CN110441442A - A kind of Poria cocos standard decoction UPLC characteristic map construction method and detection method - Google Patents

Abstract

The invention relates to a poria cocos standard decoction UPLC characteristic spectrum construction method and a detection method. The construction method of the UPLC characteristic spectrum of the tuckahoe standard decoction comprises the following steps: (1) precisely weighing Poria standard decoction, and preparing to obtain Poria standard decoction sample solution; (2) analyzing the Poria standard decoction sample solution with ultra-high performance liquid chromatograph to obtain Poria standard decoction UPLC characteristic spectrum. The method has good precision, stability and reproducibility, fully shows the chemical component characteristics of Poria standard decoction, can comprehensively and accurately control the quality of Poria standard decoction, and also provides basis for establishing quality standard of Poria medicinal material and Poria granule.

Description

A kind of Poria cocos standard decoction UPLC characteristic map construction method and detection method

technical field

The invention belongs to the field of research on quality standards of traditional Chinese medicine formula granules, and in particular relates to a construction method and a detection method of UPLC characteristic map of Poria standard decoction.

Background technique

Poria cocos is the dried sclerotia of Poria cocos (Schw.) Wolf (the 2015 edition of "Chinese Pharmacopoeia"), which is a commonly used traditional Chinese medicine. Edema, oliguria, phlegm, dizziness and palpitations, spleen deficiency, lack of food, loose stools and diarrhea, restlessness, palpitations and insomnia, etc., have a wide range of clinical applications. Poria cocos is sweet, light and flat in nature and flavor, and it is listed in the "Shen Nong's Materia Medica", which is listed as the top grade.

The main components contained in Poria cocos are polysaccharides and triterpenoids. In addition, it has been reported that Poria cocos also contains inorganic substances such as steroid components, fatty acids, proteins, adenine, amino acids, and calcium, magnesium, iron, and potassium. element. Modern pharmacological studies have shown that polysaccharides and triterpenoids have significant pharmacological activities in anti-aging, anti-cancer and immune regulation.

The quality control of Poria cocos in the 2015 edition of "Chinese Pharmacopoeia" does not involve the qualitative and quantitative determination of clear ingredients, which has certain defects in the quality control of Poria cocos medicinal materials. At present, there have been many reports on the methods for the determination of the content of the index components of Poria cocos medicinal materials and the method of characteristic finger maps. Zhang Liangqi established a method for the simultaneous determination of 6 components of Poria cocos by using UPLC, which can completely detect all its peaks within 10 minutes, the analysis time is effectively shortened, and the analysis efficiency is significantly improved. methodological basis. Ding Gang used RP-HPLC linear gradient elution and other methods to screen out the best test conditions, and studied the HPLC fingerprints of triterpene acids in Poria cocos at 210nm and 242nm, and identified 9 chromatographic peaks, which more comprehensively reflected the The composition of triterpene acids in Poria cocos provides a methodological basis for the effective control of the quality of Poria cocos medicinal materials.

According to the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules (Draft for Comment)" issued by the National Pharmacopoeia Committee in 2016, the standard decoction of traditional Chinese medicine follows the theory of traditional Chinese medicine, and the qualified decoction pieces are used to standardize the decoction method of clinical decoction. Solid-liquid separation, prepared by appropriate concentration or drying by appropriate method, as a standard reference to measure whether the traditional Chinese medicine formula granules are basically consistent with clinical decoctions.

Because the decocting solvent of Poria cocos standard decoction is water, a lot of water-insoluble triterpene components can't be reflected well in standard decoction. Therefore, the present invention establishes a method for measuring the UPLC characteristic spectrum that can fully reflect the water-soluble characteristic components in the standard decoction of Poria cocos on the basis of predecessors' research on Poria cocos medicinal materials. Four characteristic peaks of pachymic acid A and pachymic acid B were determined by using the characteristic spectrum of Poria cocos, and three of them were identified.

Contents of the invention

The purpose of the present invention is to provide a method for constructing and a detection method for the UPLC characteristic spectrum of Poria standard decoction for the defects and deficiencies in the prior art. The method has high precision, good stability and good repeatability. The constructed UPLC characteristic map of Poria standard decoction provides a rapid and comprehensive detection method for the quality control of Poria standard decoction.

The above-mentioned technical problems to be solved by the present invention are realized through the following technical solutions:

A kind of Poria cocos standard decoction UPLC characteristic map construction method, it comprises the steps:

(1) Precisely weigh the Poria standard decoction, and prepare the Poria standard decoction solution for the test;

(2) The test solution of Poria cocos standard decoction was analyzed by ultra-high performance liquid chromatography, and the UPLC characteristic spectrum of Poria cocos standard decoction was obtained.

