CN115372517B - Construction and application of UPLC characteristic spectrum of immature bitter orange, longstamen onion bulb and cassia twig decoction - Google Patents

Construction and application of UPLC characteristic spectrum of immature bitter orange, longstamen onion bulb and cassia twig decoction Download PDF

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CN115372517B
CN115372517B CN202211082618.8A CN202211082618A CN115372517B CN 115372517 B CN115372517 B CN 115372517B CN 202211082618 A CN202211082618 A CN 202211082618A CN 115372517 B CN115372517 B CN 115372517B
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cassia twig
peak
bitter orange
decoction
immature bitter
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CN115372517A (en
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祝倩倩
石海培
陈盛君
李松
王协和
浦香兰
袁健
李淑娟
周海琴
王琪
王慧敏
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Jiangyin Tianjiang Pharmaceutical Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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

Abstract

The invention discloses construction and application of a UPLC characteristic spectrum method of an immature bitter orange, longstamen onion bulb and cassia twig decoction, wherein the characteristic spectrum adopts an ultra-high performance liquid phase, and the integral profile of an immature bitter orange, longstamen onion bulb and cassia twig decoction sample is characterized and subjected to attribution analysis by an optimized detection method, so that the chromatographic information of each medicinal flavor in the immature bitter orange, longstamen onion bulb and cassia twig decoction is fully displayed, and the effective traceability and comprehensive control of the quality of prescription medicinal flavor and product quality are fully embodied. Secondly, the method is applied to detection and quality control of medicinal materials and decoction pieces of 5 medicinal herbs in a prescription, realizes characteristic transmission from medicinal materials to decoction pieces to compound preparation samples, and achieves the purposes of quality transmission and quality tracing. The method has the advantages of simple operation, time saving and high efficiency, comprehensive chromatographic information of characteristic components and strong ingredient specificity, and provides scientific and effective research basis for development of the compound preparation of the immature bitter orange, allium macrostemon and cassia twig decoction and process control of product quality.

Description

Construction and application of UPLC characteristic spectrum of immature bitter orange, longstamen onion bulb and cassia twig decoction
Technical Field
The invention relates to a construction method of a characteristic spectrum of an immature bitter orange, longstamen onion bulb and cassia twig decoction, in particular to a construction method and application of a UPLC characteristic spectrum of an immature bitter orange, longstamen onion bulb and cassia twig Shang Jizhun sample, which can represent chromatographic information of each medicine in a prescription, comprehensively, quickly and reliably.
Background
The decoction of immature bitter orange, longstamen onion bulb and cassia twig is derived from 22 nd classical prescription directory (first batch) published in 2018, and original text is from Han-Zhang Zhongjing Lloyd 'jin Kui Yao' and is a classical proved recipe for treating chest stuffiness. The original text records the prescription composition and the preparation method as follows: four immature bitter oranges, four magnolia officinalis, two magnolia officinalis, half jin of allium macrostemon, one two cassia twig, and one snakegourd fruit (pounded). Five flavors of the drugs are added, five liters of water are used for boiling immature bitter orange and magnolia bark, two liters of water are taken for removing dregs, various drugs are taken in, boiling is performed, and three times of warm taking are performed. The compatibility principle of the recipe is as follows: fructus Trichosanthis and Bulbus Allii Macrostemi are monarch drugs. Allium macrostemon Bai Xinwen, activating yang, resolving masses, resolving phlegm and dispelling cold; gua Lou is cold in nature entering lung, and has actions of resolving phlegm and resolving masses, opening chest and relieving arthralgia. Fructus Aurantii Immaturus and cortex Magnolia officinalis are ministerial drugs, and are effective in descending qi, resolving hard mass, relieving distention and fullness, eliminating dampness and phlegm, and descending qi, and relieving fullness. Ramulus Cinnamomi is used as adjuvant drug for activating yang, dispelling cold, and calming the pulse and lowering adverse-rising energy; the compatibility of the medicines can lead to the symptoms of chest yang vibration, turbid phlegm descending, yin cold eliminating and qi movement smoothening, and the symptoms of chest obstruction and qi back flushing can be removed.
The research of classical prescriptions basically requires that the quality attribute of each medicine in the prescription should be reflected, a detection method which more comprehensively reflects the quality of a reference sample or a preparation is established, and meanwhile, the established evaluation index can reflect the correlation of medicinal materials, decoction pieces, reference samples and compound preparations, so that the quantity value transmission condition in the whole development process can be illustrated, and the internal quality of the compound preparations can be better controlled.
