CN109799306B - High performance liquid chromatography method for detecting characteristic spectrums of fried cowherb seed decoction pieces, standard decoction and formula granules - Google Patents
High performance liquid chromatography method for detecting characteristic spectrums of fried cowherb seed decoction pieces, standard decoction and formula granules Download PDFInfo
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Abstract
The invention provides a high performance liquid chromatography characteristic spectrum detection method suitable for integrally controlling the quality of fried cowherb seed decoction pieces, standard decoction and formula granules. The HPLC characteristic spectrum detection method can integrally control the characteristic components in the fried cowherb seed decoction pieces, the standard decoction and the formula granules, ensures the stability of the integral quality of the fried cowherb seed decoction pieces, the standard decoction and the formula granules, and has the advantages of simple operation, high precision, good stability, good repeatability and high accuracy.
Description
Technical Field
The invention particularly relates to an HPLC detection method for characteristic spectrums of fried cowherb seed decoction pieces, standard decoction and formula granules.
Background
The cowherb seed is originally recorded in Shen nong Ben Cao Jing, is a main drug for gynecology and surgery, has wide clinical application, has the functions of activating blood and stimulating the menstrual flow, promoting lactation and reducing swelling, and inducing diuresis and treating stranguria, mainly treats amenorrhea, dysmenorrhea, galactopoiesis, acute mastitis and painful stranguria, and has obvious curative effect. However, since the seed medicine is hard and hard to be decocted to obtain effective components, it is commonly used as a processed product. The processing method of the cowherb seed in the past includes steaming, steaming with wine, stir-frying with charcoal, stir-frying with wine and the like, and the stir-frying method is used more at present. The fried cowherb seed is a main processed product of the cowherb seed, and compared with a raw product of the cowherb seed, the cowherb seed has the advantages of easier decoction of effective components, stronger dispersing power and better effects of activating blood and stimulating the menstrual flow, promoting lactation and treating stranguria.
At present, under the item of frying the cowherb seeds in the first part of the 'Chinese pharmacopoeia' 2015 edition, only a method for measuring the content of a single component of the cowherb seed flavonoid glycoside by adopting a high performance liquid chromatography is adopted, and the characteristic map control of the cowherb seed flavonoid glycoside is not carried out. Other partial literature reports establish the fried cowherb seed decoction pieces and the composition and content change before and after processing, such as Zhou national flood, research on the influence of the chemical composition and processing of the fried cowherb seed [ D ] Chinese academy of traditional Chinese medicine, 2016, 5; high performance liquid fingerprint chromatogram research of Chenlin, Huchangjiang, Fengjian, etc. formula particles of fried cowherb seed [ J ] Asia Pacific traditional medicine, 2013,9(4): 16-18; exemplary Huangyi, Ouyang, Xiaozhaoqi, etc. HPLC finger print and content determination research of parched semen Vaccariae in different producing areas [ J ] Zhongnan pharmacy, 2017(7): 879-.
However, the above reports only aim at the fingerprint spectrum research established before and after the processing of the fried cowherb seed decoction pieces and the fried cowherb seed formula granules, and the report of the standard decoction is not found. Therefore, the existing detection method is difficult to effectively compare and analyze the difference and the change of the characteristic maps of the fried cowherb seed decoction pieces, the standard decoction and the formula granules, the transmission condition of the drug effect substance basis of the fried cowherb seed in the technical process from the raw materials to the formula granule finished product is difficult to deeply know, and the technical process from the fried cowherb seed decoction pieces to the formula granule finished product is difficult to integrally evaluate and control. Therefore, the establishment of the unified determination method for the characteristic spectrums of the fried cowherb seed decoction pieces, the standard decoction and the formula granules is beneficial to the overall evaluation of the scientificity and rationality of the relevant technological process of the fried cowherb seed, and the internal quality of the fried cowherb seed decoction pieces, the standard decoction and the formula granules can be controlled more integrally, thereby ensuring the clinical curative effect of the fried cowherb seed.
Disclosure of Invention
In order to solve the problems, the invention provides an HPLC detection method suitable for determining the characteristic spectrums of the fried cowherb seed decoction pieces, the standard decoction and the formula granules.
