CN102379934B - High efficiency liquid chromatography detection method for content of raddeanin A in rhizoma anemones raddeanae - Google Patents
High efficiency liquid chromatography detection method for content of raddeanin A in rhizoma anemones raddeanae Download PDFInfo
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- CN102379934B CN102379934B CN 201010275092 CN201010275092A CN102379934B CN 102379934 B CN102379934 B CN 102379934B CN 201010275092 CN201010275092 CN 201010275092 CN 201010275092 A CN201010275092 A CN 201010275092A CN 102379934 B CN102379934 B CN 102379934B
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Abstract
The invention relates to a high efficiency liquid chromatography detection method for the content of raddeanin A in rhizoma anemones raddeanae, belonging to a detection method of rhizoma anemones raddeanae. In the method, an evaporative light-scattering detector is adopted, an external standard two-point method is used for quantifying, octadecylsilane bonded silica is taken as a filler, a mixed solution of methanol or acetonitrile and water is taken as a flowing phase, and the flow speed of the flowing phase is 0.2-1 ml per minute; and a methanol ultrasonic treatment method is adopted for sample pretreatment. The method has the advantages of high sensitivity, stability, high reproducibility and capability of effectively controlling the qualities of the rhizoma anemones raddeanae and a preparation thereof.
Description
Invention field
The present invention relates to the biapiculate detection method of Chinese medicine, particularly the detection method of content of the pointed at both ends and preparation of Chinese medicine.
Background technology
The Radde Anemone Rhizome that has another name called pointed at both ends, for pointed at both ends be the dry rhizome of cohosh anemone raddeana Regel Anemone raddeanaRegel.Mainly be distributed in ground such as three provinces in the northeast of China and Shandong, Hebei, Shanxi, maximum with Jilin Province's output.Medicinal material is excavated many summers, removes residual stem and fibrous root, dries.This medicine head is stated from safe show " Bencao Pinhui Jingyao " of the Liu Wen of the Ming Dynasty.Effect with " dispelling rheumatism subduing inflammation ".As far back as the Qing Dynasty, Wang Xugao " deciding cancer looses " is once with treatment breast cancer pointed at both ends.Now clinical in wind-cold-dampness arthralgia, brothers' contraction, arthralgia, ulcer sore pain.Many and other medicines compatibility, folk prescription is not used as medicine, and is the primary raw material of famous Chinese patent drug " veins and artery relieving pill " and " Re-creating Bolus ".This product is rich in triterpene saponin, and wherein the triterpenoid saponin anemonin A is one of antineoplastic main effective constituent.
About the anemonin A detection method in the medicinal material pointed at both ends, be in the prior art and adopt high performance liquid chromatography to use the UV-detector detection to carry out assay, owing to be saponin component, on ultra-violet absorption spectrum, have only terminal the absorption, usually ultraviolet wavelength is chosen as about 206nm as measuring wavelength, therefore the chromatographic peak response is low, poor sensitivity, and flowing, it is more serious to reach the impurity interference mutually, thereby make the entire method reappearance bad, the measurement result error is very big.Item pointed at both ends of version Chinese Pharmacopoeia in 2010 down, the sample treatment that adopts the extraction of methyl alcohol Soxhlet, ether defatting, water-saturated n-butanol to extract repeatedly, and be aided with acetonitrile and 0.1% phosphoric acid solution gradient elution mode and be the chromatographic condition of the phase that flows, just be accomplished the content that high performance liquid chromatography-UV-detector detects anemonin A, about 10～15 hours of sample preparation time, because the test sample disposal route is loaded down with trivial details, easily cause the measurement result error, the mobile phase of gradient elution mode very easily makes chromatographic peak separate unstability of base line again.And adopt above-mentioned existing method to medicinal material anemonin A assay pointed at both ends in the patent medicine, almost can't carry out.
