Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

Direct separation of the enantiomers of ramosetron on a chlorinated cellulose‐based chiral stationary phase in hydrophilic interaction liquid chromatography mode

Ramosetron is an enantiopure active pharmaceutical ingredient marketed in Japan since 1996 and later in a few Southeast Asian countries predominantly as an antiemetic for patients receiving chemotherapy. In this study, a simple and rapid high‐performance liquid chromoatography method for the separation of ramosetron and its related enantiomeric impurity by using hydrophilic interaction liquid chromatography mode is presented. Chiral resolution was performed on an analytical column (100 mm × 4.6 mm id) packed with 3 μm particles of cellulose‐based Chiralpak IC‐3 chiral stationary phase. Using a mobile phase containing acetonitrile–water–diethylamine (100:10:0.1, v/v/v) and setting the column temperature at 35°C, the resolution value was 7.35. At a flow rate of 1 mL/min, the enantioseparation was completed within 5 min. The proposed method was partially validated and it has proven to be sensitive with limit of detection and limit of quantitation of the (S)‐enantiomer impurity of 44.5 and 133.6 ng/mL.     

In‐situ photopolymerized polyhedral oligomeric silsesquioxane‐derived monolithic capillary columns with quinidine functionality for enantioseparation by nano‐liquid chromatography

The successful fabrication of monolithic capillary columns for enantiomer separations was achieved within vinylized fused silica capillaries via fast “one‐pot” photo‐initiated free radical polymerization reaction. A mixture consisting of polyhedral oligomeric silsesquioxane, O‐[2‐(methacryloyloxy)ethylcarbamoyl]‐10,11‐dihydroquinidine was copolymerized in the presence of n‐butanol, ethylene glycol and photo‐initiator 2,2‐dimethoxy‐2‐phenylacetophenone. The morphology of the resultant polymeric hybrid inorganic‐organic material and its permeability as well as porosity can be controlled by adjusting the composition of the monomers and binary porogenic solvent. The chromatographic characteristics of the columns have been investigated. Separation factors of N‐acetyl‐phenylalanine (Ac‐Phe) and dichlorprop dropped with decrease of chiral functional monomer. Permeability was better when the macroporogen ethyleneglycol was present at higher concentrations during the polymerization. In general, the chiral compounds were well separated (dichlorprop: α = 1.53, R_s up to 4.14; Ac‐Phe: α = 1.36, R_s up to 2.69) by nano‐HPLC with an optimized enantioselective monolithic capillary column which can be prepared within a few minutes.     

Enantioseparation of selected chiral sulfoxides in high‐performance liquid chromatography with polysaccharide‐based chiral selectors in polar organic mobile phases with emphasis on enantiomer elution order

The separation of the enantiomers of 17 chiral sulfoxides was studied on polysaccharide‐based chiral columns in polar organic mobile phases. Enantiomer elution order (EEO) was the primary objective in this study. Two of the six chiral columns, especially those based on amylose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(4‐chloro‐3‐methylphenylcarbamate) (Lux Cellulose‐4) proved to be most successful in the separation of the enantiomers of the studied sulfoxides. Interesting examples of EEO reversal were observed depending on the chiral selector or the composition of the mobile phase. For instance, the R‐(+) enantiomer of lansoprazole eluted before the S‐(?) enantiomer on Lux Cellulose‐1 in both methanol or ethanol as the mobile phase, while the elution order was opposite in the same eluents on amylose tris(3,5‐dimethylphenylcarbamate) with the S‐(?) enantiomer eluting before the R‐(+) enantiomer. The R‐(+) enantiomer of omeprazole eluted first on Lux Amylose‐2 in methanol but it was second when acetonitrile was used as the mobile phase with the same chiral selector. Several other examples of reversal in EEO were observed in this study. An interesting example of the separation of four stereoisomers of phenaminophos sulfoxide containing chiral sulfur and phosphor atoms is also reported here.     

