Separation and quantitative analysis of coumarin compounds from Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook. f by pressurized capillary electrochromatography

A pressurized capillary electrochromatography (pCEC) method with post-column detection cell has been developed for the therapeutically important coumarins from Angelica dahurica extract. The separation of five major coumarins (xanthotoxol, osthenol, imperatorin, oxypeucedanin hydrate, byakangelicin) was optimized with respect to composition of the mobile phase, ionic strength of buffers, pH, and applied voltage. Baseline separation was achieved for the five coumarins in less than 25 min using a mobile phase of methanol-acetonitrile-phosphate buffer (pH 4.8; 15 mM) (22.5:15:62.5, v/v/v). The method showed satisfactory retention time and peak area repeatability with the first use of post-column detection cell in the pCEC instrument. Comparing to capillary high performance liquid chromatography (capillary HPLC) and conventional high performance liquid chromatography (HPLC), higher column efficiency, and shorter analysis time were achieved in pCEC. The five coumarins in the extract samples representing different stages of traditional extraction of A. dahurica were also quantitatively analyzed by pCEC. The calibration curves were linear in the range 37-129, 36-126, 12-41, 88-306, 20-69 microg/ml of the standard solutions containing the five coumarins with correlation coefficients between 0.9976 and 0.9994.     

Chiral separation of β-blockers after derivatization with (−)-menthyl chloroformate by reversed-phase high performance liquid chromatography

Optimum conditions of chiral derivatization reaction of beta-blockers acebutolol, arotinolol, beta-xolol, bisoprolol, celiprolol, metoprolol and pindolol) with (-)-menthyl chloroformate were investigated for the resolution by HPLC. With more than 30 times molar excess of (-)-menthyl chloroformate chiral derivatization reactions were completed within one hour at room temperature except arotinolol and celiprolol. Diastereomeric derivatives of beta-blockers were well resolved on the ODS column using acetonitrile-methanol-water as a mobile phase.     

Comparison of the miniaturised techniques capillary electrochromatography and capillary liquid chromatography for the chiral separation of chlorthalidone

The aim of this study was to compare the miniaturised techniques, capillary electrochromatography (CEC) and capillary liquid chromatography (CLC), for the chiral separation of chlorthalidone. In both cases, hydroxypropyl-beta-cyclodextrin was used as a chiral selector in the mobile phase, while an achiral stationary phase was used. Earlier, this separation was already optimised in CEC. Now, the separation was optimised in CLC. The influence of the organic modifier content and the cyclodextrin concentration on the separation was studied by means of a central composite design. Optimal separation conditions were determined, after response modelling, from the response surface contour plots. When these conditions were compared with those of the CEC optimisation, we can see the potential of using CLC as a chiral separation technique since less chiral selector was used, faster separations were obtained and better repeatability was observed in comparison with its electrical-driven counterpart.     

HPLC法拆分氟比洛芬对映体

建立氟比洛芬手性药物的高效液相色谱拆分方法。方法:手性流动相添加剂HPLC法:利用C18柱,以羟丙基-β-环糊精作为手性流动相添加剂,调节有机修饰剂甲醇的比例和添加不同量的三乙胺对氟比洛芬进行拆分;手性固定相HPLC法:利用Chiral-pakAD手性柱,以正己烷-乙腈为流动相基本成分,调整两者不同比例和添加不同量的三乙胺,对氟比洛芬进行拆分。结果:手性流动相添加剂法:使用C18柱对氟比洛芬对映异构体进行拆分,调节流动相中有机修饰剂甲醇浓度、手性流动相添加剂羟丙基环糊精浓度、峰型修饰剂三乙胺的浓度等都不能使氟比洛芬对映体达到基线分离,只能部分分离。手性固定相法:氟比洛芬对映体在Chiral-pakAD手性柱上能达到较好的分离。在正己烷-乙腈流动相系统中,正己烷体积含量为90%,三乙胺体积含量为0.05%的条件下,氟比洛芬对映体得到了较好的分离,分离度为10.0。结论:建立的手性固定相法能有效拆分氟比洛芬对映体而手性流动相添加剂法不能拆分氟比洛芬对映体。     

