CN103424498A - Establishing method and application of fingerprint of detoxifying and kidney harmonizing capsule - Google Patents
Establishing method and application of fingerprint of detoxifying and kidney harmonizing capsule Download PDFInfo
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Abstract
The invention provides an establishing method of the fingerprint of a detoxifying and kidney harmonizing capsule, and an application of the fingerprint. The establishing method comprises the following steps: 1, dissolving the content of the detoxifying and kidney harmonizing capsule in an 70%(V) aqueous ethanol solution to prepare a sample solution having a concentration of 5mg/ml; and 2, detecting the sample solution by adopting a high performance liquid chromatograph-tandem mass spectrometry to obtain a total ionic chromatogram, wherein a high performance liquid chromatograph adopts a C18 reversed-phase bonded silica gel chromatography column, a mobile phase 1 and a mobile phase 2 are used for gradient elution, an electro-spray detector is used for detection, the mobile phase 1 is a 0.1%(V) aqueous solution of formic acid, and the mobile phase 2 is a 0.1%(V) formic acid-acetonitrile solution. The method has the advantages of high detection accuracy and reliability when the method is used for obtaining the fingerprint, and the obtained fingerprint can rapidly, simply, comprehensively and effectively display the effective components of the detoxifying and kidney harmonizing capsule and the contents of the effective components.
Description
Technical field
The invention belongs to the drug test field, relate to a kind of method for building up and application thereof of compound Chinese medicinal preparation finger-print, be specifically related to a kind of method for building up and application thereof of toxin clearing away and the kidney nourishing capsule fingerprint pattern.
Background technology
Compound Chinese medicinal preparation refers to the preparation that the above Chinese medicine of two flavors is carried out to the compatibility combination and be applied to disease treatment.Because of its complicated component, so it is very large accurately to detect the difficulty of its each effective constituent.
Traditional Chinese medicine fingerprint means Chinese medicine, comprise that Chinese crude drug, medicine materical crude slice, extract or Chinese patent drug are after suitably processing, chromatogram or the spectrogram that can indicate this Chinese medicine main chemical compositions characteristic of taking certain analysis means to obtain, it has the characteristics such as characteristic, globality, sxemiquantitative, has been widely used in recent years analysis and the detection of Chinese medicine.
Hydrolysis and condensation is also development in recent years a kind of high-end analytical technology rapidly, wherein liquid chromatography mass coupling technique (LC-MS) is after gas chromatography combined with mass spectrometry technology (GC-MS) widespread use, the another kind of coupling detection technique of being familiar with gradually and accepting.But, because its instrument is expensive, also be not used widely.Ultra Performance Liquid Chromatography (UHPLC)-flight time mass spectrum combined instrument can not only separate the Chinese medicine complicated ingredient fast, and it adopts ionization techniques, can solve some test problems without the uv absorption composition, can also obtain the accurate molecular weight of ionizable composition, so the Structural Identification that this detection technique is the compound Chinese medicinal preparation constituent and confirmation provide of great value reference frame.
The toxin clearing away and the kidney nourishing capsule is the new Chinese medicine of beautiful pearl group independent research, is mainly used in treating chronic renal failure, compensatory stage of renal insufficiency, azotemia phase and uremia early stage.This medicine is comprised of medicinal materials such as the Radix Astragali, rheum officinales, has tonifying speen and tonifying kidney, turbid, promoting blood circulation and removing blood stasis effect falls in logical internal organs.From 1996, treated early, mid-term Chronic Renal Failure Patients more than 1000 examples.This capsule can significantly reduce serum creatinine and urea nitrogen concentration, increase appetite and urine amount, improve renal anemia.
In the drug standards of toxin clearing away and the kidney nourishing capsule, differentiate that item is respectively with (1) Astragaloside IV reference substance; (2) icariin reference substance; (3) Paeoniflorin reference substance; (4) Ginsenosides Rg1 and Rb1 reference substance; (5) rheum officinale reference substance; (6) Radix Angelicae Sinensis, Ligusticum wallichii reference substance, according to Chinese Pharmacopoeia version in 2010, an appendix VIB carries out the thin-layered chromatography discriminating.Containing quantifier, stipulate: according to 2010 editions one appendix VID high effective liquid chromatography for measuring of Chinese Pharmacopoeia, wherein press Paeoniflorin (C containing Chinese herbaceous peony for every
23H
28O
11) calculate, must not be less than 0.45mg; Press icariin (C containing barrenwort for every
33H
40O
15) calculate, must not be less than 0.15mg.
