超高效合相色谱法拆分和测定克伦特罗对映体

doi:10.3724/SP.J.1123.2021.06045

色谱 ›› 2021, Vol. 39 ›› Issue (12): 1347-1354.DOI: 10.3724/SP.J.1123.2021.06045

超高效合相色谱法拆分和测定克伦特罗对映体

张文华¹^,²^,^*(), 洪灯¹^,², 雷美康¹, 胡晓莉², 侯建波¹^,², 谢文¹^,², 徐敦明³, 伊雄海⁴, 李优⁴

1.杭州海关技术中心, 浙江杭州 310016
2.浙江省检验检疫科学技术研究院, 浙江杭州 310016
3.厦门海关技术中心, 福建厦门 361026
4.上海海关动植物与食品检验检疫技术中心, 上海 200135

收稿日期:2021-06-27 出版日期:2021-12-08 发布日期:2021-11-23
通讯作者: 张文华
作者简介:*Tel:(0571)81100295,E-mail: zwh@zaiq.org.cn.
基金资助:
国家重点研发计划(2019YFC1605400);海关总署科研项目(2020HK196);浙江省科技计划项目(2018C37014)

Separation and determination of clenbuterol enantiomers by ultra-performance convergence chromatography

ZHANG Wenhua¹^,²^,^*(), HONG Deng¹^,², LEI Meikang¹, HU Xiaoli², HOU Jianbo¹^,², XIE Wen¹^,², XU Dunming³, YI Xionghai⁴, LI You⁴

1. Technic Center of Hangzhou Customs, Hangzhou 310016, China
2. Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China
3. Technical Center of Xiamen Customs, Xiamen 361026, China
4. Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Customs, Shanghai 200135, China

Received:2021-06-27 Online:2021-12-08 Published:2021-11-23
Contact: ZHANG Wenhua
Supported by:
National Key R&D Program of China(2019YFC1605400);Project of General Administration of Customs(2020HK196);Science and Technology Project of Zhejiang Province(2018C37014)

摘要/Abstract

摘要：

建立了基于超高效合相色谱技术(UPC²)的克伦特罗对映体拆分方法,并对所建立的方法进行了方法学考察及应用。实验考察了两种克伦特罗对映体标准溶液的稳定性,并对手性色谱柱、助溶剂、系统背压、色谱柱温度等色谱分离条件进行了优化。采用Acquity Trefoil AMY1 (150 mm×3.0 mm, 2.5 μm)手性色谱柱进行分离,以超临界CO₂-含0.5%(v/v) 10 mol/L醋酸铵的甲醇溶液为流动相,以流速2.0 mL/min梯度洗脱,检测波长为241 nm,进样体积为10 μL,系统背压为13.8 MPa,柱温为40 ℃时,两种克伦特罗对映体分离效果最好。两种克伦特罗对映体的线性范围为1.0~20.0 mg/L,相关系数均大于0.9997,仪器检出限(S/N=3)均为0.5 mg/L。10.0 mg/L混合标准工作溶液重复进样6次,(+)、(-)克伦特罗对映体峰面积的相对标准偏差(RSD, n=6)分别为0.65%和0.76%。应用该方法对市售克伦特罗外消旋体标准品进行拆分,采用外标定量法计算克伦特罗外消旋体标准中间溶液10.0 mg/L中两种克伦特罗对映体含量,其中(+)-克伦特罗的含量为5.6 mg/L, (-)-克伦特罗的含量为5.5 mg/L。该计算结果与文献报道的工业品克伦特罗外消旋体中(+)-克伦特罗与(-)-克伦特罗的比例为1.02∶1.00基本相符。该方法具有分析速度快、分离效果好、有机溶剂消耗少等特点,适用于克伦特罗对映体的拆分,为其他手性化合物的拆分、药效精细分析和产品质量评定提供了可靠的技术支持。

关键词: 超高效合相色谱, 克伦特罗, 对映体, 手性分离

Abstract:

