CN114280211B - SPE pretreatment-based semen trichosanthis feature map analysis method - Google Patents
SPE pretreatment-based semen trichosanthis feature map analysis method Download PDFInfo
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Abstract
The invention discloses a preprocessing method based on SPEIs prepared from semen Trichosanthis by analyzing characteristic spectrum. The method is based on a solid phase extraction technology, so that lipophilic compounds and large-polarity impurities in the semen trichosanthis standard decoction are effectively separated, and the interference of the compounds on characteristic patterns is reduced. The method adopts Agilent ZORBAX SB-C 18 High performance liquid chromatographic column with methanol as mobile phase A and 0.1% phosphoric acid water solution as mobile phase B, detection wavelength of 245nm and flow rate of 0.6mL min ‑1 Gradient elution was performed. Recording a chromatogram, counting retention time and relative retention time of characteristic peaks, selecting relative retention time and relative standard deviation by a reference index, and selecting a reference peak for normalization value processing. The method for detecting the characteristics of the semen trichosanthis has the advantages of simple gradient of chromatographic conditions, good reproducibility, high precision and good stability, ensures the stability of the whole quality of the semen trichosanthis, ensures the quality control technology of the semen trichosanthis to be more perfect and scientific, and provides effective basis for quality identification of traditional Chinese medicinal materials and subsequent research of formula particles.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine analysis and quality identification and control, in particular to a snakegourd seed characteristic spectrum analysis method based on SPE pretreatment.
Background
Semen Trichosanthis is a dry mature seed of fructus Trichosanthis Trichosanthes kirilowii Maxim of Cucurbitaceae, and has slight smell and light taste. Fructus trichosanthis belongs to Chinese medicinal plants with wider planting range in China, grows in sunny places, is mainly distributed in places ranging from north to Yangtze river basin in China due to limitation of climate conditions, and is mainly produced in places such as Anhui, shandong, henan and Hubei provinces. The traditional Chinese medicine has the effects of clearing heat and eliminating phlegm, relieving chest stuffiness and eliminating stagnation, and moistening dryness and lubricating intestines, so that the traditional Chinese medicine has wide application in the traditional Chinese medicine compound preparation. At present, a plurality of Chinese patent medicines such as cough relieving gold pill, wind-dispelling and lung-regulating pill, lung-heat clearing and phlegm-resolving pill, white diarrhea syrup and the like are used for clinically treating symptoms such as cough due to lung heat, yellow and thick phlegm, chest stuffiness and pain, stuffiness and fullness in chest, acute mastitis, pulmonary abscess, acute appendicitis, constipation and the like. However, the current literature report on semen trichosanthis focuses on the identification of chemical components, and no research on characteristic patterns exists.
Semen trichosanthis belongs to fruit seed parts, is rich in lipophilic compounds such as grease, and in the HPLC qualitative process, the response value of the components is low, and the problems of low spectrogram separation degree, poor repeatability, pollution to columns and detectors, difficult flushing and the like are easily caused. The method comprises the steps of preprocessing semen trichosanthis samples by adopting Solid-Phase Extraction (SPE) sample preprocessing technology, and enriching, separating and purifying the samples by adopting a selective adsorption and selective elution mode, thus being a physical Extraction process comprising a liquid Phase and a Solid Phase. Meanwhile, the sample can be eluted according to the eluent with different polarities, so that the components suitable for HPLC analysis can be effectively reserved, the interference of lipophilic compounds is reduced, and the method has the advantages of simplicity in operation, good reproducibility, solvent saving and the like. The characteristic spectrum is a quality evaluation method for reflecting the integral characteristics of chemical components of the traditional Chinese medicine, and the authenticity, the consistency and the stability of the quality of the traditional Chinese medicine and the preparation thereof can be effectively detected and controlled, and the method is common to the industry. Before the invention, the method for carrying out solid-phase extraction pretreatment on the semen trichosanthis and establishing a characteristic map by taking chemical components contained in the semen trichosanthis as indexes is not reported. Aiming at the situation, the solid phase extraction process of the semen trichosanthis sample is researched, the HPLC method is adopted for carrying out characteristic spectrum research, the quality of semen trichosanthis medicinal materials is strictly controlled, the resources of the semen trichosanthis medicinal materials are more reasonably applied, and a scientific basis is provided for the deep research and development of the medicinal materials.
Disclosure of Invention
The invention aims to provide an SPE pretreatment method and a characteristic spectrum detection method for semen trichosanthis, and the quality of semen trichosanthis medicinal materials can be comprehensively controlled by the method, so that the quality stability, consistency and controllability of semen trichosanthis products are better ensured.