As a preferred solution, the chromatographic conditions for the analysis of the ultra-high performance liquid chromatography used are: using octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, and 0.08-0.12% formic acid as mobile phase B, Gradient elution was carried out, the flow rate was 0.17-0.22ml/min, the column temperature was 28-32°C, the injection volume was 0.08-0.12μl, and the detection wavelength was 220-280nm.

As a most preferred solution, the chromatographic conditions for the analysis of the high performance liquid chromatography are as follows: using octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, and 0.1% formic acid as mobile phase B, carry out gradient For elution, the flow rate is 0.2ml/min, the column temperature is 30°C, the injection volume is 1μl, and the detection wavelength is 252nm.

As a preferred scheme, the gradient elution condition is: 0~21min, the volume fraction of mobile phase A changes to 40~99%, and the volume fraction of mobile phase B changes to 60~1%; 21~22min, mobile phase The volume fraction of A varies from 99 to 40%, and the volume fraction of mobile phase B varies from 1 to 60%.

As a preferred option, the preparation method of the Poria standard decoction is as follows: take 180-220 g of Poria decoction pieces, add water to decoct twice, add 8-10 times the amount of water to the first decoction, soak for 40-60 minutes, and boil with high heat Then decoct on low heat for 26-32 minutes, filter the decoction while it is hot, and quickly cool the filtrate with cold water; add 6.4-7.6 times the amount of water for the second time, heat it to a boil with strong fire, then decoct on low heat for 22-28 minutes, and filter the decoction while it is hot , the filtrate was quickly cooled with cold water, and the two decoctions were combined; the decoction was transferred to a container, concentrated under reduced pressure and low temperature to 120~140g of extract; stirred, divided into 8~12ml bottles, and the volume of each bottle was 1.6 ~2.4ml, freeze-dried after aliquoting, take out, and obtain.

As a most preferred solution, the preparation method of the Poria standard decoction is as follows: take 200 g of Poria decoction pieces, put them in an electric ceramic pot, add water to decoct twice, add 9 times the amount of water to the first decoction, soak for 50 minutes, and 500W After boiling with strong fire, decoct with 300W slow fire for 30 minutes. The decoction is filtered through a 400-mesh screen while it is hot. The filtrate is quickly cooled with cold water. For the second time, add 7 times the amount of water. The mesh screen is filtered while it is hot, the filtrate is quickly cooled with cold water, and the two decoctions are combined. Transfer the decoction to a round bottom flask, and use a rotary evaporator to concentrate under reduced pressure and low temperature to 130g of extract at temperature: 62°C; vacuum degree: -0.10MPa; under magnetic stirring, divide into 10ml brown vials , the volume of each bottle is 2ml, after the filling is completed, transfer to a vacuum freeze dryer to freeze dry, take it out, and roll on the aluminum cover to get it. The temperature is -45ºC, the sublimation drying temperature is -35ºC~0ºC, and the analytical drying temperature is 10ºC~20ºC.

As a preferred option, the test solution is prepared by a method comprising the following steps: take an appropriate amount of Poria cocos standard decoction, grind it finely, take the Poria cocos standard decoction powder equal to 25 ~ 35g of Poria cocos medicinal material, and accurately weigh , accurately add 9~11ml of 45~55% methanol, weigh the weight, treat with ultrasonic for 28~32 minutes, let it cool, weigh again, make up the lost weight with 45~55% methanol, shake well, centrifuge, and supernatant The solution is passed through a 0.18~0.26μm microporous membrane, and the subsequent filtrate is obtained.

As a most preferred solution, the described test solution is prepared by a method comprising the following steps: take an appropriate amount of Poria cocos standard decoction, grind it finely, take the Poria cocos standard decoction powder equal to 30g of Poria cocos medicinal material, accurately weigh, Place in a stoppered Erlenmeyer flask, accurately add 10ml of 50% methanol, weigh it, use 250W power, 40kHz ultrasonic treatment for 30 minutes, let it cool, weigh again, make up the lost weight with 50% methanol, shake Uniformly, centrifuged, the supernatant was passed through a 0.22 μm microporous membrane, and the subsequent filtrate was obtained.

The present invention also provides the detection method of Poria cocos standard decoction, comprises the steps:

(1) Precisely weigh the Poria standard decoction to be tested, and prepare the sample solution of the Poria standard decoction to be tested;

(2) Precisely absorb the sample solution of standard Poria cocos decoction to be detected, inject it into an ultra-high performance liquid chromatograph, measure it, and obtain the UPLC characteristic map of standard Poria cocos decoction to be detected;

(3) Compare the measured UPLC characteristic spectrum with the constructed Poria standard decoction UPLC characteristic spectrum. If it is consistent with the UPLC characteristic spectrum of Poria standard decoction, the quality is qualified.

