反相高效液相色谱法在药物分析中的应用研究
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摘要
高效液相色谱法(High Performance Liquid Chromatography，HPLC)是70年代迅速发展起来的一项高效、快速的分离分析技术，是现代分离测试的重要手段。鉴于其简便、快速、灵敏、准确的特点，目前，在医药、卫生、食品、环保等各个领域已得到广泛应用。
本论文建立了用反相高效液相色谱法测定中药中几种成分的方法，由综述和研究报告两部分组成，综述部分简单概述了液相色谱的历史、一般原理、仪器构成及其在药物分析中的应用，研究报告部分建立了太太口服液和蜂胶片中几种成份的测定方法，包括：反相高效液相色谱分别测定太太口服液中的阿魏酸、肉桂酸和槲皮素，反相高效液相色谱法测定蜂胶片中的咖啡酸和同时测定蜂胶片中五种组分。
1．反相高效液相色谱测定太太口服液中的阿魏酸
建立了太太美容口服液中阿魏酸含量的HPLC测定方法。色谱柱为Shim-Pack VP-ODS柱(150×4.6 mm，5μm)；流动相为甲醇-水-冰醋酸(32：66：2)；流速1 mL／min；检测波长为323 nm。阿魏酸的线性范围为0.252～5.04μg／mL，r=0.9993，平均回收率为99.9％(n=5)，RSD为1.2％。实验证明该方法简便、快速、准确，适合于太太美容口服液中阿魏酸含量的测定。
2．反相高效液相色谱法测定太太口服液中的肉桂酸
利用色谱柱Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10×4.6 mm，5.0μm)；以甲醇-水-冰醋酸(体积比)=70：29：1为流动相的条件下，实现了对太太口服液中肉桂酸的分离测定。流速：1 mL／min；280 nm处进行检测。肉桂酸的线性范围为0.012～4.80μg／mL，r=0.9998，平均回收率为99.1％(n=5)，RSD为3.26％。实验表明该方法简便、快速，应用于实际样品太太口服液中肉桂酸含量的测定，结果令人满意。
3．反相高效液相色谱法测定太太口服液中的槲皮素。
在这一节，我们建立了太太美容口服液中槲皮素含量的HPLC测定方法。色谱柱为Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10×4.6 mm，5.0μm)；流动相为甲醇-含0.2％磷酸的缓冲溶液(60：40)；流速：1 mL／min；254 nm处检测。结果表明槲皮素在0.00720～0.360μg／mL范围内，浓度与峰面积有良好的线性关系(r=0.9999)，槲皮素的回收率为93.9％～98.6％(n=5)，相对标准偏差为2.07％。应用于实际样品中槲皮素含量的测定，取得了很好的结果。
4．反相高效液相色谱法测定蜂胶片的咖啡酸
利用反相高效液相色谱法测定了蜂胶片中咖啡酸含量。色谱柱采用Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10×4.6mm，5.0μm)；在流动相为甲醇-水-甲酸(35：64：1)和流速1 mL／min条件下进行分离测定；检测波长为323 nm。咖啡酸的线性范围为0.120～24.0μg／mL，r=0.9995，平均回收率为101.0％(n=5)，RSD为2.69％。实验表明该方法简便、快速，适合于蜂胶片中咖啡酸含量的测定
5．反相高效液相色谱法同时测定蜂胶片中的五种组分
采用反相高效液相色谱法测定了蜂胶片中的阿魏酸、苯甲酸、水杨酸、肉桂酸和槲皮素五种组分。色谱柱为Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10 i．d．×4.6 mm，5.0μm)；流动相为含0.2％磷酸的缓冲溶液和甲醇，采用梯度洗脱，流速为1 ml／min，检测波长为：0～20 min，323 nm；20-25 min，230 nm；25-37 min，236 nm；37-45 min，280 nm；45-60 min，254 nm。五种组分的回收率分别介于94.2％-111.2％，实验表明在较宽的浓度范围内线性较好(r=0.9979-0.9996)。应用于蜂胶片中这五种组分的测定，结果满意。
High performance liquid chromatography (HPLC) as an important modern measurement of separation now is one of high performance and fastly speed analysis and separation technology emerging in the 1970's. Due to its' convenience, high-speed, sensitivity and fairly accuracy, it has been widely used in the areas of production of medicine, foodstuff, and environment etc.