But, the detection method of existing toxin clearing away and the kidney nourishing capsule effective constituent is perfect not enough, still can not reflect comprehensively, accurately and efficiently various effective constituent and content thereof, be unfavorable for quality control and the clinical practice of this product, can not clearly toxin clearing away and the kidney nourishing capsule and other like products be made a distinction.
Summary of the invention
Technical matters to be solved by this invention is to make up the weak point of prior art, a kind of method for building up of toxin clearing away and the kidney nourishing capsule fingerprint pattern is provided, adopt the method to obtain finger-print and there is high, the reliable advantage of detection accuracy, can show quick, easy, fully and effectively effective constituent and the content thereof of toxin clearing away and the kidney nourishing capsule.
On the one hand, the invention provides a kind of method for building up of toxin clearing away and the kidney nourishing capsule fingerprint pattern, this method for building up comprises the following steps:
(1) content of toxin clearing away and the kidney nourishing capsule is dissolved in to the 70%(volume ratio) ethanol water, make the need testing solution that concentration is 5mg/ml;
(2) adopt the HPLC-MS instrument to detect need testing solution, obtain the total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C
18The reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with the electron spray detecting device, described mobile phase 1 is the 0.1%(volume ratio) aqueous formic acid, mobile phase 2 is the 0.1%(volume ratio) the formic acid acetonitrile solution, number percent meter by volume, described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% by 95%, and mobile phase 2 at the uniform velocity becomes 20% by 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% by 80%, and mobile phase 2 at the uniform velocity becomes 40% by 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 by 60%, and mobile phase 2 at the uniform velocity becomes 100% by 40%;
18-20 minute, mobile phase 1 is 0, mobile phase 2 is 100%.
On the other hand, the invention provides a kind of detection method of toxin clearing away and the kidney nourishing capsule effective constituent, this detection method comprises the following steps:
(1) content of toxin clearing away and the kidney nourishing capsule is dissolved in to the 70%(volume ratio) ethanol water, make the need testing solution that concentration is 5mg/ml;
(2) Paeoniflorin, icariin, notoginsenoside R, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, forulic acid, calycosin glucoside, Astragaloside IV, Rhein and archen are dissolved in to the standby reference substance solution of Methanol, each concentration of component is 0.02mg/ml;
(3) adopt the HPLC-MS instrument to detect respectively need testing solution and reference substance solution, obtain the total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C
18The reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with the electron spray detecting device, described mobile phase 1 is the 0.1%(volume ratio) aqueous formic acid, mobile phase 2 is the 0.1%(volume ratio) the formic acid acetonitrile solution, number percent meter by volume, described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% by 95%, and mobile phase 2 at the uniform velocity becomes 20% by 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% by 80%, and mobile phase 2 at the uniform velocity becomes 40% by 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 by 60%, and mobile phase 2 at the uniform velocity becomes 100% by 40%;
18-20 minute, mobile phase 1 is 0, mobile phase 2 is 100%.
Wherein, the total ion current collection of illustrative plates of described need testing solution comprises the characteristic peak with following retention time and mass number: 4.4min(525.1614), 4.8min(491.1195), 5.2min(193.0506), 6.4min(977.5327), 6.9min(845.4904), 8.9min(721.2349), 10.9min(599.2997), 12.1min(829.4591), 12.8min(945.5428), 14.0min(283.0248), 16.2min(269.0455).More specifically, the total ion current collection of illustrative plates of described need testing solution comprises the characteristic peak with following retention time and mass number: 4.4min(525.1614), 4.8min(491.1195), 5.2min(193.0506), 6.4min(977.5327), 6.9min(845.4904), 7.8min(507.1508), 8.1min(463.1610), 8.3min(645.2193), 8.5min(867.2939), 8.9min(721.2349), 10.1min(623.1770), 10.9min(599.2997), 12.1min(829.4591), 12.8min(945.5428), 13.7min(871.4697), 14.0min(283.0248), 14.2min(659.2345), 15.0min(513.1766), 15.4min(913.4802), 16.2min(269.0455), 16.9min(535.3640).
