The use of SAX-HPLC-CD as a heparin screening strategy.

Heparin, a heterogeneous polysaccharide, has been widely used as an anticoagulant for decades. Recently, however, international events involving the sudden onset of allergic-type reactions following heparin administration led to numerous fatalities, and demanded the use of multiple laborious, time consuming techniques to identify an economically motivated adulterant. Using these methods cooperatively, the semi-synthetic molecule known as oversulfated chondroitin sulfate (OSCS), was found to be present at significant concentrations. Since the discovery of this adulterant, several analytical methods have been put forth or updated to advance the process of screening pharmaceutical heparins; of these, strong anion exchange high performance liquid chromatography (SAX-HPLC) methods have now become routine. In this preliminary work, we report the use of circular dichroism (CD) detection in conjunction with existing SAX-HPLC methods to quantitate various sulfated polysaccharides. The proposed strategy exploits the selectivity associated with CD detection of heparin and heparin-like polysaccharides, while taking advantage of the method's insensitivity to the use of mobile phase additives and programmed gradients. The limit of detection of heparin by CD was found to be ∼0.22 mg/mL, whereas traditional UV/Vis detection yielded a detection limit of ∼1.09 mg/mL. The success of CD detection varied for other polymers, however no significant modifications were made to the separations method to capitalize on the advantages of CD detection.

The development of an FIA-CD strategy for screening sulfated polysaccharides using antimalarial drugs and related species as probes.

Heterogeneous sulfated polysaccharides have attracted significant attention in light of their various biological activities. However, recent events involving heparin have dramatically illustrated that several analytical challenges exist in accounting for such species. In this case, tainted heparin was associated with acute reactions that lead to numerous deaths. Researchers were forced to use time-consuming, sophisticated techniques (e.g., enzymatic digestion, NMR, CE, HPLC, MS, etc.) to identify the cause of these adverse effects. Extensive investigations ultimately showed oversulfated chondroitin sulfate, a semi-synthetic sulfated polysaccharide, to be present in the contaminated samples. These events highlighted the need for a new generation of screening techniques. In this work, we report the development of a screening strategy that exploits unique circular dichroism features observed as a function of association between investigated polymers and judiciously selected probe molecules (i.e., chloroquine, N1-(7-chloro-4-quinolinyl)-N3-methyl-1,3-propanediamine, quinacrine, and N2-9-acridinyl-N1,N1-dimethyl-1,2-ethanediamine). Application of obtained spectropolarimetry results to a flow injection analysis circular dichroism platform allowed for the establishment of linear polysaccharide response curves for dextran sulfate, heparin, and oversulfated chondroitin sulfate in the low micromolar range. Lastly, through additional work with heparin, the proposed method was shown to be capable of rapidly screening sulfated polysaccharide samples for closely related impurities.

The use of circular dichroism as a simple heparin-screening strategy.

Heparin, a heterogeneous polysaccharide, is used extensively as an anticoagulant. Recently, however, tainted heparin was associated with acute reactions that lead to numerous deaths. Extensive investigations ultimately showed oversulfated chondroitin sulfate, a semi-synthetic polysaccharide, to be present in the contaminated samples. These events highlighted the need for new, convenient heparin-screening methods capable of rapidly determining sample purity. In this work, we report the use of circular dichroism spectroscopy to analyze heparin samples for the presence of heparin-like adulterants (e.g., chondroitin sulfate A, dermatan sulfate, and oversulfated chondroitin sulfate) in a simple and straightforward manner. This strategy exploits the subtle differences in the optical properties of each polymer; these differences result from structural dissimilarities. To the best of our knowledge, the findings presented here are the first report of heparin purity screening using traditional spectropolarimetry techniques.

Application of a modified linear solvation energy relationship (LSER) model to retention on a butylimidazolium-based column for high performance liquid chromatography.

Previously, a new HPLC stationary phase based on n-butylimidazolium bromide was investigated using a linear solvation energy relationship (LSER) to systematically evaluate the intermolecular interactions between 32 test solutes and the stationary phase. The results and further comparisons with conventional reversed phase systems revealed that retention properties are similar to phenyl phases in both methanol/water and acetonitrile/water mixtures. In this work, the LSER model is extended by including the degree of ionization molecular descriptor, D, which takes into account the pK(a) of ionizable analytes and the pH of the mobile phase. The D molecular descriptor has been further divided into D(+) and D(-) components that separately account for the ionization of basic and acidic solutes, respectively. This is the first study where the ionization terms for weakly acidic solutes and weakly basic solutes have been separated. LSER results obtained with the expanded solute set with and without the inclusion of the D(+) and D(-) solute descriptors were compared. The improved correlation and standard error obtained for the expanded test set in the presence and absence of the D(+) and D(-) descriptors (R(2): 0.987 vs 0.846; SE: 0.051 vs 0.163 for 60% MeOH) support inclusion of these additional terms. Further, the coefficients obtained from the multiple linear regression for the expanded test set with the D(+) and D(-) descriptors were more consistent with the coefficients obtained when the test set included just neutral analytes. In addition, the expanded LSER model did a better job of predicting elution order for the ionizable analytes. This work provides further supporting evidence for the multimodal nature of the butylimidazolium stationary phase.