The modern pharmaceutical researches of the immature bitter orange, the magnolia officinalis and the cassia twig in the immature bitter orange, the longstamen onion bulb and the cassia twig decoction are more complete, the quality standard and the related detection items are more definite, and the quality control method of corresponding content indexes is also provided under each medicinal taste item of Chinese pharmacopoeia 2010 edition. The snakegourd fruit and the allium macrostemon belong to difficult varieties in the field of traditional Chinese medicine research, the research basis is weak, and the quality standard requirements of content, characteristic patterns and the like are not specified under 2010 edition pharmacopoeia. Because the medicinal flavors of the snakegourd fruit and the allium macrostemon mainly contain volatile and fatty oil and other small polar substances, the stability of the components is poor, and the difficulty of detection analysis and quality control is high. The classical prescription research requires the traditional mode of extraction, generally adopts water decoction, and the volatile components and the small-polarity effective components in the snakegourd fruit and the allium macrostemon have very low content in the aqueous solution and are not easy to detect. How to express the active ingredients of snakegourd fruit and allium macrostemon in the water decoction becomes the key point of the research. In addition, the compound contains a plurality of medicines, the chemical components are complex, and the components with different polarities in each medicine are comprehensively represented and effectively separated on one map, so that the analysis and detection are very difficult.
In the prior art, the quality standard research on the citrus aurantium, allium macrostemon and cassia twig decoction is less, at present, only 2 patents CN111759937A, CN114062563A respectively adopt an HPLC method to establish the fingerprint spectrum and the characteristic spectrum of the citrus aurantium macrostemon and cassia twig decoction, but the spectrum characterization information of the two patents is not perfect enough, the chromatographic peaks are relatively less, and the chromatographic peak information of allium macrostemon in the prescription is not reflected. The allium macrostemon in the recipe is a monarch drug of the recipe and is also a key drug for treating chest stuffiness, and is a key for the recipe to exert clinical curative effect, so the characterization of allium macrostemon spectrum information has very important significance for the quality control of the compound preparation and the exertion of the curative effect of the product.
Disclosure of Invention
The invention aims to: the invention aims to provide a construction method of UPLC characteristic spectrum of citrus aurantium, allium macrostemon and cassia twig decoction, which is characterized comprehensively and efficiently, and solves the problems of less chromatographic peak information, incomplete drug flavor characterization, low detection efficiency, poor quality control effect and the like of the existing citrus aurantium macrostemon and cassia twig decoction quality control method; the invention further aims to provide an application of the method in detection and quality control of corresponding medicinal materials and decoction pieces in a prescription, and an evaluation method for quality transmission and quality tracing is realized, namely, consistency of quality of samples in a plurality of links of an industrial chain is evaluated by adopting a set of method, and quality tracing and control of the whole process of a product are achieved.
The technical scheme is as follows: the construction method of the UPLC characteristic spectrum of the immature bitter orange, the allium macrostemon and the cassia twig decoction comprises the following steps:
(1) Adding solvent into the reference substance to obtain reference substance solution;
(2) Taking the decoction of the immature bitter orange, the allium macrostemon and the cassia twig as a test sample, adding a solvent for treatment, and preparing a test sample solution;
(3) Detecting by liquid chromatography;
(4) Establishing a characteristic map: the peak values are 22, and 10 characteristic peaks are identified by reference substances, namely uridine, adenosine, guanosine, rutin naringin, hesperidin, neohesperidin, cinnamaldehyde, honokiol and magnolol.
Further, the relative retention time of each of the common peaks in step (4) is as follows:
the relative retention times of the 8 consensus peaks with guanosine as reference peak S1 are as follows: 0.68 (±10%), 0.75 (+ -10%), 0.94 (+ -10%), 1.00 (+ -10%), 1.82 (+ -10%), 2.34 (+ -10%), 3.01 (+ -10%).
The relative retention times of 14 common peaks with naringin as reference peak S2 are as follows: 0.81 (±10%), 0.85 (+ -10%), 0.88 (+ -10%), 0.94 (+ -10%), 1.00, 1.06 (+ -10%), 1.13 (+ -10%), 1.23 (+ -10%), 1.33 (+ -10%), 1.37 (+ -10%), 1.44 (+ -10%), 1.47 (+ -10%), 1.52 (+ -10%), and 1.56 (+ -10%).
