CN109856274A - Open the discrimination method of Medicated Leaven ingredient in spleen ball and Qipi oral liquid - Google Patents
Open the discrimination method of Medicated Leaven ingredient in spleen ball and Qipi oral liquid Download PDFInfo
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- CN109856274A CN109856274A CN201910092575.3A CN201910092575A CN109856274A CN 109856274 A CN109856274 A CN 109856274A CN 201910092575 A CN201910092575 A CN 201910092575A CN 109856274 A CN109856274 A CN 109856274A
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Abstract
The invention discloses a kind of discrimination methods for opening Medicated Leaven ingredient in spleen ball and Qipi oral liquid, the discrimination method is using amarogentin as reference substance, it whether is opened in spleen ball and Qipi oral liquid sample using the method detection of high performance liquid chromatography tandem mass spectrum containing amarogentin, is fed intake underproof problem to identify and open in spleen ball and Qipi oral liquid sample with the presence or absence of Medicated Leaven.High performance liquid chromatography tandem mass spectrum method is quick, sensitive, can accurately identify in sample whether contain amarogentin, establish the discrimination method of Medicated Leaven, and the quality of raising Medicated Leaven, control are opened feeding intake for Medicated Leaven in spleen ball and Qipi oral liquid and is of great significance.
Description
Technical field
The present invention relates to the authentication technique field of Chinese patent drug, it is specifically a kind of open in spleen ball and Qipi oral liquid Medicated Leaven at
The discrimination method divided.
Background technique
Spleen ball (big honeyed bolus) is opened by ginseng, stir-baked RHIZOMA ATRACTYLDIS MACROCEPHALAE in bran, Poria cocos, Radix Glycyrrhizae, dried orange peel, Chinese yam, lotus seeds (stir-fry), stir-baked FRUCTUS CRATAEGI, six
Fine medicinal material powder adds refined honey to be mixed to prepare to Divine Comedy (stir-fry), coloured malt, rhizoma alismatis etc. ten simply.Spleen ball (water-bindered pill) is opened by ginseng, bran
The medicinal material simply of rhizoma atractylodis macrocephalae, Poria cocos, Radix Glycyrrhizae, dried orange peel, Chinese yam, lotus seeds (stir-fry), stir-baked FRUCTUS CRATAEGI, Medicated Leaven (stir-fry), coloured malt, rhizoma alismatis etc. ten
Fine powder is sieved, and mixes, and with water pill, polishing, dry be made is formed.Qipi oral liquid is by ginseng, stir-baked RHIZOMA ATRACTYLDIS MACROCEPHALAE in bran, Poria cocos, sweet
Medicinal material forms grass, dried orange peel, Chinese yam, stir-fry lotus seeds, stir-baked FRUCTUS CRATAEGI, stir-fry Medicated Leaven, coloured malt etc. ten simply.70% alcohol reflux of ginseng
It extracts three times, 3 hours for the first time, second 2 hours, third time 3 hours, extracting solution recycles ethanol, and the another extracting container of medical fluid is received
Collection;Dried orange peel, stir-baked RHIZOMA ATRACTYLDIS MACROCEPHALAE in bran extraction volatile oil are spare;Aqueous solution after the dregs of a decoction and distillation merges with eight taste medicines such as remaining Poria cocos, adds
Water decocts 2 times, 2 hours for the first time, second 1 hour, decocting liquid filtration, merging filtrate, be concentrated into relative density be 1.12~
1.20(85 DEG C) medicinal extract, add ethyl alcohol to alcohol content up to 70%, stir evenly, stand 24 hours, take supernatant recycle ethyl alcohol, filtration, sugarcane
Simple syrup is made in sugared 150g, and potassium sorbate 1.5g is dissolved with water, and above-mentioned volatile oil is added thereto, and mixes, and adjusting pH to 4.0~
5.0,1000mL is made, stirs evenly, filter, sterilizing, packing to get.'Qipi ' has the effect of strengthening the spleen and stomach;It is clinically used for taste
Weakness, indigestion, abdominal distension loose stool.
