CN108459100A - A kind of detection method of radix cyathulae medicinal material - Google Patents
A kind of detection method of radix cyathulae medicinal material Download PDFInfo
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- CN108459100A CN108459100A CN201810236236.3A CN201810236236A CN108459100A CN 108459100 A CN108459100 A CN 108459100A CN 201810236236 A CN201810236236 A CN 201810236236A CN 108459100 A CN108459100 A CN 108459100A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of detection methods of radix cyathulae medicinal material, using high effective liquid chromatography for measuring sample HPLC collection of illustrative plates, and compare the similarity and characteristic peak difference of gained collection of illustrative plates and radix cyathulae HPLC finger-prints.Detection method uses multi-wavelength handoff technique, solves the contradiction that multicomponent can not all be detected because absorbing wavelength difference is big under single wavelength, and the true and false of the detection method for radix cyathulae medicinal material differentiates, high sensitivity, efficiently and accurately.
Description
Technical field
The present invention relates to a kind of detection methods of radix cyathulae medicinal material, and in particular to a kind of finger-print detection side of radix cyathulae
Method belongs to drug abuse test field.
Background technology
Radix cyathulae be amaranthaceous plant radix cyathulae (Cyathula officinalis Kuan) dry root, record in《China
Pharmacopeia》2015 editions one, there is dispelling stasis of blood and stimulating the menstrual flow, it is easing joint movement, the effect of inducing diuresis for treating strangurtia.
Radix cyathulae is famous Genuine crude drugs in Sichuan, and main product is in Sichuan Jinkouhe District, Baoxing County and enshi city, Xuanen County
Etc. ground.Radix cyathulae is multi-component complex system, existing《Pharmacopeia》Cup amaranth steroid in radix cyathulae is only measured to the quality control of radix cyathulae
Ketone content, the measurement of one-component are difficult to reflect the total quality under radix cyathulae multicomponent system, and the common adulterant head of radix cyathulae
Flower cup amaranth and the miscellaneous root of bidentate achyranthes are not only similar to radix cyathulae on mode of appearance, and cyasterone content can also reach《Pharmacopeia》Limitation refers to
Mark, it is existing《Pharmacopeia》Method is difficult to radix cyathulae and adulterant, mixes adulterant and effectively distinguish.In the market radix cyathulae fake mostly with
It is blended in radix cyathulae based on headdress flower cup amaranth and/or the sale of the miscellaneous root of bidentate achyranthes, has severely impacted the clinical application safety and road of radix cyathulae
The utilization of ground medicinal material.
It is badly in need of a kind of capable of accurately differentiating radix cyathulae certified products, radix cyathulae adulterant and the discrimination method for mixing adulterant radix cyathulae.
Invention content
To solve the above problems, the present invention provides a kind of detection methods of radix cyathulae medicinal material.
The detection method of radix cyathulae medicinal material of the present invention, is detected using HPLC finger-prints, including following operating procedure：
(1) prepared by test solution：Take medicinal material to be checked, extract, filtration, take subsequent filtrate to get.
(2) it draws test solution and injects high performance liquid chromatograph, chromatographic condition is as follows：
Chromatographic column：Using octadecylsilane chemically bonded silica as filler；
Mobile phase：Using acetonitrile as mobile phase A, using 0.1-0.3% phosphate aqueous solutions as Mobile phase B.
Wherein, test solution preparation method described in step (1) is：Medicinal powder to be checked is taken, methanol aqueous solution is added to carry
Take, filter, take subsequent filtrate to get.
Further, the concentration of the methanol aqueous solution is 30~60%；And/or the methanol aqueous solution addition is
5~80v/w times of medicinal powder.
Further, the concentration of the methanol aqueous solution is 40~50%；And/or the methanol aqueous solution addition is
10~40v/w times of medicinal powder.
Further, the concentration of the methanol aqueous solution is 45%；And/or the methanol aqueous solution addition is medicinal material
20v/w times of powder.
Wherein, described to be extracted as ultrasonic extraction in step (1), the ultrasonic extraction time is 0~2 hour.
Further, the ultrasonic extraction time is 1 hour.
Wherein, in step (2), the chromatographic column is Agilent ZORBAX SB-C18 columns.
Further, institute's chromatographic column specification is 250mm × 4.6mm, 5 μm.
Wherein, Mobile phase B described in step (2) is 0.2% phosphate aqueous solution.
