CN110412163A - An a kind of survey for ZHENNAONING JIAONANG comments detection method - Google Patents
An a kind of survey for ZHENNAONING JIAONANG comments detection method Download PDFInfo
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- CN110412163A CN110412163A CN201910713888.6A CN201910713888A CN110412163A CN 110412163 A CN110412163 A CN 110412163A CN 201910713888 A CN201910713888 A CN 201910713888A CN 110412163 A CN110412163 A CN 110412163A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention relates to drug tests, in particular to a kind of one for ZHENNAONING JIAONANG surveys comments detection method more.The method is surveyed comprising 3 kinds of tanshinone component HPLC mono- comments content assaying method more, uses Tanshinone I I in testAAs single calibration object, it is measured with external standard correction up factorization method.The present invention develops accurate and reliable, simple and easy measuring method, can be used as the detection means of ZHENNAONING JIAONANG tanshinone component.The use of the tanshinone IIA for easily preparing and having good stability is reference substance, while measuring 3 kinds of tanshinone components, method quickly, accurately, can more fully reflect the quality condition of preparation.Experiment shows that this method specificity is good.It is repeated good.Accuracy is good.Reproducibility precision is good.Test solution has good stability within 48 hours.One established surveys comments suitability of the method in Cryptotanshinone, salvia miltiorrhiza bge I composition detection good more.This method provides effective accurate quality control method for ZHENNAONING JIAONANG effective substance Quality Control.
Description
Technical field
The present invention relates to drug tests, in particular to a kind of one for ZHENNAONING JIAONANG surveys comments detection method more.
Background technique
In the prior art, ZHENNAONING JIAONANG record in 2015 the version Pharmacopoeia of the People's Republic of China one.Function master
It controls to control and disturbing caused headache, nausea and vomiting, blurring of vision, extremity numbness, tinnitus in ailment said due to cold or exposure；Angioneurotic headache, hypertension,
Artery sclerosis is shown in above-mentioned disease person.
ZHENNAONING JIAONANG is the Chinese materia medica preparation to be processed from strand by Chinese medicine, Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae, pig brain, Rhizoma Gastrodiae, asarum etc.,
The tanshinone compound contained in middle preparation has specific antiatherosclerosis, reduction myocardial oxygen consumption and antibacterial, resists
The multiple biological activities such as oxidation, anti-inflammatory, are the primary efficacy ingredients of ZHENNAONING JIAONANG.Tanshinone compound ingredient mainly wraps
Include Tanshinone IIA, Cryptotanshinone and tanshinone Ι.The version Pharmacopoeia of the People's Republic of China one peaceful glue of town brain recorded in 2015
The content assaying method that tanshinone compound has been formulated in capsule quality standard, using Syrups by HPLC Tanshinone IIAOne
The content of a ingredient, this Testing index are not enough to reflect comprehensively the reliability and stability of ZHENNAONING JIAONANG.In conjunction with town
Brain Yiganning capsule effective substance data should add both effectiveness ingredient Cryptotanshinone relevant to ZHENNAONING JIAONANG indication
With tanshinone Ι as quality evaluation index.And the former mark of detection time comparison of 3 kinds of ingredients is separately measured using traditional detection method
Quasi- detection time takes a long time；And the stability of Cryptotanshinone and tanshinone Ι reference substance solution is poor, is not easy to store up for a long time
It deposits, and the expensive testing cost that will cause improves；The reference substance of especially tanshinone Ι is not easy to buy, and factors above all can be right
Tanshinone detection causes to perplex in ZHENNAONING JIAONANG.
Included to survey using one in Chinese Pharmacopoeia comments method to carry out quality evaluation, the key technology of this method to complex component more
It is characterized in that how passing through the suitably a certain typical component of experimental selection as internal reference object, and establishes corresponding test method, with
Realize that separating degree and detection limit reach analysis method requirement between internal reference object and other components to be measured；Being determined by experimental study should
Correction factor of each component to be measured of method relative to internal reference object measures containing for component to be measured to realize through relative correction factor
Amount.Properly whether ZHENNAONING JIAONANG is compound preparation, and constituent is complicated, one is surveyed, method is commented to be used for wherein more, and how
Carrying out wherein analysis of effective component becomes a problem, and the present invention has carried out experimental study to this, select pharmacological activity clear and
Comparision contents Tanshinone II abundantA, Cryptotanshinone and tanshinone Ι constituents are measured.
Summary of the invention
In view of this, the present invention provides tanshinone compound measuring method in a kind of ZHENNAONING JIAONANG.The detection method
By to an index components (Tanshinone IIA) detected, reach to the multiple ingredients of same type (Cryptotanshinone, tanshinone Ι)
While measure, method practicability is high, easy to operate, save the cost, realizes while detecting 3 kinds of tanshinones in ZHENNAONING JIAONANG
The detection method of constituents.
The technical scheme is that a kind of one for ZHENNAONING JIAONANG surveys and comments detection method more, its step are as follows:
1) test fluid is made in ZHENNAONING JIAONANG.
2) with Tanshinone II in high performance liquid chromatograph measurement test fluidAContent.
3) content of Cryptotanshinone and tanshinone Ι in sample is calculated using correction factor.
Wherein, ZHENNAONING JIAONANG 1) is prepared into test fluid.Method is as follows: it takes 20 intragranular of ZHENNAONING JIAONANG tolerant, mixes,
About 1.5-3.0g is taken, it is accurately weighed, it sets in stuffed conical flask, methanol 30-60ml is added in precision, and weighed weight is ultrasonically treated (function
Rate 250W, frequency 42kHz) 10-40 minutes, it lets cool, in weighed weight, the weight of less loss is supplied with methanol, is shaken up, filter, take
Subsequent filtrate to get.
Preferably, the method is as follows: it takes 20 intragranular of ZHENNAONING JIAONANG tolerant, mixes, take about 2.4g, it is accurately weighed, set tool plug
In conical flask, 50ml, weighed weight is added in precision, and ultrasonic treatment (power 250W, frequency 42kHz) 30 minutes is let cool, weighed
Weight is supplied the weight of less loss with methanol, is shaken up, filtration, take subsequent filtrate to get.
Wherein, 2) with Tanshinone II in high performance liquid chromatograph measurement test fluidAContent.Method is as follows, with height
Effect liquid phase chromatogram instrument measures Tanshinone II in solution to be measured and reference substance solution respectivelyAContent, using one point external standard method or
Working curve method calculates Tanshinone IIAContent, the Tanshinone II that wherein one point external standard method is 20 μ g/ml using concentrationAAs control
Product, working curve method use concentration range in the Tanshinone II of at least five gradients of 9-230 μ g/mlAAs reference substance solution.
Instrument: high performance liquid chromatograph.
