Abstract Objective: Simultaneous determination of ferulic acid and adenosine in Angelica by micellar electrokinetic capillary chromatography. Method: A fused silica capillary column (50 μm × 66.5 cm, effective length 58 cm) was used as the separation channel; a solution of 50 mmol/L borax and 10 mmol/L sodium deoxycholate and 2% methanol (pH 9.6) was used as the running buffer. Operating voltage 24kV; capillary column temperature 20 ° C; detection wavelength: 210 nm. RESULTS: With sodium pentobarbital as internal standard, the linear relationship between ferulic acid and adenosine in the concentration range of 20-320 μg/mL and 10-160 μg/mL was good (r>0.998); the recovery rate was 96.8%. ~97.4% and 93.2% to 95.0%; the RSD of the repeatability test was 4.7% and 6.8% (=5), respectively. Conclusion: This method is simple, rapid, and has good recovery rate and good reproducibility.
Key words: micellar electrokinetic capillary chromatography; Angelica; Adenine adenosine is the dry root of the genus Angelica sinensis (Oliv.) Diels, is a famous blood-activating blood medicine. It is reported in the literature that it contains volatile oil and water-soluble components. Ferulic acid is one of its main active ingredients. It has anti-oxidation and anti-free radical action, and can effectively inhibit platelet aggregation and thrombus formation. The content of the method is determined by high performance liquid chromatography L2. And high performance capillary electrophoresis . Adenosine is also one of the components of Angelica sinensis. It has the effects of inhibiting platelet aggregation and regulating adenylate cyclase activity. However, the authors have not reported the determination of adenosine in Angelica. Due to the complexity of the ingredients in Angelica medicinal herbs, the column is highly susceptible to contamination by volatile oils when analyzed by HPLC , resulting in a shortened column life. In this study, micellar electrokinetic capillary chromatography was used to simultaneously determine the content of ferulic acid and adenosine in Angelica sinensis. It has the characteristics of high efficiency, short analysis time and low cost. The experimental results show that the sample is easy to pretreat and recover. Good, good reproducibility.
1 Instruments and reagents
HP3 G16OOA high performance capillary electrophoresis apparatus, HP ChemStation, diode array detector, autosampler; Japan Shimadzu LIBROR AEL-160 electronic balance; Beijing Autoscience AS5150A ultrasonic cleaner; ferulic acid (batch 0773-9910), adenosine The control materials were purchased from China National Institute for the Control of Pharmaceutical and Biological Products; sodium deoxycholate was analytically pure (Fluka, Switzerland); methanol was chromatographically pure, and other reagents were of analytical grade; Angelica medicinal materials were purchased from Shenyang Pharmaceutical Company, and passed through Shenyang Pharmaceuticals. Professor Sun Qishi of the University of Medicinal Plants identified the dried roots of the genus Angelica sinensis (Oliv.) Diels.
2 Capillary electrophoresis conditions uncoated quartz capillary 5Om × 66.5cm, effective length 58cm (Hebei Yongnian Ruiqi Chromatography Devices Co., Ltd.); detection wavelength: 210nm; pressure injection: 5kPa, 5s; separation voltage: 24kV; capillary temperature: 20 °C; running buffer: 50 mmol/L borax and 10 mmol/L sodium deoxycholate, and containing 2% methanol (pH 9.6). The capillary column was washed successively with 1 mol/L sodium hydroxide solution, double distilled water and running buffer for 5, 5, 8 min before use, and rinsed with running buffer for 8 min after each electrophoresis. Under these conditions, the separation of ferulic acid and adenosine.
3 Preparation of the solution
3.1 The reference solution was accurately weighed 40 mg of ferulic acid reference substance and 20 mg of adenosine reference substance, dissolved in 60% methanol, and made up to 50.0 mL, and mixed for use.
3.2 The test solution was accurately weighed 2.0 g of dried Angelica powder at 60 ° C, ultrasonically extracted with 20 mL of 20% ethanol for 20 min, centrifuged at 3000 r/min for 10 min, and the supernatant was taken. The residue was extracted twice with the same method and combined. The supernatant solution was recovered under reduced pressure, and the solution was adjusted to a volume of 10 mL with 60% methanol, and then filtered through a 0.45 μm filter to obtain a test solution.
