CN109001365A - A kind of detection method of Radix Salviae Miltiorrhizae - Google Patents
A kind of detection method of Radix Salviae Miltiorrhizae Download PDFInfo
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Abstract
The present invention provides a kind of detection methods of Radix Salviae Miltiorrhizae.Detection method includes: thin-layer chromatography detection, the detection of tanshinone content of material, content of danshinolic acid B detection and finger-print detection.In detection method, by the optimization to thin-layer chromatography solvent and chromatographic condition, so that each impurity peaks separating effect is more preferable, testing result is more acurrate；Meanwhile detection method tolerance, reproducibility and specificity are more preferable.
Description
Technical field
The present invention relates to traditional Chinese medicine quality detection fields, in particular to a kind of detection method of Radix Salviae Miltiorrhizae.
Background technique
Radix Salviae Miltiorrhizae is the dry root and rhizome of Lamiaceae plant Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bge.), in China
Most area is all distributed.It has effects that promoting blood circulation, inducing meastruation to relieve menalgia, relieving restlessness and restlessness, cool blood to disappear carbuncle, clinically
It is chiefly used in treatment of cardiovascular disease, such as Fufang Danshen Pian, compound danshen dripping pills, DANQI PIAN, GUANXIN DANSHEN PIAN etc., all faces
The common medicament of bed.
Quality standard detection for Radix Salviae Miltiorrhizae, in the standard methods such as pharmacopeia, also both provides a variety of corresponding methods.So
And in existing standard detecting method, although can satisfy standard requirements, deviation is larger, testing result accuracy and precision
It is still to be improved.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of detection method of Radix Salviae Miltiorrhizae, have good tolerance, reproducibility and
Specificity, testing result are accurate.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of detection method of Radix Salviae Miltiorrhizae, comprising: (a) takes Radix Salviae Miltiorrhizae control medicinal material that control medicinal material solution is made；Take tanshinone IIA
Corresponding reference substance solution is respectively obtained with tanshin polyphenolic acid B；
Using ethyl acetate-butanone-glacial acetic acid as solvent, Radix Salviae Miltiorrhizae test solution is examined using thin layer chromatography
It surveys；
(b) high performance liquid chromatography is used, the tanshinone content of material in Radix Salviae Miltiorrhizae is detected；Wherein, the height
In effect liquid phase chromatogram method, gradient elution is carried out using following condition:
Wherein, mobile phase A is acetonitrile；Mobile phase B is the buffer solution of pH3.2~3.6 and the mixed liquor of acetonitrile, is buffered molten
The ratio of liquid and acetonitrile is 93~95:5~7；
(c) method for using high performance liquid chromatography carries out content detection to the tanshin polyphenolic acid B in Radix Salviae Miltiorrhizae, using following condition
Carry out gradient elution:
Wherein, mobile phase A is acetonitrile, and Mobile phase B is the buffer solution of pH3.0~4.0.
Compared with prior art, the invention has the benefit that
In detection method, by the optimization to chromatographic condition, so that each impurity peaks separating effect is more preferable, detection knot
Fruit is more acurrate；Meanwhile detection method tolerance, reproducibility and specificity are more preferable.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is 1 thin-layer chromatography control test result figure of the embodiment of the present invention；
Fig. 2 is 2 linear standard curve of the embodiment of the present invention；
Fig. 3 is 3 linear standard curve of the embodiment of the present invention；
Fig. 4 is precision similarity evaluation system matches figure in the embodiment of the present invention 4；
Fig. 5 is repeated similarity evaluation system matches figure in the embodiment of the present invention 4；
Fig. 6 is stability similarity evaluation system matches figure in the embodiment of the present invention 4；
Fig. 7 is 15 batches of red rooted salvia similarity evaluation system matches figures in the embodiment of the present invention 4；
Fig. 8 is red rooted salvia reference fingerprint in the embodiment of the present invention 4.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Radix Salviae Miltiorrhizae detection method provided by the present invention, be carried out on the basis of the standard detecting methods such as pharmacopeia it is excellent
Change, by solvent used in being separated for thin-layer chromatography, the tune of eluant, eluent and elution process used in high performance liquid chromatography
Whole and optimization, improves impurity separating effect, improves the accuracy of testing result.
