CN103630614B - The high-efficiency liquid chromatography method for detecting of blood-nourishing and brain-refreshing granules - Google Patents
The high-efficiency liquid chromatography method for detecting of blood-nourishing and brain-refreshing granules Download PDFInfo
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- 239000008187 granular material Substances 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004811 liquid chromatography Methods 0.000 title claims description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 78
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- 238000001514 detection method Methods 0.000 claims abstract description 16
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- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 claims description 23
- 235000001368 chlorogenic acid Nutrition 0.000 claims description 23
- 229940074393 chlorogenic acid Drugs 0.000 claims description 23
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 claims description 23
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 claims description 23
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 claims description 22
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims description 22
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- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims description 22
- 235000001785 ferulic acid Nutrition 0.000 claims description 22
- 229940114124 ferulic acid Drugs 0.000 claims description 22
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims description 22
- 229940074391 gallic acid Drugs 0.000 claims description 22
- 235000004515 gallic acid Nutrition 0.000 claims description 22
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 229930182478 glucoside Natural products 0.000 claims description 16
- 239000013558 reference substance Substances 0.000 claims description 15
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 13
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Abstract
The invention provides and a kind of detect the method for chemical composition in blood-nourishing and brain-refreshing granules, the detection sample of blood-nourishing and brain-refreshing granules uses methanol supersound extraction, use high performance liquid chromatography mass spectrum/mass spectrometry to analyze the chemical composition of preparation, use the index components of high performance liquid chromatography quantitative analysis preparation.The present invention has the most linear, repeatability, repeatability and the response rate, contributes to controlling the quality of blood-nourishing and brain-refreshing granules more comprehensively.
Description
Technical field
The invention belongs to medicine, chemical field, be specifically related to chemical composition in modern Chinese medicine product blood-nourishing and brain-refreshing granules
Detection method.
Background technology
Blood-nourishing and brain-refreshing granules is by Radix Angelicae Sinensis, Rhizoma Chuanxiong, the Radix Paeoniae Alba, Radix Rehmanniae Preparata, Ramulus Uncariae Cum Uncis, Caulis Spatholobi, Spica Prunellae, Semen Cassiae, Margarita
Mother, Rhizoma Corydalis, the compound preparation of Herba Asari 11 taste Chinese medicine composition, have effect of the suppressing the hyperactive liver that nourishes blood, activating collaterals to relieve pain, be mainly used in treatment
Headache caused by blood deficiency and excessive liver-YANG, stagger, susceptible to lose temper due to restlessness, insomnia and dreamful sleep etc..It it is the herbal species by special-protection-by-the-State.
Although blood-nourishing and brain-refreshing granules clinical efficacy is definite, but the material base research for said preparation needs deeply: at present
14 kinds of compositions in the at most the most qualitative blood-nourishing and brain-refreshing granules in document (Li Wenbo, Han Jianping, Gao Jun, etc. blood-nourishing and brain-refreshing granules
Efficient liquid-phase chromatograph finger print atlas is studied. analytical chemistry, and 2011,39 (3): 387-391), for the big compound recipe of 11 taste medicines, its base
This material base needs to be studied further, further to define the constituent of said preparation.Additionally, for blood-nourishing and brain-refreshing granules
Quality control be left to be desired: at present national standard (YBZ29552005-2009Z) only carries out quality to one composition of peoniflorin
Control, it is impossible to control while realizing multi-flavor medical material multicomponent quality.
For controlling blood-nourishing and brain-refreshing granules quality more comprehensively, set up the quality control standard meeting the modernization of Chinese medicine, need
One can the multiple chemical composition of the most qualitative blood-nourishing and brain-refreshing granules, and quantitatively in blood-nourishing and brain-refreshing granules many indexes composition point
Analysis method.
Summary of the invention
In order to realize foregoing invention purpose, the invention provides and a kind of detect multiple chemical composition in blood-nourishing and brain-refreshing granules
Method.