Structure‐enantioselectivity relationships for the study of chiral recognition in peptide enantiomer separation on cinchona alkaloid‐based chiral stationary phases by HPLC: Influence of the N‐terminal protecting group

Eleven different N‐terminal protecting groups (acetyl, benzoyl, FMOC, etc.) were employed for the HPLC separation of oligoalanine peptide enantiomers containing up to six amino acids. Isocratic HPLC separations were performed using a hydro‐organic buffered mobile phase and 4 mm ID columns containing three different chiral anion exchange stationary phases based on cinchona alkaloid‐derived chiral selectors. For most peptides successful separations could be obtained with all protecting groups, although those comprising aromatic moieties were found to yield higher enantioselectivities than those with aliphatic residues, since they are capable of undergoing favourable π‐π interactions with the selector. Systematic investigations concerning the presence or absence of structural features of related protecting groups showed that the use of protecting groups that are optimally adjusted to the binding pocket of the chiral selector effects a significant gain in enantioselectivity. At the same time these studies provided new insights into the chiral recognition mechanism.     

HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide‐based chiral columns

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide‐based chiral HPLC columns with various normal‐phase (NP), polar organic, and reversed‐phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co‐elution of enantiomers. Additionally, in organic‐aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.     

Chiral separation of novel diazenes on a polysaccharide‐based stationary phase in the reversed‐phase mode

Chiral high‐performance liquid chromatography separation of two recently synthesized liquid crystalline materials C1 and C2 was studied in the reversed‐phase mode. Both materials have an azo‐moiety and one chiral center in their molecular structures. They were available in racemic and pure S forms. For the enantiomeric separations, a Chiralpak AY‐RH stationary phase based on amylose tris(5‐chloro‐2‐methylphenylcarbamate) coated on 5 μm silica was used. The compounds were analyzed in both of their possible forms, the more thermodynamically stable E form and the labile Z form. The conditions and time scale of the UV‐induced E to Z transition were briefly evaluated. Under the optimized conditions, we were able to baseline separate S and R enantiomers of both of the studied materials not only in their E forms, but also in their Z forms. In comparison to the separation in the normal‐phase mode, which we have reported recently, the resolution in the reversed‐phase mode is significantly better. Interestingly, peak reversal was noticed for the S and R enantiomers when the separation was carried out with E versus Z forms of both compounds.     

Liquid chromatographic resolution of racemic rasagiline and its analogues on a chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A liquid chromatographic chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was applied to the resolution of 15 analytes, including racemic rasagiline, a chiral drug for the treatment of Parkinson's disease, and its analogues. The composition of mobile phase was optimized to be ethanol/acetonitrile/acetic acid/triethylamine (80:20:0.2:0.3, v/v/v/v) by evaluating the chromatographic results for the resolution of five selected analytes under various mobile phase conditions. Under the optimized mobile phase conditions, racemic rasagiline was resolved quite well with a separation factor of 1.48 and resolution of 2.71 and its 14 analogues were also resolved reasonably well with separation factors of 1.06–1.54 and resolutions of 0.54–2.11. Among 15 analytes, racemic rasagiline was resolved best except for just one analyte. The analyte structure–enantioselectivity relationship indicated that racemic rasagiline has the most appropriate structural characteristics for resolution on the chiral stationary phase.     

新型含苯并噻唑α-氨基膦酸酯类化合物在淀粉类手性固定相上的液相色谱分离

本文运用涂敷型(Chiralpak AD-H)和键合型(Chiralpak IA)两种淀粉类手性固定相高效液相色谱法,进行了新型含苯并噻唑α-氨基膦酸酯类化合物的手性分离。从色谱分离的保留因子(k)、分离系数(α)和分离度(Rs)三个方面考察了两种类型色谱柱的分离性能,上述化合物在Chiralpak IA柱上能够得到较好的基线分离。同时,讨论了温度、流动相极性和目标分析物的结构等因素对Chiralpak IA柱分离性能的影响。由于键合型固定相较稳定的性能,使某些非常规的溶剂(如THF)成功地应用于手性α-氨基膦酸酯类化合物的分离。     