In detection method, mainly by the method for thin-layer chromatography and high performance liquid chromatography, to phase in Radix Salviae Miltiorrhizae
Substance is closed to be detected.
It is specific:
(1) thin-layer chromatography detects
Thin-layer chromatography can analyze multiple samples simultaneously on lamellae, and chromatography color image characteristic is strong, is easy to flat
Row observation recognizes and compares, and is one of TCD identificafion common method.
In existing standard method, mainly use using Radix Salviae Miltiorrhizae ethanol extract centrifuged supernatant as test solution, then
With chloroform-toluene-ethyl acetate-methyl alcohol-formic acid (6:4:8:1:4) for solvent, exhibition to about 4cm is taken out, is dried, then
With petroleum ether (60~90 DEG C)-ethyl acetate (4:1) for solvent, expansion, exhibition to about 8cm is taken out, is dried, respectively in daylight
And the method inspected under ultraviolet lamp (365nm) is detected.The method operation is relatively complicated, needs successively in different solvents
It is middle to be repeatedly unfolded, and need to be monitored for a long time.
It is then to cross No. three sieves after being crushed raw material Radix Salviae Miltiorrhizae to be detected and obtain Danshen Root in the present invention；Then, will
Danshen Root, which is added in ether, is ultrasonically treated 6~10min, and filtration removes filter residue, after gained filtrate is evaporated, is carried out with ethyl alcohol molten
Solution, as Radix Salviae Miltiorrhizae test solution；
Meanwhile it is Radix Salviae Miltiorrhizae control medicinal material system is molten according to the obtained Radix Salviae Miltiorrhizae control medicinal material of the identical method of Radix Salviae Miltiorrhizae test solution
Liquid；
Tanshinone IIA and tanshin polyphenolic acid B add ethyl alcohol that corresponding reference substance solution is made respectively.
Then, by Radix Salviae Miltiorrhizae test solution, Radix Salviae Miltiorrhizae control medicinal material solution, tanshinone IIA contrast solution and tanshin polyphenolic acid B
Contrast solution is respectively the ethyl acetate of 8:1:2, the mixing of butanone and glacial acetic acid with volume ratio under same environmental conditions
Solution is unfolded；About 8cm is unfolded, takes out, dries, compare inspect under daylight and ultraviolet lamp (365nm) respectively.
By thin-layer chromatography method of the present invention as described above it is found that detection method be not necessarily in different solutions into
Row repeatedly expansion, and solvent for use is also free of benzene, toluene etc. for human body with the organic solvent compared with major injury, safety
It is good.
(2) detection of the high performance liquid chromatography to tanshinone content of material in Radix Salviae Miltiorrhizae sample
Reference substance solution preparation method is with reference to as follows: tanshinone IIA reference substance is appropriate, accurately weighed, sets in measuring bottle, adds first
Alcohol be made every 1ml containing 20 μ g solution to get；
Sample solution preparation method is with reference to as follows: Danshen Root (crossing No. three sieves) about 0.3g to be detected is placed in plug taper
In bottle, methanol 50ml, weighed weight is added in precision, and ultrasonic treatment (power 140W, frequency 42kHz) 30 minutes is let cool, then weighed
Weight is supplied the weight of less loss with methanol, shaken up, filtration, take subsequent filtrate to get；
Chromatographic condition are as follows: using octadecylsilane chemically bonded silica as filler；20 DEG C of column temperature；Detection wavelength is 270nm；Reason
60000 should be not less than by calculating by plate number by tanshinone IIA peak；
Using acetonitrile as mobile phase A；With volume ratio for (93~95): citric acid-citric acid of pH3.2~3.6 of (5~7)
The mixed liquor of sodium buffer solution and acetonitrile is Mobile phase B, carries out gradient elution according to following condition:
0~5min, mobile phase A 10 → 20%, Mobile phase B 90 → 80% (volume ratio, sum of the two 100%, under
Together)；5~22min, mobile phase A 20 → 60%, Mobile phase B 80 → 40%；22~22.5min, mobile phase A 60 → 10%, stream
Dynamic phase B 40 → 90%；22.5~25min, mobile phase A 10%, Mobile phase B 90%.