As a preferred solution, the chromatographic conditions for the analysis of the ultra-high performance liquid chromatography used are: using octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, and 0.08-0.12% formic acid as mobile phase B, Gradient elution was carried out, the flow rate was 0.17-0.22ml/min, the column temperature was 28-32°C, the injection volume was 0.08-0.12μl, and the detection wavelength was 220-280nm.

As a most preferred solution, the chromatographic conditions for the analysis of the high performance liquid chromatography are as follows: using octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, and 0.1% formic acid as mobile phase B, carry out gradient For elution, the flow rate is 0.2ml/min, the column temperature is 30°C, the injection volume is 1μl, and the detection wavelength is 252nm.

As a preferred scheme, the gradient elution condition is: 0~21min, the volume fraction of mobile phase A changes to 40~99%, and the volume fraction of mobile phase B changes to 60~1%; 21~22min, mobile phase The volume fraction of A varies from 99 to 40%, and the volume fraction of mobile phase B varies from 1 to 60%.

As a preferred option, the preparation method of the Poria standard decoction to be detected is as follows: take 180-220 g of Poria decoction pieces, decoct twice with water, add 8-10 times the amount of water for the first decoction, soak for 40-60 minutes, After boiling with strong fire, decoct with slow fire for 26~32 minutes, filter the decoction while it is hot, and cool the filtrate quickly with cold water; Filtrate hot, cool the filtrate quickly with cold water, combine the two decoctions; transfer the decoction to a container, concentrate under reduced pressure and low temperature to 120~140g of extract; stir, divide into 8~12ml bottles, and the volume of each bottle It is 1.6~2.4ml, freeze-dried after aliquoting, take out, and obtain immediately.

As a most preferred solution, the preparation method of the Poria standard decoction to be detected is: take 200 g of Poria decoction pieces, put them in an electric ceramic pot, add water to decoct twice, add 9 times the amount of water to the first decoction, and soak for 50 minutes , 500W strong fire boiled, 300W slow fire decocted for 30 minutes, the decoction was filtered through a 400-mesh sieve while it was hot, the filtrate was quickly cooled with cold water, and 7 times the amount of water was added for the second time, heated to a strong fire and boiled, then simmered for 25 minutes, the decoction Filter while hot with a 400-mesh sieve, cool the filtrate quickly with cold water, and combine the two decoctions. Transfer the decoction to a round bottom flask, and use a rotary evaporator to concentrate under reduced pressure and low temperature to 130g of extract at temperature: 62°C; vacuum degree: -0.10MPa; under magnetic stirring, divide into 10ml brown vials , the volume of each bottle is 2ml, after the filling is completed, transfer to a vacuum freeze dryer to freeze dry, take it out, and roll on the aluminum cover to get it. The temperature is -45ºC, the sublimation drying temperature is -35ºC~0ºC, and the analytical drying temperature is 10ºC~20ºC.

As a preferred option, the Poria standard decoction sample solution to be detected is prepared by a method comprising the following steps: take an appropriate amount of Poria cocos standard decoction, grind it finely, and take Poria cocos standard decoction powder equal to 25 ~ 35g of Poria cocos medicinal material , accurately weighed, accurately added 45~55% methanol 9~11ml, weighed, ultrasonically treated for 28~32 minutes, allowed to cool, and weighed again, supplemented the lost weight with 45~55% methanol, shaked well, Centrifuge, pass the supernatant through a 0.18-0.26 μm microporous membrane, and take the subsequent filtrate.

As a most preferred solution, the Poria standard decoction sample solution to be detected is prepared by a method comprising the following steps: take an appropriate amount of Poria cocos standard decoction, grind it finely, take the Poria cocos standard decoction powder equal to 30g of Poria cocos medicinal material, Accurately weighed, put in a stoppered Erlenmeyer flask, accurately add 10ml of 50% methanol, weigh, use power 250W, frequency 40kHz ultrasonic treatment for 30 minutes, let cool, weigh again, make up for the loss with 50% methanol Shake well, centrifuge, pass the supernatant through a 0.22 μm microporous membrane, and take the subsequent filtrate.

Beneficial effects: (1) The present invention constructs the UPLC characteristic map and detection method of the Poria standard decoction for the first time; (2) The UPLC characteristic map of the Poria standard decoction constructed by the present invention fully demonstrates the chemical composition characteristics of the Poria standard decoction (3) The feature spectrum constructed by the present invention comprehensively reflects the feature peak information of the sample, and the method is stable, with high precision and good reproducibility; (4) The assay method of the Poria standard decoction of the present invention is The quality control of Poria cocos standard decoction provides rapid and comprehensive detection means; (5) The present invention can comprehensively and accurately perform quality control on Poria cocos standard decoction, and also provides a basis for the establishment of quality standards for Poria cocos medicinal materials and Poria cocos formula granules.