Summary of the invention
The invention provides the plain A content of a kind of Chinese medicine middle Radde Anemone Rhizome pointed at both ends high-efficiency liquid chromatography method for detecting, to solve the low problem of complicated operation, poor reproducibility, sensitivity that exists in the prior art, this method is applicable to the quality control of medicinal material pointed at both ends, preparation pointed at both ends.The technical scheme that the present invention takes is: adopt evaporative light-scattering detector, quantitative with the external standard two-point method, the employing octadecylsilane chemically bonded silica is filling agent, and as mobile phase, the flow velocity of the phase that flows is per minute 0.2～1ml with the mixed liquor of methyl alcohol or acetonitrile and water; The ultrasonic facture of methyl alcohol is adopted in sample pretreatment.
The inventive method may further comprise the steps:
1, chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; Flowing is 80～93: 20～7 acetonitrile-water or 82～95 mutually: 18～5 methanol-water; Evaporative light-scattering detector detects; The flow velocity of the phase that flows is per minute 0.2～1ml; The drift tube temperature that evaporative light-scattering detector detects: 60～130 ℃, air velocity: 1.0～3.1L/min.
2, the preparation of reference substance solution: it is an amount of that precision takes by weighing the plain A reference substance of Radde Anemone Rhizome, adds methyl alcohol and make the solution that every 1ml contains 0.4mg, namely;
3, the preparation of need testing solution: get Radde Anemone 2～8g, put in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims decide weight, and ultrasonic processing 0.5～1 hour is put coldly, claims to decide weight again, adds methyl alcohol and supplies the weight that subtracts mistake, shakes up filtration; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely;
4, determination method: accurate reference substance solution 5 μ l, the 15 μ l of drawing, need testing solution 10 μ l inject liquid chromatograph, measure, and calculate with the two points external standard method logarithmic equation, namely;
Beneficial effect: the present invention adopts high performance liquid chromatography-evaporative light-scattering detection method to measure the content of the plain A of Chinese medicine middle Radde Anemone Rhizome pointed at both ends, and it is very simple that method of operating is compared existing method.The methodology checking shows, this method good separation, and methodological studies such as method linearity, stability, reappearance, average recovery all meet the requirement of quantitative measurement.Can more effectively control the product quality of the pointed at both ends and preparation of Chinese medicine.About 0.5 hour of sample preparation time, improved detection efficiency greatly, reduced environmental pollution, saved the detection cost, method is easy, quick, accurate.
The present invention also is fit to the preparation as one of bulk drug or bulk drug pointed at both ends, comprises clinical or pharmaceutically acceptable common formulations such as tablet, granula.
Description of drawings
Fig. 1 is that the plain A evaporative light-scattering detector of reference substance Radde Anemone Rhizome detects collection of illustrative plates;
Fig. 2 is that medicinal material evaporative light-scattering detector pointed at both ends detects collection of illustrative plates;
Fig. 3 is that the qianlietong tablets evaporative light-scattering detector detects collection of illustrative plates.
Embodiment
Instrument: day island proper Tianjin LC-10Atvp high performance liquid chromatograph, Zhejiang University's 2010 binary channels chromatographic work stations; The U.S.'s safe ELSD2000ES evaporative light-scattering detector difficult to understand; The plum Teller. holder benefit AB265-S electronic balance.
Reagent: the plain A chemical reference substance of Radde Anemone Rhizome, assay usefulness, Nat'l Pharmaceutical ﹠ Biological Products Control Institute, purity meets the technical requirement that assay is used chemical reference substance on inspection, and content is 〉=98%; Methyl alcohol: chromatographically pure, water: self-control distilled water;
Sample: Chinese medicine medicinal material pointed at both ends.Qianlietong tablets, lot number: 20100225 (Guangzhou Zhongyi Medicine Industry Co., Ltd)
Embodiment 1
(1), chromatographic condition and system suitability test: think filling agent; Phase flows: acetonitrile-water 80: 20; Evaporative light-scattering detector detects; Flow velocity: 0.2ml/min; The drift tube temperature that evaporative light-scattering detector detects: 60 ℃, air velocity: 1.0L/min.
