FI-CE在测定草药活性组分中新方法的研究及应用
摘要
各种分析仪器的联用技术是目前分析化学发展的主要趋势之一。流动注射-毛细管电泳联用技术正是这一发展趋势的体现，该技术集中了流动注射和毛细管电泳的优势。流动注射的强大样品预处理能力不仅可以拓宽毛细管电泳的应用领域，而且还能从根本上解决毛细管电泳不能连续进样的缺点。然而，迄今为止，流动注射的强大样品预处理能力不仅未能在流动注射-毛细管电泳中得到充分地应用，而且此联用技术在中草药活性组分的分离测定中仍有许多理论和应用需要深入研究。此外，浓度灵敏度低仍是制约流动注射-毛细管电泳联用技术进一步发展的瓶径之一。为了充分发挥流动注射-毛细管电泳联用技术的优势和解决毛细管电泳浓度灵敏度低及由于电动进样而引起的重现性较差的问题，本论文在综述前人工作的基础上，开展了如下具有创新性的研究工作：
1．利用流动注射高的进样频率，首次建立了分离测定决明子、大黄及其制剂中的四种活性葸醌类化合物的流动注射-毛细管区带电泳新方法，并对样品基体的影响进行了详细地探讨。
2．利用流动注射-毛细管胶束电动色谱联用技术分离测定了不稳定化合物山姜素和小豆蔻明(酸性或碱性条件下均互相转化)。
3．建立了以三甲氧苄二氨嘧啶为内标的分离测定中草药中粉防己碱和防己诺灵碱的流动注射-毛细管胶束电动色谱联用技术新方法，该方法的精密度得到了显著的提高。
4．利用电渗流引入水柱的柱头场强放大样品进样的富集手段，建立了流动注射—毛细管胶束电动色谱联用新技术，并以防己诺灵碱和粉防己碱为模型化合物验证了方法的可行性。
本论文共分为七章。
第一章：在对流动注射-毛细管电泳联用的必要性、联用的基本原理、分流接口和装置及应用等方面做了详细介绍的基础上，综述了毛细管电泳中常用的富集方法及在流动注射与毛细管电泳联用技术中的应用。
第二章：采用有效长度4.8cm未涂层的石英毛细管作为分离毛细管，建立了同时测定决明子、大黄及其制剂中大黄酚、大黄素、大黄素甲醚和芦荟大黄素的流动注射与毛细管区带电泳联用新方法。样品溶剂由100mM NaOH和乙腈的混合溶液组成(1：1 v／v)，缓冲体系由15mM硼砂、12.5mM磷酸二氢钠和42％乙腈(v／v)组成。该方法简单、快速、准确度高。在最佳条件下，四种分析物可以在6分钟内获得基线分离，进样频率可达12样／小时。大黄素甲醚、大黄酚、芦荟大黄素和大黄素的峰面积和峰高的相对标准偏差分别为1.62％、0.89％、2.49％、2.19％和4.45％、4.44％、4.34％、0.61％。
第三章：采用内径50μm，有效长度10.3cm的未涂层石英毛细管作为分离毛细管，4mM硼砂、8mM磷酸二氢钠、8mM十二烷基硫酸钠和19％(体积比)的乙醇作为缓冲溶液，建立了同时测定草豆蔻中山姜素和小豆蔻明的流动注射-毛细管胶束电动色谱联用新方法。在最佳条件下，体系的进样频率可达11-24样／小时。山姜素和小豆蔻明的峰面积和峰高的相对标准偏差分别为3.0％、2.5％和2.5％、3.1％，其峰面积与浓度间的相关系数(r)分别为0.9996和0.9997。该方法已用于草豆蔻中山姜素和小豆蔻明的测定，回收率分别为90-92％和99-105％。
第四章：首次将场强放大样品进样方式引入流动注射-毛细管胶束电动色谱联用技术，并以防己诺灵碱和粉防己碱为模型化合物考察了影响分离和富集的条件。在最佳条件下，粉防己碱和防己诺灵碱的富集倍数可达6.8-8.9倍，峰面积和峰高的相对标准偏差分别为3.6％、0.94％和4.8％、4.4％，进样频率可达50样／小时。
第五章：采用内径50μm、有效长度4.5cm的未涂层石英毛细管作为分离毛细管，建立了一种可以同时分离测定厚朴及其制剂中厚朴酚与和厚朴酚的流动注射-毛细管区带电泳新方法。最佳的样品溶解条件为150mM NaOH、最佳的缓冲溶液组成为10mM硼砂、10mM磷酸二氢钠，最佳的pH为12。在最佳条件下，厚朴酚与和厚朴酚的峰面积与浓度间的线性相关系数(r)分别为0.9991和0.9998，峰面积、峰高和迁移时间的相对标准偏差分别为2.0％、3.6％、2.0％和1.6％、2.0％、1.4％。二者可以在5分钟内达到基线分离，进样频率为28样／小时。
第六章：采用三甲氧苄二氨嘧啶为内标，建立了分离测定粉防己碱和防己诺灵碱的流动注射-毛细管胶束电动色谱新方法。最佳的缓冲溶液组成为15mM醋酸钠、15mM醋酸、3％(v／v)吐温20和5％(v／v)的甲醇、最佳的pH为5.5。在最佳条件下，其峰面积与浓度间的线性相关系数(r)分别为0.9996和0.9991，进样频率可达19-40样／小时。该方法简单、快速、重现性好并已用于防己及其制剂中粉防己碱和防己诺灵碱的分离测定，回收率分别为95-109％、92-106％。
第七章：建立了分离测定黄连及其制剂中药根碱、小檗碱和巴马汀的场强放大样品进样和流动注射—毛细管胶束电动色谱在线联用的新方法。考察了缓冲溶液的离子强度和样品环体积对富集效果的影响。利用流动注射的16通自动进样阀的结构特点，通过电渗流在样品进入毛细管前注入一段水柱，可使样品注入时的电场强度提高，以便更有效地堆积分析物离子。该体系缓冲溶液的组成为30％(v／v)乙醇-2％(v／v)吐温20-240mM醋酸铵溶液，最佳pH为4.7。在最佳条件下，富集倍数可达64-86倍，进样频率可达24样／小时。小檗碱、巴马汀和药根碱的检测限分别为27、26、22 ng／mL(S／N=3)，峰面积和峰高的相对标准偏差分别为1.6％、3.3％、3.8％和1.2％、2.7％、3.1％。
