阅读说明：本技术 基于hplc-uv法同时测定赤松茸中黄嘌呤、鸟嘌呤、腺嘌呤和次黄嘌呤含量的方法 (Method for simultaneously determining contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake based on HPLC-UV method ) 是由 王志尧 王韬 常霞 魏磊 景炳年 周雍 姜晓 谢晓阳 梁雅辉 王伟 于 2021-09-03 设计创作，主要内容包括：本发明公开了一种基于HPLC法同时测定赤松茸干品中黄嘌呤、鸟嘌呤、腺嘌呤和次黄嘌呤含量的方法。通过HPLC-UV联用检测技术,建立了一测多评含量测定的方法,实现了仅采用其中一种嘌呤标准品,确定该嘌呤标品与赤松茸供试样品中其它三种嘌呤成分之间的相对校正因子和相对保留时间,通过计算便捷地对赤松茸供试样品中的四个嘌呤成分的含量进行了测定。本发明操作简单,灵敏度高,降低了标准品成本,简化了实验步骤,能够准确地评价赤松茸产品的品质。目前还赤松茸及其相关产品还没有检测标准,本发明可以用于赤松茸的质量控制,对赤松茸的研究与产业发展具有重要的意义。(The invention discloses a method for simultaneously determining the content of xanthine, guanine, adenine and hypoxanthine in a dried product of tricholoma matsutake based on an HPLC method. By using an HPLC-UV combined detection technology, a method for measuring the content of multiple scores is established, the relative correction factor and the relative retention time between the purine standard and other three purine components in the tricholoma matsutake test sample are determined by only using one purine standard, and the content of four purine components in the tricholoma matsutake test sample is conveniently measured by calculating. The method is simple to operate and high in sensitivity, reduces the cost of the standard product, simplifies the experimental steps, and can accurately evaluate the quality of the tricholoma matsutake products. At present, no detection standard exists for the tricholoma matsutake and related products thereof, and the invention can be used for quality control of the tricholoma matsutake and has important significance for research and industrial development of the tricholoma matsutake.)

1. A method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake based on an HPLC-UV method is characterized by comprising the following steps:

1) preparing a standard solution of a reference substance: taking methanol as a solvent, and respectively preparing a series of standard solutions of xanthine, guanine, adenine and hypoxanthine reference substances with different concentrations;

2) preparing a test solution:

taking 0.2g of dried, crushed and sieved tricholoma matsutake or a derivative sample thereof, adding 8-12ml of 60-70% strong oxidizing acid, heating in a water bath at 90-110 ℃, refluxing and hydrolyzing for 20-40min, placing in an ice water bath for cooling, adjusting to be neutral by using alkali liquor, filtering, adjusting the pH of filtrate to 3.8-4.2, centrifuging, taking supernatant, and fixing the volume by using purified water to a 50ml brown volumetric flask to obtain a test solution;

3) calculation of relative correction factor and relative retention time:

precisely sucking the reference solutions of the xanthines, guanines, adenines and hypoxanthines with different concentrations prepared in the step 1) for liquid chromatography determination to obtain an HPLC-UV chromatogram, measuring and recording peak areas of chromatographic peaks of the purines, and respectively calculating relative correction factors of the xanthines, the guanines and the hypoxanthines to the adenines by taking the adenines as standard substances (the relative correction factors are respectively calculated by the aid of the adeninesf _s/x ) And relative retention time: (t _Rx ）；

The relative correction factor (f _s/x ) The calculation formula is as follows:

in the formula:f _s/x is a purine to be detectedxThe relative correction factor of (a) is,A _s is the peak area of the adenine chromatographic peak,C _s is the mass concentration of the adenine, and is,A _x is a purine to be detectedxThe peak area of the chromatographic peak of (a),C _x is a purine to be detectedxMass concentration of (d);

the relative retention time: (t _Rx ) The calculation formula is as follows:

in the formula:t _Rx indicating the purine to be detectedxRelative retention time of (d);t _x indicating the purine to be detectedxRetention time of chromatographic peak;t _s represents the retention time of an adenine chromatographic peak;