Direct high-performance liquid chromatographic determination of the enantiomeric purity of levodopa and methyldopa: comparison with pharmacopoeial polarimetric methods

Chiral high-performance liquid chromatography was employed for determination of the enantiomeric purity of levodopa and methyldopa. The determination of D-DOPA in levodopa was accomplished using a chiral ligand-exchange chromatograpy with an ordinary C18 column and a chiral mobile phase containing N,N-dimethyl-L-phenylalanine and Cu(II) acetate or by means of LC on a teicoplanin column in conjunction with ethanol-water (65:35, v/v). Both methods gave good performance, however, the latter was faster and more convenient and suitable for routine analyses. For the determination of D-methyldopa a LC method based on the use of a teicoplanin column in polar organic mode with methanol-acetic acid-triethylamine (1,000:0.05:0.05, v/v/v) mobile phase was developed. The precision, accuracy, linearity and selectivity were satisfactory. In comparison with pharmacopoeial polarimetric methods (according to the European Pharmacopoeia and the Pharmacopoea Bohemoslovaca), the LC methods proved to be much more sensitive giving detection limits 0.04% of D-DOPA and 0.3% of D-methyldopa.     

Chiral separation of β-blockers after derivatization with (-)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride by gas chromatography

Gas chromatographic method was investigated for the chiral separation of several beta-blockers(atenolol, betaxolol, bisoprolol, metoprolol and pindolol) using (-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride as a chiral derivatizing agent for amino group. Prior to N-acylation, hydroxyl group was converted into O-silyl ethers by react with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The reaction was selective and rapid and the diastereomeric derivatives were well separated by capillary gas chromatography. (R)-isomers were eluted faster than (S)-isomers when (-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride was used as the chiral derivatizing agent. But in the opposite sequence when (+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride was used. No racemization was found during the reaction.     

手性流动相HPLC法拆分氟比洛芬对映体研究

目的:建立氟比洛芬手性药物的高效液相色谱拆分方法.方法:利用C18柱,以β-环糊精及其衍生物作为手性流动相添加剂,调节有机修饰剂比例和添加不同量的峰型修饰剂对氟比洛芬对映体进行拆分.结果:采用单6-L-脯氨酸-β-环糊精作为手性流动相添加剂,利用C18柱可直接拆分S,R-氟比洛芬对映体.最佳色谱分离条件为:流动相为1.4％的6 -L - proline -β - CD甲醇溶液(w/v):pH为4.0,体积分数1.0％的三乙胺水溶液(V/V) =25:75(V/V);柱温t=25℃;流速1.0ml/min;进样量10ul,检测波长254nm.S,R-氟比洛芬对映体获得了良好分离,分离度为1.65.结论:建立的手性流动相添加剂法能有效拆分氟比洛芬对映体.     

Beta 1-selectivity of bisoprolol, a new beta-adrenoceptor antagonist, in anesthetized dogs and guinea pigs

The beta-adrenoceptor activity of the newly synthesized antagonist bisoprolol [+/-)-1-[4-(2-isopropoxyethoxymethyl)-phenoxy]-3-isopropylamino-2- propranol, hemifumarate), has been compared with the effect of several reference compounds in anesthetized dogs and guinea pigs. In anesthetized, bivagotomized dogs, isoprenaline dose-response relations for increase in heart rate and decrease in diastolic blood pressure were established. Bisoprolol had the largest beta 1/beta 2 ratio, i.e., 147 (102-292). Practolol showed a beta 1/beta 2 ratio greater than 17; betaxolol 6-15; acebutolol, atenolol, and metoprolol 1.1-3.2; mepindolol 0.6-1 and propranolol 0.2. In artificially ventilated guinea pigs, the activity of bisoprolol on histamine-induced increase in tracheal lateral pressure (TLP) and basal heart rate (HR) was tested: using doses taken at TLP (30 mm Hg) and HR (250 beats/min), bisoprolol exhibited the most pronounced ratio TLP/HR of 124 +/- 59, followed by atenolol 33 +/- 23, metoprolol 25 +/- 15, betaxolol 12 +/- 4, propranolol 1 +/- 0.3, and celiprolol 0.23 +/- 0.19. These experiments indicate that bisoprolol possesses a pronounced beta 1-selectivity, which seems to be superior to that of known beta 1-selective antagonists.     

