CN115078566B - Feature spectrum detection method and quality control method of exocarpium citri rubrum formula particles - Google Patents
Feature spectrum detection method and quality control method of exocarpium citri rubrum formula particles Download PDFInfo
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- 238000000034 method Methods 0.000 claims abstract description 38
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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
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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
- G01N30/86—Signal analysis
- G01N30/8651—Recording, data aquisition, archiving and storage
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides a characteristic spectrum detection method and a quality control method of orange formula particles, wherein the characteristic spectrum detection method of the orange formula particles comprises the steps of detecting by adopting a high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler; detection wavelength: 280-320 nm; acetonitrile is taken as a mobile phase A, a phosphoric acid aqueous solution with the concentration of 0.05-0.2% is taken as a mobile phase B, and gradient elution is adopted for elution. According to the characteristic spectrum detection method of the orange formula particles, the characteristic peaks in the characteristic spectrum obtained by the method are more, and the detection time is shorter; therefore, the method is more suitable for quality control of the orange formula particles.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine detection and quality control, in particular to a characteristic spectrum detection method and a quality control method of orange formula particles.
Background
The exocarpium Citri rubrum mainly contains flavonoid chemical components, and the flavonoid compounds are regarded as main bioactive components, and have various pharmacological activities of resisting oxidation, resisting inflammation and cancer, regulating cardiovascular system function, protecting nervous system and the like. The most common flavonoid compounds in exocarpium Citri rubrum comprise hesperidin, naringin, nobiletin, hesperetin, etc.
The orange formula granule is prepared by taking orange as a raw material, extracting with water, concentrating and drying. The high performance liquid chromatography method for the exocarpium citri rubrum formula particles disclosed in the prior art has the problems of few characteristic peaks and long detection time in the characteristic spectrum obtained by detection, and greatly wastes resources.
Disclosure of Invention
Therefore, the invention aims to solve the problems that the characteristic spectrum obtained by the high performance liquid chromatography method disclosed in the prior art has few characteristic peaks and long detection time; the invention provides a characteristic spectrum detection method of orange formula particles, which has the advantages that the characteristic peaks in the characteristic spectrum obtained by the method are more, and the detection time is shorter; therefore, the method is more suitable for quality control of the orange formula particles.
A characteristic spectrum detection method of exocarpium Citri rubrum formula granule adopts high performance liquid chromatography to detect, and the chromatographic conditions of the high performance liquid chromatography are as follows:
chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler; detection wavelength: 280-320 nm; acetonitrile is taken as a mobile phase A,0.05-0.2% phosphoric acid aqueous solution is taken as a mobile phase B, and elution is carried out according to the following gradient elution program:
t1 is 37-45, t2 is 45-55, t3 is 52-60, t2 is at least 5 larger than t1, and t3 is at least 3 larger than t 2;
the A1 is 3-5, the A2 is 8-9, and the A3 is 20-30.
In the gradient elution program, t1 is 45, t2 is 55, and t3 is 60; and A3 is 20-25.
In the high performance liquid chromatography, the column temperature of the chromatographic column is 25-35 ℃, the flow rate is 0.9-1.2 ml/min, and the theoretical plate number is not lower than 2000 calculated according to the hesperidin peak.
The column temperature of the chromatographic column is 28-32 ℃ and the flow rate is 1.1ml/min.
The mobile phase B is 0.05% phosphoric acid aqueous solution, and the detection wavelength is 300nm; the column was a Waters corecs T3.
The gradient elution procedure was:
the preparation process of the sample solution adopted in the detection is as follows: and (3) taking the orange formula particles, precisely weighing, adding a solvent, weighing, sealing, heating for refluxing, cooling, weighing again, supplementing the reduced weight with the solvent, uniformly mixing, filtering, and obtaining the subsequent filtrate as the sample solution.
The solvent is 50% methanol, and the heating reflux time is 30min.