4) detection of purines in the test sample:

performing liquid chromatography determination on a series of adenine reference substance standard solutions with different concentrations prepared in the step 1) to obtain an HPLC-UV chromatogram, measuring and recording peak areas of adenine chromatographic peaks, drawing a standard curve by adopting an external standard method with the peak areas of adenine as vertical coordinates and the concentration of adenine as horizontal coordinates, and calculating to obtain a linear equation of adenine;

performing liquid chromatography determination on the sample solution prepared in the step 2) to obtain an HPLC-UV chromatogram, measuring and recording peak areas of chromatographic peaks of adenine, guanine, xanthine and hypoxanthine, substituting the peak area of the adenine into a linear equation of the adenine, and calculating to obtain the content of the adenine in the sample;

wherein, the content of guanine, xanthine and hypoxanthine is calculated according to the following formula:

(ii) a In the formula:

indicating the purine to be detectedxMass concentration in a sample of tricholoma matsutake or a derivative thereof;

indicating the purine to be detectedxMass concentration in the test solution;

representing the mass concentration of the tricholoma matsutake or derivative samples thereof in the test solution;

indicating the purine to be detected in the test solutionxPeak area of chromatographic peak (c).

2. The method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake based on the HPLC-UV method as claimed in claim 1, wherein the conditions of the liquid chromatography are as follows:

a reverse phase C18 packed chromatography column; the mobile phase consists of 98-100% of mobile phase A and 0-2% of mobile phase B, and KH is 0.02 +/-0.001 mol/L₂PO₄-H₃PO₄Taking the buffer solution as a mobile phase A and taking methanol or acetonitrile as a mobile phase B, and carrying out isocratic elution; column temperature: 25-35 ℃, flow rate: 0.8-1.2 mL/min; wavelength of ultraviolet detector: 254 +/-2 nm.

3. The method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake based on the HPLC-UV method as claimed in claim 1, wherein in the step 1), standard solutions of xanthine, guanine, adenine and hypoxanthine controls with different concentrations of 0.05, 0.50, 5.00, 20.00 and 30.00 μ g/ml are prepared.

4. The method for simultaneously measuring the content of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake based on an HPLC-UV method as claimed in claim 1, wherein in the step 2), the strong diluted acid oxide is nitric acid, perchloric acid or concentrated sulfuric acid.

5. The method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake based on the HPLC-UV method as claimed in claim 1, wherein, in the step 2), the alkali solution is potassium hydroxide or sodium hydroxide aqueous solution with a concentration of 8-12 mol/L.

6. The method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake based on the HPLC-UV method as claimed in any one of claims 1 to 5, wherein in the step 2), 1mol/L phosphoric acid is added to the filtrate to adjust the pH to 3.8-4.2.

Technical Field

The invention belongs to the technical field of content determination of purine components in tricholoma matsutake, and particularly relates to a method for simultaneously determining the content of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake and derivatives thereof based on an HPLC-UV method.

Background

The tricholoma matsutake (Stropharia rugoso-annulata), also known as crinkled Stropharia rugoso-annulata, rugoso-annulata vinega, rugoso-annulata and Pseudoblack fizea, is one of mushroom species recommended to developing countries by grain and crop organizations of the United nations due to delicious taste and rich nutrition, is successfully introduced and cultivated manually in China in 1980, and is one of ten large varieties in the international mushroom trading market at present. The prior literature reports that the tricholoma matsutake contains polysaccharide, sterol, flavone, phenolic substances, agglutinin and other bioactive components, and has the pharmacological effects of resisting oxidation, inhibiting bacteria, inhibiting tumors, reducing blood sugar and the like.