Further, the concentrations of the reference substance and the solution thereof in the step (1) are as follows: 10-50 mu g/mL of uridine, 5-50 mu g/mL of adenosine, 5-50 mu g/mL of guanosine, 30-100 mu g/mL of rutin, 0.1-1.0mg/mL of naringin, 0.1-1.0mg/mL of hesperidin, 0.1-1.0mg/mL of neohesperidin, 5-50 mu g/mL of cinnamaldehyde, 10-50 mu g/mL of honokiol and 10-100 mu g/mL of magnolol.
Further, the test sample is an extracting solution (decoction), a reference sample (freeze-dried powder), a preparation intermediate product or a preparation finished product of the immature bitter orange, the allium macrostemon and cassia twig decoction.
Further, the specific steps of the solvent adding treatment in the step (2) are as follows: taking water or methanol-water solution as a solvent, adding 1g of a test sample into each 30-50 mL, carrying out ultrasonic treatment and dissolving.
Further, the chromatographic conditions in step (3) are specifically: octadecylsilane chemically bonded silica chromatographic column, mobile phase a: acetonitrile, mobile phase B: 0.05-1% of acid solution by volume fraction, detection wavelength: 210-360 nm, flow rate: 0.25-0.35 mL/min; column temperature: 20-40 ℃; elution was performed according to the following elution gradient:
further, the length of the chromatographic column is 100-150 mm, and the particle size of the chromatographic column packing is 1.7-1.9 mu m.
Further, the sample injection amount of the test sample solution detection is 1-2 mu L.
The construction method of the UPLC characteristic spectrum of the immature bitter orange, the allium macrostemon and the cassia twig decoction can be applied to the chromatographic information for representing 5 medicinal flavors of snakegourd fruit, allium macrostemon, immature bitter orange, magnolia officinalis and cassia twig, and the quality of the immature bitter orange, allium macrostemon and cassia twig decoction extract and the preparation thereof is detected and controlled. Wherein, the preparation process of the sample solution of corresponding medicinal materials and decoction pieces of snakegourd fruit, allium macrostemon, immature bitter orange, magnolia officinalis and cassia twig (5 medicinal flavors) comprises the following steps: taking a proper amount of medicinal materials or decoction piece powder, adding 15-50 ml of methanol with the volume fraction of 10-50%, carrying out ultrasonic treatment for 30 minutes, cooling and shaking uniformly, filtering, and taking a subsequent filtrate as a sample solution.
Further, among the 22 common peaks, peaks 2-4,6,8 are assigned to allium macrostemon, peaks 2-8 are assigned to trichosanthes kirilowii Maxim, peaks 1-4,6,8-15,17-20 are assigned to immature bitter orange, peaks 21-22 are assigned to magnolia officinalis, and peak 16 is assigned to cassia twig; wherein, peak 5,7 is the exclusive ingredient of fructus Trichosanthis, peak 1,9-15,17-20 is the exclusive ingredient of fructus Aurantii Immaturus, peak 21-22 is the exclusive ingredient of cortex Magnolia officinalis, and peak 16 is the exclusive ingredient of ramulus Cinnamomi.
The UPLC characteristic spectrum established by the invention can be used for detecting and controlling the quality of medicinal materials and decoction pieces of snakegourd fruit, allium macrostemon, immature bitter orange, magnolia officinalis, cassia twig, and the like, realizes the magnitude transfer from the medicinal materials to the decoction pieces to the immature bitter orange allium macrostemon and cassia twig decoction compound preparation, and ensures the consistency of characteristic peaks and stable quality of the compound preparation.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) The construction of the characteristic spectrum method fully considers the compatibility principle of the prescription, overcomes the difficulties of large polarity difference, large separation difficulty and the like of the active ingredients of each medicine, particularly characterizes, attributes and identifies the ingredients in the monarch medicine allium macrostemon, and solves the phenomenon that the allium macrostemon information is lost or cannot be controlled in the prior art; (2) The method constructs a characteristic spectrum of the citrus aurantium, allium macrostemon and cassia twig decoction for representing the full medicinal taste information of the prescription. 22 characteristic peaks are presented, the characteristic component information is rich, the specificity is strong, and the overall characteristics and the product quality of the compound can be comprehensively reflected; (3) The quality condition of the product can be rapidly detected, and the method is suitable for the requirements of continuous production. Compared with an HPLC detection method, the ultra-high performance liquid chromatography is more rapid and efficient, realizes rapid quality detection to meet the industrial production requirement and improves the production efficiency; (4) The method is simple and convenient to operate, is widely applicable, and is suitable for quality evaluation of extracts, reference samples, preparation intermediate products and finished products of the immature bitter orange, the allium macrostemon and cassia twig decoction; (5) The method is applied to the whole process quality detection and control of medicinal materials, decoction pieces, intermediates and finished products, realizes the whole process quantity value transmission and monitoring of characteristic peaks from the medicinal materials, decoction pieces to the production of the products, and simultaneously is used as powerful judgment and basis for product quality tracing to ensure the stability and consistency of the quality among batches of the immature bitter orange, allium macrostemon and cassia twig compound preparation; (6) The invention provides a firm and sufficient scientific basis for the research and development of the compound preparation of the classic formula of the immature bitter orange, the allium macrostemon and the cassia twig decoction.
