两种药物的手性分离和立体选择性药物动力学研究
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摘要
手性化合物的对映体分离是手性新药研制工作的基础，寻找高效、灵敏、快速、简便的手性分离方法是药物分析领域的重要课题。本文分别采用HPCE法和HPLC法对西孟坦和马布特罗对映体进行了分离，并对马布特罗对映体在大鼠体内的药物动力学行为进行了初步研究。
1．西孟坦对映体的毛细管电泳分离方法研究
以β—环糊精(β-CD)为手性选择剂，采用毛细管区带电泳对西孟坦进行了手性分离研究。考察了背景电解质中硼砂缓冲液的pH及浓度、β-CD浓度、有机改性剂甲醇含量和分离电压对手性分离的影响。建立了毛细管电泳分离西孟坦对映体的方法。最佳分离条件为：20mmol／L硼砂缓冲液(pH11.0，含12mmol／Lβ-CD)-甲醇(50∶50，v／v)，分离电压20kV，在此条件下西孟坦对映体可达基线分离。
2．毛细管电泳法测定马布特罗离解常数
利用毛细管电泳中不同pH条件下有效淌度的倒数(1／μ_e)与缓冲溶液中氢离子浓度的倒数(1／[H~+])之间存在着线性关系的原理，测定了马布特罗的pK_a值。以1％丙酮为中性标记物，离子强度为0.09的不同pH的缓冲液为运行缓冲液，检测波长为254nm，分离电压为10kV。结果马布特罗的pK_a为10.14，与另一种方法—等摩尔浓度分光光度法的测得值相比较，结果相近。
3．毛细管电泳法同时分离分析马布特罗及其结构类似物的对映体
以羟丙基-β-环糊精(HP-β-CD)为手性选择剂，应用毛细管电泳法同时对马布特罗、克仑特罗、SPFF、班布特罗等四种β_2受体激动剂类手性药物进行了对映体分离。考察了HP-β-CD浓度和背景电解质pH对分离效果的影响。最佳分离条件为100mmol／L柠檬酸—10mmol／L磷酸氢二钠—120mmol／L HP-β-CD，pH2.5，分离电压20kV，检测波长210nm，柱温为室温。在此条件下，四种药物对映体均得到良好的分离结果。对方法学进行了考察，并测定了其中两种药物的对映体纯度。
4．马布特罗对映体体内分析方法的建立及其在大鼠体内的立体选择性药物动力学研究
将非手性、手性色谱系统相结合，建立了大鼠血浆中马布特罗对映体的测定方法。首先用非手性色谱法将药物与血浆内源性杂质分离，然后收集检测器后药物色谱峰流分，应用大环抗生素固定相柱(Chirobiotic V)，以甲醇—冰醋酸—三乙胺(100∶0.01∶0.01，v／v／v)为流动相，将两个对映体完全分离。根据血浆中马布特罗的浓度和l／d-对映体浓度比值，计算得到l-和d-马布特罗的血浆药物浓度。采用本法研究了6只大鼠单剂量灌胃给予马布特罗消旋体后，两对映体的药物动力学。结果表明，马布特罗两对映体有相似的t_(max)(5.33±2.1h，l-马布特罗；5.67±1.51h，d-马布特罗)和C_(max)(266.8±37.9ng／ml，l-马布特罗；277.9±44.6ng／ml，d-马布特罗)，但配对t检验表明，两对映体的AUC_(0-∞)、t_(1／2(k))、CL／F和MRT存在显著性差异，提示l-马布特罗比d-马布特罗作用更有效、更持久。推测两对映体的代谢存在立体选择性。
Enantioseparation of chiral compound is the foundation of new chiral drug's research and development. Searching for high efficient, sensitive, rapid and simple chiral separation method in laboratory is important in pharmaceutical analysis field. Chiral separation of simendan and mabuterol was investigated in the present work by using high performance capillary electrophoresis (HPCE) and high performance liquid chromatography (HPLC). The pharmacokinetics of mabuterol enantiomers in rats were also studied by using the developed HPLC method.1. Chiral separation of simendan enantiomers by capillary zone electrophoresisThe enantiomers of simendan were separated by capillary zone electrophoresis (CZE) usingβ-cyclodextrin (β-CD) as the chiral selector. The influences of the concentration and pH of borate buffer solution,β-CD concentration, methanol content and separation voltage were investigated. A baseline separation of simendan enantiomers was achieved with background electrolyte of 20mmol/L borate buffer (pH 11.0) containing 12mmol/Lβ-CD-methanol (50:50, v/v) at a voltage of 20 kV.2. Determination of the dissociation constant (pK_a) value of mabuterol by capillary electrophoresisKnowledge of dissociation constant is important for prediction and understanding of the migration behavior of analytes in capillary electrophoresis. A CZE method was used to determine the pK_a value of mabuterol based on the linear relation between the reciprocal ofeffective mobility of the solute (1/μ_e) and the reciprocal of [H~+] of the buffer solution. Acetoneof 1% was chosen as the neutral marker. The running buffer was sodium phosphate with different pH with same ion strength of 0.09. The UV detector was set at 254nm; electrophoresis was performed at a voltage of 10 kV. The dissociation constant was determined to be 10.14, which was similar with that of comparative method (Iso-molarity UV spctrophotometry). Based on this result, chiral separation of mabuterol was investigated by HPCE.3. The simultaneous enantiomeric separation of mabuterol and its analogue by HPCE The capillary electrophoretic separation of four adrenergicβ_2-agonist (mabuterol,clenbuterol, SPFF, bambuterol) enantiomers were studied with Hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The effects of buffer pH and concentration of HP-β-CD on the electrophoretic resolution were investigated. Separation conditions were optimized to be: 100mmol/L citric acid-10mmol/L Na_2HPO_4-120mmol/L HP-β-CD buffer solution (pH 2.5), applied voltage of 20kV, detection at 210nm. Four adrenergicβ_2-agonist enantiomers were satisfactorily separated simultaneously under this condition. The methodology was investigated and this method was used in the determination of enantiomeric purity of SPFF and mabuterol.