Chromatographic column: octadecylsilane chemically bonded silica is filler.
Mobile phase: acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase.
Flow velocity: 0.8-1.1ml/min.
Column temperature: 25-35 DEG C.
Sample volume: 1-25 μ l.
Detector wavelength: 268-272nm.
The preferred detection method of step 2 are as follows:
Chromatographic column: Cosmosil C18-MS- II (5 μm, 4.6 × 250mm).
Mobile phase: acetonitrile: 0.02% phosphoric acid solution (61:39).
Flow velocity: 0.8ml/min.
Column temperature: 30 DEG C.
Sample volume: 5 μ l.
Detector wavelength: 270nm.
Wherein, step 3) calculates the content of Cryptotanshinone and tanshinone Ι in sample using correction factor.
Qualitative fashion: Tanshinone IIARelative retention time relative to Cryptotanshinone and Tanshinone I is respectively 0.50-
0.60 and 0.55-0.66.The retention time calculation formula of test substance are as follows:
Wherein, tRxAnd tRsRespectively represent component to be measured and Tanshinone II in test sampleARetention time, Rx/sRepresent opposite retain
Time.
Quantitative manner: Tanshinone IIARelative correction factor relative to Cryptotanshinone and Tanshinone I is respectively 1.10-
1.20 and 1.18-1.38.The concentration calculation formula of test analyte are as follows:
Wherein, AxAnd AsRespectively represent component to be measured and Tanshinone II in test sampleAPeak area, CxAnd CsIt respectively represents for examination
Component to be measured and Tanshinone II in productAConcentration, fx/sRepresent Tanshinone II in test sampleATo the correction factor of ingredient to be measured.
Relative retention time and relative correction factor are preferred:
It is qualitative: with Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows: 0.54 He
0.58。
It is quantitative: with Tanshinone IIAFor single subject matter, the relative correction factor of Cryptotanshinone and Tanshinone I is respectively as follows:
1.14 with 1.30.
Technical term of the present invention is explained:
One surveys comment more: one kind of substitute reference substance content assaying method utilizes effective component of chinese medicine intrinsic function relationship and ratio
Example relationship, only one ingredient (the reference substance person of can be obtained) of measurement realizes the same of multiple ingredients (reference substance be difficult to obtain or difficult supply)
Pacing is fixed.
HPLC: high performance liquid chromatograph.
The beneficial effects of the present invention are: 1, currently, there has been no one for ZHENNAONING JIAONANG tanshinone component to survey comment more
Measuring method, the present invention develop accurate and reliable, simple and easy measuring method, can be used as ZHENNAONING JIAONANG tanshinone component
Detection means.The use of the tanshinone IIA for easily preparing and having good stability is reference substance, while measuring 3 kinds of tanshinone components, side
Method quickly, accurately, can more fully reflect the quality condition of preparation.2, mobile phase with -0.02% phosphoric acid solution of acetonitrile (61:
39) each chromatographic peak is kept completely separate under isocratic condition, and each ingredient chromatographic peak purity is high to be measured, analysis time is relatively
Short, separating effect is significant.3, experiment shows that this method specificity is good.It is repeated good.Accuracy is good.Reproducibility is accurate
Degree is good.Test solution has good stability within 48 hours.One established surveys comments method in Cryptotanshinone, salvia miltiorrhiza bge I ingredient more
Suitability in detection is good.
Embodiments of the present invention are described in further detail below in conjunction with embodiment and attached drawing.
Detailed description of the invention
Fig. 1 red rooted salvia test map.
The ZHENNAONING JIAONANG Fig. 2 feminine gender sample test map.
The ZHENNAONING JIAONANG Fig. 3 test map.
Fig. 4 red rooted salvia test map.
The ZHENNAONING JIAONANG Fig. 5 test map.
The ZHENNAONING JIAONANG Fig. 6-1. test map (mobile phase 54:46, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-2. test map (mobile phase 61:39, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-3. test map (mobile phase 56:44, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-4. test map (mobile phase 57:43, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-5. test map (mobile phase 58:42, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-6. test map (mobile phase 59:41, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-7. test map (mobile phase 60:40, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-8. test map (mobile phase 55:45, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-9. test map (mobile phase 62:38, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-10. test map (mobile phase 63:37, detection device Dionex Ultimate3000).
The ZHENNAONING JIAONANG Fig. 6-11. test map (mobile phase 61:39, detection device waters).
Fig. 7 Cryptotanshinone reference substance ultraviolet spectra.
Fig. 8 salvia miltiorrhiza bge I reference substance ultraviolet spectra.
Fig. 9 tanshinone IIA reference substance ultraviolet spectra.
Figure 10 Cosmosil C18-MS- II (5 μm, 4.6 × 250 mm) chromatogram.
Figure 11 Capcell PAK C18 (5 μm, 4.6 × 250 mm) chromatogram.
Figure 12 .Waters Symmetry C18 (5 μm, 4.6 × 250 mm) chromatogram.
Figure 13 Phenomenex Gemini C18 (5 μm, 4.6 × 250 mm) chromatogram.
Figure 14 YMC-Pack ODS-A C18 (5 μm, 4.6 × 250 mm) chromatogram.
Figure 15 Thermo BDS HYPERSIL C18 (5 μm, 4.6 × 250 mm) chromatogram.
3000 high performance liquid chromatograph of Figure 16 Dionex Ultimate.
Figure 17 .Agilent 1200(DAD) liquid chromatograph chromatogram.
Figure 18 .Waters liquid chromatograph chromatogram.
Figure 19 Cryptotanshinone, salvia miltiorrhiza bge I and tanshinone IIA reference substance HPLC map (270 nm).
The ZHENNAONING JIAONANG Figure 20 sample HPLC map (270 nm).
Figure 21 negative sample HPLC map (270 nm).
Figure 22 Radix Salviae Miltiorrhizae control medicinal material HPLC map (270 nm).
Figure 23 solvent HPLC map (270 nm).
Figure 24 Cryptotanshinone reference substance detection limit.
Figure 25 Cryptotanshinone reference substance quantitative limit.
Figure 26 Tanshinone I reference substance detection limit.
Figure 27 Tanshinone I reference substance quantitative limit.
Figure 28 tanshinone IIA reference substance detection limit.
Figure 29 tanshinone IIA reference substance quantitative limit.
Figure 30 Cryptotanshinone reference substance canonical plotting.
Figure 31 Tanshinone I reference substance canonical plotting.
Figure 32 tanshinone IIA reference substance canonical plotting.