Drawings
Fig. 1 is a chromatogram specific to the spectrum of citrus aurantium, allium macrostemon and cassia twig Shang Tezheng of example 1;
fig. 2 is an experiment for prolonging the characteristic spectrum of the decoction of immature bitter orange, longstamen onion bulb and cassia twig in example 1;
FIG. 3 is a characteristic overlay map of 15 batches of reference samples in example 1;
FIG. 4 is a reference sample identification chromatogram of example 1;
FIG. 5 is a comparative characteristic map of the decoction of Zhi Xie Bai Gui Zhi;
FIG. 6 is a characteristic map of the fructus Trichosanthis medicinal material of example 3;
FIG. 7 is a characteristic spectrum of the allium macrostemon herb of example 3;
FIG. 8 is a characteristic map of immature bitter orange medicinal material in example 3;
FIG. 9 is a characteristic map of Magnolia officinalis of example 3;
fig. 10 is a characteristic map of cassia twig medicinal material of example 3;
FIG. 11 shows the characteristic peak assignment of the fructus Trichosanthis in example 3;
FIG. 12 is a characteristic peak assignment of the allium macrostemon in example 3;
FIG. 13 is the characteristic peak assignment of immature bitter orange in example 3;
FIG. 14 is a characteristic peak assignment of Magnolia officinalis of example 3;
FIG. 15 shows the characteristic peak assignment of ramulus Cinnamomi in example 3.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1
Feature map methodology validation
The preparation method of the citrus aurantium and allium macrostemon cassia twig decoction preparation (reference sample) comprises the following steps:
soaking fructus Aurantii Immaturus and cortex Magnolia officinalis in 1000mL water for 30-60min, decocting for 30-60min, filtering, and removing residue to obtain decoction; adding Bulbus Allii Macrostemi, fructus Trichosanthis, and ramulus Cinnamomi into the decoction, decocting for 10-20 min, filtering, and lyophilizing.
1 instrument and reagent
Agilent Technologies 1290 Infinicity ultra-high performance liquid chromatograph; 1290DAD FS diode array detector; 1290MCT column incubator; 1290Vial sample autosampler; 1290Fiexible Pump quaternary Pump; open LAB 2.3 chromatographic workstation; an electronic analytical balance; KQ-250B ultrasonic cleaner; a pure water system; AS165W type centrifuge.
Determination of 2 chromatographic conditions
Octadecyl bonded silica gel is used as filler (column length is 150mm, inner diameter is 2.1mm, and granularity is 1.8 μm); acetonitrile is taken as a mobile phase A, a formic acid solution with the volume fraction of 0.1 percent is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 280nm.
Preparation of reference solution
Preparation of a reference solution for a control: taking a proper amount of uridine reference substance, adenosine reference substance, guanosine reference substance and rutin reference substance, precisely weighing, adding 10% volume fraction methanol water solution to prepare a solution containing 15 mug of uridine, 10 mug of adenosine, 10 mug of guanosine and 50 mug of rutin per 1ml, and shaking uniformly; taking appropriate amounts of naringin reference substance, hesperidin reference substance, neohesperidin reference substance, cinnamaldehyde reference substance, honokiol reference substance and magnolol reference substance, precisely weighing, adding methanol to dissolve and prepare a solution containing 0.5mg of naringin, 0.5mg of hesperidin, 0.5mg of neohesperidin, 10 mug of cinnamaldehyde, 25 mug of honokiol and 30 mug of magnolol per 1ml, and shaking uniformly; all were used as reference solutions for the control.
