Use of neutral capillaries for the enantioseparation of N-benzoylated amino acids by capillary electrophoresis with bromobalhimycin as chiral selector.

In this study, the partial filling technique on both polycationic polymer hexadimethrine bromide (HDB) modified capillary and eCAP neutral capillary were systematically compared in order to enhance the enantioseparation ability of bromobalhimycin as CE additive. The separation conditions, such as pH, the plug length, and the concentration of bromobalhimycin, etc., were optimized in order to obtain satisfactory separations. As expected, for all tested 28 N-benzoylated amino acids, up to five times higher enantioresolutions were obtained on the eCAP neutral capillary compared to that on the polycationic polymer hexadimethrine bromide modified capillary. Moreover, 26 of 28 tested racemic compounds were almost baseline- resolved without observing any interference from the front of the plug of bromobalhimycin. Although the limitation of longer running time on the neutral capillary, it allows the use of higher content of bromobalhimycin in the running buffer without any interference on the detection of analytes when enantioseparations are more difficult to obtain.

Evaluation of chlorine containing cellulose-based chiral stationary phases for the LC enantioseparation of basic pharmaceuticals using polar non-aqueous mobile phases.

The discrimination ability of three cellulose-based chiral stationary phases (CSPs) was evaluated towards the enantiomers of basic drugs, using ACN as the main solvent in polar organic mobile phases. The study was focused on CSPs containing cellulose tris(3-chloro-4-methylphenylcarbamate) (3-Cl-4-MePC), cellulose tris(4-chloro-3-methylphenylcarbamate) (4-Cl-3-MePC) or cellulose tris(3,5-dichlorophenylcarbamate) (3,5-diClPC) as the chiral selector. The behaviour of these CSPs was studied systematically in order to investigate the influence of the presence and position of the chlorine substituents on the phenylcarbamate moieties on the retention and resolution of the enantiomers. The evaluation was made with three different generic mobile phases, namely ACN/0.1%DEA/0.1% TFA (DEA, diethylamine), ACN/0.1%DEA/0.2% FA and ACN/0.1%DEA/0.2%AcA, deduced from the previous study. The nature of the acidic additive and of the chiral selector was found to be particularly important for the retention and enantioresolution of these basic compounds. High-resolution values could be obtained for most studied enantiomers with these CSPs, clearly demonstrating the interest of using them in combination with polar organic mobile phases. However, significant differences in enantioresolution between the CSPs have been observed for many compounds, indicating that these phases seem to be quite complementary.

Development of acidic phospholipid containing immobilized artificial membrane column to predict drug-induced phospholipidosis potency.

Drug-induced phospholipidosis (DIPLD) represents a big concern for both regulatory authorities and pharmaceutical companies in drug discovery. Many researches pointed out that the negatively charged intralysosomal lipids play an important role in the formation of DIPLD. To better mimic this negatively charged lipid surface, a novel immobilized artificial membrane (IAM) column was prepared via in situ copolymerization of 12-methacryloyl n-dodecylphosphocholine (MDPC) and 12-methacryloyl n-dodecylphosphoric acid (MDPA). By introducing MDPA, the surface of the resulting monolithic column can be maintained negatively charged over a broad pH range. Scanning electron microscopy, elemental analysis and nano-HPLC experiments were carried out to characterize the physicochemical properties and chromatographic performance of the obtained monolithic IAM column. The results of ζ-potential and retention mechanism studies indicate that both hydrophobic and electrostatic interactions contribute greatly to the retention of cation analytes owing to the existence of the negatively charged MDPA under acidic conditions. To better assess the DIPLD potency of drug, the molar ratio between MDPC and MDPA in the monolithic column was carefully optimized. The results show that the poly(MDPC70PA30-co-EDMA) column has the best predictability with only two false-positives (donepezil, flecainide) in qualitative analysis of 61 drugs.

Development of zirconium modified adenosine triphosphate functionalized monolith for specific enrichment of N-glycans.