Clenbuterol enantiomers differ greatly in their bioactivities. By optimizing the conditions for chromatographic separation and method validation, ultra-performance convergence chromatography (UPC²) was adopted to separate the enantiomers of clenbuterol. Standard solutions of (+)-clenbuterol and (-)-clenbuterol were stored at -18 ℃ for 1, 3, 5, 7, 14, 30, and 60 d, and then, their stability was monitored. The impacts of different chromatographic columns, cosolvents, system backpressure, and chromatographic column temperature on the separation of the two enantiomers were investigated. Acquity Trefoil AMY1 (150 mm×3.0 mm, 2.5 μm) was used for separation, and CO₂-0.5% (v/v) ammonium acetate was used as the mobile phase. Gradient elution at a flow rate of 2.0 mL/min was adopted. The detection wavelength was set to 241 nm, and the injection volume was set to 10 μL. The backpressure was set to 13.8 MPa, and the column temperature was maintained at 40 ℃. The two enantiomers showed good linear relationships in the range of 1.0 to 20.0 mg/L with correlation coefficients greater than 0.9997. The limits of detection (LODs, S/N=3) of (+)-clenbuterol and (-)-clenbuterol were both 0.5 mg/L. The relative standard deviation (RSD, n=6) for the peak area of the 10.0 mg/L mixed standard working solution with six replicate injections ranged from 0.65% to 0.76%. The effectiveness and practicability of this method were demonstrated by using it to detect standard clenbuterol racemate. The (+)-clenbuterol and (-)-clenbuterol contents were 5.6 mg/L and 5.5 mg/L, respectively, in the standard clenbuterol racemates, as determined by the external standard method of quantification. The detection results suggested that the content ratio of (+)-clenbuterol and (-)-clenbuterol was close to 1.02∶1.00, which is consistent with the literature data. The established method has the advantages of rapid analysis, good separation effect, and low consumption of organic solvents, and it is suitable for the separation of clenbuterol enantiomers. This method can also provide technical support for the separation of other chiral drugs, analysis of the effects of chiral drugs, and assessment of product quality.

Key words: ultra-performance convergence chromatography (UPC²), clenbuterol, enantiomers, chiral separation

中图分类号:

O658

张文华, 洪灯, 雷美康, 胡晓莉, 侯建波, 谢文, 徐敦明, 伊雄海, 李优. 超高效合相色谱法拆分和测定克伦特罗对映体[J]. 色谱, 2021, 39(12): 1347-1354.

ZHANG Wenhua, HONG Deng, LEI Meikang, HU Xiaoli, HOU Jianbo, XIE Wen, XU Dunming, YI Xionghai, LI You. Separation and determination of clenbuterol enantiomers by ultra-performance convergence chromatography[J]. Chinese Journal of Chromatography, 2021, 39(12): 1347-1354.

图/表 10

图1 克伦特罗两种对映体的结构式

Fig. 1 Chemical structures of two clenbuterol enantiomers

图2 克仑特罗对映体标准溶液的光谱图

Fig. 2 Spectra of the standard solution of clenbuterol enantiomers

图3 不同色谱柱对(+)-克伦特罗和(-)-克伦特罗分离效果的影响

Fig. 3 Effect of different chromatographic columns on the separation of (+)-clenbuterol and (-)-clenbuterol

图4 不同助溶剂对(+)-克伦特罗和(-)-克伦特罗分离效果的影响

Fig. 4 Effect of different organic solvent mobile phases on separation of (+)-clenbuterol and (-)-clenbuterol

图5 不同系统背压对(+)-克伦特罗和(-)-克伦特罗对映体分离效果的影响

Fig. 5 Effect of different system backpressure on the separation of (+)-clenbuterol and (-)-clenbuterol

图6 不同色谱柱温度对(+)-克伦特罗和(-)-克伦特罗分离效果的影响

Fig. 6 Effect of different column temperatures on the separation of (+)-clenbuterol and (-)-clenbuterol

图7 不同定溶试剂对(+)-克伦特罗和(-)-克伦特罗分离效果的影响

Fig. 7 Effect of different constant volume reagents on the separation of (+)-clenbuterol and (-)-clenbuterol

表1 克伦特罗对映体的线性范围、线性方程、相关系数和检出限

Table 1 Linear ranges, linear equations, correlation coefficients (r2), and limits of detection (LODs) of clenbuterol enantiomers

