CN101601805A - The detection method of peimine and peiminine in the Chinese medicine Bulbus Fritillariae Cirrhosae extract - Google Patents
The detection method of peimine and peiminine in the Chinese medicine Bulbus Fritillariae Cirrhosae extract Download PDFInfo
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Abstract
The invention provides the detection method of peimine and peiminine in the Chinese medicine Bulbus Fritillariae Cirrhosae extract, adopt the method for capillary electrophoresis electrochemical light-emitting.Detectability to peimine and peiminine is respectively 1.25 * 10
-10Mol/L and 1.0 * 10
-10Mol/L, it is 1.0 * 10 that the range of linearity is respectively
-8-1.0 * 10
-6Mol/L and 5.0 * 10
-8-1.0 * 10
-6Mol/L; Than low 4 orders of magnitude of evaporation light detection method detectability commonly used, than low 2 orders of magnitude of mass spectrum detection detectability, the range of linearity is crossed over two orders of magnitude; Runtime buffer effects of ion liquid B MImBF
4Realize the high efficiency separation of peimine and peiminine; Receive upgrading sample size and do not need testing sample carried out post before, post-column derivation, Chinese medicine Bulbus Fritillariae Uninbracteatae extracting solution is directly used in detection; Whole electrophoresis process is in 12min.
Description
Technical field
The invention belongs to capillary electrophoresis electrochemical light-emitting detection technique field, be specifically related to the method that capillary electrophoresis electrochemical light-emitting detects peimine and peiminine in the Chinese medicine Bulbus Fritillariae Cirrhosae extract.
Background technology
Because various active component have important pharmacological action in the Chinese medicine, so the detection of active ingredient of Chinese herbs has become the hot fields of present analysis chemical research.In order to improve the international competitiveness of Chinese medicine industry, the natural drug development of China must integrate with mutually with advanced person's detection technique, and the analyzing and testing of Chinese medicine must be scientific.Bulbus Fritillariae Uninbracteatae is a kind of famous Chinese herbal medicine that is used for relieving cough and resolving phlegm.Two kinds of main active peimines and peiminine in the Bulbus Fritillariae Uninbracteatae have important pharmacological action to relieving cough and resolving phlegm.People are studying always and are exploring the new method that active component detects in the Bulbus Fritillariae Uninbracteatae, to the important topic that peimine in the Bulbus Fritillariae Cirrhosae extract and peiminine effective separates, Sensitive Detection is chemical analysis field.
Because peimine and peiminine do not have chromophore, therefore often need derive and just can realize analyzing and testing it, detection major part to peimine and peiminine in the Chinese medicine Bulbus Fritillariae Uninbracteatae concentrates on chromatograph-evaporation light coupling method [1-3] at present, comprises high performance liquid chromatography-mass spectrometric analysis method [4] in addition.
Peimine and peiminine derived often needs complicated operation sequence, tediously long analysis time, and evaporated the restriction of optical detection technique sensitivity, be difficult to satisfy the detection demand of low content active component in the Chinese medicine Bulbus Fritillariae Cirrhosae extract.High performance liquid chromatography-mass spectrography not only has the structural information that higher sensitivity can also provide analyte simultaneously, is that Chinese medicine extract detects important, a conventional analytical method.But instrument and equipment costliness, complicated operation that chromatography-mass spectroscopy needs, this is the technology material obstacle that this technology is difficult to promote the use of.
Capillary electrophoresis is a kind of means of compartment analysis efficiently, and the sample demand is little, high efficiency separation, and instrument is cheap, is applicable to the analysis of various charged ions and neutral substance, has a wide range of applications in fields such as analytical chemistry, bioanalysis chemistry.The pyridine ruthenium electrochemical is luminous to be a kind of sensitive detection technique, its principle is to produce some special materials by electrochemical method at electrode surface, between these materials or and system in form excited state by electron transport between other component, excited state turns back to ground state and produces luminescence phenomenon, the luminous intensity that produces is proportional to analyte concentration, thereby analyte obtains detecting.Luminous need not of pyridine ruthenium electrochemical derived to testing sample, need not other extra light source, avoided the interference of background light source, and the luminescence-producing reaction of pyridine ruthenium electrochemical is efficiently reversible, and itself can be recycled reagent, is a kind of detection means of sensitive and economic.Capillary electrophoresis and the coupling mutually of pyridine ruthenium electrochemical luminescence technology bring out the best in each other the advantage of the two, and it is low, simple to operate that capillary electrophoresis electrochemical light-emitting detects multiple techniques instrument cost, can realize the high efficiency separation of testing sample, Sensitive Detection.(list of references [1] Z.Liu, Y.Jin, P.Shen, J.Wang, Y.Shen, Talanta 71 (2007) 1873.[2] Y.Jin, P.Shen, J.Zhang, C.Zhuo, C.Xu, Y.Yu, Research﹠amp; Informationon Traditional Chinese Medicine 7 (2005) 13.[3] S.-L.Li, G.Lin, S.-W.Chan, P.Li, J.Chromatogr.A 909 (2001) 207.[4] J.-L.Zhou, P.Li, H.-J.Li, Y.Jiang, M.-T.Ren, Y.Liu, J.Chromatogr.A 1177 (2008) 126.)
