CN107831233A - The method that a variety of colouring agents synchronously detect in Chinese medicine preparation - Google Patents
The method that a variety of colouring agents synchronously detect in Chinese medicine preparation Download PDFInfo
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- CN107831233A CN107831233A CN201711208085.2A CN201711208085A CN107831233A CN 107831233 A CN107831233 A CN 107831233A CN 201711208085 A CN201711208085 A CN 201711208085A CN 107831233 A CN107831233 A CN 107831233A
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- chinese medicine
- medicine preparation
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- colouring agents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses the method that a variety of colouring agents synchronously detect in Chinese medicine preparation, is measured using method associated with liquid chromatography mass, and its detection method comprises the following steps：（1）The preparation of mixed reference substance solution；（2）The preparation of sample solution；（3）Carry out combined gas chromatography mass spectrometry analysis；（4）Obtain a result.A variety of colouring agents include sunset yellow, acid red, methyl orange, gold orange II, auramine O, erythrosine and Basic Orange.The method of the invention has the advantages of easy to operate quick, the degree of accuracy is high, stability is good, and suitable for quality control of the laboratory for Chinese medicine preparation, this method can be detected in batches, and detection efficiency is high.
Description
Technical field
The present invention relates to field of traditional Chinese medicine detection, the method that specifically a variety of colouring agents synchronously detect in Chinese medicine preparation.
Background technology
Chinese medicine preparation and Manufacture of medicinal slices of TCM are the sources that Chinese patent drug production and operation use, in Chinese medicine preparation and the prepared slices of Chinese crude drugs
Middle dyeing is adulterated to bring serious harm to follow-up Chinese medicine production, clinical formulation.With the popularization of traditional Chinese medicine and Chinese medicine system
The demand of the development of agent, city's field alignment medicine preparation and the prepared slices of Chinese crude drugs increasingly increases, rise in price.Under the driving of economic interests,
The phenomenon of adulterated fraud happens occasionally, and adulterated dyeing is common means of playing tricks.In order to further control the product matter of Chinese medicine preparation
Amount, ensures the drug safety of the public, present invention Liquid Chromatography-Mass Spectrometry is to sunset yellow, acid red, first in Chinese medicine preparation
The dyestuffs such as base orange, gold orange II, auramine O, erythrosine and Basic Orange carry out examination, have great importance.
At present, dye the detection method of adulterated product mainly have experience differential method, physics and chemistry differential method, tlc identification method,
High performance liquid chromatography, near infrared spectrum differential method, spectrum and its combination method etc., wherein LC-MS-MS is most
For accurately and fast, stably, and more conventional method.The liquid chromatograph mass spectrography for the detection synthetic colourant reported
（LC-MS/MS）In method, typically it is related to the industries such as food, cosmetics and chemical article, also without blanket inspection
Survey method.
The detection method of coloring agent is also related in open source literature at present, here is the pertinent literature detected：
1st, Chinese patent：Method that is a kind of while detecting six kinds of dyeing agent contents in grape wine；Application number：
201610535407.3；The applying date： 2016.07.08；Applicant：Jiangsu King's Luck Brewery Co., Ltd.；Summary：This hair
It is bright to disclose method that is a kind of while detecting six kinds of dyeing agent contents in grape wine, comprise the following steps：A, the preparation of sample, amount
The wine sample to be measured of certain volume is taken, boils to dry to the greatest extent and removes, just volume is settled to ultra-pure water, crosses miillpore filter；B, coloring agent mixes
The preparation of standardization solution：A certain amount of coloring agent standard substance is weighed respectively, is settled to certain volume with ultra-pure water dilution, is made
Required strength solution is diluted to step by step after used time mixing, crosses aqueous phase miillpore filter；C, high performance liquid chromatography-level Four bar-quiet is used
Electric field orbit ion trap quality analysis combined instrument carries out qualitative and quantitative analysis.The present invention is to 7 kinds of coloring agents in grape wine sample
Detection accuracy is good, high sensitivity, accuracy are good, and the rate of recovery meets analysis testing requirements, is applicable to various wines from grape cultivars
The detection and analysis of middle coloring agent.