Chiral purity test of bevantolol by capillaryelectrophoresis and high performance liquid chromatography

Two methods for the chiral purity determination of bevantolol were developed, namely capillary electrophoresis (CE) using carboxymethyl-beta-cyclodextrin (CM-beta-CD) as a chiral selector and high-performance liquid chromatography (HPLC) using a chiral stationary phase. In the HPLC method, the separation of bevantolol enantiomers was performed on a Chiralpak AD-H column by isocratic elution with n-hexane-ethanol-diethylamine (10:90:0.1, v/v/v) as mobile phase. In the CE method, bevantolol enantiomers were separated on an uncoated fused silica capillary with 50 mM amonium phosphate dibasic adjusted to a pH 6.5 with phosphoric acid containing 15 mM CM-beta-CD as running buffer. Validation data such as linearity, recovery, detection limit, and precision of the two methods are presented. The detection limits of S-(-)-bevantolol were 0.1% and 0.05% for CE and HPLC method, respectively and R-(+)-bevantolol were 0.15% and 0.05% for CE and HPLC method, respectively. There was generally good agreement between the HPLC and CE results.     

同时分析酒石酸美托洛尔和富马酸比索洛尔与阿替洛尔的气相色谱-质谱联用法研究

目的建立同时分析酒石酸美托洛尔、富马酸比索洛尔和阿替洛尔的气相色谱-质谱联用法。方法样品经N,O-双（三甲基硅烷）三氟乙酰胺衍生化后,用气相色谱-质谱联用仪进行分离与分析。结果3种心脏病常用药物酒石酸美托洛尔、富马酸比索洛尔和阿替洛尔的检出限分别为16．73、33．58、21．46mg／L,对应的线性范围均为0．05—1．00g／L,回收率为91．2％、98．8％、88．4％。结论建立了同时分离分析3种心脏病药物的方法,该方法样品处理简便,色谱分离完全,结果准确可靠,为后期心脏病患者尿液中的药物代谢组学研究提供了基础。     

LC-ESI-MS method for the determination of bisoprolol in human plasma

A sensitive liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the determination of bisoprolol in human plasma, using metoprolol as internal standard (I.S.). After alkalization with sodium hydroxide, the samples were extracted with ethyl acetate and separated by HPLC on a ZORBAX SB-C18 column with a mobile phase of 10 mM ammonium acetate buffer containing 0.1% formic acid-methanol (32:68, v/v) at a flow rate of 1 ml/min. The chromatographic separation was achieved in less than 5 min. The linearity was established over the concentration range of 0.05-120 ng/ml. The intra- and inter-run standard deviation was less than 3.8 and 7.5%, respectively. The method had been successfully applied to study the relative bioavailability of bisoprolol fumarate tablets in healthy Chinese volunteers. The pharmacokinetic parameters of the reference and test tablets have been compared.     

Use of teicoplanin stationary phase for the enantiomeric resolution of atenolol in human urine by nano-liquid chromatography-mass spectrometry