Blood-nourishing and brain-refreshing granules high-efficiency liquid chromatography method for detecting according to the present invention, detection sample uses the first of 10% ~ 50%
Alcohol extraction, chromatographic condition is for using C18Chromatographic column, flowing is phosphoric acid solution-acetonitrile mutually, and detector is diode array detector.
High-efficient liquid phase color is used on the basis of High Performance Liquid Chromatography/Mass Spectrometry/mass spectrometry analyzes the chemical composition of preparation
The index components of spectrum quantitative analysis preparation.
According to one of embodiment of the present invention, detection sample uses 25% methanol supersound extraction.Inventor is experimentally verified that,
Said extracted condition extracts active ingredients efficiency is the highest.
One of according to the embodiment of the present invention, chromatogram flow phase A phase is 0.01% ~ 0.05% phosphoric acid solution, and B phase is second
Nitrile.Preferably, chromatogram flow phase A phase is 0.02% phosphoric acid solution, and B phase is acetonitrile, and gradient elution method is:
0～10min, 5%B phase → 10%B phase；
10～15min, 10%B phase → 12%B phase；
15～25min, 12%B phase → 15%B phase；
25～35min, 15%B phase → 18%B phase；
35～40min, 18%B phase → 19%B phase；
40～43min, 19%B phase.
According to another embodiment of the present invention, chromatogram flow phase flow velocity is 0.5-1.5mL/min, it is preferable that flowing phase
Flow velocity is 0.9-1.2mL/min.
According to a further embodiment of the present invention, column temperature is 25 ~ 35 DEG C.Preferably, column temperature is 30 DEG C.Inventor investigates
The impact of separating effect is used by column temperature (not temperature control, 25 DEG C, 30 DEG C, 35 DEG C), in the case of result shows column temperature 30 DEG C, and preparation
In component peak shape to be measured symmetrical, separating degree is high and analysis result is stable.
According to one of embodiment of the present invention, chromatography column feed materials amount is 2.5 ~ 25 μ L.
According to one of embodiment of the present invention, diode array detector is used to become length scanning, chromatographic detector wavelength
For 210-350nm.Preferably, chromatographic detector wavelength is chosen as 280nm, 320nm and 230nm successively.
Experiment proves: gallic acid and protocatechuic acid optimum absorb wavelength are 280nm, chlorogenic acid, caffeic acid, and ferulic acid is
Good absorbing wavelength is 320nm, and lactone glucoside of Radix Paeoniae and peoniflorin optimum absorb wavelength are 230nm.Therefore employing photodiode array detection
Device (DAD) becomes length scanning, can realize the detection at optimal wavelength of multiple composition in once analyzing.
According to one of embodiment of the present invention, before high performance liquid chromatography detects, still further comprise employing mass spectrum pair
Chemical composition in blood-nourishing and brain-refreshing granules carries out the step detected, and testing conditions is: ion source is ESI source, and spray voltage is set to
4kV, sheath atmospheric pressure is 35psi, assists N2Flow velocity is 5L/min, and heated capillary temperature is 300 DEG C, and in source, CID is 10V, just
Anion sweeps m/z:100-1200 entirely.
According to the detection method of the present invention, gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, Chinese herbaceous peony
The assay result of medicine lactone glycoside is respectively 0.376-0.434mg g-1、0.094-0.137mg·g-1、0.611-
0.871mg·g-1、0.113-0.149mg·g-1、0.484-2.194mg·g-1、4.139-5.543mg·g-1And 0.261-
0.326mg·g-1。
The present invention utilizes HPLC to determine the content of 7 kinds of index components in blood-nourishing and brain-refreshing granules, comes relative to national standard
Say, blood-nourishing and brain-refreshing granules quality can be controlled more comprehensively.The present invention has the most linear, repeatability, repeatability and the response rate,
Contribute to controlling the quality of blood-nourishing and brain-refreshing granules more comprehensively.By experiment in vivo it has been determined that peoniflorin and lactone glucoside of Radix Paeoniae are for entering
Blood component, therefore said determination method can be later stage medicine offers reference for the determination of experimental administration amount.