4. Analysis of enantiomers in vivo and stereoselective pharmacokinetics of mabuterol in rats
An achiral-chiral sequential method to determine the enantiomers of mabuterol in rat plasma was developed. Mabuteol was separated with endogenesis substances in plasma earlier by achiral chromatography system. Then the fraction of mabuterol from the achiral system was collected. The two enantiomers of mabuterol were separated by the chiral chromatography system, which was composed of Chirobiotic V column and mobile phase of MeOH-HAc-TEA (100/0.01/0.01, v/v/v). Based on the concentration of mabuterol and the l/d ratios in the same sample, the plasma levels for l-and d-mabuterol were calculated. The method described above was used for the stereoselective pharmacokinetics study of mabuterol in six Wistar rats after intra gastric administraiton of 10mg/kg mabuterol racemate. The results showed that both enantiomers were absorbed slowly with a similar t_(max) of 5.33±2.1h and 5.67±1.51, and C_(max), of 266.8±37.9ng/ml and 277.9±44.6ng/ml for l-mabuteol and d-mabuteol, respectively (p＞0.05). Significant differences of AUC_(0-∞)、t_(1／2(k))、CL/F and MRT were found between two enantiomers of mabuterol by paired Student's t-test, which indicated that l-mabuterol was more effective and persisting longer than its antipode, showed the metabolic stereoselectivity of mabuterol. The developed method should be fundamental contributions for the preclinical study of single mabuterol enantiomer.
1．西孟坦对映体的毛细管电泳分离方法研究
以β—环糊精(β-CD)为手性选择剂，采用毛细管区带电泳对西孟坦进行了手性分离研究。考察了背景电解质中硼砂缓冲液的pH及浓度、β-CD浓度、有机改性剂甲醇含量和分离电压对手性分离的影响。建立了毛细管电泳分离西孟坦对映体的方法。最佳分离条件为：20mmol／L硼砂缓冲液(pH11.0，含12mmol／Lβ-CD)-甲醇(50∶50，v／v)，分离电压20kV，在此条件下西孟坦对映体可达基线分离。
2．毛细管电泳法测定马布特罗离解常数
利用毛细管电泳中不同pH条件下有效淌度的倒数(1／μ_e)与缓冲溶液中氢离子浓度的倒数(1／[H~+])之间存在着线性关系的原理，测定了马布特罗的pK_a值。以1％丙酮为中性标记物，离子强度为0.09的不同pH的缓冲液为运行缓冲液，检测波长为254nm，分离电压为10kV。结果马布特罗的pK_a为10.14，与另一种方法—等摩尔浓度分光光度法的测得值相比较，结果相近。
3．毛细管电泳法同时分离分析马布特罗及其结构类似物的对映体
以羟丙基-β-环糊精(HP-β-CD)为手性选择剂，应用毛细管电泳法同时对马布特罗、克仑特罗、SPFF、班布特罗等四种β_2受体激动剂类手性药物进行了对映体分离。考察了HP-β-CD浓度和背景电解质pH对分离效果的影响。最佳分离条件为100mmol／L柠檬酸—10mmol／L磷酸氢二钠—120mmol／L HP-β-CD，pH2.5，分离电压20kV，检测波长210nm，柱温为室温。在此条件下，四种药物对映体均得到良好的分离结果。对方法学进行了考察，并测定了其中两种药物的对映体纯度。
4．马布特罗对映体体内分析方法的建立及其在大鼠体内的立体选择性药物动力学研究
将非手性、手性色谱系统相结合，建立了大鼠血浆中马布特罗对映体的测定方法。首先用非手性色谱法将药物与血浆内源性杂质分离，然后收集检测器后药物色谱峰流分，应用大环抗生素固定相柱(Chirobiotic V)，以甲醇—冰醋酸—三乙胺(100∶0.01∶0.01，v／v／v)为流动相，将两个对映体完全分离。根据血浆中马布特罗的浓度和l／d-对映体浓度比值，计算得到l-和d-马布特罗的血浆药物浓度。采用本法研究了6只大鼠单剂量灌胃给予马布特罗消旋体后，两对映体的药物动力学。结果表明，马布特罗两对映体有相似的t_(max)(5.33±2.1h，l-马布特罗；5.67±1.51h，d-马布特罗)和C_(max)(266.8±37.9ng／ml，l-马布特罗；277.9±44.6ng／ml，d-马布特罗)，但配对t检验表明，两对映体的AUC_(0-∞)、t_(1／2(k))、CL／F和MRT存在显著性差异，提示l-马布特罗比d-马布特罗作用更有效、更持久。推测两对映体的代谢存在立体选择性。
Enantioseparation of chiral compound is the foundation of new chiral drug's research and development. Searching for high efficient, sensitive, rapid and simple chiral separation method in laboratory is important in pharmaceutical analysis field. Chiral separation of simendan and mabuterol was investigated in the present work by using high performance capillary electrophoresis (HPCE) and high performance liquid chromatography (HPLC). The pharmacokinetics of mabuterol enantiomers in rats were also studied by using the developed HPLC method.1. Chiral separation of simendan enantiomers by capillary zone electrophoresisThe enantiomers of simendan were separated by capillary zone electrophoresis (CZE) usingβ-cyclodextrin (β-CD) as the chiral selector. The influences of the concentration and pH of borate buffer solution,β-CD concentration, methanol content and separation voltage were investigated. A baseline separation of simendan enantiomers was achieved with background electrolyte of 20mmol/L borate buffer (pH 11.0) containing 