Wherein, high performance liquid chromatography described in step (2) is using gradient elution, elution program：
Wherein, Detection wavelength switches for multi-wavelength in high performance liquid chromatography described in step (2)：0~15min
220nm, 15~20min 200nm, 20~25min 250nm, 25~90nm 220nm, 90~109min 250nm, 109~
116min 200nm, 116~170min 220nm.
Wherein, flow velocity 0.5-1.5mL/min in high performance liquid chromatography described in step (2)；Sample size 10-40 μ L；Column
Warm 15-30 DEG C.
Further, flow velocity 1.0mL/min in high performance liquid chromatography described in step (2)；20 μ L of sample size；Column temperature 20
℃。
Wherein, the similarity and/or Characteristic chromatographic peak difference of comparative sample HPLC collection of illustrative plates and radix cyathulae HPLC finger-prints
Differentiate the radix cyathulae true and false.
Further, the radix cyathulae HPLC finger-prints have 18 shared chromatographic peaks, are reference peak with No. 9 peaks, each total
There is the relative retention time of chromatographic peak to be followed successively by：0.037、0.055、0.064、0.149、0.210、0.259、0.484、0.722、
1.000,1.030,1.082,1.112,1.146,1.153,1.170,1.279,1.281,1.289, each chromatographic peak is opposite to be retained
Time tolerance is ± 5%；
It is arranged by retention time ascending order, each chromatographic peak is respectively：No. 1 peak：Retention time 3.8min；No. 2 peaks：Retention time
5.7min；No. 3 peaks：Retention time 6.6min；No. 4 peaks：Retention time 15.3min；No. 5 peaks：Retention time 21.6min；No. 6
Peak：Retention time 26.6min；No. 7 peaks：Retention time 49.7min；No. 8 peaks：Retention time 74.1min；No. 9 peaks：Retention time
102.6min；No. 10 peaks：Retention time 105.7min；No. 11 peaks：Retention time 111.0min；No. 12 peaks：Retention time
114.2min；No. 13 peaks：Retention time 117.7min；No. 14 peaks：Retention time 118.4min；No. 15 peaks：Retention time
120.0min；No. 16 peaks：Retention time 131.2min；No. 17 peaks：Retention time 131.5min；No. 18 peaks：Retention time
132.3min, each chromatographic peak retention time tolerance are ± 0.5min.
Further, the similarity criterion is：Institute's sample product HPLC collection of illustrative plates and radix cyathulae HPLC finger-print phases
It is radix cyathulae certified products to be more than 0.9 like degree, and similarity is less than 0.5 for radix cyathulae adulterant or mixes adulterant radix cyathulae.Using Chinese medicine chromatography
(2004A editions) above-mentioned similarities of evaluation of fingerprint similarity evaluation system.
Further, the Characteristic chromatographic peak diversity judgement standard is：
(1) No. 17 peak, retention time 131.5min, nearby a small amount of low-response value tag chromatographic peak inspection without chromatographic peak or only
Go out, then institute's sample product are radix cyathulae certified products, specific as follows：
Peak area is 0 at retention time 128.6min；Peak area is 0~81.4 at retention time 130.7min；When reservation
Between at 131.2min peak area be 0~61.3；Peak area is 0~68.9 at retention time 133.5min；Retention time
Peak area is 0 at 134.3min；Peak area is 0 at retention time 137.2min；At retention time 138.7min peak area be 0~
56.2；Peak area is 0 at retention time 142.3min；Peak area is 0 at retention time 148.8min；
Each chromatographic peak retention time tolerance is ± 0.5min, and each chromatographic peak peak area tolerance is ± 10%；
(2) No. 17 peaks, retention time 131.5min nearby have a large amount of high response Characteristic chromatographic peak detections, then institute's sample
Product are radix cyathulae adulterant, specific as follows：
Peak area is more than 146.9 at retention time 128.6min；Peak area is more than 568.0 at retention time 130.7min；
Peak area is more than 137.2 at retention time 131.2min；Peak area is more than 1254.4 at retention time 133.5min；Retention time
Peak area is more than 171.5 at 134.3min；Peak area is more than 2499.8 at retention time 137.2min；Retention time 138.7min
Locate peak area and is more than 353.0；Peak area is more than 17.1 at retention time 142.3min；Peak area is big at retention time 148.8min
In 40.3；
Each chromatographic peak retention time tolerance is ± 0.5min, and each chromatographic peak peak area tolerance is ± 10%；
(3) there is chromatographic peak detection at retention time 133.5min and 137.2min, then institute's sample product mix adulterant river ox for 5%
Knee, it is specific as follows：
Peak area is 92.5~180.2 at retention time 133.5min；Peak area is 137.5 at retention time 137.2min
~220.5；
Adulterant Characteristic chromatographic peak detection quantity and peak area size are proportionate with pseudo- proportion is mixed in sample HPLC collection of illustrative plates, institute
State adulterant Characteristic chromatographic peak include retention time be 128.6,130.7,131.2,133.5,134.3,137.2,138.7,
142.3, the peak of 148.8min；
Each chromatographic peak retention time tolerance is ± 0.5min, and each chromatographic peak peak area tolerance is ± 10%.