Aspect and advantage that the present invention adds will part be given in the following description, and part will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage will become from the following description of the accompanying drawings of embodiments
Substantially with easy to understand, wherein:
The high-efficient liquid phase chromatogram of Fig. 1 reference substance；
The high-efficient liquid phase chromatogram of Fig. 2 test sample.
Detailed description of the invention
Embodiments of the invention are described below in detail, and the example of described embodiment is shown in the drawings.Below with reference to
The embodiment that accompanying drawing describes is exemplary, is only used for explaining the present invention, and is not construed as limiting the claims.
Experimental example one methodological study and sample determination
1 instrument and reagent
XS205DU electronic balance (Mettler Toledo Inc. of Switzerland), (city of Kunshan surpasses KQ-250VDB ultrasonic cleaner
Sound Instrument Ltd.), liquid chromatograph-mass spectrometer: Thermo Electron Corp.'s Finnigan Surveyor liquid phase systems (bag
Include quaternary gradient pump, vacuum degassing machine, automatic sampler, post constant temperature system and DAD detector)；Finnigan LCQ ion trap
Mass spectrograph, model is LCQ Advantage MAX, Agilent1200 chromatograph of liquid, the AgilentDAD detector (U.S.
Agilent company), gallic acid (lot number: 110831-200803), protocatechuic acid (lot number: 110809-200604), chlorogenic acid
(lot number: 110753-200413), caffeic acid (lot number: 110885-200102), ferulic acid (lot number: 110773-200611), Chinese herbaceous peony
Medicine glycosides (lot number: 110736-200630, assay is used) is provided by Nat'l Pharmaceutical & Biological Products Control Institute, lactone glucoside of Radix Paeoniae
Glycosides (lot number: 10081445, Tianjin one side Science and Technology Ltd.), acetonitrile (chromatographically pure, Merck company), phosphoric acid (chromatographically pure, sky
Tianjin recovery fine chemistry industry institute), water is that MilliQ prepares ultra-pure water.
The preparation of 2 solution
The preparation of 2.1 reference substance solution
Precision weighs gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, and lactone glucoside of Radix Paeoniae compares
Product are appropriate, add 25% methanol and are respectively prepared every 1ml containing gallic acid 3.77 μ g, protocatechuic acid 1.63 μ g, chlorogenic acid 4.05 μ g, coffee
Coffee acid 1.33 μ g, peoniflorin 29.36 μ g, lactone glucoside of Radix Paeoniae 12.95 μ g, the mixed solution of ferulic acid 2.43 μ g, to obtain final product.
The preparation of 2.2 need testing solutions
Take blood-nourishing and brain-refreshing granules appropriate, finely ground, take about 200mg, accurately weighed, put in 25ml measuring bottle, add 25% methanol
25ml, supersound process 30 minutes, let cool, add 25% methanol to scale, shake up, with 0.22 μm filtering with microporous membrane, take subsequent filtrate,
Obtain.
3 mass spectral analyses
Ion source is ESI source, and spray voltage is set to 4kV, and sheath gas (N2) pressure is 35psi, and auxiliary gas (N2) flow velocity is 5L/
Min, heated capillary temperature is 300 DEG C.In source, CID is 10V.Negative ions sweeps m/z:100-1200 entirely.
Utilize HPLC-DADMS/MS to identify 41 kinds of compositions altogether, be shown in Table 1:
The qualitative table of table 1 chemical composition
On the basis of above HPLC-DAD-MSn analyzes, index in therein 7 is become gallic acid, former catechu
Acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, lactone glucoside of Radix Paeoniae carries out HPLC quantitative analysis.
4. chromatographic condition and system suitability
4.1 chromatographic condition
It is chromatographic column with Agilent ZorbaxSB-C18 (250mm × 4.6mm, 5 μm)；With 0.02% phosphoric acid solution (A)-
Acetonitrile (B) carries out gradient elution (0～10min, 5%B → 10%B mutually for flowing；10～15min, 10%B → 12%B；15～
25min, 12%B → 15%B；25～35min, 15%B → 18%B；35～40min, 18%B → 19%B；40～43min, 19%B)；Stream
Speed is 0.95ml min-1；Column temperature is 30 DEG C；Detection wavelength is 230nm, 280nm and 320nm；Sample size is 10 μ l.