Preferably, above-mentioned detection method also comprises step (4): the characteristic peak that the retention time of take in the total ion current figure of need testing solution is 4.4min is the S peak, calculates the relative retention time of each characteristic peak; The corresponding peak-to-peak peak area ratio of feature of retention time in the total ion current figure of calculating need testing solution and the total ion current figure of reference substance solution.
More preferably, described step (4) comprises the described characteristic peak in the total ion current figure that extracts need testing solution, and the characteristic peak that the retention time of take is 4.4min is the S peak, calculates the relative retention time of each characteristic peak; The corresponding peak-to-peak peak area ratio of feature of retention time in the total ion current figure of calculating need testing solution and the total ion current figure of reference substance solution.
Preferably, in above-mentioned two kinds of methods, described step (1) comprises the following steps: get the content of toxin clearing away and the kidney nourishing capsule, mix porphyrize, weighing 0.5g, being placed in the 100ml volumetric flask, in volumetric flask, adding the 70%(volume ratio) ethanol water dissolves described content, then to the 70%(volume ratio that adds surplus in volumetric flask) ethanol water is to scale, shake up, filter, get subsequent filtrate, obtain need testing solution.
Preferably, in above-mentioned two kinds of methods, it is 0.3ml/min that the high performance liquid chromatography in described step (2) detects the flow rate of mobile phase adopted, and column temperature is 30 ℃, and sample size is 5 μ l; Described Mass Spectrometer Method adopts level Four bar-flight time tandem mass spectrometer; Described Mass Spectrometer Method adopts the electron spray ionisation source, in the negative ion mode, detects, and atomization gas pressure is 2.39kPa, and the dry gas temperature is 350 ℃, and the dry gas flow velocity is 10 liter/mins, and the kapillary high voltage is 3500V, and capillary outlet voltage is 100V.
In existing toxin clearing away and the kidney nourishing capsule quality standard, " discriminating " requires take Astragaloside IV, icariin, Paeoniflorin, Ginsenosides Rg1 and Rb1 reference substance and rheum officinale, Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material is contrast, carries out the thin layer discriminating; " assay " regulation: the paeoniflorin content in employing high effective liquid chromatography for measuring white Peony Root and the Icariin content in epimedium herb, not to its intrinsic chemical composition carry out comprehensively, systematic research, it is single that it detects index, complex operation, can't show effective constituent and the content thereof of toxin clearing away and the kidney nourishing capsule accurately, all sidedly.Therefore the present invention proposes the method for building up of toxin clearing away and the kidney nourishing capsule fingerprint pattern, and apply it in the middle of the detection of toxin clearing away and the kidney nourishing capsule effective constituent.The feature that has that it's too late by total peak in finger-print, can determine effectively, all sidedly effective constituent and content thereof, the stability of monitoring production technology, guarantee constant product quality, homogeneous, controlled, method of the present invention also has stability and favorable reproducibility, operability advantages of higher simultaneously.
The accompanying drawing explanation
The total ion current collection of illustrative plates that Fig. 1 is toxin clearing away and the kidney nourishing capsule need testing solution, 1 in figure is to 21 numberings that are characteristic peak.
The extraction ion current collection of illustrative plates that Fig. 2 is toxin clearing away and the kidney nourishing capsule need testing solution, 1 in figure is to 21 numberings that are characteristic peak.
The extraction ion current collection of illustrative plates that Fig. 3 is reference substance solution, 1 in figure, 2,3,4,5,10,12,13,14,16,20 indicates the characteristic peak of Paeoniflorin, calycosin glucoside, forulic acid, notoginsenoside R, ginsenoside Rg1, icariin, ginsenoside Rb1, Astragaloside IV, ginsenoside Rd, Rhein, archen successively.
