Method for measuring content of peimine and peiminine in Huangshi rattle buccal tablets
1. A method for measuring the content of peimine and peiminine in Huangshi rattle buccal tablets comprises the following steps:
1) control solution: precisely weighing peimine reference substance and peiminine reference substance, and adding methanol to obtain mixed solution;
2) test solution: adding a mixed solution of concentrated ammonia test solution and ethanol (1: 2) into the Huangshi rattle buccal tablets, adding diethyl ether: trichloromethane: dissolving the mixed solution of ethanol (50: 16: 5), filtering, and adding the filter residue into the mixture of diethyl ether: trichloromethane: washing with mixed solution of ethanol (50: 16: 5), mixing the filtrate and washing solution, volatilizing, dissolving the residue with methanol, and filtering;
3) and (3) determination: precisely absorbing the reference solution, injecting the test solution into a high performance liquid chromatograph to obtain a chromatogram, and respectively calculating the content of the peimine and the content of the peimine by using an external standard two-point method logarithmic equation according to the peak area of the chromatogram.
2. The method according to claim 1, characterized by the steps of:
1) control solution: precisely weighing peimine reference substance and peiminine reference substance, and adding methanol to obtain mixed solution containing peiminine 0.2-0.3mg and peiminine 0.1-0.2mg per 1 ml;
2) test solution: weighing 7-9g of Huangshi rattle buccal tablet, precisely weighing, dripping 10ml of a mixed solution of concentrated ammonia test solution and ethanol (1: 2) for moistening, sealing, standing, adding diethyl ether: trichloromethane: mixing the solution with ethanol (50: 16: 5), ultrasonic treating for 25-35min, filtering, and treating the filter residue with diethyl ether: trichloromethane: washing with mixed solution of ethanol (50: 16: 5), mixing filtrates and washing solutions, volatilizing in water bath, dissolving residue with methanol, transferring to measuring flask, diluting to scale, shaking, and filtering;
3) and (3) determination: precisely sucking reference substance solutions, respectively 10-20 μ l of the test solution, injecting into a high performance liquid chromatograph to obtain a chromatogram, and respectively calculating the content of peimine and the content of peimine by using an external standard two-point method logarithmic equation according to the peak area of the chromatogram.
3. The method according to claim 1, characterized by the steps of:
1) control solution: precisely weighing peimine reference substance and peiminine reference substance, and adding methanol to obtain mixed solution containing peiminine 0.25mg and peiminine 0.15mg per 1 ml;
2) test solution: weighing 8g of Huangshi rattle buccal tablet, precisely weighing, adding 10ml of concentrated ammonia solution-ethanol (1: 2) mixed solution dropwise for moistening, sealing, standing for 20min, adding diethyl ether: trichloromethane: mixing 25ml of ethanol (50: 16: 5), performing ultrasonic treatment (power 250W, frequency 50Hz, water bath temperature below 30 ℃) for 30min, filtering, and adding diethyl ether into filter residues: trichloromethane: washing with appropriate amount of ethanol (50: 16: 5) mixed solution, mixing filtrates and washing solutions, volatilizing in 60 deg.C water bath, dissolving residue with methanol, transferring to 5ml measuring flask, diluting with methanol to desired volume, shaking, and filtering;
3) and (3) determination: precisely sucking 20 mu l of reference solution and 20 mu l of test solution, injecting into a high performance liquid chromatograph to obtain a chromatogram, and respectively calculating the content of peimine and the content of peiminine by using an external standard two-point method logarithmic equation according to the peak area of the chromatogram;
wherein, the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph, ELSD detector
A chromatographic column: phenomenex Gemini-NX C18(4.6mm X150 mm X5 μm)
Mobile phase: acetonitrile: water: diethylamine 65: 35: 0.035
A detector: temperature of the drift tube: 70 ℃; gas flow rate: 1.4 ml/min; gain parameters: 1.
column temperature: 30 ℃; flow rate: 1.0 ml/min; sample introduction amount: 20 mu l of the mixture; operating time: and 20 min.
Background
The HUANGSHISONGQI buccal tablet is a compound preparation, and is prepared from herba Menthae, Bulbus Fritillariae Thunbergii, semen Scaphii Lychnophori, periostracum Cicadae, radix et rhizoma Rhei, fructus forsythiae, radix Platycodi, Catechu, fructus Chebulae, rhizoma Ligustici Chuanxiong, Glycyrrhrizae radix, Mentholum, etc. Has the effects of relieving sore throat, relieving voice, clearing heat, eliminating phlegm, and relieving swelling and pain. Can be used for treating syndrome of wind-heat affecting lung, manifested by hoarseness, phonation pain, and dry throat; acute laryngitis and acute attack of chronic laryngitis are marked by the above symptoms.
The thunberg fritillary bulb is a monarch drug in the prescription, can detoxify, eliminate stagnation and cure carbuncle, mainly comprises peimine (thunbergine) and peiminine (dehydropeimine), the peiminine has no ultraviolet absorption, the peiminine only has terminal absorption, and is not suitable for being detected by an ultraviolet detector, and the HPLC-ELSD method is adopted for analysis, so the effect is better.
