CN115963196B - Construction method and application of HPLC (high Performance liquid chromatography) characteristic spectrum of kelp product - Google Patents
Construction method and application of HPLC (high Performance liquid chromatography) characteristic spectrum of kelp product Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a construction method of HPLC characteristic spectrum of kelp product and application thereof, wherein the construction method comprises the following steps of; s1, preparing a kelp sample solution; s2, preparing a reference substance solution; s3, preparing a reference substance solution; s4, high performance liquid chromatography determination: sucking the kelp sample solution prepared in the step S1, the reference solution prepared in the step S2 and the reference solution prepared in the step S3, and injecting into high performance liquid chromatography to obtain an HPLC characteristic spectrum; the HPLC characteristic spectrum at least comprises a uridine peak, an adenosine peak, a uracil peak and a guanosine peak. The construction method can be used for quantitatively controlling the contents of uridine, guanosine, uracil and adenosine at the same time, and can effectively control and comprehensively evaluate the quality of kelp products.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a construction method and application of an HPLC characteristic spectrum of a kelp product.
Background
The thallus laminariae is dry leaf of thallus laminariae of Laminaria japonica of Laminariaceae or thallus laminariae Ecklonia kurome okam of Alternariaceae, has cold nature, salty taste, and effects of resolving phlegm, softening and resolving hard mass, inducing diuresis and relieving swelling, and can be used for treating goiter, scrofula, testis swelling and pain, and phlegm fluid edema.
As a medicine and food dual-purpose traditional Chinese medicine with long application history, a plurality of researches on chemical composition, pharmacological action and clinical application of kelp are carried out by former people. Only the content of iodine and polysaccharide is measured in the quality standard of thallus laminariae loaded in one part of Chinese pharmacopoeia of 2020 edition, and the accurate description and evaluation of the whole quality of thallus laminariae products cannot be realized. Li Rong the first established HPLC reference fingerprint of kelp can be used for identifying the authenticity of kelp medicine (see Li Rong, tang Xuli, zhang Min, li Guojiang, wang Changyun, guan Hua poem. HPLC chemical fingerprint research of marine pharmaceutical biology series I. HPLC chemical fingerprint of kelp medicine [ J ]. University of sea of China (Nature science edition), 2009, 39 (S1): 37-41.). Then Huang Huahua the quality evaluation of thallus laminariae by UPLC fingerprint spectrum and chemical pattern recognition technique (see Huang Huahua, zhang Yi, lv Shishi. Thallus laminariae UPLC fingerprint spectrum and chemical pattern recognition research [ J ]. Chinese medicinal materials 2020, 43 (07): 1651-1655.). The existing method has fewer effective separated components, and the constructed characteristic spectrum has fewer characteristic peaks, so that the quality of the kelp product can not be monitored comprehensively. The existing method can not detect characteristic patterns of components with larger polarity in thallus laminariae and preparations thereof, for example, the standard thallus laminariae decoction is freeze-dried powder prepared by decocting thallus laminariae medicinal materials with fixed preparation process water after processing, so that most of the components with larger polarity in the standard thallus laminariae decoction are extracted with water and can not be detected by the characteristic pattern method.
The kelp characteristic spectrum detection method is only suitable for detecting the authenticity of kelp decoction pieces, and kelp standard decoction is freeze-dried powder prepared by decocting kelp medicinal materials with fixed preparation process water after processing, and the contained components have high polarity, so that the kelp decoction pieces cannot be detected by the existing method. Moreover, the existing method for preparing the sample by using the characteristic spectrum method has the defects of too long time, complicated operation and no attribution of the identification characteristic peak, and can not effectively detect and control the authenticity and the quality consistency and the stability of the kelp and the preparation thereof. Therefore, it is necessary to establish a quality control method of kelp products, which provides a basis for effectively controlling and comprehensively evaluating the quality of kelp and its preparations.
Disclosure of Invention
In order to solve the technical problems, the invention provides a construction method of HPLC characteristic spectrum of kelp products and application thereof, which can effectively control and comprehensively evaluate the quality of kelp products.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The construction method of HPLC characteristic spectrum of thallus laminariae product comprises the following steps;
S1, preparing a kelp sample solution;
s2, preparing a reference substance solution;
s3, preparing a reference substance solution;
s4, high performance liquid chromatography determination: sucking the thallus laminariae sample solution prepared in step S1, the reference solution prepared in step S2 and the reference solution prepared in step S3, and injecting into high performance liquid chromatography to obtain HPLC characteristic spectrum.
The kelp sample in the invention comprises kelp medicinal materials, kelp decoction pieces, kelp standard decoction, kelp dry extract powder or kelp formula granules.
The preparation method of the kelp sample solution comprises the following steps: weighing thallus laminariae sample, and precisely adding 10% methanol to obtain thallus laminariae sample solution. Specifically, the preparation method comprises the following steps: precisely weighing thallus laminariae sample, precisely adding 10% methanol by volume, ultrasonic treating, taking out, cooling, weighing, adding 10% methanol by volume to make up the weight, shaking, filtering, and collecting filtrate.
Preferably, the preparation of the kelp test sample solution comprises the following steps: precisely weighing thallus laminariae sample, precisely adding 10% methanol 20-50mL, ultrasonic treating for 30-90 min with ultrasonic power of 250-800W, taking out, cooling, weighing, adding 10% methanol to make up the weight of the filtrate, shaking, and filtering to obtain filtrate.