In this study, to selectively enrich N-glycans from complex biological samples, a novel Zr(IV) modified adenosine triphosphate (Zr(IV)-ATP) functionalized monolith was prepared through a facile approach. Well-defined macroporous structure was observed in the ATP functionalized monolith, which allows rapid mass transfer under low backpressure and is beneficial for the enrichment of N-glycans. After being modified with Zr(IV), the resulting Zr(IV)-ATP functionalized monolith could selectively capture N-glycans through the specific interactions between the sulfonate groups of 1-aminopyrene-3,6,8-trisulfonic acid (APTS) labeled N-glycans and Zr(IV). An APTS labeled maltooligosaccharide ladder was used to optimize the enrichment conditions for APTS labeled N-glycans, and capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detector was employed to evaluate the enrichment efficiency. The results show that the APTS labeled maltooligosaccharides could be enriched under the selected conditions and the signal amplify factors of the maltooligosaccharides were between 7.4 and 19.5 with RSDs for reproducibility from 4.0% to 8.3% (n = 3). Finally, the proposed method was successfully used for the enrichment and detection of N-glycans released from Ribonuclease B.

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector and polar non-aqueous mobile phases.

The resolving power of a new commercial polysaccharide-based chiral stationary phase, Sepapak-4, with cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n-hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face-centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n-hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.

Rapid preparation of 1-vinylimidazole based non-affinity polymers for the highly-selective purification of antibodies from multiple biological sources.

There is an urgent need for developing advanced purification techniques with the merits of low cost and satisfactory capacity in order to meet the challenges in the current downstream purification of monoclonal antibodies (mAbs). Herein, a simple and inexpensive nitrogen heterocycle molecule, 1-vinylimidazole (VIM), was proposed as the capture ligand of antibodies for the first time. The corresponding VIM-based non-affinity polymeric material (polyVIM) was then fabricated via a one-step polymerization for use in the highly selective purification of antibodies. Compared to the previously reported materials, this novel material exhibited many advantages without clearly sacrificing selectivity, such as a simpler and faster fabrication (within 1.5 h), comparable or even higher binding capacity (saturated static adsorption capacity > 190 mg/g polymer, dynamic binding capacity about 31.62 mg/g polymer), lower non-specific protein adsorption, and much lower cost. Notably, the polyVIM can effectively purify the antibodies from multiple biological sources with high purity (95.4% for mAbs in the cell culture medium, 93.3% for hIgG in the human serum), with an acceptable recovery (91.6% for mAbs, 77.0% for hIgG), and good reusability (> 10 times). Moreover, the target ELISA binding assay and NFAT-luc reporter gene assay demonstrated that the enriched antibodies can well maintain their binding activity and bioactivity during the whole purification process. The excellent performance of the polyVIM material may be attributed to the high recognition ability of VIM for antibodies, as well as the biocompatible and antifouling properties of the porous polymer. This study provides a promising alternative material for the purification of mAbs in downstream processes and the enrichment of hIgG in human serum.

Hydrophilic polymeric monoliths containing choline phosphate for separation science applications.

In this research, a hydrophilic polymeric monolith containing choline phosphate (CP) was fabricated through the thermally initiated free-radical polymerization of 2-{2-(methacryloyloxy) ethyldimethylammonium}ethyl n-butyl phosphate (MBP) with ethylene dimethacrylate (EDMA) using isopropanol and tetrahydrofuran as the porogenic system. Excellent mechanical strength, permeability, stability and reproducibility were obtained on the optimized poly(MBP-co-EDMA) monolith. Column efficiency as high as 107,500 plates/m was achieved for the analysis of thiourea at a flow velocity of 0.2 mm/s on the monolith. Both hydrophilic and electrostatic interactions were observed for the retention of charged analytes on the poly(MBP-co-EDMA) monolith. It is worth noting that the resulting monolith exhibits higher selectivity and efficiency than the classical 2-(methacryloyloxy)ethyl phosphorylcholine functionalized polymeric monolith for the chromatographic separation of polar analytes. Also, the novel monolithic column was successfully applied to enrich N-glycopeptides from tryptic digest of human IgG. In a word, the versatile MBP functionalized polymeric monolith not only opens up interesting possibilities for reverse zwitterionic CP derivative-based polymeric materials in separation science, but also represents a promising hydrophilic interaction chromatography stationary phase.