Purines are widely present in plants and animals and are important substances constituting the life code, nucleic acid. Many edible fungi contain purine chemical components, but the purine content of different varieties of fungi is greatly different. Uric acid (Uric acid, structure shown in figure 1), which is the final product of purine metabolism, is trioxypurine, is slightly soluble in water, and is easily crystallized. If the uric acid produced in the body is too much to be excreted or the excretion mechanism of uric acid is degraded, the uric acid in the body is too much retained, and the body fluid of the human body is acidified when the blood uric acid concentration is more than 7 mg/dl, so that gout can be caused if the human body is left for a long time. In addition, the urate concentration in blood is increased to reach an oversaturated state, and urate crystals are deposited on the kidney, so that gout and nephropathy can be caused. Therefore, the high purine diet is not good for the health of people, and especially for gout patients, the purine intake in the diet should be controlled. Four common purines are xanthine, guanine, adenine and hypoxanthine (see figure 1 for structure). However, it has not been reported whether or not the Tricholoma matsutake contains purine components. Therefore, with the growing area of the tricholoma matsutake growing, the tricholoma matsutake is used as food material to enter more and more household dining tables and even used as raw material of commercial food, and the determination of the content of purine is of great significance to the development of the tricholoma matsutake industry.

Through research, four purines, namely xanthine, guanine, adenine and hypoxanthine, are similar in structural formula and high in polarity, the content of the four purines in the tricholoma matsutake is low, and the four purines are difficult to separate from each other in a baseline manner through a conventional chromatographic method. At present, no detection method and quality control method for purine components in tricholoma matsutake and derivatives thereof are reported. Although other edible fungi have purine content detection methods, the defects of complicated sample pretreatment method, complex detection operation process and the like exist mostly. Meanwhile, each purine needs the same high-purity standard, and the high-purity standard conforming to the content determination is difficult to obtain, so the detection cost is high. The invention discloses a technical method for simultaneously determining the qualitative and quantitative detection of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake and derivatives thereof based on an HPLC (high performance liquid chromatography) -UV (ultraviolet) combined detection technology, and establishes a method for determining the multi-score content by one detection, so that the qualitative and quantitative determination of four representative purine components in a sample to be tested of tricholoma matsutake and derivatives thereof is conveniently and simultaneously realized by only adopting one purine standard substance. The detection method provided by the invention is simple to operate and high in sensitivity, only one standard substance is needed, the cost of the standard substance is reduced, the experimental steps are simplified, and the quality of the tricholoma matsutake product can be accurately evaluated. Aiming at the current situation that a method for qualitatively and quantitatively detecting purine substances in tricholoma matsutake and related products thereof does not exist at present, the method can be used for identifying and controlling the tricholoma matsutake, and has important significance for research and industrial development of the tricholoma matsutake.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and provides a method for simultaneously, qualitatively and quantitatively determining four purines such as xanthine, guanine, adenine, hypoxanthine and the like in a dry tricholoma matsutake product only by using one standard product aiming at the current situation that a detection method and a quality control method for purine components in the tricholoma matsutake and derivatives thereof do not exist at present.

In order to achieve the above object, the technical solution of the present invention mainly includes the following contents:

a method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake and derivatives thereof based on an HPLC-UV method comprises the following steps:

1) preparing a standard solution of a reference substance: taking methanol as a solvent, and respectively preparing a series of standard solutions of xanthine, guanine, adenine and hypoxanthine reference substances with different concentrations;

2) preparing a test solution:

taking 0.2g of dried, crushed and sieved tricholoma matsutake or derivative samples thereof, putting the tricholoma matsutake or derivative samples into a 50ml round flask, adding 8-12ml of 60-70% strong oxidizing acid, heating in a water bath at 90-110 ℃, refluxing, hydrolyzing for 20-40min, quickly putting the mixture into an ice water bath for cooling, adjusting the mixture to be neutral by using alkali liquor, filtering, adjusting the pH of filtrate to 3.8-4.2, centrifuging, taking supernate, fixing the volume by using purified water into a 50ml brown volumetric flask to obtain a sample solution, and storing the sample solution in a refrigerator at 4 ℃ for later use;