2nd, Chinese patent：38 kinds of methods for limiting the use of coloring agent while detection in cosmetics；Application number：
201410539619.X；The applying date： 2014.10.14；Applicant：Food Inspection institute of Daliang City；Summary：38 in a kind of cosmetics
The method that kind limits the use of coloring agent while detection, the coloring agent are amaranth, Acid Blue 74, famille rose, Indian yellow 1, acid orange
10th, sunset yellow, azogeramine, the red, fast green of temptation, acid blue 9, acid red 50, acid yellow 11, acid black 1, Blue VRS, acid
Property orange 7, acid red 52, acid green 22, patent blue v, acid red 92, acid green 9, acid red 88, pigment red 49, bromocresol green,
Acid violet 43, acid violet 9, Acid blue 62, Acid Blue 7, alkalescence purple 2, acid violet 50, solvent yellow 33, solvent yellow 21, paratonere
64th, solvent red 3, solvent yellow 16, solvent yellow 29, pigment red 4, solvent green 3, alkali blue 26, detecting step are as follows：Solution is matched somebody with somebody
System, the preparation of various Standard Stock solutions, the preparation of sample solution, liquid chromatography qualitative analysis, quantitative determination.The present invention can be same
When detect in cosmetics 38 kinds and limit the use of coloring agent, detection efficiency is high, and testing cost is low.
3rd, Chinese patent：A kind of method for detecting nine kinds of synthetic colourant concentration in reconstituted tobacco auxiliary material；Application number
201510707623.7；The applying date： 2015.10.27；Applicant：Yunnan Reascend Tobacco Tech.(Group) Co., Ltd., Yunnan
Detection technique limited company of Tontru；Summary：A kind of side for detecting nine kinds of synthetic colourant concentration in reconstituted tobacco auxiliary material
Method, the inventive method are to prepare lemon yellow, amaranth, indigo, carmine, sunset yellow, the red, light blue of temptation, acid red, erythrosine
Nine kinds of synthetic colourant mixing mother liquors and sample solution, prepare standard working solution, using the triple level Four bars of liquid chromatography-tandem
Mass spectrometer, analysis detection obtain nine kinds of synthetic colourant concentration in sample solution.The inventive method accurately and reliably, sensitivity
Height, it is especially suitable for the analysis of nine kinds of synthetic colourants in complex matrices reconstituted tobacco auxiliary material.
The content of the invention
It is an object of the invention to provide the method that a variety of colouring agents in a kind of Chinese medicine preparation synchronously detect, to make up existing skill
The deficiency of art.The method is easy to operate, and detection efficiency is high, and the method has the advantages that accuracy rate height, favorable reproducibility, stability are good.
The present invention is achieved through the following technical solutions：
The method that a variety of colouring agents synchronously detect in Chinese medicine preparation, its detection method comprise the following steps：
（1）The preparation of mixed reference substance solution：Precision weighs sunset yellow, acid red, methyl orange, gold orange II, auramine O, red respectively
Moss is red and Basic Orange reference substance is appropriate, adds methanol and is configured to the mixing reference substance that each composition quality fraction is 0.1 μ g/ml
Solution, it is kept in dark place under the conditions of 4~6 DEG C；
（2）The preparation of sample solution：Chinese medicine preparation sample 0.4~0.6g is taken, it is accurately weighed, add 50~60ml of methanol, ultrasound
Processing 30~40 minutes, weighed weight, is filtered after the weight of less loss is supplied with methanol；Precision draws subsequent filtrate 1ml, puts 10ml amounts
In bottle, methanol dilution is added to cross 0.2 μm of filter membrane to scale, you can to obtain testing sample solution, lucifuge is protected under the conditions of 4~6 DEG C
Deposit；
（3）Carry out chromatograph-mass spectrometer coupling analysis：The accurate mixed reference substance solution and sample solution for drawing equal volume respectively,
HPLC-MS instrument is injected, is measured according to certain chromatographic condition and Mass Spectrometry Conditions, obtains HPLC-MS
Figure；
（4）Obtain a result：By the spectrogram of sample solution compared with the spectrogram of mixed reference substance solution, analysis is obtained a result.
The supersound process condition is：500~550W of power, 70~80kHz of frequency.
