by competing for sites of electrostatic interaction. In this study, circular dichroism (CD) and UV/Visible (UV/Vis) absorption spectroscopy techniques were used to investigate the interactions between N-methyl-N'-(7-chloro-4-quinolyl)-1,3-diaminopropane (QD), an achiral, bicyclic compound similar to previously investigated antimalarial drugs, and heparin, a complex GAG that is frequently used as a clinical anticoagulant. Relatively intense heparin-induced CD features were observed for QD and were noted to be radically different from previous studies using related chiral drugs, underscoring the importance of the Pfieffer effect on this and similar heparin research. Additionally, the induced CD for QD was observed to be highly dependent upon drug concentration, heparin concentration, system pH, equilibration time, and ionic strength. These results, in connection with recent work, provide new insight into the nature of the association between GAGs and antimalarial species.

Characterization of surface-confined ionic liquid stationary phases: impact of cation and anion identity on retention.

A series of surface-confined ionic liquid (SCIL) stationary phases for high-performance liquid chromatography were synthesized in-house. The synthesized phases were characterized by the linear solvation energy relationship (LSER) method to determine the effect of residual linking ligands and the role of the cation and the anion on retention. Statistical analysis was utilized to determine whether the system coefficients returned from multiple linear regression analysis of chromatographic retention data for a set of 28 neutral aromatic probe solutes were significantly different. Examination of the energetics of retention via kappa-kappa plots agrees with the results obtained from the LSER analysis. Residual linking ligands were determined to contribute reversed-phase-type retention character to the chromatographic system. Furthermore, retention on the SCIL phases was observed to be more profoundly affected by the identity of the anion than by that of the cation.

Affinity capillary electrophoresis and isothermal titration calorimetry for the determination of fatty acid binding with beta-cyclodextrin.

Affinity capillary electrophoresis (ACE) and isothermal titration calorimetry (ITC) were used to investigate the binding interaction between several fatty acids (FAs) and beta-cyclodextrin (beta-CD). Within each method, steps taken to obtain accurate binding constants are discussed. The stoichiometry of interaction was revealed to be 1:1 regardless of FA chain length. The binding constants obtained using ACE were: octanoate, 6.4x10(2); 2-octenoate, 4.7x10(2); decanoate, 3.7x10(3); 9-decenoate, 1.8x10(3) and dodecanote, 1.4x10(4). The binding constants obtained from ITC were of the same order of magnitude, but were consistently greater than those from ACE. Thermodynamic data obtained using ITC are used to explain the observed trends in binding strength.

Retention characteristics of a new butylimidazolium-based stationary phase. Part II: anion exchange and partitioning.

A surface-confined ionic liquid (SCIL) and a commercial quaternary amine silica-based stationary phase were characterized employing the linear solvation energy relationship (LSER) method in binary methanol/water mobile phases. The retention properties of the stationary phases were evaluated in terms of intermolecular interactions between 28 test solutes and the stationary phases. The comparison reveals a difference in the hydrophobic and hydrogen bond acceptance interaction properties between the two phases. The anion exchange retention mechanism of the SCIL phase was demonstrated using nucleotides. The utility of the SCIL phase in predicting logk (IL/water) values by chromatographic methods is also discussed.

Study of injection bias in a simple hydrodynamic injection in microchip CE.

The electrokinetically pinched method is the most commonly used mode for sample injection in microchip capillary electrophoresis (microCE) due to its simplicity and well-defined sample volume. However, the limited injection volume and the electrophoretic bias of the pinched injection may limit its universal usage to specific applications. Several hydrodynamic injection methods in microCE have been reported; however, almost all claimed that their methods are bias-free without considering the dispensing bias. To investigate the dispensing bias, a simple hydrodynamic injection was developed in single-T and double-T glass microchips. The sample flow was produced by hydrostatic pressure generated by the liquid level difference between the sample reservoir and the other reservoirs. The reproducibility of peak area and peak area ratio was improved to a significant extent using large-surface reservoirs for the buffer reservoir and the sample waste reservoir to reduce the Laplace pressure effect. Without a voltage applied on the sample solution, the voltage-related sample bias was eliminated. The dispensing bias was analyzed theoretically and studied experimentally. It was demonstrated that the dispensing bias existed and could be reduced significantly by appropriately setting up the voltage configuration and by controlling the appropriate liquid level difference.

Mobile phase effects on retention on a new butylimidazolium-based high-performance liquid chromatographic stationary phase.

A new HPLC stationary phase based on n-butylimidazolium bromide has been characterized by a linear solvation energy relationship (LSER) approach in the binary acetonitrile/water mobile phases. The retention properties of the stationary phase were systematically evaluated in terms of intermolecular interactions between 28 test solutes and the stationary phase. The results and further comparisons with conventional reversed phase system confirm that retention properties are similar to phenyl phases in acetonitrile/water mixtures. The results obtained with acetonitrile/water mixtures are also compared with results obtained using methanol/water mixtures.

Analytical performance of polymer-based microfluidic devices fabricated by computer numerical controlled machining.