The technical scheme of the invention is as follows: a semen trichosanthis characteristic spectrum analysis method based on SPE pretreatment comprises the following steps:
(1) Preparation of test solution:
adding pure water into the semen trichosanthis standard decoction freeze-dried powder for full dissolution, wherein the mass volume ratio of the semen trichosanthis standard decoction freeze-dried powder to the pure water is 0.3:10, carrying out ultrasonic treatment and centrifugation, and taking an oil layer and water layer mixed solution as a solid phase extraction test sample; adding methanol into solid-phase extraction packing of semen trichosanthis for full soaking, wherein the mass-volume ratio of the solid-phase extraction packing of semen trichosanthis to the methanol is 1:10, and filling the column; leaching and activating by methanol; eluting and balancing with pure water; sampling a solid phase extraction test sample; eluting with pure water for the first time; eluting for the second time with 30-50% methanol eluent, collecting methanol eluent, evaporating to dryness, redissolving, filtering, and collecting the subsequent filtrate to obtain sample solution;
(2) Preparation of a control solution:
weighing guanosine reference substance, preparing into reference substance solution with concentration of 3.0 μg/ml by taking methanol with volume concentration of 10% as solvent, filtering, and taking subsequent filtrate as reference substance solution;
(3) Liquid phase analysis process:
respectively taking the sample solution and the reference substance solution, respectively injecting into a high performance liquid chromatograph, measuring, and establishing an HPLC characteristic spectrum to obtain the semen Trichosanthis characteristic spectrum.
Further, the methanol eluent with the volume concentration of 30% is preferable in the step (1).
Further, the semen trichosanthis solid phase extraction filling material in the step (1) comprises SP-120-30/50-ODS-RPS, ODS-A-HG and C8-HG.
Further, the solid-phase extraction filler of semen Trichosanthis in the step (1) is preferably SP-120-30/50-ODS-RPS with particle size of 12nm-50 μm.
Further, the preparation conditions of the semen trichosanthis standard decoction freeze-dried powder in the step (1) are as follows: mashing semen Trichosanthis decoction pieces, adding water, and soaking thoroughly, wherein the mass ratio of semen Trichosanthis decoction pieces to water is 1:8; heating and boiling, decocting for 45-60 min, sieving to obtain medicinal residue, adding 7 times of water into the medicinal residue, heating and boiling, decocting for 30-60 min, filtering while hot, mixing the two medicinal liquids to obtain semen Trichosanthis standard decoction, taking standard decoction sample, lyophilizing, and obtaining lyophilized powder.
Further, the power of the ultrasonic treatment in the step (1) is 250W, and the frequency is 40kHz; the rotational speed of the centrifugation is 4000r/min, and the time is 10min; the rinsing rate is not more than 1ml/min.
Further, the evaporating in the step (1) specifically comprises the following steps: vacuum concentration is adopted, the vacuum degree is 90mbar, the rotating speed is 100rpm, and the water bath temperature is 60 ℃ for evaporating.
Further, the filtering in the step (2) specifically includes: passing through a microporous filter membrane of 0.22 μm.
Further, the conditions of the high performance liquid chromatography in the step (3) are specifically as follows: the chromatographic column is Agilent ZORBAX SB-C 18 (4.6 mm. Times.250 mm,5.0 μm); column temperature is 30-40 ℃; the flow rate is 0.4-0.8 mL.min -1 The method comprises the steps of carrying out a first treatment on the surface of the The detection wavelength is 230-285 nm; the sample injection amount is 10 mu L; methanol is a mobile phase A, and phosphoric acid water with volume concentration of 0.1% is a mobile phase B; gradient elution was performed.
Further, the conditions of the high performance liquid chromatography in the step (3) are preferably: the chromatographic column is Agilent ZORBAX SB-C 18 (4.6 mm. Times.250 mm,5.0 μm); column temperature is 35 ℃; flow rate 0.6mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Detection wavelength 245nm; the sample injection amount is 10 mu L; methanol is a mobile phase A, and 0.1% phosphoric acid water is a mobile phase B; gradient elution was performed.
Further, the semen trichosanthis feature map in the step (3) includes 6 feature peaks, the peak corresponding to the guanosine reference is taken as an S2 peak, and the relative retention time of the peak 2 and the peak 1, the peak 3, the peak 4, the peak 5 and the peak 6 is calculated, wherein the relative retention time is within + -10% of a specified value, and the specified value is 0.831 (peak 1), 2.284 (peak 3), 2.447 (peak 4), 3.046 (peak 5) and 3.412 (peak 6).
Compared with the prior art, the invention has the following effective results:
(1) According to the invention, the HPLC characteristic spectrum of semen trichosanthis is established, 6 common peaks are calibrated, chromatographic peaks in the constructed HPLC characteristic spectrum can be well separated, the characteristic spectrum information is rich, the chromatographic peak type is good, and the detection method has good stability and repeatability.