The characteristic spectrum of the exocarpium citri rubrum formula particle comprises characteristic peaks corresponding to vitamin E-2, ferulic acid, narirutin, hesperidin, nobiletin, 3,5,6,7,8,3',4', -heptamethoxy flavone and hesperetin, wherein peak 4 is vitamin E-2, peak 7 is narirutin, peak 8 is hesperidin, peak 10 is nobiletin, peak 11 is 3,5,6,7,8,3',4' -heptamethoxy flavone and peak 12 is hesperetin; the peak 8 is taken as an S peak, the relative retention time of other characteristic peaks relative to the peak 8 is + -10% of a specified value, and the specified value of each characteristic peak is:
peak 1:0.36, peak 2:0.41, peak 3:0.50, peak 4:0.65, peak 5:0.69, peak 6:0.70, peak 7:0.92, peak 9:1.28, peak 10:1.38, peak 11:1.41, peak 12:1.42.
The technical scheme of the invention has the following advantages:
the characteristic spectrum detection method of the orange formula particle can obtain the characteristic spectrum with more stable base line and more characteristic peaks, and the separation effect of each characteristic peak is better. Therefore, the characteristic spectrum can comprehensively reflect the characteristic peak information of the orange formula particles, and is suitable for the overall quality control of the orange formula particles.
In addition, the invention shortens the detection and analysis time and improves the separation degree of chromatographic peaks while guaranteeing the information quantity of the chromatographic peaks of the characteristic spectrum.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a comparison characteristic spectrum of the exocarpium Citri rubrum formulation granule of the present invention.
FIG. 2 is a fingerprint of the localization of the vitamin C-2 and exocarpium Citri rubrum formulation in the present invention.
FIG. 3 is a fingerprint of the location of ferulic acid and exocarpium Citri rubrum formulation in the present invention.
FIG. 4 is a fingerprint of the location of narirutin and exocarpium Citri Grandis formulation in the present invention.
FIG. 5 is a fingerprint of the location of hesperidin and exocarpium Citri rubrum formula particles in the present invention.
FIG. 6 is a fingerprint of the location of nobiletin and exocarpium Citri rubrum formula particles in the present invention.
FIG. 7 is a plot of the location fingerprints of 3,5,6,7,8,3',4', -heptamethoxyflavone and exocarpium Citri rubrum formulations in the present invention.
FIG. 8 is a localized fingerprint of hesperetin and exocarpium Citri rubrum formulation particles of the present invention.
FIG. 9 is a characteristic map obtained by the gradient elution procedure of Table 15 in accordance with the present invention.
FIG. 10 is a characteristic map obtained by the gradient elution procedure of Table 20 in accordance with the present invention.
FIG. 11 is a characteristic map obtained by the gradient elution procedure of Table 21 in accordance with the present invention.
FIG. 12 is a profile obtained using the gradient elution procedure of Table 22 in accordance with the present invention.
FIG. 13 is a characteristic map obtained by the gradient elution procedure of Table 23 in accordance with the present invention.
FIG. 14 is a characteristic map obtained by the gradient elution procedure of Table 24 in accordance with the present invention.
FIG. 15 is a characteristic map obtained by the gradient elution procedure of Table 25 in accordance with the present invention.
FIG. 16 is a characteristic spectrum obtained by using a Kromasil 100-5-C18 column according to the present invention.
FIG. 17 is a characteristic spectrum obtained by using an Agilent SB-C18 chromatographic column in accordance with the invention.
FIG. 18 is a characteristic spectrum obtained by using a Agilent Eclipse Plus C chromatographic column according to the invention.
FIG. 19 is a schematic representation of the invention employing Waters
Characteristic spectrum obtained by T3 chromatographic column.
Detailed Description
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or instruments used are not noted by the manufacturer and are all conventional reagent products available commercially, and the percentages not noted in the invention are all volume percentages.
Instrument:
the chromatograph 1 is a Waters 2695 chromatographic system, comprising a quaternary gradient infusion pump (Alliance 2695), a 120-bit high-performance automatic sampler, an original-installed import chromatographic column incubator, a Waters 2998 diode array ultraviolet detector and an Empower chromatographic management system;
chromatograph 2: the Shimadzu chromatographic system comprises an LC-20AT type quaternary pump, an SIL-20AC type autosampler, an SPD-M20A type PDA diode array detector, a CTO-20AC type column incubator, a CBM-20A system controller and an Empower chromatographic management system;
chromatograph 3: the UltiMate 3000 chromatographic system comprises an LPG-3400A quaternary pump, a WPS-3000TSL automatic sampler, a PDA diode array detector and a chromatographic workstation;
XS204, XS205, XSE205 one ten thousandth balance (mertrehler switzerland), ME36S one ten thousandth level (mertrehler switzerland), sonicator (Shanghai guide sonicator limited), water bath.