Specific embodiment
Embodiment 1
An a kind of survey for ZHENNAONING JIAONANG comments detection method, and its step are as follows:
1, ZHENNAONING JIAONANG is prepared into test fluid
It takes 20 intragranular of ZHENNAONING JIAONANG tolerant, mixes, take about 2.4g, it is accurately weighed, it sets in stuffed conical flask, methanol is added in precision
50ml, weighed weight, ultrasonic treatment (power 250W, frequency 42kHz) 30 minutes let cool, then weighed weight, are supplied and subtracted with methanol
The weight of mistake, shakes up, filtration, take subsequent filtrate to get.
2, with total-tanshinone content in high performance liquid chromatograph measurement test fluid
Take Tanshinone IIAAppropriate reference substance, it is accurately weighed, it sets in brown measuring bottle, adds methanol that the solution that every 1ml contains 20 μ g is made,
To obtain the final product.
Sample measurement, the peak face of target component in sample solution and reference substance solution is measured using high performance liquid chromatograph
Product.
Chromatographic condition
Chromatographic column: Cosmosil C18-MS- II (4.6mm × 250mm, 5 μm)；Mobile phase: with acetonitrile: 0.02% phosphoric acid solution
(61:39) is mobile phase；
Column temperature: 30 DEG C；
Flow velocity: 0.8ml/min；
Detection wavelength is 270nm；
Sample volume: 5 μ l；
3, Tanshinone II in sample is calculated using relative correction factorA, Cryptotanshinone, Tanshinone I content:
With Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows:
0.54 and 0.58；
With Tanshinone IIAFor single subject matter, the relative correction factor of Cryptotanshinone and Tanshinone I is respectively as follows: 1.14 and 1.30.
Qualitative fashion: the retention time calculation formula of test substance are as follows:
Wherein, tRxAnd tRsRespectively represent component to be measured and Tanshinone II in test sampleARetention time, Rx/sRepresent opposite retain
Time；
Quantitative manner: the concentration calculation formula of test substance are as follows:
Wherein, AxAnd AsRespectively represent component to be measured and Tanshinone II in test sampleAPeak area, CxAnd CsIt respectively represents for examination
Component to be measured and Tanshinone II in productAConcentration, fx/sRepresent Tanshinone II in test sampleATo the correction factor of ingredient to be measured.
By measurement, the content of tanshinone is 0.309mg in the determinand-1。
Embodiment 2
An a kind of survey for ZHENNAONING JIAONANG comments detection method, and its step are as follows:
1, ZHENNAONING JIAONANG is prepared into test fluid
It takes 20 intragranular of ZHENNAONING JIAONANG tolerant, mixes, take about 2.4g, it is accurately weighed, it sets in stuffed conical flask, methanol is added in precision
50ml, weighed weight, ultrasonic treatment (power 250W, frequency 42kHz) 30 minutes let cool, then weighed weight, are supplied and subtracted with methanol
The weight of mistake, shakes up, filtration, take subsequent filtrate to get.
2, with total-tanshinone content in high performance liquid chromatograph measurement test fluid
Take Tanshinone IIAAppropriate reference substance, it is accurately weighed, it sets in brown measuring bottle, adds methanol that the solution that every 1ml contains 20 μ g is made,
To obtain the final product.
Sample measurement, the peak face of target component in sample solution and reference substance solution is measured using high performance liquid chromatograph
Product.
Chromatographic condition
Chromatographic column: Cosmosil C18-MS- II (4.6mm × 250mm, 5 μm)；
Mobile phase: with acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase；
Column temperature: 25 DEG C；
Flow velocity: 0.8ml/min；
Detection wavelength is 268nm；
Sample volume: 5 μ l；3, Tanshinone II in sample is calculated using relative correction factorA, Cryptotanshinone, Tanshinone I content:
With Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows:
0.54 and 0.58；
With Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows: 1.08 and 1.25.
Qualitative fashion: the retention time calculation formula of test substance are as follows:
Wherein, tRxAnd tRsRespectively represent component to be measured and Tanshinone II in test sampleARetention time, Rx/sRepresent opposite retain
Time；
Quantitative manner: the concentration calculation formula of test substance are as follows:
Wherein, AxAnd AsRespectively represent component to be measured and Tanshinone II in test sampleAPeak area, CxAnd CsIt respectively represents for examination
Component to be measured and Tanshinone II in productAConcentration, fx/sRepresent Tanshinone II in test sampleATo the correction factor of ingredient to be measured.
By measurement, the content of tanshinone is 0.314mg in the determinand-1。
Embodiment 3
An a kind of survey for ZHENNAONING JIAONANG comments detection method, and its step are as follows:
1, ZHENNAONING JIAONANG is prepared into test fluid
It takes 20 intragranular of ZHENNAONING JIAONANG tolerant, mixes, take about 2.4g, it is accurately weighed, it sets in stuffed conical flask, methanol is added in precision
50ml, weighed weight, ultrasonic treatment (power 250W, frequency 42kHz) 30 minutes let cool, then weighed weight, are supplied and subtracted with methanol
The weight of mistake, shakes up, filtration, take subsequent filtrate to get.
2, with total-tanshinone content in high performance liquid chromatograph measurement test fluid
Take Tanshinone IIAAppropriate reference substance, it is accurately weighed, it sets in brown measuring bottle, adds methanol that the solution that every 1ml contains 20 μ g is made,
To obtain the final product.
Sample measurement, the peak face of target component in sample solution and reference substance solution is measured using high performance liquid chromatograph
Product.
Chromatographic condition
Chromatographic column: Cosmosil C18-MS- II (4.6mm × 250mm, 5 μm)；
Mobile phase: with acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase；
Column temperature: 35 DEG C；
Flow velocity: 1.0ml/min；
Detection wavelength is 272nm；
Sample volume: 5 μ l；
3, Tanshinone II in sample is calculated using relative correction factorA, Cryptotanshinone, Tanshinone I content:
With Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows:
0.54 and 0.58；
With Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows: 1.17 and 1.31.
Qualitative fashion: the retention time calculation formula of test substance are as follows:
Wherein, tRxAnd tRsRespectively represent component to be measured and Tanshinone II in test sampleARetention time, Rx/sRepresent opposite retain
Time；
Quantitative manner: the concentration calculation formula of test substance are as follows:
Wherein, AxAnd AsRespectively represent component to be measured and Tanshinone II in test sampleAPeak area, CxAnd CsIt respectively represents for examination
Component to be measured and Tanshinone II in productAConcentration, fx/sRepresent Tanshinone II in test sampleATo the correction factor of ingredient to be measured.
By measurement, the content of tanshinone is 0.313mg in the determinand-1。
Embodiment 4
An a kind of survey for ZHENNAONING JIAONANG comments detection method, and its step are as follows:
1, ZHENNAONING JIAONANG is prepared into test fluid
It takes 20 intragranular of ZHENNAONING JIAONANG tolerant, mixes, take about 2.4g, it is accurately weighed, it sets in stuffed conical flask, methanol is added in precision
50ml, weighed weight, ultrasonic treatment (power 250W, frequency 42kHz) 30 minutes let cool, then weighed weight, are supplied and subtracted with methanol
The weight of mistake, shakes up, filtration, take subsequent filtrate to get.