Most preferably, the preparation of the kelp test sample solution comprises the following steps: precisely weighing thallus laminariae sample, precisely adding 50mL of 10% methanol, performing ultrasonic treatment for 30 min with power of 250W, taking out, cooling, weighing, adding 10% methanol to make up the weight, shaking, filtering, and collecting the filtrate.
The reference substance in the invention is kelp reference medicinal material, and the preparation of the reference substance solution comprises the following steps: adding 10% methanol to thallus laminariae control material to obtain reference solution. Specifically, the preparation method comprises the following steps: adding thallus laminariae reference material into conical flask with plug, adding 10% methanol, ultrasonic treating, taking out, cooling, shaking, filtering, and collecting filtrate as reference material solution.
Preferably, the preparation of the reference solution is: 1g of kelp reference medicine is taken and placed in a conical flask with a plug, 30mL of 10% methanol is added, ultrasonic treatment is carried out for 30 minutes, ultrasonic power is 250W, the kelp reference medicine is taken out, cooled, shaken uniformly and filtered, and the subsequent filtrate is taken as reference medicine solution.
The reference substances in the invention are uridine, adenosine, uracil and guanosine, and the preparation of the reference substance solution is as follows: taking a proper amount of uridine, adenosine, uracil and guanosine reference substances, adding 10% methanol by volume percent to prepare a mixed solution serving as a reference substance solution. Specifically, the preparation method comprises the following steps: taking appropriate amounts of uridine, adenosine, uracil and guanosine reference substances, adding 10% methanol to obtain mixed solutions containing 10 μg per 1mL, and making into reference substance solution.
Preferably, in the step S4, the chromatographic column of the high performance liquid chromatography uses octadecylsilane chemically bonded silica as a filler, and the volume ratio is 3:2, the acetonitrile and methanol mixed solution is a mobile phase A, and the phosphoric acid solution with the volume percentage of 0.1 percent is a mobile phase B for gradient elution.
Further preferably, the conditions of the gradient elution are as follows:
preferably, the chromatographic conditions of the high performance liquid chromatography in step S4 are: the flow rate is 0.7-0.9mL/min per minute; the column temperature is 23-27 ℃; the detection wavelength is 230-320nm.
Further preferably, the chromatographic conditions of the high performance liquid chromatography in step S4 are: octadecylsilane chemically bonded silica is used as a filler, the column length of a chromatographic column is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; in the volume ratio of 3:2, taking acetonitrile and methanol mixed solution as a mobile phase A, and taking 0.1% phosphoric acid solution as a mobile phase B for gradient elution; the flow rate is 0.7-0.9mL/min per minute; the column temperature is 23-27 ℃; the detection wavelength is 230-320nm; the theoretical plate number should be not less than 5000 as calculated by adenosine peak.
Most preferably, the chromatographic conditions of the high performance liquid chromatography in step S4 are: octadecylsilane chemically bonded silica is used as a filler, the column length of a chromatographic column is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; in the volume ratio of 3:2, taking acetonitrile and methanol mixed solution as a mobile phase A, and taking 0.1% phosphoric acid solution as a mobile phase B for gradient elution; the flow rate is 0.8mL/min per minute, and the column temperature is 25 ℃; the detection wavelength is 260nm, and the theoretical plate number is not less than 5000 calculated according to the adenosine peak.
Preferably, in step S4, a comparison characteristic spectrum composed of 6 characteristic peaks is generated by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation software, wherein the peak No. 2 is uracil peak, the peak No. 4 is uridine peak, the peak No. 5 is adenosine peak, the peak No. 6 is guanosine peak, and the peak No. 1 and the peak No. 3 are common peaks.
Further preferably, the adenosine peak is taken as an S peak, and the ratio of the relative retention time of each characteristic peak to the S peak is: 0.37-Peak 1, 0.39-Peak 2, 0.52-Peak 3, 0.87-Peak 4, 1.32-Peak 6.
The invention also provides application of the construction method in kelp product quality control.
Preferably, the kelp product in the invention is kelp medicinal material, kelp decoction pieces, kelp standard decoction, kelp dry extract powder or kelp formula granules.
Preferably, the use allows quantitative control of the quality of the kelp product with respect to the uridine, guanosine, uracil and adenosine contents at the same time.
The beneficial effects of the invention are as follows:
(1) The construction method of the HPLC characteristic spectrum of the kelp product provided by the invention simultaneously realizes the common control of all evaluation index components of uridine, guanosine, uracil and adenosine, and meanwhile, the constructed contrast characteristic spectrum contains 6 common characteristic peaks, so that the number of the characteristic peaks is obviously improved, the accuracy of the characteristic spectrum of the kelp product can be effectively improved, and the aim of controlling the overall quality of the kelp product is fulfilled.
(2) According to the construction method of the HPLC characteristic spectrum of the kelp product, disclosed by the invention, the preparation process of the solution of the test sample is optimized, 10% methanol is added into the test sample for complete extraction, the content of detected components is relatively high, the separation effect of each characteristic peak is relatively good, and the peak shape is good.
(3) According to the construction method of the HPLC characteristic spectrum of the kelp product, provided by the invention, chromatographic conditions for obtaining the characteristic spectrum are further optimized, including optimization of a mobile phase and optimization of a gradient elution program, acetonitrile-methanol (3:2) -0.1% phosphoric acid solution is finally selected as the mobile phase for gradient elution, uracil, uridine, adenosine and guanosine are used as reference substances, so that the peak shape can be effectively improved, the chromatographic peak separation degree is improved, the precision is high, the stability is good, the repeatability is good, the accuracy is high, the analysis rate is fast, and the quality of the kelp product can be comprehensively, clearly and effectively controlled.
