CN103529137B - Measuring method for medicine composition fingerprint - Google Patents
Measuring method for medicine composition fingerprint Download PDFInfo
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- CN103529137B CN103529137B CN201210352838.8A CN201210352838A CN103529137B CN 103529137 B CN103529137 B CN 103529137B CN 201210352838 A CN201210352838 A CN 201210352838A CN 103529137 B CN103529137 B CN 103529137B
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Abstract
The invention discloses a measuring method for a medicine composition fingerprint, and can measure a medicine composition fingerprint through a high efficiency liquid phase chromatography method so as to effectively monitor the medicine quality. The method comprises the steps of: (a) test sample solution preparation: adopting a proper amount of medicine to be put into a measuring bottle, adding a mixed solution of a methyl alcohol and water to carry out ultrasonic dissolution, metering volume, filtering, and thus obtaining a test sample solution; (b) setting of chromatographic condition: utilizing octadecyl silane bonding silica gel as a padding for a chromatographic column, adopting gradient elution, utilizing a moving phase A as a methanoic acid water solution, and utilizing a moving phase B as acetonitrile, wherein the detection wavelength is 240-300nm; (c) measurement: absorbing the test sample solution to be fed into a liquid phase chromatograph, and measuring according to a high efficiency liquid phase chromatography, thus obtaining the medicine composition fingerprint. The detection method provided by the invention has the advantages that the operation is convenient, the efficiency is high, the repeatability and the stability are good, and the detection method is easy to control.
Description
Technical field
The present invention relates to medical art, be specifically related to a kind of assay method of finger-print of pharmaceutical composition.
Background technology
Traditional Chinese medicine fingerprint is spectrogram or the chromatogram of the chemical composition of Chinese materia medica obtained by means of wave spectrum or chromatographic technique, being a kind of comprehensively, quantifiable discriminating means, is the current evaluation Chinese medicine authenticity, one of the stability and conforming quality control model that meet characteristics of Traditional Chinese Medicine.At present, U.S. FDA, Britain and India herbal medicine allusion quotation, German medicinal plant association, Canadian medicinal plant association all accept the quality control method of chromatographic fingerprinting.Because Chinese medicine compound prescription finger-print whole collection of illustrative plates is made up of different peak group, each peak group represents one group of biological information, and the mutually collaborative and antagonism between various biological information, just in time embody compatibility and the prescription theory of Chinese medicine compound prescription.In addition, by the treatment to the main disease of body and main symptom between these peaks group or peak group, embody and control certain Chinese medicine compound prescription finger-print, just can guarantee its corresponding drug effect.The method for building up of current traditional Chinese medicine fingerprint mainly contains: spectroscopic methodology, as ultraviolet spectroscopy (UV), infra-red sepectrometry (IR); Chromatography, as thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), capillary electrophoresis; And additive method, as x-ray diffraction approach, nuclear magnetic resonance method etc.Wherein to have analysis speed fast for high performance liquid chromatography (HPLC), quantitative precision is high, detecting device kind is many, the features such as good stability, be not subject to the restriction of sample volatile grade and thermal stability, wider than additive method ranges of application such as vapor-phase chromatographies, be one of main method building finger-print.
Depression is a kind of common mental illness, and main manifestations is depressed, interest attenuating, pessimism, retardation of thinking, self-accusation are poor from crime, diet sleep, worry or feel whole body many places discomfort, suicidal thought and behavior can appear in severe patient.At present, depression becomes rapidly the second important diseases causing serious burden in global disease to the mankind, the misery that patient and family members thereof are caused and to the loss that society causes be other diseases incomparable.Capsule for reliving liver and reliving upset is the Chinese patent drug being used for the treatment of depression of domestic first SFDA approval.Main effect is spleen-benefiting mind-tranquilizing, be applicable to light, the moderate unipolar depression person that belongs to syndrome of stagnation of liver qi and spleen deficiency, disease sees that few indigestion and loss of appetite, uncomfortable in chest, the fatigue and weak of depressed, interest decline, sluggishness, difficulty falling asleep, early awakening, dreaminess, nervous, being irritable and getting angry easily, food, hidrosis, pain, tongue fur are white or greasy, arteries and veins string or thin.This medicine is on the experiential basis drawing traditional Chinese medical science associated therapy depression, uses for reference the newest research results formula of domestic and international Modern medical therapy depression and obtains.The mechanism of action of capsule for reliving liver and reliving upset produces important protective effect by its effective constituent through the biochemistry imbalance of blood-brain barrier to the atrophy of hippocampal neuron and maincenter local organization.Having clinical report capsule for reliving liver and reliving upset efficient to light modest depression is 68.10%, high far beyond 29.0% of placebo, difference has statistical significance, Clinical Global curative effect evaluation also demonstrate that capsule for reliving liver and reliving upset antidepression curative effect is obviously better than placebo, and well [capsule for reliving liver and reliving upset treats the randomized double-blind placebo-controlled study of light moderate depressive patients, Sun Xinyu etc., clinical research for its security and tolerance, 2009,18 (5)].Therefore, capsule for reliving liver and reliving upset is all good pure Chinese medicinal preparation of a kind of efficacy and saferry.
Capsule for reliving liver and reliving upset is made up of Herba Hyperici Monogyni, wilsonii two taste Chinese medicine.Herba Hyperici Monogyni has had the use history of 2400, is placed on antidepression effect aspect in recent years to the study hotspot of Hypericum Chinense.The principal ingredient of Herba Hyperici Monogyni alcohol extracting is 6 classes: naphthodianthrones (naphthoi-anthrones), phloroglucinol derivatives (phloroglucinols), flavonoids (flavonoids), bisflavones (biflavones), derived from phenyl acrylic acid (phenylpropanes), procyanidin (proanthocyanidins).In addition, a small amount of tannic acid, xanthone, volatile oil and amino acid etc. are also had.Wilsonii first appeared in Shennong's Herbal, 2010 version " Chinese Pharmacopoeia " record it and have effect of " replenish qi to invigorate the spleen, tonify the kidney to relieve mental strain ".Its main chemical composition of bibliographical information has acanthopanax senticosus saponins, flavonoids, polysaccharide, triterpene saponin, lignanoid and coffee mesitoyl quinine acid etc.The medicinal material Herba Hyperici Monogyni used due to capsule for reliving liver and reliving upset and wilsonii all have complicated chemical composition.Therefore, set up a kind of finger print measuring method of capsule for reliving liver and reliving upset, particularly by high effective liquid chromatography for measuring finger-print information, give its quality control from the angle of drug substance group entirety and have very important significance.