Medicated Leaven has the function of strengthening the spleen and stomach, in the tune that helps digestion, and is used for weakness of the spleen and the stomach, retention of food and drink, feeling of stuffiness in chest abdominal distension, children
Dyspepsia.Each province's concocted specification or Chinese medicine standard have been recorded, and composition includes flour, wheat bran, semen armeniacae amarae, red small
Beans, sweet wormwood, polygonum flaccidum, Siberian cocklebur grass, but each medicinal material dosage ratio is not quite similar, it is quite different.The adulterant of Medicated Leaven is more, predominantly
Use inferior materials and turn out substandard goods in production and processing, do not add semen armeniacae amarae and rde bean raw material mostly, after the fermented processing of these medicinal materials, appearance with just
Product Medicated Leaven is very much like, and identification difficulty is big, and illegal retailer often pretends to be Medicated Leaven with this fake and poor products, illegally speculates, seriously
Influence quality of medicinal material, curative effect and the drug safety of Medicated Leaven.Medicated Leaven quality currently on the market there is a problem of very big, pass through
Traditional method cannot achieve the purpose that accurately to identify.It opens Medicated Leaven in spleen ball prescription and feeds intake the ten of only all medicinal material input amounts
/ mono-, and semen armeniacae amarae proportion also very little in Medicated Leaven.Since content is low in the sample for amarogentin, use is general
Constant discrimination method is more difficult to identify.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides one kind to open Medicated Leaven ingredient in spleen ball and Qipi oral liquid
Discrimination method opened using the method detection of high performance liquid chromatography tandem mass spectrum using amarogentin as reference substance and spleen ball and open spleen
Whether contain amarogentin in oral solution sample, feeds intake in spleen ball and Qipi oral liquid sample with the presence or absence of Medicated Leaven to identify to open
Underproof problem opens feeding intake for Medicated Leaven in spleen ball and Qipi oral liquid for control and provides technical support.
Technical solution of the present invention is summarized as follows:
The discrimination method of Medicated Leaven ingredient in spleen ball and Qipi oral liquid is opened, specific method includes following content:
(1) chromatographic condition: using octadecyl silane as filler (chromatography column internal diameter 2.1mm, length 100mm, 1.8 μ of partial size
M), 35 DEG C of column temperature;Using 0.1% aqueous formic acid as mobile phase A, acetonitrile is Mobile phase B;Gradient elution is carried out by the regulation of table 1.
(2) Mass Spectrometry Conditions: mass spectrum assembles electrospray ionisation source (ESI), dry temperature degree: 350 DEG C;Nebulizer pressure: 40
Psi;Dry gas stream speed: 5 L/min, sheath stream temperature degree: 350 DEG C;Sheath stream gas velocity: 10 L/min, High pressure RF:
150v;Low pressure RF:60v;Capillary voltage: 4000 v;Spray nozzle voltage: 500 V;Residence time: 60ms;Detection
Mode: positive ion detection, scanning mode: multiple-reaction monitoring (MRM).Mass spectrometry parameters are shown in Table 2.
(3) preparation of reference substance solution: being made certain density stock solution for amarogentin with methanol, and when use is diluted to
Concentration is the standard solution of 100 ng/mL.
(4) preparation of test solution:
1) spleen ball is opened as test sample: being taken and is opened spleen ball sample and shred, is mixed, is weighed 2 g, add 1 g of diatomite, grind well, and first is added
50 mL of alcohol is ultrasonically treated 30min, and with 0.2 μm of membrane filtration, filtrate is concentrated into 5 mL, then with 0.2 μm of membrane filtration, takes continuous filter
Liquid to get;
2) Qipi oral liquid is as test sample: take Qipi oral liquid cross 0.2 μm of filter membrane to get.
(5) prepared by negative sample solution:
1) it takes and lacks the amygdalate spleen ball negative sample that opens and shred, mix, weigh 2 g, add 1 g of diatomite, grind well, methanol 50 is added
ML is ultrasonically treated 30min, and with 0.2 μm of membrane filtration, filtrate is concentrated into 5 mL, then with 0.2 μm of membrane filtration, takes subsequent filtrate, i.e.,
Spleen ball negative sample solution must be opened.