Summary of the invention the purpose of this invention is to provide a kind of sensitivity, simply, capillary electrophoresis electrochemical light-emitting detects the method for peimine and peiminine in the Bulbus Fritillariae Cirrhosae extract fast.
1.1 the preparation of product to be checked
1.1.1 Chinese medicine Bulbus Fritillariae Uninbracteatae
1.1.2 the preparation of peimine standard solution and peiminine standard solution
In peimine standard substance and the peiminine standard substance dissolving methanol, make peimine standard substance storing solution and peiminine standard substance storing solution respectively, the concentration of described storing solution is for being respectively 5.0mmol/L, and storing solution is hidden 4 ℃ of refrigerator and cooled and preserved;
Use methanol respectively, it is 5.0 * 10 that peimine standard substance storing solution and peiminine standard substance storing solution are diluted to concentration
-4The peimine standard solution of mol/L and peiminine standard solution;
1.1.3 the preparation of Bulbus Fritillariae Cirrhosae extract related solution
A. the preparation of Bulbus Fritillariae Cirrhosae extract solution
With the Bulbus Fritillariae Uninbracteatae grind into powder, cross 40 mesh sieve, take by weighing 0.2g Chinese medicine Bulbus, with the alkalization of 0.2mL ammonia, in ultrasonic pond, use 3.0mL chloroform ultrasonic extraction 30min then, extracting operation repeats 2 times, combining extraction liquid at room temperature vapors away chloroform, obtains Chinese medicine Bulbus Fritillariae Cirrhosae extract dry; The dry that is extracted by 0.2g Chinese medicine Bulbus is dissolved in the 1.0mL methanol, with the membrane filtration of 0.45 μ m, obtains Bulbus Fritillariae Cirrhosae extract solution;
B. the preparation of Bulbus Fritillariae Cirrhosae extract dilute solution
The Bulbus Fritillariae Cirrhosae extract solution that is obtained by steps A dilutes with methanol, obtains the Bulbus Fritillariae Cirrhosae extract dilute solution, and the volume that methanol adds is 20 or 120 times of steps A methanol volume;
C. the preparation of Bulbus Fritillariae Cirrhosae extract dilution mark-on solution
Adding the concentration that is obtained by step 1.1.2 in the Bulbus Fritillariae Cirrhosae extract solution that steps A obtains respectively is 5.0 * 10
-5The peimine standard solution of mol/L and concentration are 5.0 * 10
-5The peiminine standard solution of mol/L obtains Bulbus Fritillariae Cirrhosae extract dilution mark-on solution with the methanol dilution, and the volume that methanol adds is 20 or 120 times of steps A methanol volume;
1.2 detect step and condition
1.2.1 apparatus
Internal diameter 50 μ m not coating melt silicon capillary; Diameter 500 μ m platinum dish working electrodes; ± 30kV DC high-voltage power supply, U.S. Spellman high-pressure electronic company produces; The electrochemiluminescence detection system, the auspicious analytical tool advanced in years in Xi'an Co., Ltd produces; CHI 832 type electrochemical analysers, Shanghai occasion China instrument company produces;
1.2.2 reagent
NaH
2PO
4, Na
2HPO
4, NaOH, ammonia, chloroform, methanol, 1-butyl-3-methyl imidazolium tetrafluoroborate (BMImBF
4), ruthenie six chloride monohydrates of terpyridyl (Ru (bpy)
3 2+), the standard substance of peimine and peiminine, secondary water; Agents useful for same is analytical pure; The solution for preparing before using all through 0.45 μ m membrane filtration;
1.2.3 the preparation of solution
1.2.3.1 the preparation of background electrolyte
(1) preparation of phosphate buffered solution
Compound concentration is the NaH of 200.0mmol/L
2PO
4With concentration be the Na of 200.0mmol/L
2HPO
4, will be made into the phosphate buffered solution that concentration is 100.0mmol/L with the two mixing reuse secondary water dilution of concentration, adjusting the phosphate buffered solution pH value is 7.48;
(2) Ru (bpy)
3 2+The preparation of solution
With secondary water preparation 20.0mmol/L Ru (bpy)
3 2+Solution;
(3) preparation of background electrolyte
With the phosphate buffered solution of step (1) preparation and the Ru (bpy) of step (2) preparation
3 2+Solution mixes, with secondary water dilution preparation background electrolyte; Phosphate concn is 50.0mmol/L in the background electrolyte, Ru (bpy)
3 2+Concentration is 5.0mmol/L;
(4) contain the background electrolyte of peimine and contain the preparation of the background electrolyte of peiminine
Earlier with the phosphate buffered solution of step (1) preparation and the Ru (bpy) of step (2) preparation