There is not been reported for the current mensuration for capsule for reliving liver and reliving upset finger-print.Because the active constituents of medicine in capsule for reliving liver and reliving upset medicinal material used wilsonii and hypericum perforatum is all many, therefore the finger-print obtained when these two kinds of medicines detect (namely detecting capsule for reliving liver and reliving upset) simultaneously clear performance must go out effective active composition respective in wilsonii and hypericum perforatum, avoid the peak of heterogeneity to occur overlapping, this brings no small difficulty with regard to giving the foundation of its finger-print.The detection method of a large amount of report wilsonii or hypericum perforatum finger-print is had in prior art, but, we learn that prior art can not be used for the mensuration of capsule for reliving liver and reliving upset finger-print about wilsonii or hypericum perforatum HPLC finger-print testing conditions by experiment, its existence effectively can not be separated effective active composition respective in wilsonii and hypericum perforatum, detection time is long, the shortcomings such as efficiency is low, complicated operation.Such as CN1796992A discloses a kind of Radix Et Caulis Acanthopanacis Senticosi injection fingerprint atlas detection method, use 0.1% phosphate aqueous solution for mobile phase A, 30% acetonitrile solution is that B carries out gradient elution, in the method, wilsonii principle active component Syringin went out peak at 35 minutes later, and required detection time is longer.[foundation of Radix Et Caulis Acanthopanacis Senticosi injection finger-print and the application in quality control thereof such as Luo Guoan, Luo Guoan etc., Chinese patent drug, 2006, the 28th volume, 1st phase] Radix Et Caulis Acanthopanacis Senticosi injection finger-print is studied, use 0.2% aqueous formic acid for mobile phase A, 35% acetonitrile solution is that B carries out gradient elution, determined wavelength 270nm, in the method, main effectively one-tenth Syringin B went out peak at about 34 minutes, and required detection time is also longer.The method for detecting fingerprint of acanthopanax of Chinese Pharmacopoeia Commission in " the Radix Et Caulis Acanthopanacis Senticosi injection quality standard draft " of publicity in 2011 with acetonitrile-water (30:70) for mobile phase A, 0.5% formic acid solution is that Mobile phase B carries out gradient elution, determined wavelength 270 nm, its Syringin went out peak at about 40 minutes, appointed required detection time so longer.Liu is towards Son [Master's thesis, the structure of hypericum perforatum extraction process and quality control system, Zhongshan University, 2004] in Hypericum Perforatum P.E finger-print is studied, employing methyl alcohol is mobile phase A, water buffer salt (94.9% water, 5% methyl alcohol, 0.1% phosphoric acid, pH=4.50 adjusted by triethylamine) be Mobile phase B, acetonitrile is that mobile phase C carries out gradient elution, determined wavelength 254nm, active component in Hypericum Perforatum P.E is effectively separated by the method, but its mobile phase preparation relative complex.CN200710118972.0 discloses the HPLC fingerprint atlas detection method of the preparation that a kind of hypericum perforatum total flavones is prepared into, use acetonitrile is mobile phase A, 0.1-1.0% phosphate aqueous solution is that Mobile phase B carries out gradient elution, the effective constituent rutin that in hypericum perforatum total flavones described in the method, content is maximum went out peak at about 23 minutes, Quercetin went out peak at about 52 minutes, and whole detection time is longer.The elegant virtue of fiber crops waits [capsule of weeping forsythia medicinal material chemical component fingerprint research, Shanxi Forestry science and technology, 2011,40th volume, the 1st phase] the hypericum perforatum medicinal material chemical component fingerprint research carried out, adopt 1% aqueous acetic acid to be mobile phase A, acetonitrile solution containing 1% acetic acid is formic acid solution is Mobile phase B, determined wavelength 265 nm, its rutin went out peak at about 46 minutes, and its detection time is also longer.
Summary of the invention
The invention provides a kind of assay method of medicine composition fingerprint, comprise the following steps:
A prepared by () need testing solution: thing of getting it filled is appropriate, is placed in measuring bottle, adds the mixed solution ultrasonic dissolution of methyl alcohol and water, constant volume, filters, is need testing solution;
(b) chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler; Adopt gradient elution, mobile phase A is aqueous formic acid, and Mobile phase B is acetonitrile, determined wavelength 240-300nm;
C () measures: draw above-mentioned need testing solution injection liquid chromatography, according to high effective liquid chromatography for measuring, obtain this medicine composition fingerprint;
Described pharmaceutical composition is selected from capsule for reliving liver and reliving upset, Hypericum Perforatum P.E or siberian Ginseng P.E.
In described (a) step, preferred detection wavelength is 240-300nm, and flow velocity is 0.5ml/min, and column temperature is 25-35 DEG C, and sampling volume is 1-10ul; Further preferred detection wavelength is 265nm, and flow velocity is 0.5ml/min, and column temperature is 30 DEG C, and sampling volume is 5ul;
In described (a) step, the mixed solution of particular methanol and water is the methanol solution of 20% ~ 75%.
In described (a) step, preferred need testing solution preparation method is: get it filled thing 0.20 ~ 0.25g, is placed in 25ml measuring bottle, adds 50%CH
3oH solution, ultrasonic 30min, dissolves, adds 50%CH
3oH is settled to scale, filters, obtains test product sample solution.
In described (a) step, preferred color of choice spectral condition is: chromatographic column take octadecylsilane chemically bonded silica as filler; Adopt gradient elution, mobile phase A is 0.1% aqueous formic acid, and Mobile phase B is acetonitrile;
Gradient elution program is:
During 0-3min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
During 10min, mobile phase A is 0.1% aqueous formic acid of 90%, and Mobile phase B is the acetonitrile of 10%;
During 20min, mobile phase A is 0.1% aqueous formic acid of 80%, and Mobile phase B is the acetonitrile of 20%;
During 30min, mobile phase A is 0.1% aqueous formic acid of 65%, and Mobile phase B is the acetonitrile of 35%;
During 40min, mobile phase A is 0.1% aqueous formic acid of 45%, and Mobile phase B is the acetonitrile of 55%;
During 45min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 50min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 51-60min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
Account for the total fingerprint peaks of total peak area number percent more than 1% containing 13 unimodal areas in the capsule for reliving liver and reliving upset finger-print recorded, be respectively:
No. 1 peak, retention time RT is 12.45, RSD is 0.08%, and peak area is 365.49, RSD is 28.82%;
No. 2 peaks, retention time RT is 13.85, RSD is 0.14%, and peak area is 272.90, RSD is 31.95%;
No. 4 peaks, retention time RT is 17.40, RSD is 0.09%, and peak area is 535.48, RSD is 25.40%;
No. 5 peaks, retention time RT is 17.81, RSD is 0.09%, and peak area is 543.92, RSD is 33.10%;
No. 6 peaks, retention time RT is 18.31, RSD is 0.08%, and peak area is 279.93, RSD is 35.21%;
No. 9 peaks, retention time RT is 25.02, RSD is 0.06%, and peak area is 2136.24, RSD is 13.35%;
No. 10 peaks, retention time RT is 25.70, RSD is 0.05%, and peak area is 1934.51, RSD is 25.88%;
No. 11 peaks, retention time RT is 25.97, RSD is 0.05%, and peak area is 2221.12, RSD is 13.79%;
No. 13 peaks, retention time RT is 27.18, RSD is 0.01%, and peak area is 150.16, RSD is 19.44%;
No. 14 peaks, retention time RT is 27.60, RSD is 0.05%, and peak area is 804.87, RSD is 12.59%;
No. 15 peaks, retention time RT is 27.94, RSD is 0.05%, and peak area is 416.84, RSD is 19.74%;
No. 19 peaks, retention time RT is 33.70, RSD is 0.04%, and peak area is 1757.78, RSD is 27.66%;
No. 20 peaks, retention time RT is 37.51, RSD is 0.04%, and peak area is 307.31, RSD is 16.64%; Also account for the total fingerprint peaks of total peak area number percent more than 1% containing 13 unimodal areas in measured capsule for reliving liver and reliving upset finger-print, be respectively:
No. 3 peaks, retention time RT is 16.15, and peak area is 79.84, and accounting for total peak area number percent is 0.62%;
No. 7 peaks, retention time RT is 21.56, and peak area is 95.12, and accounting for total peak area number percent is 0.76%;
No. 8 peaks, retention time RT is 22.81, and peak area is 30.48, and accounting for total peak area number percent is 0.21%;
No. 12 peaks, retention time RT is 26.78, and peak area is 45.62, and accounting for total peak area number percent is 0.35%;