The invention relates to a high performance liquid chromatography characteristic spectrum detection method for integrally controlling fried cowherb seed decoction pieces, standard decoction and formula granules, which comprises the following operation steps:
1) preparation of a reference solution: dissolving the reference substance to obtain reference substance solution;
2) preparing a reference decoction piece solution: extracting control decoction pieces of parched semen Vaccariae to obtain control medicinal solution;
3) preparing a test solution: extracting parched semen Vaccariae decoction pieces, standard decoction, and formula granule to obtain test solution;
3) respectively sucking the reference solution, the reference decoction piece solution and the sample solution, injecting into a high performance liquid chromatograph under the following chromatographic conditions:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 240-270 nm; mobile phase: acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, the gradient elution procedure was as follows:
wherein, the reference substance in the step 1) comprises: eight reference substances including 5-hydroxymethyl furfural, cowherb seed flavonoid glycoside, protocatechuic acid, erythrine, saporin, cowherb seed cyclic peptide A, cowherb seed cyclic peptide B and isovitexin-2' -O-arabinoside.
Wherein, the dissolving in the step 1) is dissolving by adding methanol or 70% methanol solution.
Wherein, the extraction in the step 2) refers to: adding 70% methanol solution, and ultrasonic extracting.
Wherein, the extraction in the step 3) refers to: adding 70% methanol solution, and ultrasonic extracting.
Preferably, the extraction method is as follows:
adding 10 times of 70% methanol into parched semen Vaccariae decoction pieces, or adding 100 times of 70% methanol into parched semen Vaccariae standard decoction powder, or grinding parched semen Vaccariae formula granule into fine powder, and adding 50 times of 70% methanol; ultrasonic extracting for 30min, cooling, adding 70% methanol to make up the lost weight, shaking, filtering, and collecting the filtrate to obtain the sample solution of parched semen Vaccariae decoction pieces, standard decoction or formula granule; the power of the ultrasonic extraction is 600W, and the frequency is 40 kHz.
Wherein the chromatographic column is ZORBAX SB-Aq C18 chromatographic column with specification of 4.6mm × 250mm and 5 μm.
Wherein the detection wavelength is 270 nm.
Wherein the chromatographic conditions further comprise: the flow rate was 1.0mL/min, the column temperature was 30 ℃ and the amount of sample was 10. mu.L.
The detection method of the high performance liquid chromatography characteristic spectrum of the fried cowherb seed decoction pieces, the standard decoction and the formula granules can integrally control the characteristic components in the fried cowherb seed decoction pieces, the standard decoction and the formula granules, ensure the stability of the integral quality of the fried cowherb seed decoction pieces, the standard decoction and the formula granules, and has the advantages of simple operation, high precision, good stability, good repeatability and high accuracy.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Fig. 1 shows a contrast map of decoction pieces of cowherb seed, peak 2: 5-hydroxymethylfurfural; peak 4: protocatechuic acid; peak 5: erythrine; peak 6 (S): cowherb seed flavonoid glycoside; peak 7: saponin of saponarin; peak 9: isovitexin-2 "-O-arabinoside; peak 12: vaccaria cypeptide B; peak 13: and (3) the vaccaria segetalis cyclic peptide A.
Fig. 2 shows a contrast map of the standard decoction of the fried cowherb seed, peak 2: 5-hydroxymethylfurfural; peak 4: protocatechuic acid; peak 5: erythrine; peak 6 (S): cowherb seed flavonoid glycoside; peak 7: saponin of saponarin; peak 9: isovitexin-2 "-O-arabinoside; peak 12: vaccaria cypeptide B; peak 13: and (3) the vaccaria segetalis cyclic peptide A.
Fig. 3 shows a contrast map of the formula granules of the fried cowherb seed, peak 2: 5-hydroxymethylfurfural; peak 4: protocatechuic acid; peak 5: erythrine; peak 6 (S): cowherb seed flavonoid glycoside; peak 7: saponin of saponarin; peak 9: isovitexin-2 "-O-arabinoside; peak 12: vaccaria cypeptide B; peak 13: and (3) the vaccaria segetalis cyclic peptide A.
FIG. 4 is a superposition diagram of the comparative characteristic spectra of decoction pieces, standard decoction and formula granules, peak 2: 5-hydroxymethylfurfural; peak 4: protocatechuic acid; peak 5: erythrine; peak 6 (S): cowherb seed flavonoid glycoside; peak 7: saponin of saponarin; peak 9: isovitexin-2 "-O-arabinoside; peak 12: vaccaria cypeptide B; peak 13: and (3) the vaccaria segetalis cyclic peptide A.
Detailed Description
Instrument and reagent
1 apparatus
High performance liquid chromatograph: agilent 1260 type HPLC, waters e2695 type HPLC, Shimadzu 30AD type HPLC;
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: KQ-600DB model (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);
a chromatographic column: ZORBAX Eclipse XDB-C18Analytical 4.6X 250mm 5-Micron, Diamonsil 5. mu. m C18(2) 250X 4.6mm, Shim-pack GIST 5. mu. m C184.6.6X 250 mm.
2 reagent
Acetonitrile and phosphoric acid are chromatographically pure, water is ultrapure water, and other reagents are analytically pure.