The existing methods for detecting the content of peimine and peiminine have been reported, for example: the Chinese pharmacopoeia 2020 edition carries a method for measuring the content of peimine and peiminine in Huangshi Sound pills, and the Chinese patent ZL202110444762.0 also reports a method for measuring the content of peiminine A and peiminine B in Huangshi Sound tea.
The method for determining peimine and peiminine in Huangshi Sound pills in the prior art comprises the following basic steps:
the prior art has the following disadvantages:
the extraction time is long, and the soaking time and the ultrasonic time reach 1.5 hours; the extraction efficiency is low; the detection time is longer.
The prior art lacks a method for measuring the content of peimine and peiminine in Huangshi rattle buccal tablets.
The invention develops a method for measuring the content of peimine and peiminine in Huangshi rattle buccal tablets. On the basis of the prior art, the extraction time and the detection time are optimized, and the working efficiency is greatly improved.
Disclosure of Invention
The invention provides a method for measuring the content of peimine and peiminine in Huangshi rattle buccal tablets, which comprises the following steps:
1) control solution: precisely weighing peimine reference substance and peiminine reference substance, and adding methanol to obtain mixed solution;
2) test solution: adding a mixed solution of concentrated ammonia test solution and ethanol (1: 2) into the Huangshi rattle buccal tablets, adding diethyl ether: trichloromethane: dissolving the mixed solution of ethanol (50: 16: 5), filtering, and adding the filter residue into the mixture of diethyl ether: trichloromethane: washing with mixed solution of ethanol (50: 16: 5), mixing the filtrate and washing solution, volatilizing, dissolving the residue with methanol, and filtering;
3) and (3) determination: precisely absorbing the reference solution, injecting the test solution into a high performance liquid chromatograph to obtain a chromatogram, and respectively calculating the content of the peimine and the content of the peimine by using an external standard two-point method logarithmic equation according to the peak area of the chromatogram.
Preferably, the method comprises the following steps:
1) control solution: precisely weighing peimine reference substance and peiminine reference substance, and adding methanol to obtain mixed solution containing peiminine 0.2-0.3mg and peiminine 0.1-0.2mg per 1 ml;
2) test solution: weighing 7-9g of Huangshi rattle buccal tablet, precisely weighing, dripping 10ml of a mixed solution of concentrated ammonia test solution and ethanol (1: 2) for moistening, sealing, standing, adding diethyl ether: trichloromethane: mixing the solution with ethanol (50: 16: 5), ultrasonic treating for 25-35min, filtering, and treating the filter residue with diethyl ether: trichloromethane: washing with mixed solution of ethanol (50: 16: 5), mixing filtrates and washing solutions, volatilizing in water bath, dissolving residue with methanol, transferring to measuring flask, diluting to scale, shaking, and filtering;
3) and (3) determination: precisely absorbing reference substance solutions, injecting the test substance solutions into a high performance liquid chromatograph with 10-20 μ l each, obtaining a chromatogram, and respectively calculating the content of peimine and the content of peimine by using an external standard two-point method logarithmic equation according to the peak area of the chromatogram;
most preferably, the method comprises the following steps:
1) control solution: precisely weighing peimine reference substance and peiminine reference substance, and adding methanol to obtain mixed solution containing peiminine 0.25mg and peiminine 0.15mg per 1 ml;
2) test solution: weighing 8g of Huangshi rattle buccal tablet, precisely weighing, adding 10ml of concentrated ammonia solution-ethanol (1: 2) mixed solution dropwise for moistening, sealing, standing for 20min, adding diethyl ether: trichloromethane: mixing 25ml of ethanol (50: 16: 5), performing ultrasonic treatment (power 250W, frequency 50Hz, water bath temperature below 30 ℃) for 30min, filtering, and adding diethyl ether into filter residues: trichloromethane: washing with appropriate amount of ethanol (50: 16: 5) mixed solution, mixing filtrates and washing solutions, volatilizing in 60 deg.C water bath, dissolving residue with methanol, transferring to 5ml measuring flask, diluting with methanol to desired volume, shaking, and filtering;
3) and (3) determination: precisely sucking 20 mu l of reference solution and 20 mu l of test solution, injecting into a high performance liquid chromatograph to obtain a chromatogram, and respectively calculating the content of peimine and the content of peiminine by using an external standard two-point method logarithmic equation according to the peak area of the chromatogram;
wherein, the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph, ELSD detector
A chromatographic column: phenomenex Gemini-NX C18(4.6mm X150 mm X5 μm)
Mobile phase: acetonitrile: water: diethylamine 65: 35: 0.035
A detector: temperature of the drift tube: 70 ℃; gas flow rate: 1.4 ml/min; gain parameters: 1.
column temperature: 30 ℃; flow rate: 1.0 ml/min; sample introduction amount: 20 mu l of the mixture; operating time: and 20 min.