(2) The solid phase extraction pretreatment method is good, can effectively separate the lipophilic compound, reduces the interference of the lipophilic compound on a spectrogram, and ensures that the characteristic peak separation degree of the characteristic spectrogram is better and the identification degree is higher.
(3) The HPLC characteristic spectrum of the semen trichosanthis constructed by the invention fills the defect of quality control of the semen trichosanthis in the prior art and provides technical support for subsequent researches on semen trichosanthis formula particles and the like.
(4) The invention establishes the semen trichosanthis characteristic map based on the high performance liquid chromatography combined with the SPE sample pretreatment technology, monitors 6 characteristic peaks, can effectively ensure the stability of the whole quality of semen trichosanthis, and provides scientific basis for quality identification of Chinese medicinal materials rich in grease.
Drawings
FIG. 1 is a flowchart of a method for analyzing characteristics of semen Trichosanthis in example 1;
FIG. 2 is a graph of the comparison characteristics of the semen Trichosanthis standard decoction of example 1, wherein peak 2 is guanosine;
FIG. 3 is a chromatogram of the results of the investigation of the elution solvent in example 2;
FIG. 4 is a chromatogram of the results of solid phase extraction of the filler study in example 2;
FIG. 5 is a chromatogram of the wavelength investigation result in example 3;
FIG. 6 is a chromatogram of the results of the column temperature investigation in example 3;
FIG. 7 is a chromatogram of the flow rate investigation result in example 3;
FIG. 8 is a chromatogram of the results of the durability test of the column in example 4.
Detailed Description
Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the invention. Rather, they are merely examples of apparatus and methods consistent with aspects of the invention as detailed in the accompanying claims.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.
It should be understood that although the terms first, second, third, etc. may be used herein to describe various information, these information should not be limited by these terms. These terms are only used to distinguish one type of information from another. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of the invention. The word "if" as used herein may be interpreted as "at … …" or "at … …" or "responsive to a determination", depending on the context.
The method for analyzing the semen trichosanthis characteristic spectrum based on SPE pretreatment provided by the invention is further described below with reference to the accompanying drawings and specific embodiments, but the protection scope of the invention is not limited to the method.
In the following embodiments, the instrument involved comprises: agilent1260 type high performance liquid chromatograph, agilent ZORBAX SB-C 18 Chromatographic column (4.6 mm. Times.250 mm,5.0 μm), SQP electronic balance (Sidoriko instruments Co., ltd.), KQ-250DB ultrasonic cleaner (Kunskia ultrasonic instruments Co., ltd.).
In the following examples, the reagents involved include: methanol (Anhui Tiandi high purity solvent Co., ltd., chromatographic purity), purified water (Waha Co., ltd.), methanol (national medicine group chemical Co., ltd.).
In the following examples, the test articles involved include: semen trichosanthis standard decoction freeze-dried powder lot number: GLZ-190902, GLZ-190904, GLZ-190905, GLZ-190910.
In the following examples, the reference substances involved include: guanosine (PS 1215-0100, chenopodium biotechnology Co., ltd.).
Example 1
The embodiment provides a snakegourd seed characteristic spectrum analysis method, which comprises the following steps:
(1) Preparation of test solutions
Taking 0.3g of semen trichosanthis standard decoction freeze-dried powder, adding 10ml of pure water, performing ultrasonic treatment at the power of 250W and the frequency of 40kHz for 30 minutes, centrifuging (the rotating speed is 4000r/min, the time is 10 min), and taking the mixed solution of an oil layer and a water layer as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of solid phase extraction test sample, and loading the sample into a semen trichosanthis solid phase extraction column, wherein the semen trichosanthis solid phase extraction column comprises: SBEQ-CR0001 with inner diameter of 5.5mm and length of 57mm was eluted with 5ml of pure water for the first time; eluting with 5ml 30% methanol for the second time, collecting 30% methanol eluate, evaporating to dryness (vacuum concentration mode, vacuum degree of 90mbar, rotation speed of 100rpm, water bath temperature of 60deg.C), redissolving with 1ml 30% methanol, filtering, and collecting filtrate as sample solution;
the preparation conditions of the snakegourd seed standard decoction freeze-dried powder are as follows: taking 100g of semen trichosanthis decoction pieces by adopting a decoction kettle device, mashing, adding 8 times of water by mass, fully soaking for 30min, heating and boiling, then decocting for 45 min-60 min, passing through a 120-mesh screen while hot, adding 7 times of water into the residues, heating and boiling, then decocting for 30 min-60 min, filtering while hot, and combining the two liquid medicines, namely the semen trichosanthis standard decoction. Taking a proper amount of standard decoction, and freeze-drying to obtain freeze-dried powder;
(2) Preparation of control solution
Precisely weighing a proper amount of guanosine reference substance, precisely weighing, preparing a reference substance solution with a concentration of 3 mug/ml by taking methanol with a volume concentration of 10% as a solvent, passing through a microporous filter membrane with a concentration of 0.22 mu m, and taking a subsequent filtrate as the reference substance solution;