Clenbuterol enantiomer	Linear range/(mg/L)	Linear equation	r²	LOD/(mg/L)
(+)-Clenbuterol	1.0-20.0	Y=1.48×10⁴X+4.34×10³	0.9997	0.5
(-)-Clenbuterol	1.0-20.0	Y=1.30×10⁴X+5.26×10²	0.9998	0.5

图8 (+)-克伦特罗和(-)-克伦特罗标准溶液60天内的稳定性考察(乙腈溶液)

Fig. 8 Stability test for (+)-clenbuterol and (-)- clenbuterol in acetonitrile during 60 d

图9 克伦特罗外消旋体的拆分

Fig. 9 Separation of standard clenbuterol racemate

参考文献

[1]	Wu J S. Pharmacology. Beijing: People’s Sanitary Publishing Press, 1998
[1]	吴景时. 药理学. 北京: 人民卫生出版社, 1998
[2]	Huang K L, Hu W G, Wang W L, et al. Journal of Analytical Science, 2007, 23(1):9
[2]	黄可龙, 胡卫国, 王蔚玲, 等. 分析科学学报, 2007, 23(1):9
[3]	Thevis M, Thomas A, Beuck S, et al. Rapid Commun Mass SP, 2013, 27(4):507
[4]	Gausepopl G J, Blaschke B. Biomed Sci Appl, 1998, 713(2):443
[5]	Abou-Basha L I, Aboul-Enein H Y. Biomed Chromatogr, 1996, 10(2):69
[6]	Waldeck B, Windmark E. Acta Pharmacol Toxicol, 1985, 56:221
[7]	Martin P, Puech A J P, Brochet D, et al. Eur J Pharmacol, 1985, 117:127
[8]	Wu Y L, Yang T, Shan J H, et al. Chinese Journal of Analytical Chemistry, 2010, 38(6):833
[8]	吴银良, 杨挺, 单吉浩, 等. 分析化学, 2010, 38(6):833
[9]	Tang Q, Song H, Fu C, et al. Chinese Journal of Analytical Chemistry, 2004, 32(6):755
[9]	唐琴, 宋航, 付超, 等. 分析化学, 2004, 32(6):755
[10]	Lv L L, Wang L J, Li J, et al. J Pharmaceut Biomed, 2017, 145:399
[11]	Chen G, Liu Y J, Lü Y, et al. Meat Research, 2015, 29(5):22
[11]	陈国, 刘勇军, 吕燕, 等. 肉类研究, 2015, 29(5):22
[12]	Wang Z L, Zhang J L, Zhao Y, et al. Chinese Journal of Sports Medicine, 2015, 34(12):1186
[12]	王占良, 张建丽, 赵宇, 等. 中国运动医学杂志, 2015, 34(12):1186
[13]	Benjamín V B, Jahir B, Martha E R, et al. J Anal Toxicol, 2020, 44(3):237
[14]	Liu Y J, Xu W, Zhang H, et al. Electrophoresis 2019, 40(21):2828
[15]	Ultra-Performance Convergence Chromatography(UPC²): New Categories of Chromatography Have Empowered Scientists With New Imaginations.(2012-05-15) [2021-06-27]. https://www.antpedia.com/index.php?action-viewnews-itemid-212253-php-1
[15]	UPC²超高效合相色谱: 色谱新品类赋予科学家新的想象力. (2012-05-15) [2021-06-27]. https://www.antpedia.com/index.php?action-viewnews-itemid-212253-php-1
[16]	Zhang W H, Xie W, Hou J B, et al. Chinese Journal of Chromatography, 2019, 37(12):1356
[16]	张文华, 谢文, 侯建波, 等. 色谱, 2019, 37(12):1356
[17]	Jiang H, Yang L, Xing X D, et al. J Pharmaceut Biomed Anal, 2018, 153:117
[18]	Yu W S, Liu X, Zhang Y Z, et al. Anal Lett, 2020, 53(10):1654
[19]	Chang X Q, Sun P, Ma Y, et al. Molecules, 2020, 25(3):502
[20]	Yashima E, Okamoto Y. Angew Chem Int Ed, 1998, 37(8):1020
[21]	William H P, Thomas C P. J Chem Rev, 1989, 89:347
[22]	GB/T 32465-2015
[23]	Separation Degree. [2021-07-10]. https://baike.baidu.com/item/%E5%88%86%E7%A6%BB%E5%BA%A6/11007800?fr=Aladdin
[23]	分离度. [2021-07-10]. https://baike.baidu.com/item/%E5%88%86%E7%A6%BB%E5%BA%A6/11007800?fr=Aladdin

