几种手性药物的高效液相色谱分析方法研究

【作者】 栾燕；

【导师】 钟大放；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2001， 硕士

【摘要】 药物的手性问题已成为当今生物医药领域所面临的一个重要问题。对映体的拆分和测定在分离科学上曾被认为是最困难的工作之一，随着各种分离技术的发展，尤其是高效液相色谱法在手性药物分析领域的应用越来越广泛，为解决手性拆分问题提供了有效的手段。本文采用柱前手性衍生化高效液相色谱和手性液相色谱-质谱-质谱联用等分析技术，对几种药物进行了手性拆分研究，所建立的方法已被成功地应用于药品质量控制或药物动力学研究。1 柱前手性衍生化-反相高效液相色谱法拆分酮洛芬对映异构体的方 法研究 建立了一种用柱前手性衍生化-反相高效液相色谱法分析酮洛芬对映异构体的方法。采用二氯亚砜和S-（－）-1-（1-萘基）乙胺作为衍生化试剂，S-（+）-酮洛芬和R-（-）-酮洛芬衍生化后生成一对非对映异构体。选用Hypersil C₁₈柱，以乙腈-水-乙酸-三乙胺（体积比为58:42:0.1:0.02）为流动相，在254nm下检测。色谱流份经电喷雾离子阱多级质谱分析验证。非对映异构体色谱峰的保留时间分别为t_R=7.3min和t_R=8.5min，分离度为1.9，经电喷雾离子阱多级质谱分析验证了衍生物结构。衍生化产物稳定，方法灵敏度高，重现性好，操作简单，可用于酮洛芬的药品质量控制。2 柱前手性衍生化-反相高效液相色谱法拆分麻黄碱、伪麻黄碱对映 异构体的方法研究 建立了一种用柱前手性衍生化-反相高效液相色谱法分析麻黄碱、伪麻黄碱对映异构体的方法。以（－）-氯甲酸薄荷醇酯作为手性衍生化试剂，（－）-麻黄碱、（+）-麻黄碱、（+）-伪麻黄碱和（－）-伪麻黄碱衍生化后生成四个非对映异构体。选用Hypersil C₁₈柱，以乙腈-水-乙酸-三乙胺（体积比为29:20:0.2:0.01）为流动相，在254nm下检测。色谱流份经电喷雾离子阱多级质谱分析验证。麻黄碱衍生化后生成的一对非对映异构体的保留时间分别为t_R=17.1min和t_R=18.3min，分离度为1.6； 摘 要 二 伪麻黄a生化后生成的一对非对映异构体的保留时间分别为 tR＝ ZI．3 min和 tR＝ 23．2 dn，分离度为 1．9。经电喷雾离子啡多级质谱分析驾h正 了衍生物的结构。本方法操作简单、快速、重现性好，已被批准用于 麻黄碱、伪麻黄碱的药品质量控制。 3 柱前衍生化一正相高效涪相色谱法拆分甲基麻黄碱的方法研究 建立了一种用柱前手性衍生化－正相高效液相色谱法分析甲基麻 黄献映异构体的方法。以(＋氯甲酸薄荷醇酯作为手性衍生化试剂， 卜卜甲基麻黄碱与(个甲基麻黄碱衍生化后生咸一对非对映异构体。选 用 LIChros。h St 60柱，以正己烷－异丙醇－三乙胺（体积比为 94：6： 0刀2）为流动相，在220 urn下检测。非对映异构体色肿的保留时间 分别为 tR＝ 3．6 dn和 tR＝ 4．3dn，分离度为 1．8。经电喷雾离子阶多级 质谱分析验证了衍生物结构。本方法操作简单、重现性好，已被批准 用于甲基麻黄碱的药品质量控制。 4 氨氯地平的对映体选择性药物动力学研究 采用非手性、手性色谱系统相结合的方法，建立了测定血浆中氨 氯地平对映体浓度的手性液相色普质谱－质谱联用法。将收集的非手 性色谱系统中氨氯地平的流份碱化后用乙醚－二氯甲烷提取，分取有机 相吹干，残余物溶解后注入手性溯目色谱－质谱－质谱系统分析。根据 血浆中氨氯地平的浓度和（－帜）－体浓度比值，计脓到(－和仰－氨氯 地平的血浆药物浓度。色谱柱为手性AGP柱，流动相为正丙醇－0刀1 moVL醋酸接(1．2：10①，电喷雾离子化四极串联质谱，以选择离子反 应监测方式（S删）进行检测。闪－氨氯地平的线性范围为l．54．5 Ilg／mL，(＞氨氯地平的线性范围为 0．75刁刀吟InL。采用本法研究了 6名赈受试者单剂量。服氨氯地平 10 mg后，两对映体的药物动力学， 发现血浆中闪－体的浓度始终高于仰－体，队氨氯地平和伏－氨氯地平 的Cu比值为 1二5，AUC－t的比值为 137。更多还原