Reagent:
hesperidin (lot number: 110721-202019, calculated as 95.3%) was purchased from China food and drug verification institute
Chuanchenyisu (batch No. 112055-202102, for content determination, calculated as 99.7%) is purchased from Chinese food and drug inspection institute
Hesperetin (lot number: 112054-202102, for content determination, calculated as 99.7%) was purchased from China food and drug inspection institute
Ferulic acid (batch No. 110773-201915, 99.4% for content determination) was purchased from Chinese food and drug testing institute
Vietnamine-2 (lot number: DST210901-023 for content determination, calculated as 98%) was purchased from Chenopodium medical science and technology Co., ltd
Narirutin (lot number 250107-202110, 98% for content determination) was purchased from Shanghai Yongsheng Biotechnology Co., ltd
3,5,6,7,8,3',4', -heptamethoxyflavone (lot number 170106-202111, 98% calculated for content determination) was purchased from Shanghai Yongsheng Biotechnology Co., ltd.)
Exocarpium Citri rubrum formula granule (batch number: 1902001Y, 1902002Y, 1902003Y) is prepared by the following steps: taking 2300g of exocarpium citri rubrum decoction pieces, adding water to decoct for 2 times, adding 10 times of water to soak for 30 minutes at the first time, decocting for 30 minutes, filtering with 200-mesh filter cloth, adding 8 times of water at the second time, decocting for 20 minutes, filtering with 200-mesh filter cloth, mixing filtrates, concentrating to fluid extract with the relative density of 1.24-1.30 g/ml (40-60 ℃) at 70-80 ℃, adding 3% maltodextrin auxiliary material, mixing uniformly, carrying out belt drying (the temperature of the first area is 100-120 ℃, the temperature of the second area is 80-100 ℃, the temperature of the third area is 25-30 ℃), crushing, the plaster yield is 30.0-40.0% (the auxiliary material amount is subtracted), adding a proper amount of maltodextrin auxiliary material, mixing uniformly, granulating, preparing 1000g, and packaging.
Reagent: acetonitrile is chromatographic pure, water is ultrapure water, and other reagents are all analytical pure.
Example 1
A characteristic spectrum detection method of exocarpium Citri rubrum formula granule adopts High Performance Liquid Chromatography (HPLC) to detect, and the chromatographic conditions of the HPLC are as follows:
chromatographic column: octadecylsilane chemically bonded silica is used as filler (Waters)
T3, particle size 2.7 μm, specification 4.6mm.times.150mm); acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, and performing gradient elution according to the following specification in Table 1; the detection wavelength is 300nm; column temperature is 30 ℃; the flow rate was 1.1ml per minute. The theoretical plate number is not less than 2000 calculated as hesperidin peak. .
TABLE 1
Preparation of reference solution: taking appropriate amounts of reference substances such as vitamin E-2, ferulic acid, narirutin, hesperidin, nobiletin, 3,5,6,7,8,3',4', -heptamethoxy flavone, hesperetin, etc., placing into a measuring flask, and adding methanol to prepare different reference substance solutions containing 50 μg of reference substance per 1ml.
Preparation of test solution: grinding exocarpium Citri rubrum formula granule, precisely weighing about 0.2g, placing into conical flask with plug, precisely adding 50ml of 50% methanol, weighing, heating and refluxing for 30min, taking out, cooling, weighing again, supplementing the weight of loss with 50% methanol, mixing, filtering, and collecting filtrate.
Assay: respectively precisely sucking 10 μl of reference solution and sample solution, and injecting into liquid chromatograph for measurement.