Description of drawings

Figure 1 is an overlay of UPLC characteristic spectra of 25 batches of Poria standard decoction.

Figure 2 is the UPLC control characteristic spectrum of Poria standard decoction.

Figure 3 is the UPLC characteristic spectrum of Poria reference medicinal material.

Figure 4 is the PDA chromatogram and mass spectrometry total ion chromatogram of Poria standard decoction.

Figure 5 is the UPLC characteristic spectrum of Poria standard decoction and mixed reference solution.

Figure 6 is the low-energy and high-energy mass spectrum of peak 2 pachymic acid B in negative ion mode.

Figure 7 is the low-energy and high-energy mass spectrum of peak 2 pachymic acid B in positive ion mode.

Figure 8 is a chemical structural formula diagram of pachymic acid B.

Figure 9 is the low-energy and high-energy mass spectrum of peak 3 pachymic acid A in negative ion mode.

Figure 10 is the low-energy and high-energy mass spectrum of peak 3 pachymic acid A in negative ion mode.

Figure 11 is a chemical structural formula diagram of pachymic acid A.

Figure 12 is the low-energy and high-energy mass spectrum of peak 4 pyripolic acid C in negative ion mode.

Figure 13 is the low-energy and high-energy mass spectrum of peak 4 pyripolic acid C in negative ion mode.

Fig. 14 is a diagram showing the chemical structural formula of pyripolic acid C.

Figure 15 is the UPLC characteristic spectrum of Poria standard decoction.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. the embodiment. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Example 1

Construction of characteristic map of Poria standard decoction

See Table 1, Table 2, Table 3, and Table 4 for the main instruments, reagents, reference substance information, and source of Poria standard decoction, respectively.

Table 1 Main Instruments

Table 2 Main Reagents

Table 3 Reference substance information

Table 4 Origin information of 25 batches of Poria standard decoction

experiment method

Preparation of reference solution

Accurately weigh an appropriate amount of pachymic acid B reference substance, add methanol to make a solution containing 20 μg of pachymic acid B per 1 ml, and obtain it.

Preparation of Poria standard decoction

Take 200g of Poria decoction slices, put them in an electric ceramic pot, add water to decoct twice, add 9 times the amount of water to the first decoction, soak for 50 minutes, boil at 500W strong fire, then decoct at 300W slow fire for 30 minutes, and the decoction passes through a 400-mesh sieve Filtrate while it is hot, cool the filtrate with cold water quickly, add 7 times the amount of water for the second time, heat it to a boil with strong fire, then decoct with slow fire for 25 minutes, filter the decoction while it is hot with a 400 mesh screen, cool the filtrate with cold water quickly, combine two decoctions liquid. Transfer the decoction to a round bottom flask, and use a rotary evaporator to concentrate under reduced pressure and low temperature to 130g of extract at temperature: 62°C; vacuum degree: -0.10MPa; under magnetic stirring, divide into 10ml brown vials , each bottle has a subpackage volume of 2ml, after subpackaging, transfer it to a vacuum freeze dryer to freeze-dry, take it out, roll on an aluminum cap, and get it. The eutectic point test result of Poria cocos vacuum freeze dryer is -32.5°C, the pre-freezing temperature is -45°C, the sublimation drying temperature is -35°C~0°C, the analytical drying temperature is 10°C~20°C, and the total drying time is 47 hours.

Chromatographic conditions

Use octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, and 0.1% formic acid as mobile phase B, carry out gradient elution according to the provisions in Table 5; the flow rate is 0.2ml per minute; the column temperature is 30°C ; The detection wavelength is 252nm.

Table 5 Gradient elution table

Preparation of Poria standard decoction for test solution

Take an appropriate amount of Poria cocos standard decoction, grind it finely, take Poria cocos standard decoction powder equal to 30g of Poria cocos medicinal material, accurately weigh it, put it in a stoppered Erlenmeyer flask, accurately add 10ml of 50% methanol, weigh it, use a power of 250W, Ultrasonic treatment with a frequency of 40kHz for 30 minutes, let cool, and then weighed, supplemented the lost weight with 50% methanol, shaken, centrifuged, the supernatant was passed through a 0.22μm microporous membrane, and the subsequent filtrate was obtained.

Preparation of reference medicinal solution

Take 3 g of Poria cocos reference medicinal material, accurately weighed, put in a stoppered Erlenmeyer flask, accurately add 25 ml of 50% methanol, weigh it, shake it in a constant temperature oscillator for 2 hours, and use a power of 250W and a frequency of 40kHz for 60 minutes of ultrasonic treatment. Allow to cool, weigh again, make up for the lost weight with 50% methanol, shake well, centrifuge, pass the supernatant through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the final product.