3) calculation of relative correction factor and relative retention time:

precisely sucking the reference solutions of the xanthines, guanines, adenines and hypoxanthines with different concentrations prepared in the step 1) for liquid chromatography determination to obtain an HPLC-UV chromatogram, measuring and recording peak areas of chromatographic peaks of the purines, and respectively calculating relative correction factors (f) of the xanthines, the guanines and the hypoxanthines to the adenines by taking the adenines as standard substances_s/x) And relative retention time (t)_Rx)；

The relative correction factor (f)_s/x) The calculation formula is as follows:

in the formula: f. of_s/xRelative correction factor for purine x to be measured, A_sIs the peak area of adenine chromatographic peak, C_sIs adenine mass concentration, A_xIs the peak area of chromatographic peak of purine x to be measured, C_xThe mass concentration of purine x to be detected;

the relative retention time (t)_Rx) The calculation formula is as follows:

in the formula:expressing the ratio of the retention time of the purine x chromatographic peak to be detected to the retention time of the adenine chromatographic peak, namely the relative retention time of the purine x to be detected; t is t_xRepresenting the retention time of the purine x chromatographic peak to be detected; t is t_sRepresents the retention time of an adenine chromatographic peak;

4) detection of purines in the test sample:

performing liquid chromatography determination on a series of adenine reference substance standard solutions with different concentrations prepared in the step 1) to obtain an HPLC-UV chromatogram, measuring and recording peak areas of adenine chromatographic peaks, drawing a standard curve by adopting an external standard method with the peak areas of adenine as vertical coordinates and the concentration of adenine as horizontal coordinates, and calculating to obtain a linear equation of adenine;

performing liquid chromatography determination on the sample solution prepared in the step 2) to obtain an HPLC-UV chromatogram, measuring and recording peak areas of chromatographic peaks of adenine, guanine, xanthine and hypoxanthine, substituting the peak area of the adenine into a linear equation of the adenine, and calculating to obtain the content of the adenine in the sample;

wherein, the content of guanine, xanthine and hypoxanthine is calculated according to the following formula:

in the formula:representing the mass concentration of the purine x to be detected in the sample of the tricholoma matsutake or the derivative thereof; c'_xRepresenting the mass concentration of the purine x to be detected in the test solution; c₀Representing the mass concentration of the tricholoma matsutake or derivative samples thereof in the test solution; a'_xShowing the peak area of chromatographic peak of purine x to be detected in the test solution.

Further, in the determination process, the liquid chromatography determination conditions used are as follows:

a reverse phase C18 packed chromatography column; the mobile phase consists of 98-100% of mobile phase A and 0-2% of mobile phase B, and KH is 0.02 +/-0.001 mol/L₂PO₄-H₃PO₄Taking buffer solution (pH 4.0 +/-0.2) as mobile phase A, taking methanol or acetonitrile as mobile phase B, and performing isocratic elution; column temperature: 25-35 ℃; flow rate: 0.8-1.2 mL/min; wavelength of ultraviolet detector: 254 plus or minus 2 nm; sample introduction amount:2-20μL。

the reversed phase C18 filler chromatographic column bonded with different groups has obvious influence on the separation effect of purine components. In view of the physical and chemical properties of the purine components, which are relatively polar and weakly basic, it is preferable to use an inverse phase C18 packed chromatographic column that has some tolerance to low pH and low concentration organic phases: waters Sunfire C18; waters Atlantis T3, Waters Sunfier C18 chromatography column.

Further, in the step 1), reference solutions of xanthine, guanine, adenine and hypoxanthine controls with different concentrations of 0.05, 0.50, 5.00, 20.00 and 30.00 mu g/ml are prepared. Specifically, the preparation method comprises respectively taking appropriate amount of xanthine, guanine, adenine and hypoxanthine, precisely weighing, and adding methanol to obtain solutions containing 100 μ g per ml as standard stock solutions. A proper amount of a stock solution of xanthine, guanine, adenine and hypoxanthine standard substances is precisely measured and diluted by methanol to prepare a reference substance solution containing 0.05, 0.50, 5.00, 20.00 and 30.00 mu g of xanthine, guanine, adenine and hypoxanthine per milliliter.

