CN111505156A - Fingerprint spectrogram quality determination method for herba Cirsii formulation granules - Google Patents
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Abstract
The invention discloses a fingerprint spectrogram quality determination method for herba cepbalanoplosis segeti formula granules, which comprises the steps of putting the herba cepbalanoplosis segeti formula granules into methanol, performing ultrasonic treatment, cooling and filtering to obtain a test solution; mixing linarin control, apigenin control, caffeic acid control, chlorogenic acid control, and protocatechuic acid control, and dissolving with methanol to obtain reference solution; and respectively sucking the test solution and the reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph, and measuring by adopting gradient elution to obtain a fingerprint spectrogram of the herba cepbalanoplosis segeti formula particles. The fingerprint spectrogram of the herba cepbalanoplosis segeti formula particles is constructed by a gradient elution mode through a high performance liquid chromatography, is used for determining various effective substances of linarin, apigenin, caffeic acid, chlorogenic acid and protocatechuic acid in the herba cepbalanoplosis segeti formula particles, can be used as an accurate quality control method of the herba cepbalanoplosis segeti formula particles, and has the characteristics of good separation effect and high sensitivity.
Description
Technical Field
The invention relates to a fingerprint spectrogram quality determination method for herba cepbalanoplosis segeti formula granules, in particular to a fingerprint spectrogram quality determination method for herba cepbalanoplosis segeti formula granules, which is constructed by adopting a high performance liquid chromatography, and belongs to the field of traditional Chinese medicine quality control.
Background
Herba Cephalanoploris is selected from Cephalanoploris (Cynara cardunculus) of CompositaeCirsium setosum(Wild.) MB) has the effects of cooling blood, stopping bleeding, removing blood stasis and relieving swelling, and is used for treating epistaxis, hematemesis, hematochezia, hematuria, metrorrhagia, metrostaxis, traumatic hemorrhage, carbuncle, swelling, sore and toxin, and the like. Modern researches find that the common cephalanoplos herb also has various pharmacological activities of reducing blood pressure, resisting bacteria, tumors, resisting oxidation and the like. The herba cepbalanoplosis segeti formula granules are prepared by processing dried overground part of herba cepbalanoplosis segeti medicinal materials and preparing the dried overground part of the herba cepbalanoplosis segeti into formula granules according to the main quality standard of standard decoction. Modern pharmacological research finds that various active ingredients can be extracted from the herba cepbalanoplosis segeti, such as caffeic acid lipids (chlorogenic acid and caffeic acid) and flavonoids (rutin, linarin and the like), and also protocatechuic acid, protocatechuic aldehyde and the like. However, due to the influence of the factors of the medicinal materials such as different producing areas, climates, ecological environments and the like, and the factors of process conditions and the like of each link in the preparation process of the formula granules, the difference of the main components contained in the common cephalanoplos herb formula granules in different batches or production processes is often larger. Therefore, in order to keep the property, taste and efficacy of the herba cepbalanoplosis segeti formula granules consistent with those of the original medicines, a quality control system of the herba cepbalanoplosis segeti formula granules is necessarily required to be established so as to carry out qualitative and quantitative analysis on the herba cepbalanoplosis segeti formula granules or certain effective ingredients or index ingredients in the herba cepbalanoplosis segeti formula granules.
Hedonrich et al, in HP L C for simultaneous determination of contents of linarin and apigenin in herba Cephalanoploris granule (China modern applied medicine, 34 rd 3 rd Vol. 3 rd 3 nd 2007, 410-412), described the adoption of HP L C as high performance liquid chromatographyA method for simultaneously measuring the contents of linarin and apigenin in a herba cepbalanoplosis segeti formula particle by a spectrometry method comprises the following detection conditions: Diamonsil-C18(4.6 mm × 250mm, 5 μm) chromatographic column, using acetonitrile-1% glacial acetic acid water solution as mobile phase to carry out gradient elution with flow rate of 1.0m L min-1The detection wavelength is 335nm, and the column temperature is 30 ℃. The sampling amount of linarin and apigenin is in a good linear relation with the peak area in 0.3-1.5 mu g (r = 0.9994) and 0.075-0.375 (r = 0.9997) respectively; the sample recovery rate was 99.19% and 99.12%, respectively, and the RSD was 1.27% and 1.26%, respectively.