Determination of Common Peaks in Characteristic Spectrum of Poria Standard Decoction

(1) Take 25 batches of Poria cocos standard decoction, inject the test solution under specified chromatographic conditions, obtain UPLC characteristic spectra of 25 batches of Poria cocos standard decoction (see Figure 1), and carry out common peak identification to establish Poria cocos standard Decoction UPLC control characteristic map (Figure 2).

(3) Experimental results: The common peaks with better separation and purer chromatographic peaks are the characteristic peaks of the characteristic spectrum of Poria standard decoction. 25 batches of Poria standard decoction have 4 characteristic peaks (see Figure 1), and 4 The characteristic peaks can all be found in the characteristic spectrum of the Poria cocos reference medicinal material (see Figure 3), indicating that the four characteristic peaks can be stably transferred from the Poria cocos medicinal material to the Poria standard decoction; therefore, the above four common peaks are selected as the UPLC of the Poria cocos standard decoction The characteristic peaks of the characteristic spectrum, and the peak 2 (poria cocos B) was used as the reference peak for standard research, and tried to use UPLC/MS Q-TOF technology to identify the chemical components of the characteristic peaks, and determined that peak 2 was Poria cocos Acid B, peak 3 is pachymic acid A, peak 4 is poricic acid C.

Study on Stability of Poria Standard Decoction Test Sample Solution

Take the Poria cocos standard decoction to prepare the test solution. Under the specified chromatographic conditions, inject samples at 0, 2, 4, 8, 12, 16, 20, and 24 hours respectively, with an injection volume of 1 μl. Taking pachymic acid B chromatographic peak as reference peak S, calculate the relative retention time and relative peak area of each characteristic peak, see table 6 and table 7, experimental result shows, the relative retention time and relative peak area RSD of each characteristic peak in 24 hours Values are all less than 2.0%, and the test solution is stable and good within 24 hours.

Table 6 Stability investigation results of characteristic spectrum of Poria standard decoction (relative retention time)

Table 7 Stability investigation results of characteristic spectrum of Poria standard decoction (relative retention peak area)

Study on the Characteristic Map of Poria Standard Decoction

In order to strictly control the quality of Poria Formula Granules and provide more comprehensive quality control parameters for the production process of Poria Formula Granules, the characteristic map of Poria Standard Decoction was established, and the limit was set for the relative peak area of the characteristic peaks in the characteristic map of Poria Standard Decoction standard. This study is based on the full representativeness of 25 batches of Poria standard decoction, and the maximum and minimum values are taken as the basis for limiting the relative peak area; select peak 2 with moderate peak area and better resolution as the reference peak, calculate and The relative peak area ranges of other characteristic peaks are stipulated, so the relative peak area ranges of each characteristic peak established are quantitative. The established upper and lower limits of the peak area have variety characteristics, which can avoid misjudgment of some qualified batches in large-scale production, and can also provide important quality control parameters for large-scale production processes, especially for the quality control of unstable characteristic peak components, thereby improving Product quality and quality control level.

In the study, four common peaks of Poria standard decoction were determined, and PLC/MS Q-TOF technology was used to identify the chemical components of the characteristic peaks. The results of the four characteristic peaks were preliminarily speculated to be: peak 1 was 3-ketone Base-6,16α-dihydroxy-lanoster-7,-9(11),-24(31)-triene-21-acid; peak 2 is pachymic acid B; peak 3 is 16α-hydroxyl-3,- 4 ring-opening-lanoster-4(28),-7,9(11), 4(31)-tetraene-3,21-dioic acid (namely pachymic acid A); peak 4 is pigolic acid C. After comparison with the reference substance, the identification results of peak 2, peak 3 and peak 4 were confirmed.

The determination method of the characteristic map was established by high performance liquid chromatography and the methodological investigation was carried out. The characteristic map of 25 batches of Poria standard decoction was determined, and finally the standard of the characteristic map of Poria standard decoction was determined: four characteristics should be presented in the test sample chromatogram. Peak, with peak 2 as the S peak, calculate the relative retention time and relative peak area of each characteristic peak and S peak, stipulate the range of each characteristic peak, the results are as follows:

Table 8 Relative retention time of characteristic spectra of 25 batches of Poria standard decoction

Table 9 The relative peak areas of characteristic spectra of 25 batches of Poria standard decoction

Table 10 The relative retention time and relative peak area range of each characteristic peak of Poria standard decoction

The Identification of the Characteristic Peaks of the Characteristic Spectrum of Poria Standard Decoction

Experimental conditions

Instruments: Waters ACQUITY UPLC ^TM I-Class liquid phase system, Xevo G2-XS Q-TOF mass spectrometry system (Waters Technology Co., Ltd.); Waters Hss T3 C ₁₈ (2.1mm×100mm, 1.8μm) chromatographic column.