5-hydroxymethylfurfural (Dopperphy Biotechnology, Inc., lot number 17011104); cowherb seed flavonoid glycoside (China institute for testing and testing food and drug; lot number: 111853) -201704, content is 96.9%); protocatechuic acid (China institute for testing and testing food and drug; lot number 110809-; erythrine (Szechwan, Vickqi Biotech Co., Ltd., Sichuan province, lot number: wkq18012302, content is more than or equal to 98%); saponin (vickers biotechnology limited, sikawa, batch No.:
wkq 18011711); vaccaria segetalis cyclic peptide A (Dowman der Biotech, Inc., lot number: MUST-18033102, content 99.24%); vaccaria segetalis cyclic peptide B (Dowman Stokes Biotech, Inc., lot number: MUST-18051007, content 99.6%); isovitexin-2 "-O-arabinoside (Sichuan Weickqi Biotech limited, batch No. wkq18010308, content ≥ 98%); semen Vaccariae preparata reference decoction pieces (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd.).
Frying cowherb seed decoction pieces: (batch number: CWBLX180601, CWBLX180602, CWBLX180603, CWBLX180604, CWBLX180606, CWBLX180607, CWBLX180615, CWBLX180616, CWBLX180617, CWBLX180618, CWBLX180619, CWBLX180620, CWBLX180621, CWBLX180622, CWBLX180623, CWBLX180624, CWBLX180625, CWBX 180626, CWBLX180627, CWBLX180628, CWBLX180629, CWBLX180630)
Frying standard decoction of cowherb seed: (Sichuan New Green pharmaceutical science and technology Co., Ltd., batch No.: CWBBLXBT 180601, CWBBLXBT 180602, CWBBLXBT 180603, CWBBLXBT 180604, CWBBLXBT 180606, CWBXBT 180607, CWBXBT 180615, CWBBLXBT 180616, CWBBLXBT 180617, CWBXBT 180618, CWBXBT 180619, CWBXBT 180620, CWBXBT 180621, CWBXBT 180622, CWBXBT 623 180180180180180180180180180624, CWBXBT 180624, CWBXBT 180625, CWBXBT 180626, CWBBT 180627, CWBXBT 180628, CWBXBT 180629, CWBBT 180630)
The formula particle of the fried cowherb seed comprises the following components: (Sichuan New Green pharmaceutical science and technology development Co., Ltd., lot Nos. SY1806001, SY1806002, SY 1806003).
Example 1 the detection method of the invention is used for detecting the high performance liquid characteristic spectrum of the standard decoction of the fried cowherb seed
1 control reference solution preparation: taking appropriate amount of cowherb seed flavonoid glycoside reference substance, erythrina indica alkali reference substance and sapogenin reference substance, precisely weighing, and adding 70% methanol to obtain solution containing cowherb seed flavonoid glycoside 0.1mg, erythrina indica alkali 60 μ g and sapogenin 50 μ g per 1 ml. And adding appropriate amount of 5-hydroxymethylfurfural reference substance, protocatechuic acid reference substance, cowherb seed cyclic peptide A reference substance, cowherb seed cyclic peptide B reference substance and isovitexin-2 '-O-arabinoside reference substance into methanol to obtain solution containing 5-hydroxymethylfurfural 20 μ g, protocatechuic acid 60 μ g, cowherb seed cyclic peptide A60 μ g, cowherb seed cyclic peptide B60 μ g and isovitexin-2' -O-arabinoside 30 μ g per 1ml, to obtain the final product.
2, preparing reference solution of reference decoction pieces: precisely weighing 2.5g of parched semen Vaccariae control decoction pieces, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing the plug, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 30min, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking, filtering, and collecting the subsequent filtrate.
3, preparation of a test solution: taking 0.5g of fried cowherb seed standard decoction, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, sealing the plug, weighing, performing ultrasonic treatment (power 600W and frequency 40kHz) for 20 minutes, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
4, detection: precisely sucking 10 μ l of each of the reference solution, reference decoction piece solution and sample solution, injecting into liquid chromatograph, and measuring.
Octadecylsilane chemically bonded silica was used as a filler (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 270 nm. The number of theoretical plates is not less than 3000 calculated according to the flavonoid glycoside peak of cowherb seed.