No. 16 peaks, retention time RT is 28.43, and peak area is 18.87, and accounting for total peak area number percent is 0.13%;
No. 17 peaks, retention time RT is 28.78, and peak area is 33.07, and accounting for total peak area number percent is 0.26%;
No. 18 peaks, retention time RT is 29.69, and peak area is 55.36, and accounting for total peak area number percent is 0.42%;
No. 21 peaks, retention time RT is 48.26, and peak area is 2.90, and accounting for total peak area number percent is 0.02%;
No. 22 peaks, retention time RT is 50.50, and peak area is 29.03, and accounting for total peak area number percent is 0.20%;
No. 23 peaks, retention time RT is 51.12, and peak area is 1.45, and accounting for total peak area number percent is 0.01%;
No. 24 peaks, retention time RT is 51.75, and peak area is 52.26, and accounting for total peak area number percent is 0.36%;
No. 26 peaks, retention time RT is 52.35, and peak area is 2.90, and accounting for total peak area number percent is 0.01%;
No. 25 peaks, retention time RT is 52.10, and peak area is 1.45, and accounting for total peak area number percent is 0.02%;
In measured extract of hypericum perforatum finger-print, total fingerprint peaks is:
No. 1 peak, retention time RT is 12.58min, and peak area is 139.52, and accounting for total peak area number percent is 2.06%;
No. 2 peaks, retention time RT is 14.02min, and peak area is 49.23, and accounting for total peak area number percent is 0.73%;
No. 3 peaks, retention time RT is 18.07min, and peak area is 122.54, and accounting for total peak area number percent is 1.81%;
No. 4 peaks, retention time RT is 18.50min, and peak area is 36.08, and accounting for total peak area number percent is 0.53%;
No. 5 peaks, retention time RT is 25.19min, and peak area is 1345.87, and accounting for total peak area number percent is 19.83%;
No. 6 peaks, retention time RT is 25.90min, and peak area is 1224.44, and accounting for total peak area number percent is 18.04%;
No. 7 peaks, retention time RT is 26.15min, and peak area is 1307.68, and accounting for total peak area number percent is 19.27%;
No. 8 peaks, retention time RT is 27.78min, and peak area is 505.83, and accounting for total peak area number percent is 7.45%;
No. 9 peaks, retention time RT is 28.09min, and peak area is 215.12, and accounting for total peak area number percent is 3.17%;
No. 10 peaks, retention time RT is 29.01min, and peak area is 7.30, and accounting for total peak area number percent is 0.11%;
No. 11 peaks, retention time RT is 29.81min, and peak area is 41.98, and accounting for total peak area number percent is 0.62%;
No. 12 peaks, retention time RT is 33.87min, and peak area is 940.09, and accounting for total peak area number percent is 13.85%;
No. 13 peaks, retention time RT is 37.72min, and peak area is 168.23, and accounting for total peak area number percent is 2.48%;
No. 14 peaks, retention time RT is 48.24min, and peak area is 2.22, and accounting for total peak area number percent is 0.03%;
No. 15 peaks, retention time RT is 50.72min, and peak area is 0.35, and accounting for total peak area number percent is 0.01%;
No. 16 peaks, retention time RT is 51.07min, and peak area is 1.58, and accounting for total peak area number percent is 0.02%;
No. 17 peaks, retention time RT is 51.83min, and peak area is 19.72, and accounting for total peak area number percent is 0.29%;
No. 18 peaks, retention time RT is 52.48min, and peak area is 1.55, and accounting for total peak area number percent is 0.02%;
No. 19 peaks, retention time RT is 52.96min, and peak area is 1.94, and accounting for total peak area number percent is 0.03%;
In measured siberian Ginseng P.E finger-print, total fingerprint peaks is:
No. 1 peak, retention time RT is 12.60min, and peak area is 123.82, and accounting for total peak area number percent is 5.47%;
No. 2 peaks, retention time RT is 14.02min, and peak area is 266.82, and accounting for total peak area number percent is 11.79%;
No. 3 peaks, retention time RT is 16.36min, and peak area is 88.02, and accounting for total peak area number percent is 3.89%;
No. 4 peaks, retention time RT is 17.57min, and peak area is 409.19, and accounting for total peak area number percent is 18.08%;
No. 5 peaks, retention time RT is 18.06min, and peak area is 318.40, and accounting for total peak area number percent is 14.07%;
No. 6 peaks, retention time RT is 18.49min, and peak area is 291.64, and accounting for total peak area number percent is 12.88%;
No. 7 peaks, retention time RT is 21.81min, and peak area is 128.95, and accounting for total peak area number percent is 5.70%;
No. 8 peaks, retention time RT is 22.88min, and peak area is 32.84, and accounting for total peak area number percent is 1.45%;
No. 9 peaks, retention time RT is 25.83min, and peak area is 31.10, and accounting for total peak area number percent is 1.37%;
No. 10 peaks, retention time RT is 27.37min, and peak area is 48.01, and accounting for total peak area number percent is 2.12%;
No. 11 peaks, retention time RT is 28.97min, and peak area is 19.72, and accounting for total peak area number percent is 0.87%.
Can be found out by above capsule for reliving liver and reliving upset finger-print, extract of hypericum perforatum finger-print, siberian Ginseng P.E finger-print information: the capsule for reliving liver and reliving upset finger-print that detection method obtains contains the total absorption peak in extract of hypericum perforatum finger-print and siberian Ginseng P.E finger-print, illustrate adopt this method detects the capsule for reliving liver and reliving upset finger-print that obtains can objective reaction capsule for reliving liver and reliving upset inherence effective constituent content and with or without, be convenient to control product quality comprehensively.
Advantage of the present invention is as follows:
1, the present invention set up capsule for reliving liver and reliving upset HPLC fingerprint atlas detection method and finger-print, Efficient Characterization is carried out for its effective constituent Herba Hyperici Monogyni and wilsonii major part pharmacological active substance, has achieved maximum possible its chemical composition is detected.Provide a kind of method capsule for reliving liver and reliving upset quality evaluated by overall finger-print information, avoid and only measure wherein once the one-sidedness that, two chemical compositions judge capsule for reliving liver and reliving upset total quality, make capsule for reliving liver and reliving upset quality control more objective and accurate;
2, its wilsonii principle active component of fingerprint atlas detection method provided by the present invention went out peak before 22 minutes, and hypericum perforatum principal ingredient went out peak after 24 minutes, and the peak separation property of two kinds of medicinal materials effective constituent is separately good;
3, detection time needed for fingerprint atlas detection method provided by the present invention is reasonable, and the peak of main active went out peak in 40 minutes, and peak intensity is all comparatively large, therefore makes detection efficiency efficient;
4, its repeatability and good stability of fingerprint atlas detection method provided by the present invention;
5, fingerprint atlas detection method provided by the present invention avoids and use phosphoric acid in mobile phase, in direct and mass spectrometer coupling after high performance liquid chromatography detection, can effectively determine the structure of related substances, better the quality of monitoring related drugs.
Accompanying drawing illustrates:
Fig. 1 effective constituent reference substance HPLC chromatogram
Wherein: peak 1 is Syringin, peak 2 is chlorogenic acid, and peak 3 is rutin, and peak 4 is Hyperoside, and peak 5 is Isofraxidin, and peak 6 is quercitin, and peak 7 is Quercetin.
Fig. 2 blank baseline HPLC chromatogram
Fig. 3 capsule for reliving liver and reliving upset HPLC finger-print
Fig. 4 capsule for reliving liver and reliving upset HPLC finger-print (partial enlarged drawing)
Fig. 5 capsule for reliving liver and reliving upset HPLC finger-print (partial enlarged drawing)
Fig. 6 extract of hypericum perforatum HPLC finger-print
Fig. 7 siberian Ginseng P.E HPLC finger-print
Fig. 8 different batches capsule for reliving liver and reliving upset HPLC finger-print (10 batches)
Fig. 9 embodiment three HPLC chromatogram
Embodiment
The mensuration of embodiment one Herba Hyperici Monogyni finger-print
1, reagent: acetonitrile (HPLC, Sigma aldrich Company Inc.USA), formic acid for analyze pure (Sigma aldrichCompany Inc.USA), pure water through Milli-Q pure water system self-control (Millipore Bedford, MA, USA).
2, instrument: Agilent Infinity 1290 (CA, USA) and Finnigan LTQ Ion Trap Mass Spectrometer (SanJose, CA, USA) coupling, software is respectively Agilent Chemstation and Xcalibur.