Reagents: analytically pure methanol (Guangzhou Chemical Reagent Factory); formic acid (Merck Co., Ltd.), acetonitrile (Thermo Fisher Co., Ltd.), methanol (Thermo Fisher Co., Ltd.) for HPLC Chromatographic grade, water was laboratory ultrapure water system (Merck & Co., Ltd., Milli-Q Direct).

Mass spectrometry conditions: double-spray ESI ion source, acquisition mode: MSE data acquisition under ESI (+/-); acquisition mass range: 50-1200Da; calibration solution: sodium formate, leucine enkephalin; spray voltage: 2.0/2.0kv ; Cone voltage: 40V; Source temperature: 120 degrees; Desolvation temperature: 450°C; Desolvation gas flow: 800L/hr Cone gas flow: 50L/hr.

Chromatographic conditions: Chromatographic column: Waters Hss T3 C ₁₈ (2.1mm×100mm, 1.8μm)

Flow rate: 0.3 ml/min Column temperature: 40°C Detection wavelength: 252nm, injection volume: 1μl, mobile phase: acetonitrile as mobile phase A, 0.1% formic acid as mobile phase B, carry out the gradient as specified in Table 11 Elution:

Table 11 Characteristic peak identification mobile phase gradient

Preparation of reference solution

Take an appropriate amount of reference substances of pachymic acid A, pachymic acid B, and pigolic acid C, accurately weigh them, and add methanol to make a mixed control solution containing 50 μg each of pachymic acid A, pachymic acid B, and pigolic acid C per 1 ml.

Preparation of the test solution

Take an appropriate amount of Poria cocos standard decoction, grind it finely, take Poria cocos standard decoction powder equal to 30g of Poria cocos medicinal material, put it in a stoppered conical flask, add 10ml of 50% methanol precisely, weigh it, and use an ultrasonic wave with a power of 250W and a frequency of 40kHz Treat for 30 minutes, let cool, weigh again, make up for the lost weight with 50% methanol, shake well, centrifuge, pass the supernatant through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the final product.

Injection Analysis

Precisely draw 1 µl each of the above-mentioned reference solution and the test solution, inject them into the LC-MS system, use the UNIFI software to analyze the collected data, obtain the accurate mass number from the high-resolution system, analyze the elemental composition and Get the chemical formula of the compound, combine fragment analysis and online database search, and compare the chromatographic peak retention line of the compound with the reference substance.

result

The retention times of peak 2, peak 3, and peak 4 of the reference substance mixed with the reference substance can all correspond to the retention times of the reference substances pachymic acid B, pachymic acid A, and pigolic acid C (see Figure 5).

Peak 2: In the negative ion mode, m/z 483.3115 is the [MH]- peak; in the positive ion mode, there is m/z 485.3262, which is the [M+H]+ peak, and the base peak is m/z 467.3159, which is the [MH]+ peak. +HH ₂ O]+ peak (see Figure 6, Figure 7). The elemental composition analysis shows that the neutral molecular formula is: C ₃₀ H ₄₄ O ₅ , combined with literature (Zhu L, Xu J, Zhang S, et al. Qualitatively and quantitatively comparing secondary metabolites in three medicinal parts derived from Poria cocos (Schw.) Wolf using UHPLC-QTOF-MS/MS-based chemical profiling. [J]. J Pharm Biomed Anal, 2017, 150:278-286.). The results conform to the following three points: 1. Negative ions have no obvious neutral loss of -98; 2. Negative ions have obvious neutral loss of -74; 3. Positive ions have m/z325. Based on the above analysis, it is determined to be pachymic acid B ( Figure 8)

Peak 3: In the negative ion mode, m/z 497.3278 is the [MH]- peak; in the positive ion mode, there is m/z 499, which is the [M+H]+ peak, and the base peak m/z 481.3325 is the [M+ HH ₂ O]+ peak (see Figure 9, Figure 10). The elemental composition analysis shows that the neutral molecular formula is: C ₃₁ H ₄₆ O ₅ , combined with the literature (Li Ke. Separation and purification of triterpenoid chemical components in Poria cocos, structural identification and research on the fingerprints of Poria cocos medicinal materials[D]. Hubei Zhong University of Medicine, 2013.). The results conform to the following two points: 1. The negative ion has obvious -74, indicating that it is a C3, C4 ring-opening triterpene; 2. The positive ion has m/z 325. Based on the above analysis, it is determined to be pachymic acid A (Figure 11).