In the embodiment, 15 batches of exocarpium citri rubrum decoction piece standard decoction (freeze-dried powder) and 3 batches of exocarpium citri rubrum formula particles are adopted to generate a characteristic map. The preparation process of the standard decoction (freeze-dried powder) of the exocarpium citri rubrum decoction pieces comprises the following steps: taking the medicinal material of the exocarpium citri rubrum, removing impurities according to the specification of decoction pieces [ processing ] under the "exocarpium Citri rubrum" item of the 2020 edition of Chinese pharmacopoeia, cutting into pieces, and processing into the exocarpium citri rubrum decoction pieces meeting the requirements. About 100g of decoction pieces are taken, put into a marmite, added with 1000mL of purified water for soaking for 30 minutes, boiled with strong fire (500W), decocted with slow fire (300W) for 30 minutes, filtered with 200-mesh filter cloth while hot, and the filtrate is rapidly cooled to room temperature; adding 800mL of purified water into the residue, boiling with strong fire (500W), decocting with slow fire (200W) for 25 min, filtering with 200 mesh filter cloth, rapidly cooling the filtrate to room temperature, mixing the two filtrates, and weighing the filtrate. Concentrating the filtrate under reduced pressure (55-65 ℃) to obtain a concentrated extract with the relative density of 1.06-1.12 g/ml (60 ℃ +/-5 ℃), weighing the concentrated extract, measuring the solid content of the concentrated extract, and calculating the extract yield of the standard decoction of the red tangerine peel decoction pieces. Placing the concentrated extract in a freeze dryer, freeze drying (-80deg.C, 0 Mpa) to dry state, taking out, weighing, pulverizing, and packaging in penicillin bottle to obtain exocarpium Citri rubrum decoction piece standard decoction (lyophilized powder).
Based on the characteristic patterns generated by the obtained multi-batch orange decoction piece standard decoction (freeze-dried powder) and orange formula particles, fitting by adopting a mode of 'multi-point correction and MARK peak matching' by adopting fingerprint similarity evaluation software '2012 edition of a traditional Chinese medicine fingerprint similarity evaluation system' compiled by pharmacopoeia committee, and generating a comparison characteristic pattern to obtain an orange formula particle comparison characteristic pattern; after the chromatographic matching, 12 common characteristic peaks with proper response values (peak areas), good separation degree and high purity are obtained, and the chromatographic peak numbers of the chromatographic peaks are rearranged according to the sequence of the chromatographic peaks and are from peak 1 to peak 12, as shown in figure 1. Wherein the relative retention times and relative peak areas of the control profile are shown in tables 2 and 3 below.
TABLE 2
TABLE 3 Table 3
The retention time and peak area of the 12 characteristic peaks are extracted respectively for characteristic maps generated by 15 batches of exocarpium citri rubrum decoction piece standard decoction (freeze-dried powder) and 3 batches of exocarpium citri rubrum formula particles, and the relative retention time and relative peak area of each characteristic peak are obtained, and the results are shown in the following tables 4 and 5.
TABLE 4 Table 4
TABLE 5
The characteristic spectrum characteristic peaks of the orange formula particles are analyzed by a high performance liquid chromatography-high resolution mass spectrometry (HPLC-Q-TOF/MS), a natural product high resolution mass spectrum database and related documents are combined according to the multi-stage mass spectrum information of the samples, and the HPLC is adopted to locate the reference substances, wherein the specific related results are shown in figures 2-8 and table 6.
TABLE 6 HPLC-Q-TOF/MS analysis results of orange formula particles
From the above results, it was determined that peak 4 was vitamin ning-2, peak 7 was narirutin, peak 8 was hesperidin, peak 10 was nobiletin, peak 11 was 3,5,6,7,8,3',4' -heptamethoxy flavone, and peak 12 was hesperetin. Because hesperidin is an index component for content measurement, the relative retention time and the relative peak area of each characteristic peak are better evaluated by using hesperidin as a reference peak.
From the results shown in tables 2 and 4, 12 characteristic peaks should be present in the sample chromatogram and should correspond to retention times of 12 characteristic peaks in the control characteristic spectrum, wherein peak 4, peak 6, peak 7, peak 8, peak 10, peak 11 and peak 12 respectively correspond to vitamin E-2, ferulic acid, narirutin, hesperidin, nobiletin, 3,5,6,7,8,3',4' -heptamethoxyflavone, hesperetin reference substances, peak corresponding to the hesperidin reference substance peak is S peak, and the relative retention time of each characteristic peak and the S peak is calculated and should be within + -10% of the specified value. The specified value is: 0.36 (Peak 1), 0.41 (Peak 2), 0.50 (Peak 3), 0.65 (Peak 4), 0.69 (Peak 5), 0.70 (Peak 6), 0.92 (Peak 7), 1.28 (Peak 9), 1.38 (Peak 10), 1.41 (Peak 11), 1.42 (Peak 12).