3, extract of hypericum perforatum sample preparation: the extract of hypericum perforatum that Example five prepares, weighs 0.22g, is placed in 25ml measuring bottle, adds 50%CH
3oH solution, ultrasonic 30min, dissolves, adds 50%CH
3oH is settled to scale, filters, obtains extract of hypericum perforatum sample solution;
4, chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler; Adopt gradient elution, mobile phase A is aqueous formic acid, and Mobile phase B is acetonitrile, and gradient elution program is:
During 0-3min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
During 10min, mobile phase A is 0.1% aqueous formic acid of 90%, and Mobile phase B is the acetonitrile of 10%;
During 20min, mobile phase A is 0.1% aqueous formic acid of 80%, and Mobile phase B is the acetonitrile of 20%;
During 30min, mobile phase A is 0.1% aqueous formic acid of 65%, and Mobile phase B is the acetonitrile of 35%;
During 40min, mobile phase A is 0.1% aqueous formic acid of 45%, and Mobile phase B is the acetonitrile of 55%;
During 45min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 50min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 51-60min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
Determined wavelength 265nm, flow velocity is 0.5ml/min, and column temperature is 30 DEG C, and sampling volume is 5ul;
5, measure: draw above-mentioned extract of hypericum perforatum sample solution injection liquid chromatography, according to high effective liquid chromatography for measuring, obtain extract of hypericum perforatum finger-print (see accompanying drawing 6), wherein total fingerprint peaks is:
No. 1 peak, retention time RT is 12.58min, and peak area is 139.52, and accounting for total peak area number percent is 2.06%;
No. 2 peaks, retention time RT is 14.02min, and peak area is 49.23, and accounting for total peak area number percent is 0.73%;
No. 3 peaks, retention time RT is 18.07min, and peak area is 122.54, and accounting for total peak area number percent is 1.81%;
No. 4 peaks, retention time RT is 18.50min, and peak area is 36.08, and accounting for total peak area number percent is 0.53%;
No. 5 peaks, retention time RT is 25.19min, and peak area is 1345.87, and accounting for total peak area number percent is 19.83%;
No. 6 peaks, retention time RT is 25.90min, and peak area is 1224.44, and accounting for total peak area number percent is 18.04%;
No. 7 peaks, retention time RT is 26.15min, and peak area is 1307.68, and accounting for total peak area number percent is 19.27%;
No. 8 peaks, retention time RT is 27.78min, and peak area is 505.83, and accounting for total peak area number percent is 7.45%;
No. 9 peaks, retention time RT is 28.09min, and peak area is 215.12, and accounting for total peak area number percent is 3.17%;
No. 10 peaks, retention time RT is 29.01min, and peak area is 7.30, and accounting for total peak area number percent is 0.11%;
No. 11 peaks, retention time RT is 29.81min, and peak area is 41.98, and accounting for total peak area number percent is 0.62%;
No. 12 peaks, retention time RT is 33.87min, and peak area is 940.09, and accounting for total peak area number percent is 13.85%;
No. 13 peaks, retention time RT is 37.72min, and peak area is 168.23, and accounting for total peak area number percent is 2.48%;
No. 14 peaks, retention time RT is 48.24min, and peak area is 2.22, and accounting for total peak area number percent is 0.03%;
No. 15 peaks, retention time RT is 50.72min, and peak area is 0.35, and accounting for total peak area number percent is 0.01%;
No. 16 peaks, retention time RT is 51.07min, and peak area is 1.58, and accounting for total peak area number percent is 0.02%;
No. 17 peaks, retention time RT is 51.83min, and peak area is 19.72, and accounting for total peak area number percent is 0.29%;
No. 18 peaks, retention time RT is 52.48min, and peak area is 1.55, and accounting for total peak area number percent is 0.02%;
No. 19 peaks, retention time RT is 52.96min, and peak area is 1.94, and accounting for total peak area number percent is 0.03%.
The mensuration of embodiment two siberian Ginseng P.E finger-print
1, reagent: acetonitrile (HPLC, Sigma aldrich Company Inc.USA), formic acid for analyze pure (Sigma aldrichCompany Inc.USA), pure water through Milli-Q pure water system self-control (Millipore Bedford, MA, USA).
2, instrument: Agilent Infinity 1290 (CA, USA) and Finnigan LTQ Ion Trap Mass Spectrometer (SanJose, CA, USA) coupling, software is respectively Agilent Chemstation and Xcalibur.
3, siberian Ginseng P.E sample preparation: the siberian Ginseng P.E that Example six prepares, weighs 0.20g, is placed in 25ml measuring bottle, adds 50%CH
3oH solution, ultrasonic 30min, dissolves, adds 50%CH
3oH is settled to scale, filters, obtains siberian Ginseng P.E sample solution;
4, chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler; Adopt gradient elution, mobile phase A is aqueous formic acid, and Mobile phase B is acetonitrile, and gradient elution program is:
During 0-3min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
During 10min, mobile phase A is 0.1% aqueous formic acid of 90%, and Mobile phase B is the acetonitrile of 10%;
During 20min, mobile phase A is 0.1% aqueous formic acid of 80%, and Mobile phase B is the acetonitrile of 20%;
During 30min, mobile phase A is 0.1% aqueous formic acid of 65%, and Mobile phase B is the acetonitrile of 35%;
During 40min, mobile phase A is 0.1% aqueous formic acid of 45%, and Mobile phase B is the acetonitrile of 55%;
During 45min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 50min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 51-60min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
Determined wavelength 265nm, flow velocity is 0.5ml/min, and column temperature is 30 DEG C, and sampling volume is 5ul;
5, measure: draw above-mentioned siberian Ginseng P.E sample solution injection liquid chromatography, according to high effective liquid chromatography for measuring, obtain siberian Ginseng P.E finger-print (see accompanying drawing 7), wherein total fingerprint peaks is:
No. 1 peak, retention time RT is 12.60min, and peak area is 123.82, and accounting for total peak area number percent is 5.47%;
No. 2 peaks, retention time RT is 14.02min, and peak area is 266.82, and accounting for total peak area number percent is 11.79%;
No. 3 peaks, retention time RT is 16.36min, and peak area is 88.02, and accounting for total peak area number percent is 3.89%;
No. 4 peaks, retention time RT is 17.57min, and peak area is 409.19, and accounting for total peak area number percent is 18.08%;
No. 5 peaks, retention time RT is 18.06min, and peak area is 318.40, and accounting for total peak area number percent is 14.07%;
No. 6 peaks, retention time RT is 18.49min, and peak area is 291.64, and accounting for total peak area number percent is 12.88%;
No. 7 peaks, retention time RT is 21.81min, and peak area is 128.95, and accounting for total peak area number percent is 5.70%;
No. 8 peaks, retention time RT is 22.88min, and peak area is 32.84, and accounting for total peak area number percent is 1.45%;
No. 9 peaks, retention time RT is 25.83min, and peak area is 31.10, and accounting for total peak area number percent is 1.37%;
No. 10 peaks, retention time RT is 27.37min, and peak area is 48.01, and accounting for total peak area number percent is 2.12%;
No. 11 peaks, retention time RT is 28.97min, and peak area is 19.72, and accounting for total peak area number percent is 0.87%;
Embodiment three capsule for reliving liver and reliving upset finger-print detects
1, sample: capsule for reliving liver and reliving upset sample prepared by Example four, lot number: 110902.
2, reagent: acetonitrile (HPLC, Sigma aldrich Company Inc.USA), formic acid for analyze pure (Sigma aldrichCompany Inc.USA), pure water through Milli-Q pure water system self-control (Millipore Bedford, MA, USA), ammonium acetate (1Guanghua Chemical Plant Co., Ltd., Guangdong, China).
3, instrument: Agilent Infinity 1290 (CA, USA) and Finnigan LTQ Ion Trap Mass Spectrometer (SanJose, CA, USA) coupling, software is respectively Agilent Chemstation and Xcalibur.
4, capsule for reliving liver and reliving upset sample solution preparation: get capsule for reliving liver and reliving upset sample capsules content 0.25g in 25ml measuring bottle, add 50%CH3OH and be about 20ml, ultrasonic 30min, add 50%CH3OH to scale, shake up, get appropriate solution and cross 0.45um filter membrane, get subsequent filtrate, to obtain final product.
5, chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler (Eclipse Plus C18,5um, 250*4.6mm, Agilent); Mobile phase is with 0.5% formic acid and 20mM ammonium acetate aqueous solution (0.5%HCOOH, 20mMNH4Ac) for mobile phase A, and be acetonitrile mobile phase B, carry out gradient elution, gradient condition is:
During 0-3min, the mobile phase A of 95%, the Mobile phase B of 5%
During 20min, the mobile phase A of 85%, the Mobile phase B of 15%
During 40min, the mobile phase A of 80%, the Mobile phase B of 20%
During 55min, the mobile phase A of 55%, the Mobile phase B of 45%
During 65min, the mobile phase A of 30%, the Mobile phase B of 70%
During 75min, the mobile phase A of 0%, the Mobile phase B of 100%
During 85min, the mobile phase A of 0%, the Mobile phase B of 100%
During 86-94min, the mobile phase A of 95%, the Mobile phase B of 5%
Flow velocity is 1.0ml/min, column temperature: 30 DEG C, and UV determined wavelength is 265nm, sampling volume: 5ul.
6, result: above-mentioned condition detection method needs longer gradient timetable just can detect all effective constituent, and T.T. reaches 94min.Testing result is shown in Fig. 7.
The method contrasts in table 1 with experimental technique of the present invention:
Table 1 comparative example contrasts with detection method
As apparent from upper table can: these two principal ingredient peak areas are basically identical, but peak height aspect the inventive method is at least 2 times of right left sides of comparative example method, and the preparation of the inventive method mobile phase is simple, and formic acid ratio is 0.1%, very little to Mass Spectrometer Method interference.