(3) Liquid phase analysis process
Respectively taking 10 μl of the sample solution and the reference solution, injecting into high performance liquid chromatograph, measuring, and establishing HPLC characteristic spectrum (flow chart is shown in figure 1, and liquid chromatogram is shown in figure 2);
the conditions of the high performance liquid chromatography are as follows: the chromatographic column is Agilent ZORBAX SB-C 18 (4.6 mm. Times.250 mm,5.0 μm); column temperature is 35 ℃; flow rate 0.6mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Detection wavelength 245nm; feeding inA sample amount of 10. Mu.L; methanol is a mobile phase A, and 0.1% phosphoric acid water is a mobile phase B; the gradient elution procedure was:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 5～20 | 95～80 |
| 10～35 | 20～45 | 80～55 |
| 35～53 | 45～95 | 55～5 |
| 53～65 | 95～100 | 5～0 |
Finally, the following steps are provided: the semen trichosanthis characteristic spectrum should show 6 characteristic peaks, the peak corresponding to the guanosine reference of the peak 2 is taken as an S peak, and the relative retention time of the peak 1, the peak 3, the peak 4, the peak 5 and the peak 6 and the peak 2 (S peak) is calculated, wherein the relative retention time is within +/-10% of a specified value, and the specified values are 0.831 (peak 1), 2.284 (peak 3), 2.447 (peak 4), 3.046 (peak 5) and 3.412 (peak 6).
Example 2
Based on example 1, the eluting solvent and the solid phase extraction filler in the preparation of the sample solution are examined in this example, and the technical scheme is further described.
(1) Investigation of elution solvent
0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902) is taken, precisely weighed, placed in a conical bottle with a plug, added with 10ml of pure water, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuged, and taken as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, and eluting with 5ml of pure water for the first time; eluting with 5ml of 10% methanol for the second time, collecting 10% methanol eluate, evaporating to dryness, redissolving with 1ml of 10% methanol, filtering, and collecting the filtrate as sample solution; the results obtained are shown as a in fig. 3.
0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902) is taken, precisely weighed, placed in a conical bottle with a plug, added with 10ml of pure water, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuged, and taken as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, and eluting with 5ml of pure water for the first time; eluting with 5ml of 20% methanol for the second time, collecting 20% methanol eluate, evaporating to dryness, redissolving with 1ml of 20% methanol, filtering, and collecting the filtrate as sample solution; the results obtained are shown as B in fig. 3.
0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902) is taken, precisely weighed, placed in a conical bottle with a plug, added with 10ml of pure water, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuged, and taken as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, and eluting with 5ml of pure water for the first time; eluting with 5ml of 30% methanol for the second time, collecting 30% methanol eluate, evaporating to dryness, redissolving with 1ml of 30% methanol, filtering, and collecting the filtrate as sample solution; the results obtained are shown as C in fig. 3.
0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902) is taken, precisely weighed, placed in a conical bottle with a plug, added with 10ml of pure water, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuged, and taken as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, and eluting with 5ml of pure water for the first time; eluting with 5ml of 40% methanol for the second time, collecting 40% methanol eluate, evaporating to dryness, redissolving with 1ml of 40% methanol, filtering, and collecting the filtrate as sample solution; the results obtained are shown as D in fig. 3.
0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902) is taken, precisely weighed, placed in a conical bottle with a plug, added with 10ml of pure water, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuged, and taken as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, and eluting with 5ml of pure water for the first time; eluting with 5ml of 50% methanol for the second time, collecting 50% methanol eluate, evaporating to dryness, redissolving with 1ml of 50% methanol, filtering, and collecting the filtrate as sample solution; the results obtained are shown as E in FIG. 3.
0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902) is taken, precisely weighed, placed in a conical bottle with a plug, added with 10ml of pure water, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuged, and taken as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, and eluting with 5ml of pure water for the first time; eluting with 5ml of 80% methanol for the second time, collecting 80% methanol eluate, evaporating to dryness, redissolving with 1ml of 80% methanol, filtering, and collecting the filtrate as sample solution; the results obtained are shown as F in fig. 3.
0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902) is taken, precisely weighed, placed in a conical bottle with a plug, added with 10ml of pure water, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuged, and taken as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol for activation, leaching at a leaching speed not exceeding 1ml/min; eluting with 5ml pure water at a rate not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, and eluting with 5ml of pure water for the first time; eluting with 5ml of methanol for the second time, collecting methanol eluate, evaporating to dryness, redissolving with 1ml of methanol, and filtering to obtain filtrate as sample solution; the results obtained are shown as G in fig. 3.