3 2+Solution mixes; Part in the mixed solution that obtains is added the peimine standard solution that obtains by step 1.1.2, another part in the mixed solution that obtains is added the peiminine standard solution that obtains by step 1.1.2, respectively with the dilution of secondary water, obtain containing the background electrolyte and the background electrolyte that contains peiminine of peimine respectively then; Contain the background electrolyte of peimine and contain that phosphate concn is 50.0mmol/L, Ru (bpy) in the background electrolyte of peiminine
3 2+Concentration is 5.0mmol/L, and the concentration of peimine and peiminine is 1.0 * 10
-6Mol/L;
1.2.3.2 the preparation of runtime buffer solution
(a) preparation of phosphate buffered solution
Compound concentration is the NaH of 200.0mmol/L
2PO
4With concentration be the Na of 200.0mmol/L
2HPO
4, will be with the two mixing of concentration, the dilution of reuse secondary water is made into the phosphate buffered solution that concentration is 100.0mmol/L, and adjusting the phosphate buffered solution pH value is 8.00;
(b) BMImBF
4The preparation of solution
With BMImBF
4Be made into the solution that concentration is 100.0mmol/L with the dilution of secondary water;
(c) preparation of runtime buffer solution
The BMImBF that phosphate buffered solution that step (a) is obtained and step (b) obtain
4Solution mixes, and the dilution of reuse secondary water obtains runtime buffer solution; Phosphate concn is 8.0mmol/L in the runtime buffer solution, BMImBF
4Concentration is 40.0mmol/L;
1.2.4 detection step
1.2.4.1 the processing of separation capillary
In order to guarantee peimine and peiminine high efficiency separation, the repeatability of Sensitive Detection and analysis result needs capillary tube is done following processing:
New capillary column uses the activation of 0.1mol/L sodium hydroxide to spend the night, and before, capillary column washes 5min with the sodium hydroxide of 0.1mol/L concentration, and reuse secondary water flushing 5min uses the runtime buffer solution equilibria 5min that is obtained by step 1.2.3.2 then;
Owing to contain a large amount of coexisting substances in the Chinese medicine Bulbus Fritillariae Cirrhosae extract capillary tube inner wall is produced absorption, therefore, need capillary tube is carried out following activation processing between twice sample introduction: capillary column washes 30s with the sodium hydroxide of 0.1mol/L concentration, reuse secondary water flushing 30s uses the runtime buffer solution equilibria 30s that is obtained by step 1.2.3.2 then.
1.2.4.2 the cyclic voltammetry experiment of peimine standard solution and peiminine standard solution
In scan round 3-10 week in the background electrolyte that step 1.2.3.1 (3) obtains, the scanning potential region is 0-1.30V, writes down steady periodic cyclic voltammetry curve; Use scan round 3-10 week in background electrolyte that contains peimine that is obtained by step 1.2.3.1 (4) and the background electrolyte that contains peiminine then respectively, sweep interval is similarly 0-1.30V, writes down steady periodic cyclic voltammetry curve;
To compare with the cyclic voltammetry curve that background electrolyte obtains respectively by background electrolyte that contains peimine and the cyclic voltammetry curve that the background electrolyte that contains peiminine obtains, have electro-chemical activity and electro-chemical activity interval thereof to determine peimine and peiminine;
1.2.4.3 the detection of Chinese medicine Bulbus Fritillariae Cirrhosae extract
The dilution mark-on solution of the Bulbus Fritillariae Cirrhosae extract that Bulbus Fritillariae Cirrhosae extract dilute solution that the step B among the step 1.1.3 is obtained and the step C among the 1.1.3 obtain detects by following condition respectively:
Detect current potential 1.20V; Sample injection time 3s; Sample introduction high pressure 16kV; Electrophoretic separation high pressure 16kV; Add the background electrolyte of step 1.2.3.1 preparation in the detection cell; The runtime buffer solution that electrophoresis adopts step 1.2.3.2 to obtain; Three-electrode system comprises that diameter 500 μ m platinum dish working electrodes, platinum filament are to the utmost point and Ag/AgCl reference electrode; Capillary column internal diameter 50 μ m; Electrochemiluminescence adopts the styletable detecting pattern; The photomultiplier tube bias voltage is set in 800V; Obtain the electrophoresis pattern that the Chinese medicine Bulbus Fritillariae Uninbracteatae detects.
About the detection electrophoresis pattern of Hupeh Fritillary Bulb and Fritillaria walujewii Regel is seen Fig. 2 and Fig. 3 respectively.