4 Linear relationship study Precision draw reference solution 0.25,0.5,1.0,1.5,2.0,4.0mL in a 10mL volumetric flask, add 60% methanol to the scale, mix and then separately injected, record the respective peak area, to Awei The peak area ratio of acid, adenosine and pentobarbital sodium is ordinate, and the concentration X (μg/mL) is linearly regression on the abscissa. The regression equations of ferulic acid and adenosine are:
Y=160.3X +0.1540 r=0.9992
Y=9256X +0.1989 r=0.9986
The linear range of the two is 20-320 μg / mL, 10 ~ 160 μg / mL.
5 precision test Take 1.5mL of the reference solution in a 10mL volumetric flask, add 60% methanol to the mark, mix and sample for 5 times, according to the peak area ratio of ferulic acid and internal standard, the RSD is 4.4%; The peak area ratio of adenosine to internal standard was calculated to be 3.2% RSD.
6 Repeatability test Weighed 5 parts of Angelica powder dried at 60 ° C, each serving 2.0 g, prepared the test solution according to the law, according to the "capillary electrophoresis conditions", the RSD calculated by ferulic acid content was 4.7%; The RSD was calculated to be 6.8% based on the content of adenosine.
7 Recovery test Precision draw reference solution 0.25mL and 1.25mL. Add to 2.0g of known content of Angelica sinensis (ferrifre acid: 0.269mg / g, adenosine: 0.202 mg / g), prepare the test solution according to the law, according to the "capillary electrophoresis conditions" to determine, The average recovery of Wei acid was between 96.8% and 97.4%, the average recovery of adenosine was between 93.2% and 95.0%, and the RSD was within 6.4%, n=3.
8 Determination of samples Take 10 samples of Angelica sinensis from different origins, and prepare the test solution for determination.
9 Discussion
9.1 Selection of extraction method The parallel test was used to compare the reflux method, decoction method and ultrasonic method. The results showed that the extraction rate of decoction method was low by the content of ferulic acid and adenosine. The extraction rate of the ultrasonic method is basically the same. Since the ultrasonic method is simple and time-saving, the ultrasonic method is used in this experiment.
9.2 Optimization of extraction process Ferulic acid is soluble in hot water, alcohol, ethyl acetate and ether. Adenosine is easily soluble in water, so it is extracted with ethanol solution containing a certain amount of water. The extraction process of Angelica sinensis was optimized by orthogonal design. The effects of extraction time, solvent dosage and solvent concentration on the extraction efficiency were investigated. The content of ferulic acid and adenosine was used as the index to evaluate the orthogonal test results. Preferably, the extraction process conditions are as follows: 60 mL of 60% ethanol, ultrasonic extraction for 20 min, a total of 3 times.
9.3 Selection of running buffer In the experiment, 10~100mmol/L borax buffer was firstly investigated and subjected to capillary zone electrophoresis. It was found that ferulic acid and adenosine could not be separated from adjacent peaks and then changed. 5～100mmol/L sodium deoxycholate was added to 50mmol/L borax and subjected to micellar electrokinetic capillary chromatography. The results were as follows: buffer 50mmol/L borax, 10mmol/L sodium deoxycholate and 2% methanol separation. The effect is good.
9.4 Effect of Buffer pH on Separation The effect of buffer pH between 3.5 and 11.0 on the resolution of ferulic acid, adenosine and adjacent peaks was investigated. When the pH is below 8.5, it is almost impossible to Separated from adjacent peaks, and pH between 9.2 and 9.8, ferulic acid, adenosine and adjacent peaks can reach baseline separation.
9.5 Selection of operating voltage When the operating voltage rises to 28 kV, the migration rate of adenosine and ferulic acid increases, but the resolution decreases. When the operating voltage drops to 20 kV, the migration time is extended, and the resolution is considered comprehensively. And the impact of migration time, this paper chooses the operating voltage is 24 kV.