2) it takes and lacks amygdalate Qipi oral liquid negative sample to cross 0.2 μm of filter membrane molten to get Qipi oral liquid negative sample
Liquid.
(6) it measures: it is accurate respectively to draw reference substance solution, negative sample solution and each 2 μ L of test solution, inject liquid matter
Combined instrument detected to get.
The beneficial effects of the present invention are:
Discrimination method of the invention opens spleen using amarogentin as reference substance, using the method detection of high performance liquid chromatography tandem mass spectrum
Whether contain amarogentin in ball and Qipi oral liquid sample, opens in spleen ball and Qipi oral liquid sample to identify with the presence or absence of six
Divine Comedy feeds intake underproof problem.The adulterant of Medicated Leaven is more, predominantly uses inferior materials and turns out substandard goods in production and processing, does not add bitter apricot mostly
Renhe rde bean raw material, and the present invention then can detecte out using amarogentin as reference substance used in Medicated Leaven ingredient whether
It is added to semen armeniacae amarae, is showed to obtain and open to whether there is in spleen ball and Qipi oral liquid sample using what adulterant Medicated Leaven fed intake
As.High performance liquid chromatography tandem mass spectrum method is quick, sensitive, can accurately identify in sample whether contain amarogentin, solve
The problem of general constant discrimination method cannot being used to identify due to amarogentin content is few, establish the identification side of Medicated Leaven
Method opens feeding intake for Medicated Leaven in spleen ball and Qipi oral liquid and is of great significance to the quality of raising Medicated Leaven, control.
Detailed description of the invention
Fig. 1 is the thin-layer chromatogram for opening spleen ball, and spleen ball analog sample is opened in 1 expression in figure, and spleen ball opens in 2 expression J pharmaceutical factories
(lot number: 17013074), 3 indicate that opening spleen ball lacks semen armeniacae amarae negative sample to sample, and 4 indicate that amarogentin reference substance, A are blackish green
Spot;
Fig. 2 is that the HPLC-MS MRM of amarogentin reference substance schemes;
Fig. 3 is to open spleen ball sample (D company, lot number: 1711042) HPLC-MS MRM figure;
Fig. 4 is the HPLC-MS MRM figure for opening spleen ball sample (E company, lot number: A16280);
Fig. 5 is to open spleen ball sample (company A, lot number: 817031) HPLC-MS MRM figure;
Fig. 6 is Qipi oral liquid sample (K company, lot number: 20171155) HPLC-MS MRM figure;
Fig. 7 is the HPLC-MS MRM figure for opening spleen ball and lacking semen armeniacae amarae negative sample.
Specific embodiment
For the more detailed introduction present invention, below with reference to embodiment, the present invention will be further described.
Embodiment, content are as follows:
Instrument: highly effective liquid phase chromatographic system (including 1290 G4220B of Agilent pump, 1290 G4226A autosamplers
1290 column ovens, Agilent Technologies, the U.S.), mass spectrometer: 6460 QQ-TOF mass spectrometry instrument of Agilent (U.S.'s peace
Jie Lun scientific & technical corporation);BP211D type electronic analytical balance (Sartorius AG);Milli-Q type ultrapure water preparing instrument
(Millipore company, the U.S.).
Reagent: acetonitrile (chromatographic grade, Merck), methanol (analyze pure, Sinopharm Chemical Reagent Co., Ltd.), and formic acid is (excellent
Grade is pure, and fine chemistry industry research institute is recovered in Tianjin), ultrapure water (is prepared) by Milli-Q pure water system, amarogentin (Zhong Jian institute,
Lot number 110820-201607).
Test sample is replaced with letter from 11 different 'Qipi ' production units, the title of production unit, and one
A corresponding organization of letter, the several patents applied on the same day are consistent herein.