(4) The invention also provides application of the construction method in kelp product quality control, and the application can be used for quantitatively controlling the contents of uridine, guanosine, uracil and adenosine at the same time. In addition, the invention can also effectively realize the quality control of the substance transmission process, and specifically comprises the following steps: the quality control of intermediate products in the preparation process of the formula particles can be effectively realized, and the components of the finally prepared kelp formula particles can be basically consistent with those in kelp medicinal materials, decoction pieces, standard decoction, extract, extractum and dry paste powder.
Drawings
FIG. 1 is a comparative profile of example 1;
FIG. 2 is an HPLC profile for example 2.1 extraction solvent investigation;
FIG. 3 is an HPLC profile for the extraction method of example 2.2, the extraction time of example 2.3 and the ultrasonic power investigation;
FIG. 4 is an HPLC profile for examining the amount of extraction solvent in example 2.4;
FIG. 5 is an ultraviolet absorption spectrum of adenosine of example 3.1;
FIG. 6 is a graph showing ultraviolet absorption spectrum of uridine in example 3.1;
FIG. 7 is a graph showing the ultraviolet absorption spectrum of guanosine in example 3.1;
FIG. 8 is a chart showing the ultraviolet absorption spectrum of uracil in example 3.1;
FIG. 9 is a HPLC characteristic spectrum of the kelp standard decoction of example 3.1 at different wavelengths;
FIG. 10 is a HPLC characteristic spectrum of a standard decoction of thallus laminariae (herba Zosterae Marinae) of example 3.2 under column temperature examination;
FIG. 11 is a HPLC characteristic spectrum of a standard decoction flow rate survey of thallus laminariae (herba Zosterae Marinae) of example 3.3;
FIG. 12 is a HPLC characteristic spectrum of a standard kelp (kelp) decoction sample size examination of example 3.4;
FIG. 13 is an HPLC profile of chromatographic condition 1 of example 3.5;
FIG. 14 is an HPLC profile of chromatographic condition 2 of example 3.5;
FIG. 15 is an HPLC profile of chromatographic condition 3 of example 3.5;
FIG. 16 is an HPLC profile of chromatographic condition 4 of example 3.5;
FIG. 17 is an HPLC profile of chromatographic condition 5 of example 3.5;
FIG. 18 is a graph of the chromatographic peak assignment of example 4.1;
FIG. 19 is a graph of the results of durability testing of the chromatographic column of example 4.5;
FIG. 20 is an HPLC characteristic spectrum of 15 batches of kelp (kelp) standard decoction of example 6;
Wherein R is a contrast characteristic map; s2 to S16 are in turn ：20220725-1、20220725-2、20220725-3、20220725-4、20220725-5、20220725-6、20220725-7、20220725-8、20220726-1、20220726-2、20220726-3、20220726-4、20220726-5、20220726-6、20220726-7;
FIG. 21 is a comparative profile of example 6;
FIG. 22 is an HPLC characteristic spectrum of 15 batches of kelp (kelp) medicinal material of example 7;
Wherein R is a contrast characteristic map; s2 to S16 are in turn ：W20220501、W20220502、W20220503、W20220504、W20220505、S20220501、S20220502、S20220503、S20220504、S20220505、P20220501、P20220502、P20220503、P20220504、P20220505;
FIG. 23 is a comparative profile of example 7;
FIG. 24 is an HPLC characteristic spectrum of 15 batches of kelp (kelp) decoction pieces of example 8;
Wherein R is a contrast characteristic map; s2 to S16 are in turn ：220501、220502、220503、220504、220505、220506、220507、220508、220509、220510、220511、220512、220513、220514、220515;
FIG. 25 is a comparison feature map of example 8.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, mass ratios or percentages herein are volume ratios or percentages unless otherwise specified.
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 apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Instrument and reagent
High performance liquid chromatograph: agilent 1260 type high performance liquid chromatograph, waters e2695 type high performance liquid chromatograph, waters ACQUITY ARC-2489UV type high performance liquid chromatograph;
an electronic balance: sidoris BSA2235, sidoris SECURA D-1CN, sidoris MSA3.6P-OCE.DM, shanghai Tianmei balance instruments Co., ltd. FA2204C;
ultrapure water machine: HHITECH MASTER Touch-DUVT (Shanghai and Thai instruments Co., ltd.);
ultrasonic cleaner: DTC-33 (500W, 40KHz; dingtai (Hubei) Biochemical technology device manufacturing Co., ltd.);
chromatographic column: shim-PACK GIST C, 18-AQ 250X 4.6mm,5 μm, waters X SELECT HSST3, 250X 4.6mm,5 μm.
Acetonitrile and methanol are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity.
Adenosine (China food and drug inspection institute, batch No. 110879-201703, content of 99.7%).
Guanosine (Chinese food and drug assay institute, lot number: 111977-201501, content of 93.6%).
Uracil (China food and drug inspection institute, batch No. 100469-201302, content of 99.6%).
Uridine (China food and drug inspection institute, lot number: 110887-202104, content of 99.6%).
Kelp (kelp) control drug (Tiandi Heng Yi pharmaceutical Co., ltd., lot number W20220504).