Preparation and evaluation of 400µm I.D. polymer-based hydrophilic interaction chromatography monolithic columns with high column efficiency.

The quest for higher column efficiency is one of the major research areas in polymer-based monolithic column fabrication. In this research, two novel polymer-based HILIC monolithic columns with 400µm I.D.×800µm O.D. were prepared based on the thermally initiated co-polymerization of N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine (SPP) and ethylene glycol dimethacrylate (EDMA) or N,N'-methylenebisacrylamide (MBA). In order to obtain a satisfactory performance in terms of column permeability, mechanical stability, efficiency and selectivity, the polymerization parameters were systematically optimized. Column efficiencies as high as 142, 000 plates/m and 120, 000 plates/m were observed for the analysis of neutral compounds at 0.6mm/s on the poly(SPP-co-MBA) and poly(SPP-co-EDMA) monoliths, respectively. Furthermore, the Van Deemter plots for thiourea on the two monoliths were compared with that on a commercial silica based ZIC-HILIC column (3.5µm, 200Å, 150mm×300µm I.D.) using ACN/H2O (90/10, v/v) as the mobile phase at room temperature. It was noticeable that the Van Deemter curves for both monoliths, particularly the poly(SPP-co-MBA) monolith, are significantly flatter than that obtained for the ZIC-HILIC column, which indicates that in spite of their larger internal diameters, they yield better overall efficiency, with less peak dispersion, across a much wider range of usable linear velocities. A clearly better separation performance was also observed for nucleobases, nucleosides, nucleotides and small peptides on the poly(SPP-co-MBA) monolith compared to the ZIC-HILIC column. It is particularly worth mentioning that these 400µm I.D. polymer-based HILIC monolithic columns exhibit enhanced mechanical strength owing to the thicker capillary wall of the fused-silica capillaries.

Liquid chromatography separation of the chiral prodrug eslicarbazepine acetate and its main metabolites in polar organic mode. Application to their analysis after in vitro metabolism.

A LC method using a chiral stationary phase (CSP) with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector in polar organic mode (POM) was developed for the separation of the biopharmaceutic classification system (BCS) class II chiral prodrug eslicarbazepine acetate (ESL) and its main metabolites, namely eslicarbazepine, its optical antipode, (R)-licarbazepine, and the achiral oxcarbazepine (OXC). The percentage of methanol (MeOH) in the mobile phase containing acetonitrile (ACN) as the main solvent was found to significantly influence analyte retention and resolution. A reversal of elution order of OXC and (R)-licarbazepine was observed, depending on the MeOH percentage in the mobile phase. The optimized mobile phase consisted of ACN/MeOH/acetic acid/diethylamine (95/5/0.2/0.07; v/v/v/v). The potential of this chemo- and enantioselective LC method combined with solid-phase extraction (SPE) was then evaluated for in vitro metabolism studies using ESL as a model case. Only eslicarbazepine could be detected after incubation of ESL in human liver microsome systems.

Influence of the linking spacer length and type on the enantioseparation ability of ß-cyclodextrin functionalized monoliths.

In order to investigate the effect of the linking spacer on the enantioseparation ability of ß-cyclodextrin (ß-CD) functionalized polymeric monoliths, three ß-CD-functionalized organic polymeric monoliths with different spacer lengths were prepared by using three amino-ß-CDs, i.e. mono-6-amino-6-deoxy-ß-CD, mono-6-ethylenediamine-6-deoxy-ß-CD, mono-6-hexamethylenediamine-6-deoxy-ß-CD, as starting materials. These amino-ß-CDs reacted with glycidyl methacrylate to produce functional monomers which were then copolymerized with ethylene dimethacrylate. The enantioseparation ability of the three monoliths was evaluated using 14 chiral acidic compounds, including mandelic acid derivatives, nonsteroidal anti-inflammatory drugs, N-derivatized amino acids, and chiral herbicides under optimum chromatographic conditions. Notably, the poly(GMA-NH2-ß-CD-co-EDMA) column provides higher enantioresolution and enantioselectivity than the poly(GMA-EDA-ß-CD-co-EDMA) and poly(GMA-HDA-ß-CD-co-EDMA) columns for most tested chiral analytes. Furthermore, the enantioseparation performance of triazole-linker containing monoliths was compared to that of ethylenediamine-linker containing monoliths. The results indicate that the enantioselectivity of ß-CD monolithic columns is strongly related to the length and type of spacer tethering ß-CD to the polymeric support.