5, results: 13 characteristic peaks are presented in the characteristic map of the test sample and correspond to the retention time of 13 characteristic peaks in the reference chromatogram of the reference decoction piece, wherein 8 peaks are respectively the same as the retention time of the corresponding reference peaks, the peak corresponding to the reference peak of the cowherb seed flavonoid glycoside is an S peak, the relative retention time of the other characteristic peaks and the S peak is calculated except for the peak 2, the peak 4, the peak 5, the peak 12 and the peak 13, wherein the relative retention time of the peak 1 and the peak 3 is within +/-10% of a specified value, and the relative retention time of the peaks 6-11 is within +/-5% of the specified value. The specified values are: 0.217 (peak 1), 0.320 (peak 3), 1.000 (peak 6S), 1.051 (peak 7), 1.142 (peak 8), 1.206 (peak 9), 1.429 (peak 10), 1.501 (peak 11).
Example 2 the detection method of the invention is used for detecting the high performance liquid characteristic spectrum of the fried cowherb seed formula particle
1 control reference solution preparation: the preparation method is the same as that of example 1.
2, preparing reference solution of reference decoction pieces: the preparation method is the same as that of example 1.
3, preparation of a test solution: taking a proper amount of the formula particles of the fried cowherb seeds, grinding, taking 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, sealing the plug, weighing, ultrasonically treating (with the power of 600W and the frequency of 40kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the cowherb seed powder.
4, detection: respectively sucking 10 μ L of reference solution of control, reference solution of control decoction pieces and sample solution, and injecting into high performance liquid chromatograph under the following chromatographic conditions:
octadecylsilane chemically bonded silica was used as a filler (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm); acetonitrile is used as a mobile phase A, 0.1 percent phosphoric acid solution is used as a mobile phase B, and the gradient elution procedure is the same as that of the example 1; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 270 nm. The number of theoretical plates is not less than 3000 calculated according to the flavonoid glycoside peak of cowherb seed.
5, results: 13 characteristic peaks are presented in the characteristic map of the test sample and correspond to the retention time of 13 characteristic peaks in the reference chromatogram of the reference decoction piece, wherein 8 peaks are respectively the same as the retention time of the corresponding reference peaks, the peak corresponding to the reference peak of the cowherb seed flavonoid glycoside is an S peak, the relative retention time of the other characteristic peaks and the S peak is calculated except for the peak 2, the peak 4, the peak 5, the peak 12 and the peak 13, wherein the relative retention time of the peak 1 and the peak 3 is within +/-10% of a specified value, and the relative retention time of the peaks 6-11 is within +/-5% of the specified value. The specified values are: 0.217 (peak 1), 0.320 (peak 3), 1.000 (peak 6S), 1.051 (peak 7), 1.142 (peak 8), 1.206 (peak 9), 1.429 (peak 10), 1.501 (peak 11).
Embodiment 3 the detection method of the invention is used for detecting the high performance liquid characteristic spectrum of the fried cowherb seed decoction pieces
1 control reference solution preparation: the preparation method is the same as that of example 1.
2, preparing reference solution of reference decoction pieces: the preparation method is the same as that of example 1.
3, preparation of a test solution: precisely weighing 2.5g of parched semen Vaccariae decoction pieces, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing the plug, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 30min, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking, filtering, and collecting the subsequent filtrate.
4, detection: respectively sucking 10 μ L of reference solution of control, reference solution of control decoction pieces and sample solution, and injecting into high performance liquid chromatograph under the following chromatographic conditions:
octadecylsilane chemically bonded silica was used as a filler (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm); acetonitrile is used as a mobile phase A, 0.1 percent phosphoric acid solution is used as a mobile phase B, and the gradient elution procedure is the same as that of the example 1; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 270 nm. The number of theoretical plates is not less than 3000 calculated according to the flavonoid glycoside peak of cowherb seed.
5, results: 13 characteristic peaks are presented in the characteristic map of the test sample and correspond to the retention time of 13 characteristic peaks in the reference chromatogram of the reference decoction piece, wherein 8 peaks are respectively the same as the retention time of the corresponding reference peaks, the peak corresponding to the reference peak of the cowherb seed flavonoid glycoside is an S peak, the relative retention time of the other characteristic peaks and the S peak is calculated except for the peak 2, the peak 4, the peak 5, the peak 12 and the peak 13, wherein the relative retention time of the peak 1 and the peak 3 is within +/-10% of a specified value, and the relative retention time of the peaks 6-11 is within +/-5% of the specified value. The specified values are: 0.217 (peak 1), 0.320 (peak 3), 1.000 (peak 6S), 1.051 (peak 7), 1.142 (peak 8), 1.206 (peak 9), 1.429 (peak 10), 1.501 (peak 11).
Experimental example 1 chromatographic conditions and System suitability test
Selecting a mobile phase: the separation effect of 3 different mobile phases is examined, which respectively comprises the following steps: (1) gradient elution with acetonitrile-0.1% formic acid; (2) gradient elution with acetonitrile-0.1% formic acid; (3) acetonitrile-0.1% phosphoric acid gradient elution. The result shows that the chromatographic peak separation degree, the number of theoretical plates and the symmetry are better under the condition of acetonitrile-0.1 percent phosphoric acid gradient elution, so that the acetonitrile-0.1 percent phosphoric acid gradient elution is used as a mobile phase of the determination method of the characteristic spectrum of the fried cowherb seed.