Besides, the method for simultaneously determining the content of 5 active ingredients in herba Cephalanoploris formula granules by using HP L C-DAD method (Chinese pharmacist, 21 vol.6, 1125-1127) by using HP L C-DAD method, and the method for simultaneously determining the content of the active ingredients in chlorogenic acid, baicalin, rutin, linarin and apigenin 5 in herba Cephalanoploris formula granules by using ThermoHypersil BDS C-DAD method18(250 mm × 4.6.6 mm, 5 μm) chromatographic column, mobile phase acetonitrile (A) -1% phosphoric acid water solution (B), flow rate 1.0m L. min-1Gradient elution, detection wavelength 326nm, column temperature 35 ℃. The mass concentrations of chlorogenic acid, baicalin, rutin, linarin and apigenin are respectively 0.072-1.446 mu g (r = 0.9997), 0.043-0.864 mu g (r = 0.9995), 0.094-1.884 mu g (r = 0.9997), 0.150-3.000 mu g (r = 0.9998) and 0.043-0.856 mu g (r = 0.9996), and the linear relation with the peak areas is good. The sample recovery rates of chlorogenic acid, baicalin, rutin, linarin and apigenin were 97.67%, 98.19%, 97.92%, 98.12%, 98.02% and 1/17/5 (n = 6), respectively.
From the above, it can be known that the herba cepbalanoplosis segeti formula granules contain a plurality of active ingredients, and in the determination standard established by the existing HP L C (-DAD) method, the measured ingredients are mostly only index ingredients of 2-3 types of substances, but not all main active ingredients related to the curative effect of the herba cepbalanoplosis segeti formula granules.
Disclosure of Invention
The invention aims to provide a fingerprint spectrogram quality determination method for herba cepbalanoplosis segeti formula particles, which is used for determining various effective substances such as linarin, apigenin, caffeic acid, chlorogenic acid and protocatechuic acid in the herba cepbalanoplosis segeti formula particles by constructing the fingerprint spectrogram of the herba cepbalanoplosis segeti formula particles through a high performance liquid chromatography in a gradient elution mode, can be used as an accurate quality control method for the herba cepbalanoplosis segeti formula particles, and has the characteristics of good separation effect and high sensitivity.
The invention is realized by the following technical scheme: a fingerprint spectrum quality determination method for herba Cephalanoploris formula granules comprises the following steps:
a: dissolving herba Cephalanoploris granule in methanol, ultrasonic treating, cooling, and filtering to obtain test solution;
b: mixing linarin control, apigenin control, caffeic acid control, chlorogenic acid control, and protocatechuic acid control, and dissolving with methanol to obtain reference solution;
c: and (3) determination: and respectively sucking the test solution and the reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph, and measuring by adopting gradient elution to obtain a fingerprint spectrogram of the herba cepbalanoplosis segeti formula particles.
In the step A, 0.1-0.3 g of herba cepbalanoplosis segeti formula particle powder is added into 10-30 ml of 40-60% methanol by mass concentration, ultrasonic treatment is carried out for 20-40 min, cooling is carried out to 5-20 ℃, and a test solution is prepared after filtration.
In the step B, the mass concentration of the methanol is 5-100%, and each 1ml of reference substance solution contains 10-50 μ g of linarin, 10-40 μ g of apigenin, 5-50 μ g of caffeic acid, 5-50 μ g of chlorogenic acid and 5-50 μ g of protocatechuic acid.
In the step C, the detection conditions of the high performance liquid chromatograph meet the following conditions:
filling agent: octadecylsilane chemically bonded silica gel, and a silane,
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent phosphoric acid solution, gradient elution is carried out,
temperature of the column: 30-40 ℃,
flow rate: 0.8 to 1.2ml/min,
detection wavelength: 320-336 nm.
The gradient elutes as follows:
0-10 min: 10% mobile phase A, 90% mobile phase B,
10-20 min: 10 to 15% of mobile phase A, 90 to 85% of mobile phase B,
20-55 min: 15-40% of mobile phase A, 85-60% of mobile phase B,
55-60 min: 40 to 50% of mobile phase A, 60 to 50% of mobile phase B,
60-80 min: 50-75% of mobile phase A and 50-25% of mobile phase B.