Kelp (kelp) standard decoction freeze-dried powder (prepared by Tiandi Hengyi pharmaceutical Co., ltd., lot number) ：20220725-1、20220725-2、20220725-3、20220725-4、20220725-5、20220725-6、20220725-7、20220725-8、20220726-1、20220726-2、20220726-3、20220726-4、20220726-5、20220726-6、20220726-7).
Kelp (kelp) medicinal material (prepared by Tiandi Hengyi pharmaceutical Co., ltd., lot number) ：W20220501、W20220502、W20220503、W20220504、W20220505、S20220501、S20220502、S20220503、S20220504、S20220505、P20220501、P20220502、P20220503、P20220504、P20220505).
Kelp decoction pieces (prepared by Tiandi Heng Yi pharmaceutical Co., ltd., lot number) ：220501、220502、220503、220504、220505、220506、220507、220508、220509、220510、220511、220512、220513、220514、220515).
Example 1
(One) preparation of a test solution:
(1) Preparing a medicinal material and decoction piece sample solution: taking about 1.5g of medicinal materials and decoction piece test sample powder (sieving with a third sieve), precisely weighing, placing into a conical bottle with a plug, precisely adding 50mL of 10% methanol, sealing, weighing, performing ultrasonic treatment (power 250w, frequency 40 kHz) for 30 minutes (shaking once every 5 minutes), taking out, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
(2) Preparation of standard decoction test sample solution: taking a proper amount of standard decoction sample, grinding, taking about 1.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 50mL of 10% methanol, sealing, weighing, performing ultrasonic treatment (power 250w, frequency 40 kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
(II) preparation of a reference solution: 1g of kelp (kelp) reference medicine is taken, placed in a conical flask with a plug, added with 30mL of 10% methanol, subjected to ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, taken out, cooled, shaken uniformly, filtered, and the subsequent filtrate is taken as reference substance solution of the reference medicine.
(III) preparation of a control solution: and respectively taking appropriate amounts of uridine, adenosine, uracil and guanosine reference substances, and adding 10% methanol to prepare mixed solutions containing 10 mug of each 1mL of the mixed solutions as reference substance solutions.
(IV) high performance liquid chromatography determination:
Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); 3:2 (v/v) acetonitrile-methanol as mobile phase A, and phosphoric acid solution with volume percentage of 0.1% as mobile phase B; elution was performed according to the gradient in table 1; the flow rate was 0.8mL per minute; the column temperature is 25 ℃; the detection wavelength was 260nm. The theoretical plate number should be not less than 5000 as calculated by adenosine peak.
Table 1 example 1 gradient elution table
Assay: respectively precisely sucking 10 μl of the sample solution, the reference solution and the reference solution, and injecting into a liquid chromatograph for measurement to obtain HPLC characteristic spectrum.
According to the obtained HPLC characteristic spectrum, a comparison characteristic spectrum (shown in figure 1) is obtained by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.1 version), and identification of common peaks is carried out, wherein the total of 6 common peaks are identified, 4 peaks respectively correspond to the retention time of the corresponding reference peaks, the No. 2 peak is uracil peak, the No. 4 peak is uridine peak, the No. 5 peak is adenosine peak, the No. 6 peak is guanosine peak, and the No. 1 peak and the No. 3 peak are common peaks. The adenosine peak is taken as an S peak, and the ratio of the relative retention time of each characteristic peak to the S peak is as follows: 0.37 (Peak 1), peak 2 (0.39), 0.52 (Peak 3), peak 4 (0.87), peak 6 (1.32).
Example 2 extraction method investigation
2.1 Extraction solvent investigation
About 1.0g of kelp decoction pieces (batch number 220515) is taken, precisely weighed, placed in a conical flask with a plug, respectively added with 20mL of water, 10% of methanol, 30% of methanol, 50% of methanol and 100% of methanol for extraction, subjected to ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain each sample solution. The results of the measurement according to the chromatographic conditions of experimental example 1 are shown in FIG. 2.
The results of different extraction solvent researches show that when 10% methanol is used for preparing samples, the content of detection components is relatively high, the separation effect of each characteristic peak is relatively good, and the peak shape is good, so that 10% methanol is used as the optimal extraction solvent of kelp (kelp) raw medicinal material and the characteristic map of the kelp preparation.
2.2 Extraction method investigation
About 1.0g of kelp decoction pieces (batch number: 220515) is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 20mL of 10% methanol, and the test sample extraction method is respectively reflux and ultrasonic investigation, the extraction time is 30min, cooled, weighed again, the weight is complemented by 10% methanol, shaken uniformly, filtered, and the subsequent filtrate is taken to obtain each test sample solution. The results of the measurement according to the chromatographic conditions of experimental example 1 are shown in FIG. 3.
The results of different extraction methods show that the effects of respectively carrying out ultrasonic extraction and reflux extraction on the sample are consistent. However, from the viewpoints of convenient operation and time and cost saving, the ultrasonic extraction operation is simpler and more convenient, so the method for extracting the sample is determined to be ultrasonic extraction.
2.3 Extraction time and ultrasound Power investigation
About 1.0g of kelp decoction pieces (batch number: 220515) is taken, precisely weighed, placed in a conical flask with a plug, added with 20mL of 10% methanol, subjected to ultrasonic treatment (respectively examined when ultrasonic power of a test sample is 250W, 500W and 800W), respectively examined when the extraction time of the test sample is 30 minutes, 60 minutes and 90 minutes, cooled, weighed again, complemented by the weight of the loss by 10% methanol, shaken uniformly, filtered, and obtained from the subsequent filtrate. The results of the measurement according to the chromatographic conditions of experimental example 1 are shown in FIG. 3.