Wavelength selection: on the basis of the experimental conditions, a diode array detector is utilized to respectively carry out full-band scanning on a 5-hydroxymethylfurfural reference solution, a cowherb seed flavonoid glycoside reference solution, a cowherb seed cyclic peptide A reference solution, a cowherb seed cyclic peptide B reference solution, an erythrine reference solution, a saponin reference solution, an isovitexin-2' -O-arabinoside reference solution, a protocatechuic acid reference solution and a sample solution, and chromatograms of a comparison sample at wavelengths of 250nm, 260nm, 270nm and 280nm show that the peak shapes and the symmetry of various chromatograms are better at a detection wavelength of 270nm, the information content of the whole chromatogram is larger, and the detection wavelength is determined to be 270 nm. Is consistent with the characteristic spectrum method of the standard decoction of the fried cowherb seed.
Column temperature investigation: based on the experimental conditions set forth above, the column temperatures were examined at 25 ℃, 30 ℃ and 35 ℃. The results show that under the three column temperature conditions, all the spectral peaks can be well separated, so the subsequent investigation is carried out by referring to the characteristic spectrum method of the standard decoction of the roasted cowherb seed, and the provisional column temperature is 30 ℃.
And (3) flow rate investigation: on the basis of the experimental conditions set forth above, the flow rates were examined at 0.8ml/min, 1ml/min, and 1.2ml/min, respectively. The result shows that under the condition of three flow rates, the chromatographic peaks can be well separated, so that the subsequent investigation is carried out by referring to the characteristic spectrum method of the standard decoction of the roasted cowherb seed, wherein the provisional flow rate is 1.0 ml/min.
Delayed test
On the basis of the experimental conditions formulated above, the chromatogram acquisition time was extended to 120 min.
The result shows that after the chromatogram is collected for 60 minutes, only one chromatographic peak appears in about 74 minutes, and the peak is not caused by the test sample after being confirmed by inserting a blank needle under the same condition. Therefore, when the chromatogram is collected for 60 minutes, the chromatographic peak is completely collected. The chromatogram acquisition time was determined to be 60 minutes.
In conclusion, the chromatographic conditions and the systematic adaptability test of the characteristic spectrum of the fried cowherb seed formula particles are determined as follows: octadecylsilane chemically bonded silica was used as a filler (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 270 nm. The number of theoretical plates is not less than 3000 calculated according to the flavonoid glycoside peak of cowherb seed. See table 9.
Table 9 set forth mobile phase gradients
Experimental example 2 preparation examination of test solution
Investigation of the extraction method: precisely weighing about 1.0g of the product (lot No. 1804039), placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, sealing the plug, weighing, examining the extraction method of the sample by reflux and ultrasound respectively, extracting for 30min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate.
The results show that the test samples have consistent effects when being subjected to ultrasonic extraction and reflux extraction respectively. Because the ultrasonic extraction operation is simpler and more convenient, the method for extracting the test sample is determined to be ultrasonic extraction.
Investigation of extraction solvent: precisely weighing about 1.0g of the product (lot No. 1804039), placing in a conical flask with a stopper, respectively examining the extraction solvents of the sample, namely methanol, 70% methanol and water, wherein the addition amount of the solvent is 50ml, sealing the stopper, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss by the extraction solvent, shaking up, filtering, and taking the subsequent filtrate. The results show that the extraction effect of water and 70% methanol is good, no obvious difference exists, and the tentative extraction solvent is 70% methanol.
And (3) extracting time investigation: precisely weighing about 1.0g of the product (batch No. 1804039), placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, sealing the plug, weighing, performing ultrasonic treatment (power 600W and frequency 40kHz), respectively examining the extraction time of the sample at 20min, 30min and 40 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate.
The results show that there is no significant difference when the samples are processed at the three extraction times, and the provisional sample extraction time is determined to be 30 minutes.
In conclusion, the preparation method of the test solution of the characteristic spectrum of the fried cowherb seed formula particles is determined as follows: taking a proper amount of the product, grinding, precisely weighing about 1.0g, placing into a conical flask with a plug, precisely adding 50ml of 70% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 600W and frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
EXAMPLE 3 precision test
Taking about 1.0g of parched semen Vaccariae formula granule (batch number: 1804039), precisely weighing 1 part, preparing and measuring according to a proposed experimental method, and continuously feeding sample for 6 times, 10 μ l each time, and measuring. The result shows that the RSD of the retention time of each characteristic peak is 0.01-0.21%, the RSD of the relative peak area of each characteristic peak is 0.05-1.88%, and the precision of the instrument is good.