12mmol/Lβ-CD-methanol (50:50, v/v) at a voltage of 20 kV.2. Determination of the dissociation constant (pK_a) value of mabuterol by capillary electrophoresisKnowledge of dissociation constant is important for prediction and understanding of the migration behavior of analytes in capillary electrophoresis. A CZE method was used to determine the pK_a value of mabuterol based on the linear relation between the reciprocal ofeffective mobility of the solute (1/μ_e) and the reciprocal of [H~+] of the buffer solution. Acetoneof 1% was chosen as the neutral marker. The running buffer was sodium phosphate with different pH with same ion strength of 0.09. The UV detector was set at 254nm; electrophoresis was performed at a voltage of 10 kV. The dissociation constant was determined to be 10.14, which was similar with that of comparative method (Iso-molarity UV spctrophotometry). Based on this result, chiral separation of mabuterol was investigated by HPCE.3. The simultaneous enantiomeric separation of mabuterol and its analogue by HPCE The capillary electrophoretic separation of four adrenergicβ_2-agonist (mabuterol,clenbuterol, SPFF, bambuterol) enantiomers were studied with Hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The effects of buffer pH and concentration of HP-β-CD on the electrophoretic resolution were investigated. Separation conditions were optimized to be: 100mmol/L citric acid-10mmol/L Na_2HPO_4-120mmol/L HP-β-CD buffer solution (pH 2.5), applied voltage of 20kV, detection at 210nm. Four adrenergicβ_2-agonist enantiomers were satisfactorily separated simultaneously under this condition. The methodology was investigated and this method was used in the determination of enantiomeric purity of SPFF and mabuterol.
4. Analysis of enantiomers in vivo and stereoselective pharmacokinetics of mabuterol in rats
An achiral-chiral sequential method to determine the enantiomers of mabuterol in rat plasma was developed. Mabuteol was separated with endogenesis substances in plasma earlier by achiral chromatography system. Then the fraction of mabuterol from the achiral system was collected. The two enantiomers of mabuterol were separated by the chiral chromatography system, which was composed of Chirobiotic V column and mobile phase of MeOH-HAc-TEA (100/0.01/0.01, v/v/v). Based on the concentration of mabuterol and the l/d ratios in the same sample, the plasma levels for l-and d-mabuterol were calculated. The method described above was used for the stereoselective pharmacokinetics study of mabuterol in six Wistar rats after intra gastric administraiton of 10mg/kg mabuterol racemate. The results showed that both enantiomers were absorbed slowly with a similar t_(max) of 5.33±2.1h and 5.67±1.51, and C_(max), of 266.8±37.9ng/ml and 277.9±44.6ng/ml for l-mabuteol and d-mabuteol, respectively (p＞0.05). Significant differences of AUC_(0-∞)、t_(1／2(k))、CL/F and MRT were found between two enantiomers of mabuterol by paired Student's t-test, which indicated that l-mabuterol was more effective and persisting longer than its antipode, showed the metabolic stereoselectivity of mabuterol. The developed method should be fundamental contributions for the preclinical study of single mabuterol enantiomer.
引文
[1] Nerurkar SG, Dighe SV, Wiliams RL. Bioequivalence of racemic drugs. J Clin Pharmacol, 1992, 32(10): 935-943.
[2] Howe R Shanks RG. Optical Isomers of Propranolol. Nature, 1966, 210(43): 1336-1338.
[3] Nation RL. Chirality in new drug development: Clinical pharmacokinetic consideration. Clin Pharmacokinet, 1994, 27: 249-255.
[4] Wilson H, Camp De. Chiral drugs: The FDA perspective on manufacturing and control. J Pharm Biomed Anal, 1993, 11(11/12): 1167-1172.