Beneficial effect: the detection method of two kinds of active component peimines and peiminine relatively has following characteristics with other detection method in the Chinese medicine Bulbus Fritillariae Uninbracteatae involved in the present invention:
1) highly sensitive, the range of linearity is wide.Method of the present invention is respectively 1.25 * 10 to the detectability of peimine and peiminine
-10Mol/L and 1.0 * 10
-10Mol/L is than low 4 orders of magnitude of evaporation light detection method detectability commonly used, than low 2 orders of magnitude of mass spectrum detection detectability.It is 1.0 * 10 that the range of linearity of peimine and peiminine is respectively
-8-1.0 * 10
-6Mol/L and 5.0 * 10
-8-1.0 * 10
-6Mol/L crosses over two orders of magnitude.
2) separation function is powerful.Ionic liquid has special physics, chemical property, runtime buffer effects of ion liquid B MImBF
4Use, make the peimine of structural similarity and the separation selectivity of peiminine that material alterations take place, can realize the high efficiency separation of the two.
3) amount of samples is few, and detection time is short.The sample size of upgrading received can satisfy the separation detection demand of capillary electrophoresis electrochemical light-emitting system.Before detection method of the present invention does not need testing sample carried out post, post-column derivation, the Chinese medicine Bulbus Fritillariae Cirrhosae extract can be directly used in the capillary electrophoresis separation electrochemiluminescence and detect, whole electrophoresis process can be controlled in the 12min.
4) analysis cost is low, and is simple to operate.Capillary tube, high voltage power supply, potentiostat are the basic operation platforms that capillary electrophoresis electrochemical light-emitting detects, and more auxiliary laboratory common chemical reagent can be finished the detection task; The pyridine ruthenium electrochemical is luminous reversible, efficient, greatly reduces the consumption of reagent, reduces and detects cost.Compare with some very sensitive detection methods such as mass spectrums, method of the present invention is more convenient, and instrument cost is relatively low simultaneously.
The present invention is applied to the capillary electrophoresis electrochemical light-emitting detection technique detection of peimine and peiminine in the Chinese medicine Bulbus Fritillariae Cirrhosae extract first, adopts ionic liquid BMImBF first
4Active component to Chinese medicine separates.
Description of drawings
Fig. 1 is the electrophoresis spectrogram that Bulbus Fritilariae Hupehensis extract is detected.Wherein a is the Bulbus Fritillariae Cirrhosae extract dilute solution electrochemiluminescence electrophoresis spectrogram of Hupeh Fritillary Bulb, and b is the Bulbus Fritillariae Cirrhosae extract dilution mark-on solution electrophoresis spectrogram that adds peimine standard substance and peiminine standard substance.Peimine standard substance and peiminine standard substance mark-on concentration are 2.5 * 10
-7Mol/L.1,2 peaks are respectively the electrophoresis peak of peiminine and peimine.
Fig. 2 is the electrophoresis spectrogram that the Fritillaria walujewii Regel extract is detected.Wherein a is the Bulbus Fritillariae Cirrhosae extract dilute solution electrochemiluminescence electrophoresis spectrogram of Fritillaria walujewii Regel, and b is the Bulbus Fritillariae Cirrhosae extract dilution mark-on solution electrophoresis spectrogram that adds the peimine standard substance.Peimine standard substance mark-on concentration is 1.0 * 10
-6Mol/L.1 peak is the electrophoresis peak of peimine.