This thesis establishes several detection methods by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC). It includes two sections—review and research report. In the review section, information about high performance liquid chromatography such as history, principles, HPLC equipment and the applications in the medicine analysis was summarized. In the next research report section, analysis methods of several compounds in the taita beauty essence and beeswax tablets were reported. That is: Respective determination of ferulic acid, cinnamic acid and quercetin in taita beauty essence by reversed-phase high-performance liquid chromatography; Determination of caffeic acid in beeswax tablets and simultaneous determinations of five compositions in beeswax tablets by reversed-phase high-performance liquid chromatography.
1. Determination of Ferulic Acid in Taita Beauty Essence by Reversed-Phase High-Performance Liquid Chromatography
The HPLC method for the determination of ferulic acid in Taita Beauty Essence was established. A Shim-pack VP-ODS column ( 150 x 4.6 mm, 5 urn ) was used, with the mobile phase of methanol-water-acetic acid (32: 66: 2) at the detection wavelength of 323 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.252～5.04μg/ml (r=0.9993 ). The average recovery was 99.9 % ( n=5 ) and RSD was 1.2 %. It was proved that the method was convenient, rapid, accurate and suitable for the determination of ferulic acid in Taita Beauty Essence.
2 Determination of Cinnamic Acid in Taita Beauty Essence by Reversed-Phase High-Performance Liquid Chromatography
A method using HPLC was developed for determination of cinnamic acid in taita beauty essence. The separation was carried on a Shim-pack VP-ODS column (150×4.6 mm, 5μm ) and a guard column (10×4.6 mm, 5.0μm), with the mobile phase of methanol-water-acetic acid=( 70: 29: 1,V/V ) at the detection wavelength of 280 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.012～4.80μg/mL, (r=0.9998 ). The average recovery was 99.1 % (n=5 ) and RSD was 3.26 %. The method has been successfully applied to determination, which was proved to be simple and rapid.
3 Determination of Quercetin in Taita Beauty Essence by Reversed-Phase High-Performance Liquid Chromatography
In this chapter, a HPLC method was developed for the determination of quercetin in Taita Beauty Essence. A Shim-pack VP-ODS column (150×4.6 mm, 5μm ) and a guard column (10×4.6 mm, 5.0μm) were used, The analysis was carried under the condition of mobile phase of methanol-0.2 % (v/v) phosphoric acid in water ( 60:40) and at the detection wavelength of 254 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.00720～0.360μg/mL (r=0.9999 ). The average recovery was 93.9 %～98.6 % (n=5) and the relative standard deviation was 2.07 %. The method was suitable for the determination of quercetin in Taita Beauty Essence. A good result was obtained.
4. Determination of Caffeic Acid in Beeswax Tablets by Reversed-Phase High-Performance Liquid Chromatography
The HPLC method for the determination of caffeic acid in beeswax tablets was established. A Shim-pack VP-ODS column (150×4.6 mm, 5μm) and a guard column (10×4.6 mm, 5.0μm) were used, with the mobile phase of methanol-water-formic acid (35: 64: 1) at the detection wavelength of 323 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.120～24.0μg/mL (r=0.9995 ). The average recovery was 101.0 % (n=5) and the relative standard deviation was 2.69 %. It was proved that the method was convenient, rapid and suitable for the determination of caffeic acid in Taita Beauty Essence.
5. Simultaneous Determinations of Five Components in Beeswax Tablets by Reversed-Phase High-Performance Liquid Chromatography
A reversed high-performance liquid chromatographic method was employed to the determination of ferulic acid, benzoic acid, salicylic acid, cinnamic acid and quercetin in beeswax tablets. These five compounds were analyzed simultaneously with a shim-pack VP-ODS (150×4.6 mm, 5.0μm) and a guard column (10×4.6 mm, 5.0μm) by gradient elution using 0.2 % (v/v) phosphoric acid in water-methanol as the mobile phase. The flow rate was 1 ml/min and the detection wavelength was performed according to time program: 0-20 min, 323 nm; 20-25 min, 230 run; 25-37 min, 236 nm; 37-45 min, 280 nm; 45-60 min, 254 nm. The recovery of the method was in the range of 94.2 %-111.2 %, and all the compounds showed good linearity (r-0.9979-0.9996) in a relatively wide concentration range. The method has been successfully applied to determination of the five compounds in beeswax tablets.