The fingerprint spectrum of the herba cepbalanoplosis segeti formula particle has 9 characteristic peaks, the linarin chromatographic peak is taken as a reference substance S peak, and the relative retention time of the 9 characteristic peaks is respectively as follows: the relative retention time of the characteristic peaks is within. + -. 8% of the specified value, namely 0.176 for peak 1, 0.332 for peak 2, 0.355 for peak 3, 0.422 (S peak) for peak 4, 0.624 for peak 5, 0.861 for peak 6, 1.000 for peak 7, 1.168 for peak 8 and 2.546 for peak 9.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) aiming at the quality control problem of the conventional herba cepbalanoplosis segeti formula particles, the invention takes various effective substances including linarin, apigenin, caffeic acid, chlorogenic acid and protocatechuic acid in the herba cepbalanoplosis segeti formula particles as reference substances, and adopts a high performance liquid chromatography to construct a fingerprint of the herba cepbalanoplosis segeti formula particles, thereby not only providing scientific experimental basis for the research of a herba cepbalanoplosis segeti formula particle quality evaluation system, but also realizing the exclusive identification of the quality of the herba cepbalanoplosis segeti formula.
(2) The method adopts the methods of methanol dissolution and ultrasonic treatment to extract the herba cepbalanoplosis segeti formula particles, and can comprehensively detect various main effective components in the herba cepbalanoplosis segeti formula particles, so that the fingerprint of the herba cepbalanoplosis segeti formula particles is constructed, and an effective method is provided for the overall quality evaluation of the herba cepbalanoplosis segeti formula particles.
(3) The method obtains the characteristic spectrum of the herba cepbalanoplosis segeti formula granules under specific detection conditions, takes the linarin chromatographic peak as a reference substance S peak, has 9 chromatographic peaks in total, and has uniform chromatographic peak distribution.
(4) The method adopts gradient elution to carry out determination by adopting the high performance liquid chromatography, and has the advantages of simple operation, good separation degree, high precision, good repeatability, reasonable detection time and the like.
(5) The characteristic spectrum of the herba cepbalanoplosis segeti formula particles constructed by the method is highly consistent with the characteristic spectrum of a reference medicinal material, and chromatographic peak information is complete.
Drawings
FIG. 1 is a graph of the UV absorption spectrum of a linarin control.
Fig. 2 is an ultraviolet absorption spectrum of an apigenin control.
Fig. 3 is an ultraviolet light absorption spectrum of a caffeic acid control.
FIG. 4 is the UV absorption spectrum of a chlorogenic acid control.
FIG. 5 is an ultraviolet absorption spectrum of a protocatechuic acid control.
FIG. 6 is a 3D chromatogram of standard decoction of Cirsium setosum.
FIG. 7 is a chromatogram of standard decoction of herba Cephalanoploris at different wavelengths.
FIG. 8 is a chromatogram for column temperature investigation.
FIG. 9 is a chromatogram for flow rate investigation.
Fig. 10 is a chromatogram for solvent extraction investigation.
FIG. 11 is a chromatogram for examination of the extraction method.
Fig. 12 is an extraction time survey chromatogram.
FIG. 13 is a chromatogram peak of a Cirsium setosum formula granule.
FIG. 14 is an instrumental survey chromatogram.
FIG. 15 is a chromatogram for column investigation.
Fig. 16 is a verification chart of the feature map of 3 batches of herba cepbalanoplosis segeti formula granules.
Figure 17 is a control profile of a cephalanoplos formulation granule.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
The following examples relate to the following test instruments and materials (all in mass concentration):
high performance liquid chromatograph: waters 2695-2996 high performance liquid chromatograph, Agilent 1260-type high performance liquid chromatograph and Shimadzu 20-AD high performance liquid chromatograph;
an electronic balance: ME204E/02, MS205DU, XP26 (Mettler-Tollido instruments, Inc.);
an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);
an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);
agilent extended-C18250 × 4.6.6 mm, Kromasil 100-5-C184.6 × 250mm, Phenomenex L una 5um C18(2)100A 4.6 × 250 mm.