One, the applicant is first tested using thin-layered chromatography, specifically when Medicated Leaven ingredient in spleen ball is opened in identification
Content is as follows:
(1) it taking and opens spleen ball sample 9g, add diatomite 5g finely ground, add methanol 30mL, be ultrasonically treated 30 minutes, filtration, filtrate is evaporated,
Residue is evaporated, and residue adds water 5mL to make to dissolve, by D101 type macroporous absorbent resin (internal diameter 1.5cm, long 8cm), with dilute ammoniacal liquor (4
→ 100) 30mL is eluted, and is discarded ammoniacal liquor, then eluted with water 20mL, is discarded aqueous, then eluted with 20% ethyl alcohol 30mL, collection is washed
De- liquid, is evaporated, residue adds methanol 1mL to make to dissolve, as test solution.
(2) take lack it is amygdalate open spleen ball negative sample 9g, add diatomite 5g finely ground, add methanol 30mL, ultrasonic treatment 30
Minute, filtration, filtrate is evaporated, and residue is evaporated, and residue adds water 5mL to make to dissolve, and passes through D101 type macroporous absorbent resin (internal diameter
1.5cm, long 8cm), it is eluted with dilute ammoniacal liquor (4 → 100) 30mL, discards ammoniacal liquor, then eluted with water 20mL, discard aqueous, then use
20% ethyl alcohol 30mL elution, collects eluent, is evaporated, residue adds methanol 1mL to make to dissolve, as negative sample solution.
(3) amarogentin reference substance is taken, the solution of every 1mL amarogentin containing 1mg is made of methanol, it is molten as reference substance
Liquid.
(4) test solution, negative sample solution, each 10 μ L of reference substance solution are drawn, is put respectively in same silica G thin layer
On plate, with acetate-methanol-water (20:5:3) for solvent, it is unfolded, takes out, dry, sprays with 5% vanillin-sulfuric acid solution,
It is clear that spot development is heated at 105 DEG C.In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, identical face should be shown
The spot of color.
(5) thin-layered chromatography test result is shown in Fig. 1, does not find to open spleen ball sample and amarogentin reference substance in thin-layer chromatography
Corresponding position shows same blob.
The analysis of causes: opening Medicated Leaven in spleen ball prescription and feed intake 1/10th of only all medicinal material input amounts, and semen armeniacae amarae
The proportion also very little in Medicated Leaven.It is difficult using general constant discrimination method since content is low in the sample for amarogentin
To identify.The above reason is equally applicable to Qipi oral liquid, it is therefore desirable to and the higher discrimination method of sensitivity is identified,
By the study found that with higher sensitivity high performance liquid chromatography tandem mass spectrum method can be used to be identified.
Two, the discrimination method for opening Medicated Leaven ingredient in spleen ball and Qipi oral liquid, using amarogentin as reference substance, using height
Whether the method detection of effect liquid phase chromatogram tandem mass spectrum opens in spleen ball and Qipi oral liquid sample containing amarogentin, opens to identify
It feeds intake underproof problem in spleen ball and Qipi oral liquid sample with the presence or absence of Medicated Leaven, specific method includes following content:
(1) chromatographic condition: using octadecyl silane as filler (chromatography column internal diameter 2.1mm, length 100mm, 1.8 μ of partial size
M), 35 DEG C of column temperature;Using 0.1% aqueous formic acid as mobile phase A, acetonitrile is Mobile phase B;Gradient elution is carried out by the regulation of table 1.
(2) Mass Spectrometry Conditions: mass spectrum assembles electrospray ionisation source (ESI), dry temperature degree: 350 DEG C;Nebulizer pressure: 40
Psi;Dry gas stream speed: 5 L/min, sheath stream temperature degree: 350 DEG C;Sheath stream gas velocity: 10 L/min, High pressure RF:
150v;Low pressure RF:60v;Capillary voltage: 4000 v;Spray nozzle voltage: 500 V;Residence time: 60ms;Detection
Mode: positive ion detection, scanning mode: multiple-reaction monitoring (MRM).Mass spectrometry parameters are shown in Table 2.
(3) preparation of reference substance solution: being made certain density stock solution for amarogentin with methanol, and when use is diluted to
Concentration is the standard solution of 100 ng/mL.