The preparation of embodiment four capsule for reliving liver and reliving upset
Capsule for reliving liver and reliving upset involved in the present invention is primarily of extract of hypericum perforatum and siberian Ginseng P.E composition.The preparation method of this medicine is preferably Herba Hyperici Monogyni 1800g, wilsonii 1500g, above two taste medicinal materials, Herba Hyperici Monogyni adds 75% alcohol reflux and extracts twice, for the first time 10 times amount, second time 8 times amount, each 1 hour, merge extract, filter, filtrate reduced in volume to relative density is 1.10(70 DEG C), spraying dry obtains dry extract; Wilsonii adds 8 times of water gagings, decocts three times, each 2 hours, collecting decoction, filter, filtrate is concentrated into relative density and is about 1.18(70 DEG C) medicinal extract, spraying dry obtains dry extract.Get above-mentioned two kinds of dry extracts, add appropriate amount of auxiliary materials (pregelatinized starch 52g, talcum powder 18g, dolomol 2g) mixing, incapsulate, make 1000, to obtain final product.
The preparation of embodiment five extract of hypericum perforatum
The preparation of extract of hypericum perforatum: extract twice with 75% alcohol reflux after Herba Hyperici Monogyni medicinal material 1800g pulverizes, 10 times amount 75% ethanol for the first time, second time 8 times amount 75% ethanol, each 1 hour, merge extract, filter, filtrate reduced in volume to relative density is 1.10(70 DEG C), spraying dry obtains extract of hypericum perforatum
The preparation of embodiment six siberian Ginseng P.E
The preparation of siberian Ginseng P.E: get wilsonii medicinal material 1500g and pulverize, add 8 times of water gagings, decoct three times, each 2 hours, collecting decoction, filter, filtrate is concentrated into relative density and is about 1.18(70 DEG C) medicinal extract, spraying dry obtains siberian Ginseng P.E.
Embodiment seven capsule for reliving liver and reliving upset finger-print detects
(1) mensuration of the finger-print of capsule for reliving liver and reliving upset
1, sample: capsule for reliving liver and reliving upset sample prepared by Example four, lot number: 110902.
2, reagent: acetonitrile (HPLC, Sigma aldrich Company Inc.USA), formic acid for analyze pure (Sigma aldrichCompany Inc.USA), pure water through Milli-Q pure water system self-control (Millipore Bedford, MA, USA)
3, instrument: Agilent Infinity 1290 (CA, USA) and Finnigan LTQ Ion Trap Mass Spectrometer (SanJose, CA, USA) coupling, software is respectively Agilent Chemstation and Xcalibur.
4, capsule for reliving liver and reliving upset sample solution preparation: get capsule for reliving liver and reliving upset sample contents 0.25g in 25ml measuring bottle, add 50%CH
3oH is about 20ml, and ultrasonic 30min, adds 50%CH
3oH, to scale, shakes up, and gets appropriate solution and crosses 0.45um filter membrane, get subsequent filtrate, to obtain final product.
5, chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler (Eclipse Plus C18,5um, 250*4.6mm, Agilent); Mobile phase A: 0.1% aqueous formic acid, Mobile phase B: acetonitrile, by gradient elution, elution program is as follows:
During 0-3min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
During 10min, mobile phase A is 0.1% aqueous formic acid of 90%, and Mobile phase B is the acetonitrile of 10%;
During 20min, mobile phase A is 0.1% aqueous formic acid of 80%, and Mobile phase B is the acetonitrile of 20%;
During 30min, mobile phase A is 0.1% aqueous formic acid of 65%, and Mobile phase B is the acetonitrile of 35%;
During 40min, mobile phase A is 0.1% aqueous formic acid of 45%, and Mobile phase B is the acetonitrile of 55%;
During 45min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 50min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 51-60min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
Flow velocity is 1.0ml/min, column temperature: 30 DEG C, and UV determined wavelength is 265nm, sampling volume: 5ul.
6, finger-print data:
Account for the total fingerprint peaks of total peak area number percent more than 1% containing 13 unimodal areas in the capsule for reliving liver and reliving upset finger-print recorded, be respectively:
No. 1 peak, retention time RT is 12.48, and peak area is 371.40, and accounting for total peak area number percent is 2.76%;
No. 2 peaks, retention time RT is 13.83, and peak area is 272.07, and accounting for total peak area number percent is 2.12%;
No. 4 peaks, retention time RT is 17.41, and peak area is 541.52, and accounting for total peak area number percent is 4.13%;
No. 5 peaks, retention time RT is 17.80, and peak area is 543.00, and accounting for total peak area number percent is 4.23%;
No. 6 peaks, retention time RT is 18.31, and peak area is 279.62, and accounting for total peak area number percent is 2.19%;
No. 9 peaks, retention time RT is 25.02, and peak area is 2125.66, and accounting for total peak area number percent is 16.24%;
No. 10 peaks, retention time RT is 25.71, and peak area is 1948.00, and accounting for total peak area number percent is 14.50%;
No. 11 peaks, retention time RT is 25.97, and peak area is 2228.14, and accounting for total peak area number percent is 16.83%;
No. 13 peaks, retention time RT is 27.19, and peak area is 150.37, and accounting for total peak area number percent is 1.14%;
No. 14 peaks, retention time RT is 27.61, and peak area is 802.03, and accounting for total peak area number percent is 6.05%;
No. 15 peaks, retention time RT is 27.95, and peak area is 418.13, and accounting for total peak area number percent is 3.13%;
No. 19 peaks, retention time RT is 33.72, and peak area is 1770.37, and accounting for total peak area number percent is 13.16%;
No. 20 peaks, retention time RT is 37.55, and peak area is 305.84, and accounting for total peak area number percent is 2.32%;
Also account for the total fingerprint peaks of total peak area number percent more than 1% containing 13 unimodal areas in measured capsule for reliving liver and reliving upset finger-print, be respectively:
No. 3 peaks, retention time RT is 16.19, and peak area is 80.94, and accounting for total peak area number percent is 0.63%;
No. 7 peaks, retention time RT is 21.56, and peak area is 94.02, and accounting for total peak area number percent is 0.75%;
No. 8 peaks, retention time RT is 22.81, and peak area is 26.89, and accounting for total peak area number percent is 0.21%;
No. 12 peaks, retention time RT is 26.77, and peak area is 45.73, and accounting for total peak area number percent is 0.34%;
No. 16 peaks, retention time RT is 28.43, and peak area is 18.36, and accounting for total peak area number percent is 0.14%;
No. 17 peaks, retention time RT is 28.78, and peak area is 32.90, and accounting for total peak area number percent is 0.26%;
No. 18 peaks, retention time RT is 29.69, and peak area is 54.41, and accounting for total peak area number percent is 0.41%;
No. 21 peaks, retention time RT is 48.25, and peak area is 5.21, and accounting for total peak area number percent is 0.05%;
No. 22 peaks, retention time RT is 50.49, and peak area is 22.19, and accounting for total peak area number percent is 0.17%;
No. 23 peaks, retention time RT is 51.21, and peak area is 1.05, and accounting for total peak area number percent is 0.01%;
No. 24 peaks, retention time RT is 51.75, and peak area is 39.80, and accounting for total peak area number percent is 0.31%;
No. 25 peaks, retention time RT is 52.39, and peak area is 2.69, and accounting for total peak area number percent is 0.02%;
No. 26 peaks, retention time RT is 52.84, and peak area is 36.58, and accounting for total peak area number percent is 0.29%.
(2) qualification of effective constituent in capsule for reliving liver and reliving upset finger-print
The present invention is also studied the structure of compound in capsule for reliving liver and reliving upset finger-print obtained above, contrast by liquid chromatography-Electrospray ion trap mass spectrometry analytical approach and with effective constituent reference substance Liquid Chromatography data, identify the chemical composition of 22 total absorption peaks in above-mentioned capsule for reliving liver and reliving upset finger-print, namely by the retention time of the retention time of chromatographic peak with effective constituent reference substance is contrasted, and the mass spectrometric data of its mass spectrometric data with document is contrasted, final its structure of confirmation.Wherein there are 6 compounds by carrying out contrasting thus confirming structure with effective constituent reference substance retention time.