As can be seen from the results in tables 3 and 5, the peak area response values of the peaks 4, 10, 11 and 12 were small in the multi-batch samples, and thus the values of the relative peak areas were also small, while the values of the relative peak areas of the peak 7 were moderate, indicating that the peak 7 (narirutin) was relatively large in the characteristic map. And in combination with analysis of the results of the following methodological verification, it was determined that the relative peak areas of peak 7 were less disturbed by different influencing factors. Therefore, the peak area of peak 7 (narirutin) can be calculated by taking the peak corresponding to the hesperidin reference substance as the reference peak, and the semi-quantitative control of the flavonoid components in the orange formula particles is improved, so that the standard control level is improved. Wherein, the relative peak area mean value of peak 7 in the characteristic spectrum of the multi-batch orange formula particle is 0.45, SD is 0.15, the actual measurement range is 0.26-0.62, and the mean value + -2SD range is 0.15-0.74. Thus, the relative peak area of peak 7 (naringin) must not be less than 0.15 as specified in-2 SD.
Example 2
This example differs from example 1 in that the chromatographic conditions are different, in particular as follows:
1. detection wavelength
An appropriate amount of the orange formulation particles (lot number: 1902001Y) was taken and ground, and a test solution was prepared according to the preparation method of the test solution in example 1, and the test solutions were measured under the same other conditions as in example 1 using different detection wavelengths (280 nm, 290nm, 300nm, 310nm, 320 nm), and the detection results are shown in Table 7.
TABLE 7
The investigation results of different wavelengths show that: at the wavelength of 280-320nm, the baseline of the characteristic spectrum becomes unstable gradually along with the increase of the wavelength, the peak information amount of the characteristic spectrum is more at the wavelength of 300nm, the response value of each peak is relatively higher, and the response of each peak is relatively balanced. In comprehensive consideration, the detection wavelength of the characteristic spectrum of the orange formula particle is preferably 300 nm.
2. Flow rate
An appropriate amount of the orange formulation particles (lot number: 1902001Y) was taken and ground, and test solutions were prepared according to the preparation method of the test solution in example 1, and the results were measured under the same other conditions as in example 1 at different flow rates (0.9 ml/min, 1.0ml/min, 1.1ml/min, 1.2 ml/min) respectively, as shown in tables 8 to 10 below.
TABLE 8
TABLE 9
Table 10
The peak numbers in the above table 8 are normal chromatographic peaks labeled sequentially based on the time sequence of the peak, and the peaks in the tables 9 and 10 are characteristic peaks labeled corresponding to the characteristic peaks in the control map in example 1.
The results of different flow rate investigation show that: different flow rates (0.9 ml/min, 1.0ml/min, 1.1ml/min, 1.2 ml/min) are examined respectively, and have certain influence on the separation condition of each peak and surrounding small peaks, but the flow rates in the range meet the requirement of system applicability, wherein the baseline of 1.1ml/min is more stable at 20-30min, so that the baseline is preferably 1.1ml/min.
3. Column temperature
An appropriate amount of the orange peel formulation particles (lot number: 1902001Y) was ground, and the test solutions were prepared according to the preparation method of the test solution in example 1, and were measured by using different column temperatures (25 ℃, 28 ℃, 30 ℃, 32 ℃ and 35 ℃) under the same other conditions as in example 1, and the detection results are shown in tables 11 to 13 below.
TABLE 11
Table 12
TABLE 13
The peak numbers in the above table 11 are normal chromatographic peaks labeled sequentially based on the time sequence of the peak, and the peaks in the tables 12 and 13 are characteristic peaks labeled corresponding to the characteristic peaks in the control map in example 1.