The methanol and the acetonitrile are chromatographically pure, the water is ultrapure water, and the other reagents are analytically pure.
A linarin reference substance (China institute for testing and testing food and drug; batch No. 111528-201710, content is 96.6%),
chlorogenic acid reference substance (China institute for testing food and drug; batch No. 110753-201716, content in 99.3%),
apigenin reference (China institute for testing and testing food and drug; batch No.: 111901-201603, the content is 99.2%),
protocatechuic acid reference substance (China institute for testing and testing food and drug; batch No. 110809-,
caffeic acid control (China institute for testing and testing food and drug, batch No. 110885-201703, content is 99.7%).
Herba cepbalanoplosis segeti formula granules SY1809001, SY1809002 and SY 1809003.
Example 1:
the embodiment relates to a fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules.
Preparation of a test solution: taking 0.1g of herba Cirsii formulation granule powder, placing in a conical flask, adding 10ml of 40% methanol precisely, weighing, ultrasonically treating for 20min, with power of 600W and frequency of 25kHz, cooling to 5 deg.C, weighing again, supplementing the lost weight with the methanol, shaking, and filtering to obtain the test solution.
Preparation of reference solutions: taking a linarin reference substance, an apigenin reference substance, a caffeic acid reference substance, a chlorogenic acid reference substance and a protocatechuic acid reference substance respectively, precisely weighing, mixing, dissolving with 5% methanol by mass concentration, and making into solutions containing 10 μ g linarin, 10 μ g apigenin, 5 μ g caffeic acid, 5 μ g chlorogenic acid and 5 μ g protocatechuic acid in each 1ml as reference substance solutions.
And (3) high performance liquid chromatography detection: respectively and precisely sucking 10 mul of the test solution, the reference solution and the reference medicinal material solution, injecting into a high performance liquid chromatograph, and measuring by adopting gradient elution, wherein the detection conditions are as follows:
filling agent: octadecylsilane chemically bonded silica (column length 250mm, inner diameter 4.6mm, particle size 5 μm),
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent phosphoric acid solution, gradient elution is carried out,
temperature of the column: at a temperature of 30 c,
flow rate: 1.2ml/min of the mixture is added,
detection wavelength: 320 nm.
Gradient elution was as follows:
measuring according to the method to obtain the characteristic spectrum of the herba Cephalanoploris formula granules. The characteristic map shows 9 characteristic peaks, wherein the peak corresponding to the linarin reference is an S peak, and the relative retention time of each characteristic peak and the S peak is calculated and is within +/-8% of a specified value.
Example 2:
the embodiment relates to a fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules.
Preparation of a test solution: taking 0.3g of herba cepbalanoplosis segeti formula particle powder, placing the powder in an erlenmeyer flask, precisely adding 30ml of methanol with the mass concentration of 60%, weighing, carrying out ultrasonic treatment for 40min, carrying out power 600W and frequency 25kHz, cooling to 20 ℃, weighing again, supplementing the lost weight with the methanol, shaking up, and filtering to obtain a test solution.
Preparation of reference solutions: taking a linarin reference substance, an apigenin reference substance, a caffeic acid reference substance, a chlorogenic acid reference substance and a protocatechuic acid reference substance respectively, precisely weighing, mixing, dissolving with 100% methanol by mass concentration, and making into solutions containing 50 μ g linarin, 40 μ g apigenin, 50 μ g caffeic acid, 50 μ g chlorogenic acid and 50 μ g protocatechuic acid in each 1ml as reference substance solutions.
And (3) high performance liquid chromatography detection: respectively and precisely sucking 10 mul of the test solution, the reference solution and the reference medicinal material solution, injecting into a high performance liquid chromatograph, and measuring by adopting gradient elution, wherein the detection conditions are as follows:
filling agent: octadecylsilane chemically bonded silica (column length 250mm, inner diameter 4.6mm, particle size 5 μm),
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent phosphoric acid solution, gradient elution is carried out,
temperature of the column: at a temperature of 40 c,
flow rate: 0.8ml/min of the mixture is added,
detection wavelength: 336 nm.