Wherein the high-performance liquid chromatogram determination method of reference substance is:
1) effective constituent reference substance: be respectively Hyperoside (lot number 111521-2001004), rutin (lot number 100080-200707), Syringin (lot number 111574-200502), chlorogenic acid (lot number 110753-200413), Quercetin (lot number 100081-200406), quercitin (lot number 111538-200302) and Isofraxidin (lot number 110837-201005), all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
2) preparation of effective constituent reference substance solution: get appropriate reference substance respectively, put in the EP pipe adding 50% methanol-water 1ml, ultrasonic 10min, to obtain final product.
3) liquid phase chromatogram condition is identical with assay method with the liquid phase chromatogram condition in " mensuration of the finger-print of capsule for reliving liver and reliving upset " in the present embodiment with assay method.
4) effective constituent reference substance HPLC chromatogram refers to accompanying drawing 1.
Wherein liquid chromatography-Electrospray ion trap mass spectrometry analytical approach is: by there being the UV-detector of high performance liquid chromatograph solution out to pass through shunting, entering mass spectrometer and carrying out material survey; Mass spectrometer testing conditions is preferably: mass spectrometer flow velocity is 0.2ml/min.Electron spray ionisation, positive ion mode and negative ion mode detect, and nitrogen is as sheath gas, assisted gas and sweep gas.Under positive ion mode: spray voltage 3.6kV: sheath gas is 15arb, assisted gas is 5arb, and sweep gas is 0arb; Capillary temperature: 275 DEG C, capillary voltage is 10V, and lens voltage is 80V.Under negative ion mode: spray voltage 5kV: sheath gas is 15arb, assisted gas is 5arb, and sweep gas is 0arb; Capillary temperature: 275 DEG C, capillary voltage is-10V, and lens voltage is-100V.Full scan mass range: 210-1000amu.Second order ms data dependence type collection, 3 peaks that in full scan collection of illustrative plates, responsiveness intensity is the highest are used for second mass analysis, and high-purity helium is used as collision gas, and collision energy is 35ev.
Described liquid chromatography-Electrospray ion trap mass spectrometry analytical approach measurement result refers to table 2.
Table 2 capsule for reliving liver and reliving upset compound identification table
Note: "/" represents the no response under corresponding ion mode of this compound, and " a " represents that compound determines structure by contrasting with reference substance.
Certified 22 compound structures are as follows:
Wherein above-claimed cpd name is called: 1, neochlorogenic acid (Neochlorogenic acid); 2, Syringin (Eleutheroside B/syringoside); 3, Chlorogenic acid(chlorogenic acid); 4,1 '-caffeoylquinic acids (1 '-O-caffeoylquinic acid); 5,3 ', 5 '-dicaffeoylquinic acid (3 ', 5 '-O-dicaffeoylquinic acid); 6, eleutheroside E (Eleutheroside E); 7, rutin (Rutin); 8, Hyperoside (Hyperoside); 9, isoquercitrin (Isoquercitrin); 10, Isofraxidin (Isofaxidin); 11,1 ', 5 '-dicaffeoylquinic acid (1 ', 5 '-O-dicaffeoylquinic acid); 12, Quercetin-3-β-D-R (Quercetin-3-β-D-arabinose); 13, quercitin (Quercitrin); 14,4 ', 5 '-dicaffeoylquinic acid (4 ', 5 '-O-dicaffeoylquinicacid); 15, acetoxyl group Hyperoside (Acetyl-hyperoside); 16, Quercetin (Quercetin); 17, I3, II8 amentoflavone (I3, II8-Biapigenin); 18, former pseudohypericin (Protopseudohypericin); 19, hyperforine (Hyperforin); 20, adhyperforin (Adhyperforin); 21, hypericin (Hypericin); 22, pseudohypericin (Pseudohypericin).
Embodiment eight capsule for reliving liver and reliving upset finger-print detects
1, sample: the capsule for reliving liver and reliving upset prepared according to embodiment four, lot number 100701.
2, reagent: acetonitrile (HPLC, Sigma aldrich Company Inc.USA), formic acid for analyze pure (Sigma aldrichCompany Inc.USA), pure water through Milli-Q pure water system self-control (Millipore Bedford, MA, USA)
3, instrument: Agilent Infinity 1290 (CA, USA) and Finnigan LTQ Ion Trap Mass Spectrometer (SanJose, CA, USA) coupling, software is respectively Agilent Chemstation and Xcalibur.
4, capsule for reliving liver and reliving upset sample solution preparation: get capsule for reliving liver and reliving upset sample contents 0.25g in 25ml measuring bottle, add 50%CH
3oH is about 20ml, and ultrasonic 30min, adds 50%CH
3oH, to scale, shakes up, and gets appropriate solution and crosses 0.45um filter membrane, get subsequent filtrate, to obtain final product.
5, chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler (Eclipse Plus C18,5um, 250*4.6mm, Agilent); Mobile phase A: 0.1% aqueous formic acid, Mobile phase B: acetonitrile, by gradient elution, elution program is as follows:
During 0-3min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
During 10min, mobile phase A is 0.1% aqueous formic acid of 90%, and Mobile phase B is the acetonitrile of 10%;
During 20min, mobile phase A is 0.1% aqueous formic acid of 80%, and Mobile phase B is the acetonitrile of 20%;
During 30min, mobile phase A is 0.1% aqueous formic acid of 65%, and Mobile phase B is the acetonitrile of 35%;
During 40min, mobile phase A is 0.1% aqueous formic acid of 45%, and Mobile phase B is the acetonitrile of 55%;
During 45min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 50min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 51-60min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
Flow velocity is 1.0ml/min, column temperature: 30 DEG C, and UV determined wavelength is 265nm, sampling volume: 5ul.
6, detect
(1) stability of solution experiment
According to the method described above, measure and extract rear solution under 4 DEG C of conditions, place the stability of solution of 0,4,16 and 48h, measurement result is in table 3:
Table 3 stability of solution experimental result
The RSD%<3% of above result display all the components peak area, proves that this solution is stable in 48h under 4 DEG C of conditions.
(2) reappearance experiment
By above-mentioned test method, replicate determination 6 parts, measurement result is in table 4:
Table 4 reappearance experimental result
| Peak number | Average residence time | Average residence time RSD% | Average peak area | Peak area RSD% | Unimodally account for total peak area number percent |
| 1 | 12.47min | 0.03% | 248.88 | 1.55% | 1.96% |
| 2 | 13.89min | 0.01% | 330.14 | 1.81% | 2.60% |
| 3 | 16.2min | 0.02% | 120.63 | 2.33% | 0.95% |
| 4 | 17.42min | 0.04% | 694.57 | 2.54% | 5.47% |
| 5 | 17.82min | 0.01% | 864.72 | 1.98% | 6.81% |
| 6 | 18.31min | 0.01% | 444.43 | 1.57% | 3.50% |
| 7 | 21.44min | 0.03% | 151.10 | 1.83% | 1.19% |
| 8 | 22.8min | 0.02% | 27.94 | 2.01% | 0.22% |
| 9 | 25.04min | 0.04% | 1890.71 | 2.51% | 14.89% |
| 10 | 25.73min | 0.01% | 1762.46 | 0.67% | 13.88% |
| 11 | 25.98min | 0.01% | 1823.41 | 0.84% | 14.36% |
| 12 | 26.75min | 0.03% | 44.44 | 1.62% | 0.35% |
| 13 | 27.2min | 0.04% | 153.64 | 2.80% | 1.21% |
| 14 | 27.63min | 0.01% | 754.25 | 0.76% | 5.94% |
| 15 | 27.94min | 0.01% | 275.54 | 0.47% | 2.17% |
| 16 | 28.42min | 0.03% | 21.59 | 2.20% | 0.17% |
| 17 | 28.78min | 0.01% | 55.87 | 2.76% | 0.44% |
| 18 | 29.69min | 0.01% | 58.41 | 1.77% | 0.46% |
| 19 | 33.71min | 0.03% | 1285.02 | 2.73% | 10.12% |
| 20 | 37.52min | 0.01% | 252.69 | 0.57% | 1.99% |
| 21 | 48.23min | 0.01% | 5.71 | 1.97% | 0.05% |
| 22 | 50.48min | 0.03% | 23.29 | 2.43% | 0.18% |
| 23 | 51.22min | 0.01% | 1.16 | 2.57% | 0.01% |
| 24 | 51.74min | 0.01% | 42.02 | 1.72% | 0.33% |
| 25 | 52.44min | 0.03% | 2.95 | 2.78% | 0.02% |
| 26 | 52.83min | 0.01% | 35.02 | 1.57% | 0.28% |
The RSD%<3% of the parallel 6 parts of sample room peak areas of above result display, proves that the method reappearance is good.