(2), the preparation of reference substance solution: it is an amount of that precision takes by weighing the plain A reference substance of Radde Anemone Rhizome, adds methyl alcohol and make the solution that every 1ml contains 0.4mg, namely;
(3), the preparation of need testing solution: get Radde Anemone 2g, put in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims decide weight, ultrasonic processing 0.5 hour is put coldly, claims to decide weight again, adds methyl alcohol and supplies the weight that subtracts mistake, shakes up filtration; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely;
(4), determination method: precision is drawn reference substance solution 5 μ l, 15 μ l, and need testing solution 10 μ l inject liquid chromatograph, measure, and calculate with the two points external standard method logarithmic equation, namely;
Embodiment 2
(1), chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; Phase flows: acetonitrile-water 86: 14; Evaporative light-scattering detector detects; Flow velocity: 0.6ml/min; The drift tube temperature that evaporative light-scattering detector detects: 90 ℃, air velocity: 2.0L/min.
(2), the preparation of reference substance solution: it is an amount of that precision takes by weighing the plain A reference substance of Radde Anemone Rhizome, adds methyl alcohol and make the solution that every 1ml contains 0.4mg, namely;
(3), the preparation of need testing solution: get Radde Anemone 5g, put in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims decide weight, ultrasonic processing 0.8 hour is put coldly, claims to decide weight again, adds methyl alcohol and supplies the weight that subtracts mistake, shakes up filtration; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely;
(4), determination method: precision is drawn reference substance solution 5 μ l, 15 μ l, and need testing solution 10 μ l inject liquid chromatograph, measure, and calculate with the two points external standard method logarithmic equation, namely;
Embodiment 3
(1), chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; Phase flows: acetonitrile-water 93: 7; Evaporative light-scattering detector detects; Flow velocity: 1ml/min; The drift tube temperature that evaporative light-scattering detector detects: 130 ℃, air velocity: 3.1L/min.
(2), the preparation of reference substance solution: it is an amount of that precision takes by weighing the plain A reference substance of Radde Anemone Rhizome, adds methyl alcohol and make the solution that every 1ml contains 0.4mg, namely;
(3), the preparation of need testing solution: get Radde Anemone 8g, put in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims decide weight, ultrasonic processing 1 hour is put coldly, claims to decide weight again, adds methyl alcohol and supplies the weight that subtracts mistake, shakes up filtration; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely;
(4), determination method: precision is drawn reference substance solution 5 μ l, 15 μ l, and need testing solution 10 μ l inject liquid chromatograph, measure, and calculate with the two points external standard method logarithmic equation, namely;
Embodiment 4
(1), chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; Phase flows: methanol-water 82: 18; Evaporative light-scattering detector detects; Flow velocity: 0.2ml/min; The drift tube temperature that evaporative light-scattering detector detects: 60 ℃, air velocity: 1.0L/min.
(2), the preparation of reference substance solution: it is an amount of that precision takes by weighing the plain A reference substance of Radde Anemone Rhizome, adds methyl alcohol and make the solution that every 1ml contains 0.4mg, namely;
(3), the preparation of need testing solution: get Radde Anemone 2g, put in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims decide weight, ultrasonic processing 0.5 hour is put coldly, claims to decide weight again, adds methyl alcohol and supplies the weight that subtracts mistake, shakes up filtration; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely;
(4), determination method: precision is drawn reference substance solution 5 μ l, 15 μ l, and need testing solution 10 μ l inject liquid chromatograph, measure, and calculate with the two points external standard method logarithmic equation, namely;
(1), chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; Phase flows: methanol-water 95: 5; Evaporative light-scattering detector detects; Flow velocity: 1ml/min; The drift tube temperature that evaporative light-scattering detector detects: 130 ℃, air velocity: 3.1L/min.