Gradient elution was as follows:
measuring according to the method to obtain the characteristic spectrum of the herba Cephalanoploris formula granules. The characteristic map shows 9 characteristic peaks, wherein the peak corresponding to the linarin reference is an S peak, and the relative retention time of each characteristic peak and the S peak is calculated and is within +/-8% of a specified value.
Example 3:
the embodiment relates to a fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules.
Preparation of a test solution: taking 0.2 of herba Cirsii formulation particle powder, placing in a conical flask, adding 20ml of 50% methanol precisely, weighing, ultrasonically treating for 30min at a power of 600W and a frequency of 25kHz, cooling to 15 ℃, weighing again, supplementing the lost weight with the methanol, shaking up, and filtering to obtain the test solution.
Preparation of reference solutions: taking a linarin reference substance, an apigenin reference substance, a caffeic acid reference substance, a chlorogenic acid reference substance and a protocatechuic acid reference substance respectively, precisely weighing, mixing, dissolving with 50% methanol by mass concentration, and making into solutions containing 40 μ g linarin, 30 μ g apigenin, 40 μ g caffeic acid, 40 μ g chlorogenic acid and 50 μ g protocatechuic acid in each 1ml as reference substance solutions.
And (3) high performance liquid chromatography detection: respectively and precisely sucking 10 mul of the test solution, the reference solution and the reference medicinal material solution, injecting into a high performance liquid chromatograph, and measuring by adopting gradient elution, wherein the detection conditions are as follows:
filling agent: octadecylsilane chemically bonded silica (column length 250mm, inner diameter 4.6mm, particle size 5 μm),
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent phosphoric acid solution, gradient elution is carried out,
temperature of the column: at a temperature of 35 c,
flow rate: 1.0ml/min of the mixture is added,
detection wavelength: 330 nm.
Gradient elution was as follows:
measuring according to the method to obtain the characteristic spectrum of the herba Cephalanoploris formula granules. The characteristic map shows 9 characteristic peaks, wherein the peak corresponding to the linarin reference is an S peak, and the relative retention time of each characteristic peak and the S peak is calculated and is within +/-8% of a specified value.
Example 4: chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filler (the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 μm); acetonitrile is used as a mobile phase A; using 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the specification in the following table; the column temperature was 35 ℃; the flow rate is 1.0ml per minute, the detection wavelength is 330nm, and the number of theoretical plates is not less than 5000 calculated according to the linarin peak.
(one) wavelength selection
Respectively scanning the linarin reference substance, the apigenin reference substance, the caffeic acid reference substance, the chlorogenic acid reference substance, the protocatechuic acid reference substance and the test solution in full wave band by using a diode array detector, and respectively extracting chromatograms of the test solution at wavelengths of 280nm, 300nm, 330nm and 350 nm.
Fig. 1 to 5 are ultraviolet absorption spectra of a linarin reference, an apigenin reference, a caffeic acid reference, a chlorogenic acid reference and a protocatechuic acid reference, respectively, fig. 6 is a 3D chromatogram of a herba cepbalanoplosis segeti standard decoction, and fig. 7 is a chromatogram of different wavelengths of the herba cepbalanoplosis segeti standard decoction.
As a result, when the detection wavelength was 330nm, the amount of information on the chromatographic peak was large, and the base line of the chromatogram was more stable, so that the detection wavelength was determined to be 330 nm.
(II) column temperature examination
The column temperatures were 30 ℃, 35 ℃ and 40 ℃ respectively, and the results are shown in FIG. 8 and Table 1 below.
TABLE 1
The result shows that when the column temperature is 30-40 ℃, the chromatogram has symmetrical peak shapes and good separation degree, but has great influence on the relative retention time of the peak 2 and the peak 3, so the column temperature is selected to be 35 ℃.
(III) investigation of flow velocity
The flow rates were 0.8ml/min, 1.0ml/min, and 1.2ml/min, respectively, and were examined as shown in FIG. 9 and Table 2 below.
TABLE 2
The result shows that when the flow rates are respectively 0.8ml/min, 1.0ml/min and 1.2ml/min, the relative retention time RSD of each characteristic peak is 0-9.54%, and when the flow rate is 1.0ml/min, the chromatogram has good peak shape and better separation degree. Therefore, the flow rate was determined to be 1.0 ml/min.