The specific embodiment
Following examples adopt capillary electrophoresis electrochemical light-emitting analyzing and testing system.Detect current potential 1.20V; Sample injection time 3s; Sample introduction high pressure 16kV; Electrophoretic separation high pressure 16kV; Add the background electrolyte of step 1.2.3.1 preparation in the detection cell; The runtime buffer solution that electrophoresis adopts step 1.2.3.2 to obtain; Three-electrode system comprises that diameter 500 μ m platinum dish working electrodes, platinum filament are to the utmost point and Ag/AgCl reference electrode; Capillary column internal diameter 50 μ m; Electrochemiluminescence adopts the styletable detecting pattern; The photomultiplier tube bias voltage is set in 800V;
The detection of peimine and peiminine in the embodiment 1 Chinese medicine Bulbus Fritilariae Hupehensis extract
1.1 the preparation of product to be checked
1.1.1 the Chinese medicine Bulbus Fritillariae Uninbracteatae is the Chinese medicine Hupeh Fritillary Bulb
1.1.2 the preparation of peimine standard solution and peiminine standard solution
In peimine standard substance and the peiminine standard substance dissolving methanol, make peimine standard substance storing solution and peiminine standard substance storing solution respectively, the concentration of described storing solution is for being respectively 5.0mmol/L, and storing solution is hidden 4 ℃ of refrigerator and cooled and preserved;
Use methanol respectively, it is 5.0 * 10 that peimine standard substance storing solution and peiminine standard substance storing solution are diluted to concentration
-4The peimine standard solution of mol/L and peiminine standard solution;
1.1.3 the preparation of Bulbus Fritillariae Cirrhosae extract solution
The preparation of A Bulbus Fritillariae Cirrhosae extract solution
With the Bulbus Fritillariae Uninbracteatae grind into powder, cross 40 mesh sieve, accurately take by weighing 0.2g Chinese medicine Bulbus, alkalize with 0.2mL ammonia, use 3.0mL chloroform ultrasonic extraction 30min then in ultrasonic pond, extracting operation repeats 2 times, combining extraction liquid, at room temperature vapor away chloroform, obtain Chinese medicine Bulbus Fritillariae Cirrhosae extract dry; The dry that is extracted by 0.2g Chinese medicine Bulbus is dissolved in the 1.0mL methanol, with the membrane filtration of 0.45 μ m, obtains Bulbus Fritillariae Cirrhosae extract solution;
The preparation of B Bulbus Fritillariae Cirrhosae extract dilute solution
The Bulbus Fritillariae Cirrhosae extract solution that is obtained by A dilutes with methanol, obtains the Bulbus Fritillariae Cirrhosae extract dilute solution, and the volume that methanol adds is 120 times of steps A methanol volume;
The preparation of C Bulbus Fritillariae Cirrhosae extract dilution mark-on solution
Adding the concentration that is obtained by step 1.1.2 in the Bulbus Fritillariae Cirrhosae extract solution that steps A obtains respectively is 5.0 * 10
-5The peimine standard solution of mol/L and concentration are 5.0 * 10
-5The peiminine standard solution of mol/L obtains Bulbus Fritillariae Cirrhosae extract dilution mark-on solution with the methanol dilution, and the volume that methanol adds is 120 times of steps A methanol volume;
1.2 detect step and condition
1.2.1 apparatus
Internal diameter 50 μ m not coating melt silicon capillary; Diameter 500 μ m platinum dish working electrodes; ± 30kV DC high-voltage power supply, U.S. Spellman high-pressure electronic company produces; The electrochemiluminescence detection system, the auspicious analytical tool advanced in years in Xi'an Co., Ltd produces; CHI 832 type electrochemical analysers, Shanghai occasion China instrument company produces;
1.2.2 reagent
NaH
2PO
4, Na
2HPO
4, NaOH, ammonia, chloroform, methanol, 1-butyl-3-methyl imidazolium tetrafluoroborate (BMImBF
4), ruthenie six chloride monohydrates of terpyridyl (Ru (bpy)
3 2+), the standard substance of peimine and peiminine, secondary water; Agents useful for same is analytical pure; The solution for preparing before using all through 0.45 μ m membrane filtration;
1.2.3 the preparation of solution
1.2.3.1 the preparation of background electrolyte
(1) preparation of phosphate buffered solution
Compound concentration is the NaH of 200.0mmol/L
2PO
4With concentration be the Na of 200.0mmol/L
2HPO
4, will be made into the phosphate buffered solution that concentration is 100.0mmol/L with the two mixing reuse secondary water dilution of concentration, adjusting the phosphate buffered solution pH value is 7.48;
(2) Ru (bpy)
3 2+The preparation of solution
With secondary water preparation 20.0mmol/L Ru (bpy)
3 2+Solution;
(3) preparation of background electrolyte
With the phosphate buffered solution of step (1) preparation and the Ru (bpy) of step (2) preparation
3 2+Solution mixes, with secondary water dilution preparation background electrolyte; Phosphate concn is 50.0mmol/L in the background electrolyte, Ru (bpy)
3 2+Concentration is 5.0mmol/L;
(4) contain the background electrolyte of peimine and contain the preparation of the background electrolyte of peiminine
Earlier with the hydrochlorate buffer solution of step (1) preparation and the Ru (bpy) of step (2) preparation
3 2+Solution mixes, and adds the peimine standard solution and the peiminine standard solution that are obtained by step 1.1.2 more respectively, then with the dilution of secondary water, and the background electrolyte that obtains containing the background electrolyte of peimine respectively and contain peiminine; Contain the background electrolyte of peimine and contain that phosphate concn is 50.0mmol/L, Ru (bpy) in the background electrolyte of peiminine
3 2+Concentration is 5.0mmol/L, and the concentration of peimine and peiminine is 1.0 * 10
-6Mol/L;