The results of different column temperature investigation show that: the different column temperatures (25 ℃, 28 ℃, 30 ℃, 32 ℃ and 35 ℃) are examined respectively, and have certain influence on the retention time of each peak, the total area of chromatographic peaks, the separation degree, the peak shape and the like, but the column temperatures in the range meet the requirement of system applicability. Wherein, in the range of 28 ℃ to 32 ℃, when the column temperature changes, the deviation of the relative retention time of each characteristic chromatographic peak is within 3%, and the overall effect of chromatographic peak separation is considered, preferably 30 ℃.
4. Mobile phase
An appropriate amount of the orange formulation particles (lot number: 1902001Y) was taken and ground, and a sample solution was prepared according to the preparation method of the sample solution in example 1, and the same other conditions as in example 1 were used to determine the same mobile phases B (0.1% aqueous phosphoric acid solution, 0.1% aqueous formic acid solution, 0.1% aqueous acetic acid solution) respectively.
The mobile phase phosphoric acid, formic acid and acetic acid are compared and researched, the peak information amount of the characteristic spectrum of acetonitrile-0.1% phosphoric acid aqueous solution is more, and the separation degree between chromatographic peaks is obviously better than that of acetic acid and formic acid with the same concentration; therefore, the mobile phase system with acetonitrile-0.1% phosphoric acid aqueous solution as the characteristic spectrum of the orange formula granule is preferable. And on the basis, the concentration of phosphoric acid is optimally compared to obtain a more proper mobile phase system, and the specific process is as follows:
an appropriate amount of the orange peel formulation particles (lot number: 1902001Y) was ground, and a test solution was prepared according to the method for preparing a test solution in example 1, and the test solutions were measured using phosphoric acid aqueous solutions (0.05%, 0.1%, 0.15%, 0.2%) having different concentrations under the same other conditions as in example 1, and the measurement results are shown in Table 14 below.
TABLE 14
The peak numbers in table 14 are common chromatographic peaks labeled sequentially based on the order of the peak emission times.
The results of different mobile phase investigation show that: the pH of the mobile phase is not obviously influenced on the whole, when acetonitrile-0.05% phosphoric acid is adopted as a mobile phase system, the total area of chromatographic peaks is higher than that of other mobile phase systems, and the damage to a chromatographic column is larger in consideration of the fact that the higher the concentration of phosphoric acid is, the lower the pH value of the mobile phase is, and the mobile phase system of the characteristic spectrum method of the orange formula particles is comprehensively considered, wherein the acetonitrile-0.05% phosphoric acid solution is preferably adopted.
5. Chromatographic column type
Test substances (lot number: 1902001Y) were taken and a test substance solution was prepared in the same manner as in example 1, and the measurement was carried out using the gradient elution program shown in Table 15 below and using different types of chromatographic columns under the same other conditions as in example 1.
TABLE 15
Different types of chromatographic columns include:
chromatographic column 1: kromasil 100-5-C18 (5 μm,4.6 mm. Times.150 mm);
chromatographic column 2: agilent SB-C18 (5 μm,4.6 mm. Times.150 mm);
chromatographic column 3: agilent Eclipse Plus C18 (5 μm,4.6 mm. Times.150 mm);
chromatographic column 4: waters
T3(2.7μm，4.6mm×150mm)；
The detection results of the chromatographic columns of different brands are shown in the figure16-FIG. 19 shows that, as a result of detection, different brands of columns have a certain influence on the measurement result, but the influence is not remarkable, and Waters is preferably used in combination with the separation effect of each column
T3 chromatographic column.
Meanwhile, the measurement was carried out by using the gradient elution program in example 1 and using the following different types of columns under the same other conditions as in example 1, and the results are shown in tables 16 to 17.
Chromatographic column 1: agilent SB-C18 (5 μm,4.6 mm. Times.150 mm);
chromatographic column 2: CAPCRLL CORE C18 (5 μm,4.6 mm. Times.150 mm);
chromatographic column 3: waters
T3(2.7μm，4.6mm×150mm)；
Table 16
TABLE 17
From the above tables 16-17, it can be seen that: the different brands of chromatographic columns have certain influence on the measurement result, but the influence is not obvious, and the separation effect of each chromatographic column is combined, preferably Waters is adopted
T3 chromatographic column.