超高效合相色谱法拆分和测定克伦特罗对映体

Separation and determination of clenbuterol enantiomers by ultra-performance convergence chromatography

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

扫码分享

图/表 10

参考文献

相关文章 15

编辑推荐

Metrics

[1]	刘华林, 李亚楠, 字敏, 陈政, 段爱红, 袁黎明. 基于共价有机框架材料TpPa-NH₂-Glu的高效液相色谱固定相用于分离手性化合物[J]. 色谱, 2023, 41(2): 187-194.
[2]	郑康妮, 秦该照, 蒋雪菲, 章俊辉, 袁黎明. 多孔有机笼的制备及其用作毛细管电色谱手性固定相[J]. 色谱, 2023, 41(10): 929-936.
[3]	陈炎, 黄聪灵, 江薰垣, 陈志廷, 王刚, 万凯, 唐雪妹. 新型手性杀虫剂唑虫酯异构体拆分及其在蔬菜中的分析方法建立[J]. 色谱, 2022, 40(7): 634-643.
[4]	贾文燕, 唐明华, 章俊辉, 袁黎明. 多孔有机笼毛细管电色谱手性柱的制备及应用[J]. 色谱, 2022, 40(4): 391-398.
[5]	黄永鹏, 唐慧, 孟祥燕, 陈博, 钟辉, 邹志云. 直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相拆分布地奈德对映体及其制剂含量的测定[J]. 色谱, 2022, 40(3): 296-301.
[6]	李佳晨, 曹玲, 方方, 施海蔚, 黄青, 谭力, 段巧莲, 冯有龙. 超高效合相色谱法快速测定保健食品中10种脂溶性维生素[J]. 色谱, 2022, 40(12): 1136-1142.
[7]	齐艳丽, 高婧, 王伟荣, 金静, 吕莹, 秦曙. 基于手性固定相的超高效液相色谱-串联质谱法检测小麦及其加工制品中的腈菌唑对映体[J]. 色谱, 2021, 39(7): 702-707.
[8]	熊婉淇, 彭博, 段爱红, 袁黎明. 基于L-谷氨酸的手性硅胶球的制备及其应用[J]. 色谱, 2021, 39(6): 607-613.
[9]	汤文川, 常靖, 王元凤, 王爱华, 王瑞花. 苯丙胺类、氯胺酮、卡西酮类毒品手性分离的研究进展[J]. 色谱, 2021, 39(3): 271-280.
[10]	张旭, 董妙雪, 徐银, 王利娟, 乔晓强. 手性非水毛细管电泳法测定沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体[J]. 色谱, 2021, 39(12): 1355-1361.
[11]	汤雯淇, 孟莎莎, 徐铭, 古志远. 基于金属有机骨架材料固定相的气相色谱分离应用[J]. 色谱, 2021, 39(1): 57-68.
[12]	张琪. 毛细管电泳新型手性分离体系研究进展[J]. 色谱, 2020, 38(9): 1028-1037.
[13]	王翠杰, 高煜, 李星林, 赵无垛, 于阿娟, 张书胜. 以氧化锌为锌源的手性金属-有机骨架的合成及其用于联糠醛外消旋体的选择性吸附[J]. 色谱, 2020, 38(5): 516-521.
[14]	普青, 何宇雨, 袁黎明. 手性有序无机介孔硅用作气相色谱固定相[J]. 色谱, 2020, 38(4): 484-489.
[15]	刘明霞, 李向军, 白玉, 刘虎威. 毛细管电泳在手性化合物分离分析中的研究进展[J]. 色谱, 2020, 38(3): 317-323.