Fig. 4 shows the comparative result of the total ion current collection of illustrative plates of toxin clearing away and the kidney nourishing capsule and NIAODUQING KELI need testing solution, the total ion current collection of illustrative plates that wherein Fig. 4-a is the toxin clearing away and the kidney nourishing capsule, the total ion current collection of illustrative plates that Fig. 4-b is NIAODUQING KELI.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.
1 material and instrument
1.1 material: the toxin clearing away and the kidney nourishing capsule is provided by Livzon Group Limin Pharmaceutical Factory.In experiment, agents useful for same acetonitrile, formic acid are chromatographically pure, and water is ultrapure water.
1.2 instrument: Agilent company LC-MS instrument (1290UHPLC binary gradient pump, internally provided with vacuum degasser, 100 automatic samplers, intelligent pole incubator, G6520B high precision quadrupole rod time-of-flight mass spectrometry instrument system); Chromatographic column: Agilent C
18Reversed-phase bonded silica chromatographic column (Angilent Zorbax Eclipse Plus C18), specification: 2.1 * 100mm, packing material size 1.8 μ m.
2 experimental techniques and result
2.1 the preparation of need testing solution: get 10 of toxin clearing away and the kidney nourishing capsules, remove capsule shells, collect content, and content is mixed to porphyrize, accurately weighed 0.5g, be placed in the 100ml volumetric flask, add the 70%(volume ratio) ethanol water 80ml, ultrasonic processing 30 minutes, take out, let cool, adding again the 70%(volume ratio) ethanol water is to scale, shakes up, filter, get subsequent filtrate, obtain need testing solution.
2.2 the preparation of reference substance solution: precision takes Paeoniflorin respectively, icariin, notoginsenoside R, the ginsenoside Rg1, the ginsenoside Rb1, the ginsenoside Rd, forulic acid, the calycosin glucoside, Astragaloside IV, Rhein, archen reference substance 2mg, with methyl alcohol dissolve and constant volume in the 100ml measuring bottle, make in every ml methanol solution and contain respectively Paeoniflorin, icariin, notoginsenoside R, the ginsenoside Rg1, the ginsenoside Rb1, the ginsenoside Rd, forulic acid, the calycosin glucoside, Astragaloside IV, Rhein, the reference substance solution of each 0.02mg of archen.
2.3 chromatographic condition: chromatographic column is Agilent C
18Reversed-phase bonded silica chromatographic column (Agilent Zorbax Eclipse Plus C18), specification 2.1mm * 100mm, filling material 1.8 μ m; Mobile phase 1 is the 0.1%(volume ratio) aqueous formic acid, mobile phase 2 be the 0.1%(volume ratio) the formic acid acetonitrile solution of volume; Flow velocity is 0.3ml/min; Column temperature is 30 ℃; Sample introduction 5 μ l; Adopt the gradient elution mode (at the uniform velocity) of table 1:
Table 1 gradient elution table
2.4 mass spectrum condition: ion gun is electron spray ESI source, and the negative ion mode detects; Atomization gas pressure: 2.39kPa(35psig), the dry gas temperature: 350 ℃, dry gas flow velocity: 10L/min, kapillary high voltage: 3500V, capillary outlet voltage: 100V.
2.5 determination method: precision is drawn reference substance solution and each 5 μ l of need testing solution respectively, and injection liquid matter combined instrument, measure, and records the collection of illustrative plates of 20 minutes.