4.2 linear relationships are investigated
Precision draws mixing reference substance solution 2.5,5,10,15,20,25 μ l respectively, and sample introduction measures its peak area.Reference substance
Chromatogram is shown in that Fig. 1,1-7 are respectively gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, lactone glucoside of Radix Paeoniae, peoniflorin and Resina Ferulae
Acid.With peak area value Y as vertical coordinate, reference substance concentration (μ g/ml) is abscissa mapping, carries out linear regression analysis, draws foster
The standard curve equation of 7 index components, correlation coefficient and the range of linearity (being shown in Table 2) in blood brain-refreshing granules.Result shows, 7
Composition is in the respective range of linearity, and peak area value and sample size have good linear relationship.
The regression equation of 7 kinds of compositions and the range of linearity in table 2 blood-nourishing and brain-refreshing granules
4.3 precision test
Accurate draw mixing reference substance solution 10 μ l, by above-mentioned chromatographic condition continuous sample introduction 6 times, measure peak area, no food
Son acid, protocatechuic acid, chlorogenic acid, caffeic acid, lactone glucoside of Radix Paeoniae, the RSD of peoniflorin and ferulic acid be respectively 0.271%,
1.947%, 0.356%, 0.387%, 0.342%, 0.508% and 0.506%, show that this instrument precision is good.
4.4 replica test
Take with 6 parts of a batch of sample, prepare need testing solution, measure by above-mentioned chromatographic condition, gallic acid, former catechu
The RSD of acid, chlorogenic acid, caffeic acid, lactone glucoside of Radix Paeoniae, peoniflorin and ferulic acid peak area is respectively 1.813%, 2.364%,
2.870%, 2.231%, 2.484%, 0.475% and 0.726%, show that the repeatability of this method is good.
4.5 recovery test
Precision weighs the blood-nourishing and brain-refreshing granules powder about 100mg of known content, and totally 6 parts, precision adds mixing comparison respectively
Product solution, preparation test solution also measures, gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, lactone glucoside of Radix Paeoniae, peoniflorin and
The response rate meansigma methods of ferulic acid is respectively 99.7%, 95.4%, 100.7%, 101.8%, 103.8%, 97.0% and 104.7%, RSD
It is respectively 2.124%, 1.696%, 1.123%, 4.917%, 2.723% and 2.821%.
The mensuration of 5 samples
The need testing solution of test agent in preparing 10 batches, sample introduction measures, and the accurate 10 μ l that draw inject chromatograph of liquid, measure
Peak area (test sample chromatogram is shown in Fig. 2), calculating gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin,
Lactone glucoside of Radix Paeoniae peak content, the results are shown in Table 3.Gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, Radix Paeoniae
The average content of lactone glycoside is respectively 0.395,0.108,0.709,0.134,1.759,4.602 and 0.291mg g-1.
Table 3 blood-nourishing and brain-refreshing granules assay result (mg g-1)
The optimization experiment of experimental example two Extraction solvent
Respectively using 10% methanol, 25% methanol, 50% methanol as Extraction solvent, prepare need testing solution, measure 7 kinds effectively
One-tenth gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, peoniflorin, lactone glucoside of Radix Paeoniae, the peak area of ferulic acid, and
25% methanol processing each component area in sample and is designated as 100%, obtain different solvents extraction ratio, result is as shown in table 4:
The impact on extraction ratio of the table 4 variable concentrations methanol
From experimental result, using the methanol extraction of 25%, the extraction ratio of 7 kinds of compositions to be measured is the highest, is that optimum extraction is dense
Degree.