The hybrid techniques of the analysis instruments are one of the trends of the development of analytical chemistry. The combination of flow injection with capillary electrophoresis (FI-CE) is representative of the trend. The combined technique that integrates the essential favorable merits of FI and CE can significantly expand the application scope of CE by exploring the various on-line sample pretreatments, and overcomes the shortcoming of discontiguous injection of CE. However, hitherto, the ability of on-line sample pretreatments of FI is not widely applied, and many theories and applications need to deeply study in separation and determination of active compound in herbal medicine by FI-CE. Additional, the poor concentration sensitivity is one of the reasons of confined development of FI-CE. To exert the advantages of FI-CE and overcome the shortcoming brought by electrokinetic means in CE, the following major innovative work are carried out in this dissertation on the basis of the previous literatures:
1. A Novel method on separation and determination of active compound in Juemingzi, Rhubarb and related herbal preparations by FI-capillary zone electrophoresis (FI-CZE) was developed for the first time, and the effect of matrix was discussed in detail.
2. The changeful alpinetin and cardamonin in Alpinia Katsumadai were firstly separated and determined by FI-micellar electrokinetic chromatography (FI-MEKC).
3. In order to improve the repeatability of the combination of FI-MEKC, the internal method was used, and a new method for the separation and determination of tetrandrine and fangchinoline was founded.
4. A head-column field amplified sample injection (HC-FASI) in FI-MEKC was developed using electroosmotic flow to introduce a short water plug. Tetrandrine and fangchinoline were selected as model compound to demonstrate this stacking method. This dissertation consists of seven chapters.
In chapter 1, the combination of FI and CE was discussed from the necessity of the combined technique, the principle of this technique, split-flow interfaces and the basic FI-CE system setup, as well as novel applications to a variety of analytical problems, and so on. More importantly, the methods of stacking in CE and FI-CE were reviewed in detail.