The results of different extraction time surveys show that the samples can be completely extracted in 30, 60 and 90 minutes, and the detection results of the characteristic patterns have no obvious difference in combination with the consideration of time saving and cost, so that 30 minutes is selected as the optimal extraction time.
The results of different ultrasonic power surveys show that the ultrasonic power can be fully extracted at the ultrasonic power of 250W. The ultrasonic power of the test sample was determined to be 250W.
2.4 Investigation of the extraction solvent quantity
About 1.0g of kelp decoction pieces (batch number: 220515) is taken, precisely weighed, placed into a conical flask with a plug, respectively added with 20mL, 30mL and 50mL of 10% methanol, sealed, weighed, subjected to ultrasonic treatment (power 250W and frequency 40 kHz) for 30min, cooled, weighed again, supplemented with 10% methanol to the reduced weight, shaken uniformly, filtered, and obtained from the subsequent filtrate to obtain each sample solution. The results of the measurement according to the chromatographic conditions of experimental example 1 are shown in FIG. 4.
The results of investigation of the amounts of different extraction solvents show that the amount of the extraction solvents is more when the amount of the extraction solvents is 50mL, the content is relatively higher than that of other groups, and the detection results of the combined characteristic spectrum have no obvious difference, so that 50mL is selected as the optimal amount of the extraction solvents.
EXAMPLE 3 chromatographic condition investigation
3.1 Wavelength investigation
Based on the experimental conditions set forth in example 1, the diode array detector was used to scan all bands of adenosine, uracil, uridine, guanosine and kelp (kelp) standard decoction sample solutions, and to extract chromatograms of kelp (kelp) standard decoction sample solutions at wavelengths of 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm and 320nm, respectively, wherein the ultraviolet absorption spectrograms of adenosine, uracil, uridine and guanosine are shown in fig. 5-8, and the chromatograms of kelp (kelp) standard decoction sample solutions at different wavelengths are shown in fig. 9.
The result shows that the information amount of the chromatographic peak is larger when the detection wavelength is 260nm, and the base line of the chromatogram is more stable, so the detection wavelength is determined to be 260nm.
3.2 Column temperature investigation
Based on the experimental conditions set forth in example 1, the column temperatures were examined at 23℃and 25℃and 27℃respectively, and the results are shown in FIG. 10. Test lot number: 20220822 batches.
The result of the column temperature investigation shows that when the column temperature is 25 ℃, the peak shape of the chromatogram is symmetrical, and the separation degree is better, so that the column temperature is finally determined to be 25 ℃.
3.3 Flow rate investigation
Based on the experimental conditions set forth in example 1, the flow rates were examined at 0.7mL/min, 0.8mL/min, and 0.9mL/min, respectively, and the results are shown in FIG. 11. Test lot number: 20220822 batches.
The flow rate investigation result shows that the chromatogram peak shape is good and the separation degree is moderate when the flow rate is 0.8mL/min. The flow rate was determined to be 0.8mL/min.
3.4 Sample injection amount investigation
Based on the experimental conditions set forth in example 1, the sample injection amounts were examined to be 10. Mu.L and 20. Mu.L, respectively, and the results are shown in FIG. 12. Test lot number: 20220822 batches.
The sample injection amount investigation result shows that when the sample injection amount is 10 mu L, the chromatogram peak shape is good, and the separation degree is moderate. The amount of sample introduced was determined to be 10. Mu.L.
3.5 Chromatographic Condition optimization
Chromatographic condition 1
Preparation of test solution: about 1.5g of kelp decoction piece powder is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 50mL of 10% methanol, sealed, weighed, subjected to ultrasonic treatment (power 250w and frequency 40 kHz) for 30 minutes, taken out, cooled, weighed again, complemented with 10% methanol for the loss of weight, shaken uniformly, filtered, and obtained to obtain subsequent filtrate.
Assay: precisely sucking 10 μl of the sample solution, injecting into liquid chromatograph, and measuring.
Octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile as mobile phase a and 0.1% phosphoric acid as mobile phase B, and gradient elution was performed as specified in table 2; the column temperature is 30 ℃; the detection wavelength is 217nm; the flow rate was 0.3mL/min.
TABLE 2 gradient elution table for chromatographic condition 1
Chromatographic condition 2
Preparation of test solution: about 0.2g of kelp decoction piece powder is taken, precisely weighed, placed in a hydrolysis tube with a plug, precisely added with 10mL of 9mol/L hydrochloric acid solution, weighed, hydrolyzed for 3 hours at 150 ℃, taken out, cooled, weighed again, supplemented with 9mol/L hydrochloric acid solution to reduce weight, uniformly mixed, filtered, precisely measured with 5mL of filtrate, transferred to an evaporation dish, evaporated to dryness, the residue is dissolved with 0.1mol/L hydrochloric acid solution, transferred to a 25mL measuring flask, added with 0.1mol/L hydrochloric acid solution to scale, and shaken uniformly to obtain the kelp decoction piece.
