Fast turnaround bioanalysis in discovery and early clinical development

Summary The difect and indirect separation of enantiomers of secondary amino acids was studied by high-performance liquid chromatography. Direct separation was by using a macrocyclic glycopeptide, teicoplanin or ristocetin A, as chiral stationary phase. Indirect separation was via pre-column derivatization with (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester [(S)-NIFE] as a new chiral derivatizing agent. Both direct and indirect separations were performed by means of reversed-phase HPLC. Conditions for separations were optimized. The methods described offer good enantioselectivity for synthetic chiral imino acids. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Achiral and chiral separations using MEKC,polyelectrolyte multilayer coatings,and mixed mode separation techniques with molecular micelles

Mixed mode (MM) separation using a combination of MEKC and polyelectrolyte multilayer (PEM) coatings is herein reported for the separation of achiral and chiral analytes. Many analytes are difficult to separate by MEKC and PEM coatings alone. Therefore, the implementation of a MM separation provides several advantages for overcoming the limitations of these well‐established methods. In this study, it was observed that achiral separations using MEKC and PEM coatings individually resulted in partial resolution of eight very similar aryl ketones when the molecular micelle (sodium poly(N‐undecanoyl‐L ‐glycinate)) concentration was varied from 0.25 to 1.00% w/v and the bilayer number varied from 2 to 4. However, when MM separation was introduced, baseline resolution was achieved for all eight analytes. In the case of chiral separations, temazepam, aminoglutethimide, benzoin, benzoin methyl ether, and coumachlor were separated using the three separation techniques. For chiral separations, the chiral molecular micelle, sodium poly(N‐undecanoyl‐L ‐leucylvalinate), was employed at concentrations of 0.25–1.50% w/v for both MEKC and PEM coatings. Overall, the results revealed partial separation with MEKC and PEM coatings individually. However, MM separation enabled baseline separation of each chiral mixture. The separation of achiral and chiral compounds from different compound classes demonstrates the versatility of this MM approach.     

Comparison of Separation Efficiency of Macrocyclic Glycopeptide-Based Chiral Stationary Phases for the LC Enantioseparation of β-Amino Acids

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eighteen unnatural β-amino acids, including several β-3-homo-amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V and V2), and ristocetin A (Chirobiotic R) as chiral selectors. The effects of the organic modifier, mobile phase composition and pH on the separations were investigated. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic T2 column exhibited better selectivity than the Chirobiotic T column for the separation of β-3-homo-amino acid enantiomers; vancomycin or ristocetin A exhibited lower selectivity. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers, with the exception of the Chirobiotic R column, where the elution sequence R < S was observed.     

Effect of urea on analyte complexation by 2,6‐dimethyl‐β‐CD in peptide enantioseparations by CE

The aim of the present study was the investigation of the effect of urea on analyte complexation in CD‐mediated separations of peptide enantiomers by CE in the pH range of about 2–5. pH‐independent complexation and mobility parameters in the absence and presence of 2 M urea were obtained by three‐dimensional, non‐linear curve fitting of the effective analyte mobility as a function of pH and heptakis‐(2,6‐di‐O‐methyl)‐β‐CD concentration. Urea led to decreased binding strength of the CD towards the protonated and neutral analyte enantiomers as well as to decreased mobilities of the free analytes. In contrast, mobilities of the fully protonated enantiomer–CD complexes as well as the pK_a values of the free and complexed analytes increased. The effect of urea on separation efficiency varied with pH and CD concentration. In the case of Ala‐Tyr and Ala‐Phe, separations improved in the presence of urea at pH 2.2. In contrast, separations were impaired by urea at pH 3.8 and low concentrations of the CD. Decreased separation efficiency was noted for Asp‐PheOMe and Glu‐PheNH₂ at low CD concentrations when urea was added but separations improved at higher CD concentrations over the entire pH range studied. The effect of urea on analyte complexation appeared to be primarily non‐stereoselective. Furthermore, the pH‐dependent reversal of the enantiomer migration order observed for Ala‐Tyr and Ala‐Phe can be rationalized by the complexation and mobility parameters.     