Meanwhile, the characteristic spectrum method is found to have poor durability on different types of chromatographic columns in the research and development process, so that the types of the chromatographic columns need to be fixed, and Waters are preferably adopted in the invention
T3 chromatographic column, and examine the same type chromatographic column of different batches, the investigation process is: taking the same batch of sample solution, setting up chromatographic column Waters +.>
Batch 3 (SN: 01313100716709, SN:01313100716709, SN: 01393132315615) having different T3 groups was examined for the relative retention time and relative peak area of each characteristic peak and the reference S peak (No. 8 peak) when the column group number was changed, and the results of the examination are shown in tables 18 and 19 below.
TABLE 18
TABLE 19
From the above results, the average deviation of the relative retention time of the multi-batch chromatographic columns was less than 2%. The results showed that the retention time of each characteristic chromatographic peak did not change much with the change of the batch number of the different chromatographic columns, thus determining the Waters of the fixed chromatographic column
T3 (150 mm. Times.4.6 mm,2.7 μm) can be used for the feature pattern detection of the orange formula particles.
6. Elution gradient
An appropriate amount of the orange formulation particles (lot number: 1902001Y) was taken and ground, and a test solution was prepared according to the preparation method of the test solution in example 1, and the same other conditions as in example 1 were used for the test solution by the elution gradient procedure of Table 15 and tables 20 to 25 below, respectively.
Table 20
Table 21
Table 22
Table 23
Table 24
Table 25
The chromatograms obtained by the respective detection in the gradient elution procedures of tables 19 to 25 are shown in FIGS. 9 to 15. The characteristic peaks 1-12 can be effectively separated by adopting the gradient elution program, but in the obtained spectrogram of the gradient elution program in the table 23, the chromatographic information is rich, the separation degree of the chromatographic peaks is good, the base line is stable, and the gradient elution program in the table 23 is preferred.
Example 3
The method of example 1 was examined for precision and stability, and the verification procedure was as follows:
1. precision of
1.1 Instrument precision test
The same sample solution of the orange formula particle was taken and repeatedly sampled 6 times under the chromatographic conditions of example 1, and the relative retention time and the relative peak area of each common peak were measured, and the detection results are shown in table 26.
Table 26
The results show that: the relative retention time of each characteristic peak and the reference S peak (No. 7 peak) is less than 2% and the relative peak area is less than 2%, which indicates that the method has good instrument precision.
1.2 method repeatability test
6 parts of the same orange formula particles were repeatedly prepared according to the test preparation method under the chromatographic conditions of example 1, and the relative retention time and the relative peak area of each common peak were measured according to the chromatographic conditions, and the detection results are shown in Table 27.
Table 27
The results show that: the relative retention time of each characteristic peak to the reference S peak (peak No. 7) was less than 2% RSD, while the relative peak area was less than 2% RSD. Indicating that the reproducibility of the method is good.
1.3 intermediate precision (different operators)
Three inspectors respectively took the same orange formula particles at different times, prepared samples according to the preparation method of the test sample under the chromatographic condition of example 1, and measured the relative retention time and the relative peak area of each common peak by the same equipment, and the detection results are shown in table 28.
Table 28
The results show that: the deviation of the relative peak areas of each characteristic peak and the reference S peak (peak No. 7) was less than 2%. Indicating that the intermediate precision of the method is good.
1.4 intermediate precision (different high Performance liquid chromatography)
Samples were prepared by the same inspector at different times from the same orange formula particles according to the test preparation method under the chromatographic conditions of example 1, and the relative retention time and relative peak area of each common peak were measured by three different devices (waters, daim, shimadzu), and the detection results are shown in table 29.
Table 29
The results show that: the chromatographic peak can reappear on the liquid phase, the deviation of the relative retention time of the peak 1, the peak 2 and the other characteristic peaks is smaller than 2%, and the deviation of the relative peak areas of the peak 4, the peak 5 and the peak 11 is larger.
1.5 stability investigation
The same sample solution of the orange formula particle was sampled at 0, 2, 4, 8, 12, 18 and 24 hours according to the chromatographic conditions of example 1, and the relative retention time and the relative peak area of each common peak were measured, and the detection results are shown in table 30.