Embodiment 1
The preparation of reference substance solution: precision weighs gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, Radix Paeoniae
Glycosides, lactone glucoside of Radix Paeoniae reference substance is appropriate, adds 25% methanol and is respectively prepared every 1ml containing gallic acid 3.77 μ g, protocatechuic acid 1.63 μ
G, chlorogenic acid 4.05 μ g, caffeic acid 1.33 μ g, peoniflorin 29.36 μ g, lactone glucoside of Radix Paeoniae 12.95 μ g, ferulic acid 2.43 μ g mixed
And solution, to obtain final product.
The preparation of need testing solution: take blood-nourishing and brain-refreshing granules appropriate, finely ground, take about 200mg, accurately weighed, put 25ml amount
In Ping, add 25% methanol 25ml, supersound process 30 minutes, let cool, add 25% methanol to scale, shake up, with 0.22 μm microporous filter membrane
Filter, take subsequent filtrate, to obtain final product.
Chromatographic condition: be chromatographic column with Agilent ZorbaxSB-C18 (250mm × 4.6mm, 5 μm)；With 0.02% phosphoric acid
Solution (A)-acetonitrile (B) carries out gradient elution (0～10min, 5%B → 10%B mutually for flowing；10～15min, 10%B → 12%B；
15～25min, 12%B → 15%B；25～35min, 15%B → 18%B；35～40min, 18%B → 19%B；40～43min, 19%
B)；Flow velocity is 0.95ml min-1；Column temperature is 30 DEG C；Detection wavelength is 230nm, 280nm and 320nm；Sample size is 10 μ l.
The mensuration of sample: the need testing solution of test agent in preparing 10 batches, sample introduction measures, and the accurate 10 μ l that draw inject liquid phase
Chromatograph, measures peak area, calculates gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, peony lactone
Glycosides peak content, the average content of 7 kinds of compositions is respectively 0.395,0.108,0.709,0.134,1.759,4.602 and
0.291mg·g-1。
Embodiment 2
The preparation of reference substance solution is with embodiment 1.
The preparation of need testing solution: take blood-nourishing and brain-refreshing granules appropriate, finely ground, take about 200mg, accurately weighed, put 25ml amount
In Ping, add 10% methanol 25ml, reflow treatment 30 minutes, let cool, add 10% methanol to scale, shake up, with 0.22 μm microporous filter membrane
Filter, take subsequent filtrate, to obtain final product.
Chromatographic condition: be chromatographic column with Agilent ZorbaxSB-C18 (150mm × 4.6mm, 5 μm)；With 0.01% phosphoric acid
Solution (A)-acetonitrile (B) carries out gradient elution (0～10min, 5%B → 10%B mutually for flowing；10～15min, 10%B → 12%B；
15～25min, 12%B → 15%B；25～35min, 15%B → 18%B；35～40min, 18%B → 19%B；40～43min, 19%
B)；Flow velocity is 0.5ml min-1；Column temperature is 25 DEG C；Detection wavelength is 210nm, 256nm and 300nm；Sample size is 2.5 μ l.
The mensuration of sample: the need testing solution of test agent in preparing 10 batches, sample introduction measures, and the accurate 10 μ l that draw inject liquid phase
Chromatograph, measures peak area, calculates gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, peony lactone
Glycosides peak content, the average content of 7 kinds of compositions is respectively 0.382,0.096,0.695,0.123,1.741,4.596 and
0.280mg·g-1。
Embodiment 3
The preparation of reference substance solution is with embodiment 1.
The preparation of need testing solution: take blood-nourishing and brain-refreshing granules appropriate, finely ground, take about 200mg, accurately weighed, put 25ml amount
In Ping, add 25% methanol 25ml, reflow treatment 30 minutes, let cool, add 25% methanol to scale, shake up, with 0.22 μm microporous filter membrane
Filter, take subsequent filtrate, to obtain final product.
Chromatographic condition: be chromatographic column with Waters XBridge-C18 (250mm × 4.6mm, 5 μm)；Molten with 0.05% phosphoric acid
Liquid (A)-acetonitrile (B) carries out gradient elution (0～10min, 5%B → 10%B mutually for flowing；10～15min, 10%B → 12%B；15
～25min, 12%B → 15%B；25～35min, 15%B → 18%B；35～40min, 18%B → 19%B；40～43min, 19%B)；
Flow velocity is 1.2ml min-1；Column temperature is 35 DEG C；Detection wavelength is 240nm, 300nm and 350nm；Sample size is 25 μ l.