Enantiomeric separation of racemic sulphoxides on chiral stationary phases by gas and liquid chromatography

Summary The enantiomeric resolution of seven racemic sulphoxides on chiral stationary phases has been investigated by gas and liquid chromatography. In gas chromatography the separations were performed on octakis-(2,6-di-O-pentyl-3-O-butyryl)-γ-cyclodextrin (FS Lipodex-E) and heptakis-(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin (DMP-β-CD). Both stationary phases were suitable for separation of the enantiomers of the sulphoxides. With one exception for each series all racemetes could be resolved on both stationary phases; FS Lipodex-E was more enantioselective than DMP-β-CD, whereas the latter seemed more generally applicable. Liquid chromatographic separations with Chiralcel-OB as stationary phase were significantly improved by optimization of mobile phase composition and temperature. Resolution factors up to R_s=6 were achieved indicating that the improved separations could now be easily used for preparative purposes.     

Synthesis and chromatographic evaluation of new polymeric chiral stationary phases based on three (1<Emphasis Type="Italic">S</Emphasis>,2<Emphasis Type="Italic">S</Emphasis>)-(−)-1,2-diphenylethylenediamine derivatives in HPLC and SFC

Three new polymeric chiral stationary phases were synthesized based on (1S,2S)-1,2-bis(2,4,6-trimethylphenyl)ethylenediamine, (1S,2S)-1,2-bis(2-chlorophenyl)ethylenediamine, and (1S,2S)-1,2-di-1-naphthylethylenediamine via a simple free-radical-initiated polymerization in solution. These monomers are structurally related to (1S,2S)-1,2-diphenylethylenediamine which is the chiral monomer used for the commercial P-CAP-DP polymeric chiral stationary phase (CSP). The performance of these three new chiral stationary phases were evaluated in normal phase high-performance liquid chromatography (HPLC) and supercritical fluid chromatography and the results were compared with those of the P-CAP-DP column. All three new phases showed enantioselectivity for a large number of racemates with a variety of functional groups, including amines, amides, alcohols, amino acids, esters, imines, thiols, and sulfoxides. In normal phase, 68 compounds were separated with 28 baseline separations (Rs ≥ 1.5) and in SFC, 65 compounds were separated with 24 baseline separations. In total 72 out of 100 racemates were separated by these CSPs with 37 baseline separations. Complimentary separation capabilities were observed for many analytes. The new polymeric CSPs showed similar or better enantioselectivities compared with the commercial column in both HPLC and SFC. However, faster separations were achieved on the new stationary phases. Also, it was shown that these polymeric stationary phases have good sample loading capacities while maintaining enantioselectivity.     

Reversed-Phase Liquid Chromatographic Separation, on Cellulose Chiral Stationary Phases, of the Stereoisomers of Methoxytetra-hydronaphthalene Derivatives, New Agonist and Antagonist Ligands for Melatonin Receptors

A stereoselective high-performance liquid chromatographic method has been established for chiral separation of melatoninergic derivatives with one or two chiral centers, new agonist and antagonist ligands for melatonin receptors. Reversed-phase separations were performed on cellulose-based chiral stationary phases—tris-3,5-dimethylphenylcarbamate (Chiralcel OD-RH) or tris-methylbenzoate (Chiralcel OJ-R). Water–modifier (methanol or acetonitrile) mixtures in different proportions were used as mobile phases. The effects of organic mobile-phase modifier concentration, temperature, and compound structure were examined. Baseline separation (R_S > 1.5) was readily obtained for many of the compounds.     