[5] 丁慈．手性药物的开发与前景．药学进展，1996，20(3)：147-150．
[6] 曾苏，沈向忠．体内手性药物的测定．国外医药—合成药．生化药．制剂分册，1994，15(1)：41-43．
[7] 王普善．加速手性技术的开发，迎接世界制药工业的手性挑战．中国医药情报，1998，4(1)：9-19．
[8] 慈薇，柴逸峰，刘荔荔等．毛细管电泳非环糊精体系拆分手性药物的研究进展．药学学报，2002，37(1)：75-80．
[9] 林炳承．手性分子的色谱分离．色谱，1990，8(6)：363-367．
[10] 曾苏．高效液相色谱手性试剂衍生化法及其应用．色谱，1994，12(6)：406-410．
[11] 曾苏．高效液相色谱手性流动相添加剂法分离药物对映体．色谱，1995，13(1)：21-25．
[12] 彭清涛，胡文祥，谭生键．药物对映体HPLC分离测定研究新进展．药学学报，1998，33(10)：793-800．
[13] 李新，曾苏．手性配位体交换流动相添加剂法拆分对映体．色谱，1996，14(5)：354-359．
[14] 吕湘林，杨宪桂．光学异构体的手性HPLC拆分法及其药学应用研究的进展(续)．药学学报，1988，23(1)：67-79．
[15] Aboul-Enein HY, Ali I. Macrocyclic antibiotics as effective chiral selectors for enantiomeric resolution by liquid chromatography and capillary electrophoresis. Chromatographia, 2000, 52: 679-691.
[16] Biaschke G, Chakvetadze B. Enantiomer separation of drugs by capillary electromigration techniques. J.Chromatogr. A. 2000, 875: 3-25.
[17] Evans CE and Stalcup AM. Comprehensive strategy for chiral separations using sulfated cyclodextrins in capillary electrophoresis. Chirality, 2003, 15: 709-723
[18] Innes CA and Wagstaff AJ. Levosimendan: a review of its use in the management of acute decompensated heart failure. Drugs, 2003, 63: 2651-2671.
[19] Sorsa T, Pollesello P, Rosevear PR, Drakenberg T and Kilpelainen I. Stereoselective binding of levosimendan to cardiac troponin C causes Ca~(2+)-sensitization. Euro J Pharmacol, 2004, 486: 1-8.
[20] Hasenfuss G, Pieske B, Castell M, et al. Influence of the novel inotropic agent levosimendan on isomeric tension and calcium cycling in failing human myocardium. Circulation, 1998, 98(20): 2141-2147.
[21] Papp JG. Introduction: positive inotropy by calcium sensitization an evolving approach for the treatment of end-stage heart failure. Am J Cardio, 1999, 83(123): 1-3.
[22] Lilleberg J, Antila S, Karlsson M, Nieminen M. S, Pentikainen P. J. Pharmacokinetics and pharmacodynamics of simendan, a nvel calcium sensitizer in healthy volunteers. Clin. Pharmacol. Ther, 1994, 56: 554-563.
[23] Sandell E. P, Hayha M, Antila S, Heikkinen P, Ottoila P, Lehtonen L. A, Pentikainen P. J. Pharmacokinetics of levosimendan in healthy volunteers and patients with congestive heart failure. J. Cardorvasc. Pharmacol. 1995, 26(suppl.1): S57-S62.
[24] Wikberg T, Korkolainen T, Karlsson M. Enantiomeric bioanalysis of simendan and levosimendan by chiral high-performance liquid chromatography. Chirality, 1996, 8(7): 511-517.
[25] 刘学良，黄晓佳，郝卫强，王俊德，于亿年．高效液体相色谱法直接拆分药物西孟旦对映体．药物分析杂志，2002，22(4)：261-264．
[26] Soini H, Stefansson M, Riekkola M. L, Novotny, M. V. Maltooligosaccharides as chiral selectors for the separation of pharmaceuticals by capillary electrophoresis. Anal. Chem., 1994, 66 (20): 3477-3484.
[27] 张丹丹，郭兴杰，袁波，李发美．高效液相色谱手性流动相添加剂分离西孟坦对映体．分析化学，2004，32(2)：229-231．
[28] Murai T., Maejima T., Sanai K., Osada E. Pharmacological studies of mabuterol, a new selective 132-stimulant Ⅰ: bronchodilating effect. Arzneim Forsch. 1984, 34: 1633-1640.
[29] 王学娅，赵怀清，王美惠，郭季安，马布特罗对豚鼠支气管平滑肌松弛作用和平喘作用的影响．中国新药与临床杂志，2003，22(5)：276-279．
[30] Aboul-Enein HY., Imran Ali. Chiral resolution of clenbuterol, cimaterol, and mabuterol on Chirobiotic V, T and TAG columns. J Sep Sci, 2002, 25(13): 851-855.
[31] Yuge T, Hase T, Takayanagi Y, Kamasuka T, Amemiya K, Horiba M. Pharmacokinetic studies of mabuterol, a new selective β_2-stimulant Ⅰ: studies on the absorption, distribution and excretion in rats. Arzneim Forsch, 1984, 34: 1659-1667.
[32] Yanamoto I, Matsuura E, Horiba M, Akima K, Nomura K, Aizawa T. Enzyme immunoassay for mabuterol, a selective β_2-adrenergic stimulant in the trachea. J Immunoassay, 1985, 6(3): 261-276.