Present inventor investigates and knows simultaneously, when employing formic acid water-acetonitrile system detects capsule for reliving liver and reliving upset as being more suitable for HPLC-MS during mobile phase, in capsule for reliving liver and reliving upset, wilsonii can reach comparatively ideal separation with the main active substances of hypericum perforatum, there is no overlap, and peak shape is excellent.
Present inventor detects liver soothing and depression relief absorbing wavelength by diode array detector (detection of DAD detecting device), find that each composition in liver soothing and depression relief has absorption within the scope of 200-400nm, it all has absorption at 240-300nm place, all be suitable as determined wavelength condition, each composition wherein when determined wavelength is 265nm in sample all has absorption and main compound has and absorbs more by force.
Present inventor has also investigated different extract to the impact of the extraction effect of product to be tested on testing result, comprises water, ethanol water and methanol aqueous solution.The effect that result display ethanol water extracts is not as water and methanol aqueous solution: such as 75%CH
3each peak-to-peak height of OH extraction sample and peak area are all obviously greater than 75%CH
3cH
2the sample that OH extracts.When using water or the aqueous solution containing methyl alcohol, (content comprises 75%CH
3oH, 50%CH
3oH, 25%CH
3oH) time, the testing result of sample is all passable, wherein as use 50%CH
3during OH, its Detection results is best.
The finger-print of embodiment nine capsule for reliving liver and reliving upset
1, sample: each batch sample of capsule for reliving liver and reliving upset (lot number 100601,100603,100701,100702,100703,100704,100705,100801,100802,100901,100902,101002,110412,110601,110511,110705,110803,110903,111003,111111,111113,111122,111203,111204,111205,111206,111207,111208,111211,111212) prepared by embodiment four
2, reagent: acetonitrile (HPLC, Sigma aldrich Company Inc.USA), formic acid for analyze pure (Sigma aldrichCompany Inc.USA), pure water through Milli-Q pure water system self-control (Millipore Bedford, MA, USA)
3, instrument: Agilent Infinity 1290 (CA, USA) and Finnigan LTQ Ion Trap Mass Spectrometer (SanJose, CA, USA) coupling, software is respectively Agilent Chemstation and Xcalibur.
4, capsule for reliving liver and reliving upset need testing solution preparation: capsule for reliving liver and reliving upset each batch sample content 0.25g of Example four preparation respectively, in 25ml measuring bottle, adds 50%CH
3oH is about 20ml, and ultrasonic 30min, adds 50%CH
3oH, to scale, shakes up, and gets appropriate solution and crosses 0.45um filter membrane, get subsequent filtrate, to obtain final product.
5, chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler (Eclipse Plus C18,5um, 250*4.6mm, Agilent); Mobile phase A: 0.1% aqueous formic acid, Mobile phase B: acetonitrile, by gradient elution, elution program is as follows:
During 0-3min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
During 10min, mobile phase A is 0.1% aqueous formic acid of 90%, and Mobile phase B is the acetonitrile of 10%;
During 20min, mobile phase A is 0.1% aqueous formic acid of 80%, and Mobile phase B is the acetonitrile of 20%;
During 30min, mobile phase A is 0.1% aqueous formic acid of 65%, and Mobile phase B is the acetonitrile of 35%;
During 40min, mobile phase A is 0.1% aqueous formic acid of 45%, and Mobile phase B is the acetonitrile of 55%;
During 45min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 50min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 51-60min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
Flow velocity is 1.0ml/min, column temperature: 30 DEG C, and UV determined wavelength is 265nm, sampling volume: 5ul.
6, data analysis
Carried out finger-print detection to 30 batches of capsule for reliving liver and reliving upset altogether, its similarity is more than 90%.All collection of illustrative plates Average residence time, peak area mean value and RSD data are in table 5.Wherein ten batches of detection spectrograms are shown in Fig. 8.
Table 5 capsule for reliving liver and reliving upset finger-print testing result (30 batches)
Document
[1]Determination of eleutheroside E and eleutheroside B in rat plasma and tissue by high-performanceliquid chromatography using solid-phase extraction and photodiode array detection Shi lanFeng*,Fang diHu,Jianxiong Zhao,Xi Liu,Y‘un Li.European Journal of Pharmaceutics and Biopharmaceutics,62(2006)315–320
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Sampo Mattlla,1 Terttu Ka¨ma¨ra¨inen2 and Jorma Jalonen1，Identification of Isomeric Dicaffeoylquinic Acidsfrom Eleutherococcus senticosus usingHPLC-ESI/TOF/MS and 1H-NMR Methods.PHYTOCHEMICAL ANALYSIS,Phytochem.Anal.13,316–328(2002)
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[6]J.Zhao,I.A.Khan,Hypericum perforatum-Chemical Profiling and Quantitative Results of St.John’sWort Products by an Improved High Performance Liquid Chromatography Method,M.Ganzera.Journal ofPharmaceutical Science,2002,91(3):623-630
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Claims (9)
1. an assay method for medicine composition fingerprint, is characterized in that comprising the following steps:
A prepared by () need testing solution: thing of getting it filled is appropriate, is placed in measuring bottle, adds the mixed solution ultrasonic dissolution of methyl alcohol and water, constant volume, filters, is need testing solution;
(b) chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as filler; Adopt gradient elution, mobile phase A is aqueous formic acid, and Mobile phase B is acetonitrile, determined wavelength 240-300nm;
C () measures: draw above-mentioned need testing solution injection liquid chromatography, according to high effective liquid chromatography for measuring, obtain this medicine composition fingerprint;
Described pharmaceutical composition is selected from capsule for reliving liver and reliving upset, Hypericum Perforatum P.E or siberian Ginseng P.E containing hypericum perforatum and wilsonii, and described gradient elution program is:
During 0-3min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%;
During 10min, mobile phase A is 0.1% aqueous formic acid of 90%, and Mobile phase B is the acetonitrile of 10%;
During 20min, mobile phase A is 0.1% aqueous formic acid of 80%, and Mobile phase B is the acetonitrile of 20%;
During 30min, mobile phase A is 0.1% aqueous formic acid of 65%, and Mobile phase B is the acetonitrile of 35%;
During 40min, mobile phase A is 0.1% aqueous formic acid of 45%, and Mobile phase B is the acetonitrile of 55%;
During 45min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 50min, mobile phase A is 0.1% aqueous formic acid of 0%, and Mobile phase B is the acetonitrile of 100%;
During 51-60min, mobile phase A is 0.1% aqueous formic acid of 95%, and Mobile phase B is the acetonitrile of 5%.
2. medicine composition fingerprint assay method according to claim 1, is characterized in that: determined wavelength described in (a) step is 240-300nm, and flow velocity is 0.5ml/min, and column temperature is 25-35 DEG C, and sampling volume is 1-10ul.
3. medicine composition fingerprint assay method according to claim 2, is characterized in that: determined wavelength described in (a) step is 265nm, and flow velocity is 0.5ml/min, and column temperature is 30 DEG C, and sampling volume is 5ul.
4. medicine composition fingerprint assay method according to claim 1, is characterized in that: the mixed solution of methyl alcohol and water described in (a) step is the methanol solution of 20% ~ 75%.
5. medicine composition fingerprint assay method according to claim 1, is characterized in that: described in (a) step, need testing solution preparation method is: get it filled thing 0.20 ~ 0.25g, is placed in 25ml measuring bottle, adds 50%CH3OH solution, ultrasonic
30min, dissolves, adds 50%CH3OH and be settled to scale, filters, obtains test product sample solution.