Wherein, the radix cyathulae adulterant includes headdress flower cup amaranth and/or the miscellaneous root of bidentate achyranthes；It is described to mix adulterant radix cyathulae to be blended with
5% or more headdress flower cup amaranth and/or the radix cyathulae of the miscellaneous root of bidentate achyranthes.
In practical applications, each chromatographic peak retention time of gained radix cyathulae finger-print and radix cyathulae HPLC fingerprints of the present invention
Whether each chromatographic peak retention time of collection of illustrative plates is consistent, not important, as long as the opposite reservation between each chromatographic peak of gained finger-print
Time is consistent with the present invention, or in the error range that allows of the present invention, all should at last protection scope of the present invention it
Interior, described relative retention time herein can be a chromatographic peak being arbitrarily designated in finger-print for reference to calculating institute behind peak
The relative retention time obtained is not limited to relative retention time listed in this specification.
Detection method provided by the invention uses multi-wavelength handoff technique based on 220nm wavelength, in part-time section,
Solves the contradiction that multicomponent can not all be detected because absorbing wavelength difference is big under single wavelength.
Detection method provided by the invention can detect to mix pseudo- proportion and mix adulterant radix cyathulae 5% or more, and without pair
Peak information carries out statistical disposition, directly observation comparison be it could be assumed that, taste the mirror such as flavour compared to traditional observation appearance, mouth
Other method has the advantages that high sensitivity, efficiently and accurately.
Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific implementation mode of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Description of the drawings
Fig. 1 is radix cyathulae HPLC finger-prints；
Fig. 2 is adulterant headdress flower cup amaranth, mixes the comparison of pseudo- radix cyathulae and radix cyathulae finger-print, wherein S1 is radix cyathulae, S2
To mix pseudo- radix cyathulae, S3 headdress flower cup amaranths；
Fig. 3 is adulterant, mixes the comparison of pseudo- radix cyathulae and radix cyathulae finger-print, wherein A01~A10 is radix cyathulae, C01
~C08 be mix pseudo- radix cyathulae, B01~B04 is headdress flower cup amaranth, B05~B08 is the miscellaneous root of bidentate achyranthes；
Fig. 4 is that difference mixes pseudo- ratio radix cyathulae HPLC chromatogram, and wherein D01~D10, which is followed successively by, to be mixed pseudo- ratio and be respectively
0%, 1%, 2%, 5%, 10%, 20%, 30%, 50%, 75%, 100% mixes pseudo- radix cyathulae.
Specific implementation mode
The structure of 1 radix cyathulae HPLC finger-prints of embodiment and evaluation
1, instrument and material
High performance liquid chromatograph (1260 types of Agilent, Anjelen Sci. ＆ Tech. Inc of the U.S.)；Methanol, acetonitrile, phosphoric acid
For chromatographically pure；Water is ultra-pure water；Radix cyathulae medicinal material is acquired from Jinkouhe District, Baoxing County, Enshi City and Xuanen County and other places, identified
It is the dry root of amaranthaceous plant radix cyathulae (Cyathula officinalis Kuan).
2, construction method
(1) prepared by test solution：Radix cyathulae medicinal powder (crossing No. three sieves) 1.0g is taken, it is accurately weighed, set tool plug taper
In bottle, 45% methanol aqueous solution 20mL, close plug is added in precision, and weighed weight is ultrasonically treated 1h, lets cool, then weighed weight, uses
45% methanol aqueous solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
(2) chromatographic condition：Chromatographic column：Agilent ZORBAX SB-C18 250mm × 4.6mm, 5 μm；It is stream with acetonitrile
Dynamic phase A, using 0.2% phosphate aqueous solution as Mobile phase B；Gradient elution program is as follows：
Detection wavelength switches for multi-wavelength：0~15min 220nm, 15~20min 200nm, 20~25min 250nm,
25~90nm 220nm, 90~109min 250nm, 109~116min 200nm, 116~170min 220nm；Flow velocity
1.0ml/min；20 μ L of sample size；20 DEG C of column temperature.