Specifically, in step 2), the strong dilute acid oxide is preferably nitric acid, perchloric acid, concentrated sulfuric acid, or the like.

Specifically, in the step 2), the alkali liquor is preferably potassium hydroxide or sodium hydroxide aqueous solution with the concentration of 8-12 mol/L.

Specifically, in the step 2), 1mol/L phosphoric acid is preferably added into the filtrate to adjust the pH to 3.8-4.2.

The invention adopts adenine as a reference substance to construct a one-test-and-multiple-evaluation method for purine components in tricholoma matsutake or a derivative thereof test sample, because adenine is a composition component of nucleic acid and coenzyme, participates in the synthesis of DNA and RNA in vivo, is an essential component for maintaining the metabolic function of organisms, widely exists in a natural life body, has stable structure, is similar to xanthine, guanine and hypoxanthine, and has relatively low price and easy acquisition of standard substances, and finally, adenine is preferably used as the reference substance.

Aiming at the problems that the node types of the determined purine alkaloid components are very similar, the components are alkalescent, the polarity and the water solubility are extremely strong, the properties are similar, and the conventional chromatographic conditions are difficult to realizeEffective separation, the invention preferably selects 0.02 +/-0.001 mol/L KH₂PO₄-H₃PO₄The buffer solution is a water phase (mobile phase A), the methanol or the acetonitrile is an organic phase (mobile phase B) which forms a mobile phase, and the mobile phase B has an extremely low content (0-2%) in the composition of the mobile phase.

The invention aims at negative nutritional ingredients of tricholoma matsutake and derivatives thereof as purine ingredients, and particularly relates to the application of the tricholoma matsutake and the derivatives thereof to food populations with gout and high uric acid by simultaneously performing one-test and multiple-evaluation and qualitatively and quantitatively determining four representative purine ingredients of the tricholoma matsutake and the derivatives thereof, namely xanthine, guanine, adenine and hypoxanthine. By using an HPLC-UV combined detection technology, a content determination method for one-time multi-evaluation is established, and the method realizes that only one purine standard product is adopted, the relative correction factor and the relative retention time between the purine standard product and other three purine components in the tricholoma matsutake test sample are determined, and the four purine components in the tricholoma matsutake test sample are conveniently and quantitatively determined by calculation. And different chromatographic systems (Shimadzu LC-20AT, Agilent 1260) and different chromatographic columns (Waters Atlantis T3C 18, 4.6mm X250 mm, 5 μm; Waters Sunfire C18, 4.6mm X250 mm, 5 μm; Waters X-Bridge C18, 4.6mm X150 mm, 3.5 μm) are used for the relative correction factor (f, RSD) provided by the invention<3%) and relative retention time (t)_R，RSD<5%) good durability.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a technical method for simultaneously, qualitatively and quantitatively determining four representative purine components including xanthine, guanine, adenine and hypoxanthine aiming at tricholoma matsutake and derivatives thereof based on one-time evaluation for the first time. The method can be used for the identification and quality control of the tricholoma matsutake, can solve the bottleneck that no qualitative and quantitative detection method exists for the tricholoma matsutake and the derivatives thereof at present, and has important significance for the research and development and industrial development of the tricholoma matsutake and the derivatives thereof.

Drawings

FIG. 1 is a structural formula of uric acid and purines xanthine, guanine, adenine and hypoxanthine;

FIG. 2 is a liquid chromatogram of purine standard (A) and intact strain of Pinus densiflora (B) in example 1; in the figure, 1, guanine; 2. hypoxanthine; 3. adenine; 4. xanthine;

FIG. 3 is a standard curve for xanthine, guanine, adenine and hypoxanthine in example 1;

FIG. 4 is a liquid chromatogram of a sample solution of caps of Pinus densiflora in example 2;

FIG. 5 is a liquid chromatogram of a sample solution of the tricholoma matsutake mycelia in example 3.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