The results show that: the impurities in the traditional Chinese medicine standard decoction are mostly water-soluble components such as starch, polysaccharide, protein, mucilage, tannin pigment, inorganic salt and the like and low-density grease components, so that pure water is firstly adopted for first elution in the SPE process, and the interference of the water-soluble impurities on a liquid spectrogram is reduced. According to the similar principle of miscibility, the eluting power of the components with large polarity gradually decreases and the eluting power of the components with small polarity gradually increases with the increase of the proportion of methanol in the eluting solvent. When the eluting solvent is 30% methanol to 50% methanol, other large polar components except for small polar components such as mashed matters, fatty acids and the like are reserved in the solid phase extraction small column, and the large polar components are eluted. According to the liquid-phase spectrogram analysis, when the second eluting solvent is 30% methanol, the chromatographic peak information is rich, the separation degree of each chromatographic peak is good, and the response is high, so that the optimal chromatographic analysis effect can be achieved by adopting 30% methanol as the second eluting solvent in the SPE pretreatment process of the semen trichosanthis standard decoction.
(2) Inspection of solid phase extraction packing
Taking 0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902), precisely weighing, placing into a conical bottle with a plug, adding 10ml of pure water, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuging, and taking an oil layer and water layer mixed solution as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, soaking in 5ml methanol for 30min, loading into column, and leaching with 5ml methanol for activation at a speed of not more than 1ml/min; eluting with 5ml pure water at a speed not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, performing first elution with 5ml of pure water, performing second elution with 5ml of 30% methanol, collecting 30% methanol eluent, re-dissolving with 1ml of 30% methanol, filtering, and taking the subsequent filtrate as a test sample solution; the results obtained are shown as a in fig. 4.
Taking 0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902), precisely weighing, placing into a conical bottle with a plug, adding 10ml of pure water, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuging, and taking an oil layer and water layer mixed solution as a solid phase extraction test sample; weighing 0.5g ODS-A-HG, soaking in 5ml methanol for 30min, loading on column, and eluting with 5ml methanol for activation at se:Sup>A speed of not more than 1ml/min; eluting with 5ml pure water at a speed not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, performing first elution with 5ml of pure water, performing second elution with 5ml of 30% methanol, collecting 30% methanol eluent, re-dissolving with 1ml of 30% methanol, filtering, and taking the subsequent filtrate as a test sample solution; the results obtained are shown in FIG. 4B.
Taking 0.3g of semen trichosanthis freeze-dried powder (batch number: GLZ-190902), precisely weighing, placing into a conical bottle with a plug, adding 10ml of pure water, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuging, and taking an oil layer and water layer mixed solution as a solid phase extraction test sample; weighing C8-HG, adding 5ml of methanol, soaking for 30min, loading on a column, and leaching and activating with 5ml of methanol at a speed not exceeding 1ml/min; eluting with 5ml pure water at a speed not exceeding 1ml/min; taking 250 μl of a solid phase extraction test sample, performing first elution with 5ml of pure water, performing second elution with 5ml of 30% methanol, collecting 30% methanol eluent, re-dissolving with 1ml of 30% methanol, filtering, and taking the subsequent filtrate as a test sample solution; the results are shown in FIG. 4C. Analysis of results: the three types of fillers and their characteristics used in this study are shown in table 1.
TABLE 1 solid phase extraction packing details table
The average pH value of the semen trichosanthis standard decoction is 5.4, so three acid-resistant solid-phase extraction fillers are selected in the study. According to spectrogram information analysis, the SP-120-30/50-ODS-RPS and ODS-A-HG fillers have large chromatographic peak information quantity relative to the C8-HG fillers, and meanwhile, the SP-120-30/50-ODS-RPS fillers have the characteristics of high coverage rate, high repetition rate, strong stability and the like, are suitable for se:Sup>A characteristic spectrum analysis process, are relatively low in price, and are determined to be SP-120-30/50-ODS-RPS in se:Sup>A solid phase extraction process from the economic point of view.
In summary, the pretreatment method of the sample solution of the semen trichosanthis feature map comprises the following steps: taking 0.3g of semen trichosanthis freeze-dried powder, precisely weighing, placing into a conical bottle with a plug, adding 10ml of pure water, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, centrifuging, and taking the mixed solution of an oil layer and a water layer as a solid phase extraction test sample; weighing 0.5g SP-120-30/50-ODS-RPS, adding 5ml methanol, soaking for 30min, loading on column, leaching with 5ml methanol, and activating at a speed not exceeding 1ml/min; eluting with 5ml pure water at a speed not exceeding 1ml/min; taking 250 μl of solid phase extraction test sample, eluting with 5ml of pure water for the first time, eluting with 5ml of 30% methanol for the second time, collecting 30% methanol eluate, redissolving with 1ml of 30% methanol, filtering, and collecting the subsequent filtrate.