Preparation of sample solution
(1) Method for preparing sample solution
The preparation method is finally determined as follows: taking a proper amount of the product, about 0.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of 50% volume fraction methanol aqueous solution, sealing, weighing, performing ultrasonic treatment (250W, 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% volume fraction methanol aqueous solution, shaking uniformly, filtering, and taking subsequent filtrate.
(2) Investigation of specificity
The extraction solvent has no interference to the measurement of characteristic spectrum of sample of the citrus aurantium, allium macrostemon and cassia twig Shang Jizhun, and the result is shown in figure 1.
(3) Integrity inspection
On the same chromatographic conditions, the elution gradient when the organic phase ratio is highest is maintained, the elution time is doubled, and the chromatogram is recorded.
The result shows that no obvious chromatographic peak flows out after the original gradient elution is finished, the chromatographic condition basically meets the principle of maximum information quantity, and the result is shown in the figure 2.
(4) Investigation of precision
Preparing a sample with a method batch number, preparing a test solution according to a test solution preparation method, continuously injecting the sample for 6 times, 1 μl each time, and recording a chromatogram. The relative retention time of peak 1 to peak 8 was calculated with peak 4 corresponding to guanosine as reference peak S1, and peak 13 corresponding to naringin as reference peak S2, and the relative retention time of peak 9 to peak 22 was calculated, with the results shown in the following table.
TABLE 1 investigation of precision
TABLE 2 investigation of precision
Note that: f2-uridine, F3-adenosine, F4-guanosine, F12-naringin, F13-naringin, F15-neohesperidin, F16-cinnamaldehyde, F21-honokiol and F22-magnolol.
Conclusion: the relative retention time of each characteristic peak has RSD less than 1% and good precision.
(5) Stability investigation
Taking a lot number sample, preparing a test solution according to a test solution preparation method, respectively injecting samples at 0h,2h,4h,6h,8h and 12h, and recording a chromatogram with 1 μl of each sample injection. The relative retention time of peak 1 to peak 8 was calculated with peak 4 corresponding to guanosine as reference peak S1, and peak 13 corresponding to naringin as reference peak S2, and the relative retention time of peak 9 to peak 22 was calculated, with the results shown in the following table.
TABLE 3 examination of stability
Table 4 examination of stability
Note that: f2-uridine, F3-adenosine, F4-guanosine, F12-naringin, F13-naringin, F15-neohesperidin, F16-cinnamaldehyde, F21-honokiol and F22-magnolol.
Conclusion: the test solution has good stability (RSD% < 1.0%) within 24 hours.
(6) Repeatability investigation
Preparing a sample with a method batch number, preparing a test solution according to a test solution preparation method, preparing 6 parts in parallel, feeding 1 μl each time, and recording a chromatogram. The relative retention time of peak 1 to peak 8 was calculated with peak 4 corresponding to guanosine as reference peak S1, and peak 13 corresponding to naringin as reference peak S2, and the relative retention time of peak 9 to peak 22 was calculated, with the results shown in the following table.
Table 5 examination of repeatability
Table 6 examination of repeatability
Note that: f2-uridine, F3-adenosine, F4-guanosine, F12-naringin, F13-naringin, F15-neohesperidin, F16-cinnamaldehyde, F21-honokiol and F22-magnolol.
Conclusion: the results show that the relative retention time RSD of each characteristic peak is less than 1%, and the repeatability of the test solution is good.
(7) Determination of sample and determination of common Peak
According to the preparation method and chromatographic conditions of the sample to be tested of the characteristic spectrum determined above, the characteristic spectrum of the samples of the multiple batches of the immature bitter orange allium macrostemon and the cassia twig Shang Jizhun is measured, 1 μl is sampled each time, and a UPLC chromatogram is recorded. And (3) carrying out result analysis by adopting a traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system (2012 edition) recommended by the national formulary committee, selecting a common peak according to the peak attribution condition, calculating the relative retention time of each common peak, fitting a common peak superposition spectrum, and generating a characteristic comparison spectrum of a common mode, wherein the result is shown in figure 3.
TABLE 7 results for multiple batches of test samples (relative retention time)
TABLE 8 results for multiple batches of samples (relative retention time)
Note that: f2-uridine, F3-adenosine, F4-guanosine, F12-naringin, F13-naringin, F15-neohesperidin, F16-cinnamaldehyde, F21-honokiol and F22-magnolol.