Assay: precisely measuring 5mL of a sample solution, placing the sample solution into a 25mL measuring flask, adding 2.5mL of acetonitrile solution of 0.1mol/L Phenyl Isothiocyanate (PITC) and acetonitrile solution of 1mol/L triethylamine, shaking uniformly, standing at room temperature for 1 hour, adding 50% acetonitrile to the scale, and shaking uniformly. Taking 10mL of the solution, adding 10mL of n-hexane, shaking, standing for 10 minutes, taking the lower layer solution, filtering, taking 5 mu L of each of the subsequent filtrate, injecting into a liquid chromatograph, and measuring to obtain the composite.
Octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); sodium acetate at 0.1mol/L (acetic acid ph=6.5): acetonitrile=93: 7 is mobile phase a, acetonitrile: water = 4:1 is mobile phase B, gradient elution is performed as specified in table 3; the column temperature is 30 ℃; the detection wavelength is 254nm; the flow rate is 0.5mL/min; sample injection volume: 5. Mu.L.
TABLE 3 gradient elution table for chromatographic condition 2
Chromatographic condition 3
The sample solution was prepared as in chromatographic condition 1.
Octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); gradient elution was performed as specified in table 4 with acetonitrile as mobile phase a and 0.1% phosphoric acid as mobile phase B; the column temperature is 30 ℃; the detection wavelength is 260nm; the flow rate was 0.8mL/min.
Assay: precisely sucking 10 μl of the sample solution, injecting into liquid chromatograph, and measuring.
TABLE 4 chromatographic condition 3 gradient elution table
Chromatographic condition 4
The sample solution was prepared as in chromatographic condition 1.
Octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); gradient elution was performed with methanol as mobile phase a and 0.1% phosphoric acid as mobile phase B, as specified in table 5; the column temperature is 30 ℃; the detection wavelength is 260nm; the flow rate was 0.8mL/min.
Assay: precisely sucking 10 μl of the sample solution, injecting into liquid chromatograph, and measuring.
TABLE 5 chromatographic condition 4 gradient elution table
Chromatographic condition 5
The sample solution was prepared as in chromatographic condition 1.
Octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); acetonitrile: methanol=3: 2 (v/v) as mobile phase A and 0.1% phosphoric acid as mobile phase B, gradient elution was performed as specified in Table 6; the column temperature is 25 ℃; the detection wavelength is 260nm; the flow rate was 0.8mL/min.
Assay: precisely sucking 10 μl of the sample solution, injecting into liquid chromatograph, and measuring.
TABLE 6 chromatographic condition 5 gradient elution table
The detection results of the 5 chromatographic conditions are shown in figures 13-17, and the HPLC chromatogram of chromatographic condition 1 has smaller chromatographic peak of thallus laminariae (herba Zosterae Marinae), poorer separation degree and inclusion peak. The HPLC profile of chromatographic condition 2 had fewer kelp (kelp) chromatographic peaks and had severe baseline lifting. The baseline rise of kelp (kelp) chromatographic peak in the HPLC profile of chromatographic condition 3 was severe. The rise in the baseline of the kelp (kelp) chromatographic peak in the HPLC profile of chromatographic condition 4 was severe. The HPLC chromatogram of chromatographic condition 5 has stable chromatographic peak baseline of thallus laminariae (herba Zosterae Marinae), more chromatographic peaks, and good separation degree. And when the organic phase is simply methanol or acetonitrile, the chromatographic base line is seriously lifted, and when the methanol and the acetonitrile are in a certain proportion, the chromatographic peak is good in shape and stable in base line, so that the organic phase is acetonitrile: methanol=3: 2 (v/v).
EXAMPLE 4 methodology investigation
4.1 Chromatographic peak assignment
Preparation of a standard decoction (batch number: 20220822) of thallus laminariae (herba Zosterae Marinae) test sample solution: taking appropriate amount of thallus laminariae (herba Zosterae Marinae) standard decoction, grinding, taking about 1.5g, precisely weighing, placing into conical flask with plug, precisely adding 50mL of 10% methanol, sealing, weighing, ultrasonic treating (power 250w, frequency 40 kHz) for 30 min, taking out, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking, filtering, and collecting filtrate.
Preparation of reference solution: 1g of kelp (kelp) reference medicine is taken, placed in a conical flask with a plug, added with 30mL of 10% methanol, subjected to ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, taken out, cooled, shaken uniformly, filtered, and the subsequent filtrate is taken as reference substance solution of the reference medicine.
Preparation of a control solution: and respectively taking appropriate amounts of uridine, adenosine, uracil and guanosine reference substances, and adding 10% methanol to prepare mixed solutions containing 10 mug of each 1mL of the mixed solutions as reference substance solutions.
Preparation of negative control solution: according to the experimental conditions, preparing a kelp (kelp) deficiency standard decoction negative control solution.
The characteristic spectrum peak of the kelp (kelp) standard decoction is positioned, and the result is shown in figure 18.
4.2 Precision investigation
The control reference solution was sampled 6 times in series by the experimental method of example 1, 10. Mu.L each time, and the relative retention time and the relative peak area of each control peak were calculated, and the results are shown in tables 7 and 8.
TABLE 7 precision investigation-retention time
TABLE 8 precision investigation-peak area
The precision investigation result shows that the peak retention time RSD of each reference substance is 0.03%, the peak area RSD is 0.08% -0.09%, and the instrument precision is good.