For the elution process disclosed in the prior arts such as pharmacopeia, in detection method, by for
The adjustment of eluant, eluent and elution process can further improve impurity separating effect, improve the precision and accuracy of detection.
(3) detection of the high performance liquid chromatography to content of danshinolic acid B in Radix Salviae Miltiorrhizae sample
Reference substance solution is accurately weighed the preparation method is as follows: tanshin polyphenolic acid B reference substance is appropriate, sets in measuring bottle, adds methanol-water
(8:2) mixed solution be made solution of every 1ml containing 0.10mg to get.
The preparation method of test solution is with reference to as follows: Danshen Root (crossing No. three sieves) to be detected is placed in plug conical flask,
Radix Salviae Miltiorrhizae sample powder to be measured (crossing No. three sieves) about 0.15g is taken, it is accurately weighed, it sets in stuffed conical flask, methanol-water is added in precision
(8:2) mixed solution 50ml, close plug, weighed weight, ultrasonic treatment (power 140W, frequency 42kHz) 30 minutes are let cool, then claim
Determine weight, the weight of less loss is supplied with methanol-water (8:2) mixed solution, is shaken up, filter, precision measures subsequent filtrate 5ml, moves to
In 10ml measuring bottle, add methanol-water (8:2) mixed solution to be diluted to scale, shake up, filter, take subsequent filtrate to get.
Chromatographic condition are as follows: using octadecylsilane chemically bonded silica as filler；Column temperature is 20 DEG C；Flow velocity is per minute
1.2ml, Detection wavelength 286nm；Number of theoretical plate is calculated by tanshin polyphenolic acid B peak should be not less than 6000；
Using acetonitrile as mobile phase A, disodium hydrogen phosphate-sodium citrate buffer of pH3.0~4.0 is Mobile phase B, according to
Following condition is eluted:
0~15min, mobile phase A 10 → 15%, Mobile phase B 90 → 85% (volume ratio, sum of the two 100%, under
Together)；15~30min, mobile phase A 15 → 35%, Mobile phase B 85 → 65%；30~40min, mobile phase A 35 → 10%, stream
Dynamic phase B 65 → 90%；40~45min, mobile phase A 10%, Mobile phase B 90%.
Different from the isocratic elution method disclosed in the prior arts such as pharmacopeia, in the present invention, using the side of gradient elution
Formula carries out the detection of tanshin polyphenolic acid B.Likewise, can further improve impurity by the adjustment for eluant, eluent and elution process
Separating effect improves the precision and accuracy of detection.
(4) further, it in detection method, can further include for impurity, moisture content, total ash, acid
Property insoluble ash, metal and harmful element and extract etc. at least one of index the step of being detected, to realize
Globality detection for Radix Salviae Miltiorrhizae quality.
1 Radix Salviae Miltiorrhizae sample thin-layer chromatography of embodiment
Raw material Radix Salviae Miltiorrhizae to be detected (crossing No. three sieves) 1g is taken, add diethyl ether about 10ml, is ultrasonically treated 8min, and filtration removes filter residue,
It after gained filtrate is evaporated, is dissolved with 3ml ethyl alcohol, as Radix Salviae Miltiorrhizae test solution；
Radix Salviae Miltiorrhizae control medicinal material 1g is taken, by the same way, Radix Salviae Miltiorrhizae control medicinal material solution is made；
Tanshinone IIA reference substance is taken, adds ethyl alcohol that the tanshinone IIA contrast solution that content is 0.5mg/ml is made；
Tanshin polyphenolic acid B reference substance is taken, adding ethyl alcohol that content is made is 1.5mg/ml danshinolic acid reference substance solution.