2.6 the common characteristic peak is determined: need testing solution and reference substance solution are carried out to stratographic analysis.By the total ion current figure (TIC) to 10 batches of toxin clearing away and the kidney nourishing capsules, analyze relatively, find out its common characteristic peak, specifically see Fig. 1, extract and obtain extracting the ion flow graph by these common characteristic peak mass number, specifically see Fig. 2, mark the retention time Rt at each common characteristic peak, obtain the toxin clearing away and the kidney nourishing capsule standard fingerprint, its common characteristic peak is 21, and its retention time Rt value and accurate mass number are respectively 4.4min(525.1614), 4.8min(491.1195), 5.2min(193.0506), 6.4min(977.5327), 6.9min(845.4904), 7.8min(507.1508), 8.1min(463.1610), 8.3min(645.2193), 8.5min(867.2939), 8.9min(721.2349), 10.1min(623.1770), 10.9min(599.2997), 12.1min(829.4591), 12.8min(945.5428), 13.7min(871.4697), 14.0min(283.0248), 14.2min(659.2345), 15.0min(513.1766), 15.4min(913.4802), 16.2min(269.0455), 16.9min(535.3640).By the characteristic peak with reference substance solution extraction ion current, compare, result shows that wherein the Rt value is Paeoniflorin, calycosin glucoside, forulic acid, notoginsenoside R, ginsenoside Rg1, icariin, ginsenoside Rb1, Astragaloside IV, ginsenoside Rd, Rhein, archen for 4.4min, 4.8min, 5.2min, 6.4min, 6.9min, 8.9min, 10.9min, 12.1min, 12.8min, 14.0min, 16.2min chromatographic peak through conclusive evidence; With corresponding peak, icariin object of reference peak be the S peak, calculate the relative retention time of each characteristic peak, its relative retention time all setting ± 5% within, setting is followed successively by 0.50,0.54,0.59,0.72,0.78,0.88,0.92,0.94,0.96,1.00,1.14,1.23,1.37,1.44,1.54,1.58,1.61,1.69,1.74,1.82,1.91; Paeoniflorin, ginsenoside Rg1, icariin, ginsenoside Rb1's peak area and corresponding object of reference peak area ratio should be between 0.5～1.5.
2.7 precision test: get same toxin clearing away and the kidney nourishing capsule need testing solution continuous sample introduction 6 times, extract characteristic peak, mark retention time, No. 10 peak icariin of take are the S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value all is less than 1%, and the precision of instrument is good.Precision the results are shown in Table 2:
Table 2 precision result
2.8 study on the stability: get same toxin clearing away and the kidney nourishing capsule need testing solution, respectively at 0,1,2,4,8,12 hour sample introduction, extract characteristic peak, mark retention time, No. 10 peak icariin of take are the S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value all is less than 1%, need testing solution place in 12 hours stable.Stability result is in Table 3.
Table 3 stability result
2.9 replica test: get with 6 parts of a collection of toxin clearing away and the kidney nourishing capsules, according to the need testing solution preparation method, prepare, the difference sample introduction, extract characteristic peak, mark retention time, No. 10 peak icariin of take are the S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time relative standard deviation value all is less than 1%, and method is reproducible.Repeatability the results are shown in Table 4:
The repeated result of table 4
2.10 middle precision: get with a collection of toxin clearing away and the kidney nourishing capsule, under the variable such as same date, different analysts condition not, measure respectively in accordance with the law, result is as follows.
2.10.1 different analysis times: get with a collection of toxin clearing away and the kidney nourishing capsule, press need testing solution preparation method preparation respectively at same date not, parallel three parts, sample introduction, extract characteristic peak, marks retention time, No. 10 peak icariin of take are the S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value all is less than 1%, the results are shown in Table 5:
Table 5 different analysis time of result
2.10.2 different analysts: get with a collection of toxin clearing away and the kidney nourishing capsule, different personnel press need testing solution preparation method preparation, parallel three parts, sample introduction, extract characteristic peak, marks retention time, No. 10 peak icariin of take are the S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value all is less than 1%, the results are shown in Table 6:
The different analyst's results of table 6
2.11 toxin clearing away and the kidney nourishing capsule fingerprint pattern detection method application example
At present, be used for the treatment of clinically the early stage medicine of chronic renal failure, compensatory stage of renal insufficiency, azotemia phase and uremia and mainly contain NIAODUQING KELI, its prescription and effect all approach with the toxin clearing away and the kidney nourishing capsule.
Apply toxin clearing away and the kidney nourishing capsule fingerprint pattern detection method of the present invention, can differentiate quickly and accurately the true and false of toxin clearing away and the kidney nourishing capsule, toxin clearing away and the kidney nourishing capsule and NIAODUQING KELI are distinguished, both finger-prints relatively see Fig. 4-a and Fig. 4-b.