(3) accurate test solution of drawing injects high performance liquid chromatograph, measure to get.
According to the method described above, the HPLC collection of illustrative plates of 10 batches of radix cyathulae medicinal materials is measured, chromatogram is referring to A01~A10 in Fig. 3.
(4) fingerprint map construction：10 batches of radix cyathulae medicinal material HPLC collection of illustrative plates of gained import chromatographic fingerprints of Chinese materia medica similarity
Evaluation system (2004A editions), using median method, retention time window is 0.5min, and Auto-matching common characteristic peaks generate river
Root of bidentate achyranthes HPLC finger-prints.Finger-print is referring to Fig. 1.
3, HPLC finger-prints
The radix cyathulae HPLC finger-prints of generation have 18 common characteristic peaks, are arranged by retention time ascending order, each chromatography
Peak is respectively：No. 1 peak：Retention time 3.8min；No. 2 peaks：Retention time 5.7min；No. 3 peaks：Retention time 6.6min；No. 4
Peak：Retention time 15.3min；No. 5 peaks：Retention time 21.6min；No. 6 peaks：Retention time 26.6min；No. 7 peaks：Retention time
49.7min；No. 8 peaks：Retention time 74.1min；No. 9 peaks：Retention time 102.6min；No. 10 peaks：Retention time 105.7min；
No. 11 peaks：Retention time 111.0min；No. 12 peaks：Retention time 114.2min；No. 13 peaks：Retention time 117.7min；No. 14
Peak：Retention time 118.4min；No. 15 peaks：Retention time 120.0min；No. 16 peaks：Retention time 131.2min；No. 17 peaks：It protects
Stay time 131.5min；No. 18 peaks：Retention time 132.3min.
4, similarity analysis
It is that control collection of illustrative plates carries out similarity analysis with gained radix cyathulae HPLC finger-prints, 10 batch radix cyathulae samples are similar
Degree be respectively 0.972,0.997,0.949,0.965,0.996,0.981,0.997,0.946,0.995,0.956, each sample with
Radix cyathulae reference fingerprint similarity is all higher than 0.9.Explanation：Radix cyathulae HPLC finger-prints obtained by the method for the present invention have very
Strong representativeness.
The application detection method of embodiment 2 differentiates headdress flower cup amaranth and the miscellaneous root of bidentate achyranthes
1, instrument and material
High performance liquid chromatograph (1260 types of Agilent, Anjelen Sci. ＆ Tech. Inc of the U.S.)；Methanol, acetonitrile, phosphoric acid
For chromatographically pure；Water is ultra-pure water；8 screwdriver bits spend cup amaranth and the miscellaneous root of bidentate achyranthes, are collected in Jinkouhe District, Baoxing County, the counties E Bian, are identified as
The root of the root and the miscellaneous root of bidentate achyranthes (C.officinalis*C.capitata) of headdress flower cup amaranth (C.capitata Moq.).
2, differentiate
(1) prepared by test solution：Measuring samples powder (crossing No. three sieves) 0.5g is taken, it is accurately weighed, set conical flask with cover
In, 30% methanol aqueous solution 20mL, close plug is added in precision, and weighed weight is ultrasonically treated 0.5h, lets cool, then weighed weight, uses
30% methanol aqueous solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
(2) chromatographic condition：Chromatographic column：Agilent ZORBAX SB-C18 250mm × 4.6mm, 5 μm；It is stream with acetonitrile
Dynamic phase A, using 0.1% phosphate aqueous solution as Mobile phase B；Gradient elution program is as follows：
Detection wavelength switches for multi-wavelength：0~15min 220nm, 15~20min 200nm, 20~25min 250nm,
25~90nm 220nm, 90~109min 250nm, 109~116min 200nm, 116~170min 220nm；Flow velocity
1.5mL/min；40 μ L of sample size；15 DEG C of column temperature.
(3) accurate test solution of drawing injects high performance liquid chromatograph, measure to get.
According to the above method, measure 8 screwdriver bits flower cup amaranth and miscellaneous root of bidentate achyranthes HPLC collection of illustrative plates respectively, chromatogram referring to B01 in Fig. 3~
B08, wherein B01~B04 are headdress flower cup amaranth, B05~B08 is the miscellaneous root of bidentate achyranthes.