Peak 4: In the negative ion mode, m/z 481.3321 is the [MH]- peak; in the positive ion mode, there is m/z 483.3467, which is the [M+H]+ peak, and the base peak m/z 465.3363 is the [MH]+ peak. +HH ₂ O]+ peak (see Figure 12, Figure 13). The element composition analysis shows that the neutral molecular formula is: C ₃₁ H ₄₆ O ₄ , combined with the literature (Li Ke. Separation and purification of triterpenoid chemical components in Poria cocos, structural identification and research on the fingerprints of Poria cocos medicinal materials[D]. Hubei Zhong University of Medicine, 2013.). The results are consistent with the following two points: 1. There is no obvious -74 in negative ions; 2. There is m/z 309 in positive ions. Based on the above analysis, it was determined to be 3-keto-16α-hydroxy-lanoster-7,9(11),24(31)-trien-21-acid, ie, kinetolic acid C (Figure 14).

Example 2

Poria standard decoction detection method, detection steps are as follows:

(1) Precisely weigh the Poria standard decoction to be tested, and prepare the sample solution of the Poria standard decoction to be tested;

(2) Precisely absorb the sample solution of standard Poria cocos decoction to be detected, inject it into an ultra-high performance liquid chromatograph, measure it, and obtain the UPLC characteristic map of standard Poria cocos decoction to be detected;

(3) Compare the measured UPLC characteristic spectrum with the constructed Poria standard decoction UPLC characteristic spectrum. If it is consistent with the UPLC characteristic spectrum of Poria standard decoction, the quality is qualified.

Chromatographic conditions

Use octadecylsilane bonded silica gel as filler (column length is 150mm, inner diameter is 2.1mm, particle size is 1.6μm); acetonitrile is used as mobile phase A, 0.1% formic acid is used as mobile phase B, as specified in Table 12 Carry out gradient elution; the flow rate is 0.2ml per minute; the column temperature is 30°C; the detection wavelength is 252nm.

Table 12 Gradient elution table

Preparation of reference solution

Accurately weigh an appropriate amount of pachymic acid B reference substance, add methanol to make a solution containing 20 μg of pachymic acid B per 1 ml, and obtain it.

Preparation of the reference drug solution

Take 3 g of Poria cocos reference medicinal material, accurately weighed, put in a stoppered Erlenmeyer flask, accurately add 25 ml of 50% methanol, weigh it, shake it in a constant temperature oscillator for 2 hours, and use a power of 250W and a frequency of 40kHz for 60 minutes of ultrasonic treatment. Allow to cool, weigh again, make up for the lost weight with 50% methanol, shake well, centrifuge, pass the supernatant through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the final product.

Preparation of the test solution

Take an appropriate amount of Poria cocos standard decoction and grind it finely, take Poria cocos standard decoction powder equal to 30g of Poria cocos medicinal material, accurately weigh it, put it in a stoppered conical flask, accurately add 10ml of 50% methanol, weigh it, use power 250W, frequency Ultrasonic treatment at 40kHz for 30 minutes, let cool, weigh again, make up the lost weight with 50% methanol, shake well, centrifuge, pass the supernatant through a 0.22μm microporous membrane, and take the subsequent filtrate to obtain the final product.

Determination: Precisely draw 10 μl of the control medicinal material solution, 2 μl of the reference substance solution and the test solution, inject it into the liquid chromatograph, measure it, and obtain it.

The measurement results

Table 13 The relative retention time of each characteristic peak of Poria standard decoction and the specified range of relative peak area