1.2.3.2 the preparation of runtime buffer solution
(1) preparation of phosphate buffered solution
Compound concentration is the NaH of 200.0mmol/L
2PO
4With concentration be the Na of 200.0mmol/L
2HPO
4, will be with the two mixing of concentration, the dilution of reuse secondary water is made into the phosphate buffered solution that concentration is 100.0mmol/L, and adjusting the phosphate buffered solution pH value is 8.00;
(2) BMImBF
4The preparation of solution
With BMImBF
4Be made into the solution that concentration is 100.0mmol/L with the dilution of secondary water;
(3) preparation of runtime buffer solution
The BMImBF that runtime buffer solution that step (1) is obtained and step (2) obtain
4Solution mixes, and the dilution of reuse secondary water obtains runtime buffer solution; Phosphate concn is 8.0mmol/L in the runtime buffer solution, BMImBF
4Concentration is 40.0mmol/L;
1.2.4 detection step
1.2.4.1 the processing of separation capillary
In order to guarantee peimine and peiminine high efficiency separation, the repeatability of Sensitive Detection and analysis result needs capillary tube is done following processing:
New capillary column uses the activation of 0.1mol/L sodium hydroxide to spend the night, and before, capillary column washes 5min with the sodium hydroxide of 0.1mol/L concentration, and reuse secondary water flushing 5min uses the runtime buffer solution equilibria 5min that is obtained by step 1.2.3.2 then; Owing to contain a large amount of coexisting substances in the Chinese medicine Bulbus Fritillariae Cirrhosae extract capillary tube inner wall is produced absorption, therefore, need capillary tube is carried out following activation processing between twice sample introduction: capillary column washes 30s with the sodium hydroxide of 0.1mol/L concentration, reuse secondary water flushing 30s uses the runtime buffer solution equilibria 30s that is obtained by step 1.2.3.2 then.
1.2.4.2 the cyclic voltammetry experiment of peimine standard solution and peiminine standard solution
In scan round 3-10 week in the background electrolyte that step 1.2.3.1 (3) obtains, the scanning potential region is 0-1.30V, writes down steady periodic cyclic voltammetry curve; Use scan round 3-10 week in background electrolyte that contains peimine that is obtained by step 1.2.3.1 (4) and the background electrolyte that contains peiminine then respectively, sweep interval is similarly 0-1.30V, writes down steady periodic cyclic voltammetry curve;
To compare with the cyclic voltammetry curve that background electrolyte obtains respectively by background electrolyte that contains peimine and the cyclic voltammetry curve that the background electrolyte that contains peiminine obtains, have electro-chemical activity and electro-chemical activity interval thereof to determine peimine and peiminine;
1.2.4.3 the detection of Chinese medicine Bulbus Fritillariae Cirrhosae extract
The dilution mark-on solution of the Bulbus Fritillariae Cirrhosae extract that obtains among the Bulbus Fritillariae Cirrhosae extract dilute solution that obtains among the step 1.1.3B and the 1.1.3C is detected by following condition respectively:
Detect current potential 1.20V; Sample injection time 3s; Sample introduction high pressure 16kV; Electrophoretic separation high pressure 16kV; Add the background electrolyte of step 1.2.3.1 preparation in the detection cell; The runtime buffer solution that electrophoresis adopts step 1.2.3.2 to obtain; Three-electrode system comprises that diameter 500 μ m platinum dish working electrodes, platinum filament are to the utmost point and Ag/AgCl reference electrode; Capillary column internal diameter 50 μ m; Electrochemiluminescence adopts the styletable detecting pattern; The photomultiplier tube bias voltage is set in 800V; Obtain the electrophoresis spectrogram (see figure 1) of Chinese medicine Bulbus Fritilariae Hupehensis extract.
The detection of peimine and peiminine in the embodiment 2 Chinese medicine Fritillaria walujewii Regel extracts
The detection of Chinese medicine Fritillaria walujewii Regel extract, the detection step that is adopted are all adopted detection step and the condition identical with embodiment 1 except that step 1.1.3.
1.1.3 the preparation of Bulbus Fritillariae Cirrhosae extract solution
The preparation of A Bulbus Fritillariae Cirrhosae extract solution
With the Bulbus Fritillariae Uninbracteatae grind into powder, cross 40 mesh sieve, accurately take by weighing 0.2g Chinese medicine Bulbus, alkalize with 0.2mL ammonia, use 3.0mL chloroform ultrasonic extraction 30min then in ultrasonic pond, extracting operation repeats 2 times, combining extraction liquid, at room temperature vapor away chloroform, obtain Chinese medicine Bulbus Fritillariae Cirrhosae extract dry; The dry that is extracted by 0.2g Chinese medicine Bulbus is dissolved in the 1.0mL methanol, with the membrane filtration of 0.45 μ m, obtains Bulbus Fritillariae Cirrhosae extract solution;
The preparation of B Bulbus Fritillariae Cirrhosae extract dilute solution
The Bulbus Fritillariae Cirrhosae extract solution that is obtained by A dilutes with methanol, obtains the Bulbus Fritillariae Cirrhosae extract dilute solution, and the volume that methanol adds is 20 times of steps A methanol volume;
The preparation of C Bulbus Fritillariae Cirrhosae extract dilution mark-on solution
Adding the concentration that is obtained by step 1.1.2 in the Bulbus Fritillariae Cirrhosae extract solution that steps A obtains is 5.0 * 10
-5The peimine standard solution of mol/L, the Bulbus Fritillariae Cirrhosae extract that obtains Fritillaria walujewii Regel with the methanol dilution dilutes mark-on solution, and the volume that methanol adds is 20 times of steps A methanol volume; Obtain the electrophoresis spectrogram (see figure 2) of Chinese medicine Fritillaria walujewii Regel extract.