Chiral separation of acidic compounds using an O-9-(tert-butylcarbamoyl)quinidine functionalized monolith in micro-liquid chromatography.

An O-9-(tert-butylcarbamoyl) quinidine (t-BuCQD) functionalized polymeric monolithic capillary column was prepared by the in situ copolymerization method. The physicochemical properties of the optimized monolithic column were characterized by scanning electron microscopy and micro-LC. Satisfactory column permeability, efficiency, stability and reproducibility were obtained for this monolithic column. The chiral recognition ability of the resulting monolith was also evaluated using 47 N-derivatized amino acids, eight N-derivatized dipeptides, and two herbicides. Under the selected conditions, the enantiomers of all chiral analytes were baseline separated with exceptionally high selectivity and resolution using micro-LC. It is worth noting that this chiral stationary phase (CSP) containing quinidine with a tert-butyl carbamate residue as chiral selector exhibits much higher enantioselectivity and diastereoselectivity than the previously developed O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine (MQD) based CSP for N-derivatized amino acids and dipeptides. These results indicate that this novel quinidine-based polymeric monolith can be used as an effective tool for the enantioseparation of chiral acidic compounds.

One-pot preparation of a sulfamethoxazole functionalized affinity monolithic column for selective isolation and purification of trypsin.

A facile and efficient "one-pot" copolymerization strategy was used for the preparation of sulfonamide drug (SA) functionalized monolithic columns. Two novel SA-immobilized methacrylate monolithic columns, i.e. poly(GMA-SMX-co-EDMA) and poly(GMA-SAA-co-EDMA) were prepared by one-pot in situ copolymerization of the drug ligand (sulfamethoxazole (SMX) or sulfanilamide (SAA)), the monomer (glycidyl methacrylate, GMA) and the cross-linker (ethylene dimethacrylate, EDMA) within 100 µm i.d. capillaries under optimized polymerization conditions. The physicochemical properties and column performance of the fabricated monolithic columns were characterized by elemental analysis, scanning electron microscopy and micro-HPLC. Satisfactory column permeability, efficiency and separation performance were obtained on the optimized poly(GMA-SMX-co-EDMA) monolithic column for small molecules, such as a standard test mixture and eight aromatic ketones. Notably, it was found that the poly(GMA-SMX-co-EDMA) monolith showed a selective affinity to trypsin, while the poly(GMA-SAA-co-EDMA) monolith containing sulfanilamide did not exhibit such affinity at all. This research not only provides a novel monolith for the selective isolation and purification of trypsin, but it also offers the possibility to easily prepare novel drug functionalized methacrylate monoliths through a one-pot copolymerization strategy.

Enantiomeric separation of N-protected amino acids by non-aqueous capillary electrophoresis with dimeric forms of quinine and quinidine derivatives serving as chiral selectors.

A non-aqueous capillary electrophoretic method with quinine and tert.-butyl carbamoylated quinine as chiral selectors was developed previously for the enantioseparation of N-protected amino acids. This system was here applied as a screening tool for a fast evaluation of the chiral discrimination potential of six new dimeric forms of carbamoylated quinine and quinidine derivatives as chiral additives: 1,3-phenylene-bis(carbamoylated quinine), 1,6-hexamethylene-bis(carbamoylated quinine), 1,6-hexamethylene-bis(carbamoylated quinidine), trans-1,4-cyclohexylene-bis(carbamoylated quinine), trans-1,4-cyclohexylene-bis(carbamoylated-11-dodecylthio-dihydroquinine) and trans-1,4-cyclohexylene-bis(carbamoylated-11-dodecylsulfinyl-dihydroquinine). A series of 24 chiral acids, as various benzoyl, 3,5-dinitrobenzoyl (DNB) and 3,5-dinitrobenzyloxycarbonyl amino acid derivatives were investigated with regards to enantioselectivity employing these different dimeric chiral selectors. The composition of the background electrolyte was 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM chiral selector in an ethanol-methanol (60:40, v/v) mixture and the enantioseparations were performed at 15 degrees C and in the reversed polarity mode at -25 kV. With these dimeric chiral selectors, higher enantioselectivity values, compared to those obtained with monomeric derivatives, were usually achieved, especially with the dimers containing dodecyl substituents. For example, an alpha value of 4 and a resolution value of 78 were obtained for DNB-phenylalanine, using trans-1,4-cyclohexylene-bis(carbamoylated-11-dodecylthio-dihydroquinine) as selector.