2, with total-tanshinone content in high performance liquid chromatograph measurement test fluid
Take Tanshinone IIAAppropriate reference substance, it is accurately weighed, it sets in brown measuring bottle, adds methanol that the solution that every 1ml contains 20 μ g is made,
To obtain the final product.
Sample measurement, the peak face of target component in sample solution and reference substance solution is measured using high performance liquid chromatograph
Product.
Chromatographic condition
Chromatographic column: YCM-Pack ODS-A(4.6mm × 250mm, 5 μm)；
Mobile phase: with acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase；
Column temperature: 30 DEG C；
Flow velocity: 0.8ml/min；
Detection wavelength is 270nm；
Sample volume: 10 μ l；
3, Tanshinone II in sample is calculated using relative correction factorA, Cryptotanshinone, Tanshinone I content:
Relative correction factor and relative retention time
With Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows:
0.54 and 0.58；
With Tanshinone IIAFor single subject matter, the relative retention time of Cryptotanshinone and Tanshinone I is respectively as follows: 1.13 and 1.26.
Qualitative fashion: the retention time calculation formula of test substance are as follows:
Wherein, tRxAnd tRsRespectively represent component to be measured and Tanshinone II in test sampleARetention time, Rx/sRepresent opposite retain
Time；
Quantitative manner: the concentration calculation formula of test substance are as follows:
Wherein, AxAnd AsRespectively represent component to be measured and Tanshinone II in test sampleAPeak area, CxAnd CsIt respectively represents for examination
Component to be measured and Tanshinone II in productAConcentration, fx/sRepresent Tanshinone II in test sampleATo the correction factor of ingredient to be measured.
By measurement, the content of tanshinone is 0.312mg in the determinand-1。
When commenting index to carry out assay using a survey, since analyte content is obtained by relative correction factor calculating
, therefore the stability of the determination of relative correction factor and relative correction factor influences whether the accurate of final assay result
Property.ZHENNAONING JIAONANG complicated component, the separating effect of impurity and target component is the pass for influencing accuracy in detection in ingredient to be measured
Key factor, the present inventor have conducted extensive research screening to it, including tanshinone imitates ingredient enrichment method, constituent analysis chromatography
Condition and correction factor determination and verifying.Different enrichment modes and analysis chromatographic condition combination, target component obtained and
Impurity composition situation is all different.With reference first to one red rooted salvia tanshinone content determination item of " Chinese Pharmacopoeia " version in 2015
Lower chromatographic condition is groped, and using acetonitrile, 0.02% phosphoric acid solution as mobile phase, adjusts 6 different gradient proportions, as a result hidden pellet
Ginseng ketone and salvia miltiorrhiza bge I are unable to reach are kept completely separate always, and chromatogram is as shown in Figs. 1-5, the impurity composition of ZHENNAONING JIAONANG finished product
It is different from red rooted salvia, there is the undesirable (separation of impurity peaks separation at the retention time appearance of Tanshinone I using the method
Spend < 1.5), therefore be changed to be groped using isocratic elution." one surveys comment 4 in method measurement Radix Salviae Miltiorrhizae to successive reference literature more
Kind tanshinone component " and " HPLC method measures tanshinone in ZHENNAONING JIAONANG ", with acetonitrile, 0.2% phosphoric acid solution or methanol, water
(86:14) is that mobile phase is detected, and carries out mobile phase ratio adjustment, and salvia miltiorrhiza bge I is unable to reach always with impurity to be divided completely
From.Carry out isocratic elution by largely flowing phase composition and ratio screening and grope, is with separating degree, tailing factor and detection time
Preliminary screening index, initial option uses -0.02% phosphoric acid solution of acetonitrile to carry out isocratic elution for mobile phase, and is with 1% acetonitrile
Amplification adjusts mobile phase ratio, and -0.02% phosphoric acid of acetonitrile (50:50) → (65:35), as shown in Fig. 6, -0.02% phosphoric acid of acetonitrile is molten
Each chromatographic peak is kept completely separate under the conditions of liquid (61:39), each ingredient chromatographic peak purity is high to be measured, and analysis time is relatively short.
Final confirmation -0.02% phosphoric acid solution of acetonitrile (61:39) is mobile phase, on this basis, carries out correction factor confirmation research, in detail
See summary of the invention, sees Fig. 1 red rooted salvia test map.1: Fig. 1 detection method of note is version " People's Republic of China's medicine in 2015
Allusion quotation " Radix Salviae Miltiorrhizae quality standard, sample sample weighting amount is 0.3092g.Chromatographic condition are as follows: octadecylsilane chemically bonded silica is filler；
Using acetonitrile as mobile phase A, using 0.02% phosphoric acid solution as Mobile phase B, by 0~6min 61:39,6~20min, 61 → 90:39 →
10,20~20.5min 90 → 61:10 → 39,20.5~25min 61:39；Column temperature is 20 DEG C；Detection wavelength is 270nm, reason
Tanshinone II is pressed by plate numberAPeak is calculated as 81 974.See the ZHENNAONING JIAONANG Fig. 2 feminine gender sample test map.The note detection side 2: Fig. 2
Method is version Pharmacopoeia of the People's Republic of China Radix Salviae Miltiorrhizae quality standard in 2015, and sample sample weighting amount is 2.4057g.Chromatographic condition are as follows:
Octadecylsilane chemically bonded silica is filler；Using acetonitrile as mobile phase A, using 0.02% phosphoric acid solution as Mobile phase B, by 0~
6min 61:39,6~20min 61 → 90:39 → 10,20~20.5min 90 → 61:10 → 39,20.5~25min 61:
39；Column temperature is 20 DEG C；Detection wavelength is 270nm.See the ZHENNAONING JIAONANG Fig. 3 test map.3: Fig. 3 detection method of note is 2015
Year version Pharmacopoeia of the People's Republic of China Radix Salviae Miltiorrhizae quality standard, sample sample weighting amount are 2.4066g.Chromatographic condition are as follows: octadecyl
Silane group silica gel is filler；Using acetonitrile as mobile phase A, using 0.02% phosphoric acid solution as Mobile phase B, by 0~6min 61:
39,6~20min 61 → 90:39 → 10,20~20.5min 90 → 61:10 → 39,20.5~25min 61:39；Column temperature is
20℃；Detection wavelength is 270nm, and number of theoretical plate presses Tanshinone IIAPeak is calculated as 81 354.See Fig. 4 red rooted salvia detection figure