Nano-liquid chromatography (nano-LC) was used for the enantiomeric resolution of atenolol employing a teicoplanin modified silica stationary phase prepared in our laboratory. Experiments were carried out in a fused silica capillary of 75 microm i.d. packed with chiral modified silica particles of 5 microm diameter. Separated enantiomers were revealed by on-line UV detector at 205 nm or electrospray-ion-trap mass spectrometer (ESI-MS). Atenolol enantiomers were eluted utilizing a mobile phase with the following composition: 500 mM ammonium acetate pH 4.5/methanol/acetonitrile 1:60:39 (v/v/v) allowing to achieve good enantioresolution in a reasonable analysis time (about 8 min) with a flow rate of about 900 nL/min. After comparing the sensitivity of the nano-LC method using a conventional UV detector for capillary electrophoresis, a zeta cell (3 nL volume) employed in nano-LC and the ion-trap MS the method was validated with the MS detector offering the highest sensitivity (limit of detection (LOD)=50 ng/mL; limit of quantification (LOQ)=400 ng/mL for each atenolol enantiomer). (-)-Psi-Nor-ephedrine was used as the internal standard. The method was successfully applied to the analysis of atenolol enantiomers present in human urine samples of a patient under atenolol therapy.     

Enantiomeric separation of beta-blockers by HPLC using (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid as chiral stationary phase

Direct liquid chromatographic separations of the enantiomers of metoprolol and bisoprolol have been developed, using (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid as chiral stationary phase (CSP). The separations were achieved in a normal phase system employing a mobile phase containing n-hexane, 1,2-dichloroethane and methanol. Column efficiency was strongly dependent on the composition of the mobile phase. The eluent contents of methanol and of 1,2-dichloroethane were optimized, and so was flow-rate and column temperature. Under the optimal conditions, linear responses for (R)-metoprolol and (S)-metoprolol are obtained in the range of 0.079–1.38 and 0.015–5.80 mg/ml, with detection limits of 0.008 and 0.002 mg/ml, respectively. As for bisoprolol, the linear ranges of (R)-isomer and (S)-isomer are 0.05–1.31 and 0.02–1.00 mg/ml with detection limits of 0.001 and 0.008 mg/ml, respectively. The relative standard deviation (R.S.D.) of each enantiomer did not exceed 0.90%. The method has been successfully applied to the determination of enantiomers in pharmaceuticals.     

Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs transport across Caco-2 cell monolayers

This study aimed to determine the transepithelial transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers, a model of human intestinal epithelial membrane. Six chiral aryloxy enantiomers (atenolol, sotalol, celiprolol, carvedilol, metoprolol and propafenone) were tested in bi-directional transport studies. The separation and quantitation of these enantiomers were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) using 2, 3, 4, 6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate (GITC) as a pre-column derivatizing agent. Bi-directional transport studies demonstrated that celiprolol and carvedilol exhibited significant enantioselectivity in polarized transport at the concentration range tested. The efflux ratio (ER) for (R)-(+)-celiprolol was 8.96, but it was much lower for (S)-(-)-celiprolol which is 3.42 at the concentration of 96.0 μM; carvedilol had the same transport behavior as celiprolol while the difference between the ER values of two enantiomers was not as significant as celiprolol at the concentration of 5.0 μM. They are 2.41 for (R)-(+)-carvedilol and 1.98 for (S)-(-)-carvedilol. But in the transport studies of racemic atenolol, sotalol, metoprolol and propafenone, no enantioselective transport were observed over the concentration range tested. Because P-glycoprotein (P-gp) is highly expressed in Caco-2 cells, we inferred that P-gp might participate in the transport processes of celiprolol and carvedilol in chirally discriminative ways.     

焦谷氨酸对映体的手性高效液相色谱分离

目的:采用大环抗生素游壁菌素键合硅胶手性分离柱 Chirobiotic T 对焦谷氨酸手性对映体进行分离。方法:以 Chirobi-otic T 柱为色谱柱,流动相为甲醇-三乙胺缓冲溶液(醋酸调 pH)体系和甲醇-醋酸-三乙胺极性有机溶剂体系,检测波长为214 nm,考察了焦谷氨酸对映体的分离,并与大环抗生素万古霉素键合硅胶手性分离柱 Chirobiotie V 进行了比较。结果:在甲醇-0.1%三乙胺(80:20,以醋酸调 pH 4.0)为流动相时,焦谷氨酸对映体在 Chirobiotic T 柱上可以实现基线分离,但有系统峰干扰;而甲醇-醋酸-三乙胺极性有机溶剂体系则更利于焦谷氨酸对映体的分离,并可对实际样品进行定量分析,无系统峰干扰。而在这2种条件下,Chirobiotic V 柱无法基线拆分焦谷氨酸对映体。结论:Chirobiotie T 柱对焦谷氨酸手性对映体有很好的拆分能力,可用于实际样品的定量分析。     

Comparative study of the enantiomeric resolution of chiral antifungal drugs econazole, miconazole and sulconazole by HPLC on various cellulose chiral columns in normal phase mode.