Table 30
The results show that: the deviation of the relative retention time and the relative peak area of each chromatographic peak and the reference peak is less than 2%, which shows that the sample solution is stable within 24 hours and meets the measurement requirements.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. The characteristic spectrum detection method of the exocarpium citri rubrum formula particles is characterized by adopting a high performance liquid chromatography for detection, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:
chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler, particle size of 2.7 μm, specification of 4.6mm×150mm; detection wavelength: 280-320 nm; acetonitrile is taken as a mobile phase A,0.05-0.2% phosphoric acid aqueous solution is taken as a mobile phase B, and elution is carried out according to the following gradient elution program:
t1 is 37-45, t2 is 45-55, t3 is 52-60, t2 is at least 5 larger than t1, and t3 is at least 3 larger than t 2;
the A1 is 3-5, the A2 is 8-9, and the A3 is 20-30;
the characteristic spectrum of the exocarpium Citri rubrum formula granule comprises characteristic peaks corresponding to vitamin adopted-2, ferulic acid, narirutin, hesperidin, nobiletin, 3,5,6,7,8,3',4', -heptamethoxyl flavone and hesperetin.
2. The method for detecting the characteristic spectrum of the exocarpium citri rubrum formula granule according to claim 1, wherein t1 is 45, t2 is 55 and t3 is 60 in the gradient elution program; and A3 is 20-25.
3. The method for detecting the characteristic spectrum of the exocarpium citri rubrum formula granule according to claim 1 or 2, wherein the column temperature of a chromatographic column in the high performance liquid chromatography is 25-35 ℃, the flow rate is 0.9-1.2 ml/min, and the theoretical plate number is not lower than 2000 calculated according to the hesperidin peak.
4. The method for detecting the characteristic spectrum of the exocarpium citri rubrum formula granules according to claim 3, wherein the column temperature of the chromatographic column is 28-32 ℃ and the flow rate is 1.1ml/min.
5. A method according to claim 1 or 2The characteristic spectrum detection method of the exocarpium citri rubrum formula particles is characterized in that the mobile phase B is 0.05% phosphoric acid aqueous solution, and the detection wavelength is 300nm; the chromatographic column is Waters Cortecs ® T3。
6. The method for detecting the characteristic spectrum of the orange formula granules according to claim 1 or 2, wherein the gradient elution procedure is as follows:
7. the method for detecting the characteristic spectrum of the orange formula granules according to claim 1 or 2, wherein the preparation process of the sample solution adopted in the detection is as follows: and (3) taking the orange formula particles, precisely weighing, adding a solvent, weighing, sealing, heating for refluxing, cooling, weighing again, supplementing the reduced weight with the solvent, uniformly mixing, filtering, and obtaining the subsequent filtrate as the sample solution.
8. The method for detecting the characteristic spectrum of the exocarpium citri rubrum formula granules according to claim 7, wherein the solvent is 50% methanol, and the heating reflux time is 30min.
9. The method for quality control by adopting the characteristic spectrum constructed by the characteristic spectrum detection method of the orange formula particle according to any one of claims 1-8, which is characterized in that the characteristic spectrum of the orange formula particle comprises characteristic peaks corresponding to vitamin-2, ferulic acid, narirutin, hesperidin, nobiletin, 3,5,6,7,8,3',4', -heptamethoxyflavone and hesperetin, wherein peak 4 is vitamin-2, peak 7 is narirutin, peak 8 is hesperidin, peak 10 is nobiletin, peak 11 is 3,5,6,7,8,3',4' -heptamethoxyflavone and peak 12 is hesperetin; the peak 8 is taken as an S peak, the relative retention time of other characteristic peaks relative to the peak 8 is + -10% of a specified value, and the specified value of each characteristic peak is:
peak 1:0.36, peak 2:0.41, peak 3:0.50, peak 4:0.65, peak 5:0.69, peak 6:0.70, peak 7:0.92, peak 9:1.28, peak 10:1.38, peak 11:1.41, peak 12:1.42.
10. the method of claim 9, wherein peak 8 is defined as S peak and the relative peak area value of peak 7 is not less than 0.15.
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