Example 3
Based on the embodiment 1-2, the chromatographic conditions (wavelength, column temperature and flow velocity) in the construction of the characteristic spectrum are examined in the embodiment, and the technical scheme is further described.
(1) Investigation of elution solvent
The sample solution of semen Trichosanthis was subjected to full-band scanning by using a diode array detector, chromatograms of the sample solution at wavelengths of 230nm, 245nm, 265nm and 285nm were extracted, and the other chromatograms were measured under the same conditions as in example 1, and the results are shown in FIG. 5 (5A, 5B, 5C and 5D).
The results show that: when the detection wavelength is 245nm, the information amount of the chromatographic peak is large, the base line of the chromatographic peak is stable, and the separation degree between the chromatographic peaks is good, so that the detection wavelength is determined to be 245nm.
(2) Column temperature investigation
The column temperatures were set at 30℃and 35℃and 40℃respectively, and the other chromatographic conditions were the same as in example 1, and the results were shown in FIG. 6 (6A, 6B, 6C).
The results show that: when the column temperature is 35 ℃, the chromatographic peak type is symmetrical, and the separation degree is moderate, so the column temperature is set to be 35 ℃.
(3) Flow rate investigation
The flow rates were set to 0.4ml/min, 0.6ml/min and 0.8ml/min, respectively, and the other chromatographic conditions were the same as in example 1, and the results were shown in FIG. 7 (7A, 7B and 7C).
The results show that: when the flow rate was 0.6ml/min, each characteristic peak-to-peak type was good, and the degree of separation was moderate, so the flow rate was determined to be 0.6ml/min.
In summary, the chromatographic conditions of the semen trichosanthis characteristic spectrum and the systematic adaptability test determine that the method is preferable: the chromatographic column was Agilent ZORBAX SB-C18 (4.6mm. Times.250 mm,5.0 μm); column temperature is 35 ℃; the flow rate is 0.6mL min < -1 >; detection wavelength 245nm; sample injection amount is 10 μl; methanol is a mobile phase A, and 0.1% phosphoric acid water is a mobile phase B; the gradient elution procedure was:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 5～20 | 95～80 |
| 10～35 | 20～45 | 80～55 |
| 35～53 | 45～95 | 55～5 |
| 53～65 | 95～100 | 5～0 |
Example 4
Based on example 1, this example also performed the following methodology investigation, specifically including:
(1) Precision investigation
Sample solutions of semen Trichosanthis samples were continuously taken six times, and the peak area and the relative peak area, the retention time and the relative retention time of each characteristic peak were calculated, and the obtained results were as shown in tables 2 to 5 below.
Table 2 results of precision investigation (Peak area)
Table 3 results of precision investigation (relative peak area)
TABLE 4 precision investigation results (retention time)
TABLE 5 precision investigation results (relative retention time)
The results show that: the relative retention time RSD and relative peak area RSD of the common peak are less than 5%, and the instrument has good precision.
(2) Repeatability investigation
Six parts of semen trichosanthis freeze-dried powder are taken, the preparation and the measurement of a sample solution are carried out according to the construction method of the semen trichosanthis characteristic spectrum, the peak area and the relative peak area of each characteristic peak are calculated, the retention time and the relative retention time are calculated, and the obtained results are shown in the following tables 6-9.
TABLE 6 repeatability test results (peak area)
TABLE 7 repeatability test results (relative peak area)
TABLE 8 repeatability test results (retention time)
TABLE 9 repeatability test results (relative retention time)
The results show that: the relative retention time RSD and relative peak area RSD of the common peak are all less than 5%, and the method has good repeatability.
(3) Stability investigation
Based on the construction method of the semen trichosanthis characteristic spectrum, the same semen trichosanthis sample solution is taken and measured at the same time of 0h, 2h, 4h, 8h, 12h and 24h respectively, the peak area and the relative peak area of each characteristic peak are calculated, the retention time and the relative retention time are calculated, and the obtained results are shown in tables 10-13 below.
Table 10 stability investigation results (peak area)
TABLE 11 stability study results (relative peak area)
TABLE 12 stability investigation results (retention time)
TABLE 13 stability investigation results (relative retention time)
The results show that: the relative retention time RSD and relative peak area RSD of the common peak are less than 5%, and the sample solution is stable within 24 hours.