In chapter 2, an unmodified effective length of 4.8 cm fused-silica capillary was used as separation capillary. A novel method for the separation and determination of physcion, chrysophanol, aloe-emodin and emodin in Rhubarb, Juemingzi and Chinese herbal preparations was developed by FI-CZE for the first time. The sample solvent was consisted of NaOH (100 mmol/L) and acetonitrile (ACN) (1:1 v/v), and a running buffer was composed of 15 mmol/L sodium borate -12.5 mmol/L sodium dihydrogen phosphate-42% (v/v) ACN . Under the optimized conditions, separation of the compounds could be achieved within 6 min. The sample throughput rate could reach up to 12 h~(-1). The repeatability (defined as relative standard deviation, RSD) was 4.45%, 4.44%, 4.34%, 0.61% with peak height evaluation and 1.62%, 0.89%, 2.49%, 2.19% with peak area evaluation for physcion, chrysophanol, aloe-emodin and emodin, respectively.
In chapter 3, a 50μm I.D. and unmodified effective length of 10.3 cm fused-silica was used as separation capillary. The background electrolyte (BGE) containing 4 mM sodium borate-8 mM NaH_2PO_4 (pH 8.1)-8 mM Sodium dodecyl sulfate (SDS)-19% (v/v) ethanol was used. A novel method for the separation and determination of alpinetin and cardamonin in Alpinia Katsumadai Hayata was developed by FI-MEKC for the first time. Under the optimized conditions, the sample throughput rate could reach up to 12 h~(-1). The repeatabilities (n = 4) reached RSD of 3.0 and 2.5% for the peak areas and 2.5 and 3.1% for peak heights of alpinetin and cardamonin, respectively. Regression equations revealed linear relationships (r: 0.9996-0.9997) between the peak area of each analyte and the concentration. Recoveries were in the range 90-92% and 99-105% for alpinetin and cardamonin, respectively.
In chapter 4, a FASI was first successfully combined with the FI-MEKC system. Fangchinoline and tetrandrine were selected as model mixture to demonstrate this stacking method. The parameters influencing the separation and stacking were studied. Under the optimum conditions, 6.8- to 8.9-fold improvement in concentration sensitivity relative to conventional FI-CE methods was achieved, and sample throughput rate can reach up to 50 h~(-1) . The repeatability (RSD) was 4.8, 4.4% with peak height evaluation and 3.6, 0.94% with peak area evaluation for tetrandrine and fangchinoline, respectively.
In chapter 5, a 50μm I.D. and unmodified effective length of 4.5 cm fused-silica was used as separation capillary. A novel method for the separation and determination of honokiol and magnolol in Magnolia officinalis and related herbal medicines was developed by combination of FI-CZE. The sample solvent consisted of 150 mM NaOH, a running buffer composed of 10 mM sodium tetraborate-10 mM sodium dihydrogenphosphate (NaH_2PO_4) at pH 12 was applied for the separation of the analytes. Under the optimized conditions, regression equations revealed linear relationships (r: 0.9991-0.9998) between the peak area of each analyte and the concentration. The repeatability (RSD) was 2.0%, 1.6% with peak area evaluation, 3.6%, 2.0% with peak height evaluation and 2.0%, 1.4% with migration time evaluation for honokiol and magnolol, respectively. The separation could be achieved within 5 min and sample throughput rate can reach up to 28 h~(-1).
In chapter 6, a novel method for the separation and determination of fangchinoline and tetrandrine was developed by combination of FI-MEKC system with trimethoprim as internal standard. A running buffer composed of 15 mM acetic acid-15 mM sodium acetate-3% (v/v) Tween 20-5% (v/v) methanol at pH 5.5 was applied for the separation of the analytes. Under the optimized conditions, the method resulted in excellent linearity with correlation coefficient of regression equation of 0.9996 and 0.9991 for TET and FAN, respectively. The real sample throughput was 19-40 samples per hour. The method was simple, rapid, reproducible, and has been successfully used for the determination of fangchinoline and tetrandrine in various herbal medicines. Recoveries were in the range 95-109% and 92-106% for tetrandrine and fangchinoline, respectively.
In chapter 7, a novel method for the separation and determination of berberine, palmatine and jatrorrhizine in Coptidis Rhizoma and related herbal medicines was developed in this paper by combination of HC-FASI-FI-MEKC. The effect ionic strength of BGE and volume of sample loop were studied. Based on the characteristic of the 16-way auto-switching valve, a short water plug was introduced by the electroosmotic flow (EOF) at the capillary inlet end prior to sample introduction. The BGE containing 240 mM ammonium acetate (pH 4.7)-30 % (v/v) ethanol-2 % (v/v) Tween 20 was used. Under the optimized conditions, 64 to 86-fold improvements in the detection sensitivity was obtained for the analytes and the sample throughput can reach up to 24 h~(-1). The limit of detection (LOD) for the berberine, palmatine and jatrorrhizine was found to be 27, 26, 22 ng mL~(-1) (S/N = 3). The repeatabilities (n = 4) reached RSDs of 1.2,2.7 and 3.1 % for the peak areas and 1.6, 3.3 and 3.8 % for peak heights of berberine, palmatine and jatrorrhizine, respectively.