[33] Qureshi GA, Eriksson A. Determination of clenbuterol and mabuterol in equine plasma by ion-pair liquid chromatography with electrochemical detection. J. Chromatogr, 1988, 441: 197-205.
[34] Ramos F, Santos C, Silva A, da Silveira MI. β_2- adrenergic agonist residues: simultaneous methyl- and butylboronic derivatization for confirmatory analysis by gas chromatography-mass spectrometry. J Chromatogr. B, 1998, 716: 366-370.
[35] Brambilla G, Fiori M, Rizzo B, Crescenzi V, Masei G. Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices. J Chromatogr. B, 2001, 759: 27-32.
[36] Li F, Zhang D, Lu X, Wang Y, Xiong Z. Enantiomeric analysis of simendan by CE with beta-CD as chiral selector compared with CMPA-HPLC. Biomed Chromatogr, 2004, 18(10): 866-71.
[37] 范瑞芳，史雪岩，顾峻岭，傅若农．应用二元环糊精体系的毛细管区带电泳手性拆分．分析化学，2003，31(5)：559-561．
[38] Yang J and Hage DS. Chiral separations in capillary electrophoresis using human serum albumin as a buffer additive. Anal Chem, 1994, 66: 2719-2725.
[39] 余丽宁，李发美．人血清白蛋白亲和毛细管电泳法分离马来酸曲美布汀对映体．分析化学，2001，29(7)：785-787．
[40] Nishi H and Kuwahara Y. Enantiomer separation by capillary electrophoresis utilizing carboxymethyl derivatives of polysaccharides as chiral selectors. J Pharma Biomed Anal, 2002, 27: 577-585.
[41] Nishi H, Fukuyama T, Matsuo M and Terabe S. Chiral separation of diltiazem, trimetoquinol and related compoumds by micellar electrokinetic chromatography with bile salts. J Chromatog, 1990, 515: 233-243.
[42] Kang J, Wistuba D, Schurig V. Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique. Electrophoresis, 2003, 24(15): 2674-2679.
[43] Ward TJ, Farris AB 3rd. Chiral separations using the macrocyclic antibiotics: a review. J Chromatogr A, 2001, 906: 73-89.
[44] Kim E, Koob YM, Chung DS. Chiral counter-current chromatography of gemifloxacin guided by capillary electrophoresis using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector. J Chromatogr A, 2004, 1045(1-2): 119-124.
[45] Tanaka Y, Otsuka K, Terabe S. Separation of enantiomers by capillary electrophoresis-mass spectrometry employing a partial filling technique with a chiral crown ether. J Chromatogr A. 2000, 875(1-2): 323-330.
[46] Sarmini K, Kenndler E. Influence of organic solvents on the separation selectivity in capillary electrophoresis. J Chromtogr. A, 1997, 792: 3-11.
[47] 主沉浮，林秀丽，魏云鹤，林秀玲．新型环糊精衍生物单6—O一苯基胺甲酰基—β—环糊精的合成及其在手性药物毛细管区带电泳拆分中的作用．分析化学，2003，31(4)：389-394．
[48] Mrestani Y, Neubert R, Munk A, Wiese M. Determination of dissociation constants of cephalosporins by capillary zone electrophoresis. J Chromatogr A, 1998, 803: 273-278.
[49] 王德先，寇素如等．毛细管电泳法测定淫羊藿甙的pKa值及其在中药淫羊藿中的含量．色谱，2001，19(1)：64-67．
[50] 晁若冰，伍朝篑，贺晓英．弱酸弱碱性药物pK_a值的分光光度测定法．华西药学杂志，1990，5(2)：104-106．
[51] 沈建民，武振卿．药物结构与制剂，北京，中国药物科技出版社，1989．
[52] 张远杏，武凤兰．莫索尼定的电离常数和分配系数的测定．沈阳药科大学学报，2001，18(1)：37-39．
[53] 刘迎春，赵兵．电位滴定法测定盐酸青藤碱的电离常数．沈阳药科大学学报，1999，16(3)：217-218．
[54] 杨世昕．甲硝唑解离常数的测定．华西药学杂志．1999，14(1)：9-10．
[55] 王长虹，孙殿甲．盐酸骆驼蓬解离常数的等摩尔浓度分光光度法测定．中国医药工业杂志，2002，33(1)：558-559．
[56] 袁波，范毅．pH薄层色谱法测定槐定碱和拉马宁碱的离解常数．沈阳药科大学学报．1999，16(1)：44-47
[57] Waldeck B. β-Adrenoceptor agonists and asthma-100 years of development. Euro. J Pharmacol. 2002, 445(1-2): 1-12.
[58] Toussaint B, Hubert PH, Tjaden UR, van der Greef J, Crommen J. Enantiomeric separation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection new optimization technique for transient isotachophoresis. J Chromatogr A, 2000, 871 (1-2): 173-180.
[59] Toussaint B, Grysler J, Hubert PH, Tjaden UR, Vander Greef J, Crommen J. In-line concentration and enantioseparation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection. Biomed Chromatogr, 2000, 14: 32-33.