9.6 Peak Purity Detection The peaks of ferulic acid and adenosine are identified by comparing the online UV absorption spectrum with the addition of the reference solution. At the same time, the peak purity check function in the HP ChemStation is used to select ferulic acid and gland. The purity of the chromatographic peak of the glycoside was evaluated. When the matching factor of the ultraviolet spectrum was greater than 950, the quantification was performed to avoid the error caused by the impure purity of the chromatographic peak.
references
1 ZHENGHu-zhan (Zheng Huzhan), DONG ze-hong (Dong Zehong), SHE Jing (Yu Jing). Modem Research and Application of Traditional Chinese Medicine (Modern Research and Application of Traditional Chinese Medicine). Beijing (Beijing): Xueyan Press (Xueyuan Press), 1998.206
Key words: micellar electrokinetic capillary chromatography; Angelica; Adenine adenosine is the dry root of the genus Angelica sinensis (Oliv.) Diels, is a famous blood-activating blood medicine. It is reported in the literature that it contains volatile oil and water-soluble components. Ferulic acid is one of its main active ingredients. It has anti-oxidation and anti-free radical action, and can effectively inhibit platelet aggregation and thrombus formation. The content of the method is determined by high performance liquid chromatography L2. And high performance capillary electrophoresis . Adenosine is also one of the components of Angelica sinensis. It has the effects of inhibiting platelet aggregation and regulating adenylate cyclase activity. However, the authors have not reported the determination of adenosine in Angelica. Due to the complexity of the ingredients in Angelica medicinal herbs, the column is highly susceptible to contamination by volatile oils when analyzed by HPLC , resulting in a shortened column life. In this study, micellar electrokinetic capillary chromatography was used to simultaneously determine the content of ferulic acid and adenosine in Angelica sinensis. It has the characteristics of high efficiency, short analysis time and low cost. The experimental results show that the sample is easy to pretreat and recover. Good, good reproducibility.
1 Instruments and reagents
HP3 G16OOA high performance capillary electrophoresis apparatus, HP ChemStation, diode array detector, autosampler; Japan Shimadzu LIBROR AEL-160 electronic balance; Beijing Autoscience AS5150A ultrasonic cleaner; ferulic acid (batch 0773-9910), adenosine The control materials were purchased from China National Institute for the Control of Pharmaceutical and Biological Products; sodium deoxycholate was analytically pure (Fluka, Switzerland); methanol was chromatographically pure, and other reagents were of analytical grade; Angelica medicinal materials were purchased from Shenyang Pharmaceutical Company, and passed through Shenyang Pharmaceuticals. Professor Sun Qishi of the University of Medicinal Plants identified the dried roots of the genus Angelica sinensis (Oliv.) Diels.
2 Capillary electrophoresis conditions uncoated quartz capillary 5Om × 66.5cm, effective length 58cm (Hebei Yongnian Ruiqi Chromatography Devices Co., Ltd.); detection wavelength: 210nm; pressure injection: 5kPa, 5s; separation voltage: 24kV; capillary temperature: 20 °C; running buffer: 50 mmol/L borax and 10 mmol/L sodium deoxycholate, and containing 2% methanol (pH 9.6). The capillary column was washed successively with 1 mol/L sodium hydroxide solution, double distilled water and running buffer for 5, 5, 8 min before use, and rinsed with running buffer for 8 min after each electrophoresis. Under these conditions, the separation of ferulic acid and adenosine.
3 Preparation of the solution
3.1 The reference solution was accurately weighed 40 mg of ferulic acid reference substance and 20 mg of adenosine reference substance, dissolved in 60% methanol, and made up to 50.0 mL, and mixed for use.
3.2 The test solution was accurately weighed 2.0 g of dried Angelica powder at 60 ° C, ultrasonically extracted with 20 mL of 20% ethanol for 20 min, centrifuged at 3000 r/min for 10 min, and the supernatant was taken. The residue was extracted twice with the same method and combined. The supernatant solution was recovered under reduced pressure, and the solution was adjusted to a volume of 10 mL with 60% methanol, and then filtered through a 0.45 μm filter to obtain a test solution.
4 Linear relationship study Precision draw reference solution 0.25,0.5,1.0,1.5,2.0,4.0mL in a 10mL volumetric flask, add 60% methanol to the scale, mix and then separately injected, record the respective peak area, to Awei The peak area ratio of acid, adenosine and pentobarbital sodium is ordinate, and the concentration X (μg/mL) is linearly regression on the abscissa. The regression equations of ferulic acid and adenosine are:
Y=160.3X +0.1540 r=0.9992
Y=9256X +0.1989 r=0.9986
The linear range of the two is 20-320 μg / mL, 10 ~ 160 μg / mL.