Claims (1)
1, the detection method of peimine and peiminine in the Chinese medicine Bulbus Fritillariae Cirrhosae extract is characterized in that step and condition are as follows:
1.1 the preparation of product to be checked
1.1.1 Chinese medicine Bulbus Fritillariae Uninbracteatae
1.1.2 the preparation of peimine standard solution and peiminine standard solution
In peimine standard substance and the peiminine standard substance dissolving methanol, make peimine standard substance storing solution and peiminine standard substance storing solution respectively, the concentration of described storing solution is for being respectively 5.0mmol/L, and storing solution is hidden 4 ℃ of refrigerator and cooled and preserved;
Use methanol respectively, it is 5.0 * 10 that peimine standard substance storing solution and peiminine standard substance storing solution are diluted to concentration
-4The peimine standard solution of mol/L and peiminine standard solution;
1.1.3 the preparation of Bulbus Fritillariae Cirrhosae extract related solution
A. the preparation of Bulbus Fritillariae Cirrhosae extract solution
With the Bulbus Fritillariae Uninbracteatae grind into powder, cross 40 mesh sieve, take by weighing 0.2g Chinese medicine Bulbus, with the alkalization of 0.2mL ammonia, in ultrasonic pond, use 3.0mL chloroform ultrasonic extraction 30min then, extracting operation repeats 2 times, combining extraction liquid at room temperature vapors away chloroform, obtains Chinese medicine Bulbus Fritillariae Cirrhosae extract dry; The dry that is extracted by 0.2g Chinese medicine Bulbus is dissolved in the 1.0mL methanol, with the membrane filtration of 0.45 μ m, obtains Bulbus Fritillariae Cirrhosae extract solution;
B. the preparation of Bulbus Fritillariae Cirrhosae extract dilute solution
The Bulbus Fritillariae Cirrhosae extract solution that is obtained by steps A dilutes with methanol, obtains the Bulbus Fritillariae Cirrhosae extract dilute solution, and the volume that methanol adds is 20 or 120 times of steps A methanol volume;
C. the preparation of Bulbus Fritillariae Cirrhosae extract dilution mark-on solution
Adding the concentration that is obtained by step 1.1.2 in the Bulbus Fritillariae Cirrhosae extract solution that steps A obtains respectively is 5.0 * 10
-5The peimine standard solution of mol/L and concentration are 5.0 * 10
-5The peiminine standard solution of mol/L obtains Bulbus Fritillariae Cirrhosae extract dilution mark-on solution with the methanol dilution, and the volume that methanol adds is 20 or 120 times of steps A methanol volume;
1.2 detect step and condition
1.2.1 apparatus
Internal diameter 50 μ m not coating melt silicon capillary; Diameter 500 μ m platinum dish working electrodes; ± 30kV DC high-voltage power supply, U.S. Spellman high-pressure electronic company produces; The electrochemiluminescence detection system, the auspicious analytical tool advanced in years in Xi'an Co., Ltd produces; CHI 832 type electrochemical analysers, Shanghai occasion China instrument company produces;
1.2.2 reagent
NaH
2PO
4, Na
2HPO
4, NaOH, ammonia, chloroform, methanol, 1-butyl-3-methyl imidazolium tetrafluoroborate (BMImBF
4), ruthenie six chloride monohydrates of terpyridyl (Ru (bpy)
3 2+), the standard substance of peimine and peiminine, secondary water; Agents useful for same is analytical pure; The solution for preparing before using all through 0.45 μ m membrane filtration;
1.2.3 the preparation of solution
1.2.3.1 the preparation of background electrolyte
(1) preparation of phosphate buffered solution
Compound concentration is the NaH of 200.0mmol/L
2PO
4With concentration be the Na of 200.0mmol/L
2HPO
4, will be made into the phosphate buffered solution that concentration is 100.0mmol/L with the two mixing reuse secondary water dilution of concentration, adjusting the phosphate buffered solution pH value is 7.48;
(2) Ru (bpy)
3 2+The preparation of solution
With secondary water preparation 20.0mmol/L Ru (bpy)
3 2+Solution;
(3) preparation of background electrolyte
With the phosphate buffered solution of step (1) preparation and the Ru (bpy) of step (2) preparation
3 2+Solution mixes, with secondary water dilution preparation background electrolyte; Phosphate concn is 50.0mmol/L in the background electrolyte, Ru (bpy)
3 2+Concentration is 5.0mmol/L;
(4) contain the background electrolyte of peimine and contain the preparation of the background electrolyte of peiminine
Earlier with the phosphate buffered solution of step (1) preparation and the Ru (bpy) of step (2) preparation