Microchip electrophoresis of N-glycans on serpentine separation channels with asymmetrically tapered turns

We designed and fabricated microfluidic devices with serpentine separation channels and asymmetrically tapered turns, thus allowing high efficiency separations and minimizing band broadening associated with the “racetrack” effect. We evaluated the performance of these devices by measuring the variation in separation efficiency with separation length, electric field strength, taper ratio of the turns, and number of turns. N‐Glycans derived from ribonuclease B and labeled with 8‐aminopyrene‐1,3,6‐trisulfonic acid were electrophoretically separated on serpentine channels with separation lengths of 11, 18, 22, and 36 cm at electric field strengths from 750 to 1750 V/cm. Separations on the 36‐cm channel produced plate numbers up to 940 000 with an analysis time under 3.1 min, whereas separations on the 22‐cm channel had a shorter analysis time (less than 1.25 min), still with respectable efficiencies (up to 600 000 plates). Turn‐induced dispersion was minimized with taper ratios 2 and 3, whereas having two or four 180° turns along with the separation length did not impact the overall efficiency. The developed device was used to analyze native and desialylated N‐glycans derived from the blood serum of an ovarian cancer patient and a disease‐free individual. Separation efficiencies similar to that achieved with the model glycans from ribonuclease B were attained for these biological samples.     

Efficient chromatographic separation of intact proteins derivatized with a fluorogenic reagent for proteomics analysis

To achieve more efficient separation of intact proteins for proteomics applications, three columns of differing diameters (4.0, 4.6 and 6.0 mm internal diameter) were chosen for comparison and investigated to identify optimal conditions. The column with the largest diameter gave the largest peak capacity, showing the efficient separation of intact proteins, such as two protein standards, glutathione S‐transferase and β‐lactoglobulin. On the other hand, a low‐molecular‐weight compound was separated effectively on the smaller diameter column, demonstrating that the separation mechanism seems to differ between high‐ and low‐molecular‐weight compounds. Finally, using the 6.0 mm i.d. column, 680 protein peaks were observed in mouse liver extracts, demonstrating that a wider diameter separation column is effective for intact protein separations. Copyright © 2013 John Wiley & Sons, Ltd.     

Electro‐kinetic Separation of Rare Earth Elements Using a Redox‐Active Ligand

Purification of rare earth elements is challenging due to their chemical similarities. All of the deployed separation methods rely on thermodynamic properties, such as distribution equilibria in solvent extraction. Rare‐earth‐metal separations based on kinetic differences have not been examined. Herein, we demonstrate a new approach for rare‐earth‐element separations by exploiting differences in the oxidation rates within a series of rare earth compounds containing the redox‐active ligand [{2‐(t BuN(O))C₆H₄CH₂}₃N]³⁻. Using this method, a single‐step separation factor up to 261 was obtained for the separation of a 50:50 yttrium–lutetium mixture.     

A comparison of the direct and indirect LC methods for separating enantiomers of unusual glycine and alanine amino acid analogues

Summary Reversed-phase high-performance liquid chromatographic methods were developed for the separation of the enantiomers of five glycine and twelve alanine analogues. The enantioselective separation involved two methods. The direct separations were performed on chiral stationary phases containing a macrocyclic glycopeptide antibiotic: teicoplanin (Chirobiotic T column), ristocetin A (Chirobiotic R column) or chiral crown ether (Crownpak CR(+) column). The indirect methods involved pre-column derivatization with the chiral derivatizing agents 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate andN-α-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent). The different methods were compared in systematic chromatographic examinations. The effects of organic modifier content, mobile phase composition, pH and flow rate on the separation were investigated. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Comparative study of cylindrical and parallel‐plate electrophoretic separations for the removal of ions and sub‐23 nm particles

Cylindrical and parallel‐plate electrophoretic separations for the removal of ions and sub‐23 nm particles were compared in this study. First, COMSOL Multiphysics® software was utilized to simulate the ion and particle trajectories inside both electrophoretic separations. The results show that ions and sub‐23 nm particles are removed simultaneously and that all particles can pass through both electrophoretic separations smoothly at a trap voltage of 25 V. The experimental results show that ion losses become smaller with increasing ion flow rates, and ion losses of the cylindrical and parallel‐plate electrophoretic separations range from 56.2 to 71.6% and from 43.8 to 59.6%, respectively, at ion flow rates ranging from 1–3 L/min. For the removal of ions and sub‐23 nm particles, the collection efficiency of both electrophoretic separations can reach 100%, but the parallel‐plate electrophoretic separation requires a lower trap voltage. The particle loss of the parallel‐plate electrophoretic separation is under approximately 10%, which is lower than that of the cylindrical electrophoretic separation. In particular, for large particles (800–2500 nm), the particle losses inside the cylindrical electrophoretic separation are approximately two times higher than those inside the parallel‐plate electrophoretic separation. The parallel‐plate electrophoretic separation is beneficial for the removal of ions and sub‐23 nm particles.     