The chiral resolution of (+/-)-econazole, (+/-)-miconazole and (+/-)-sulconazole on the columns containing different cellulose derivatives namely Chiralcel OD, OJ, OB, OK, OC and OF in normal phase mode has been described. The mobile phase used was hexane-2-propanol-diethylamine (425:74:1, v/v/v). The flow rates of the mobile phase used were 0.50, 1.00 and 1.50 ml/min. The values of the separation factor (alpha) of the resolved enantiomers of econazole, miconazole and sulconazole on chiral phases were ranged from 1.07 to 2.50 while the values of resolution factor (R(s)) varied from 0.17 to 3.90. The chiral recognition mechanisms between the analytes and the chiral selectors are discussed.     

Different effects of the beta-adrenoceptor antagonists celiprolol and metoprolol on vascular structure and function in long-term type I diabetic rats

An intriguing problem of diabetes mellitus is the development of generalized angiopathy and concomitant hypertension. However, there is still a controversy whether beta-adrenoceptor antagonists can be used as antihypertensive agents in diabetes. Four groups of rats were investigated: nondiabetic controls, diabetes mellitus, diabetes + celiprolol (250 mg/kg body weight/day), diabetes + metoprolol (125 mg/kg body weight/day) after 6 months. Diabetes was induced by i.v. streptozotocin injection. We examined vascular structure and function histologically and by an in vitro microvideoangiometry of isolated perfused mesenterium. Additionally, we investigated the effects of hyperglycemia and celiprolol on NO release in cultivated aortic endothelial cells and the effect of celiprolol on transendothelial paracellular permeability. Diabetes resulted in endothelial dysfunction, characterized by a reduced response to acetylcholine and L-N(G)-nitro-arginine and an unchanged response to sodium nitroprusside (SNP). These effects were significantly antagonized by celiprolol but were not influenced by metoprolol treatment. This was supported by the finding of typical vascular changes associated with diabetes like media thickening, reduced cardiac capillary/muscle fiber ratio, and glomerulosclerosis, which were significantly reduced by celiprolol but not influenced by metoprolol treatment. Ketonuria improved after celiprolol treatment, whereas blood glucose, lipids, and body weight were not different between the diabetic groups. In cultured cells, celiprolol did not induce direct NO release but reversed the impairment of stimulated NO release caused by hyperglycemia. Furthermore, celiprolol reduced endothelial paracellular permeability. We conclude that celiprolol can exert antiangiopathic effects in diabetic rats and that both beta-adrenoceptor antagonists did not aggravate diabetic angiopathy and metabolic derangement.     

Beta-1 antagonist and cardiostimulatory effects of celiprolol in anesthetized dogs