本论文建立了用反相高效液相色谱法测定中药中几种成分的方法，由综述和研究报告两部分组成，综述部分简单概述了液相色谱的历史、一般原理、仪器构成及其在药物分析中的应用，研究报告部分建立了太太口服液和蜂胶片中几种成份的测定方法，包括：反相高效液相色谱分别测定太太口服液中的阿魏酸、肉桂酸和槲皮素，反相高效液相色谱法测定蜂胶片中的咖啡酸和同时测定蜂胶片中五种组分。
1．反相高效液相色谱测定太太口服液中的阿魏酸
建立了太太美容口服液中阿魏酸含量的HPLC测定方法。色谱柱为Shim-Pack VP-ODS柱(150×4.6 mm，5μm)；流动相为甲醇-水-冰醋酸(32：66：2)；流速1 mL／min；检测波长为323 nm。阿魏酸的线性范围为0.252～5.04μg／mL，r=0.9993，平均回收率为99.9％(n=5)，RSD为1.2％。实验证明该方法简便、快速、准确，适合于太太美容口服液中阿魏酸含量的测定。
2．反相高效液相色谱法测定太太口服液中的肉桂酸
利用色谱柱Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10×4.6 mm，5.0μm)；以甲醇-水-冰醋酸(体积比)=70：29：1为流动相的条件下，实现了对太太口服液中肉桂酸的分离测定。流速：1 mL／min；280 nm处进行检测。肉桂酸的线性范围为0.012～4.80μg／mL，r=0.9998，平均回收率为99.1％(n=5)，RSD为3.26％。实验表明该方法简便、快速，应用于实际样品太太口服液中肉桂酸含量的测定，结果令人满意。
3．反相高效液相色谱法测定太太口服液中的槲皮素。
在这一节，我们建立了太太美容口服液中槲皮素含量的HPLC测定方法。色谱柱为Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10×4.6 mm，5.0μm)；流动相为甲醇-含0.2％磷酸的缓冲溶液(60：40)；流速：1 mL／min；254 nm处检测。结果表明槲皮素在0.00720～0.360μg／mL范围内，浓度与峰面积有良好的线性关系(r=0.9999)，槲皮素的回收率为93.9％～98.6％(n=5)，相对标准偏差为2.07％。应用于实际样品中槲皮素含量的测定，取得了很好的结果。
4．反相高效液相色谱法测定蜂胶片的咖啡酸
利用反相高效液相色谱法测定了蜂胶片中咖啡酸含量。色谱柱采用Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10×4.6mm，5.0μm)；在流动相为甲醇-水-甲酸(35：64：1)和流速1 mL／min条件下进行分离测定；检测波长为323 nm。咖啡酸的线性范围为0.120～24.0μg／mL，r=0.9995，平均回收率为101.0％(n=5)，RSD为2.69％。实验表明该方法简便、快速，适合于蜂胶片中咖啡酸含量的测定
5．反相高效液相色谱法同时测定蜂胶片中的五种组分
采用反相高效液相色谱法测定了蜂胶片中的阿魏酸、苯甲酸、水杨酸、肉桂酸和槲皮素五种组分。色谱柱为Shim-Pack VP-ODS柱(150×4.6 mm，5μm)和预柱Shim-Pack VP-ODS柱(10 i．d．×4.6 mm，5.0μm)；流动相为含0.2％磷酸的缓冲溶液和甲醇，采用梯度洗脱，流速为1 ml／min，检测波长为：0～20 min，323 nm；20-25 min，230 nm；25-37 min，236 nm；37-45 min，280 nm；45-60 min，254 nm。五种组分的回收率分别介于94.2％-111.2％，实验表明在较宽的浓度范围内线性较好(r=0.9979-0.9996)。应用于蜂胶片中这五种组分的测定，结果满意。
High performance liquid chromatography (HPLC) as an important modern measurement of separation now is one of high performance and fastly speed analysis and separation technology emerging in the 1970's. Due to its' convenience, high-speed, sensitivity and fairly accuracy, it has been widely used in the areas of production of medicine, foodstuff, and environment etc.
