Determination of competitive adsorption isotherm parameters of pindolol enantiomers on alpha1-acid glycoprotein chiral stationary phase

In this paper, inverse method (IM) was used to determine the binary competitive adsorption isotherm of pindolol enantiomers by a least-square fitting of the proposed model to the experimentally measured elution curves of racemic pindolol. The isotherm parameters were determined by minimizing the least-square error using an adaptation of genetic algorithm, non-dominated sorting genetic algorithm with jumping genes (NSGA-II-JG). An equilibrium dispersive (ED) model combined with bi-Langmuir isotherm was used in predicting the elution profiles. The determined parameters show good agreement with the experimental profiles at various experimental conditions such as sample volume, concentration and flow rates of the racemic mixture. Robustness and validity of the isotherm parameters were also verified by frontal analyses at various step inputs. Results from both the pulse tests and the frontal analysis indicate that adsorption isotherm derived from the inverse method is quite reliable. This method requires relatively less number of experiments to be performed and therefore, lower experimental costs confirming that inverse method is an attractive alternative approach of experimental technique in determining the competitive adsorption isotherm for binary systems.     

Investigation of chiral recognition mechanism on chicken alpha(1)-acid glycoprotein using separation system

Chicken alpha1-acid glycoprotein (alpha1-AGP) consists of 183 amino acid residues and has only one Trp residue at the 26 position. In this study, the Trp26 residue was modified with 2-nitrophenylsulfenyl chloride and chiral separation of neutral, acidic and basic compounds was examined on chicken alpha1-AGP and Trp-modified chicken alpha1-AGP columns. Chiral separation of propranolol, alprenolol and oxprenolol was lost on the Trp-modified chicken alpha1-AGP column, while chlorpheniramine, ketoprofen and benzoin were still enantioseparated on the Trp-modified chicken alpha1-AGP column despite of lower enantioselectivity than that on the chicken alpha1-AGP column. These results suggest that the Trp26 residue could be responsible for chiral recognition of these compounds. Competition studies using N,N-dimethyl-n-octylamine (DMOA) as a competitor indicated that propranolol, alprenolol and oxprenolol competed with DMOA on a single binding site near the Trp26 region and that further bindings of chlorpheniramine, ketoprofen and benzoin occurred at the secondary binding site in a non-competitive fashion with DMOA.     

Retention processes on alpha 1-acid glycoprotein-bonded stationary phase.

Stereoselective separations of charged enantiomers on CHIRAL-AGP can be controlled by varying the pH and adding charged and uncharged additives to the mobile phase. The interaction with the selector, alpha 1-acid glycoprotein, was studied by monitoring the effects of the variables on retention and by indirect detection, in part using a simple multivariate design. The stereoselectivity is due to simultaneous retention processes involving ion-exchange and ion-pairing mechanisms. The predominant mode of interaction for different solutes was elucidated from variables that promote or counteract either of the processes. Considerable improvements in the stereoselectivity were achieved with chiral or achiral anionic and cationic additives that act in a synergistic or competitive mode.     

High-performance liquid chromatographic separation of the enantiomers of hydroxychloroquine and its major metabolites in biological fluids using an alpha 1-acid glycoprotein stationary phase.

An enantioselective two-stage off-line assay has been developed for the analysis of hydroxychloroquine and its three major metabolites in biological fluids. The first non-stereoselective stage of the assay (PRP-1 column) separates and quantitates parent drug and metabolites. Fractions containing hydroxychloroquine and each of the metabolites are collected manually, evaporated, reconstituted in mobile phase and re-injected onto an alpha 1-acid glycoprotein column to separate and determine proportions of individual enantiomers. Preliminary results from patients samples indicate that the disposition of hydroxychloroquine and its major metabolites is enantioselective. p6     

Optical resolution of a series of potential cholecystokinin antagonist 4(3H)-quinazolone derivatives by chiral liquid chromatography on alpha1-acid glycoprotein stationary phase

Optical resolution of the enantiomers of new 4(3H)-quinazolone derivatives is investigated using the alpha1-acid glycoprotein chiral stationary phase (Chiral-AGP). Stereoselective separation of the model compounds can be controlled by varying the pH and adding uncharged organic modifiers (acetonitrile and 2-propanol) to the mobile phase. For the majority of quinazolone derivatives, Chiral-AGP is proved to be an excellent enantioselector, because optimized chromatographic conditions allow for the baseline separation of the enantiomers. Separation factors between 1.19 and 1.85 are obtained. The effects of acetonitrile and 2-propanol on the chromatographic behavior of the model compounds are quite different because of their different hydrophobic- and hydrogen-bonding properties. The eluent pH and organic modifier concentration also contributes to the chiral recognition by altering the protein environment. The analysis of the experimental results leads to new information about the chromatographic mechanism on a Chiral-AGP surface.     