The chromatographic condition is：Chromatographic column HypersilGOLDC18,100mm × 2.1mm, 3 μm；30~38 DEG C of column temperature,
0.4~0.8ml/min of flow velocity；Using 0.02mol/L ammonium acetates as mobile phase A, using acetonitrile as Mobile phase B, gradient elution is carried out, is entered
The μ l of sample amount 1~2.
The program of the gradient elution is：0~5min, 10%A+90%B；5~10min, 90%A+10%B；10~25min,
5%A+95%B；25~30min, 10%A+90%B；Wherein, percentage represents the volume proportion of mobile phase A and Mobile phase B.
The Mass Spectrometry Conditions are：Use electron spray ionisation source（ESI）, it is 200 DEG C to dry temperature degree；Nebulizer pressure
20Psi；Dry gas stream 10~12L/min of speed, sheath stream temperature degree：350℃ ；Sheath stream gas velocity：10L/min；Spray nozzle voltage 500
V；Residence time：20 ms；Detection pattern：Negative ions detect, scan mode：Multiple-reaction monitoring（MRM）.
Described a variety of coloring agents include sunset yellow, acid red, methyl orange, gold orange II, auramine O, erythrosine and Basic Orange.
The testing conditions and method that the present invention uses are on the basis of lot of documents is consulted, and explore and grind by many experiments
Study carefully what is screened afterwards, the purpose that the degree of accuracy is high, stability is good can be reached.
Beneficial effects of the present invention：
1st, the method that a variety of colouring agents synchronously detect in Chinese medicine preparation of the present invention, can make up in the prior art for Chinese medicine system
The deficiency of agent quality control, the method is easy to operate, and detection efficiency is high, once crosses post, once analyzes process, can synchronously detect a variety of
Coloring agent, reduce testing cost.
2nd, the method that a variety of colouring agents synchronously detect in Chinese medicine preparation of the present invention, can detect simultaneously in seven kinds of colouring agents
One or more, suitable for quality control of the laboratory for Chinese medicine preparation, can be detected in batches, detection efficiency is high, accurate
Exactness is high, stability is good.
3rd, for Chinese medicine preparation there is presently no the examination criteria of correlation in terms of coloring agent, detection method of the present invention can
Reference frame is provided with the foundation for company standard or professional standard.
Brief description of the drawings
Fig. 1 is the HPLC-MS MRM figures of mixed reference substance solution.
Fig. 2 is the HPLC-MS MRM figures of Gegen Qinlian Tablets.
Embodiment
The present invention is introduced in order to more detailed, with reference to embodiment, the present invention will be further described.
Instrument and reagent are as follows：Liquid chromatograph-mass spectrometer；Sunset yellow reference substance, acid red reference substance, methyl
Orange reference substance, the reference substance of gold orange II, the reference substance of auramine 0, red scarlet reference substance, Basic Orange reference substance, by Chinese medicine biology
Product calibrating is provided；Gegen Qinlian Tablets Chinese medicine preparation is chosen as test sample；Acetonitrile used is chromatographically pure, and water is high-purity
Water, other reagents are that analysis is pure.
Embodiment 1
The method that a variety of colouring agents synchronously detect in Chinese medicine preparation, its detection method comprise the following steps：
（1）The preparation of mixed reference substance solution：Precision weighs sunset yellow, acid red, methyl orange, gold orange II, auramine O, red respectively
Moss is red and Basic Orange reference substance is appropriate, adds methanol and is configured to the mixing reference substance that each composition quality fraction is 0.1 μ g/ml
Solution, it is kept in dark place under the conditions of 4~6 DEG C；
（2）The preparation of sample solution：Gegen Qinlian Tablets sample 0.5g is taken, grinding is accurately weighed, addition methanol 50ml, at ultrasound
Reason 40 minutes, weighed weight, is filtered after the weight of less loss is supplied with methanol；Precision draws subsequent filtrate 1ml, puts in 10ml measuring bottles,
Methanol dilution is added to cross 0.2 μm of filter membrane, you can obtain testing sample solution, be kept in dark place under the conditions of 4~6 DEG C to scale；
（3）Carry out chromatograph-mass spectrometer coupling analysis：The accurate mixed reference substance solution and sample solution for drawing equal volume respectively,
HPLC-MS instrument is injected, is measured according to certain chromatographic condition and Mass Spectrometry Conditions, obtains HPLC-MS
Figure；
（4）Obtain a result：By the spectrogram of sample solution compared with the spectrogram of mixed reference substance solution, analysis is obtained a result.