4.3 Repeatability investigation
6 Parts of kelp (kelp) standard decoction (lot number: 20220822) was precisely weighed, prepared and measured according to the experimental method of example 1, and the retention time and peak area of each characteristic peak were calculated, and the results are shown in tables 9 and 10.
Table 9 repeatability investigation-relative retention time
Table 10 repeatability investigation-relative peak area
The repeatability investigation result shows that the relative retention time RSD of each characteristic peak of the repeatability is 0.03-0.07%, and the relative peak area RSD is 0.13-6.90%, and the method has good repeatability.
4.4 Intermediate precision investigation
Based on the experimental conditions set forth in example 1, 6 parts of kelp (kelp) standard decoction was precisely weighed, and test solutions were prepared and measured on Agilent 1260 and Waters e2695 type high performance liquid chromatographs, respectively, and the results are shown in tables 11 and 12.
TABLE 11 intermediate precision-relative retention time
TABLE 12 intermediate precision-relative peak area
The intermediate precision result shows that the same sample is measured by different personnel at different times, and the method has better stability. The relative retention time RSD of each characteristic peak is 0.60% -1.43%, and the relative peak area RSD is 0.58% -4.74%, so that the method has good stability.
4.5 Column durability inspection
Based on the experimental conditions set forth in example 1, the chromatographic column was Waters Xselect HSST C18
250×4.6mm、Shim pack GIST C18 250×4.6mm(SN：21E24971)，Shim pack GIST C18
The results of examination of 250X 4.6mm (SN: 21G 27585) are shown in FIG. 19, and the relative retention time and relative peak area of each characteristic peak are calculated, and the results are shown in tables 13 and 14.
TABLE 13 column durability investigation-relative retention time
TABLE 14 column durability investigation-relative peak area
The chromatographic column durability investigation experiment results show that under the condition that octadecylsilane chemically bonded silica gel of different types is used as a filler chromatographic column, the chromatographic column has good separation degree and peak shape, and the relative retention time is stable.
4.6 Stability investigation
Based on the experimental conditions set forth above, the same sample solution was taken and measured at 0h, 23h, 37h, 57h, 89h, respectively, and the retention time and peak area results of each characteristic peak are shown in tables 15 and 16.
Table 15 stability investigation-retention time
TABLE 16 stability investigation-peak area
The stability investigation result shows that the corresponding characteristic peak retention time RSD is 0.12% -0.49%, the peak area RSD is 0.38% -31.79%, the retention time of each characteristic peak is stable, the peak area change of each characteristic peak except for the peak 3 is large, the stability investigation result shows that the stability investigation result has a stable growth trend, the rest characteristic peaks are relatively stable, and the stability test result shows that the sample solution is in 89 hours, so that the identification and content measurement of the characteristic peaks are not influenced.
Example 5 determination of characteristic peaks and establishment of control Profile
5.1 Establishment of a limit of a specified value of relative retention time: the results of each examination and verification of methodology are summarized in table 17.
Table 17 methodological results RSD% summary criteria-retention time-relative retention time for each item
In the column durability test, the RSD range of peak 1 to peak 6 was 0.03% to 1.53%, so the relative retention time of each peak was tentatively set to ±10%.
Finally, the following steps are provided: it is recommended to use a Shim-PACK GIST C18-AQ chromatographic column for analysis, wherein the sample characteristic spectrum should show 6 characteristic peaks, and the peak corresponding to the adenosine reference substance is S peak.
EXAMPLE 6 verification of batch 15 kelp (kelp) Standard decoction
The method of example 1 was used to perform a feature spectrum analysis on 15 batches of kelp standard decoction samples, a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) was used to synthesize 15 batches of kelp standard decoction, the result is shown in fig. 20, the contrast feature spectrum is shown in fig. 21, the relative retention time and the relative peak area ratio are calculated, and the results are shown in tables 18 and 19.
TABLE 18 relative Retention time of kelp (kelp) stock decoction
TABLE 19 relative peak area of the standard decoction of kelp (kelp)
According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 6 peaks with better repeatability are selected as characteristic peaks. The relative retention time of each characteristic peak and the S peak was calculated to be within ±10% of the predetermined value, and the predetermined values were 0.37 (peak 1), peak 2 (0.39), 0.52 (peak 3), peak 4 (0.87), and peak 6 (1.32).
Meanwhile, similarity calculation is carried out on 15 batches of kelp (kelp) standard decoction feature patterns, and the similarity between 15 batches of kelp (kelp) standard decoction feature patterns and the standard feature patterns is more than 0.9. The detailed results are shown in Table 20.
TABLE 20 similarity results of kelp (kelp) standard decoction of 15 batches
EXAMPLE 7 verification of batch of kelp (kelp) medicinal material
The method of example 1 was used to perform a characteristic spectrum analysis on 15 batches of kelp (kelp) medicinal material samples, a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) was used to synthesize 15 batches of kelp (kelp) medicinal material, the result is shown in fig. 22, the contrast characteristic spectrum is shown in fig. 23, the relative retention time and the relative peak area ratio are calculated, and the results are shown in tables 21 and 22.
Table 21 relative retention time of characteristic patterns of thallus laminariae (herba Zosterae Marinae)
Surface 22 characteristic map of kelp (kelp) medicinal material relative peak area
According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 6 peaks with better repeatability are selected as characteristic peaks. Finally, the following steps are provided: the sample chromatogram should show 6 characteristic peaks and correspond to the retention time of 6 characteristic peaks in the reference chromatogram of the reference material, wherein 4 peaks respectively correspond to the retention time of the reference material peaks of the corresponding reference material; the peak corresponding to the reference peak of the adenosine reference substance is an S peak, the relative retention time of the peak 1, the peak 3 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is: 0.37 (Peak 1), peak 2 (0.39), 0.52 (Peak 3), peak 4 (0.87), peak 6 (1.32).