Development and validation of a liquid chromatographic method for the stability study of a pharmaceutical formulation containing voriconazole using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector and polar organic mobile phases.

The ophthalmic solution of voriconazole, i.e. (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, made from an injection formulation which also contains sulfobutylether-ß-cyclodextrin sodium salt as an excipient (Vfend), is used for the treatment of fungal keratitis. A liquid chromatographic (LC) method using polar organic mobile phase and cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica as chiral stationary phase was successfully developed to evaluate the chiral stability of the ophthalmic solution. The percentage of methanol (MeOH) in the mobile phase containing acetonitrile (ACN) as the main solvent significantly influenced the retention and resolution of voriconazole and its enantiomer ((2S,3R)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol). The optimized mobile phase consisted of ACN/MeOH/diethylamine/trifluoroacetic acid (80/20/0.1/0.1; v/v/v/v). The method was found to be selective not only regarding the enantiomer of voriconazole but also regarding the specified impurities described in the monograph from the European Pharmacopoeia. The LC method was then fully validated applying the strategy based on total measurement error and accuracy profiles. Under the selected conditions, the determination of 0.1% of voriconazole enantiomer could be performed. Finally, a stability study of the ophthalmic solution was conducted using the validated LC method.

Liposome electrokinetic chromatography based in vitro model for early screening of the drug-induced phospholipidosis risk.

Drug-induced phospholipidosis (PLD) is a storage disorder of lysosomes characterized by the excessive accumulation of phospholipids as a result of improper medical treatments. Although few evidences have supported that PLD can induce significant pathological consequences, this potential toxicity indeed can put off the drug discovery process. In this research, a high-throughput liposome electrokinetic chromatography (LEKC) method was validated to evaluate the PLD risk of drug candidates by screening drug-phospholipid interaction, which correlates to the phospholipidosis inducing risk. A statistical analysis based on the Spearman's correlation test showed that the retention factors (log k) of the tested drugs in the LEKC system and the literature reported in vivo and in vitro PLD data were highly correlated. In order to investigate the predictability of LEKC, the effect of liposome composition such as the molar ratio of phospholipids and the addition of cholesterol were also discussed in this study. The results indicated that the LEKC method could offer a fast, reliable and cost-effective screening tool for early prediction of the PLD inducing potential of drug candidates.

Enantioresolution of basic pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral stationary phase and polar organic mobile phases.

A polysaccharide-based chiral stationary phase (Sepapak-4), with cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector, has been investigated in liquid chromatography (LC). Its enantioresolution power was evaluated towards 13 basic amino-drugs with widely different structures and polarities, using polar organic mobile phases. After preliminary experiments, acetonitrile was selected as the main mobile phase component, to which a low concentration of diethylamine (0.1%) was systematically added in order to obtain efficient and symmetrical peaks. Different organic solvents were first added in small proportions (5-10%) to acetonitrile to modulate analyte retention. Polar organic modifiers were found to decrease retention and enantioresolution while hexane had the opposite effect, indicating normal-phase behaviour under these conditions. The addition of an organic acid (formic, acetic or trifluoroacetic acid) was found to strongly influence the retention of the basic amino drugs in these nonaqueous systems. The nature and proportion of the acidic additive in the mobile phase had also deep impact on enantioresolution. Therefore, the studied compounds could be subdivided in three groups in respect to the acidic additive used. All analytes could be enantioseparated in relatively short analysis times (10-20 min) using these LC conditions.