CN103424498B - 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 drug tests, 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 carries out compatibility combination by the above Chinese medicine of two tastes and is applied to the preparation of disease treatment.Because of its complicated component, so the difficulty accurately detecting its each effective constituent is very large.
Traditional Chinese medicine fingerprint means Chinese medicine, comprise Chinese crude drug, medicine materical crude slice, extract or Chinese patent drug after suitably processing, the chromatogram that can indicate this Chinese medicine main chemical compositions characteristic taking certain analysis means to obtain or spectrogram, it has the features such as characteristic, globality, sxemiquantitative, has been widely used in the analysis and resolution of Chinese medicine in recent years.
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 be familiar with gradually and accepted.But because its instrument price is expensive, be not also used widely.Ultra Performance Liquid Chromatography (UHPLC)-flight time mass spectrum combined instrument can not only by Chinese medicine complicated ingredient quick separating, and it adopts ionization techniques, some test problems without uv absorption composition can be solved, the accurate molecular weight of ionizable composition can also be obtained, so this detection technique is the Structural Identification of compound Chinese medicinal preparation constituent and confirmation provide of great value reference frame.
The new Chinese medicine of toxin clearing away and the kidney nourishing capsule Shi Li pearl group independent research, be mainly used in treatment chronic renal failure, compensatory stage of renal insufficiency, azotemia phase and uremia early stage.This medicine is made up of the medicinal material such as the Radix Astragali, rheum officinale, has tonifying speen and tonifying kidney, ventilation quality, effect promoting blood circulation and removing blood stasis.From 1996, treated early, mid-term Chronic Renal Failure Patients more than 1000 example.This capsule significantly can reduce serum creatinine and urea nitrogen concentration, increase appetite and urine volume, 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, carries out thin-layered chromatography discriminating according to Chinese Pharmacopoeia version in 2010 annex VIB.Specify containing quantifier: according to Chinese Pharmacopoeia 2010 editions one annex VID high effective liquid chromatography for measuring, wherein press Paeoniflorin (C containing Chinese herbaceous peony for every
23h
28o
11) calculate, must not 0.45mg be less than; Icariin (C is pressed for every containing barrenwort
33h
40o
15) calculate, must not 0.15mg be less than.
But, the detection method of existing toxin clearing away and the kidney nourishing capsule is perfect not enough, still can not reflect various effective constituent and content thereof comprehensively, accurately and efficiently, 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 the weak point making up 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, effective constituent and the content thereof of toxin clearing away and the kidney nourishing capsule can be shown quick, easy, fully and effectively.
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 70%(volume ratio) ethanol water, make the need testing solution that concentration is 5mg/ml;
(2) adopt HPLC-MS instrument to detect need testing solution, obtain total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C
18reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with electron spray detecting device, described mobile phase 1 is 0.1%(volume ratio) aqueous formic acid, mobile phase 2 is 0.1%(volume ratio) formic acid acetonitrile solution, by volume percentages, described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, and mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% from 95%, and mobile phase 2 at the uniform velocity becomes 20% from 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% from 80%, and mobile phase 2 at the uniform velocity becomes 40% from 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 from 60%, and mobile phase 2 at the uniform velocity becomes 100% from 40%;
18-20 minute, mobile phase 1 is 0, and 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, this detection method comprises the following steps:
(1) content of toxin clearing away and the kidney nourishing capsule is dissolved in 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 Methanol for reference substance solution, each concentration of component is 0.02mg/ml;
(3) adopt HPLC-MS instrument to detect need testing solution and reference substance solution respectively, obtain total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C
18reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with electron spray detecting device, described mobile phase 1 is 0.1%(volume ratio) aqueous formic acid, mobile phase 2 is 0.1%(volume ratio) formic acid acetonitrile solution, by volume percentages, described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, and mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% from 95%, and mobile phase 2 at the uniform velocity becomes 20% from 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% from 80%, and mobile phase 2 at the uniform velocity becomes 40% from 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 from 60%, and mobile phase 2 at the uniform velocity becomes 100% from 40%;
18-20 minute, mobile phase 1 is 0, and 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 being 4.4min with the retention time in the total ion current figure of need testing solution is S peak, calculates the relative retention time of each characteristic peak; Calculate the peak-to-peak peak area ratio of feature that in the total ion current figure of need testing solution and the total ion current figure of reference substance solution, retention time is corresponding.