(4) result
8 sample HPLC collection of illustrative plates of gained are imported into similarity evaluation (2004A editions), with reality
Apply 1 gained radix cyathulae HPLC finger-prints of example be control collection of illustrative plates, carry out similarity evaluation, as a result show 8 crowdes of measuring samples B01~
The similarity difference 0.254,0.014,0.009,0.302,0.383,0.305,0.010,0.001, respectively less than 0.5 of B08；Gained
8 batches of sample HPLC chromatograms carry out characteristic peak variance analysis with 1 gained radix cyathulae HPLC finger-prints of embodiment, as a result show 8
A sample near retention time 131.5min (No. 17 peaks), retention time 128.6,130.7,131.2,133.5,134.2,
137.2,138.7,142.3, high response characteristic chromatographic peak detects at 148.8min, the peak of each batch of each Characteristic chromatographic peak of sample
Area is shown in Table 1.
18 screwdriver bit of table spends the peak area of cup amaranth and each Characteristic chromatographic peak of the miscellaneous root of bidentate achyranthes
(5) differentiate
The similarity of 8 batches of measuring samples and 1 gained radix cyathulae HPLC finger-prints of embodiment is respectively less than 0.5, and this 8 batches
Measuring samples are satisfied by near retention time 131.5min (No. 17 peaks)：Peak area is more than at retention time 128.6min
146.9；Peak area is more than 568.0 at retention time 130.7min；Peak area is more than 137.2 at retention time 131.2min；It protects
Peak area at time 133.5min is stayed to be more than 1254.4；Peak area is more than 171.5 at retention time 134.3min；Retention time
Peak area is more than 2499.8 at 137.2min；Peak area is more than 353.0 at retention time 138.7min；Retention time 142.3min
Locate peak area and is more than 17.1；Peak area is more than 40.3 at retention time 148.8min, meets adulterant river in the application detection method
The criterion of the root of bidentate achyranthes.According to detection method, which is accredited as adulterant radix cyathulae (headdress flower cup amaranth
And/or the miscellaneous root of bidentate achyranthes).Identification result is consistent with sample message.
Description of test, the application detection method can accurately differentiate adulterant radix cyathulae (headdress flower cup amaranth and the miscellaneous root of bidentate achyranthes), credible
Degree is high.
The application detection method of embodiment 3 differentiate mix pseudo- 5% headdress flower cup amaranth, the miscellaneous root of bidentate achyranthes radix cyathulae
1, instrument and material
High performance liquid chromatograph (1260 types of Agilent, Anjelen Sci. ＆ Tech. Inc of the U.S.)；Methanol, acetonitrile, phosphoric acid
For chromatographically pure；Water is ultra-pure water；Measuring samples are 8 batches and mix pseudo- 5% radix cyathulae, mix fake method be by headdress flower cup amaranth, the miscellaneous root of bidentate achyranthes by
Proportion is mixed into radix cyathulae, and crushing is to be checked, mixes pseudo- 5% radix cyathulae sample source and proportion is shown in Table 2.
28 batches 5%, table mixes pseudo- radix cyathulae and mixes fake information
2, differentiate
(1) prepared by test solution：Measuring samples powder (crossing No. three sieves) 2.0g is taken, it is accurately weighed, set conical flask with cover
In, 60% methanol aqueous solution 40mL, close plug is added in precision, and weighed weight is ultrasonically treated 2h, lets cool, then weighed weight, with 60%
Methanol aqueous solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
(2) chromatographic condition：Chromatographic column：Agilent ZORBAX SB-C18 250mm × 4.6mm, 5 μm；It is stream with acetonitrile
Dynamic phase A, using 0.3% phosphate aqueous solution as Mobile phase B；Gradient elution program is as follows：
Detection wavelength switches for multi-wavelength：0~15min 220nm, 15~20min 200nm, 20~25min 250nm,
25~90nm 220nm, 90~109min 250nm, 109~116min 200nm, 116~170min 220nm；Flow velocity
0.5mL/min；20 μ L of sample size；15 DEG C of column temperature.
(3) accurate test solution of drawing injects high performance liquid chromatograph, measure to get.
According to the above method, measures 8 batches and mix pseudo- radix cyathulae HPLC collection of illustrative plates, chromatogram is referring to C01~C08 in Fig. 3.