Spectrum.4: Fig. 4 detection method of note is version Pharmacopoeia of the People's Republic of China Radix Salviae Miltiorrhizae quality standard in 2015 after optimization, sample sample weighting amount
For 0.3092g.Chromatographic condition are as follows: octadecylsilane chemically bonded silica is filler；Using acetonitrile as mobile phase A, with 0.02% phosphoric acid
Solution is Mobile phase B, by 0~6min 61:39,90 → 61:10 of 6~28min 61 → 90:39 → 10,28~28.5min →
39,28.5~33min 61:39；Column temperature is 20 DEG C；Detection wavelength is 270nm, and number of theoretical plate presses Tanshinone IIAPeak is calculated as 65
146.See the ZHENNAONING JIAONANG Fig. 5 test map.5: Fig. 5 detection method of note is version " People's Republic of China's medicine in 2015 after optimization
Allusion quotation " Radix Salviae Miltiorrhizae quality standard.Sample sample weighting amount is 2.4066g.Chromatographic condition are as follows: octadecylsilane chemically bonded silica is filler；
Using acetonitrile as mobile phase A, using 0.02% phosphoric acid solution as Mobile phase B, by 0~6min 61:39,6~28min, 61 → 90:39 →
10,28~28.5min 90 → 61:10 → 39,28.5~33min 61:39；Column temperature is 20 DEG C；Detection wavelength is 270nm, reason
Tanshinone II is pressed by plate numberAPeak is calculated as 6 5291.See Fig. 6-1-ZHENNAONING JIAONANG-6-10 test map.Infuse 6-1-6-
10: Fig. 6-1-6-10 uses-0.02% phosphoric acid solution of acetonitrile for mobile phase isocratic elution, adjusts mobile phase by amplification of 1% acetonitrile
Ratio, -0.02% phosphoric acid of acetonitrile (54:44) → (65:35)；Column temperature is 30 DEG C；Detection wavelength is 270nm, and number of theoretical plate presses Radix Salviae Miltiorrhizae
II peak A of ketone is calculated as 2 8427.See the ZHENNAONING JIAONANG Fig. 6-11. test map.Infusing 6-11: Fig. 6-11 detection method is that town brain is peaceful
Tanshinone component one is surveyed and comments method more in capsule, and sample sample weighting amount is 2.4066g.Chromatographic condition are as follows: octadecylsilane key
Conjunction silica gel is filler；With acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase；Column temperature is 30 DEG C；Detection wavelength is
270nm, number of theoretical plate press Tanshinone IIAPeak is calculated as 2 8313.
Detection method of the invention is obtained by screening, and screening process is as follows:
1. the selection of chromatographic condition
The selection of 1 .1 Detection wavelength
Take Cryptotanshinone, salvia miltiorrhiza bge I and Tanshinone IIAReference substance methanol solution measures its ultra-violet absorption spectrum (see Fig. 7-respectively
Fig. 9).According to its maximum absorption wavelength, selecting this method Detection wavelength is 270nm.
The comparison of mobile phase
Groped referring to chromatographic condition under one red rooted salvia tanshinone content determination item of " Chinese Pharmacopoeia " version in 2015, with
Acetonitrile, 0.02% phosphoric acid solution are mobile phase, adjust 6 different gradient proportions, as a result Cryptotanshinone and salvia miltiorrhiza bge I always can not
Reach and be kept completely separate or purity does not reach requirement, therefore is changed to be groped using isocratic elution.
It uses -0.02% phosphoric acid solution of acetonitrile for mobile phase isocratic elution, adjusts mobile phase ratio by amplification of 1% acetonitrile,
- 0.02% phosphoric acid of acetonitrile (50:50) → (65:35), as a result each chromatographic peak obtains under the conditions of -0.02% phosphoric acid solution of acetonitrile (61:39)
To being kept completely separate, each ingredient chromatographic peak to be measured is with high purity, and analysis time is relatively short.With this mobile phase ratio in 6 root chromatogram columns
On tested, obtain satisfied separating effect, therefore be included in text.
The selection of Extraction solvent
According to the solubility property of three kinds of tanshinones, and related document is referred to, is handled with the following method: taking this product 30
Content (ZHENNAONING JIAONANG, lot number 160802) mixes, takes about 2.4g, accurately weighed, and methanol, ethyl alcohol, acetone three is respectively adopted
Kind solvent supersonic handles sample, and three kinds of tanshinone contents are shown in Table 1.
The result shows that: using the test sample that the test sample content of methanol ultrasound preparation is higher than ethyl alcohol, prepared by acetone.Therefore,
Selecting methanol is to extract solvent and be included in text.
The selection of 1.4 extracting modes and time
Methanol eddy, ultrasonic two kinds of extracting methods is respectively adopted, different extraction times handle sample (ZHENNAONING JIAONANG, lot number
160802), Cryptotanshinone, salvia miltiorrhiza bge I, Tanshinone II in comparative sampleAContent, the results are shown in Table 2.
The result shows that: use ultrasonic extraction, different ultrasonic time sample size no significant differences.It is mentioned using reflow method
It takes, return time is longer, and assay value is lower, shows tanshinone component to thermally labile.In view of ultrasonic extraction content compared with
It is high and stable, it can extract completely within ultrasonic treatment 30 minutes, therefore select to be ultrasonically treated 30 minutes as extracting method, side by side
Enter text.
1.5 chromatographic column serviceability tests
Same test solution is taken, using 3000 high performance liquid chromatograph of Dionex Ultimate, is drafted respectively by text
Method sample introduction investigates Cryptotanshinone, salvia miltiorrhiza bge I and Tanshinone II using 6 root chromatogram columnsANumber of theoretical plate situation.Investigate result
It is shown in Table 3-1,3-2, chromatogram is shown in Figure 10-Figure 15.
The result shows that Cryptotanshinone, salvia miltiorrhiza bge I and Tanshinone II in 6 kinds of chromatographic columnsAThe theoretical cam curve at peak is all larger than
14000.6 root chromatogram columns reach the requirement of method system applicability, this method good tolerance.
1.6 different instruments are investigated
Take same lot number test solution, Cosmosil C18- MS- II (5 μm, 4.6 × 250 mm) chromatographic column uses
3000 high performance liquid chromatograph of Dionex Ultimate, Agilent high performance liquid chromatograph and Waters high performance liquid chromatography
Instrument, the method sample introduction drafted respectively by text investigate Cryptotanshinone, salvia miltiorrhiza bge I and Tanshinone IIANumber of theoretical plate situation.
Investigation the results are shown in Table 4-1,4-2,4-3, and chromatogram is shown in Figure 16-Figure 18.