Development of an affinity silica monolith containing alpha1-acid glycoprotein for chiral separations

An affinity monolith based on silica and containing immobilized alpha(1)-acid glycoprotein (AGP) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of AGP in the silica monolith was 18% higher than that obtained with silica particles and 61% higher than that measured for a GMA/EDMA monolith. The higher surface area of the silica monolith gave materials that contained 1.5- to 3.6-times more immobilized protein per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the AGP silica monolith were evaluated by injecting two chiral analytes onto this column (i.e., R/S-warfarin and R/S-propranolol). In each case, the AGP silica monolith gave higher retention plus better resolution and efficiency than AGP columns containing silica particles or a GMA/EDMA monolith. The AGP silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with AGP as a chiral stationary phase.     

冠醚固定相的制备及手性拆分

以R-联萘酚为原料合成了R-(1,1′-二萘基)-20-冠-6,并将其涂敷于C18硅胶(平均粒径5μm,孔径120nm)上制成了可用于高效液相色谱手性拆分的R-(1,1′-二萘基)-20-冠-6冠醚固定相(CSP).在以pH=2的高氯酸溶液为流动相,流速为0.1mL·min-1,柱温为25℃的条件下,研究了CSP对9种α-氨基酸对映体的拆分能力.实验结果表明,有5种手性氨基酸(缬氨酸、苯甘氨酸、对羟基苯甘氨酸、谷氨酸、色氨酸)得到不同程度的拆分,说明CSP能对手性氨基酸进行一定的拆分.     

The acute phase protein alpha1-acid glycoprotein: a model for altered glycosylation during diseases

Glycosylation is one of the most important post-translational modifications of proteins, and has been widely acknowledged as one of the most important ways to modulate both protein function and lifespan. The acute phase proteins are a major group of serum proteins whose concentration is altered during various pathophysiological conditions. The aim of this paper is to review the structure and functions of the alpha1-acid glycoprotein (AGP). AGP belongs to the subfamily of immunocalins, a group of binding proteins that also have immunomodulatory functions. One of the most interesting features of AGP is that its glycosylation microheterogeneity can be modified during diseases. This aspect is particularly remarkable, since both the immunomodulatory and the binding properties of AGP strongly depend on its carbohydrate composition. For these reasons, AGP can be considered an outstanding model for the study of glycan pattern modification during diseases. This review is focused on the most recent studies on the occurrence of different glycoforms in plasma and tissues and how the appearance of different oligosaccharide patterns during systemic inflammation or diseases can influence AGP's biological functions. The first part of the review will describe the structure of AGP and the several biological functions identified so far for this protein. The second part will be devoted to the post-translational modifications of the oligosaccharides micro-heterogeneity of AGP caused by pathological states. A critical evaluation of the impact of different AGP glycoforms on both its transport and anti-inflammatory features, and how the modifications of the glycan pattern can be utilized in clinical biochemistry, is also discussed.     

Direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using a chiral alpha 1-acid glycoprotein column

A 10-cm long alpha 1-acid glycoprotein column is used for the enantiomeric resolution of the clinically used racemic aminoglutethimide (+/- AG) and its acetylated metabolite (+/- AAG). A direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite is accomplished without any derivatizations. Maximum resolutions of 1.37 and 0.73 are obtained for the enantiomers of aminoglutethimide and its acetylated metabolite, respectively. The effect of the 2-propanol content in mobile phase on retention and enantioselectivity of aminoglutethimide and its acetylated metabolite is demonstrated. The variation of the separation factors (alpha) with pH in enantiomeric separation of aminoglutethimide is also shown.     

Enantiomeric separation of diuretics on a novel Pirkle-type chiral stationary phase

A newly developed Pirkle-type straight chiral stationary phase (CSP), based on the 3,5-dinitrobenzoyl derivative of 1,2 diphenylethylene-diamine and known as ULMO, has been successfully applied to the direct resolution of the enantiomers of several diuretics by liquid chromatography. In this study, the effect of changes in the mobile phase and the intrinsic stereoselective properties of this CSP towards a specific racemate are determined experimentally. A mobile phase consisting of n-hexane and 2-propanol appears most appropriate for the chiral separation of the tested diuretics on the ULMO-column.     