Then, according to thin-layered chromatography, above-mentioned each 5 μ l of four kinds of solution is drawn, is put respectively on same silica gel g thin-layer plate,
Make into strips, with ethyl acetate, butanone and glacial acetic acid (8:1:2) they are solvent, and expansion opens up to about 8cm, takes out, dry,
It is inspected under daylight and ultraviolet lamp (365nm) respectively；
Wherein, solvent dosage: each 10ml；Expansion mode: double flute opens up cylinder (10 × 20cm)；Exhibition is away from 8 ㎝；Colour developing: ultraviolet
It is inspected under light lamp (365nm)；Temperature: 25 DEG C；Humidity: 55%
In sample chromatogram, on position corresponding with reference medicine chromatography and reference substance chromatography, the spot of same color is shown
Point or fluorescence spot, as a result as shown in Figure 1.
Tanshinone content of material detects in 2 Radix Salviae Miltiorrhizae sample of embodiment
1. the preparation of reference substance solution:
Tanshinone IIA reference substance is appropriate, accurately weighed, sets in measuring bottle, adds methanol that the solution that every 1ml contains 20 μ g is made, i.e.,
?.
2. the preparation of test solution:
Radix Salviae Miltiorrhizae sample powder to be detected (crossing No. three sieves) about 0.3g is taken, it is accurately weighed, it sets in stuffed conical flask, precision is added
Methanol 50ml, weighed weight, ultrasonic treatment (power 140W, frequency 42kHz) 30 minutes let cool, then weighed weight, are mended with methanol
The weight of sufficient less loss, shakes up, filtration, take subsequent filtrate to get.
3. chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A；It is the pH3.2 of 95:5 with volume ratio
Sodium citrate-citric acid buffer solution and acetonitrile mixed liquor be Mobile phase B, according to following condition carry out gradient elution:
| Time (min) | Mobile phase A (v/v%) | Mobile phase B (v/v%) |
| 0~5 | 10→20 | 90→80 |
| 5~22 | 20→60 | 80→40 |
| 22~22.5 | 60→10 | 40→90 |
| 22.5~25 | 10 | 90 |
20 DEG C of column temperature；Detection wavelength is 270nm.Number of theoretical plate is calculated by tanshinone IIA peak should be not less than 60000.
Measuring method: accurate respectively to draw reference substance solution and each 10ul of test solution.Liquid chromatograph is injected, is surveyed
It is fixed.Using tanshinone IIA reference substance as reference, with its corresponding peak for the peak S, when calculating the corresponding reservation of Cryptotanshinone, salvia miltiorrhiza bge I
Between, relative retention time should be within the scope of ± the 5% of specified value.Corresponding retention time and correction factor are shown in such as following table institute
Show:
| Ingredient (peak) to be measured | It is opposite to retain (s) | The time adjustment factor |
| Cryptotanshinone | 0.75 | 1.18 |
| Salvia miltiorrhiza bge I | 0.79 | 1.31 |
| Tanshinone IIA | 1.00 | 1.00 |
4. linear test
Take tanshinone IIA reference substance solution (23.02 μ g/ml).It is accurate respectively to measure 2,4,8,12,16,20 μ l, inject color
Spectrometer is tested by text chromatographic condition, as a result as shown in the table:
| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| Sample volume (μ g) | 0.04604 | 0.09208 | 0.18416 | 0.27624 | 0.36832 | 0.4604 |
| Peak area (A) | 318.9614 | 642.0988 | 1288.8495 | 1932.5615 | 2576.9949 | 3223.0073 |
Using peak area as ordinate (Y), sample volume is that abscissa (X) draws standard curve, is freed as shown in Figure 1.It returns
Equation are as follows: y=7006.6x-2.9372, coefficient R2=1, show tanshinone IIA within the scope of 0.04604~0.4604ug
Linear relationship is good；
5. instrument precision is tested
The reference substance solution prepared under 1. item is taken, under above-mentioned chromatographic condition, repeats sample introduction 6 times, measures tanshinone IIA peak
Area, calculate RSD% (tanshinone IIA) value be 0.24%, show that instrument precision is good, measurement result as shown in the table below:
| Number | 1 | 2 | 3 | 4 | 5 | 6 | Average value | RSD% |
| Area (A) | 1611.9132 | 1611.9429 | 1617.2340 | 1618.1431 | 1618.9027 | 1621.7645 | 1616.650067 | 0.24% |