Conclusion: through detection of 15 batches of samples, the relative retention time of characteristic peaks is stable, and RSD is less than 1%, which indicates that the characteristic spectrum constructed by the invention is reliable and stable.
(8) Reference substance identification
Taking appropriate amounts of uridine reference substance, adenosine reference substance, guanosine reference substance and rutin control substance, precisely weighing, adding 10% methanol by volume fraction to prepare solution containing 15 μg of uridine, 10 μg of adenosine, 10 μg of guanosine and 50 μg of rutin per 1ml, shaking uniformly; taking appropriate amounts of naringin reference substance, hesperidin reference substance, neohesperidin reference substance, cinnamaldehyde reference substance, honokiol reference substance and magnolol reference substance, precisely weighing, adding methanol to dissolve and prepare a solution containing 0.5mg of naringin, 0.5mg of hesperidin, 0.5mg of neohesperidin, 10 mug of cinnamaldehyde, 25 mug of honokiol and 30 mug of magnolol per 1ml, and shaking uniformly; all were used as reference solutions for the control, and the results are shown in FIG. 4.
Example 2
Establishing a characteristic map:
in order to better ensure the operability and reproducibility of the feature method, facilitate the judgment in the implementation process of the later standard, reduce the influence of different laboratories and external conditions on the repeatability of the standard, comprehensively consider that the reference substance solution of the reference substance is combined with the relative retention time, and establish a feature map in the form of a concomitant reference, and the result is shown in figure 5.
The chromatogram of the test sample should show 22 characteristic peaks, wherein peak 2, peak 3, peak 4, peak 12, peak 13, peak 14, peak 15, peak 16, peak 21, and peak 22 should correspond to the peak retention time of uridine control, adenosine control, guanosine control, rutin control, naringin control, hesperidin, neohesperidin control, cinnamaldehyde control, honokiol control, and magnolol control reference respectively. The peak corresponding to the guanosine reference peak is regarded as an S1 peak, and the relative retention time of the peak 1, the peak 5 to the peak 8 and the S1 peak is calculated, wherein the relative retention time is within a range of + -10% of a specified value, and the specified value is: 0.68 (Peak 1), 1.10 (Peak 5), 1.82 (Peak 6), 2.34 (Peak 7), 3.01 (Peak 8); the peak corresponding to the naringin reference peak is taken as an S2 peak, the relative retention time of the peak 9-11, the peak 17-20 and the S1 peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is: 0.81 (peak 9), 0.85 (peak 10), 0.88 (peak 11), 1.33 (peak 17), 1.37 (peak 18), 1.44 (peak 19), 1.47 (peak 20).
Example 3
And (3) applying a characteristic map:
(1) Feature map measurement of single medicinal material
Preparation of test solution:
weighing 1g of medicinal powder of snakegourd fruit, allium macrostemon and immature bitter orange, precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of 10% volume fraction methanol, sealing, weighing, performing ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 10% volume fraction methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution.
Weighing about 0.5g of magnolia officinalis and cassia twig medicinal material powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 50% volume fraction methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% volume fraction methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution.
Chromatographic conditions: the chromatographic conditions of the citrus aurantium, allium macrostemon and cassia twig Shang Tezheng are the same as those of example 1.
The measurement method comprises precisely sucking 1 μl of reference solution and 1 μl of sample solution, respectively, and injecting into a liquid chromatograph for measurement.
The results are shown in FIGS. 6 to 10.
(2) Characteristic peak assignment
(2.1) Compound negative home verification
The preparation method comprises the following steps: respectively taking 5 compounds of fructus trichosanthis, allium macrostemon, fructus aurantii immaturus, magnolia officinalis and cassia twig-lack decoction pieces, decocting in water, filtering while the compounds are hot, cooling, and freeze-drying to obtain the taste-lack reference sample of the fructus aurantii immaturus, allium macrostemon and cassia twig-lack decoction. Respectively taking a proper amount of each taste-lacking reference sample, precisely weighing, placing into a conical bottle with a plug, adding 25ml of 50% methanol by volume fraction, weighing, ultrasonically (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 50% methanol by volume fraction, shaking uniformly, filtering, and taking the subsequent filtrate.
Chromatographic conditions: the chromatographic conditions of the citrus aurantium, allium macrostemon and cassia twig Shang Tezheng are the same as those of example 1.