The mensuration of sample: the need testing solution of test agent in preparing 10 batches, sample introduction measures, and the accurate 10 μ l that draw inject liquid phase
Chromatograph, measures peak area, calculates gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, peony lactone
Glycosides peak content, the average content of 7 kinds of compositions is respectively 0.388,0.092,0.691,0.130,1.747,4.590 and
0.279mg·g-1。
Embodiment 4
The preparation of reference substance solution is with embodiment 1.
The preparation of need testing solution: take blood-nourishing and brain-refreshing granules appropriate, finely ground, take about 200mg, accurately weighed, put 25ml amount
In Ping, add 50% methanol 25ml, supersound process 30 minutes, let cool, add 50% methanol to scale, shake up, with 0.22 μm microporous filter membrane
Filter, take subsequent filtrate, to obtain final product.
Chromatographic condition: be chromatographic column with Shimadzu WondaSil-C18 (250mm × 4.6mm, 5 μm)；With 0.02% phosphoric acid solution
(A)-acetonitrile (B) carries out gradient elution (0～10min, 5%B → 10%B mutually for flowing；10～15min, 10%B → 12%B；15～
25min, 12%B → 15%B；25～35min, 15%B → 18%B；35～40min, 18%B → 19%B；40～43min, 19%B)；Stream
Speed is 1.5ml min-1；Column temperature is 30 DEG C；Detection wavelength is 230nm, 280nm and 320nm；Sample size is 5 μ l.
The mensuration of sample: the need testing solution of test agent in preparing 10 batches, sample introduction measures, and the accurate 10 μ l that draw inject liquid phase
Chromatograph, measures peak area, calculates gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, peony lactone
Glycosides peak content, the average content of 7 kinds of compositions is respectively 0.397,0.102,0.703,0.130,1.756,4.593 and
0.286mg·g-1。
Embodiment 5
The preparation of reference substance solution is with embodiment 1.
The preparation of need testing solution: take blood-nourishing and brain-refreshing granules appropriate, finely ground, take about 200mg, accurately weighed, put 25ml amount
In Ping, add 25% methanol 25ml, stirring cold extraction 60 minutes, add 25% methanol to scale, shake up, with 0.22 μm filtering with microporous membrane,
Take subsequent filtrate, to obtain final product.
Chromatographic condition: be chromatographic column with Agilent ZorbaxSB-C18 (250mm × 4.6mm, 5 μm)；With 0.04% phosphoric acid
Solution (A)-acetonitrile (B) carries out gradient elution (0～10min, 5%B → 10%B mutually for flowing；10～15min, 10%B → 12%B；
15～25min, 12%B → 15%B；25～35min, 15%B → 18%B；35～40min, 18%B → 19%B；40～43min, 19%
B)；Flow velocity is 0.95ml min-1；Column temperature is room temperature；Detection wavelength is 230nm, 280nm and 320nm；Sample size is 15 μ l.
The mensuration of sample: the need testing solution of test agent in preparing 10 batches, sample introduction measures, and the accurate 10 μ l that draw inject liquid phase
Chromatograph, measures peak area, calculates gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, peony lactone
Glycosides peak content, the average content of 7 kinds of compositions is respectively 0.375,0.082,0.683,0.114,1.737,4.581 and
0.272mg·g-1。
Embodiment 6
The preparation of reference substance solution is with embodiment 1.
The preparation of need testing solution: take blood-nourishing and brain-refreshing granules appropriate, finely ground, take about 200mg, accurately weighed, put 25ml amount
In Ping, add 35% methanol 25ml, supersound process 30 minutes, let cool, add 35% methanol to scale, shake up, with 0.22 μm microporous filter membrane
Filter, take subsequent filtrate, to obtain final product.