Enantioselective separation of chiral vicinal diols in capillary electrophoresis using a mono‐6A‐aminoethylamino‐β‐cyclodextrin as a chiral selector

This paper describes an improved access to mono‐6^A‐aminoethylamino‐β‐CD (β‐CDen), a very efficient cationic chiral selector for CZE in the separation of eight chiral aromatic vicinal diols. The β‐CDen concentration has a strong influence on the efficiency of enantioseparation. The effects of the pH and concentration of the BGE, the capillary temperature, and the applied voltage on the resolution and separation selectivity have been studied. Excellent chiral resolution was achieved under the optimal conditions of β‐CDen 10 mM, pH 10, 200 mM borate buffer at 15 kV and 20°C within 20 min. Moreover, the developed method was successfully applied to the determination of the enantiomeric purity of the catalytic asymmetric dihydroxylation (AD) reaction products.     

Determination of Amantadine Residue in Honey by Solid‐phase Extraction and High‐performance Liquid Chromatography with Pre‐column Derivatization and Fluorometric Detection

Amantadine (AMA) is an anti‐viral drug used in apiculture to protect honeybee against the sacbrood virus (Morator aetatulae). This study described a reliable high‐performance liquid chromatographic (HPLC) method for analyzing AMA in honey using a solid‐phase extraction (SPE) cartridge (Plexa PCX) for purification, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F) as a pre‐column derivatization agent, and fluorometric detection (λ_ex=470 nm, λ_em=530 nm). The chromatographic separation was performed on an XDB C18 column (150×4.6 mm i.d.) using 0.1% trifluoroacetic acid/acetonitrile (35:65,V/V) as the mobile phase at a flow rate of 1.0 mL·min⁻¹ with a run time of 20 min. Under these optimal conditions, a linear relationship was observed in the range of 0.025–1.0 µg· mL⁻¹ with a good correlation coefficient (0.998) and low limit of detection (0.0080 µg·g⁻¹), the recoveries were all above 90%, and the intra‐day and inter‐day precision (RSD) ranged from 3.4%–5.1%.     

Resolution of beta-amino acids on a high performance liquid chromatographic doubly tethered chiral stationary phase containing N-CH3 amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) containing N-CH(3 )amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied to the resolution of various beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were consistent with those on the corresponding singly tethered CSP while the chiral recognition ability of the doubly tethered CSP was generally greater in terms of both the separation (alpha) and the resolution factors (R(S)) than that of the corresponding singly tethered CSP. From these results, it was concluded that attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the CSP improves the chiral recognition ability for the resolution of beta-amino acids without any change in the chiral recognition mode. The retention factors (k(1)) on the doubly tethered CSP were larger than those on the corresponding singly tethered CSP and these retention factors were found to be controllable with the variation of the type and the content of the organic and/or acidic modifier in the aqueous mobile phase without significant change in the separation and the resolution factors.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of α‐amino acids and proton pump inhibitors on a ligand exchange chiral stationary phase

A new ligand exchange chiral stationary phase (new CSP) containing residual silanol group‐protecting n‐octyl groups on the silica surface was prepared by treating a ligand exchange CSP (original CSP) based on sodium N‐[(R)‐2‐hydroxy‐1‐phenylethyl]‐N‐undecylaminoacetate bonded to silica gel with excess n‐octyltriethoxysilane. The new and original CSPs containing an identical amount of chiral selector were applied to the resolution of α‐amino acids and proton pump inhibitors (PPIs) including omeprazole, pantoprazole, lansoprazole, and rabeprazole. The separation factors (α) and resolutions (R_S) were greater on the new CSP than on the original CSP except for the resolution of asparagine. The trends of the retention factors (k₁) for the resolution of α‐amino acids on the new and original CSPs with the variation of the organic modifier content in aqueous mobile phase were opposite to those for the resolution of PPIs. Removal of the nonenantioselective interactions between the residual silanol groups and the analytes and the improved lipophilicity of the new CSP were proposed to be responsible for the improved chiral recognition ability of the new CSP and the different retention behaviors of the enantiomers between the new and original CSPs.     