(2), the preparation of reference substance solution: it is an amount of that precision takes by weighing the plain A reference substance of Radde Anemone Rhizome, adds methyl alcohol and make the solution that every 1ml contains 0.4mg, namely;
(3), the preparation of need testing solution: get Radde Anemone 8g, put in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims decide weight, ultrasonic processing 1 hour is put coldly, claims to decide weight again, adds methyl alcohol and supplies the weight that subtracts mistake, shakes up filtration; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely;
(4), determination method: precision is drawn reference substance solution 5 μ l, 15 μ l, and need testing solution 10 μ l inject liquid chromatograph, measure, and calculate with the two points external standard method logarithmic equation, namely;
Embodiment 6
(1), chromatographic condition and system suitability test: chromatographic column: octadecylsilane chemically bonded silica chromatographic column Diamonsil C
18, 250 * 4.6mm; 5 μ; Phase flows: methanol-water 85: 15; Flow velocity: 0.8ml/min; The drift tube temperature that evaporative light-scattering detector detects: 80 ℃, air velocity: 2.0L/min; Column temperature: room temperature.
(2), that the preparation precision of reference substance solution takes by weighing the plain A reference substance of Radde Anemone Rhizome is an amount of, add methyl alcohol and make the solution that every 1ml contains 0.42mg, namely.
(3), the preparation of need testing solution gets Chinese medicine Radde Anemone 5g, crosses sieve No. three, accurately claims surely, puts in the tool plug conical flask the accurate methyl alcohol 50ml that adds, close plug claims to decide weight, and ultrasonic processing 30 minutes is put cold, claim again to decide weight, add methyl alcohol and supply the weight that subtracts mistake, shake up, filter; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely;
(4), determination method: accurate absorption reference substance solution each 5 μ l, 15 μ l, need testing solution 10 μ l inject liquid chromatograph respectively.
The plain A of Radde Anemone Rhizome and other component reach baseline separation under this condition, degree of separation R＞1.5, and theoretical cam curve is pressed the plain A of Radde Anemone Rhizome and is calculated greater than 6000, and the plain A retention time of Radde Anemone Rhizome is about 14 minutes.
Methanol-water and different mobile phase ratio and different flow rate of mobile phase such as acetonitrile-water have been investigated, 20～7) or methanol-water (82～95: 18～5) it is acetonitrile-water (80～93: mutually that the result flows, flow velocity is per 1 minute 0.2ml to 1ml, and the plain A of Radde Anemone Rhizome all can separate; To flow be mutually acetonitrile-water (80～93: 20～7) or methanol-water (82～95: 18～5), flow velocity is per 1 minute 0.2ml to 0.8ml, and effect is more excellent.
Linear relationship is investigated: it is an amount of that precision takes by weighing the plain A reference substance of Radde Anemone Rhizome, adds methyl alcohol and make the solution that contains the plain A0.42mg of Radde Anemone Rhizome among every 1ml, product solution in contrast.Precision is drawn 4,8,12,16,20 μ l respectively, inject high performance liquid chromatograph, measure by above-mentioned condition, natural logarithm with peak area is ordinate, the natural logarithm of the plain A sample size of Radde Anemone Rhizome is horizontal ordinate, linear regression gets regression equation and is: LnA=60243LnC+1.531, r=0.9993.Available external standard two-point method logarithmic equation is measured and is calculated.
The precision test: get same reference substance solution, continuous sample introduction 5 times, the record peak area calculates precision.
Stability test: accurate draw same need testing solution, at regular intervals sample introduction once, comparative measurements result's stability.
Reappearance test: get same lot number sample, carry out 5 parallel tests (being n=5), calculate relative standard deviation (RSD%), relatively reappearance.
Recovery test: the application of sample absorption method is adopted in this test, and precision takes by weighing 5 parts in the sample of known content, and the accurate plain A reference substance of a certain amount of Radde Anemone Rhizome that adds is measured by content determination respectively, calculates the recovery of the plain A of Radde Anemone Rhizome.
The result: experimental result shows that the detection of the plain A of Radde Anemone Rhizome is limited to 10ng, and the range of linearity of the plain A of Radde Anemone Rhizome is 1.68～8.4 μ g; Precision is 0.23%; The plain A of Radde Anemone Rhizome is stable in 12 hours, and RSD is 1.21%; This assay method has good reappearance, and RSD is 1.02%; The average recovery of the plain A of Radde Anemone Rhizome is that 99.01%, RSD is 1.55%; The plain A content of Radde Anemone Rhizome is 0.34% in the Chinese medicine medicinal material pointed at both ends after measured.