The measurement method comprises precisely sucking 1 μl of reference solution and 1 μl of sample solution, respectively, and injecting into a liquid chromatograph for measurement.
(2.2) assignment of single drug to assignment results
The attribution results of the components according to the single drug positive and corresponding negative control are shown in the following table.
Table 9 results of assignment of individual drugs to each chromatographic peak
Peak assignment is performed on snakegourd fruit, longstamen onion bulb, immature bitter orange, magnolia officinalis and cassia twig through the characteristic spectrum research of single medicine, compound negative and reference substance assignment. The chromatogram of the reference sample shows 22 characteristic peaks, wherein 5 peaks are attributed to allium macrostemon, 7 peaks are attributed to snakegourd fruit (wherein peak 5 and peak 7 are exclusive characteristic peaks of snakegourd fruit), 17 peaks are attributed to immature bitter orange (wherein peak 1, peak 9-15 and peak 17-20 are exclusive characteristic peaks of immature bitter orange), 2 peaks are attributed to magnolia officinalis (namely, peak 21 and 22 are exclusive characteristic peaks of magnolia officinalis), 1 peak is attributed to cassia twig (namely, peak 16 is exclusive characteristic peak of cassia twig), and the result is shown in figures 11-15.

Claims (4)

1. The construction method of the UPLC characteristic spectrum of the immature bitter orange, longstamen onion bulb and cassia twig decoction is characterized by comprising the following steps:
(1) Adding solvent into the reference substance to obtain reference substance solution;
(2) Taking the decoction of the immature bitter orange, the allium macrostemon and the cassia twig as a test sample, taking water or methanol-water solution as a solvent, adding 1g of the test sample into each 30-50 mL of the solvent, carrying out ultrasonic treatment, and dissolving to prepare a test sample solution;
(3) Liquid chromatography detection, wherein the chromatographic conditions are as follows: octadecylsilane chemically bonded silica gel chromatographic column, chromatographic column length of 100-150 mm, chromatographic column filler particle diameter of 1.7-1.9 μm, mobile phase A: acetonitrile, mobile phase B: formic acid solution with the volume fraction of 0.05-1 percent, and detection wavelength: 210-360 nm, flow rate: 0.25-0.35 mL/min; column temperature: 20-40 ℃; elution was performed according to the following elution gradient:
(4) Establishing a characteristic map: 22 common peaks are shown, 10 characteristic peaks are identified by a reference substance, namely uridine, adenosine, guanosine, rutin naringin, hesperidin, neohesperidin, cinnamaldehyde, honokiol and magnolol, and the relative retention time of 8 common peaks with guanosine as a reference peak S1 is as follows: 0.68, 0.75, 0.94, 1.00, 1.10, 1.82, 2.34, 3.01, respectively corresponding to peaks 1 to 8; the relative retention times of 14 common peaks with naringin as reference peak S2 are as follows: 0.81, 0.85, 0.88, 0.94, 1.00, 1.06, 1.13, 1.23, 1.33, 1.37, 1.44, 1.47, 1.52, 1.56, respectively, correspond to peaks 9 to 22.
2. The method for constructing a UPLC characteristic spectrum of the immature bitter orange, longstamen onion bulb and cassia twig decoction as set forth in claim 1, wherein the reference substance and the solution concentration thereof in the step (1) are as follows: 10-50 mu g/mL of uridine, 5-50 mu g/mL of adenosine, 5-50 mu g/mL of guanosine, 30-100 mu g/mL of rutin, 0.1-1.0mg/mL of naringin, 0.1-1.0mg/mL of hesperidin, 0.1-1.0mg/mL of neohesperidin, 5-50 mu g/mL of cinnamaldehyde, 10-50 mu g/mL of honokiol and 10-100 mu g/mL of magnolol.
3. The method for constructing the UPLC characteristic spectrum of the immature bitter orange, allium macrostemon and cassia twig decoction, which is disclosed in claim 1, is characterized in that the sample injection amount of the test solution detection is 1-2 mu L.
4. The method for constructing the UPLC characteristic spectrum of the decoction of the immature bitter orange, the allium macrostemon and the cassia twig, according to claim 1, wherein peaks 5 and 7 are exclusive components of snakegourd fruit, peaks 1,9-15,17-20 are exclusive components of immature bitter orange, peaks 21-22 are exclusive components of magnolia officinalis and peak 16 is exclusive component of cassia twig in the 22 common peaks.
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