A study comparing the electrophoretic separation performance attainable from microchips molded by masters fabricated using conventional CNC machining techniques with commercial microchips, wire imprinted microchips, and microchips from LIGA molding devices is presented. An electrophoresis-based detection system using fluorescence microscopy was used to determine the analytical utility of these microchips. The separation performance of CNC microchips was comparable to commercially available microchips as well as those fabricated from LIGA masters. The important feature of the CNC machined masters is that they have rapid design-to-device times using routinely available machining tools. This low-cost prototyping approach provides a new entry point for researchers interested in thermoplastic microchips and can accelerate the development of polymer-based lab-on-a-chip devices.

An online fiber-optic UV-visible detector for continuous free-flow electrophoresis.

A PC-controlled, scanning online UV detector for continuous free-flow electrophoresis (CFFE) was designed to allow for single UV wavelength monitoring across a 1-D array of 48 longitudinal flow cells interfaced to a CFFE apparatus. In the detection scheme, the UV light is sequentially passed through each of the flow cells. The design integrates online acquisition of absorbance spectra of components separated by CFFE. Benzoic acid standard solutions were used to examine the performance of the detector. Chloroquine diphosphate and 4-nitrophenol were used as test solutes to determine the detector's ability to distinguish analytes separated in the CFFE.

Electrosynthesis and analytical performances of functionalized poly (pyrrole/beta-cyclodextrin) films.

The supramolecular complexing properties of cyclodextrins (CDs) have been investigated inside a conducting polymer environment. In this work, we report the synthesis and characterization of a polypyrrole/beta-cyclodextrin (Ppy/beta-CD) film at a glassy carbon (GC) electrode surface. The polypyrrole/beta-cyclodextrin (Ppy/beta-CD) film was prepared by a simple electropolymerization of a 20:1 mixture of the CD and the pyrrole monomer LiClO(4) supporting electrolyte. The resulting functionalized polymer film features interesting electrochemical properties such as selective, simultaneous and quantitative detection of some organic compounds of interest such as polyhydroxyphenyls and neurotransmitters derived from pyrogallol and catechol. The fabricated electrochemical sensor exhibits a fast and reversible linear response toward catechol within the concentration range of 1.5x10(-7) to 8x10(-6)M and towards pyrogallol within the concentration range of 1x10(-6) to 1x10(-5)M. The detection limit was 4x10(-7) and 1.8x10(-6)M for catechol and pyrogallol, respectively. Studies of neurotransmitters such as epinephrine, metanephrine and l-dopa (l-3,4-dihydroxyphenylalanine), showed better response toward epinephrine and l-dopa than for metanephrine. Calibration curves for these two neurotransmitters were linear over the concentration range of 1x10(-6) to 1x10(-5)M. The detection limit was 4x10(-6) and 1x10(-6), respectively. The complexation capability of the Ppy/beta-CD system is addressed here in terms of structure-electrochemical activity relationship. The mechanical stability of the film is also discussed. Measurements were performed using cyclic voltammetry (CV), scanning electron microscopy (SEM) coupled to energy dispersive analysis of X-ray (EDAX) and electrochemical impedance spectroscopy (EIS).

Comprehensive strategy for chiral separations using sulfated cyclodextrins in capillary electrophoresis.

This review focuses on the emerging role of sulfated cyclodextrins in the capillary electrophoretic (CE) separation of chiral analytes. Since being introduced as enantioselective agents for CE in 1995, these anionic additives have continued to demonstrate remarkable application universality. The broad spectrum of chiral compounds successfully separated using this approach includes acidic, basic, neutral, and zwitterionic species. This impressive array of analyte structures is derived from a growing diversity of compound classes including pharmaceuticals, plant extracts, biomarkers, herbicides, alkaloids, fungicides, and metal ions. Moreover, literature reports highlight the minimal optimization required to achieve a successful separation. Based on these findings, sulfated cyclodextrins appear to be well suited for the development of a more universal, comprehensive separation strategy for chiral compounds. This review explores this proposition by beginning with the structure and migration properties of sulfated cyclodextrins, using applications to highlight the separating power of this technique and ending with a pragmatic, comprehensive separation strategy.

Optimization of preparative electrophoretic chiral separation of ritalin enantiomers.

Continuous free flow electrophoresis (CFFE) was applied to the preparative chiral separation of ritalin enantiomers. Sulfated beta-cyclodextrin (sbeta-CD) was used as the chiral additive. Liquid chromatography-mass spectrometry (LC-MS) experiments were applied to study the time averaged concentration of sbeta-CD in the separation chamber. The distribution of sbeta-CD in the separation chamber greatly influenced resolution and the angle of deflection. To optimize the separation, several parameters (methanol, concentration of sbeta-CD in the cathodic wash and in the separation buffer, and the introduction of a low conductivity zone) were investigated. The dependence of the resolution and deflection angles of ritalin enantiomers on the concentration of sbeta-CD in both the separation buffer and in the cathode wash solution appeared to be non-linear. Under close to optimal conditions, resolution of ritalin enantiomers was about 0.8 with an average processing rate of 0.5 mg/h. Overall, the enantiomeric purity of the individual isomers was approximately 83%; however, of the 20 vials containing ritalin, the presence of both enantiomers was only detected in three vials.