Therefore, the toxin clearing away and the kidney nourishing capsule fingerprint pattern detection method that we set up, can avoid product counterfeiting effectively, check toxin clearing away and the kidney nourishing capsule.
Claims (10)
1. the method for building up of a toxin clearing away and the kidney nourishing capsule fingerprint pattern, this method for building up comprises the following steps:
(1) content of toxin clearing away and the kidney nourishing capsule is dissolved in to the ethanol water of 70% volume ratio, makes the need testing solution that concentration is 5mg/ml;
(2) adopt the HPLC-MS instrument to detect need testing solution, obtain the total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C
18The reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with the electron spray detecting device, described mobile phase 1 is the 0.1%(volume ratio) aqueous formic acid, mobile phase 2 is the 0.1%(volume ratio) the formic acid acetonitrile solution, number percent meter by volume, described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% by 95%, and mobile phase 2 at the uniform velocity becomes 20% by 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% by 80%, and mobile phase 2 at the uniform velocity becomes 40% by 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 by 60%, and mobile phase 2 at the uniform velocity becomes 100% by 40%;
18-20 minute, mobile phase 1 is 0, mobile phase 2 is 100%.
2. the detection method of a toxin clearing away and the kidney nourishing capsule effective constituent, this detection method comprises the following steps:
(1) content of toxin clearing away and the kidney nourishing capsule is dissolved in to the 70%(volume ratio) ethanol water, make the need testing solution that concentration is 5mg/ml;
(2) Paeoniflorin, icariin, notoginsenoside R, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, forulic acid, calycosin glucoside, Astragaloside IV, Rhein and archen are dissolved in to the standby reference substance solution of Methanol, each concentration of component is 0.02mg/ml;
(3) adopt the HPLC-MS instrument to detect respectively need testing solution and reference substance solution, obtain the total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C
18The reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with the electron spray detecting device, described mobile phase 1 is the 0.1%(volume ratio) aqueous formic acid, mobile phase 2 is the 0.1%(volume ratio) the formic acid acetonitrile solution, number percent meter by volume, described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% by 95%, and mobile phase 2 at the uniform velocity becomes 20% by 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% by 80%, and mobile phase 2 at the uniform velocity becomes 40% by 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 by 60%, and mobile phase 2 at the uniform velocity becomes 100% by 40%;
18-20 minute, mobile phase 1 is 0, mobile phase 2 is 100%.
3. method according to claim 1 and 2, wherein, the total ion current collection of illustrative plates of described need testing solution comprises the characteristic peak with following retention time and mass number: 4.4min(525.1614), 4.8min(491.1195), 5.2min(193.0506), 6.4min(977.5327), 6.9min(845.4904), 8.9min(721.2349), 10.9min(599.2997), 12.1min(829.4591), 12.8min(945.5428), 14.0min(283.0248), 16.2min(269.0455).
4. according to the method in any one of claims 1 to 3, wherein, the total ion current collection of illustrative plates of described need testing solution comprises the characteristic peak with following retention time and mass number: 4.4min(525.1614), 4.8min(491.1195), 5.2min(193.0506), 6.4min(977.5327), 6.9min(845.4904), 7.8min(507.1508), 8.1min(463.1610), 8.3min(645.2193), 8.5min(867.2939), 8.9min(721.2349), 10.1min(623.1770), 10.9min(599.2997), 12.1min(829.4591), 12.8min(945.5428), 13.7min(871.4697), 14.0min(283.0248), 14.2min(659.2345), 15.0min(513.1766), 15.4min(913.4802), 16.2min(269.0455), 16.9min(535.3640).
5. according to the described method of claim 3 or 4, wherein, described method also comprises step (4):
The characteristic peak that the retention time of take in the total ion current figure of need testing solution is 4.4min is the S peak, calculates the relative retention time of each characteristic peak; The corresponding peak-to-peak peak area ratio of feature of retention time in the total ion current figure of calculating need testing solution and the total ion current figure of reference substance solution.