The supersound process condition is：Power 500W, frequency 80kHz.
The chromatographic condition is：Chromatographic column HypersilGOLDC18,100mm × 2.1mm, 3 μm；35 DEG C of column temperature, flow velocity
0.6ml/min；Using 0.02mol/L ammonium acetates as mobile phase A, using acetonitrile as Mobile phase B, gradient elution, the μ l of sample size 2 are carried out.
The program of the gradient elution is：0~5min, 10%A+90%B；5~10min, 90%A+10%B；10~25min,
5%A+95%B；25~30min, 10%A+90%B；Wherein, percentage represents the volume proportion of mobile phase A and Mobile phase B.
The Mass Spectrometry Conditions are：Use electron spray ionisation source（ESI）, it is 200 DEG C to dry temperature degree；Nebulizer pressure
20Psi；Dry gas stream speed 12L/min, sheath stream temperature degree：350℃ ；Sheath stream gas velocity：10L/min；The V of spray nozzle voltage 500；
Residence time：20 ms；Detection pattern：Negative ions detect, scan mode：Multiple-reaction monitoring（MRM）.
Embodiment 2
The method that a variety of colouring agents synchronously detect in Chinese medicine preparation, its detection method comprise the following steps：
（1）The preparation of mixed reference substance solution：Precision weighs sunset yellow, acid red, methyl orange, gold orange II, auramine O, red respectively
Moss is red and Basic Orange reference substance is appropriate, adds methanol and is configured to the mixing reference substance that each composition quality fraction is 0.1 μ g/ml
Solution, it is kept in dark place under the conditions of 4~6 DEG C；
（2）The preparation of sample solution：Gegen Qinlian Tablets sample 0.5g is taken, grinding is accurately weighed, addition methanol 60ml, at ultrasound
Reason 40 minutes, weighed weight, is filtered after the weight of less loss is supplied with methanol；Precision draws subsequent filtrate 1ml, puts in 10ml measuring bottles,
Methanol dilution is added to cross 0.2 μm of filter membrane, you can obtain testing sample solution, be kept in dark place under the conditions of 4~6 DEG C to scale；
（3）Carry out chromatograph-mass spectrometer coupling analysis：The accurate mixed reference substance solution and sample solution for drawing equal volume respectively,
HPLC-MS instrument is injected, is measured according to certain chromatographic condition and Mass Spectrometry Conditions, obtains HPLC-MS
Figure；
（4）Obtain a result：By the spectrogram of sample solution compared with the spectrogram of mixed reference substance solution, analysis is obtained a result.
The supersound process condition is：Power 550W, frequency 70kHz.
The chromatographic condition is：Chromatographic column HypersilGOLDC18,100mm × 2.1mm, 3 μm；33 DEG C of column temperature, flow velocity
0.7ml/min；Using 0.02mol/L ammonium acetates as mobile phase A, using acetonitrile as Mobile phase B, gradient elution, the μ l of sample size 2 are carried out.
The program of the gradient elution is：0~5min, 10%A+90%B；5~10min, 90%A+10%B；10~25min,
5%A+95%B；25~30min, 10%A+90%B；Wherein, percentage represents the volume proportion of mobile phase A and Mobile phase B.
The Mass Spectrometry Conditions are：Use electron spray ionisation source（ESI）, it is 200 DEG C to dry temperature degree；Nebulizer pressure
20Psi；Dry gas stream speed 101L/min, sheath stream temperature degree：350℃ ；Sheath stream gas velocity：10L/min；The V of spray nozzle voltage 500；
Residence time：20 ms；Detection pattern：Negative ions detect, scan mode：Multiple-reaction monitoring（MRM）.
The HPLC-MS MRM figures of mixed reference substance solution are as shown in Figure 1；The HPLC-MS of testing result Gegen Qinlian Tablets
MRM scheme as shown in Fig. 2 result show surveyed Gegen Qinlian Tablets sample do not detect sunset yellow, acid red, methyl orange, gold orange II,
Auramine 0, erythrosine and Basic Orange etc., illustrate that surveyed Gegen Qinlian tablet quality is good, be not added with above-mentioned seven kinds of colouring agents.