Experimental example 4 repeatability test
6 parts of the fried cowherb seed formula particles (batch number: 1804039) are precisely weighed, and are prepared and measured according to a formulated experimental method. The result shows that the RSD of the relative retention time of each characteristic peak is 0.02-1.11%, and the RSD of the relative peak area of each characteristic peak is 0.51-5.82%. The method has good repeatability. The precision of the instrument is good.
Experimental example 5 investigation of column durability
On the basis of the experimental conditions thus prepared, the columns were ZORBAX eclipseXDB-C18Analytical 4.6X 250mm 5-Micron (Agilent column), Diamonsil 5. mu.mC 18(2) 250X 4.6mm (Diima column), and Shim-pack GIST 5. mu. m C184.6.6X 250mm (Shimadzu column), respectively. The results show that the RSD of each characteristic peak relative retention time is 0.11-6.11% when the 3 chromatographic columns are used for detecting the sample; the RSD of the relative peak area of each characteristic peak is 1.66-46.40%.
EXAMPLE 6 stability test
Based on the experimental conditions, the same test solution is taken and respectively measured for 0h, 2h, 4h, 8h, 16h and 24 h. The result shows that the RSD of the retention time of each characteristic peak is 0.09-2.05%, and the RSD of the peak area of each characteristic peak is 0.07-8.28%. The method has good stability of the test sample within 24 hours.
Experimental example 7 establishment of control map of sample of parched semen Vaccariae
1. Establishment of characteristic spectrum of fried cowherb seed decoction pieces
The final analysis method is adopted to determine characteristic map of the product 22 batches of parched semen Vaccariae decoction pieces (Sichuan new green pharmaceutical technology development Co., Ltd., batch number: CWBXBT 180601, CWBXBT 180602, CWBXBT 180603, CWBXBT 180604, CWBXBT 180606, CWBXBT 180607, CWBXBT 180615, CWBXBT 180616, CWBXBT 180617, CWBXBT 180618, CWBXBT 180619, CWBXBT 180620, CWBXBT 180621, CWBXBT 180622, CWBXBT 180623, CWBXBT 180624, CWBXBT 180625, CXBT 180626, CWBXBT 180627, CWBT 180628, CWB 180629, CWBXBT 180630) and calculate relative retention time and relative peak area. See fig. 2.
According to the principle that the relative retention time is stable, samples of each batch can be detected, and the peaks are relatively high, 13 peaks with good repeatability are selected as characteristic peaks. The result shows that when the peak 6 is taken as the S peak, the relative retention time RSD of the characteristic peak of the standard decoction of the 22 batches of the stir-fried cowherb seed is less than 2.0 percent. Finally, the following steps are provided: 13 characteristic peaks are presented in a characteristic spectrum of a roasted cowherb seed standard decoction sample and correspond to the retention time of 13 characteristic peaks in a reference substance chromatogram of a reference substance of a reference decoction piece, wherein 8 peaks are respectively the same as the retention time of the corresponding reference substance peak, the peak corresponding to the cowherb seed flavonoid glycoside reference substance peak is an S peak, the relative retention time of each of the rest characteristic peaks and the S peak is calculated except for the peak 2, the peak 4, the peak 5, the peak 12 and the peak 13, wherein the relative retention time of the peak 1 and the peak 3 is within +/-10% of a specified value, and the relative retention time of the peaks 6-11 is within +/-5% of the specified value. The specified values are: 0.217 (peak 1), 0.320 (peak 3), 1.000 (peak 6S), 1.051 (peak 7), 1.142 (peak 8), 1.206 (peak 9), 1.429 (peak 10), 1.501 (peak 11). The 22 batches of the fried cowherb decoction pieces are synthesized by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition), a comparison atlas of the feature atlas of the fried cowherb decoction pieces is established, and the established feature atlas detection method can more accurately and integrally control the quality of the fried cowherb decoction pieces.
2. Establishment of characteristic spectrum of standard decoction of parched semen Vaccariae
The final analysis method is adopted to determine characteristic map of the product 22 batches of the standard decoction of parched semen Vaccariae (Sichuan new Green pharmaceutical technology development Co., Ltd., batch No.: CWBBLXBT 180601, CWBBLXBT 180602, CWBXBT 180603, CWBBLXBT 180604, CWBXBT 180606, CWBXBT 180607, CWBXBT 180615, CWBXBT 180616, CWBXBT 180617, CWBXBT 180618, CWBXBT 180619, CWBXBT 180620, CWBXBT 180621, CWBXBT 180622, CWBXBT 180623, CWBBT 180624, CWBXBT 180625, CWBBT 180626, CWBXBT 180627, CWBBT 180628, CWBXBT 180629, CWBXBT 180630) and calculate relative retention time and relative peak area. See fig. 2.