[60] Toussaint B, Palmer M, Chiap PH, Hubert P, Crommen J. On-line coupling of partial filling-capillary zone electrophoresis with mass spectrometry for separation of clenbuterol enantiomers. Electrophoresis, 2001, 22(7): 1363-1372.
[61] Gausepohl C, Blaschke G. Stereoselective determination of clenbuterol in human urine by capillary electrophoresis. J Chromatogr B Biomed Sci Appl, 1998, 713(2): 443-446.
[62] Kim KH, Kim H J, Jeun EY, Seo SH, Hong SP, Kang JS, Youm JR, Lee SC. Chiral separation of beta2-agonists by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as a chiral selector. Arch Pharm Res. 2001, 24(4): 281-285.
[63] 王志，孙亦梁，孙曾培．高效毛细管电泳分离手性物质．分析测试学报．1996，15(2)：85-92．
[64] 钟大放．以加权最小二乘法建立生物分析标准曲线的若干问题．药物分析杂志．1996，16，343-346
[65] Emm. T., Lesco. L J, Leslie. J. and Perkal M. B. Determination of albuterol in human serum by reversed-phase high-performance liquid chromatography with electrochemical deterction. J. Chromatogr, 1988, 427: 188-194.
[66] Diquet B, Doare L and Simon P. Determination of clenbuterol in the high nanogram range in plasma of mice by high-performance liquid chromatography with amperometric detection. J Chromatogr, 1984, 336: 415-421.
[67] Botterblom MH, Feenstra MG and Erdtsieck-Ernste EB, Determination of propranolol, labetalol and clenbuterol in rat brain by high-performance liquid chromatography. J Chromatogr. 1993, 613: 121-126.
[68] Laila I, Abou B, Aboul-Enein HY. Direct enantioselective separation of clenbuterol by chiral HPLC in biological fluids. Biomed Chromatogr, 1996, 10(2): 69-72.
[69] Armstrong DW, Tang YB, Chen SS. Et al. Macrocyclic antibiotics as a new class of chiral selectors for liquid chromatography. Anal Chem, 1994, 66: 1473-1478.
[70] Ducharme J, Fernandez C, Gimenez F, Farinotti R. Critical issues in chiral drug analysis in biological fluids by high performance liquid chromatography. J Chromatogr B, 1996, 686: 65-75.
[2] Howe R Shanks RG. Optical Isomers of Propranolol. Nature, 1966, 210(43): 1336-1338.
[3] Nation RL. Chirality in new drug development: Clinical pharmacokinetic consideration. Clin Pharmacokinet, 1994, 27: 249-255.
[4] Wilson H, Camp De. Chiral drugs: The FDA perspective on manufacturing and control. J Pharm Biomed Anal, 1993, 11(11/12): 1167-1172.
[5] 丁慈．手性药物的开发与前景．药学进展，1996，20(3)：147-150．
[6] 曾苏，沈向忠．体内手性药物的测定．国外医药—合成药．生化药．制剂分册，1994，15(1)：41-43．
[7] 王普善．加速手性技术的开发，迎接世界制药工业的手性挑战．中国医药情报，1998，4(1)：9-19．
[8] 慈薇，柴逸峰，刘荔荔等．毛细管电泳非环糊精体系拆分手性药物的研究进展．药学学报，2002，37(1)：75-80．
[9] 林炳承．手性分子的色谱分离．色谱，1990，8(6)：363-367．
[10] 曾苏．高效液相色谱手性试剂衍生化法及其应用．色谱，1994，12(6)：406-410．
[11] 曾苏．高效液相色谱手性流动相添加剂法分离药物对映体．色谱，1995，13(1)：21-25．
[12] 彭清涛，胡文祥，谭生键．药物对映体HPLC分离测定研究新进展．药学学报，1998，33(10)：793-800．
[13] 李新，曾苏．手性配位体交换流动相添加剂法拆分对映体．色谱，1996，14(5)：354-359．
[14] 吕湘林，杨宪桂．光学异构体的手性HPLC拆分法及其药学应用研究的进展(续)．药学学报，1988，23(1)：67-79．
[15] Aboul-Enein HY, Ali I. Macrocyclic antibiotics as effective chiral selectors for enantiomeric resolution by liquid chromatography and capillary electrophoresis. Chromatographia, 2000, 52: 679-691.
[16] Biaschke G, Chakvetadze B. Enantiomer separation of drugs by capillary electromigration techniques. J.Chromatogr. A. 2000, 875: 3-25.
[17] Evans CE and Stalcup AM. Comprehensive strategy for chiral separations using sulfated cyclodextrins in capillary electrophoresis. Chirality, 2003, 15: 709-723
[18] Innes CA and Wagstaff AJ. Levosimendan: a review of its use in the management of acute decompensated heart failure. Drugs, 2003, 63: 2651-2671.
[19] Sorsa T, Pollesello P, Rosevear PR, Drakenberg T and Kilpelainen I. Stereoselective binding of levosimendan to cardiac troponin C causes Ca~(2+)-sensitization. Euro J Pharmacol, 2004, 486: 1-8.
[20] Hasenfuss G, Pieske B, Castell M, et al. Influence of the novel inotropic agent levosimendan on isomeric tension and calcium cycling in failing human myocardium. Circulation, 1998, 98(20): 2141-2147.