More preferably, described step (4) comprises the described characteristic peak in the total ion current figure extracting need testing solution, take retention time as the characteristic peak of 4.4min is S peak, calculates the relative retention time of each characteristic peak; Calculate the peak-to-peak peak area ratio of feature that in the total ion current figure of need testing solution and the total ion current figure of reference substance solution, retention time is corresponding.
Preferably, in the above described two methods, described step (1) comprises the following steps: the content getting toxin clearing away and the kidney nourishing capsule, mixing porphyrize, weigh 0.5g, be placed in 100ml volumetric flask, in volumetric flask, add 70%(volume ratio) ethanol water dissolves described content, then in volumetric flask, adds the 70%(volume ratio of surplus) ethanol water is to scale, shake up, filter, get subsequent filtrate, obtain need testing solution.
Preferably, in the above described two 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 DEG C, 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 electron spray ionisation source, and detect with negative-ion mode, atomization gas pressure is 2.39kPa, and dry gas temperature is 350 DEG C, and dry gas flow velocity is 10 liters/min, and kapillary high voltage is 3500V, and capillary outlet voltage is 100V.
In existing toxin clearing away and the kidney nourishing capsule quality standard, the requirement of " discriminating " item for contrast, carries out TLC distinguish with Astragaloside IV, icariin, Paeoniflorin, Ginsenosides Rg1 and Rb1 reference substance and rheum officinale, Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material; " assay " item specifies: adopt the paeoniflorin content in high effective liquid chromatography for measuring white Peony Root and the Icariin content in epimedium herb, not its intrinsic chemical composition is not carried out comprehensively, systematic research, its Testing index is single, complex operation, cannot 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 in the middle of the detection applying it to toxin clearing away and the kidney nourishing capsule.By the feature that has that it's too late at peak total in finger-print, effective constituent and content thereof can be determined effectively, all sidedly, the stability of monitoring production technology, ensure constant product quality, homogeneous, controlled, method of the present invention also has stability and favorable reproducibility, operability advantages of higher simultaneously.
Accompanying drawing explanation
Fig. 1 is the total ion current collection of illustrative plates of toxin clearing away and the kidney nourishing capsule need testing solution, and in figure 1 to 21 is the numbering of characteristic peak.
Fig. 2 is the extraction ion current collection of illustrative plates of toxin clearing away and the kidney nourishing capsule need testing solution, and in figure 1 to 21 is the numbering of characteristic peak.
Fig. 3 is the extraction ion current collection of illustrative plates of reference substance solution, and 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, and wherein Fig. 4-a is the total ion current collection of illustrative plates of toxin clearing away and the kidney nourishing capsule, and Fig. 4-b is the total ion current collection of illustrative plates of 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 embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1
1 material and instrument
1.1 materials: 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 instruments: 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 systems); Chromatographic column: Agilent C
18reversed-phase bonded silica chromatographic column (AngilentZorbax Eclipse Plus C18), specification: 2.1 × 100mm, packing material size 1.8 μm.
2 experimental techniques and result
The preparation of 2.1 need testing solutions: get toxin clearing away and the kidney nourishing capsule 10, removing capsule shells, collects content, and content is mixed porphyrize, accurately weighed 0.5g, be placed in 100ml volumetric flask, add 70%(volume ratio) ethanol water 80ml, ultrasonic process 30 minutes, takes out, let cool, add 70%(volume ratio again) ethanol water to scale, shake up, filter, get subsequent filtrate, obtain need testing solution.
The preparation of 2.2 reference substance solution: precision takes Paeoniflorin respectively, icariin, notoginsenoside R, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, forulic acid, calycosin glucoside, Astragaloside IV, Rhein, archen reference substance 2mg, with methyl alcohol dissolve and constant volume in 100ml measuring bottle, make in every ml methanol solution respectively containing Paeoniflorin, icariin, notoginsenoside R, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, forulic acid, calycosin glucoside, Astragaloside IV, Rhein, the reference substance solution of each 0.02mg of archen.