6. method according to claim 5, wherein, described step (4) comprises the described characteristic peak in the total ion current figure that extracts need testing solution, the characteristic peak that the retention time of take is 4.4min is the S peak, calculates the relative retention time of each characteristic peak; The corresponding peak-to-peak peak area ratio of feature of retention time in the total ion current figure of calculating need testing solution and the total ion current figure of reference substance solution.
7. according to the described method of any one in claim 1 to 6, wherein, described step (1) comprises the following steps: get the content of toxin clearing away and the kidney nourishing capsule, mix porphyrize, weighing 0.5g, being placed in the 100ml volumetric flask, in volumetric flask, adding the 70%(volume ratio) ethanol water dissolves described content, then to the 70%(volume ratio that adds surplus in volumetric flask) ethanol water is to scale, shake up, filter, get subsequent filtrate, obtain need testing solution.
8. according to the described method of any one in claim 1 to 7, wherein, it is 0.3ml/min that described high performance liquid chromatography detects the flow rate of mobile phase adopted, and column temperature is 30 ℃, and sample size is 5 μ l.
9. according to the described method of any one in claim 1 to 8, wherein, described Mass Spectrometer Method adopts level Four bar-flight time tandem mass spectrometer.
10. according to the described method of any one in claim 1 to 9, wherein, described Mass Spectrometer Method adopts the electron spray ionisation source, in the negative ion mode, detect, atomization gas pressure is 2.39kPa, and the dry gas temperature is 350 ℃, and the dry gas flow velocity is 10 liter/mins, the kapillary high voltage is 3500V, and capillary outlet voltage is 100V.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN111213882A (en) * | 2020-03-11 | 2020-06-02 | 北京康立生医药技术开发有限公司 | Plant mixed extraction process and application thereof |
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| WO2021062889A1 (en) * | 2019-09-30 | 2021-04-08 | 青岛琛蓝医药科技发展有限公司 | Method for quality control and chromatographic fingerprinting of epimedium compound product |
| CN115494181A (en) * | 2022-10-17 | 2022-12-20 | 山东省食品药品检验研究院 | Detection method for simultaneously identifying five raw material medicines in collateral-dredging and phlegm-reducing capsule |
| CN118275595A (en) * | 2024-05-29 | 2024-07-02 | 广州白云山和记黄埔中药有限公司 | Fingerprint spectrum and multi-component content determination method of nasopharynx toxicity-removing preparation |
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| CN108061762A (en) * | 2017-03-06 | 2018-05-22 | 石家庄平安医院有限公司 | The content assaying method of three kinds of flavone components in a kind of kidney tonifying eliminating toxic particle |
| CN108061762B (en) * | 2017-03-06 | 2020-07-28 | 石家庄平安医院有限公司 | Method for measuring contents of three flavone components in kidney-tonifying toxin-expelling granules |
| CN112014481A (en) * | 2019-05-29 | 2020-12-01 | 湖北梦阳药业股份有限公司 | Detection method of fingerprint spectrum of Shengbai oral liquid |
| WO2021062889A1 (en) * | 2019-09-30 | 2021-04-08 | 青岛琛蓝医药科技发展有限公司 | Method for quality control and chromatographic fingerprinting of epimedium compound product |
| CN111213882A (en) * | 2020-03-11 | 2020-06-02 | 北京康立生医药技术开发有限公司 | Plant mixed extraction process and application thereof |
| CN115494181A (en) * | 2022-10-17 | 2022-12-20 | 山东省食品药品检验研究院 | Detection method for simultaneously identifying five raw material medicines in collateral-dredging and phlegm-reducing capsule |
| CN115494181B (en) * | 2022-10-17 | 2024-05-17 | 山东省食品药品检验研究院 | Detection method for simultaneously identifying five raw materials in capsule for dredging collaterals and resolving phlegm |
| CN118275595A (en) * | 2024-05-29 | 2024-07-02 | 广州白云山和记黄埔中药有限公司 | Fingerprint spectrum and multi-component content determination method of nasopharynx toxicity-removing preparation |
| CN118275595B (en) * | 2024-05-29 | 2024-09-13 | 广州白云山和记黄埔中药有限公司 | Fingerprint spectrum and multi-component content determination method of nasopharynx toxicity-removing preparation |
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