(4) result
8 sample HPLC collection of illustrative plates of gained are imported into similarity evaluation (2004A editions), with reality
It is control collection of illustrative plates to apply 1 gained radix cyathulae HPLC finger-prints of example, carries out similarity evaluation, and as a result showing 8 batches mixes pseudo- radix cyathulae C01
The similarity of~C08 is followed successively by 0.251,0.303,0.028,0.382,0.391,0.399,0.364,0.028, respectively less than 0.5；
8 sample HPLC chromatograms of gained carry out characteristic peak variance analysis with 1 gained radix cyathulae HPLC finger-prints of embodiment, as a result show
Show that 8 samples nearby detect 2 Characteristic chromatographic peaks in retention time 131.5min (No. 17 peaks), wherein C1~C8 samples are retaining
At time 133.5min the peak area of Characteristic chromatographic peak be followed successively by 137.2,180.2,92.5,111.8,170.6,161.8,
151.1,117.1, C1~C8 samples Characteristic chromatographic peak peak area at retention time 137.2min is followed successively by, 141.7,183.4,
137.2、130.7、220.5、195.2、170.2、137.5。
(5) differentiate
The similarity of 8 batches of measuring samples and 1 gained radix cyathulae HPLC finger-prints of embodiment is respectively less than 0.5, and this 8 batches
Measuring samples are satisfied by：Peak area is 92.5~180.2 at retention time 133.5min；Peak area at retention time 137.2min
It is 137.5~220.5, meets the criterion for mixing pseudo- 5% radix cyathulae in the application detection method.According to detection side of the invention
Method, 8 batches of measuring samples are accredited as mixing pseudo- 5% radix cyathulae.Identification result mixes fake information with sample and is consistent.
Description of test, the application detection method can accurately identify that mix pseudo- ratio be 5% to mix adulterant radix cyathulae, method
It is with a high credibility.
The application detection method of embodiment 4 differentiates that difference mixed pseudo- proportion mixes adulterant radix cyathulae
1, instrument and material
High performance liquid chromatograph (1260 types of Agilent, Anjelen Sci. ＆ Tech. Inc of the U.S.)；Methanol, acetonitrile, phosphoric acid
For chromatographically pure；Water is ultra-pure water；Measuring samples are that 10 batches of differences mix the radix cyathulae of pseudo- ratio, mix fake method be by headdress flower cup amaranth,
The miscellaneous root of bidentate achyranthes is mixed by proportion in radix cyathulae, and crushing is to be checked, and difference mixes pseudo- ratio radix cyathulae sample source and proportion is shown in Table 3.
3 10 batches of differences of table mix pseudo- ratio radix cyathulae and mix fake information
2, differentiate
(1) prepared by test solution：Measuring samples powder (crossing No. three sieves) 1.0g is taken, it is accurately weighed, set conical flask with cover
In, 45% methanol aqueous solution 20mL, close plug is added in precision, and weighed weight is ultrasonically treated 1h, lets cool, then weighed weight, with 45%
Methanol aqueous solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
(2) chromatographic condition：Chromatographic column：Agilent ZORBAX SB-C18 250mm × 4.6mm, 5 μm；It is stream with acetonitrile
Dynamic phase A, using 0.2% phosphate aqueous solution as Mobile phase B；Gradient elution program is as follows：
Detection wavelength switches for multi-wavelength：0~15min 220nm, 15~20min 200nm, 20~25min 250nm,
25~90nm 220nm, 90~109min 250nm, 109~116min 200nm, 116~170min 220nm；Flow velocity
1.0mL/min；20 μ L of sample size；20 DEG C of column temperature.
(3) accurate test solution of drawing injects high performance liquid chromatograph, measure to get.
According to the above method, measures 10 batches of differences and mix pseudo- ratio radix cyathulae HPLC collection of illustrative plates, chromatogram is referring to Fig. 4.
(4) result
10 sample HPLC collection of illustrative plates of gained are imported into similarity evaluation (2004A editions), with
1 gained radix cyathulae HPLC finger-prints of embodiment are control collection of illustrative plates, carry out similarity evaluation, and as a result showing 10 batches mixes pseudo- radix cyathulae
The similarity of D01~D10 is followed successively by 0.948,0.903,0.712,0.258,0.011,0.008,0.006,0.005,0.005,
0.005, after wherein sample mixes pseudo- ratio more than or equal to 5%, HPLC collection of illustrative plates is respectively less than 0.5 with the similarity for compareing collection of illustrative plates；It will
10 sample HPLC chromatograms of gained carry out characteristic peak variance analysis with 1 gained radix cyathulae HPLC finger-prints of embodiment, obtain
10 samples are at retention time 128.6,130.7,131.2,133.5,134.2,137.2,138.7,142.3,148.8min
Characteristic peak situation, characteristic peak quantity and peak area are shown in Table 4.The result shows that the quantity and peak area of characteristic peak mix pseudo- ratio with radix cyathulae
Example is proportionate.