6. repetitive test
Precision is weighed with a collection of 6 parts of sample of Radix Salviae Miltiorrhizae, measures the content of tanshinone in sample, average content according to the above method
For 0.30%, RSD value 1.74%, show that the measuring method of this tanshinone has good repeatability；As a result as shown in the table:
7. stability test
It is a by legal system available test sample solution below 2. item, it is placed at room temperature for, by drafting under chromatographic condition in 0,2,4,6,8,
10h distinguishes sample introduction, measures peak area.Calculating RSD% is 0.19%, shows Cryptotanshinone, Tanshinone I, tanshinone IIA in 10h
It is inside relatively stable.Stability test result is as shown in the table:
| Number | 0h | 2h | 4h | 6h | 8h | 10h | Average value | RSD (%) |
| Total peak area (A) | 1342.5241 | 1342.8654 | 1345.2145 | 1347.1201 | 1341.0213 | 1340.2689 | 1343.16915 | 0.19% |
8. recovery test
Using sample-adding absorption method.Take 6 parts of the sample (content (tanshinone IIA): 1.8mg/g) of known content, every part of precision
0.15g is weighed, tanshinone IIA reference substance solution (50 μ g/ml) 5ml is separately added into, is prepared according to preparation method under 2. item.Upper
It states and is measured under chromatographic condition, it is 0.95% that measure its average recovery rate, which be 98.98%, RSD% value, as a result as shown in the table:
9. sample size measures
Assay is carried out to ten batches of samples as described above, as a result as shown in the table:
By table data as above it is found that in the present invention in Radix Salviae Miltiorrhizae detected, (the C containing tanshinone IIA19H18O3), Cryptotanshinone
(C19H20O3) and Tanshinone I (C18H12O3) total amount average value be 0.43%.
Content of danshinolic acid B detects in 3 Radix Salviae Miltiorrhizae sample of embodiment
1. the preparation of reference substance solution:
Tanshin polyphenolic acid B reference substance is appropriate, accurately weighed, sets in measuring bottle, adds methanol-water (8:2) mixed solution that every 1ml is made and contains
The solution of 0.10mg to get.
2. the preparation of test solution
Danshen Root to be detected (crossing No. three sieves) about 0.15g is taken, it is accurately weighed, it sets in stuffed conical flask, first is added in precision
Alcohol-water (8:2) mixed solution 50ml, close plug, weighed weight, ultrasonic treatment (power 140W, frequency 42kHz) 30 minutes are let cool,
Weighed weight again is supplied the weight of less loss with methanol-water (8:2) mixed solution, is shaken up, and filtration, precision measures subsequent filtrate 5ml,
Move in 10ml measuring bottle, add methanol-water (8:2) mixed solution to be diluted to scale, shake up, filter, take subsequent filtrate to get.
3. chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, disodium hydrogen phosphate-lemon of pH3.2
Sour sodium buffer solution is Mobile phase B；According to following condition, linear gradient elution is carried out:
Column temperature is 20 DEG C；Flow velocity is 1.2ml per minute, Detection wavelength 286nm.Number of theoretical plate is calculated by tanshin polyphenolic acid B peak
6000 should be not less than.
Measuring method: accurate respectively to draw reference substance solution and each 10ul of test solution；Liquid chromatograph is injected, is surveyed
It is fixed.
4. linear test
Take tanshin polyphenolic acid B reference substance solution (0.105881mg/ml).It is accurate respectively to measure 2,4,8,12,16 μ l, inject chromatography
Instrument is tested by text chromatographic condition, as a result as shown in the table:
| Number | 1 | 2 | 3 | 4 | 5 |
| Sample volume (μ g) | 0.211762 | 0.423524 | 0.847048 | 1.270572 | 1.694096 |
| Peak area (A) | 334.7132 | 673.6862 | 1342.6668 | 2012.1085 | 2679.7456 |
Using peak area as ordinate (Y), sample volume is that abscissa (X) draws standard curve, and standard curve is as shown in Figure 2；
Regression equation are as follows: y=1581.3x+2.2111, coefficient R2=1, show tanshin polyphenolic acid B 0.211762~
2.11762ug linear relationship is good in range.