Interactions between minimum run time, modifier concentration, and efficiency parameters in a high performance liquid chromatography separation.

We modeled and studied the separation of uracil, nicotinamide, resorcinol, theobromine, theophylline, and caffeine on four C-18 columns of different lengths packed with the same stationary phase using water/methanol mobile phase at one temperature. Predictions of retention times and peak widths were compared with experimental results and were found to be sufficiently accurate for performing optimization calculations. With limits set on the required resolution and on maximum values for pressure and flow rate, calculations were performed for numerous virtual column lengths seeking the smallest possible analysis time for each length while allowing methanol concentration and flow rate to vary as required to minimize run time. Predictions were experimentally verified for the column lengths actually available. These calculations revealed the dependence of best-possible analysis time on column length, modifier concentration, flow rate, and pressure for the real system that was modeled, and provided insight into parameter interactions with respect to analysis times meeting the needs and limits specified. We show that when these parameters are considered in concert, rather than individually, conventional guidelines regarding setting their values may not always lead to the optimum.

Chiral separations.

The main goal of this review is to provide a brief overview of chiral separations to researchers who are versed in the area of analytical separations but unfamiliar with chiral separations. To researchers who are not familiar with this area, there is currently a bewildering array of commercially available chiral columns, chiral derivatizing reagents, and chiral selectors for approaches that span the range of analytical separation platforms (e.g., high-performance liquid chromatography, gas chromatography, supercritical-fluid chromatography, and capillary electrophoresis). This review begins with a brief discussion of chirality before examining the general strategies and commonalities among all of the chiral separation techniques. Rather than exhaustively listing all the chiral selectors and applications, this review highlights significant issues and differences between chiral and achiral separations, providing salient examples from specific classes of chiral selectors where appropriate.

Modeling revealed circular dichroism for quinacrine in the presence of heparin.

A simple model is presented that accounts for revealed circular dichroism signals that are observed as a function of enantiopreferential drug binding to a chiral selector. According to this model, the intensity of such signals depends heavily on the differences in enantiomer-selector association constants as well as the differences in bound vs. unbound molar ellipticity values for the chromophore containing species. The proposed model is supported by circular dichroism and capillary electrophoresis results obtained using quinacrine, a tricyclic, antimalarial drug, and heparin, a highly-sulfated glycosaminoglycan. This strategy also explores the role that revealed circular dichroism may play in the optical activity observed for some drugs in the presence of heparin, as has previously been illustrated for chiral drugs in the presence of DNA.

Separation of peptides by HPLC using a surface-confined ionic liquid stationary phase.

A butylimidazolium bromide surface-confined ionic liquid stationary phase was synthesized in-house. The synthesized phase was investigated for the separation of five peptides (Gly-Tyr, Val-Tyr-Val, leucine enkephalin, methionine enkephalin, and angiotensin-II). The peptides were successfully separated in less than 5 min. The effect of trifluoroacetic acid (TFA) on the separation of peptides was evaluated with results confirming that TFA was not acting as ion-pairing agent in separation of peptides on this phase.

Rapid determination of surfactant critical micelle concentrations using pressure-driven flow with capillary electrophoresis instrumentation.

This work demonstrates a novel, convenient utilization of capillary electrophoresis (CE) instrumentation for the determination of critical micelle concentrations (CMCs). Solution viscosity differences across a range of surfactant concentrations were monitored by hydrodynamically forcing an analyte towards the detector. Upon reaching the surfactant's CMC value, migration times were observed to change drastically. CMC values for four commonly employed anionic surfactants were determined-sodium dodecyl sulfate: 8.1mM; sodium caprylate: 300 mM; sodium decanoate: 86 mM; sodium laurate: 30 mM; and found to be in excellent agreement with values previously reported in the literature. The technique was then applied to the less well-characterized nonionic surfactants poly(oxyethylene) 8 myristyl ether (CMC approximately 9 M), poly(oxyethylene) 8 decyl ether (CMC approximately 0.95 mM) and poly(oxyethylene) 4 lauryl ether.

Heparin-induced circular dichroism of chloroquine.