5 precision test Take 1.5mL of the reference solution in a 10mL volumetric flask, add 60% methanol to the mark, mix and sample for 5 times, according to the peak area ratio of ferulic acid and internal standard, the RSD is 4.4%; The peak area ratio of adenosine to internal standard was calculated to be 3.2% RSD.
6 Repeatability test Weighed 5 parts of Angelica powder dried at 60 ° C, each serving 2.0 g, prepared the test solution according to the law, according to the "capillary electrophoresis conditions", the RSD calculated by ferulic acid content was 4.7%; The RSD was calculated to be 6.8% based on the content of adenosine.
7 Recovery test Precision draw reference solution 0.25mL and 1.25mL. Add to 2.0g of known content of Angelica sinensis (ferrifre acid: 0.269mg / g, adenosine: 0.202 mg / g), prepare the test solution according to the law, according to the "capillary electrophoresis conditions" to determine, The average recovery of Wei acid was between 96.8% and 97.4%, the average recovery of adenosine was between 93.2% and 95.0%, and the RSD was within 6.4%, n=3.
8 Determination of samples Take 10 samples of Angelica sinensis from different origins, and prepare the test solution for determination.
9 Discussion
9.1 Selection of extraction method The parallel test was used to compare the reflux method, decoction method and ultrasonic method. The results showed that the extraction rate of decoction method was low by the content of ferulic acid and adenosine. The extraction rate of the ultrasonic method is basically the same. Since the ultrasonic method is simple and time-saving, the ultrasonic method is used in this experiment.
9.2 Optimization of extraction process Ferulic acid is soluble in hot water, alcohol, ethyl acetate and ether. Adenosine is easily soluble in water, so it is extracted with ethanol solution containing a certain amount of water. The extraction process of Angelica sinensis was optimized by orthogonal design. The effects of extraction time, solvent dosage and solvent concentration on the extraction efficiency were investigated. The content of ferulic acid and adenosine was used as the index to evaluate the orthogonal test results. Preferably, the extraction process conditions are as follows: 60 mL of 60% ethanol, ultrasonic extraction for 20 min, a total of 3 times.
9.3 Selection of running buffer In the experiment, 10~100mmol/L borax buffer was firstly investigated and subjected to capillary zone electrophoresis. It was found that ferulic acid and adenosine could not be separated from adjacent peaks and then changed. 5～100mmol/L sodium deoxycholate was added to 50mmol/L borax and subjected to micellar electrokinetic capillary chromatography. The results were as follows: buffer 50mmol/L borax, 10mmol/L sodium deoxycholate and 2% methanol separation. The effect is good.
9.4 Effect of Buffer pH on Separation The effect of buffer pH between 3.5 and 11.0 on the resolution of ferulic acid, adenosine and adjacent peaks was investigated. When the pH is below 8.5, it is almost impossible to Separated from adjacent peaks, and pH between 9.2 and 9.8, ferulic acid, adenosine and adjacent peaks can reach baseline separation.
9.5 Selection of operating voltage When the operating voltage rises to 28 kV, the migration rate of adenosine and ferulic acid increases, but the resolution decreases. When the operating voltage drops to 20 kV, the migration time is extended, and the resolution is considered comprehensively. And the impact of migration time, this paper chooses the operating voltage is 24 kV.
9.6 Peak Purity Detection The peaks of ferulic acid and adenosine are identified by comparing the online UV absorption spectrum with the addition of the reference solution. At the same time, the peak purity check function in the HP ChemStation is used to select ferulic acid and gland. The purity of the chromatographic peak of the glycoside was evaluated. When the matching factor of the ultraviolet spectrum was greater than 950, the quantification was performed to avoid the error caused by the impure purity of the chromatographic peak.
references
1 ZHENGHu-zhan (Zheng Huzhan), DONG ze-hong (Dong Zehong), SHE Jing (Yu Jing). Modem Research and Application of Traditional Chinese Medicine (Modern Research and Application of Traditional Chinese Medicine). Beijing (Beijing): Xueyan Press (Xueyuan Press), 1998.206
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