Chiral stationary phases for separation of intermedine and lycopsamine enantiomers from Symphytum uplandicum

Enantioseparation of the pyrrolizidine alkaloid isomers intermedine and lycopsamine, isolated from Symphytum uplandicum, is discussed. The separatory power of two immobilized carbohydrate‐based chiral HPLC columns, Chiralpak IA and IC, in different chromatographic conditions is compared. The study demonstrated the importance of solvent and column selection while developing such chiral HPLC separation methods. The baseline HPLC separation of the two alkaloid isomers in preparatory scale is reported for the first time. The optimized separations were achieved on a Chiralpak IA column with mobile phases of ACN/methanol (80:20) and methanol/methyl‐t‐butyl ether (90:10), both containing 0.1% diethylamine.     

Using capillary electrophoresis to separate trivalent f-elements based on their speciation when complexed with simple organic ligands

Capillary electrophoresis (CE) is a useful method for rapid separations of metal cations. Under ideal conditions, CE can provide complete separation of the trivalent lanthanides in less than 10 min. Simple organic ligands must be included in the electrolyte matrix to achieve good resolution between the cations. In this paper, we demonstrate how to use complexation constants from the literature to design separation protocols for the trivalent f-elements.     

Highly efficient high‐performance liquid chromatographic separation of xylene isomers and phthalate acid esters on a homemade DUT‐67(Zr) packed column

In this study, the baseline separations of xylene isomers and phthalate acid esters on a homemade DUT‐67(Zr) packed column were achieved, respectively. The high selectivity for xylene isomers and phthalate acid esters was obtained with the increase in temperature and decrease in the retention time. The hydrophobicity of xylene isomers and phthalate acid esters resulted in the different separation time on the DUT‐67(Zr) packed column. The relative standard deviation values of retention time, peak area, peak height, and half peak width for five repeat separation of the xylene isomers were 0.26–0.35, 2.11–2.26, 1.51–2.03, and 0.29–0.77%, and the values of the phthalate acid esters on DUT‐67(Zr) column were 0.1–0.4, 4.4–5.2, 3.9–6.3, and 0.6–2.1%, respectively. The thermodynamic properties indicated that the separation of xylene isomers was controlled by ΔH and ΔS, but the separation of phthalate acid esters was mainly controlled by ΔS.     

High‐performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles

The optimization of a porous structure to ensure good separation performances is always a significant issue in high‐performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high‐performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high‐performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high‐performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36 000 m^?1. Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans‐stilbene with separation factor as 7 and theoretical plate number as 76 000 m^?1 for cis‐stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long‐ established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.     

Fast fabrication of a hybrid monolithic column containing cyclic and aliphatic hydrophobic ligands via photo‐initiated thiol‐ene polymerization

Three monomers, octakis (3‐mercaptopropyl) octasilsesquioxane, 1,2,4‐trivinylcyclohexane and isophytol were employed to synthesize a novel monolithic stationary phase via photo‐initiated thiol‐ene click polymerization for reversed‐phase liquid chromatography. Several factors such as porogenic system, reaction time and the molar ratio of functional groups were investigated in detail. The resulting poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column exhibited suitable permeability for fast separation and outstanding thermal stability. Five alkylbenzenes were employed to evaluate the ability of chromatographic separation of the resulting monolithic columns at different flow rates, and showed the highest column efficiencies of 90,200–93,100 N/m (corresponding to 10.4–10.6 μm of plate height) at a velocity of 0.41 mm/s. The baseline separations of five anilines and eight phenols further proved the applicability of poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column in the separation of small molecules.     