6., according to the medicine composition fingerprint assay method in claim 1-5 described in any one, it is characterized in that institute
Account for the total fingerprint peaks of total peak area number percent more than 1% containing 13 unimodal areas in the capsule for reliving liver and reliving upset finger-print recorded, be respectively:
No. 1 peak, retention time RT is 12.45, RSD is 0.08%, and peak area is 365.49, RSD is 28.82%;
No. 2 peaks, retention time RT is 13.85, RSD is 0.14%, and peak area is 272.90, RSD is 31.95%;
No. 4 peaks, retention time RT is 17.40, RSD is 0.09%, and peak area is 535.48, RSD is 25.40%;
No. 5 peaks, retention time RT is 17.81, RSD is 0.09%, and peak area is 543.92, RSD is 33.10%;
No. 6 peaks, retention time RT is 18.31, RSD is 0.08%, and peak area is 279.93, RSD is 35.21%;
No. 9 peaks, retention time RT is 25.02, RSD is 0.06%, and peak area is 2136.24, RSD is 13.35%;
No. 10 peaks, retention time RT is 25.70, RSD is 0.05%, and peak area is 1934.51, RSD is 25.88%;
No. 11 peaks, retention time RT is 25.97, RSD is 0.05%, and peak area is 2221.12, RSD is 13.79%;
No. 13 peaks, retention time RT is 27.18, RSD is 0.01%, and peak area is 150.16, RSD is 19.44%;
No. 14 peaks, retention time RT is 27.60, RSD is 0.05%, and peak area is 804.87, RSD is 12.59%;
No. 15 peaks, retention time RT is 27.94, RSD is 0.05%, and peak area is 416.84, RSD is 19.74%;
No. 19 peaks, retention time RT is 33.70, RSD is 0.04%, and peak area is 1757.78, RSD is 27.66%;
No. 20 peaks, retention time RT is 37.51, RSD is 0.04%, and peak area is 307.31, RSD is 16.64%.
7. according to the medicine composition fingerprint assay method in claim 1-5 described in any one, it is characterized in that also accounting for the total fingerprint peaks of total peak area number percent more than 1% containing 13 unimodal areas in measured capsule for reliving liver and reliving upset finger-print, be respectively:
No. 3 peaks, retention time RT is 16.15, and peak area is 79.84, and accounting for total peak area number percent is 0.62%;
No. 7 peaks, retention time RT is 21.56, and peak area is 95.12, and accounting for total peak area number percent is 0.76%;
No. 8 peaks, retention time RT is 22.81, and peak area is 30.48, and accounting for total peak area number percent is 0.21%;
No. 12 peaks, retention time RT is 26.78, and peak area is 45.62, and accounting for total peak area number percent is 0.35%;
No. 16 peaks, retention time RT is 28.43, and peak area is 18.87, and accounting for total peak area number percent is 0.13%;
No. 17 peaks, retention time RT is 28.78, and peak area is 33.07, and accounting for total peak area number percent is 0.26%;
No. 18 peaks, retention time RT is 29.69, and peak area is 55.36, and accounting for total peak area number percent is 0.42%;
No. 21 peaks, retention time RT is 48.26, and peak area is 2.90, and accounting for total peak area number percent is 0.02%;
No. 22 peaks, retention time RT is 50.50, and peak area is 29.03, and accounting for total peak area number percent is 0.20%;
No. 23 peaks, retention time RT is 51.12, and peak area is 1.45, and accounting for total peak area number percent is 0.01%;
No. 24 peaks, retention time RT is 51.75, and peak area is 52.26, and accounting for total peak area number percent is 0.36%;
No. 26 peaks, retention time RT is 52.35, and peak area is 2.90, and accounting for total peak area number percent is 0.01%;
No. 25 peaks, retention time RT is 52.10, and peak area is 1.45, and accounting for total peak area number percent is 0.02%.
8., according to the medicine composition fingerprint assay method in claim 1-5 described in any one, it is characterized in that in measured Hypericum Perforatum P.E finger-print, total fingerprint peaks is:
No. 1 peak, retention time RT is 12.58min, and peak area is 139.52, and accounting for total peak area number percent is 2.06%;
No. 2 peaks, retention time RT is 14.02min, and peak area is 49.23, and accounting for total peak area number percent is 0.73%;
No. 3 peaks, retention time RT is 18.07min, and peak area is 122.54, and accounting for total peak area number percent is 1.81%;
No. 4 peaks, retention time RT is 18.50min, and peak area is 36.08, and accounting for total peak area number percent is 0.53%;
No. 5 peaks, retention time RT is 25.19min, and peak area is 1345.87, and accounting for total peak area number percent is 19.83%;
No. 6 peaks, retention time RT is 25.90min, and peak area is 1224.44, and accounting for total peak area number percent is 18.04%;
No. 7 peaks, retention time RT is 26.15min, and peak area is 1307.68, and accounting for total peak area number percent is 19.27%;
No. 8 peaks, retention time RT is 27.78min, and peak area is 505.83, and accounting for total peak area number percent is 7.45%;
No. 9 peaks, retention time RT is 28.09min, and peak area is 215.12, and accounting for total peak area number percent is 3.17%;
No. 10 peaks, retention time RT is 29.01min, and peak area is 7.30, and accounting for total peak area number percent is 0.11%;
No. 11 peaks, retention time RT is 29.81min, and peak area is 41.98, and accounting for total peak area number percent is 0.62%;
No. 12 peaks, retention time RT is 33.87min, and peak area is 940.09, and accounting for total peak area number percent is 13.85%;
No. 13 peaks, retention time RT is 37.72min, and peak area is 168.23, and accounting for total peak area number percent is 2.48%;
No. 14 peaks, retention time RT is 48.24min, and peak area is 2.22, and accounting for total peak area number percent is 0.03%;
No. 15 peaks, retention time RT is 50.72min, and peak area is 0.35, and accounting for total peak area number percent is 0.01%;
No. 16 peaks, retention time RT is 51.07min, and peak area is 1.58, and accounting for total peak area number percent is 0.02%;
No. 17 peaks, retention time RT is 51.83min, and peak area is 19.72, and accounting for total peak area number percent is 0.29%;
No. 18 peaks, retention time RT is 52.48min, and peak area is 1.55, and accounting for total peak area number percent is 0.02%;
No. 19 peaks, retention time RT is 52.96min, and peak area is 1.94, and accounting for total peak area number percent is 0.03%.
9., according to the medicine composition fingerprint assay method in claim 1-5 described in any one, it is characterized in that in measured siberian Ginseng P.E finger-print, total fingerprint peaks is:
No. 1 peak, retention time RT is 12.60min, and peak area is 123.82, and accounting for total peak area number percent is 5.47%;
No. 2 peaks, retention time RT is 14.02min, and peak area is 266.82, and accounting for total peak area number percent is 11.79%;
No. 3 peaks, retention time RT is 16.36min, and peak area is 88.02, and accounting for total peak area number percent is 3.89%;
No. 4 peaks, retention time RT is 17.57min, and peak area is 409.19, and accounting for total peak area number percent is 18.08%;
No. 5 peaks, retention time RT is 18.06min, and peak area is 318.40, and accounting for total peak area number percent is 14.07%;
No. 6 peaks, retention time RT is 18.49min, and peak area is 291.64, and accounting for total peak area number percent is 12.88%;
No. 7 peaks, retention time RT is 21.81min, and peak area is 128.95, and accounting for total peak area number percent is 5.70%;
No. 8 peaks, retention time RT is 22.88min, and peak area is 32.84, and accounting for total peak area number percent is 1.45%;
No. 9 peaks, retention time RT is 25.83min, and peak area is 31.10, and accounting for total peak area number percent is 1.37%;
No. 10 peaks, retention time RT is 27.37min, and peak area is 48.01, and accounting for total peak area number percent is 2.12%;
No. 11 peaks, retention time RT is 28.97min, and peak area is 19.72, and accounting for total peak area number percent is 0.87%.
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| CN107037137A (en) * | 2016-02-04 | 2017-08-11 | 四川济生堂药业有限公司 | A kind of detection method of pharmaceutical composition |
| CN107976488B (en) * | 2016-10-25 | 2020-10-30 | 四川济生堂药业有限公司 | Detection method of pharmaceutical composition |
| CN109655558B (en) * | 2019-01-08 | 2021-08-24 | 贵阳中医学院 | Method for detecting effective part group of periploca forrestii schltr |
| CN110772564A (en) * | 2019-11-14 | 2020-02-11 | 美国琛蓝营养制品股份有限公司 | Traditional Chinese medicine extract composition with depression mood regulating effect, preparation method thereof and traditional Chinese medicine preparation |
| CN110780004A (en) * | 2019-11-14 | 2020-02-11 | 美国琛蓝营养制品股份有限公司 | Method for establishing fingerprint of traditional Chinese medicine composition or preparation thereof with depression mood regulating effect |
| CN111579701A (en) * | 2020-05-19 | 2020-08-25 | 河南科高中标检测技术有限公司 | Standard fingerprint spectrum establishment method for camelina sativa seeds and method for detecting rutin content |
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