5. instrument precision is tested
The reference substance solution prepared under 1. item is taken, under above-mentioned chromatographic condition, repeats sample introduction 6 times, measures tanshin polyphenolic acid B peak face
Product, calculating RSD% (tanshin polyphenolic acid B) value is 0.70%, shows that instrument precision is good, measurement result is as shown in the table:
| Number | 1 | 2 | 3 | 4 | 5 | 6 | Average value | RSD% |
| Area (A) | 1119.0043 | 1115.1924 | 1112.9785 | 1097.9058 | 1109.6002 | 1103.6553 | 1109.72275 | 0.70% |
6. repetitive test
Precision weighs same batch of 6 parts of sample (lot number 20161101), measures tanshin polyphenolic acid B in sample according to the above method and contains
Amount, average content 7.0%, RSD value 0 show that the measuring method of this tanshin polyphenolic acid B has good repeatability；Measurement result is such as
Shown in following table:
7. stability test
It is a by legal system available test sample solution below 2. item, it is placed at room temperature for, by drafting under chromatographic condition in 0,2,4,6,8,
10h distinguishes sample introduction, measures peak area.Calculating RSD% is 0.19%, shows that tanshin polyphenolic acid B is relatively stable in 10h.Stability examination
It is as shown in the table to test result:
8. recovery test
Using sample-adding absorption method.Take 6 parts of the sample (content (tanshin polyphenolic acid B): 70mg/g) of known content, every part of accurate title
0.075g is taken, tanshin polyphenolic acid B reference substance 50.0mg is separately added into, according to 1.3.2 lower preparation method preparations.In above-mentioned chromatographic condition
Lower measurement, it is 0.98 that measure its average recovery rate, which be 98.75%, RSD% value, as a result as shown in the table:
9. sample size measures
Assay is carried out to ten batches of samples by the method that text records, as a result as shown in the table:
By upper table data it is found that the (C containing tanshin polyphenolic acid B in present invention Radix Salviae Miltiorrhizae detected36H30O16) the average value of amount be
5.0%.
4 finger-print of embodiment
(1) instrument and reagent
Instrument: high performance liquid chromatograph: Aglient 1260；Chromatographic work station: 1260 work station of Agilent；Chromatographic column:
ZORBAX SB-C18(250mm×4.6,5μm)；Electronic analytical balance.
Reagent: acetonitrile is chromatographically pure, and water is ultrapure water；Citric acid, sodium citrate and other reagents are that analysis is pure.
Sample: base harvesting red rooted salvia of improving literature
Reference substance: Rosmarinic acid reference substance, tanshin polyphenolic acid B reference substance.
(2) method
Chromatographic condition and system suitability using octadecylsilane chemically bonded silica as filler (column length 25cm, it is interior
Diameter is 4.6mm, and partial size is 5 μm)；Referring to the method for content of danshinolic acid B detection in 3 Radix Salviae Miltiorrhizae sample of embodiment, gradient elution is carried out；
Detection wavelength is 286nm；Column temperature is 20 DEG C；Flow velocity is 1.0ml per minute.
The preparation of reference solution takes Rosmarinic acid reference substance and tanshin polyphenolic acid B reference substance appropriate, accurately weighed, adds methanol
Be made every 1ml respectively the solution containing 0.2mg to get.
The preparation of test solution takes this product powder (crossing No. three sieves) about 0.2g, and accurately weighed, 50ml first is added in precision
Alcohol, ultrasonic 20min to get.
Measuring method is accurate respectively to draw reference solution and each 10 μ 1 of test solution, infuses people's liquid chromatograph, measures,
Record 75 minutes chromatogram to get.