According to the principle that the relative retention time is stable, samples of each batch can be detected, and the peaks are relatively high, 13 peaks with good repeatability are selected as characteristic peaks. The result shows that when the peak 6 is taken as the S peak, the relative retention time RSD of the characteristic peak of the standard decoction of the 22 batches of the stir-fried cowherb seed is less than 2.0 percent. Finally, the following steps are provided: 13 characteristic peaks are presented in a characteristic spectrum of a roasted cowherb seed standard decoction sample and correspond to the retention time of 13 characteristic peaks in a reference substance chromatogram of a reference substance of a reference decoction piece, wherein 8 peaks are respectively the same as the retention time of the corresponding reference substance peak, the peak corresponding to the cowherb seed flavonoid glycoside reference substance peak is an S peak, the relative retention time of each of the rest characteristic peaks and the S peak is calculated except for the peak 2, the peak 4, the peak 5, the peak 12 and the peak 13, wherein the relative retention time of the peak 1 and the peak 3 is within +/-10% of a specified value, and the relative retention time of the peaks 6-11 is within +/-5% of the specified value. The specified values are: 0.217 (peak 1), 0.320 (peak 3), 1.000 (peak 6S), 1.051 (peak 7), 1.142 (peak 8), 1.206 (peak 9), 1.429 (peak 10), 1.501 (peak 11).
A traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) is adopted to synthesize 22 batches of the fried cowherb seed standard decoction, a comparison chromatogram of the characteristic chromatogram of the fried cowherb seed standard decoction is established, and the established characteristic chromatogram detection method can accurately and integrally control the quality of the fried cowherb seed labeled decoction.
3. Establishment of characteristic spectrum of fried cowherb seed formula particles
The determination of characteristic spectrum of 3 batches of samples (Szechwan new green pharmaceutical science and technology development ltd., batch No. SY1806001, SY1806002, SY1806003) of the parched cowherb seed formula particles is carried out by a proposed method, and the relative retention time, the relative peak area and the relative peak height are calculated. See fig. 3.
The results show that the RSD of the relative peak areas of the characteristic peaks of the 3 batches of the fried cowherb seed formula particles is large, so that the quality standard text is not included, and the RSD of the relative retention time of 13 characteristic peaks of the 3 batches of the fried cowherb seed formula particles is less than 2.0%. Finally, the following steps are provided: 13 characteristic peaks are presented in the characteristic map of the test sample and correspond to the retention time of 13 characteristic peaks in the reference chromatogram of the reference decoction piece, wherein 8 peaks are respectively the same as the retention time of the corresponding reference peaks, the peak corresponding to the reference peak of the cowherb seed flavonoid glycoside is an S peak, the relative retention time of the other characteristic peaks and the S peak is calculated except for the peak 2, the peak 4, the peak 5, the peak 12 and the peak 13, wherein the relative retention time of the peak 1 and the peak 3 is within +/-10% of a specified value, and the relative retention time of the peaks 6-11 is within +/-5% of the specified value. The specified values are: 0.217 (peak 1), 0.320 (peak 3), 1.000 (peak 6S), 1.051 (peak 7), 1.142 (peak 8), 1.206 (peak 9), 1.429 (peak 10), 1.501 (peak 11).
Synthesizing the 3 batches of the fried cowherb seed formula particles by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), establishing a comparison spectrum of the characteristic spectrum of the fried cowherb seed formula particles, and accurately and integrally controlling the quality of the fried cowherb seed formula particles by the established characteristic spectrum detection method.
Experimental example 8 the detection method of the present invention is used for comparison of HPLC characteristic spectra of parched semen Vaccariae decoction pieces, formula granules and standard decoction
1, preparation of a sample solution of fried cowherb seed decoction pieces: 2.5g of each 22 batches of fried cowherb seed decoction pieces are precisely weighed, placed in a conical flask with a plug, precisely added with 25ml of 70 percent methanol, weighed, ultrasonically treated (the power is 600W, the frequency is 40kHz) for 30 minutes, cooled, weighed again, supplemented with 70 percent methanol to reduce the weight, shaken evenly and filtered, thus obtaining the cowherb seed decoction pieces.
2, preparation of a test solution of a standard decoction of the fried cowherb seeds: the 22 batches of the standard decoction of the stir-fried cowherb seed are taken and respectively taken to be 0.5g, and the preparation method is the same as the example 1.