The invention is obtained by screening, and the screening process is as follows:
the soaking time was considered to be 10, 20, 30 and 40min respectively
Soaking time min
Peimine microgram/tablet
Peimine mu g/tablet
10
4.9
5.2
20
8.7
8.9
30
9.0
8.8
40
8.9
9.0
The result shows that the results of 20min soaking of the peimine and the peiminine contents are equivalent to 40min soaking, and 20min is preferred in consideration of time cost.
Ratio of concentrated ammonia test solution to ethanol mixed solution
Ratio of
Peimine microgram/tablet
Peimine mu g/tablet
1:0.5
7.0
7.4
1:1
8.7
8.9
1:2
10.5
10.6
The results show that the extraction efficiency is relatively high when the ratio of the concentrated ammonia test solution to the ethanol mixed solution is 1: 2.
Examine the ultrasound time for 20, 30, and 60min respectively
Ultrasonic time min
Peimine microgram/tablet
Peimine mu g/tablet
20
9.1
9.0
30
10.3
10.2
60
10.5
10.6
The result shows that the results of 30min ultrasonic treatment of the content of the peimine and the content of the peiminine are equivalent to 60min ultrasonic treatment, and the time cost is preferably 30 min.
3. Investigating the influence of different chromatographic columns on the content of peimine and peiminine
The results show that the content of the peimine and the peiminine measured by different chromatographic columns have little difference, the Phenomenex Gemini-NX C18(4.6mm multiplied by 150mm multiplied by 5 mu m) chromatographic column can finish the analysis work after 20min, and the applicability of the system meets the requirements.
The present invention does not employ prior art methods, such as those described in the following references:
high performance liquid chromatography is used for determining the content of peimine and peiminine in the Zhejiang fritillaria formula granules, the content comparison research of peiminine and peiminine in the Wabract fritillary bulb and the Songbiang fritillary bulb, and the content of peiminine and peiminine in the Zhejiang fritillary bulb is detected by an HPLC-ELSD method.
But the method of the prior art is greatly improved, and the main improvement points are as follows:
the chromatographic column is replaced by Phenomenex Gemini-NX C18(4.6mm multiplied by 150mm multiplied by 5 mu m) with shorter column length from Agilent ZORBAX SB-C18(4.6mm multiplied by 250mm multiplied by 5 mu m), the running time is reduced to 20min from 40min, and the analysis time is greatly shortened.
Secondly, the ultrasonic treatment time of the sample is reduced from 1 hour to 30 minutes, the preparation time of the Huangshi dulao buccal tablet prepared by the sample is shortened, although the components of the Huangshi dulao buccal tablet are the same as those of the Huangshi dulao pill, the proportions and the process of the prescription are not consistent, and the method is a method for specially measuring the content of the peimine and the peimine in the Huangshi dulao buccal tablet.
1. The method can be used for measuring the content of peimine and peiminine in the Huangshi rattle buccal tablets.
2. The time for extracting the sample is reduced, the time for analyzing and detecting is reduced, and the detection efficiency is improved.
Drawings
FIG. 1 shows a high performance liquid chromatogram of a control (retention time 5.893min for peimine and retention time 7.068min for peiminine)
FIG. 2 high performance liquid chromatogram of the sample
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
Example 1
Determination of content of peimine and peiminine in Huangshi rattle buccal tablets
1) Control solution: precisely weighing peimine reference substance and peiminine reference substance, and adding methanol to obtain mixed solution containing peiminine 0.25mg and peiminine 0.15mg per 1 ml;
2) test solution: weighing 8g of Huangshi rattle buccal tablet, precisely weighing, adding 10ml of concentrated ammonia solution-ethanol (1: 2) mixed solution dropwise for moistening, sealing, standing for 20min, adding diethyl ether: trichloromethane: mixing 25ml of ethanol (50: 16: 5), performing ultrasonic treatment (power 250W, frequency 50Hz, water bath temperature below 30 ℃) for 30min, filtering, and adding diethyl ether into filter residues: trichloromethane: washing with appropriate amount of ethanol (50: 16: 5) mixed solution, mixing filtrates and washing solutions, volatilizing in 60 deg.C water bath, dissolving residue with methanol, transferring to 5ml measuring flask, diluting with methanol to desired volume, shaking, and filtering;
3) and (3) determination: precisely sucking 20 mu l of reference solution and 20 mu l of test solution, injecting into a high performance liquid chromatograph to obtain a chromatogram, and respectively calculating the content of peimine and the content of peiminine by using an external standard two-point method logarithmic equation according to the peak area of the chromatogram;
wherein, the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph, ELSD detector
A chromatographic column: phenomenex Gemini-NX C18(4.6mm X150 mm X5 μm)
Mobile phase: acetonitrile: water: diethylamine 65: 35: 0.035
A detector: temperature of the drift tube: 70 ℃; gas flow rate: 1.4 ml/min; gain parameters: 1.
column temperature: 30 ℃; flow rate: 1.0 ml/min; sample introduction amount: 20 mu l of the mixture; operating time: and 20 min.
And (3) detection results:
sample (I)
Peimine microgram/tablet
Peimine mu g/tablet
1
10.6
10.9
2
10.4
10.5
3
11.0
11.2
4
10.7
10.9
5
10.6
10.7