The result shows that Cryptotanshinone, salvia miltiorrhiza bge I and Tanshinone IIAPeak reaches the requirement of method system applicability.
Conclusion
The preparation method of the test solution of optimization is established according to result above:
It takes 20 intragranular of this product tolerant, mixes, take about 2.4g, it is accurately weighed, it sets in stuffed conical flask, methanol 50ml is added in precision,
Weighed weight, ultrasonic treatment (power 250W, frequency 42kHz) 30 minutes, lets cool, then weighed weight, supplies less loss with methanol
Weight shakes up, filtration, take subsequent filtrate to get.
Chromatographic condition:
Using octadecylsilane chemically bonded silica as filler (Cosmosil C18-MS- II, 4.6mm × 25mm, 5 μm or efficiency phase
When chromatographic column).With acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase；Column temperature: 30 DEG C；Flow velocity: 0.8ml/min；Detection
Wavelength is 270nm.Number of theoretical plate is calculated by tanshinone IIA peak should be not less than 6000.
Methodology validation
The test of 2.1 specificities
The ZHENNAONING JIAONANG ZHENNAONING JIAONANG content about 2.4g(is taken, lot number: 160101), accurately weighed, the confession drafted by text
Sample solution processed, sample introduction measure test sample solution preparation method in accordance with the law.
The negative control sample of scarce Radix Salviae Miltiorrhizae is taken (according to the ratio in prescription, to prepare) 2.4g by sample preparation method, it is quasi- by text
The negative control solution of Radix Salviae Miltiorrhizae, sample introduction measurement is made in fixed sample solution preparation method in accordance with the law.
The results show that in Radix Salviae Miltiorrhizae negative control chromatogram, with Cryptotanshinone, salvia miltiorrhiza bge I and Tanshinone IIAReference substance color
The corresponding retention time of spectral peak nearby occurs without chromatographic peak, is detailed in Figure 19-Figure 23.Illustrate that this method specificity is good.
Detection limit (LOD) and quantitative limit (LOQ)
Precision weighs Cryptotanshinone, salvia miltiorrhiza bge I, Tanshinone IIAAppropriate reference substance adds methanol that every 1 ml is made containing Cryptotanshinone 6
μ g, 2 μ g of salvia miltiorrhiza bge I, Tanshinone IIAThe mixed solution of 8 μ g, as reference substance solution.Reference substance solution is gradually diluted, by true
Fixed chromatographic condition measurement obtains detection limit until reference substance peak height is 3 times of corresponding noise peak height；It is phase to reference substance peak height
10 times for answering noise peak height, obtain quantitative limit, are shown in Table 5, Figure 24-Figure 29.
The preparation of 2.3 standard curves
Precision weighs Cryptotanshinone, salvia miltiorrhiza bge I, Tanshinone IIAAppropriate reference substance is set in brown measuring bottle, adds methanol that every 1ml is made
Containing 12 μ g of Cryptotanshinone, 2 μ g of salvia miltiorrhiza bge I, Tanshinone IIAThe mixed solution of 9 μ g, as reference substance solution.It is accurate respectively to draw
1,3,5,10,15,20,25 μ l, measurement.Using reference substance sample volume as abscissa, peak area is ordinate, draws standard curve
(see Figure 30, Figure 31, Figure 32), calculates regression equation and r value, data are shown in Table 6-1,6-2,6-3.
The result shows that Cryptotanshinone sample volume is in 0.011824~0.295607 μ g range, Tanshinone I sample volume exists
In 0.001758~0.04395 μ g range, Tanshinone I IASample volume is in 0.009306~0.232662 μ g range, reference substance
Sample volume is in a linear relationship with peak area.
2.4 precision test
The ZHENNAONING JIAONANG ZHENNAONING JIAONANG content about 2.4g(is taken, lot number: 160101), accurately weighed, the confession drafted by text
Sample solution processed, the chromatographic condition drafted according to text METHOD FOR CONTINUOUS DETERMINATION 6 times, measure its chromatography to test sample solution preparation method in accordance with the law
Peak area value, Cryptotanshinone RSD is 0.8%, salvia miltiorrhiza bge I RSD is 1.2%, Tanshinone IIARSD is 0.3%.The result shows that used
Instrument precision is good, is shown in Table 7.
Instrument: Dionex Ultimate 3000.Chromatographic column: Cosmosil C18-MS- II (5 μm, 4.6 × 250 mm)
(lot number K61291).Chromatographic condition: mobile phase: acetonitrile: 0.02% phosphoric acid solution (61:39)；Flow velocity: 0.8 ml/min；Column temperature:
25 ℃；Detection wavelength: 270 nm；Sample volume: 5 μ l.
Stability test
The ZHENNAONING JIAONANG ZHENNAONING JIAONANG content about 2.4g(is taken, lot number: 160101), accurately weighed, the confession drafted by text
Sample solution processed, the chromatographic condition drafted according to text are small 0,4,8,14,24,48 respectively in accordance with the law for test sample solution preparation method
When measure its chromatographic peak area value, be shown in Table 8.The result shows that test solution has good stability within 48 hours.
2.6 repetitive test
ZHENNAONING JIAONANG sample is taken (lot number: 160101), independently to measure 6 parts by the method that text is worked out, as a result Cryptotanshinone, pellet
Join ketone I and Tanshinone IIAThe sum of average content be 0.593mg/g(RSD=1.0%, n=6).The result shows that the repetition of this law
Property is good, is shown in Table 9.
Recovery test
Take the sample (lot number: 160101 for having predicted content；Content: 0.246 mg/g of Cryptotanshinone, 0.058 mg/ of salvia miltiorrhiza bge I
G, Tanshinone IIA0.289 mg/g) 6 parts, every part of about 2.4 g are accurately weighed, set in stuffed conical flask, and hidden Radix Salviae Miltiorrhizae is added in precision
Ketone, salvia miltiorrhiza bge I and Tanshinone IIA(concentration is respectively the mixed of 11.52,2.55,13.86 μ g/ml to mixing reference substance methanol solution
Close) 50 ml, according to method measurement sample is drafted, the calculating rate of recovery is shown in Table 10.
The results show that Cryptotanshinone average recovery rate is 101.79%, RSD=1.0%(n=6)；Salvia miltiorrhiza bge I average recovery rate
For 96.74%, RSD=2.6%(n=6)；Tanshinone IIA average recovery rate is 99.28%, RSD=1.1%(n=6).Show this method
Accuracy is good.