Embodiment 7: the qianlietong tablets assay
(1), chromatographic condition and system suitability test: chromatographic column: octadecylsilane chemically bonded silica chromatographic column Diamonsil C
18, 250 * 4.6mm; 5 μ; Phase flows: methanol-water 85: 15, the flow velocity of the phase that flows: 0.8ml/min; The drift tube temperature that evaporative light-scattering detector detects: 80 ℃, air velocity: 2.0L/min; Column temperature: room temperature.
(2), that the preparation precision of reference substance solution takes by weighing the plain A reference substance of Radde Anemone Rhizome is an amount of, add methyl alcohol and make the solution that every 1ml contains 0.42mg, namely.
(3), the preparation of need testing solution, get qianlietong tablets, porphyrize, mixing is got about 6g, accurate claim fixed, put in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims to decide weight, ultrasonic processing 0.5 hour, put coldly, claim again decide weight, add methyl alcohol and supply the weight that subtracts mistake, shake up filtration; Measure subsequent filtrate 10ml, water bath method, residue add methyl alcohol makes dissolving, is transferred to the 10ml measuring bottle and is diluted to scale, shakes up, with the miillpore filter filtration of 0.45 μ m, namely.
The preparation negative sample solution of negative sample solution is to take by weighing all the other auxiliary materials except Chinese medicine is pointed at both ends in the prescription ratio, make Chinese medicine negative sample pointed at both ends by method for making, get this negative sample again and do not contain the biapiculate negative sample solution of Chinese medicine by the preparation of above-mentioned " preparation of need testing solution " method.
(4), determination method: accurate absorption reference substance solution 5 μ l, 15 μ l, need testing solution reach and do not contain the biapiculate negative sample solution 10 μ l of Chinese medicine respectively, inject liquid chromatograph.The plain A of Radde Anemone Rhizome and other component reach baseline separation under this condition, degree of separation R＞1.5, theoretical cam curve are pressed the plain A of Radde Anemone Rhizome and are calculated greater than 6000, and the plain A retention time of Radde Anemone Rhizome is about 14 minutes, negative sample solution chromatogram does not have absorption peak in the relevant position, and is visible negative noiseless.
Testing result: the plain A content of Radde Anemone Rhizome is the 2.92mg/ sheet in the qianlietong tablets after measured.
Claims (1)
- A Chinese medicine pointed at both ends in the method that detects of the high performance liquid chromatography of the plain A content of Radde Anemone Rhizome, it is characterized in that adopting evaporative light-scattering detector, quantitative with the external standard two-point method, the employing octadecylsilane chemically bonded silica is filling agent, with the mixed liquor of methyl alcohol or acetonitrile and water as mobile phase, described flow is 80～93: 20～7 acetonitrile-water or 82～95 mutually: 18～5 methanol-water, the flow velocity of the phase that flows is per minute 0.2～1ml, the drift tube temperature that evaporative light-scattering detector detects: 60～130 ℃, air velocity: 1.0～3.1L/min; The ultrasonic facture of methyl alcohol is adopted in sample pretreatment.
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| CN109738545A (en) * | 2019-02-20 | 2019-05-10 | 江西医学高等专科学校 | Wine processs the processing procedure optimization method of medicinal material pointed at both ends |
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| 两头尖地上部分化学成分及其含量测定分析;刘大有;《长春中医学院学报》;20050331;第21卷(第1期);43-44 * |
| 刘大有.两头尖地上部分化学成分及其含量测定分析.《长春中医学院学报》.2005,第21卷(第1期), |
| 李顺意.高效液相色谱-质谱-质谱法快速鉴定中药竹节香附的皂苷.《湖北大学学报(自然科学版)》.2000,第22卷(第4期), |
| 高效液相色谱-质谱-质谱法快速鉴定中药竹节香附的皂苷;李顺意;《湖北大学学报(自然科学版)》;20001231;第22卷(第4期);382-384 * |
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