This thesis establishes several detection methods by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC). It includes two sections—review and research report. In the review section, information about high performance liquid chromatography such as history, principles, HPLC equipment and the applications in the medicine analysis was summarized. In the next research report section, analysis methods of several compounds in the taita beauty essence and beeswax tablets were reported. That is: Respective determination of ferulic acid, cinnamic acid and quercetin in taita beauty essence by reversed-phase high-performance liquid chromatography; Determination of caffeic acid in beeswax tablets and simultaneous determinations of five compositions in beeswax tablets by reversed-phase high-performance liquid chromatography.
1. Determination of Ferulic Acid in Taita Beauty Essence by Reversed-Phase High-Performance Liquid Chromatography
The HPLC method for the determination of ferulic acid in Taita Beauty Essence was established. A Shim-pack VP-ODS column ( 150 x 4.6 mm, 5 urn ) was used, with the mobile phase of methanol-water-acetic acid (32: 66: 2) at the detection wavelength of 323 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.252～5.04μg/ml (r=0.9993 ). The average recovery was 99.9 % ( n=5 ) and RSD was 1.2 %. It was proved that the method was convenient, rapid, accurate and suitable for the determination of ferulic acid in Taita Beauty Essence.
2 Determination of Cinnamic Acid in Taita Beauty Essence by Reversed-Phase High-Performance Liquid Chromatography
A method using HPLC was developed for determination of cinnamic acid in taita beauty essence. The separation was carried on a Shim-pack VP-ODS column (150×4.6 mm, 5μm ) and a guard column (10×4.6 mm, 5.0μm), with the mobile phase of methanol-water-acetic acid=( 70: 29: 1,V/V ) at the detection wavelength of 280 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.012～4.80μg/mL, (r=0.9998 ). The average recovery was 99.1 % (n=5 ) and RSD was 3.26 %. The method has been successfully applied to determination, which was proved to be simple and rapid.
3 Determination of Quercetin in Taita Beauty Essence by Reversed-Phase High-Performance Liquid Chromatography
In this chapter, a HPLC method was developed for the determination of quercetin in Taita Beauty Essence. A Shim-pack VP-ODS column (150×4.6 mm, 5μm ) and a guard column (10×4.6 mm, 5.0μm) were used, The analysis was carried under the condition of mobile phase of methanol-0.2 % (v/v) phosphoric acid in water ( 60:40) and at the detection wavelength of 254 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.00720～0.360μg/mL (r=0.9999 ). The average recovery was 93.9 %～98.6 % (n=5) and the relative standard deviation was 2.07 %. The method was suitable for the determination of quercetin in Taita Beauty Essence. A good result was obtained.
4. Determination of Caffeic Acid in Beeswax Tablets by Reversed-Phase High-Performance Liquid Chromatography
The HPLC method for the determination of caffeic acid in beeswax tablets was established. A Shim-pack VP-ODS column (150×4.6 mm, 5μm) and a guard column (10×4.6 mm, 5.0μm) were used, with the mobile phase of methanol-water-formic acid (35: 64: 1) at the detection wavelength of 323 nm. The flow rate was 1 ml/min. The calibration curve was linear in the range of 0.120～24.0μg/mL (r=0.9995 ). The average recovery was 101.0 % (n=5) and the relative standard deviation was 2.69 %. It was proved that the method was convenient, rapid and suitable for the determination of caffeic acid in Taita Beauty Essence.
5. Simultaneous Determinations of Five Components in Beeswax Tablets by Reversed-Phase High-Performance Liquid Chromatography
A reversed high-performance liquid chromatographic method was employed to the determination of ferulic acid, benzoic acid, salicylic acid, cinnamic acid and quercetin in beeswax tablets. These five compounds were analyzed simultaneously with a shim-pack VP-ODS (150×4.6 mm, 5.0μm) and a guard column (10×4.6 mm, 5.0μm) by gradient elution using 0.2 % (v/v) phosphoric acid in water-methanol as the mobile phase. The flow rate was 1 ml/min and the detection wavelength was performed according to time program: 0-20 min, 323 nm; 20-25 min, 230 run; 25-37 min, 236 nm; 37-45 min, 280 nm; 45-60 min, 254 nm. The recovery of the method was in the range of 94.2 %-111.2 %, and all the compounds showed good linearity (r-0.9979-0.9996) in a relatively wide concentration range. The method has been successfully applied to determination of the five compounds in beeswax tablets.
引文
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