直链淀粉手性固定相拆分联二萘酚对映体

建立了联二萘酚对映体的高效液相色谱拆分方法。使用ChiralpakAD-H手性色谱柱,考察了流动相中极性调节剂的种类和比例、柱温以及流速对拆分联二萘酚对映体的影响。确定了最佳拆分条件:流动相为V(正己烷):V(乙醇)=55:45;流速为0.8mL/min;检测波长254nm;柱温30℃。计算了联二萘酚与固定相相互作用的焓变差值Δ(ΔH0)和熵变差值Δ(ΔS0)分别为-2.10kJ/mol和-4.94J/(mol.K),并考察了在以乙醇或异丙醇为极性调节剂流动相中两对映体的出峰顺序,结果发现在这两种流动相中出峰顺序是相反的。方法可用于联二萘酚的手性分离与检测。     

直链淀粉手性固定相法分离利阿唑对映体

建立了利阿唑对映体的高效液相色谱拆分方法。采用Chiralpak AD-H手性柱在正相条件下直接拆分利阿唑对映体,考察了流动相中有机极性调节剂的种类和浓度、酸碱的种类和含量、柱温及流速等对利阿唑对映体分离的影响。确定了最佳的拆分条件:流动相为正己烷-乙醇(含0.3%二乙胺和0.1%冰醋酸)(体积比为80∶20),流速0.6 mL/min;检测波长254 nm;柱温20 ℃。在此条件下利阿唑对映体的分离度为3.4。该法简单快速,重现性好。     

分子间缩聚制备纤维素手性固定相及其手性拆分性能

以微晶纤维素、3,5-二甲基苯基异氰酸酯和3-(三乙氧基硅)丙基异氰酸酯为原料,通过区域选择法合成了6位带有三乙氧基硅基团的纤维素-二(3,5-二甲基苯基氨基甲酸酯)衍生物,利用三乙氧基硅基团的分子间缩聚将纤维素衍生物高效的固定到硅胶基质上,制备了交联型纤维素手性固定相。在正己烷-异丙醇(体积比4:1),流速1 mL/min,检测波长230 nm时,甲霜灵对映体得到了很好的分离,分离度(Rs)为4.36。此外,还探讨了流动相的配比,不同醇类改性剂对拆分效果的影响,优化了色谱条件。结果表明,交联型纤维素手性固定相对甲霜灵对映体具有较好的手性拆分能力,并且允许使用含有氯仿和四氢呋喃的流动相。     

Study on the interaction between the chiral drug of propranolol and alpha1-acid glycoprotein by fluorescence spectrophotometry

The interaction between the chiral drug of propranolol (PPL) and alpha1-acid glycoprotein (AGP, orosomucoid) has been first studied by fluorescence spectrophotometry. The fluorescence intensity of PPL increased due to the addition of AGP into PPL. The equation of Scatchard was employed to calculate the association constant and binding site number of the two enantiomers with AGP. The association constant is 2.62 x 10(5)M-1 for R-PPL and 8.57 x 10(5)M-1 for S-PPL and the binding site number is 0.41 for R-PPL and 1.17 for S-PPL at 17 degrees C respectively. The method of thermodynamics was applied to determine the binding type of S-PPL with AGP. The results suggested that the binding type is mainly van der waals force or hydrogen bond. At last the effect of three metal cations on the association constant and the binding site number of S-PPL with AGP was examined.     

Preparation and chiral separation of a novel immobilized cellulose-based chiral stationary phase in high-performance liquid chromatography

The chiral selector 6-azido-2, 3-di(p-chlorophenylcarbamoylated) cellulose was synthesized and further chemically immobilized onto 5-μm amino functionalized spherical porous silica gel. It was used as chiral stationary phase in high-performance liquid chromatography. Thirty racemates were successfully separated into enantiomers in either normal phase mode or reversed-phase mode. Good reproducibility and stability of the chiral stationary phase have been demonstrated.     

Liquid chromatographic separation of phosphoramidate diastereomers on a polysaccharide-type chiral stationary phase

To improve the therapeutic potential of anti-HIV nucleoside analogues (d4T, AZT, 3TC and ddl), the delivery of the corresponding monophosphate from neutral, membrane-permeable prodrugs has been realised by the synthesis of lipophilic phosphoramidate triester prodrugs, such as the simple phenyl-L-alaninephosphate derivatives. However, the present non-stereoselective synthesis results in a mixture of 1:1 diastereomers, which differ from the configuration of the phosphorus atom asymmetric center. Since each diastereomer may have different biological activity and pharmacokinetic profile, analytical methods have to be developed for their separation. This work aims at showing the ability of a polysaccharide-type chiral stationary phase to resolve such diastereomers in reversed-phase high-performance liquid chromatography. The influence of operating parameters has been studied to optimise the separation; a thermodynamic approach has also been investigated to gain an insight in the retention mechanism of the prodrugs. Preliminary validation study (linearity, accuracy, repeatability) has yielded good results; in addition, the feasibility of HPLC-electrospray-mass spectrometry (HPLC-ESI-MS) coupling has been demonstrated and it is expected that this will lead to lower detection limits.