2.3 chromatographic conditions: chromatographic column is Agilent C
18reversed-phase bonded silica chromatographic column (AgilentZorbax Eclipse Plus C18), specification 2.1mm × 100mm, 1.8 μm, filling material; Mobile phase 1 is 0.1%(volume ratio) aqueous formic acid, mobile phase 2 be 0.1%(volume ratio) the formic acid acetonitrile solution of volume; Flow velocity is 0.3ml/min; Column temperature is 30 DEG C; 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 Spectrometry Conditions: ion gun is electron spray ESI source, negative-ion mode detects; Atomization gas pressure: 2.39kPa(35psig), dry gas temperature: 350 DEG C, dry gas flow velocity: 10L/min, kapillary high voltage: 3500V, capillary outlet voltage: 100V.
2.5 determination methods: accurate absorption reference substance solution and each 5 μ l of need testing solution respectively, injection liquid matter combined instrument, measures, record the collection of illustrative plates of 20 minutes.
2.6 common characteristic peaks are determined: carry out stratographic analysis to need testing solution and reference substance solution.By total ion current figure (TIC) com-parison and analysis to 10 batches of toxin clearing away and the kidney nourishing capsules, find out its common characteristic peak, specifically see Fig. 1, extract by these common characteristic peak mass number and obtain extracting ion flow graph, specifically see Fig. 2, mark the retention time Rt at each common characteristic peak, obtain 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).Compared by the characteristic peak extracting ion current with reference substance solution, result show wherein Rt value 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 confirmation be Paeoniflorin, calycosin glucoside, forulic acid, notoginsenoside R, ginsenoside Rg1, icariin, ginsenoside Rb1, Astragaloside IV, ginsenoside Rd, Rhein, archen; The peak corresponding to icariin object of reference peak is 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 between 0.5 ~ 1.5.
2.7 precision tests: get same toxin clearing away and the kidney nourishing capsule need testing solution continuous sample introduction 6 times, extract characteristic peak, mark retention time, with No. 10 peak icariin for S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value is all 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, with No. 10 peak icariin for S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value is all less than 1%, and need testing solution is stable in placement 12 hours.Stability result is in table 3.
Table 3 stability result
2.9 replica tests: get with a collection of toxin clearing away and the kidney nourishing capsule 6 parts, prepare according to need testing solution preparation method, sample introduction respectively, extract characteristic peak, mark retention time, with No. 10 peak icariin for S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time relative standard deviation value is all less than 1%, and method is reproducible.Repeatability the results are shown in Table 4:
The repeated result of table 4
2.10 Intermediate precision: get with a collection of toxin clearing away and the kidney nourishing capsule, respectively under the not variable such as same date, different analysts condition, measure, result is as follows in accordance with the law.
2.10.1 different analysis time: get with a collection of toxin clearing away and the kidney nourishing capsule, need testing solution preparation method preparation is pressed respectively at not same date, parallel three parts, sample introduction, extracts characteristic peak, marks retention time, with No. 10 peak icariin for S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value is all less than 1%, the results are shown in Table 5:
Table 5 result different analysis time
2.10.2 different analyst: 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, extracts characteristic peak, marks retention time, with No. 10 peak icariin for S peak, calculate other each characteristic peak relative retention time, result shows that each characteristic peak relative retention time RSD value is all less than 1%, the results are shown in Table 6:
The different analyst's result of table 6
2.11 toxin clearing away and the kidney nourishing capsule fingerprint pattern detection method application examples
At present, the medicine being used for the treatment of chronic renal failure, compensatory stage of renal insufficiency, azotemia phase and uremia clinically early stage mainly contains NIAODUQING KELI, and its prescription and effect are all close with 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, the true and false of toxin clearing away and the kidney nourishing capsule can be differentiated quickly and accurately, 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, inspection toxin clearing away and the kidney nourishing capsule.