Circular dichroism (CD) and UV/Visible absorption (UV/Vis) spectroscopy techniques were used to investigate the interaction between heparin and chloroquine, an antimalarial drug that has shown potential as an anti-prion agent. CD spectra of rac-chloroquine upon addition of heparin provide evidence of glycosaminoglycan (GAG) binding, support recent findings suggesting that interactions between heparin and antimalarial drugs are largely due to electrostatic interactions, and represent the first reported GAG-induced CD signal of a bicyclic, aromatic compound. The association constant ( approximately 10(3)M(-1)) between chloroquine and heparin was calculated from a UV titration curve and provided additional insight into the nature of the association between these two compounds.

Characterization of a novel pyridinium bromide surface confined ionic liquid stationary phase for high-performance liquid chromatography under normal phase conditions via linear solvation energy relationships.

Utilizing linear solvation free energy relationship methodology, a novel pyridinium bromide surface confined ionic liquid (SCIL) stationary phase was characterized under normal phase high-performance liquid chromatographic conditions. A limited set of neutral aromatic probe solutes were utilized to rapidly assess the utility of the LSER model, using mobile phases of hexane modified with 2-propanol. The excellent correlation of the global fit across the mobile phase composition range used in this study for the experimental and calculated retention values (R(2)=0.994) indicates that the LSER model is an appropriate model of characterizing this polar bonded phase under normal phase conditions. For a limited subset of compounds, retention on the pyridinium bromide SCIL stationary phase is more highly correlated with that obtained on a cyano column than on a diol column under NP conditions.

Retention characteristics of a new butylimidazolium-based stationary phase.

A new HPLC stationary phase has been synthesized based on the ionic liquid n-butylimidazolium bromide. Imidazolium was covalently immobilized on a silica substrate through an n-alkyl tether and the retention characteristics of the resulting stationary phase were evaluated systematically. Using 28 small aromatic test solutes and reversed phase conditions, the linear solvation energy relationship approach was successfully used to characterize this new phase. The retention characteristics of the test solutes show remarkable similarity with phenyl stationary phases, despite the presence of a positive charge on the new imidazolium phase. Operated in the reversed phase mode, this new stationary phase shows considerable promise for the separation of neutral solutes and points to the potential for a truly multi-modal stationary phase.

Capillary electrophoretic and nuclear magnetic resonance studies of interactions between halophenols and ionic liquid or tetraalkylammonium cations.

Aqueous capillary electrophoretic studies were performed to investigate interactions between halophenols and 1-ethyl-3-methylimidazolium tetrafluoroborate or tetraethylammonium tetrafluoroborate electrolytes. In both cases, increased halogen size correlated with increased affinity for the electrolyte cation. For isomers, the ortho substituted isomer exhibited higher affinity than the para isomer. Irreproducible CE results for analyte pairs in the presence of the ionic liquid stimulated investigations of the interactions between halophenols as well as with the cations of the electrolyte. These interactions were explored by proton and fluorine one-dimensional NMR. The NMR results indicated differences in the interactions between tetraethylammonium/iodophenols and imidazolium/iodophenols. The NMR results indicate hydrophobic stacking interactions between the iodophenols and possible similar interaction among phenols and imidazolium.

Separation of cocaine stereoisomers by capillary electrophoresis using sulfated cyclodextrins.

A capillary electrophoretic method for the separation of cocaine and its stereoisomers was developed. In this study, the effect of organic modifier was also investigated. The separation was achieved using 1% sulfated cyclodextrin, 10 mmol L(-1) phosphate buffer, 10% methanol at pH 3. The method provides good reproducibility and easy application.

Capillary electrophoretic application of 1-alkyl-3-methylimidazolium-based ionic liquids.

Ionic substances with melting points at or close to room temperature are referred to as ionic liquids. Interest in ionic liquids for their potential in different chemical processes is increasing, because they are environmentally benign and are good solvents for a wide range of both organic and inorganic materials. In this study, a capillary electrophoretic method for resolving phenolic compounds found in grape seed extracts is reported. The method, in which 1-alkyl-3-methylimidazolium-based ionic liquids are used as the running electrolytes, is simple and reproducible. The separation mechanism seems to involve association between the imidazolium cations and the polyphenols. The role of the alkyl substituents on the imidazolium cations was investigated and will be discussed.

Use of dyes to investigate migration of the chiral selector in CFFE and the impact on the chiral separations.