Optimization and Validation of a Fast Liquid Gradient for Determination of Prominent Flavan‐3‐ols and Flavonols in Fresh Vegetables

A fast high‐performance liquid chromatography method was used for analysis of prominent flavan‐3‐ols and flavonols in vegetables. Gradient elution with phosphoric acid‐acetonitrile mixtures and phosphoric acid‐methanol mixtures allowed fast and complete separation of the studied phenolic compounds within analysis times less than 10 min. The development of two elution gradients using methanol and acetonitrile as modifiers proved to be an excellent approach for the verification of the real polyphenolic composition in vegetables samples because the two optimized methods allowed the separation of the same number of compounds in the same elution order. Diode‐array detection was employed for the provisional identification of phenolic compounds that were not available as standards. We preferred methanol as a modifier because it was less toxic and cheaper than acetonitrile. Detection limits ranged between 0.12 and 0.59 μg mL^–1. High recoveries of phenolics from fresh vegetables were measured in all studied cases, independent of the phenolic structure, matrix, and vegetable in question. High levels of procyanidins between 150 and 450 mg kg^–1 were found in all studied vegetables. Quantification of quercetin and kaempferol glycosides was only possible in marrow and onion, respectively.     

Poly(glycerin 1,3‐dimethacrylate)‐based monolith with a bicontinuous structure tailored as HPLC column by photoinitiated in situ radical polymerization via viscoelastic phase separation

In this article we described our new approach to the polymer monolith with its morphology tailored for HPLC application to small solutes such as drug candidates. We prepared polymer monoliths based on glycerin 1,3‐dimethacrylate, GDMA with a bicontinuous structure by in situ photoinitiated free radical polymerization (UV irradiation at 365 nm). Our photopolymerization was carried out with a monodispese ultra high molecular weight polystyrene solution in chlorobenzene uniquely formulated as a porogen. The poly‐GDMA monoliths in bulk, rod and capillary thus prepared showed a bicontinuous network‐like structure featured by their fine skeletal thickness nearly in sub μm size. This monolithic structure was considered as a time‐evolved morphology frozen by UV‐irradiation via viscoelastic phase separation induced by the said porogenic polystyrene solution. According to our μHPLC measurement with acetophenone as a model solute, the UV prepared poly‐GDMA capillary demonstrated a much shaper elution profile affording higher column efficiency and permeability as compared with the thermally prepared capillary of the same bore size. Our investigation showed experimentally that poly‐GDMA monoliths with a well‐defined bicontinuous structure could be prepared reproducibly by photoinitiated radical polymerization via viscoelastic phase separation using the said unique porogen. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 4651–4673, 2008     

Voltammetric Behavior of p‐Nitrophenol and Its Trace Determination in Human Urine by Liquid Chromatography with a Dual Reductive Mode Electrochemical Detection System