Example 5: preparation of test solution and system applicability test
(first) examination of extraction solvent
The results of examining ethanol, 50% ethanol, 70% ethanol, water, 50% methanol, 70% methanol and methanol as extraction solvents respectively show that the chromatographic peak information obtained by using 50% methanol as the extraction solvent is large and the separation degree is good, so that the same content measurement is consistent by using 50% methanol as the extraction solvent, as shown in fig. 10.
(II) examination of extraction method
The reflux extraction and the ultrasonic extraction are considered respectively, the chromatographic peak information is approximately the same, and finally, the ultrasonic with simple operation is selected for extraction, which is shown in fig. 11.
And (3) refluxing and extracting: placing 0.2g of the small product powder in a conical flask, adding 20ml of 50% methanol precisely, weighing, heating and refluxing (water bath temperature: 80 deg.C) for 20min, weighing again, supplementing the reduced weight with 50% methanol, shaking, and filtering to obtain the test solution.
(III) examination of extraction time
Ultrasonic extraction is carried out for 20 minutes, 30 minutes and 40 minutes, and finally ultrasonic extraction is carried out for 30 minutes. See fig. 12.
Finally, the preparation method of the reference substance solution is determined:
taking 0.2g of the powder, placing the powder in a conical flask with a plug, precisely adding 20ml of 50% methanol, weighing, carrying out ultrasonic treatment (power 600W and frequency 25 kHz) for 20 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, and filtering to obtain a test solution.
Example 6: methodology investigation
Identification of chromatographic peaks
Preparation of a test solution: test solutions of the Cirsium setosum formulation were prepared according to the experimental conditions set forth in example 5.
Preparation of reference solutions: taking appropriate amount of linarin control, apigenin control, caffeic acid control, chlorogenic acid control, and protocatechuic acid control, precisely weighing, and adding 50% methanol to obtain solutions containing 40 μ g, 30 μ g, 40 μ g, and 50 μ g per 1 ml.
Preparation of reference drug solution: precisely weighing 1.0g of herba Cephalanoploris as reference material, placing in a conical flask, precisely adding 20ml of water, heating and refluxing for 30min, cooling, filtering, evaporating filtrate, precisely adding 10ml of 50% methanol into residue, ultrasonic treating (power 600W, frequency 25 kHz) for 20min, cooling, and filtering.
Preparation of negative control solution: negative control solution of herba Cirsii deficient formula granule was prepared according to the experimental conditions set forth above.
And (5) locating the peak of the characteristic diagram of the herba cepbalanoplosis segeti formula particles. See fig. 13.
(II) precision test
Continuously injecting sample solution of herba Cephalanoploris formula granule (batch number: SY 1809001) for 6 times (10 μ l each time) according to a proposed experimental method, and calculating retention time and peak area of each characteristic peak. See tables 3, 4 below.
TABLE 3
TABLE 4
The results show that the instrument is accurate.
(III) repeatability examination
6 parts of herba cepbalanoplosis segeti formula particles (batch number: SY 1809001) are precisely weighed, and are prepared and measured according to a proposed experimental method. See tables 5, 6 below.
TABLE 5
TABLE 6
The results show that the method has good repeatability.
(IV) intermediate precision considerations
Investigation with different instruments: based on the experimental conditions, two parts of herba Cephalanoploris standard decoction (batch number: SY 1809001) are precisely weighed respectively to prepare test solution. The measurement was carried out on Shimadzu 20-AD, Waters 2695-2996 type, and Agilent 1260 type HPLC chromatographs, respectively. See fig. 14 and tables 7 and 8 below.
TABLE 7
TABLE 8
The results show that the RSD of each characteristic peak relative retention time is less than 1.16 percent when the test sample is detected by the 3 instruments.
Different personnel and time surveys: based on the experimental conditions, two parts of standard herba Cephalanoploris decoction (lot number: SY 1809001) are precisely weighed by different persons (A, B) at different times (T1 and T2) respectively to prepare test samples for determination. See tables 9, 10 below.