Continuous free flow electrophoresis was investigated as a tool for the preparative chiral separation of piperoxan enantiomers using sulfated beta-cyclodextrin (sbeta-CD) as the chiral additive. Bulk migration of sbeta-CD was confirmed using LC-MS analysis of the individual fractions collected and visualized with the addition of crystal violet to the separation buffer. In the absence of sbeta-CD, the crystal violet-containing buffer was reddish/purple and the crystal violet was deflected cathodically in the chamber. In the presence of sbeta-CD, the crystal violet-containing buffer was blue and was deflected anodically. However, formation of accumulation and depletion zones was apparent in both cases. The addition of sbeta-CD to the cathodic wash solution allowed for almost complete resolution of the piperoxan enantiomers with a processing rate of 0.45 mg/ h.

Quantitative determination of clenbuterol, salbutamol and tulobuterol enantiomers by capillary electrophoresis.

Enantiomers of clenbuterol, salbutamol and tulobuterol were directly separated and quantitated from a spiked sample by capillary electrophoresis (CE) using sulfaited beta-cyclodextrin (SCD) as chiral selector and phosphate as running buffer. The SCD and buffer concentration, pH and field strength were the parameters studied to optimize the separation. Optimal separation was obtained using 50 mM of phosphate monobasic at pH = 2.24, 0.25% (w/w) of sulfated cyclodextrin and a field strength of 10 kV, with 20 min total time analysis. Comparison between two different injection modes (hydrodynamic and electrokinetic) was made. In the hydrodynamic mode, repeatability (expressed as relative standard deviation, RSD) was less than 1.2% for migration times for all the analyte peaks and less than 2% for peak area percentages. With respect to reproducibility, RSD was less than 3.8% for migration time and less than 3% for peak area percentages. Calibration curves were set up for two different sample concentration ranges (1 to 10 microg mL(-1) and 160-800 ng mL(-1), of each of the racemates studied). Although the electrokinetic injection mode for an aqueous sample appeared to suffer from some enantiodiscrimination, calibration curves were linear in the range between 1 and 10 ng mL(-1) with regression coefficients ranging from 0.9996 to 0.9952. As in the case of hydrodynamic injection, the method was tested with a spiked sample.

A comparison of phosphated and sulfated beta-cyclodextrins as chiral selectors for capillary electrophoresis.

The enantioseparation capabilities of three different functionalized beta-cyclodextrins, two sulfated beta-cyclodextrins with 4 and 15 nominal degrees of substitution and a phosphated beta-cyclodextrin with 8 degrees of substitution, were compared. While anodic detection was used with both sulfated cyclodextrins, the phosphated cyclodextrin required cathodic detection suggesting either lower ionization of the phosphated cyclodextrin or generally lower affinity of the analytes for the phosphated cyclodextrin. The effects of several experimental parameters were evaluated with respect to enantioseparation. The degrees of substitution of the cyclodextrin, pH of the background electrolyte as well as the concentration of the functionalized beta-cyclodextrin, each had a significant influence on the successful enantiomeric separation of the chiral drugs investigated.

Impact of triethylamine as a mobile phase additive on the resolution of racemic amino acids on an (+)-18-crown-6-tetracarboxylic acid-derived chiral stationary phase.

Recently, a new HPLC chiral stationary phase (CSP) prepared by covalently bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid on silica gel was successfully employed in resolving various racemic natural and unnatural amino acids containing a primary amino group. Current work details on-going efforts to improve the effectiveness of this type of material. The analytes used in this study included various substituted phenylalanines, phenylglycine homologues and other primary amino acids. In an attempt to increase enantioselectivity, the effect of methanol and triethylamine modifiers was evaluated in an aqueous mobile phase containing sulfuric acid. In general. retention time increased with increasing methanol and triethylamine concentration. In addition, highest enantioselectivities were obtained with high methanol and high triethylamine; however, these conditions produced excessively long retention. All of the analytes were well resolved on the CSP with a mobile phase of 20% methanol containing 14.3 mM triethylamine and 10.0 mM sulfuric acid.

Continuous free flow electrophoresis for preparative chiral separations of piperoxan using sulfated beta-cyclodextrin.

Continuous free flow electrophoresis was investigated as a tool for the preparative chiral separation of piperoxan using a sulfated cyclodextrin chiral additive. In the absence of chiral additive, the sample stream was deflected cathodically. However, the presence of sulfated cyclodextrin in the run buffer caused anodic deflection and splitting of the sample stream into two streams, each enriched in one enantiomer. Although the sulfated cyclodextrin used was comprised of a mixture of homologues and isomers, this polydispersity did not seem to significantly impact band dispersion. Sample introduction rates ranged from approximately 0.9-7.2 mg h-1. Maximum resolution was 0.53, using an applied voltage of 220 V, buffer composition of 0.075% sulfated cyclodextrin, 7.6 mM citrate (pH 3), 4.5 degrees C.