Studies on the analytical performance of a non-covalent coating with N,N-didodecyl-N,N-dimethylammonium bromide for separation of basic proteins by capillary electrophoresis in acidic buffers in 25-and 50-μm capillaries

Capillaries (25-and 50-μm inner diameter) coated with a double-alkyl-chain cationic surfactant N,N-didodecyl-N,N-dimethylammonium bromide (DDAB) were used for the separation of four basic standard proteins in buffers of pH 4 at various ionic strengths. The choice of buffer is critical for the analytical performance. Ammonium ions must be avoided in the buffer used in the non-covalent coating procedure owing to competition with the surfactant. Phosphate buffer gave a better separation performance than some volatile buffers; the reason seems to be a complex formation between the proteins and dihydrogenphosphate ions, which decreases tendencies for adsorption to the capillary surface. The DDAB coating was easy to produce and stable enough to permit, without recoating, consecutive separations of the proteins for up to 100 min with good precision in migration times and peak areas. A strong electroosmotic flow gives rapid separations, which is of special importance when commercial instruments are used, since the choice of the length of the capillary is restricted. Figure EOF stability in 25 micrometer i.d. capillaries. Consecutive injections of mesityloxide performed after an initial coating with 1.0     

Polymeric Membrane Nanofiltration and Its Application to Separations in the Chemical Industries

The application of membrane technology, particularly water-based nanofiltration, as a separation process in the chemical industries has increased tremendously in recent years. However, the use of membranes capable of molecular separation in non-aqueous systems (e.g. nanofiltration) is a relatively new and growing application of membrane technology. The main challenge in applying polymeric nanofiltration membranes to non-aqueous systems is that the polymers developed for water-based applications are not suitable. Polyimide is a particularly interesting polymer as it has excellent chemical resistance, and membranes produced from it provide desirable separation properties – i.e. economically viable flux and good separation of nanoscale molecules. Various research works have shown that commercial polyimide organic solvent nanofiltration (OSN) membranes, trademark STARMEM™, 1 are robust and suitable for performing molecular separations. This work will discuss in detail the use of STARMEM™ in a pharmaceutical application. The EIC-OSN process was developed for separating the enantiomers of chiral compounds in pharmaceutical applications. High optical purity (94.9%) of (S)-phenylethanol from rac-phenylethanol was achieved through the use of STARMEM™122. Process simulation of the ideal eutomer-distomer system predicted that the highest theoretical resolvability from this process would be 99.2%. Other application areas of OSN are varied, including purification and fractionation in the natural products industry, homogeneous catalyst recovery, monomer separation from oligomers, etc. Currently, OSN is used in a small number of processes including a very large petrochemical application, but it has the potential to be applied to a wide range of separations across the full spectrum of the chemical industries.     

Carboxymethyl β‐cyclodextrin as chiral selector in capillary electrophoresis: Enantioseparation of 16 basic chiral drugs and its chiral recognition mechanism associated with drugs' structural features

Herein we present the enantioseparation of 10 cardiovascular agents and six bronchiectasis drugs including propranolol, carteolol, metoprolol, atenolol, pindolol, esmolol, bisoprolol, bevantolol, arotinolol, sotalol, clenbuterol, procaterol, bambuterol, tranterol, salbutamol and terbutaline sulfate using carboxymethyl‐β ‐cyclodextrin (CM‐β ‐CD) as chiral selector. To our knowledge, there is no literature about using CM‐β ‐CD for separating carteolol, esmolol, bisoprolol, bevantolol, arotinolol, procaterol, bambuterol and tranterol. During the course of work, changes in pH, CM‐β ‐CD concentration, buffer type and concentration were studied in relation to chiral resolution. Excellent enantiomeric separations were obtained for all 16 compounds, especially for procaterol. An impressive resolution value, up to 17.10, was obtained. In particular, most of them achieved rapid separations within 20 min. Given the fact that enantioseparation results rely on analytes' structural characters, the possible separation mechanisms were discussed. In addition, in order to obtain faster separation for propranolol enantiomers in practical application, the effective length of capillary was innovatively shortened from 45 to 30 cm. After the validation, the method was successfully applied to the enantiomeric purity determination of propranolol in the formulation of drug substances.