[21] Papp JG. Introduction: positive inotropy by calcium sensitization an evolving approach for the treatment of end-stage heart failure. Am J Cardio, 1999, 83(123): 1-3.
[22] Lilleberg J, Antila S, Karlsson M, Nieminen M. S, Pentikainen P. J. Pharmacokinetics and pharmacodynamics of simendan, a nvel calcium sensitizer in healthy volunteers. Clin. Pharmacol. Ther, 1994, 56: 554-563.
[23] Sandell E. P, Hayha M, Antila S, Heikkinen P, Ottoila P, Lehtonen L. A, Pentikainen P. J. Pharmacokinetics of levosimendan in healthy volunteers and patients with congestive heart failure. J. Cardorvasc. Pharmacol. 1995, 26(suppl.1): S57-S62.
[24] Wikberg T, Korkolainen T, Karlsson M. Enantiomeric bioanalysis of simendan and levosimendan by chiral high-performance liquid chromatography. Chirality, 1996, 8(7): 511-517.
[25] 刘学良，黄晓佳，郝卫强，王俊德，于亿年．高效液体相色谱法直接拆分药物西孟旦对映体．药物分析杂志，2002，22(4)：261-264．
[26] Soini H, Stefansson M, Riekkola M. L, Novotny, M. V. Maltooligosaccharides as chiral selectors for the separation of pharmaceuticals by capillary electrophoresis. Anal. Chem., 1994, 66 (20): 3477-3484.
[27] 张丹丹，郭兴杰，袁波，李发美．高效液相色谱手性流动相添加剂分离西孟坦对映体．分析化学，2004，32(2)：229-231．
[28] Murai T., Maejima T., Sanai K., Osada E. Pharmacological studies of mabuterol, a new selective 132-stimulant Ⅰ: bronchodilating effect. Arzneim Forsch. 1984, 34: 1633-1640.
[29] 王学娅，赵怀清，王美惠，郭季安，马布特罗对豚鼠支气管平滑肌松弛作用和平喘作用的影响．中国新药与临床杂志，2003，22(5)：276-279．
[30] Aboul-Enein HY., Imran Ali. Chiral resolution of clenbuterol, cimaterol, and mabuterol on Chirobiotic V, T and TAG columns. J Sep Sci, 2002, 25(13): 851-855.
[31] Yuge T, Hase T, Takayanagi Y, Kamasuka T, Amemiya K, Horiba M. Pharmacokinetic studies of mabuterol, a new selective β_2-stimulant Ⅰ: studies on the absorption, distribution and excretion in rats. Arzneim Forsch, 1984, 34: 1659-1667.
[32] Yanamoto I, Matsuura E, Horiba M, Akima K, Nomura K, Aizawa T. Enzyme immunoassay for mabuterol, a selective β_2-adrenergic stimulant in the trachea. J Immunoassay, 1985, 6(3): 261-276.
[33] Qureshi GA, Eriksson A. Determination of clenbuterol and mabuterol in equine plasma by ion-pair liquid chromatography with electrochemical detection. J. Chromatogr, 1988, 441: 197-205.
[34] Ramos F, Santos C, Silva A, da Silveira MI. β_2- adrenergic agonist residues: simultaneous methyl- and butylboronic derivatization for confirmatory analysis by gas chromatography-mass spectrometry. J Chromatogr. B, 1998, 716: 366-370.
[35] Brambilla G, Fiori M, Rizzo B, Crescenzi V, Masei G. Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices. J Chromatogr. B, 2001, 759: 27-32.
[36] Li F, Zhang D, Lu X, Wang Y, Xiong Z. Enantiomeric analysis of simendan by CE with beta-CD as chiral selector compared with CMPA-HPLC. Biomed Chromatogr, 2004, 18(10): 866-71.
[37] 范瑞芳，史雪岩，顾峻岭，傅若农．应用二元环糊精体系的毛细管区带电泳手性拆分．分析化学，2003，31(5)：559-561．
[38] Yang J and Hage DS. Chiral separations in capillary electrophoresis using human serum albumin as a buffer additive. Anal Chem, 1994, 66: 2719-2725.
[39] 余丽宁，李发美．人血清白蛋白亲和毛细管电泳法分离马来酸曲美布汀对映体．分析化学，2001，29(7)：785-787．
[40] Nishi H and Kuwahara Y. Enantiomer separation by capillary electrophoresis utilizing carboxymethyl derivatives of polysaccharides as chiral selectors. J Pharma Biomed Anal, 2002, 27: 577-585.
[41] Nishi H, Fukuyama T, Matsuo M and Terabe S. Chiral separation of diltiazem, trimetoquinol and related compoumds by micellar electrokinetic chromatography with bile salts. J Chromatog, 1990, 515: 233-243.
[42] Kang J, Wistuba D, Schurig V. Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique. Electrophoresis, 2003, 24(15): 2674-2679.
[43] Ward TJ, Farris AB 3rd. Chiral separations using the macrocyclic antibiotics: a review. J Chromatogr A, 2001, 906: 73-89.