2.8 reappearance test
In another laboratory, test solution is prepared again according to the sample solution preparation method in text by different personnel
(ZHENNAONING JIAONANG, lot number 160101), precision are drawn with a test solution, high performance liquid chromatograph are injected, according to text
The chromatographic condition drafted METHOD FOR CONTINUOUS DETERMINATION 6 times, measures its content, by the average value of assay result and another laboratory testing
The sample lots testing result (repetitive test testing result: ZHENNAONING JIAONANG lot number is 160101；Content is Cryptotanshinone
0.246 mg/g, 0.058 mg/g of salvia miltiorrhiza bge I, Tanshinone IIA0.289 mg/g) it is compared.6 testing results, hidden pellet
Ginseng ketone RSD is 0.2%, salvia miltiorrhiza bge I RSD is 0.5%, Tanshinone IIARSD is 0.5%, hidden compared with Repeatability checking test result
Tanshinone is 0.2% containing SD is measured, and salvia miltiorrhiza bge I assay SD is 4.5%, Tanshinone IIAIt is 1.4% containing SD is measured.As a result table
Bright, reproducibility precision is good, is shown in Table 11,12.
3. one surveys the durabilities for commenting method and system suitability evaluation more
The determination of 3.1 relative retention times and correction factor
Colour examining spectral peak is treated using relative retention value method to be positioned.With Tanshinone IIAReference substance is reference, according to relative correction
Factor calculation formula calculates internal reference object Tanshinone II under different affecting factorsAIt is protected relative to Cryptotanshinone and the opposite of salvia miltiorrhiza bge I
Stay time and correction factor.Calculation formula is as follows:
RCryptotanshinone/tanshinone IIA=tR Cryptotanshinone/tR tanshinone IIA
RSalvia miltiorrhiza bge I/tanshinone IIA=tR salvia miltiorrhiza bge I/tR tanshinone IIA
fi/s=fs/fi=Ci×As/(Cs×Ai)
fCryptotanshinone/tanshinone IIA = CCryptotanshinone×ATanshinone IIA/ (CTanshinone IIA ×ACryptotanshinone)
fSalvia miltiorrhiza bge I/tanshinone IIA = CSalvia miltiorrhiza bge I×ATanshinone IIA/ (CTanshinone IIA ×ASalvia miltiorrhiza bge I)
A is peak area, and C is concentration (unit: mg grain -1)
It according to the experimental condition of 2.2 above-mentioned determinations, measures four times, according to measurement result, and with reference to pharmacopeia red rooted salvia containing measurement
The pertinent regulations for determining correction factor under item determine relative retention time and correction factor, are shown in Table 13.
Influence of 3.2 instruments to relative retention time and correction factor
Influence of four kinds of model liquid chromatographs to relative retention time and correction factor is investigated, 14-1,14-2 are shown in Table.As a result
Show that Cryptotanshinone and salvia miltiorrhiza bge I are relative to internal reference object Tanshinone II under the conditions of different model liquid chromatographAOpposite reservation
The correction factor of time and Cryptotanshinone is stable, salvia miltiorrhiza bge I correction factor that Shimadzu high performance liquid chromatograph obtains and other
The hplc determination result of type is variant.
Note: chromatographic column: Cosmosil C18-MS- II (5 μm, 4.6 × 250 mm) (lot number K61291) chromatographic condition: with
Acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase；Flow velocity: 0.8 ml/min；Column temperature: 30 DEG C；Detection wavelength: 270 nm.
Influence of the chromatographic column to relative retention time and correction factor
Using three kinds of liquid chromatographic systems, investigated 5 kinds of different models, the chromatographic column of specification to relative retention time and correction because
The influence of son, is shown in Table 15.The result shows that Cryptotanshinone and Tanshinone I are relative to internal reference object Tanshinone IIARelative retention time
It is stable under the conditions of different liquid chromatographs and different chromatographic columns from correction factor.
Note: instrument 1:Dionex Ultimate 3000；Instrument 2:Agilent 1260(VWD)；Instrument 3:Waters UPLC
Chromatographic column 1:Cosmosil C18-MS- II (5 μm, 4.6 × 250 mm) (lot number K61291)；
Chromatographic column 2:Capcell PAK C18 (5 μm, 4.6 × 250 mm) (lot number 92533)；
Chromatographic column 3:Waters Symmetry C18 (5 μm, 4.6 × 250 mm) (lot number 0216371361)；
Chromatographic column 4:Phenomenex Gemini C18 (5 μm, 4.6 × 250 mm) (lot number 5520-153)；
Chromatographic column 5:YMC-Pack ODS-A C18 (5 μm, 4.6 × 250 mm) (lot number 0425067466)；
Chromatographic condition: with acetonitrile: 0.02% phosphoric acid solution (61:39) is mobile phase；Flow velocity: 0.8 ml/min；Column temperature: 25 DEG C；
Detection wavelength: 270 nm；Sample volume: 5 μ l.
Influence of the flow velocity to relative retention time and correction factor
Investigate respectively (0.8 ml/min, 1.0 ml/min, 1.1 ml/min) different in flow rate to relative retention time and correction because
The influence of son, is shown in Table 16.The result shows that Cryptotanshinone and salvia miltiorrhiza bge I are relative to internal reference object Tanshinone IIARelative retention time
It is stable under the conditions of different in flow rate with correction factor.
Note: instrument: Dionex Ultimate 3000；Chromatographic column: Cosmosil C18-MS- II (5 μm, 4.6 × 250
Mm) (lot number K61291)；Chromatographic condition: mobile phase: acetonitrile: 0.02% phosphoric acid solution (61:39)；Flow velocity: 0.8 ml/min；Column
Temperature: 25 DEG C；Detection wavelength: 270 nm；Sample volume: 5 μ l.
Influence of the column temperature to relative retention time and correction factor
Influence of the different column temperatures (25 DEG C, 30 DEG C, 35 DEG C) to relative retention time and correction factor has been investigated respectively, has been shown in Table
17。
The result shows that Cryptotanshinone and salvia miltiorrhiza bge I are relative to internal reference object Tanshinone IIARelative retention time and correction because
Son is stable under the conditions of different column temperatures.
Note: instrument: Dionex Ultimate 3000；Chromatographic column: Cosmosil C18-MS- II (5 μm, 4.6 × 250
Mm) (lot number K61291)；Chromatographic condition: mobile phase: acetonitrile: 0.02% phosphoric acid solution (61:39)；Flow velocity: 0.8 ml/min；Column
Temperature: 25 DEG C；Detection wavelength: 270 nm；Sample volume: 5 μ l.
Influence of 3.6 Detection wavelengths to relative retention time and correction factor
Different Detection wavelengths (268 nm, 270 nm, 272 nm) have been investigated respectively to the shadow of relative retention time and correction factor
It rings, is shown in Table 18.
The result shows that Detection wavelength does not influence relative retention time, have a certain impact to correction factor.Detect wave
Length is to fSalvia miltiorrhiza bge I/tanshinone IIAInfluence is smaller, to fCryptotanshinone/tanshinone IIAInfluence it is relatively large, analysis be because Cryptotanshinone ultraviolet spectra
Caused by the gradient is larger near 270nm.