4 difference of table mix pseudo- ratio radix cyathulae adulterant characteristic peak quantity and peak area ratio compared with
(5) differentiate
Sample D04~D10 and the similarity of 1 gained radix cyathulae HPLC finger-prints of embodiment are respectively less than 0.5, and sample
It is 92.5~180.2 that D04, which meets peak area at retention time 133.5min,；Peak area is 137.5 at retention time 137.2min
~220.5, meet the criterion that pseudo- 5% radix cyathulae is mixed in the application detection method, sample D05~D10 adulterant feature peak numbers
Amount is gradually increased with area.
According to detection method, what sample D04 was accredited as mixing puppet 5% mixes adulterant radix cyathulae, sample D05~D10
It is accredited as mixing that pseudo- proportion is all higher than 5% and what is be sequentially increased mixes adulterant radix cyathulae.Identification result mixes fake information with sample and is consistent.
Description of test, the present invention can detect to mix pseudo- proportion and mix adulterant radix cyathulae 5% or more, and the method for the present invention is surveyed
Fixed mixes in pseudo- radix cyathulae HPLC collection of illustrative plates, and the quantity and peak area of adulterant characteristic peak and radix cyathulae mix pseudo- ratio and be proportionate.
Detection method provided by the invention uses multi-wavelength handoff technique based on 220nm wavelength, in part-time section,
Solves the contradiction that multicomponent can not all be detected because absorbing wavelength difference is big under single wavelength.
Detection method provided by the invention can detect to mix pseudo- proportion and mix adulterant radix cyathulae 5% or more, and without pair
Peak information carries out statistical disposition, directly observation comparison be it could be assumed that, taste the mirror such as flavour compared to traditional observation appearance, mouth
Other method has the advantages that high sensitivity, efficiently and accurately.
Claims (10)
1. a kind of detection method of radix cyathulae medicinal material, it is characterised in that：The method is detected using HPLC finger-prints
, including following operating procedure：
(1) prepared by test solution：Take medicinal material to be checked, extract, filtration, take subsequent filtrate to get.
(2) it draws test solution and injects high performance liquid chromatograph, chromatographic condition is as follows：
Chromatographic column：Using octadecylsilane chemically bonded silica as filler；
Mobile phase：Using acetonitrile as mobile phase A, using 0.1-0.3% phosphate aqueous solutions as Mobile phase B.
2. detection method according to claim 1, it is characterised in that：In step (1), the test solution preparation method
For：Take medicinal powder to be checked, methanol aqueous solution added to extract, filter, take subsequent filtrate to get；
Described to be extracted as ultrasonic extraction, the ultrasonic extraction time is 0~2 hour, it is preferable that ultrasonic time is 1 hour；
The concentration of the methanol aqueous solution is 30~60%；And/or the methanol aqueous solution addition be medicinal powder 5~
80v/w times.
3. detection method according to claim 2, it is characterised in that：The concentration of the methanol aqueous solution is 40~50%；
And/or the methanol aqueous solution addition is 10~40v/w times of medicinal powder；Preferably, the concentration of the methanol aqueous solution
It is 45%；And/or the methanol aqueous solution addition is 20v/w times of medicinal powder.
4. detection method according to claim 1, it is characterised in that：In step (2), the Mobile phase B is 0.2% phosphoric acid
Aqueous solution, using gradient elution, elution program is：
5. detection method according to claim 1, it is characterised in that：In step (2), examined in the high performance liquid chromatography
Wavelength is surveyed for multi-wavelength to switch：0~15min 220nm, 15~20min 200nm, 20~25min 250nm, 25~90nm
220nm, 90~109min 250nm, 109~116min 200nm, 116~170min 220nm.
6. detection method according to claim 1, it is characterised in that：In step (2), the chromatographic column is Agilent
ZORBAX SB-C18 columns, chromatographic column specification be 250mm × 4.6mm, 5 μm；Flow velocity 0.5- in the high performance liquid chromatography
1.5mL/min, sample size 10-40 μ L, 15-30 DEG C of column temperature；Preferably, flow velocity 1.0mL/min in the high performance liquid chromatography,
20 μ L of sample size, 20 DEG C of column temperature.
7. detection method according to claim 1, it is characterised in that：Comparative sample HPLC collection of illustrative plates and radix cyathulae HPLC fingerprints
The similarity and/or Characteristic chromatographic peak difference of collection of illustrative plates differentiate the radix cyathulae true and false.