1．利用流动注射高的进样频率，首次建立了分离测定决明子、大黄及其制剂中的四种活性葸醌类化合物的流动注射-毛细管区带电泳新方法，并对样品基体的影响进行了详细地探讨。
2．利用流动注射-毛细管胶束电动色谱联用技术分离测定了不稳定化合物山姜素和小豆蔻明(酸性或碱性条件下均互相转化)。
3．建立了以三甲氧苄二氨嘧啶为内标的分离测定中草药中粉防己碱和防己诺灵碱的流动注射-毛细管胶束电动色谱联用技术新方法，该方法的精密度得到了显著的提高。
4．利用电渗流引入水柱的柱头场强放大样品进样的富集手段，建立了流动注射—毛细管胶束电动色谱联用新技术，并以防己诺灵碱和粉防己碱为模型化合物验证了方法的可行性。
本论文共分为七章。
第一章：在对流动注射-毛细管电泳联用的必要性、联用的基本原理、分流接口和装置及应用等方面做了详细介绍的基础上，综述了毛细管电泳中常用的富集方法及在流动注射与毛细管电泳联用技术中的应用。
第二章：采用有效长度4.8cm未涂层的石英毛细管作为分离毛细管，建立了同时测定决明子、大黄及其制剂中大黄酚、大黄素、大黄素甲醚和芦荟大黄素的流动注射与毛细管区带电泳联用新方法。样品溶剂由100mM NaOH和乙腈的混合溶液组成(1：1 v／v)，缓冲体系由15mM硼砂、12.5mM磷酸二氢钠和42％乙腈(v／v)组成。该方法简单、快速、准确度高。在最佳条件下，四种分析物可以在6分钟内获得基线分离，进样频率可达12样／小时。大黄素甲醚、大黄酚、芦荟大黄素和大黄素的峰面积和峰高的相对标准偏差分别为1.62％、0.89％、2.49％、2.19％和4.45％、4.44％、4.34％、0.61％。
第三章：采用内径50μm，有效长度10.3cm的未涂层石英毛细管作为分离毛细管，4mM硼砂、8mM磷酸二氢钠、8mM十二烷基硫酸钠和19％(体积比)的乙醇作为缓冲溶液，建立了同时测定草豆蔻中山姜素和小豆蔻明的流动注射-毛细管胶束电动色谱联用新方法。在最佳条件下，体系的进样频率可达11-24样／小时。山姜素和小豆蔻明的峰面积和峰高的相对标准偏差分别为3.0％、2.5％和2.5％、3.1％，其峰面积与浓度间的相关系数(r)分别为0.9996和0.9997。该方法已用于草豆蔻中山姜素和小豆蔻明的测定，回收率分别为90-92％和99-105％。
第四章：首次将场强放大样品进样方式引入流动注射-毛细管胶束电动色谱联用技术，并以防己诺灵碱和粉防己碱为模型化合物考察了影响分离和富集的条件。在最佳条件下，粉防己碱和防己诺灵碱的富集倍数可达6.8-8.9倍，峰面积和峰高的相对标准偏差分别为3.6％、0.94％和4.8％、4.4％，进样频率可达50样／小时。
第五章：采用内径50μm、有效长度4.5cm的未涂层石英毛细管作为分离毛细管，建立了一种可以同时分离测定厚朴及其制剂中厚朴酚与和厚朴酚的流动注射-毛细管区带电泳新方法。最佳的样品溶解条件为150mM NaOH、最佳的缓冲溶液组成为10mM硼砂、10mM磷酸二氢钠，最佳的pH为12。在最佳条件下，厚朴酚与和厚朴酚的峰面积与浓度间的线性相关系数(r)分别为0.9991和0.9998，峰面积、峰高和迁移时间的相对标准偏差分别为2.0％、3.6％、2.0％和1.6％、2.0％、1.4％。二者可以在5分钟内达到基线分离，进样频率为28样／小时。
第六章：采用三甲氧苄二氨嘧啶为内标，建立了分离测定粉防己碱和防己诺灵碱的流动注射-毛细管胶束电动色谱新方法。最佳的缓冲溶液组成为15mM醋酸钠、15mM醋酸、3％(v／v)吐温20和5％(v／v)的甲醇、最佳的pH为5.5。在最佳条件下，其峰面积与浓度间的线性相关系数(r)分别为0.9996和0.9991，进样频率可达19-40样／小时。该方法简单、快速、重现性好并已用于防己及其制剂中粉防己碱和防己诺灵碱的分离测定，回收率分别为95-109％、92-106％。
第七章：建立了分离测定黄连及其制剂中药根碱、小檗碱和巴马汀的场强放大样品进样和流动注射—毛细管胶束电动色谱在线联用的新方法。考察了缓冲溶液的离子强度和样品环体积对富集效果的影响。利用流动注射的16通自动进样阀的结构特点，通过电渗流在样品进入毛细管前注入一段水柱，可使样品注入时的电场强度提高，以便更有效地堆积分析物离子。该体系缓冲溶液的组成为30％(v／v)乙醇-2％(v／v)吐温20-240mM醋酸铵溶液，最佳pH为4.7。在最佳条件下，富集倍数可达64-86倍，进样频率可达24样／小时。小檗碱、巴马汀和药根碱的检测限分别为27、26、22 ng／mL(S／N=3)，峰面积和峰高的相对标准偏差分别为1.6％、3.3％、3.8％和1.2％、2.7％、3.1％。
The hybrid techniques of the analysis instruments are one of the trends of the development of analytical chemistry. The combination of flow injection with capillary electrophoresis (FI-CE) is representative of the trend. The combined technique that integrates the essential favorable merits of FI and CE can significantly expand the application scope of CE by exploring the various on-line sample pretreatments, and overcomes the shortcoming of discontiguous injection of CE. However, hitherto, the ability of on-line sample pretreatments of FI is not widely applied, and many theories and applications need to