Note: instrument: Dionex Ultimate 3000；Chromatographic column: Cosmosil C18-MS- II (5 μm, 4.6 × 250 mm)
(lot number K61291).Chromatographic condition: mobile phase: acetonitrile: 0.02% phosphoric acid solution (61:39)；Flow velocity: 0.8 ml/min；Column temperature:
25 ℃；Detection wavelength: 270 nm；Sample volume: 5 μ l.
One survey comments method compared with external standard method result
First using external standard method to Cryptotanshinone, salvia miltiorrhiza bge I, Tanshinone IIASynchronize assay, then the survey with foundation
It comments method to calculate Cryptotanshinone, salvia miltiorrhiza bge I content more, 2 kinds of method calculated results is compared.It the results are shown in Table 19.
The result shows that the tanshinone component that two methods measure does not have significant difference, relative deviation < 5%, explanation is built
Vertical method is credible.
Note: instrument: Dionex Ultimate 3000；Chromatographic column: Cosmosil C18-MS- II (5 μm, 4.6 × 250
Mm) (lot number K61291)；Chromatographic condition: mobile phase: acetonitrile: 0.02% phosphoric acid solution (61:39)；Flow velocity: 0.8 ml/min；Column
Temperature: 25 DEG C；Detection wavelength: 270 nm；Sample volume: 5 μ l.
One survey comments verifying again for method
10 batches, sample are taken, instrument and equipment and personnel are changed, prepares reference substance solution and test solution again, is surveyed according to one and comments method more
To Cryptotanshinone, salvia miltiorrhiza bge I, Tanshinone IIAAssay is carried out, measurement result is verified again with external standard method calculated result.
It is shown in Table 20.
The result shows that there was no significant difference for the Cryptotanshinone content that measures of two methods, the equal < 3% of relative deviation, salvia miltiorrhiza bge I contain
There was no significant difference for amount, the equal < 5% of relative deviation, shows that a survey of foundation more and comments method in Cryptotanshinone, salvia miltiorrhiza bge I composition detection
In suitability it is good, can be used for drafting and formulating for standard.
Claims (7)
1. a kind of one for ZHENNAONING JIAONANG surveys comments detection method more, it is characterised in that the following steps are included:
Test fluid is made in ZHENNAONING JIAONANG；
With Tanshinone II in high performance liquid chromatograph measurement test fluidAContent；
The content of Cryptotanshinone and tanshinone Ι in sample is calculated using relative correction factor；
Wherein, the measuring method of step 2 are as follows:
Measure Tanshinone II in testing sample solution and reference substance solution respectively with high performance liquid chromatographAPeak area, using outer
It marks one point method or working curve method calculates Tanshinone IIAContent；
Instrument: high performance liquid chromatograph；
Chromatographic column: octadecylsilane chemically bonded silica is filler；
Mobile phase: acetonitrile: 0.02% phosphoric acid solution is 61:39；
Flow velocity: 0.8-1.1ml/min；
Column temperature: 25-35 DEG C；
Sample volume: 1-25 μ l；
Detector wavelength: 268-272nm.
2. detection method according to claim 1, which is characterized in that
Wherein, step 1) is as follows by the method that ZHENNAONING JIAONANG is prepared into test fluid: taking town
20 intragranular of brain Yiganning capsule is tolerant, mixes, takes 1.5-3.0g, accurately weighed, sets in stuffed conical flask, and methanol is added in precision
30-60ml, weighed weight；Ultrasonic treatment 10-40 minutes, power 250W, frequency 42kHz；It lets cool, then weighed weight, uses methanol
The weight for supplying less loss, shakes up, filtration, take subsequent filtrate to get.
3. detection method as described in claim 1, which is characterized in that
Wherein, step 1) is as follows by the method that ZHENNAONING JIAONANG is prepared into test fluid: taking 20 intragranular of ZHENNAONING JIAONANG tolerant, mixes
It is even, 2.4g is taken, it is accurately weighed, it sets in stuffed conical flask, methanol 50ml is added in precision, and weighed weight is ultrasonically treated 30 minutes, function
Rate 250W, frequency 42kHz；It lets cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, filter, take subsequent filtrate, i.e.,
.
4. detection method as described in claim 1, which is characterized in that wherein one point external standard method is 20 μ g/ml's using concentration
Tanshinone IIAAs reference substance, working curve method uses concentration range in the tanshinone of at least five gradients of 9-230 μ g/ml
ⅡAAs reference substance solution.
5. detection method according to claim 1, which is characterized in that
Wherein, chromatographic condition setting is as follows in step 2:
Chromatographic column: Cosmosil C18-MS- II, 5 μm of specification, 4.6 × 250mm;
Mobile phase: acetonitrile: 0.02% phosphoric acid solution is 61:39；
Flow velocity: 0.8ml/min；
Column temperature: 30 DEG C；
Sample volume: 5 μ l；
Detector wavelength: 270nm.
6. detection method according to claim 1, which is characterized in that
Wherein, determine and calculate respectively using relative retention time and relative correction factor in step 3) in sample Cryptotanshinone and
The peak position of tanshinone Ι and content；
Qualitative fashion: Tanshinone IIARelative retention time relative to Cryptotanshinone and Tanshinone I be respectively 0.50-0.60 and
0.55-0.66；The retention time calculation formula of test analyte are as follows:
Wherein, tRxAnd tRsRespectively represent component to be measured and and Tanshinone II in test sampleARetention time, Rx/sRepresent opposite protect
Stay the time；
Quantitative manner: Tanshinone IIARelative correction factor relative to Cryptotanshinone and Tanshinone I be respectively 1.10-1.20 and
1.18-1.38；The concentration calculation formula of test analyte are as follows:
Wherein, AxAnd AsRespectively represent component to be measured and Tanshinone II in test sampleAPeak area, CxAnd CsRespectively represent test sample
In component to be measured and Tanshinone IIAConcentration, fx/sRepresent Tanshinone II in test sampleATo the correction factor of ingredient to be measured.
7. detection method according to claim 1, which is characterized in that step 3) using relative retention time and relative correction because
Son determines and calculates respectively the peak position and content of Cryptotanshinone and Tanshinone I in sample；
Described relative retention time and relative correction factor are as follows:
It is qualitative: with Tanshinone IIAFor single subject matter, Cryptotanshinone and Tanshinone I it is opposite
Retention time is respectively as follows: 0.54 and 0.58；
It is quantitative: with Tanshinone IIAFor single subject matter, Cryptotanshinone and Tanshinone I it is opposite
Correction factor is respectively as follows: 1.14 and 1.30.
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