Methodology validation
Chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler (column length 25cm, it is interior
Diameter is 4.6mm, and partial size is 5 μm)；Referring to the method for content of danshinolic acid B detection in 3 Radix Salviae Miltiorrhizae sample of embodiment, gradient elution is carried out；
Detection wavelength is 286nm；Column temperature is 30 DEG C；Flow velocity is 1.0ml per minute.Number of theoretical plate should be not less than by the calculating of Rosmarinic acid peak
20000。
Specificity: being prepared test solution (2 parts of Duplicate Samples) respectively by the preparation method of test solution, then precision respectively
Reference solution, test solution (2 parts of Duplicate Samples), blank solution and configuration each 10 μ l of solvent are drawn, liquid chromatograph is injected,
Measurement records chromatogram.
Precision: the test solution prepared by the preparation method of test solution takes with a test solution, repeats
Sample introduction 6 times, the intuitive overall picture for observing finger-print is without significant difference, using " similarity evaluation
(2004A editions) " calculate similarity, and matching figure is as shown in Figure 4.
Result as shown in Figure 4 it is found that the similarity of the chromatographic fingerprinting measured in same instrument is 1.000,
Whole similarity >=0.950 show that the precision of the instrument is good, meet finger-print testing requirements.
Repeatability: the test solution prepared by the preparation method of test solution, operation repetitive 6 times, in above-mentioned chromatography
Under the conditions of, chromatogram is recorded, using " traditional Chinese medicine fingerprint similarity evaluation system (2004 A edition) " calculating similarity,
Figure is as shown in Figure 5.
Result is it is found that the similarity of the chromatographic fingerprinting measured in same instrument is respectively similarity as shown in Figure 5
It is 0.997,0.999,0.951,0.997,0.999,1.000 respectively, similarity >=0.95 shows this method repeatability preferably,
Meet finger-print testing requirements.
Stability: taking with a sample, respectively 0,2,4,6,8,6 time points such as 10h detected, intuitive observation refers to
The overall picture of line map, no significant change are calculated similar with " similarity evaluation (2004A editions) "
Degree, matching figure are as shown in Figure 6.
By the result of Fig. 6 result it is found that the similarity of the chromatographic fingerprinting measured in same instrument is respectively
0.961,0.994,1.000,0.994,1.000,1.000, similarity >=0.950 shows the test solution within this time
Stable components meet finger-print testing requirements.
15 batches of red rooted salvias are taken, is tested according to test sample processing in (2) method, records chromatogram.Wherein, 15 batches of Radix Salviae Miltiorrhizaes
The finger-print number of medicinal material is as shown in the table:
| Number | Lot number |
| S1 | 20141101 |
| S2 | 20141102 |
| S3 | 20141103 |
| S4 | 20141104 |
| S5 | 20141105 |
| S6 | 20151101 |
| S7 | 20151102 |
| S8 | 20151103 |
| S9 | 20151104 |
| S10 | 20151105 |
| S11 | 20161101 |
| S12 | 20161102 |
| S13 | 20161103 |
| S14 | 20161104 |
| S15 | 20161105 |
Using " traditional Chinese medicine fingerprint similarity evaluation system (2004 A editions) " analysis, with the chromatogram of the place of production S1 sample
As referring to map, median method of formation, access time width is 0.10min, after Supplements, generates red rooted salvia fingerprint
Map common pattern.Its similarity see the table below:
By upper table data it is found that the overall similarity of 15 batches of red rooted salvia finger-prints is 0.954~1 (> 0.95, symbol
Standardization requirement).15 batches of red rooted salvia similarity evaluation system matches figures are as shown in Figure 7.Meanwhile 15 batches of red rooted salvias are raw
At reference fingerprint it is as shown in Figure 8；In Fig. 8, peak 2 is Rosmarinic acid characteristic peak；Peak 4 is tanshin polyphenolic acid B characteristic peak.