3 2+Solution mixes; Part in the mixed solution that obtains is added the peimine standard solution that obtains by step 1.1.2, another part in the mixed solution that obtains is added the peiminine standard solution that obtains by step 1.1.2, respectively with the dilution of secondary water, obtain containing the background electrolyte and the background electrolyte that contains peiminine of peimine respectively then; Contain the background electrolyte of peimine and contain that phosphate concn is 50.0mmol/L, Ru (bpy) in the background electrolyte of peiminine
3 2+Concentration is 5.0mmol/L, and the concentration of peimine and peiminine is 1.0 * 10
-6Mol/L;
1.2.3.2 the preparation of runtime buffer solution
(a) preparation of phosphate buffered solution
Compound concentration is the NaH of 200.0mmol/L
2PO
4With concentration be the Na of 200.0mmol/L
2HPO
4, will be with the two mixing of concentration, the dilution of reuse secondary water is made into the phosphate buffered solution that concentration is 100.0mmol/L, and adjusting the phosphate buffered solution pH value is 8.00;
(b) BMImBF
4The preparation of solution
With BMImBF
4Be made into the solution that concentration is 100.0mmol/L with the dilution of secondary water;
(c) preparation of runtime buffer solution
The BMImBF that phosphate buffered solution that step (a) is obtained and step (b) obtain
4Solution mixes, and the dilution of reuse secondary water obtains runtime buffer solution; Phosphate concn is 8.0mmol/L in the runtime buffer solution, BMImBF
4Concentration is 40.0mmol/L;
1.2.4 detection step
1.2.4.1 the processing of separation capillary
In order to guarantee peimine and peiminine high efficiency separation, the repeatability of Sensitive Detection and analysis result needs capillary tube is done following processing:
New capillary column uses the activation of 0.1mol/L sodium hydroxide to spend the night, and before, capillary column washes 5min with the sodium hydroxide of 0.1mol/L concentration, and reuse secondary water flushing 5min uses the runtime buffer solution equilibria 5min that is obtained by step 1.2.3.2 then;
Owing to contain a large amount of coexisting substances in the Chinese medicine Bulbus Fritillariae Cirrhosae extract capillary tube inner wall is produced absorption, therefore, need capillary tube is carried out following activation processing between twice sample introduction: capillary column washes 30s with the sodium hydroxide of 0.1mol/L concentration, reuse secondary water flushing 30s uses the runtime buffer solution equilibria 30s that is obtained by step 1.2.3.2 then.
1.2.4.2 the cyclic voltammetry experiment of peimine standard solution and peiminine standard solution
In scan round 3-10 week in the background electrolyte that step 1.2.3.1 (3) obtains, the scanning potential region is 0-1.30V, writes down steady periodic cyclic voltammetry curve; Use scan round 3-10 week in background electrolyte that contains peimine that is obtained by step 1.2.3.1 (4) and the background electrolyte that contains peiminine then respectively, sweep interval is similarly 0-1.30V, writes down steady periodic cyclic voltammetry curve;
To compare with the cyclic voltammetry curve that background electrolyte obtains respectively by background electrolyte that contains peimine and the cyclic voltammetry curve that the background electrolyte that contains peiminine obtains, have electro-chemical activity and electro-chemical activity interval thereof to determine peimine and peiminine;
1.2.4.3 the detection of Chinese medicine Bulbus Fritillariae Cirrhosae extract
The dilution mark-on solution of the Bulbus Fritillariae Cirrhosae extract that Bulbus Fritillariae Cirrhosae extract dilute solution that the step B among the step 1.1.3 is obtained and the step C among the 1.1.3 obtain detects by following condition respectively:
Detect current potential 1.20V; Sample injection time 3s; Sample introduction high pressure 16kV; Electrophoretic separation high pressure 16kV; Add the background electrolyte of step 1.2.3.1 preparation in the detection cell; The runtime buffer solution that electrophoresis adopts step 1.2.3.2 to obtain; Three-electrode system comprises that diameter 500 μ m platinum dish working electrodes, platinum filament are to the utmost point and Ag/AgCl reference electrode; Capillary column internal diameter 50 μ m; Electrochemiluminescence adopts the styletable detecting pattern; The photomultiplier tube bias voltage is set in 800V; Obtain the electrophoresis pattern that the Chinese medicine Bulbus Fritillariae Uninbracteatae detects.
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