Compare the Poria cocos standard decoction characteristic spectrum with the Poria cocos reference medicinal material characteristic spectrum (Fig. 15): 4 characteristic peaks identical to the Poria cocos contrast medicinal material can be detected; the data results show (Table 13), the reference peak of the sample ( Except for peak 2), the relative retention time and relative peak area range of the remaining three peaks are all within the range specified in the standard, which shows that the method for the characteristic chromatogram of the Poria standard decoction is stable and reproducible, and can accurately reflect the standard Poria cocos. Decoction quality requirements.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description are only the principles of the present invention. Variations and improvements, which fall within the scope of the claimed invention. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. a Poria cocos standard decoction UPLC feature map construction method, is characterized in that, comprises the steps: (1) Precisely weigh the Poria standard decoction, and prepare the Poria standard decoction solution for the test; (2) The test solution of Poria cocos standard decoction was analyzed by ultra-high performance liquid chromatography, and the UPLC characteristic spectrum of Poria cocos standard decoction was obtained. 2. Poria cocos standard decoction UPLC characteristic collection of illustrative plates construction method according to claim 1, is characterized in that, the chromatographic condition that described ultra-high performance liquid chromatograph analyzes is: take octadecylsilane bonded silica gel as filler, Using acetonitrile as mobile phase A and 0.08-0.12% formic acid as mobile phase B, carry out gradient elution, the flow rate is 0.17-0.22ml/min, the column temperature is 28-32℃, and the injection volume is 0.08-0.12μl. The wavelength is 220~280nm. 3. Poria standard decoction UPLC feature map construction method according to claim 1, is characterized in that, described Poria standard decoction preparation method is: get Poria decoction pieces 180 ~ 220g, add water decoction twice, decoct for the first time Boil and add 8-10 times the amount of water, soak for 40-60 minutes, boil over high heat, then decoct over low heat for 26-32 minutes, filter the decoction while it is hot, and cool the filtrate with cold water quickly; add 6.4-7.6 times the amount of water for the second time, After heating and boiling, decoct on low heat for 22-28 minutes, filter the decoction while it is hot, cool the filtrate quickly with cold water, and combine the two decoctions; transfer the decoction to a container, concentrate under reduced pressure and low temperature to 120-140g of extract; stir , divided into 8~12ml bottles, each bottle has a volume of 1.6~2.4ml, freeze-dried after filling, take it out, and get it. 4. Poria cocos standard decoction UPLC characteristic atlas construction method according to claim 1, is characterized in that, gradient elution condition is: 0～21min, the volume fraction change of mobile phase A is 40～99%, the volume fraction of mobile phase B The volume fraction changes from 60 to 1%; 21 to 22 minutes, the volume fraction of mobile phase A changes from 99 to 40%, and the volume fraction of mobile phase B changes from 1 to 60%. 5. Poria cocos standard decoction UPLC characteristic collection of illustrative plates construction method according to claim 1, is characterized in that, described need testing solution is prepared by the method for following steps: Take an appropriate amount of Poria cocos standard decoction, grind it finely, take Poria cocos standard decoction powder equal to 25~35g of Poria cocos medicinal material, accurately weigh it, add 9~11ml of 45~55% methanol accurately, weigh it, and ultrasonically treat it for 28~32 minutes , let cool, weigh again, make up for the lost weight with 45-55% methanol, shake well, centrifuge, pass the supernatant through a 0.18-0.26 μm microporous membrane, and take the subsequent filtrate to obtain the final product. 6. a detection method of Poria cocos standard decoction, is characterized in that, comprises the steps: (1) Accurately weigh the Poria standard decoction to be tested, and prepare the sample solution of the Poria standard decoction to be tested; (2) Precisely absorb the sample solution of standard Poria cocos decoction to be detected, inject it into an ultra-high performance liquid chromatograph, measure it, and obtain the UPLC characteristic map of standard Poria cocos decoction to be detected; (3) Compare the measured UPLC characteristic spectrum with the Poria standard decoction UPLC characteristic spectrum constructed by any one of the methods in claims 1 to 5, if it is consistent with the Poria standard decoction UPLC characteristic spectrum, the quality is qualified. 7. the detection method of Poria cocos standard decoction according to claim 6, is characterized in that, The chromatographic conditions analyzed by the ultra-high performance liquid chromatograph are: using octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, and 0.08-0.12% formic acid as mobile phase B for gradient elution, The flow rate is 0.17~0.22ml/min, the column temperature is 28~32°C, the injection volume is 0.8~0.12μl, and the detection wavelength is 220~280nm. 8. the detection method of Poria standard decoction according to claim 6, is characterized in that, described Poria cocos standard decoction preparation method is: get Poria cocos decoction piece 180 ~ 220g, add water decoction twice, decoct for the first time and add 8 to 10 times the amount of water, soak for 40 to 60 minutes, boil with high heat, then decoct with low heat for 26 to 32 minutes, filter the decoction while it is hot, and quickly cool the filtrate with cold water; add 6.4 to 7.6 times the amount of water for the second time, heat with high heat and boil After that, decoct on low heat for 22-28 minutes, filter the decoction while it is hot, cool the filtrate quickly with cold water, and combine the two decoctions; transfer the decoction to a container, concentrate under reduced pressure and low temperature to 120-140g of extract; stir, divide Pack into 8~12ml bottles, each bottle has a subpackage volume of 1.6~2.4ml, freeze-dry after subpackaging, take out, and get ready. 9. the detection method of Poria standard decoction according to claim 6, is characterized in that, gradient elution condition is: 0～21min, the volume fraction change of mobile phase A is 40～99%, the volume fraction of mobile phase B The change is 60~1%; 21~22min, the volume fraction of mobile phase A changes to 99~40%, and the volume fraction of mobile phase B changes to 1~60%. 10. the detection method of Poria cocos standard decoction according to claim 6, is characterized in that, described need testing solution is prepared by the method of following steps: get Poria cocos standard decoction appropriate amount, grind finely, get and be equal to 25 ~ 35g of Poria cocos standard decoction powder, accurately weighed, ultrasonically treated for 28-32 minutes, allowed to cool, and then weighed, supplemented with 45-55% methanol to make up the lost weight, shake well, centrifuge, and supernatant Pass through a 0.18~0.26μm microporous membrane, take the subsequent filtrate, and obtain it.