Celiprolol is a new cardioselective beta adrenoceptor antagonist presently undergoing worldwide clinical traits. The present study was undertaken to further evaluate the beta antagonist and hemodynamic effects of celiprolol compared to the known beta antagonists propranolol, atenolol, and dichloroisoproterenol (DCI). The results show that celiprolol up to 1 mg/kg i.v. had no significant effect on contractile force (CF), heart rate(HR) or mean, arterial pressure (AP) in normal anesthetized dogs. In these animals, however, celiprolol produced a dose-related decreases in the beta-1 response to isoproterenol (ISO; ED50=0.01 for CF and 0.06 mg/kg, i.v. for HR) while having a lesser effect on the AP (beta-2) responses to ISO (ED50 =1.8 mg/kg, i.v.). Celiprolol was equipotent to propranolol and atenolol in antagonizing the beta-1-mediated effects of ISO while being equipotent to atenolol and 300 times less potent than propranolol in antagonizing the beta-2-mediated effects. In mecamylamine-pretreated dogs, celiprolol (1 and 3 mg/kg, i.v.) produced limited but significant increases in CF (25 ± 7 and 48 ± 10%, respectively) and HR (18 ± 1 and 22 ± 2%, respectively). DCI (0.1 mg/kg, i.v.) produced a more marked increase in CF (79 ± 9%) and HR (33 ± 7%). By contrast, atenolol 3 mg/kg i.v. and propranolol 1 and 3 mg/kg i.v. significantly depressed both CF and HR. None of the treatments had consistent effects on arterial pressure or aortic flow. In mecamylamine-and propranolol-pretreated dogs, celiprolol 1 or 3 mg/kg i.v. still produced a significant increase in CF (31 ± 9 and 21 ± 6%, respectively) and HR (15 ± 2 and 17 ± 3%, respectively). By contrast, the cardiostimulatory effects of DCI were completely abolished by propranolol. It is concluded that celiprolol is a potent and cardioselective beta adrenoceptor antagonist with mild cardiostimulatory effects only partially attributable to beta receptor stimulation.     

Development of liquid chromatographic enantiomer separation methods and validation for the estimation of (R)-enantiomer in eslicarbazepine acetate

Chiral separation method development was carried out for eslicarbazepine acetate and its (R)-enantiomer on diverse chiral stationary phases. Better chiral selectivity was observed on cellulose tris-(3,5-dichlorophenylcarbamate) immobilized column (Chiralpak IC-3). Under polar organic mode (POM), with 100% acetonitrile as mobile phase and 0.5 ml/min flow, a resolution close to three was achieved. With normal phase (NP) mobile phase consisting dichloromethane:ethanol (90:10, v/v) and 1.0 ml/min flow, a resolution close to six was achieved. Detection was done by UV at 220 and 240 nm respectively. Both the methods were found to be robust and were validated with respect to robustness, precision, linearity, limit of detection, limit of quantification and accuracy. The proposed methods are suitable for the accurate estimation of (R)-enantiomer in bulk drug samples up to 0.1% when a 1 mg/ml analyte test solution is chromatographed.     

Pharmacological actions of the selective and non-selective beta-adrenoceptor antagonists celiprolol, bisoprolol and propranolol on human bronchi.

1. The pharmacological actions of the beta-adrenoceptor antagonists, celiprolol, bisoprolol and propranolol were investigated in human lung tissue by radioligand binding experiments as well as in human isolated bronchi by functional experiments in organ baths. 2. Data from lung tissue were compared to those obtained from myocardial membranes. 3. Lung tissue was obtained from 10 patients having undergone lung resection for bronchial carcinoma and myocardial tissue from a patient who had received a heart transplantation. 4. In radioligand binding experiments, celiprolol exhibited a high affinity binding to beta 1-adrenoceptors in heart and a low affinity binding to beta 2-adrenoceptors in lung tissue. The selectivity obtained for the beta 1-adrenoceptor was calculated to a factor of eleven. 5. Compared to bisoprolol and propranolol, celiprolol elicited the lowest affinity for the beta-adrenoceptor, as judged from the K1-values. 6. In the absence and presence of the guanine nucleotide Gpp(NH)p celiprolol did not affect receptor binding. 7. In functional experiments on intact bronchi, celiprolol, bisoprolol and propranolol failed to produce relaxation (+/- forskolin) or a significant difference in efficacy in antagonizing the relaxant effects of isoprenaline. However, a rank order of potencies was revealed (propranolol:bisoprolol:celiprolol = 46:12:1). 8. Plasma concentrations for celiprolol and bisoprolol usually achieved in vivo were below the IC50 value obtained in vitro. In contrast, for propranolol, plasma concentrations were nearly identical with the IC50 value. 9. It is concluded that celiprolol is a selective beta 1-adrenoceptor antagonist on human heart and has no agonistic properties on intact human bronchi.(ABSTRACT TRUNCATED AT 250 WORDS)