(2) Chromatographic column durability investigation
Based on the construction method of the semen trichosanthis characteristic spectrum, 3 chromatographic columns of ZORBAX SB-C18 (4.6 mm multiplied by 250mm,5.0 μm), ACE Excel 5C18-AR (4.6 mm multiplied by 250mm,5.0 μm) and Kromasil C18 (4.6 mm multiplied by 250mm,5.0 μm) are respectively examined, and the obtained results are shown in fig. 8 (8A, 8B and 8C).
The results show that: the chromatographic patterns of different chromatographic columns have larger difference, wherein compared with the chromatographic columns, ZORBAX SB C18 has more chromatographic peak information and better separation degree of each peak, so the chromatographic columns Aglient ZORBAX SB-C18 are fixed in the liquid phase condition to be used as analysis columns of the semen trichosanthis characteristic spectrum.
To sum up: the relative retention time of each characteristic peak and the RSD value of the relative peak area meet the requirements in the above investigation, and the analysis method can be suitable for the analysis of the semen trichosanthis characteristic spectrum.
(5) Determination of characteristic spectrum and establishment of contrast spectrum
In the embodiment of the invention, 3 batches of snakegourd seed standard decoction are utilized for verifying the result.
Based on the construction method of the semen trichosanthis characteristic spectrum, characteristic spectrum analysis is carried out on three batches of semen trichosanthis samples, the peak area and the relative peak area of each characteristic peak are calculated, the retention time and the relative retention time are calculated, and the obtained results are shown in tables 14-17 below.
Table 14 examination results (peak area) of three batches of semen Trichosanthis standard decoction
| | Peak | 1 | Peak 2 (S) | |
|
|
|
| 190904 | 127.22 | 220.98 | 149.42 | 165.9 | 606.8 | 204.11 | |
| 190905 | 125.51 | 198.54 | 131.08 | 155.15 | 616.89 | 169.12 | |
| 190910 | 132.17 | 190.18 | 115.65 | 125.76 | 610.93 | 178.17 |
Table 15 examination results (relative peak area) of three batches of semen Trichosanthis standard decoction
| | Peak | 1 | Peak 2 (S) | |
|
|
|
| 190904 | 0.576 | 1.00 | 0.676 | 0.751 | 2.75 | 0.924 | |
| 190905 | 0.632 | 1.00 | 0.660 | 0.781 | 3.11 | 0.852 | |
| 190910 | 0.695 | 1.00 | 0.608 | 0.661 | 3.21 | 0.937 | |
| RSD％ | 9.39 | 0.00 | 5.49 | 8.54 | 8.00 | 5.06 |
Table 16 examination results (retention time) of three batches of semen Trichosanthis standard decoction
| | Peak | 1 | Peak 2 (S) | |
|
|
|
| 190904 | 8.49 | 10.18 | 23.11 | 24.73 | 30.65 | 34.04 | |
| 190905 | 8.36 | 10.09 | 23.14 | 24.80 | 30.96 | 34.63 | |
| 190910 | 8.38 | 10.08 | 23.09 | 24.77 | 30.88 | 34.56 |
Table 17 examination results (relative retention time) of three batches of semen Trichosanthis standard decoction
| | Peak | 1 | Peak 2 (S) | |
|
|
|
| 190904 | 0.834 | 1.00 | 2.27 | 2.43 | 3.01 | 3.34 | |
| 190905 | 0.829 | 1.00 | 2.29 | 2.46 | 3.07 | 3.43 | |
| 190910 | 0.831 | 1.00 | 2.29 | 2.46 | 3.06 | 3.43 | |
| RSD％ | 0.30 | 0.00 | 0.51 | 0.71 | 1.06 | 1.53 |
According to the principle that the relative retention time is stable, each batch of test samples can be detected and the chromatographic peaks are relatively high, 6 chromatographic peaks are selected as characteristic peaks.
Establishing a control map: the 3 batches of semen trichosanthis standard decoction are synthesized by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and a comparison spectrum of semen trichosanthis characteristic spectrum is established, so that the quality of the semen trichosanthis can be controlled more accurately and integrally, as shown in figure 2. Wherein peak 2 is guanosine.
The above embodiments are not limited to the scope of the present invention, and all modifications or variations based on the basic idea of the present invention are within the scope of the present invention.