deeply study in separation and determination of active compound in herbal medicine by FI-CE. Additional, the poor concentration sensitivity is one of the reasons of confined development of FI-CE. To exert the advantages of FI-CE and overcome the shortcoming brought by electrokinetic means in CE, the following major innovative work are carried out in this dissertation on the basis of the previous literatures:
1. A Novel method on separation and determination of active compound in Juemingzi, Rhubarb and related herbal preparations by FI-capillary zone electrophoresis (FI-CZE) was developed for the first time, and the effect of matrix was discussed in detail.
2. The changeful alpinetin and cardamonin in Alpinia Katsumadai were firstly separated and determined by FI-micellar electrokinetic chromatography (FI-MEKC).
3. In order to improve the repeatability of the combination of FI-MEKC, the internal method was used, and a new method for the separation and determination of tetrandrine and fangchinoline was founded.
4. A head-column field amplified sample injection (HC-FASI) in FI-MEKC was developed using electroosmotic flow to introduce a short water plug. Tetrandrine and fangchinoline were selected as model compound to demonstrate this stacking method. This dissertation consists of seven chapters.
In chapter 1, the combination of FI and CE was discussed from the necessity of the combined technique, the principle of this technique, split-flow interfaces and the basic FI-CE system setup, as well as novel applications to a variety of analytical problems, and so on. More importantly, the methods of stacking in CE and FI-CE were reviewed in detail.
In chapter 2, an unmodified effective length of 4.8 cm fused-silica capillary was used as separation capillary. A novel method for the separation and determination of physcion, chrysophanol, aloe-emodin and emodin in Rhubarb, Juemingzi and Chinese herbal preparations was developed by FI-CZE for the first time. The sample solvent was consisted of NaOH (100 mmol/L) and acetonitrile (ACN) (1:1 v/v), and a running buffer was composed of 15 mmol/L sodium borate -12.5 mmol/L sodium dihydrogen phosphate-42% (v/v) ACN . Under the optimized conditions, separation of the compounds could be achieved within 6 min. The sample throughput rate could reach up to 12 h~(-1). The repeatability (defined as relative standard deviation, RSD) was 4.45%, 4.44%, 4.34%, 0.61% with peak height evaluation and 1.62%, 0.89%, 2.49%, 2.19% with peak area evaluation for physcion, chrysophanol, aloe-emodin and emodin, respectively.