Claims (6)
1. the method that a variety of colouring agents synchronously detect in Chinese medicine preparation, it is characterised in that its detection method comprises the following steps：
（1）The preparation of mixed reference substance solution：Precision weighs sunset yellow, acid red, methyl orange, gold orange II, auramine O, red respectively
Moss is red and Basic Orange reference substance is appropriate, adds methanol and is configured to the mixing reference substance that each composition quality fraction is 0.1 μ g/ml
Solution, it is kept in dark place under the conditions of 4~6 DEG C；
（2）The preparation of sample solution：Chinese medicine preparation sample 0.4~0.6g is taken, it is accurately weighed, add 50~60ml of methanol, ultrasound
Processing 30~40 minutes, weighed weight, is filtered after the weight of less loss is supplied with methanol；Precision draws subsequent filtrate 1ml, puts 10ml amounts
In bottle, methanol dilution is added to cross 0.2 μm of filter membrane to scale, you can to obtain testing sample solution, lucifuge is protected under the conditions of 4~6 DEG C
Deposit；
（3）Carry out chromatograph-mass spectrometer coupling analysis：The accurate mixed reference substance solution and sample solution for drawing equal volume respectively,
HPLC-MS instrument is injected, is measured according to certain chromatographic condition and Mass Spectrometry Conditions, obtains HPLC-MS
Figure；
（4）Obtain a result：By the spectrogram of sample solution compared with the spectrogram of mixed reference substance solution, analysis is obtained a result.
2. the method that a variety of colouring agents synchronously detect in Chinese medicine preparation according to claim 1, it is characterised in that the ultrasound
Treatment conditions are：500~550W of power, 70~80kHz of frequency.
3. the method that a variety of colouring agents synchronously detect in Chinese medicine preparation according to claim 1, it is characterised in that the chromatogram
Condition is：Chromatographic column HypersilGOLDC18,100mm × 2.1mm, 3 μm；30~38 DEG C of column temperature, 0.4~0.8ml/ of flow velocity
min；Using 0.02mol/L ammonium acetates as mobile phase A, using acetonitrile as Mobile phase B, gradient elution, the μ l of sample size 1~2 are carried out.
4. the method that a variety of colouring agents synchronously detect in Chinese medicine preparation according to claim 1, it is characterised in that described color
In spectral condition, the program of gradient elution is：0~5min, 10%A+90%B；5~10min, 90%A+10%B；10~25min, 5%A+
95%B；25~30min, 10%A+90%B；Wherein, percentage represents the volume proportion of mobile phase A and Mobile phase B.
5. the method that a variety of colouring agents synchronously detect in Chinese medicine preparation according to claim 1, it is characterised in that the mass spectrum
Condition is：Use electron spray ionisation source（ESI）, it is 190 DEG C to dry temperature degree；Nebulizer pressure 20Psi；Dry gas stream speed 10
~12L/min, sheath stream temperature degree：350℃；Sheath stream gas velocity：10L/min；The V of spray nozzle voltage 500；Residence time：20 ms；Inspection
Survey pattern：Negative ions detect, scan mode：Multiple-reaction monitoring（MRM）.
6. the method that a variety of colouring agents synchronously detect in Chinese medicine preparation according to claim 1, it is characterised in that described is more
Kind coloring agent includes sunset yellow, acid red, methyl orange, gold orange II, auramine O, erythrosine and Basic Orange.
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| CN109507343A (en) * | 2018-12-24 | 2019-03-22 | 上海医药集团股份有限公司 | UPLC method quickly measures west safflower active constituent content and identifies illegal dyeing |
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| CN109596733A (en) * | 2018-12-21 | 2019-04-09 | 康美药业股份有限公司 | The detection method of Yellow Red coloring agent in a kind of medicinal material |
| CN109596734A (en) * | 2018-12-21 | 2019-04-09 | 康美药业股份有限公司 | The detection method of Yellow Red coloring agent in a kind of prepared slices of Chinese crude drugs |
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| CN109596737A (en) * | 2018-12-21 | 2019-04-09 | 康美药业股份有限公司 | A kind of detection method of the multicomponent coloring agent of cattail pollen |
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Application publication date: 20180323 |