TABLE 9
The result shows that different people can determine the same batch number at different times, and the method has better stability.
(V) durability examination
Durability test of chromatographic column Agilent TC-C18250 × 4.6.6 mm, Kromasil 100-5-C184.6 × 250mm, Shimadzu 5um C184.6 × 250mm, Phenomenex L una 5um C18(2)100A 4.6 × 250mm were respectively tested on the basis of the experimental conditions set forth above, see FIG. 15, and tables 11 and 12 below.
TABLE 11
TABLE 12
The results show that the RSD of the characteristic peak relative to the retention time is 1.20-7.97% when the sample is detected by the 3 chromatographic columns.
And (3) stability investigation: based on the experimental conditions, the same test solution is taken and respectively measured at 0h, 3h, 6h, 9h, 15h and 24 h. See tables 13, 14 below.
TABLE 14
The result shows that the RSD of the retention time of the corresponding characteristic peak is 0.40-1.24%, and the sample solution is stable within 24 hours.
In summary, the RSD of each characteristic peak relative retention time meets the requirements in the above studies, and the method is good. The above 9 characteristic peaks were included in the subsequent examination.
(VI) determining characteristic peaks and establishing a contrast map
Verification results of 3 batches of herba cepbalanoplosis segeti formula granules:
and (3) measuring the characteristic spectrum of the 3 batches of samples of the product by a drawn-up method, and calculating the relative retention time and the relative peak area. See fig. 16, and table 15, below 16.
TABLE 16
According to the principle that the relative retention time is stable, samples of each batch can be detected, and the peaks are relatively high, 9 peaks with good repeatability are selected as characteristic peaks. The result shows that when the peak 4 is taken as an S peak, the relative retention time RSD of the characteristic peak of the 3 batches of herba cepbalanoplosis segeti formula particles is 0.07% -0.18%, and the relative retention time RSD of the 8 characteristic peaks of the 3 batches of herba cepbalanoplosis segeti formula particles is less than 3%.
Making a limit of a specified value of the relative retention time:
the methodology for each examined item and the validation results are summarized in tables 17 and 18 below.
TABLE 17
From the above table, it is clear that the instrument and column durability have a large effect on peak 2, and the specified relative retention time of each peak is temporarily set to ± 8% in order to increase the reproducibility and applicability of the method.
Finally, the following steps are provided: the test sample characteristic map should present 9 characteristic peaks, wherein the peak corresponding to the linarin reference is the S peak. The relative retention time of each characteristic peak to the S peak is calculated and should be within ± 8% of the specified value. The specified values are: 0.176 (peak 1), 0.332 (peak 2), 0.355 (peak 3), 0.422 (peak 4), 0.624 (peak 5), 0.861 (peak 6), 1.000 (peak 7 (S)), 1.168 (peak 8), and 1.428 (peak 9).
A traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition) is adopted to synthesize 3 batches of herba cepbalanoplosis segeti formula granules, and a control map of a herba cepbalanoplosis segeti formula granule characteristic map is established, which is shown in fig. 17.
In fig. 17, the peak 1: protocatechuic acid, peak 2: chlorogenic acid, peak 4: caffeic acid, peak 7 (S): linarin, peak 9: apigenin.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Claims (6)
1. A fingerprint spectrogram quality determination method for herba Cephalanoploris formula granules is characterized by comprising the following steps: the method comprises the following steps:
a: dissolving herba Cephalanoploris granule in methanol, ultrasonic treating, cooling, and filtering to obtain test solution;
b: mixing linarin control, apigenin control, caffeic acid control, chlorogenic acid control, and protocatechuic acid control, and dissolving with methanol to obtain reference solution;
c: and (3) determination: and respectively sucking the test solution and the reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph, and measuring by adopting gradient elution to obtain a fingerprint spectrogram of the herba cepbalanoplosis segeti formula particles.
2. The fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules according to claim 1, characterized in that: in the step A, 0.1-0.3 g of herba cepbalanoplosis segeti formula particle powder is added into 10-30 ml of 40-60% methanol by mass concentration, ultrasonic treatment is carried out for 20-40 min, cooling is carried out to 5-20 ℃, and a test solution is prepared after filtration.