In chapter 3, a 50μm I.D. and unmodified effective length of 10.3 cm fused-silica was used as separation capillary. The background electrolyte (BGE) containing 4 mM sodium borate-8 mM NaH_2PO_4 (pH 8.1)-8 mM Sodium dodecyl sulfate (SDS)-19% (v/v) ethanol was used. A novel method for the separation and determination of alpinetin and cardamonin in Alpinia Katsumadai Hayata was developed by FI-MEKC for the first time. Under the optimized conditions, the sample throughput rate could reach up to 12 h~(-1). The repeatabilities (n = 4) reached RSD of 3.0 and 2.5% for the peak areas and 2.5 and 3.1% for peak heights of alpinetin and cardamonin, respectively. Regression equations revealed linear relationships (r: 0.9996-0.9997) between the peak area of each analyte and the concentration. Recoveries were in the range 90-92% and 99-105% for alpinetin and cardamonin, respectively.
In chapter 4, a FASI was first successfully combined with the FI-MEKC system. Fangchinoline and tetrandrine were selected as model mixture to demonstrate this stacking method. The parameters influencing the separation and stacking were studied. Under the optimum conditions, 6.8- to 8.9-fold improvement in concentration sensitivity relative to conventional FI-CE methods was achieved, and sample throughput rate can reach up to 50 h~(-1) . The repeatability (RSD) was 4.8, 4.4% with peak height evaluation and 3.6, 0.94% with peak area evaluation for tetrandrine and fangchinoline, respectively.
In chapter 5, a 50μm I.D. and unmodified effective length of 4.5 cm fused-silica was used as separation capillary. A novel method for the separation and determination of honokiol and magnolol in Magnolia officinalis and related herbal medicines was developed by combination of FI-CZE. The sample solvent consisted of 150 mM NaOH, a running buffer composed of 10 mM sodium tetraborate-10 mM sodium dihydrogenphosphate (NaH_2PO_4) at pH 12 was applied for the separation of the analytes. Under the optimized conditions, regression equations revealed linear relationships (r: 0.9991-0.9998) between the peak area of each analyte and the concentration. The repeatability (RSD) was 2.0%, 1.6% with peak area evaluation, 3.6%, 2.0% with peak height evaluation and 2.0%, 1.4% with migration time evaluation for honokiol and magnolol, respectively. The separation could be achieved within 5 min and sample throughput rate can reach up to 28 h~(-1).
In chapter 6, a novel method for the separation and determination of fangchinoline and tetrandrine was developed by combination of FI-MEKC system with trimethoprim as internal standard. A running buffer composed of 15 mM acetic acid-15 mM sodium acetate-3% (v/v) Tween 20-5% (v/v) methanol at pH 5.5 was applied for the separation of the analytes. Under the optimized conditions, the method resulted in excellent linearity with correlation coefficient of regression equation of 0.9996 and 0.9991 for TET and FAN, respectively. The real sample throughput was 19-40 samples per hour. The method was simple, rapid, reproducible, and has been successfully used for the determination of fangchinoline and tetrandrine in various herbal medicines. Recoveries were in the range 95-109% and 92-106% for tetrandrine and fangchinoline, respectively.
In chapter 7, a novel method for the separation and determination of berberine, palmatine and jatrorrhizine in Coptidis Rhizoma and related herbal medicines was developed in this paper by combination of HC-FASI-FI-MEKC. The effect ionic strength of BGE and volume of sample loop were studied. Based on the characteristic of the 16-way auto-switching valve, a short water plug was introduced by the electroosmotic flow (EOF) at the capillary inlet end prior to sample introduction. The BGE containing 240 mM ammonium acetate (pH 4.7)-30 % (v/v) ethanol-2 % (v/v) Tween 20 was used. Under the optimized conditions, 64 to 86-fold improvements in the detection sensitivity was obtained for the analytes and the sample throughput can reach up to 24 h~(-1). The limit of detection (LOD) for the berberine, palmatine and jatrorrhizine was found to be 27, 26, 22 ng mL~(-1) (S/N = 3). The repeatabilities (n = 4) reached RSDs of 1.2,2.7 and 3.1 % for the peak areas and 1.6, 3.3 and 3.8 % for peak heights of berberine, palmatine and jatrorrhizine, respectively.
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