8. detection method according to claim 7, it is characterised in that：The radix cyathulae HPLC finger-prints have 18 to share
Chromatographic peak is with reference to peak with No. 9 peaks, and the relative retention time of each shared chromatographic peak is followed successively by：0.037、0.055、0.064、
0.149、0.210、0.259、0.484、0.722、1.000、1.030、1.082、1.112、1.146、1.153、1.170、
1.279,1.281,1.289, each chromatographic peak relative retention time tolerance is ± 5%；
Preferably, it is arranged by retention time ascending order, each chromatographic peak is respectively：No. 1 peak：Retention time 3.8min；No. 2 peaks：Retain
Time 5.7min；No. 3 peaks：Retention time 6.6min；No. 4 peaks：Retention time 15.3min；No. 5 peaks：Retention time 21.6min；6
Number peak：Retention time 26.6min；No. 7 peaks：Retention time 49.7min；No. 8 peaks：Retention time 74.1min；No. 9 peaks：When reservation
Between 102.6min；No. 10 peaks：Retention time 105.7min；No. 11 peaks：Retention time 111.0min；No. 12 peaks：Retention time
114.2min；No. 13 peaks：Retention time 117.7min；No. 14 peaks：Retention time 118.4min；No. 15 peaks：Retention time
120.0min；No. 16 peaks：Retention time 131.2min；No. 17 peaks：Retention time 131.5min；No. 18 peaks：Retention time
132.3min, each chromatographic peak retention time tolerance are ± 0.5min.
9. detection method according to claim 7, it is characterised in that：
The similarity criterion is：Institute's sample product HPLC collection of illustrative plates is more than 0.9 with radix cyathulae HPLC fingerprint similarities
Radix cyathulae certified products, similarity are less than 0.5 for radix cyathulae adulterant or mix adulterant radix cyathulae；
The Characteristic chromatographic peak diversity judgement standard is：
(1) No. 17 peak, retention time 131.5min, a small amount of low-response value tag chromatographic peak detection without chromatographic peak or only nearby,
Then institute's sample product are radix cyathulae certified products, specific as follows：
Peak area is 0 at retention time 128.6min；Peak area is 0~81.4 at retention time 130.7min；Retention time
Peak area is 0~61.3 at 131.2min；Peak area is 0~68.9 at retention time 133.5min；Retention time 134.3min
It is 0 to locate peak area；Peak area is 0 at retention time 137.2min；Peak area is 0~56.2 at retention time 138.7min；It protects
It is 0 to stay peak area at time 142.3min；Peak area is 0 at retention time 148.8min；
Each chromatographic peak retention time tolerance is ± 0.5min, and each chromatographic peak peak area tolerance is ± 10%；
(2) No. 17 peaks, retention time 131.5min nearby have a large amount of high response Characteristic chromatographic peak detections, then institute's sample product are
Radix cyathulae adulterant, it is specific as follows：
Peak area is more than 146.9 at retention time 128.6min；Peak area is more than 568.0 at retention time 130.7min；Retain
Peak area is more than 137.2 at time 131.2min；Peak area is more than 1254.4 at retention time 133.5min；Retention time
Peak area is more than 171.5 at 134.3min；Peak area is more than 2499.8 at retention time 137.2min；Retention time 138.7min
Locate peak area and is more than 353.0；Peak area is more than 17.1 at retention time 142.3min；Peak area is big at retention time 148.8min
In 40.3；
Each chromatographic peak retention time tolerance is ± 0.5min, and each chromatographic peak peak area tolerance is ± 10%；
(3) there is chromatographic peak detection at retention time 133.5min and 137.2min, then institute's sample product mix adulterant radix cyathulae for 5%,
It is specific as follows：
Peak area is 92.5~180.2 at retention time 133.5min；At retention time 137.2min peak area be 137.5~
220.5；
Adulterant Characteristic chromatographic peak detection quantity and peak area size are proportionate with pseudo- proportion is mixed in sample HPLC collection of illustrative plates, the puppet
Product Characteristic chromatographic peak include retention time be 128.6,130.7,131.2,133.5,134.3,137.2,138.7,142.3,
The peak of 148.8min；
Each chromatographic peak retention time tolerance is ± 0.5min, and each chromatographic peak peak area tolerance is ± 10%.
10. detection method according to claim 9, it is characterised in that：The radix cyathulae adulterant include headdress flower cup amaranth and/or
The miscellaneous root of bidentate achyranthes；It is described that mix adulterant radix cyathulae be to be blended with 5% or more headdress flower cup amaranth and/or the radix cyathulae of the miscellaneous root of bidentate achyranthes.
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