The determination of trace p-nitrophenol (PNP) concentrations in human urine has been successfully achieved by high performance liquid chromatography dual electrode detection (LC-DED) in the reduction-reduction mode. Initial cyclic voltammetric studies were undertaken to investigate the electrochemical behavior of PNP at a glassy carbon electrode over a wide pH range; the redox processes giving rise to the signals have been deduced. Further, deductions regarding the behavior in the flow cells were made from hydrodynamic voltammetric data. PNP eluting from the analytical LC column is first reduced to p-hydroxylaminophenol, at the first generator electrochemical cell. This species then undergoes chemical oxidation to give a quinoneimine species which is then detected at the downstream detector electrode using an applied potential of −0.1 V. The optimum chromatographic mobile phase consisted of 40% acetonitrile, 60% water, containing 25 mM o-phosphoric acid, at a flow rate of 0.5 mL/min; this was used in conjunction with a Hypersil C₁₈ column. Hydrodynamic voltammetric studies were undertaken to investigate the dual electrode behavior of PNP, and an applied potential of −2.0 V at the generator cell and −0.10 V at the detector cell were found to be optimum. The response was found to be linear over the range 7.0 ng to 500 ng on column, with an associated R² value of 0.9981; the limit of detection was found to be 1.0 ng PNP on column. No interferences were seen for a number of common drugs or for the principal electrochemically active components of human urine or serum. The developed assay was successfully applied to the determination of trace concentrations of PNP in human urine samples, exhibiting coefficients of variation of 7.1% (n=7), with a mean recovery of 94.7% for urine fortified at 522 ng mL⁻¹.     

Cu(II)‐Mediated Atom Transfer Radical Polymerization of Methyl Methacrylate via a Strategy of Thermo‐Regulated Phase‐Separable Catalysis in a Liquid/Liquid Biphasic System: Homogeneous Catalysis,Facile Heterogeneous Separation,and Recycling

A strategy of thermo‐regulated phase‐separable catalysis (TPSC) is applied to the Cu(II)‐mediated atom transfer radical polymerization (ATRP) of methyl methacrylate (MMA) in a p‐xylene/PEG‐200 biphasic system. Initiators for continuous activator regeneration ATRP (ICAR ATRP) are used to establish the TPSC‐based ICAR ATRP system using water‐soluble TPMA as a ligand, EBPA as an initiator, CuBr₂ as a catalyst, and AIBN as a reducing agent. By heating to 70 °C, unlimited miscibility of both solvents is achieved and the polymerization can be carried out under homogeneous conditions; then on cooling to 25 °C, the mixture separates into two phases again. As a result, the catalyst complex remains in the PEG‐200 phase while the obtained polymers stay in the p‐xylene phase. The catalyst can therefore be removed from the resultant polymers by easily separating the two different layers and can be reused again. It is important that well‐defined PMMA with a controlled molecular weight and narrow molecular weight distribution could be obtained using this TPSC‐based ICAR ATRP system.

     

Analysis of Synthetic Drugs in Chinese Medicine by High Performance Liquid Chromatography/Mass Spectrometry with In‐Source Collision Induced Dissociation

Sixteen chemical drugs, often found in adulterated Chinese medicine, were studied by high performance liquid chromatography/atmospheric ionization mass spectrometry. Under optimal conditions, three pairs of compounds were either coeluted or unresolved. The lack of chromatographic resolution and the lack of specificity in UV detection were overcome by a method based on high performance liquid chromatography/electrospray mass spectrometry. This method was capable of detecting the adulterants based on their retention times, molecular ions, and characteristic fragments resulting from in‐source collision induced dissociation.     

In Vivo Monitoring of the Thiols in Rat Striatum by Liquid Chromatography with Amperometric Detection at a Functionalized Multi‐Wall Carbon Nanotubes Modified Electrode

A new chemically modified electrode (CME) was fabricated, which was based on the immobilization of multi‐wall carbon nanotubes fuctionalized with carboxylic group (MWNT‐COOH). The results indicated that the CME exhibited efficiently electrocatalytic oxidation for L ‐cysteine and glutathione with relatively high sensitivity, stability and long‐life. Coupled with HPLC, the MWNT‐COOH CME was utilized for amperometric detection of the thiols. The peak currents of L ‐cysteine and glutathione were linear to their concentrations ranging from 3.0×10^?7 to 1.0×10^?3 mol/L with the calculated detection limit (S/N=3) of 1.2×10^?7, 2.2×10^?7 mol/L, respectively. The method had been successfully applied to assess the contents of L ‐cysteine and glutathione in rat striatal microdialysates.