3, preparation of a sample solution of the fried cowherb seed formula particle: the same procedures as in example 2 were repeated except that 1g of each of the 3 batches of the parched cowherb seed formulation granules was used.
4, detection: respectively sucking 10 μ L of parched semen Vaccariae decoction pieces, standard decoction, and test solution of formula granule, injecting into high performance liquid chromatograph under the following chromatographic conditions:
octadecylsilane chemically bonded silica was used as a filler (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm); acetonitrile is used as a mobile phase A, 0.1 percent phosphoric acid solution is used as a mobile phase B, and the gradient elution procedure is the same as that of the example 1; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 270 nm. The number of theoretical plates is not less than 3000 calculated according to the flavonoid glycoside peak of cowherb seed.
6, results: a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) is adopted to respectively synthesize the characteristic spectrums of 22 batches of fried cowherb decoction pieces into a decoction piece contrast spectrum, the characteristic spectrums of 22 batches of fried cowherb standard decoction into a standard decoction contrast spectrum, and the characteristic spectrums of 3 batches of fried cowherb formula particles into a formula particle contrast spectrum. And comparing the reference spectra of the above parched semen Vaccariae decoction pieces, standard decoction, and formula granule. See fig. 4. The results show that 13 characteristic peaks can be detected in the fried cowherb seed decoction pieces, the standard decoction and the formula granules, the material bases are consistent, the method can accurately and efficiently detect the characteristic components in the fried cowherb seed decoction pieces, the standard decoction and the formula granules, and the aim of integrally controlling the quality of the decoction pieces, the standard decoction and the formula granules is fulfilled.
In conclusion, the detection method for the high performance liquid chromatography characteristic spectrum of the fried cowherb seed decoction pieces, the standard decoction and the formula granules can integrally control the characteristic components in the fried cowherb seed decoction pieces, the standard decoction and the formula granules, ensures the integral stability of the quality of the fried cowherb seed decoction pieces, the standard decoction and the formula granules, and has the advantages of simple operation, high precision, good stability, good repeatability and high accuracy.
Claims (9)
1. A detection method of HPLC characteristic spectrums of fried cowherb seed decoction pieces, standard decoction and formula granules is characterized in that: the method comprises the following operation steps:
1) preparation of a reference solution: dissolving the reference substance to obtain reference substance solution; the control article comprises: eight reference substances, namely 5-hydroxymethylfurfural, cowherb seed flavonoid glycoside, protocatechuic acid, erythrine, saporin, cowherb seed cyclic peptide A, cowherb seed cyclic peptide B and isovitexin-2' -O-arabinoside;
2) preparing a reference decoction piece solution: extracting control decoction pieces of parched semen Vaccariae to obtain control decoction piece solution;
3) preparing a test solution: extracting parched semen Vaccariae decoction pieces, standard decoction, and formula granule to obtain test solution;
4) respectively sucking the reference solution, the reference decoction piece solution and the sample solution, injecting into a high performance liquid chromatograph under the following chromatographic conditions:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 240-270 nm; mobile phase: acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, the gradient elution procedure was as follows:
2. the detection method according to claim 1, characterized in that: the dissolving in the step 1) is dissolving by adding methanol or 70 percent methanol solution.
3. The detection method according to claim 1, characterized in that: step 3) the extraction refers to: adding 70% methanol solution, and ultrasonic extracting.
4. The detection method according to claim 3, characterized in that: the extraction method comprises the following steps:
adding 10 times of 70% methanol into parched semen Vaccariae decoction pieces, or adding 100 times of 70% methanol into parched semen Vaccariae standard decoction powder, or grinding parched semen Vaccariae formula granule into fine powder, and adding 50 times of 70% methanol; ultrasonic extracting for 30min, cooling, adding 70% methanol to make up the lost weight, shaking, filtering, and collecting the filtrate to obtain the sample solution of parched semen Vaccariae decoction pieces, standard decoction or formula granule; the power of the ultrasonic extraction is 600W, and the frequency is 40 kHz.
5. The detection method according to claim 1, characterized in that: in the step 4), the chromatographic column is ZORBAXeclipse XDB-C18 chromatographic column with the specification of 4.6mm multiplied by 250mm and 5 μm.
6. The detection method according to claim 1, characterized in that: in the step 4), the detection wavelength is 270 nm.
7. The detection method according to claim 1, characterized in that: in the step 4), the flow rate of the high performance liquid chromatography is 1.0 mL/min.
8. The detection method according to claim 1, characterized in that: in the step 4), the column temperature of the high performance liquid chromatography is 30 ℃.
9. The detection method according to claim 1, characterized in that: in the step 4), the sample injection amount of the high performance liquid chromatography is 10 mu L.
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