Meanwhile, similarity calculation is carried out on characteristic maps of 15 batches of kelp (kelp) medicinal materials, and the similarity between the characteristic maps of the kelp (kelp) medicinal materials and the standard characteristic maps is more than 0.9. The detailed results are shown in Table 23.
TABLE 23 similarity results of kelp (kelp) batches
EXAMPLE 8 verification of batch kelp (kelp) decoction pieces
The characteristic spectrum analysis was performed on 15 batches of kelp (kelp) decoction pieces samples by the method of example 1, the results are shown in fig. 24, the comparative characteristic spectrum is shown in fig. 25, the relative retention time and the relative peak area ratio are calculated, and the results are shown in tables 24 and 25.
Table 24 relative retention time of characteristic patterns of kelp (kelp) decoction pieces
Table 25 relative peak area of characteristic patterns of kelp decoction pieces
According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 6 peaks with better repeatability are selected as characteristic peaks. Finally, the following steps are provided: the sample chromatogram should show 6 characteristic peaks and correspond to the retention time of 6 characteristic peaks in the reference chromatogram of the reference material, wherein 4 peaks respectively correspond to the retention time of the reference material peaks of the corresponding reference material; the peak corresponding to the reference peak of the adenosine reference substance is an S peak, the relative retention time of the peak 1, the peak 3 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is: 0.37 (Peak 1), peak 2 (0.39), 0.52 (Peak 3), peak 4 (0.87), peak 6 (1.32).
Meanwhile, similarity calculation is carried out on 15 batches of kelp decoction piece characteristic patterns, and the similarity of the kelp decoction piece characteristic patterns and the standard characteristic patterns is more than 0.9. The detailed results are shown in Table 26.
TABLE 26 similarity results of kelp decoction pieces of 15 batches
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 (5)
1. The construction method of the HPLC characteristic spectrum of the kelp product is characterized by comprising the following steps of;
S1, preparing a kelp sample solution;
s2, preparing a reference substance solution;
s3, preparing a reference substance solution;
S4, high performance liquid chromatography determination: sucking the kelp sample solution prepared in the step S1, the reference solution prepared in the step S2 and the reference solution prepared in the step S3, and injecting into high performance liquid chromatography to obtain an HPLC characteristic spectrum;
The HPLC characteristic spectrum at least comprises a uridine peak, an adenosine peak, a uracil peak and a guanosine peak;
The thallus laminariae sample in step S1 comprises thallus laminariae medicinal materials, thallus laminariae decoction pieces, thallus laminariae standard decoction, thallus laminariae dry extract powder or thallus laminariae granule;
the preparation method of the kelp sample solution comprises the following steps: weighing thallus laminariae test sample, and adding methanol with volume percentage of 10% to obtain thallus laminariae test sample solution;
the preparation of the reference solution in step S2 is as follows: adding 10% methanol into thallus laminariae control medicinal material to obtain reference solution; the preparation of the reference substance solution in the step S3 comprises the following steps: taking uridine, adenosine, uracil and guanosine reference substances, adding 10% methanol by volume percent to prepare a mixed solution serving as a reference substance solution;
In the step S4, the chromatographic column of the high performance liquid chromatography takes octadecylsilane chemically bonded silica as a filler, and the volume ratio is 3:2, taking acetonitrile and methanol mixed solution as a mobile phase A, and taking phosphoric acid solution with the volume percentage of 0.1% as a mobile phase B for gradient elution;
the detection wavelength is 260nm;
the conditions for the gradient elution were as follows:
2. The method according to claim 1, wherein the chromatographic conditions of the high performance liquid chromatography in step S4 are: the flow rate is 0.7-0.9mL/min per minute; the column temperature was 23-27 ℃.
3. The construction method according to claim 1, wherein step S4 uses a traditional Chinese medicine chromatographic fingerprint similarity evaluation software to generate a comparison characteristic spectrum composed of 6 common characteristic peaks, wherein peak No. 2 is uracil peak, peak No. 4 is uridine peak, peak No. 5 is adenosine peak, peak No. 6 is guanosine peak, and peaks No. 1 and 3 are common peaks.
4. The method according to claim 3, wherein the adenosine peak is defined as an S peak, and the ratio of the relative retention time of each characteristic peak to the S peak is: 0.37-Peak 1, 0.39-Peak 2, 0.52-Peak 3, 0.87-Peak 4, 1.32-Peak 6.
5. Use of the construction method according to any one of claims 1-4 for quality control of kelp products.
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| CA2599577A1 (en) * | 2005-03-04 | 2006-09-14 | Verenium Corporation | Nucleic acids and proteins and methods for making and using them |
| CN102068553A (en) * | 2010-12-07 | 2011-05-25 | 哈尔滨泰华药业股份有限公司 | Method for constructing high performance liquid chromatographic (HPLC) fingerprint of Rupixiao preparation |
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| CN102068553A (en) * | 2010-12-07 | 2011-05-25 | 哈尔滨泰华药业股份有限公司 | Method for constructing high performance liquid chromatographic (HPLC) fingerprint of Rupixiao preparation |
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