Chromatographic condition: be chromatographic column with Agilent ZorbaxSB-C18 (250mm × 4.6mm, 5 μm)；With 0.04% phosphoric acid
Solution (A)-acetonitrile (B) carries out gradient elution (0～10min, 5%B → 10%B mutually for flowing；10～15min, 10%B → 12%B；
15～25min, 12%B → 15%B；25～35min, 15%B → 18%B；35～40min, 18%B → 19%B；40～43min, 19%
B)；Flow velocity is 0.9ml min-1；Column temperature is 30 DEG C；Detection wavelength is 230nm, 280nm and 320nm；Sample size is 5 μ l.
The mensuration of sample: the need testing solution of test agent in preparing 10 batches, sample introduction measures, and the accurate 10 μ l that draw inject liquid phase
Chromatograph, measures peak area, calculates gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, peony lactone
Glycosides peak content, the average content of 7 kinds of compositions is respectively 0.394,0.106,0.705,0.132,1.755,4.698 and
0.287mg·g-1。
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, permissible
Understand and these embodiments can be carried out multiple change without departing from the principles and spirit of the present invention, revise, replace
And modification, the scope of the present invention be defined by the appended.
Claims (6)
1. a high-efficiency liquid chromatography method for detecting for blood-nourishing and brain-refreshing granules, comprises the steps:
Step 1, the preparation of reference substance solution
Precision weighs gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, peoniflorin, and lactone glucoside of Radix Paeoniae reference substance is fitted
Amount, adds 25% methanol and is respectively prepared every 1ml containing gallic acid 3.77 μ g, protocatechuic acid 1.63 μ g, chlorogenic acid 4.05 μ g, caffeic acid
1.33 μ g, peoniflorin 29.36 μ g, lactone glucoside of Radix Paeoniae 12.95 μ g, the mixed solution of ferulic acid 2.43 μ g, to obtain final product；
Step 2, the preparation of need testing solution
Take blood-nourishing and brain-refreshing granules appropriate, finely ground, take 200mg, accurately weighed, put in 25ml measuring bottle, add 10%～50% methanol
25ml, extracts 30～60 minutes, is settled to scale, with 0.22 μm filtering with microporous membrane, take subsequent filtrate, to obtain final product；
Chromatographic condition is for using C18Chromatographic column, specification is 250mm × 4.6mm or 150mm × 4.6mm, 5 μm, and chromatograph flows
Phase flow velocity is 0.5-1.5mL/min, and chromatographic column temperature is 25-35 DEG C, and detector is diode array detector, and detector wavelength is
210-350nm, sample size 2.5-25 μ l；
Chromatogram flow phase A phase is 0.01%-0.05% phosphoric acid solution, and B phase is acetonitrile, and gradient elution method is:
0～10min, 5%B phase → 10%B phase；
10～15min, 10%B phase → 12%B phase；
15～25min, 12%B phase → 15%B phase；
25～35min, 15%B phase → 18%B phase；
35～40min, 18%B phase → 19%B phase；
40～43min, 19%B phase.
2. detection method as claimed in claim 1, it is characterised in that chromatogram flow phase A phase is 0.02% phosphoric acid solution, B phase
For acetonitrile.
3. detection method as claimed in claim 1, it is characterised in that detection sample uses 25% methanol supersound extraction.
4. the method for claim 1, it is characterised in that chromatogram flow phase flow velocity is 0.9-1.2mL/min.
5. the method for claim 1, it is characterised in that chromatographic detector wavelength be chosen as successively 280nm, 320nm and
230nm。
6. the method for claim 1, it is characterised in that before high performance liquid chromatography detects, still further comprise and adopt
The step detected the chemical composition in blood-nourishing and brain-refreshing granules with mass spectrum, testing conditions is: ion source is ESI source, spraying
Voltage is set to 4kV, and sheath atmospheric pressure is 35psi, assists gas N2Flow velocity is 5L/min, and heated capillary temperature is 300 DEG C, in source
CID is 10V, and negative ions sweeps m/z:100-1200 entirely.
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