Comparative example 1
According to the method disclosed in the prior art, the inspection of tanshinone substance is carried out with the Radix Salviae Miltiorrhizae of batch in embodiment 2
It surveys, the specific method is as follows:
Using octadecylsilane chemically bonded silica as filler；It is flowing with 0.02% phosphoric acid solution using acetonitrile as mobile phase A
Phase B, the regulation according to the form below carry out gradient elution；
| Time (minute) | Mobile phase A (acetonitrile) | Mobile phase B (0.1% phosphoric acid aqueous acid) |
| 0 | 10 | 90 |
| 15 | 20 | 80 |
| 35 | 25 | 75 |
| 45 | 30 | 70 |
| 55 | 90 | 10 |
| 70 | 90 | 10 |
20 DEG C of column temperature；Detection wavelength is 270nm.Number of theoretical plate is calculated by tanshinone IIA peak should be not less than 60000.
The selection of reference substance solution and specific measuring method and relevant parameter is same as Example 2.
The method for respectively referring to embodiment 2, carry out 5. -9. test, it is as a result as follows:
Comparative example 2
According to the method disclosed in the prior art, danshinolic acid detection is carried out with the Radix Salviae Miltiorrhizae of batch in embodiment 2；
Chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；With acetonitrile -0.1%
Phosphoric acid solution (22:78) is mobile phase；Column temperature is 20 DEG C；Flow velocity is 1.2ml per minute；Detection wavelength is 286nm.Number of theoretical plate
6000 should be not less than by calculating by tanshin polyphenolic acid B peak.
The selection of reference substance solution and specific measuring method and relevant parameter is same as Example 3.
The method for respectively referring to embodiment 3, carry out 5. -9. test, it is as a result as follows:
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (8)
1. a kind of detection method of Radix Salviae Miltiorrhizae characterized by comprising
(a)
Take Radix Salviae Miltiorrhizae control medicinal material that control medicinal material solution is made；Tanshinone IIA and tanshin polyphenolic acid B is taken to respectively obtain corresponding reference substance molten
Liquid；
Using ethyl acetate-butanone-glacial acetic acid as solvent, Radix Salviae Miltiorrhizae test solution is detected using thin layer chromatography；
(b)
Using high performance liquid chromatography, the tanshinone content of material in Radix Salviae Miltiorrhizae is detected；
Wherein, in the high performance liquid chromatography, gradient elution is carried out using following condition:
Wherein, mobile phase A is acetonitrile；Mobile phase B be pH3.2~3.6 buffer solution and acetonitrile mixed liquor, buffer solution with
The ratio of acetonitrile is 93~95:5~7；
(c)
Using the method for high performance liquid chromatography, content detection is carried out to the tanshin polyphenolic acid B in Radix Salviae Miltiorrhizae, gradient is carried out using following condition
Elution:
Wherein, mobile phase A is acetonitrile, and Mobile phase B is the buffer solution of pH3.0~4.0.
2. the detection method of Radix Salviae Miltiorrhizae according to claim 1, which is characterized in that in step (a), acetic acid second in solvent
The volume ratio of ester, butanone and glacial acetic acid is (7~10): (1~3): (1~3).
3. the detection method of Radix Salviae Miltiorrhizae according to claim 2, which is characterized in that in step (a), acetic acid second in solvent
The volume ratio of ester, butanone and glacial acetic acid is 8:1:2.
4. the detection method of Radix Salviae Miltiorrhizae according to claim 1, which is characterized in that in step (a), Radix Salviae Miltiorrhizae test solution system
It is standby to include the following steps:
Danshen Root is added diethyl ether after ultrasonic treatment, filtration is removed filter residue and dissolved after gained filtrate is evaporated with ethyl alcohol, obtains pellet
Join test solution.
5. detection method according to claim 4, which is characterized in that the time of ultrasonic treatment is 6~10min.
6. the detection method of Radix Salviae Miltiorrhizae according to claim 1, which is characterized in that in step (b), the buffer solution is lemon
Lemon acid-sodium citrate buffer.
7. the detection method of Radix Salviae Miltiorrhizae according to claim 1, which is characterized in that in step (c), the buffer solution is phosphorus
Sour disodium hydrogen-sodium citrate buffer.
8. the detection method of Radix Salviae Miltiorrhizae according to claim 1, which is characterized in that the detection method further include to impurity,
At least one index in moisture, total ash, acid-insoluble ash, heavy metal and harmful element and extract is detected
The step of.
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