(4) preparation of test solution:
1) spleen ball is opened as test sample: being taken and is opened spleen ball sample and shred, is mixed, is taken 2 g, add 1 g of diatomite, grind well, and methanol is added
50 mL are ultrasonically treated 30min, and with 0.2 μm of membrane filtration, filtrate is concentrated into 5 mL, then with 0.2 μm of membrane filtration, take continuous filter
Liquid to get;
2) Qipi oral liquid is as test sample: take Qipi oral liquid cross 0.2 μm of filter membrane to get.
(5) prepared by negative sample solution:
1) it takes and lacks the amygdalate spleen ball negative sample that opens and shred, mix, weigh 2 g, add 1 g of diatomite, grind well, methanol 50 is added
ML is ultrasonically treated 30min, and with 0.2 μm of membrane filtration, filtrate is concentrated into 5 mL, then with 0.2 μm of membrane filtration, takes subsequent filtrate, i.e.,
Spleen ball negative sample solution must be opened.
2) it takes and lacks amygdalate Qipi oral liquid negative sample to cross 0.2 μm of filter membrane molten to get Qipi oral liquid negative sample
Liquid.
(6) it measures: it is accurate respectively to draw reference substance solution, negative sample solution and each 2 μ L of test solution, inject liquid matter
Combined instrument detected to get.Testing result as illustrated in figs. 2-7,10 batches from different production units open spleen ball and 1 batch is opened spleen
Oral solution, which detects, contains amarogentin ingredient.11 batches of test sample information are shown in Table 3.
Claims (1)
1. opening the discrimination method of Medicated Leaven ingredient in spleen ball and Qipi oral liquid, which is characterized in that the discrimination method is with bitter apricot
Whether benevolence glycosides is reference substance, opened in spleen ball and Qipi oral liquid sample and contained using the method detection of high performance liquid chromatography tandem mass spectrum
Amarogentin feeds intake underproof problem to identify and open in spleen ball and Qipi oral liquid sample with the presence or absence of Medicated Leaven, specifically
Method includes following content:
(1) chromatographic condition: using octadecyl silane as filler, chromatography column internal diameter 2.1mm, length 100mm, 1.8 μ of partial size
M, 35 DEG C of column temperature;Using 0.1% aqueous formic acid as mobile phase A, acetonitrile is Mobile phase B, gradient elution 20min is carried out, when elution
Between when being 0,5,17,20min, the ratio of mobile phase is A:95%, B:5%;When elution time is 12,16.5 min, mobile phase
Ratio is A:10%, B:90%;
(2) Mass Spectrometry Conditions: mass spectrum assembles electrospray ionisation source (ESI), dry temperature degree: 350 DEG C;Nebulizer pressure: 40
Psi;Dry gas stream speed: 5 L/min, sheath stream temperature degree: 350 DEG C;Sheath stream gas velocity: 10 L/min, High pressure RF:
150v;Low pressure RF:60v;Capillary voltage: 4000 v;Spray nozzle voltage: 500 V;Residence time: 60ms;Detection
Mode: positive ion detection, scanning mode: multiple-reaction monitoring (MRM);
(3) preparation of reference substance solution: being made certain density stock solution for amarogentin with methanol, and when use is diluted to concentration
For the standard solution of 100 ng/mL;
(4) preparation of test solution:
1) spleen ball is opened as test sample: being taken and is opened spleen ball sample and shred, is mixed, is weighed 2 g, add 1 g of diatomite, grind well, and first is added
50 mL of alcohol is ultrasonically treated 30min, and with 0.2 μm of membrane filtration, filtrate is concentrated into 5 mL, then with 0.2 μm of membrane filtration, takes continuous filter
Liquid to get;
2) Qipi oral liquid is as test sample: take Qipi oral liquid cross 0.2 μm of filter membrane to get;
(5) prepared by negative sample solution: taking that lack amygdalate negative sample appropriate, by step (4) sample solution preparation method
It is made in the same way of negative sample solution;
(6) it measures: it is accurate respectively to draw reference substance solution, negative sample solution and each 2 μ L of test solution, inject LC-MS
Instrument detected to get.
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Cited By (1)
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Application publication date: 20190607 |