Claims (8)
1. The snakegourd seed characteristic spectrum analysis method based on SPE pretreatment is characterized by comprising the following steps of:
(1) Preparation of test solution:
adding pure water into the semen trichosanthis standard decoction freeze-dried powder for full dissolution, wherein the mass volume ratio of the semen trichosanthis standard decoction freeze-dried powder to the pure water is 0.3:10, carrying out ultrasonic treatment and centrifugation, and taking an oil layer and water layer mixed solution as a solid phase extraction test sample; adding methanol into solid-phase extraction packing of semen trichosanthis for full soaking, wherein the mass-volume ratio of the solid-phase extraction packing of semen trichosanthis to the methanol is 1:10, and filling the column; leaching and activating by methanol; eluting and balancing with pure water; sampling a solid phase extraction test sample; eluting with pure water for the first time; eluting for the second time by using 30-50% methanol eluent, collecting the methanol eluent, evaporating to dryness, re-dissolving, filtering, and obtaining a subsequent filtrate to obtain a sample solution;
the solid-phase extraction filler of semen trichosanthis in the step (1) comprises SP-120-30/50-ODS-RPS or ODS-A-HG;
(2) Preparation of a control solution:
weighing guanosine reference substance, preparing into reference substance solution with concentration of 3.0 μg/ml by taking methanol with volume concentration of 10% as solvent, filtering, and taking subsequent filtrate as reference substance solution;
(3) Liquid phase analysis process:
respectively taking a sample solution and a reference substance solution, respectively injecting into a high performance liquid chromatograph, measuring, and establishing an HPLC characteristic spectrum to obtain a semen trichosanthis characteristic spectrum;
the high performance liquid chromatography conditions in the step (3) are specifically as follows: the column was Agilent ZORBAX SB with a diameter of 4.6 mm. Times.250 mm and a diameter of 5.0. Mu.m-C 18 The method comprises the steps of carrying out a first treatment on the surface of the The column temperature is 30-40 ℃; the flow rate is 0.4-0.8 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the The detection wavelength is 230-284 nm; the sample injection amount is 10 mu L; methanol is a mobile phase A, and phosphoric acid water with volume concentration of 0.1% is a mobile phase B; gradient elution is carried out;
wherein, the elution procedure is:
2. the method for analyzing the characteristics of semen trichosanthis based on SPE pretreatment according to claim 1, wherein the methanol eluent with the volume concentration of 30% is preferable in the step (1).
3. The method for analyzing the semen trichosanthis characteristic spectrum based on SPE pretreatment as recited in claim 1, wherein the semen trichosanthis solid phase extraction filler in the step (1) is SP-120-30/50-ODS-RPS with the particle size of 12nm-50 μm.
4. The method for analyzing the semen trichosanthis characteristic spectrum based on SPE pretreatment as recited in claim 1, wherein the preparation conditions of the semen trichosanthis standard decoction freeze-dried powder in the step (1) are as follows: mashing semen Trichosanthis decoction pieces, and adding pure water for full soaking, wherein the mass ratio of semen Trichosanthis decoction pieces to water is 1:8; heating and boiling, decocting for 45-60 min, sieving to obtain medicinal residues, adding 7 times of water into the medicinal residues, heating and boiling, decocting for 30-60 min, filtering while hot, mixing the two medicinal liquids to obtain semen Trichosanthis standard decoction, taking standard decoction sample, and lyophilizing to obtain lyophilized powder.
5. The method for analyzing the semen trichosanthis feature map based on SPE pretreatment as recited in claim 1, wherein the power of the ultrasonic treatment in the step (1) is 250W, and the frequency is 40kHz; the rotational speed of the centrifugation is 4000r/min, and the time is 10min; the rinsing speed is not more than 1ml/min; the evaporating step comprises the following steps: vacuum concentration is adopted, the vacuum degree is 90mbar, the rotating speed is 100rpm, and the water bath temperature is 60 ℃ for evaporating.
6. The method for analyzing the semen trichosanthis feature map based on SPE pretreatment as claimed in claim 1, wherein the filtering in the step (2) specifically comprises: passing through a microporous filter membrane of 0.22 μm.
7. The method for analyzing the semen trichosanthis characteristic spectrum based on SPE pretreatment as recited in claim 1, wherein the conditions of the high performance liquid chromatography in the step (3) are as follows: the chromatographic column was Agilent ZORBAX SB-C with a diameter of 4.6mm by 250mm and a diameter of 5.0. Mu.m 18 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature is 35 ℃; flow rate 0.6mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Detection wavelength 245nm; the sample injection amount is 10 mu L; methanol is a mobile phase A, and 0.1% phosphoric acid water is a mobile phase B; gradient elution was performed.
8. The method for analyzing the characteristic pattern of the semen trichosanthis based on the SPE pretreatment as claimed in claim 1, wherein the characteristic pattern of the semen trichosanthis in the step (3) comprises 6 characteristic peaks, the peak corresponding to the guanosine reference is taken as an S2 peak, the relative retention time of the peak 2 and the peak 1, the peak 3, the peak 4, the peak 5 and the peak 6 is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is 0.831-peak 1, 2.284-peak 3, 2.447-peak 4, 3.046-peak 5 and 3.412-peak 6.
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