[44] Kim E, Koob YM, Chung DS. Chiral counter-current chromatography of gemifloxacin guided by capillary electrophoresis using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector. J Chromatogr A, 2004, 1045(1-2): 119-124.
[45] Tanaka Y, Otsuka K, Terabe S. Separation of enantiomers by capillary electrophoresis-mass spectrometry employing a partial filling technique with a chiral crown ether. J Chromatogr A. 2000, 875(1-2): 323-330.
[46] Sarmini K, Kenndler E. Influence of organic solvents on the separation selectivity in capillary electrophoresis. J Chromtogr. A, 1997, 792: 3-11.
[47] 主沉浮，林秀丽，魏云鹤，林秀玲．新型环糊精衍生物单6—O一苯基胺甲酰基—β—环糊精的合成及其在手性药物毛细管区带电泳拆分中的作用．分析化学，2003，31(4)：389-394．
[48] Mrestani Y, Neubert R, Munk A, Wiese M. Determination of dissociation constants of cephalosporins by capillary zone electrophoresis. J Chromatogr A, 1998, 803: 273-278.
[49] 王德先，寇素如等．毛细管电泳法测定淫羊藿甙的pKa值及其在中药淫羊藿中的含量．色谱，2001，19(1)：64-67．
[50] 晁若冰，伍朝篑，贺晓英．弱酸弱碱性药物pK_a值的分光光度测定法．华西药学杂志，1990，5(2)：104-106．
[51] 沈建民，武振卿．药物结构与制剂，北京，中国药物科技出版社，1989．
[52] 张远杏，武凤兰．莫索尼定的电离常数和分配系数的测定．沈阳药科大学学报，2001，18(1)：37-39．
[53] 刘迎春，赵兵．电位滴定法测定盐酸青藤碱的电离常数．沈阳药科大学学报，1999，16(3)：217-218．
[54] 杨世昕．甲硝唑解离常数的测定．华西药学杂志．1999，14(1)：9-10．
[55] 王长虹，孙殿甲．盐酸骆驼蓬解离常数的等摩尔浓度分光光度法测定．中国医药工业杂志，2002，33(1)：558-559．
[56] 袁波，范毅．pH薄层色谱法测定槐定碱和拉马宁碱的离解常数．沈阳药科大学学报．1999，16(1)：44-47
[57] Waldeck B. β-Adrenoceptor agonists and asthma-100 years of development. Euro. J Pharmacol. 2002, 445(1-2): 1-12.
[58] Toussaint B, Hubert PH, Tjaden UR, van der Greef J, Crommen J. Enantiomeric separation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection new optimization technique for transient isotachophoresis. J Chromatogr A, 2000, 871 (1-2): 173-180.
[59] Toussaint B, Grysler J, Hubert PH, Tjaden UR, Vander Greef J, Crommen J. In-line concentration and enantioseparation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection. Biomed Chromatogr, 2000, 14: 32-33.
[60] Toussaint B, Palmer M, Chiap PH, Hubert P, Crommen J. On-line coupling of partial filling-capillary zone electrophoresis with mass spectrometry for separation of clenbuterol enantiomers. Electrophoresis, 2001, 22(7): 1363-1372.
[61] Gausepohl C, Blaschke G. Stereoselective determination of clenbuterol in human urine by capillary electrophoresis. J Chromatogr B Biomed Sci Appl, 1998, 713(2): 443-446.
[62] Kim KH, Kim H J, Jeun EY, Seo SH, Hong SP, Kang JS, Youm JR, Lee SC. Chiral separation of beta2-agonists by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as a chiral selector. Arch Pharm Res. 2001, 24(4): 281-285.
[63] 王志，孙亦梁，孙曾培．高效毛细管电泳分离手性物质．分析测试学报．1996，15(2)：85-92．
[64] 钟大放．以加权最小二乘法建立生物分析标准曲线的若干问题．药物分析杂志．1996，16，343-346
[65] Emm. T., Lesco. L J, Leslie. J. and Perkal M. B. Determination of albuterol in human serum by reversed-phase high-performance liquid chromatography with electrochemical deterction. J. Chromatogr, 1988, 427: 188-194.
[66] Diquet B, Doare L and Simon P. Determination of clenbuterol in the high nanogram range in plasma of mice by high-performance liquid chromatography with amperometric detection. J Chromatogr, 1984, 336: 415-421.
[67] Botterblom MH, Feenstra MG and Erdtsieck-Ernste EB, Determination of propranolol, labetalol and clenbuterol in rat brain by high-performance liquid chromatography. J Chromatogr. 1993, 613: 121-126.
[68] Laila I, Abou B, Aboul-Enein HY. Direct enantioselective separation of clenbuterol by chiral HPLC in biological fluids. Biomed Chromatogr, 1996, 10(2): 69-72.
[69] Armstrong DW, Tang YB, Chen SS. Et al. Macrocyclic antibiotics as a new class of chiral selectors for liquid chromatography. Anal Chem, 1994, 66: 1473-1478.
[70] Ducharme J, Fernandez C, Gimenez F, Farinotti R. Critical issues in chiral drug analysis in biological fluids by high performance liquid chromatography. J Chromatogr B, 1996, 686: 65-75.