【Abstract】 Chirality of drugs is an important aspect in fields of biological medicine. The separation of chiral drugs was once recognized as one of the most difficult work. With the development of various chiral technology, especially high-performance liquid chromatography, enantiospeciflc analysis is becoming a routine analytical task. In the present work, precolunin derivatization HPLC and chiral LCIMSIMS technology were used to separate and analyse several cbiral drugs. 1. Enantiospeciflc analysis of ketoprofen by RP-HIPLC with precolumn derivatization A high-performance liquid chromatographic (HPLC) assay for the determination of the R- and S-enantiomers of ketoprofen is described. The derivatization with tbionyl chloride and S-(-)-l-(1-naphthyl)-ethylamine was followed by reversed-phase chromatography with a mobile phase of acetonitrile-water-acetic acid-triethylamine (55 45 0.1 0.02 by vol) at a flow rate of 1.0 mL/min and UV detection was performed at 254 mu. The electrospray ionization-quadrupole ion trap mass spectrometer was applied to verify the structure of derivatives of ketoprofen. The retention times for the S- and R-ketoprofen diastereomeric amides were tR= 7.3 mm and tR? 8.5 nun respectively and had a resolution factor of 1.9. The present study provides a simple, reproducible and sensitive method for the quality evaluation of S-ketoprofen. 2. Enantiospecific analysis of ephedrine by RP-HIPLC with precolumn derivatization A high-performance liquid chromatographic method has been developed for the determination of the (-)- and (+)-enantiomers of ephedrine and the (-)- and (+)-enantiomers of pseudoephedrine simultaneously. The derivatization with (-)-menthylchloroformate was followed by reversed-phase chromatography with a mobile phase of acetonitrile-water-acetic acid-triethylamine (29 20 0.2 0.01 by vol) at a 4 flow rate of 1.0 mL/min and UV detection was performed at 254 nm. The electrospray ioriization-quadnipole ion trap mass spectrometer was applied to verify the structure of derivatives of ephedrine and pseudoephedrine. The retention times for the (-)- and (+)-.ephedrine diastereomeric derivatives were tR= 17.1 and tR= 18.3 mm respectively and have a resolution factor of 1.6. The retention times for the (+)~ and (-)-pseudoephedrine diastereomenc derivatives were tR= 21.3 and tR= 23.2 mm respectively and bad a resolution factor of 1.9. The present study provides a simple, reproducible and rapid method for the quality evaluation of (-)-ephedrine and (+)-pseudoephedrine. 3. Enantiospecific Analysis of Methylephedrine by Normal-Phase HPLC with Precolumn Derivatization A high-performance liquid chromatographic method for the determination of (-)- and (+)-.enantiomers of methylephedrine is reported. The derivatization with (-)-menthyl chloroforniate give two diastereomiric derivatives. These derivatives were separated on a LiCbrosorb Si 60 column using n-hexane-2-propanol-triethylaniine (94 6: 0.02 by vol) as the mobile phase and detected by CV detection at a wavelength of 220 nm. The electrospray ionization-quadrupole ion trap mass spectrometer was applied to verify the structure of derivatives of methylephedrine. The retention times for the (+)- and (-)-methylephedrine diastereomeric derivatives were tR= 3.6 mm and t~=~ 4.3 mm respectively and had a resolution factor of 1.8. The更多还原