Claims (5)
1. a method for building up for 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 got, mixing porphyrize, weigh 0.5g, be placed in 100ml volumetric flask, the ethanol water adding volume ratio 70% in volumetric flask dissolves described content, then the ethanol water of volume ratio 70% adding surplus in volumetric flask is to scale, shake up, filter, get subsequent filtrate, obtain need testing solution;
(2) adopt HPLC-MS instrument to detect need testing solution, obtain total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C18 reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with electron spray detecting device, described mobile phase 1 is the aqueous formic acid of volume ratio 0.1%, mobile phase 2 is the formic acid acetonitrile solution of volume ratio 0.1%, by volume percentages, and described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, and mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% from 95%, and mobile phase 2 at the uniform velocity becomes 20% from 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% from 80%, and mobile phase 2 at the uniform velocity becomes 40% from 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 from 60%, and mobile phase 2 at the uniform velocity becomes 100% from 40%;
18-20 minute, mobile phase 1 is 0, and mobile phase 2 is 100%,
And the flow rate of mobile phase adopted is 0.3ml/min, and column temperature is 30 DEG C, and sample size is 5 μ l;
Described Mass Spectrometer Method adopts level Four bar-flight time tandem mass spectrometer; Adopt electron spray ionisation source, detect with negative-ion mode, atomization gas pressure is 2.39kPa, and dry gas temperature is 350 DEG C, and dry gas flow velocity is 10 liters/min, and kapillary high voltage is 3500V, and capillary outlet voltage is 100V.
2. method according to claim 1, 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.
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, 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.
4. a detection method for toxin clearing away and the kidney nourishing capsule, this detection method comprises the following steps:
(1) content of toxin clearing away and the kidney nourishing capsule is got, mixing porphyrize, weigh 0.5g, be placed in 100ml volumetric flask, the ethanol water adding volume ratio 70% in volumetric flask dissolves described content, then the ethanol water of volume ratio 70% adding surplus in volumetric flask is to scale, shake up, filter, get subsequent filtrate, obtain need testing solution;
(2) Paeoniflorin, icariin, notoginsenoside R, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, forulic acid, calycosin glucoside, Astragaloside IV, Rhein and archen are dissolved in Methanol for reference substance solution, each concentration of component is 0.02mg/ml;
(3) adopt HPLC-MS instrument to detect need testing solution and reference substance solution respectively, obtain total ion current collection of illustrative plates; Wherein, described high performance liquid chromatography detects and adopts C18 reversed-phase bonded silica chromatographic column, mobile phase 1 and mobile phase 2 carry out gradient elution, and detect with electron spray detecting device, described mobile phase 1 is the aqueous formic acid of volume ratio 0.1%, mobile phase 2 is the formic acid acetonitrile solution of volume ratio 0.1%, by volume percentages, and described gradient is as follows:
0-0.5 minute, mobile phase 1 is 95%, and mobile phase 2 is 5%;
0.5-3 minute, mobile phase 1 at the uniform velocity becomes 80% from 95%, and mobile phase 2 at the uniform velocity becomes 20% from 5%;
3-13 minute, mobile phase 1 at the uniform velocity becomes 60% from 80%, and mobile phase 2 at the uniform velocity becomes 40% from 20%;
13-18 minute, mobile phase 1 at the uniform velocity becomes 0 from 60%, and mobile phase 2 at the uniform velocity becomes 100% from 40%;
18-20 minute, mobile phase 1 is 0, and mobile phase 2 is 100%,
And the flow rate of mobile phase adopted is 0.3ml/min, and column temperature is 30 DEG C, and sample size is 5 μ l;
Described Mass Spectrometer Method adopts level Four bar-flight time tandem mass spectrometer; Adopt electron spray ionisation source, detect with negative-ion mode, atomization gas pressure is 2.39kPa, and dry gas temperature is 350 DEG C, and dry gas flow velocity is 10 liters/min, and kapillary high voltage is 3500V, and capillary outlet voltage is 100V;
And the total ion current collection of illustrative plates of the described need testing solution wherein, obtained 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) extract the described characteristic peak in the total ion current figure of need testing solution, take retention time as the characteristic peak of 4.4min be S peak, calculate the relative retention time of each characteristic peak; Calculate the peak-to-peak peak area ratio of feature that in the total ion current figure of need testing solution and the total ion current figure of reference substance solution, retention time is corresponding.
5. method according to claim 4, 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.
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| CN201210167274.0A CN103424498B (en) | 2012-05-25 | 2012-05-25 | Establishing method and application of fingerprint of detoxifying and kidney harmonizing capsule |
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| 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 |
| CN112014481B (en) * | 2019-05-29 | 2022-10-25 | 湖北梦阳药业股份有限公司 | Detection method of raw white oral liquid fingerprint spectrum |
| CN110501445B (en) * | 2019-09-30 | 2020-12-01 | 青岛琛蓝医药科技发展有限公司 | Quality control and fingerprint spectrum detection method for epimedium compound |
| 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 |
| 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 |
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