3. The fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules according to claim 1, characterized in that: in the step B, the mass concentration of the methanol is 5-100%, and each 1ml of reference substance solution contains 10-50 μ g of linarin, 10-40 μ g of apigenin, 5-50 μ g of caffeic acid, 5-50 μ g of chlorogenic acid and 5-50 μ g of protocatechuic acid.
4. The fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules according to claim 1, characterized in that: in the step C, the detection conditions of the high performance liquid chromatograph meet the following conditions:
filling agent: octadecylsilane chemically bonded silica gel, and a silane,
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent phosphoric acid solution, gradient elution is carried out,
temperature of the column: 30-40 ℃,
flow rate: 0.8 to 1.2ml/min,
detection wavelength: 320-336 nm.
5. The fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules according to claim 4, characterized in that: the gradient elutes as follows:
0-10 min: 10% mobile phase A, 90% mobile phase B,
10-20 min: 10 to 15% of mobile phase A, 90 to 85% of mobile phase B,
20-55 min: 15-40% of mobile phase A, 85-60% of mobile phase B,
55-60 min: 40 to 50% of mobile phase A, 60 to 50% of mobile phase B,
60-80 min: 50-75% of mobile phase A and 50-25% of mobile phase B.
6. The fingerprint spectrum quality determination method for herba cepbalanoplosis segeti formula granules according to claim 1, characterized in that: the fingerprint spectrum of the herba cepbalanoplosis segeti formula particle has 9 characteristic peaks, the linarin chromatographic peak is taken as a reference substance S peak, and the relative retention time of the 9 characteristic peaks is respectively as follows: the relative retention time of the characteristic peaks is within. + -. 8% of the specified value, namely 0.176 for peak 1, 0.332 for peak 2, 0.355 for peak 3, 0.422 (S peak) for peak 4, 0.624 for peak 5, 0.861 for peak 6, 1.000 for peak 7, 1.168 for peak 8 and 2.546 for peak 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105241997A (en) * | 2015-11-10 | 2016-01-13 | 广东一方制药有限公司 | Thin-layer chromatography identification method of carbon traditional Chinese medicine formula granules |
| CN105467059A (en) * | 2015-12-30 | 2016-04-06 | 云南理想药业有限公司 | Quality detecting method for traditional Chinese medicine composition for treating hematuresis |
| CN107991425A (en) * | 2017-12-07 | 2018-05-04 | 长春人民药业集团有限公司 | A kind of detection method for the Chinese medicine composition for treating traumatic injury |
-
2020
- 2020-05-08 CN CN202010382201.8A patent/CN111505156B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105241997A (en) * | 2015-11-10 | 2016-01-13 | 广东一方制药有限公司 | Thin-layer chromatography identification method of carbon traditional Chinese medicine formula granules |
| CN105467059A (en) * | 2015-12-30 | 2016-04-06 | 云南理想药业有限公司 | Quality detecting method for traditional Chinese medicine composition for treating hematuresis |
| CN107991425A (en) * | 2017-12-07 | 2018-05-04 | 长春人民药业集团有限公司 | A kind of detection method for the Chinese medicine composition for treating traumatic injury |
Non-Patent Citations (3)
| Title |
|---|
| YANHUA LU等: "Chemical Fingerprint and Quantitative Analysis of Cirsium setosum by LC", 《CHROMATOGRAPHIA》 * |
| 方红玫等: "HPLC-DAD法同时测定小蓟配方颗粒中5种活性成分的含量", 《中国药师》 * |
| 陈毓等: "小蓟及小蓟炭指纹图谱的初步研究", 《上海中医药大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114487135A (en) * | 2020-10-26 | 2022-05-13 | 四川新绿色药业科技发展有限公司 | High performance liquid detection method and identification method for herba cepbalanoplosis segeti and herba cepbalanoplosis segeti decoction pieces, standard decoction and formula granules |
| CN114487135B (en) * | 2020-10-26 | 2023-10-03 | 四川新绿色药业科技发展有限公司 | High-efficiency liquid phase detection method and identification method of common cephalanoplos herb and common cephalanoplos herb carbon decoction pieces, standard decoction and formula particles |
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