The test samples and materials used in the following examples include:

the tricholoma matsutake is purchased from a bleai tricholoma matsutake planting base;

xanthine (Vickqi Biotechnology Ltd, Sichuan province, lot number: wkq20080411),

Hypoxanthine (Vickqi Biotech, Sichuan, Ltd., batch No. wkq20072809),

Guanine (Weickqi Biotechnology Ltd, Sichuan province, lot number wkq20080610),

Adenine (Vickqi Biotech, Inc., Sichuan, lot number wkq 20061907);

perchloric acid, phosphoric acid (analytically pure, purchased from the institute of optochemical and fine chemistry, Tianjin);

potassium hydroxide (analytically pure, Hengxing chemical reagents manufacturing Co., Ltd., Tianjin);

acetonitrile (chromatographic grade, available from Fisher corporation); wahaha purified water.

The apparatus used in the following examples included:

a high-speed traditional Chinese medicine grinder (Yuean Yongshi pharmaceutical machinery, Inc., model: 111B, 400w, 220V/50Hz, 25000 r/m); agilent 1260 high performance liquid chromatograph, DAD detector; ME204 ten thousandth balance (METTLER TOLEDO); AUW220D one ten-thousandth of a balance (SHIMADZU); the chromatographic column is Waters Sunfire C18, 4.6mm × 250mm, 5 μm; waters X-Bridge C18, 4.6mm by 150mm, 3.5 μm; waters Atlantis T3C 18, 4.6mm × 250mm, 5 μm; (ii) a A centrifugal machine (Cence Xiang instrument TDZ 5-WS); XMTD-7000 electric heating constant temperature water bath (Beijing Guangming medical instruments company); KQ-500E ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); DGX-9143B drying cabinet (Shanghai Fuma laboratory Equipment Co., Ltd.).

Example 1

A method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake and derivatives thereof based on an HPLC-UV method comprises the following steps:

1.1 preparation of Standard solutions of controls

Taking a proper amount of xanthine, guanine, adenine and hypoxanthine, precisely weighing, and adding methanol to prepare solutions containing 100 mu g of xanthine per milliliter respectively to serve as standard stock solutions. A proper amount of a stock solution of the xanthine, guanine, adenine and hypoxanthine standard substances is precisely measured, and diluted by methanol to respectively prepare a series of reference standard solutions of the xanthine, guanine, adenine and hypoxanthine with the concentration of 0.05, 0.50, 5.00, 20.00 and 30.00 mu g/ml.

1.2 preparation of test solution

Drying 3 batches of red pine mushroom, respectively taking complete strains, pileus and stipe, crushing and sieving by a No. 4 sieve. Weighing 0.2g of complete strain of tricholoma matsutake, placing the strain in a 50ml round flask, adding 10ml of 70% perchloric acid, hydrolyzing for 30min by a 100 ℃ water bath heating reflux method, quickly placing the strain in an ice water bath for cooling, adjusting the pH to be neutral by using 10ml/L KOH aqueous solution, filtering the solution in vacuum by using filter paper, adding 1mol/L phosphoric acid into the filtrate to adjust the pH to be 3.8, centrifuging for 10min at 3600r/min, taking supernatant, fixing the volume by using purified water to a 50ml brown solution bottle to obtain a sample solution, and storing the sample solution in a refrigerator at 4 ℃ for later use.

1.3 chromatographic conditions

Reverse phase C18 packed chromatography column: waters Sunfire C18, 4.6mm × 250mm, 5 μm; waters X-Bridge C18, 4.6mm by 150mm, 3.5 μm; waters Atlantis T3C 18, 4.6mm × 250mm, 5 μm; one of (1);

mobile phase: at 0.02mol/L KH₂PO₄-H₃PO₄Buffer solution (pH 4.0) mobile phase a: methanol is used as a mobile phase B for isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 99: 1; column temperature: 30 ℃; flow rate: 1.0 mL/min; wavelength of ultraviolet detector: 254 nm; sample introduction amount: 10 μ L.

1.4 System Adaptation experiments

And (3) sampling the standard solution of the control substance and the solution of the test substance, analyzing, and performing chromatographic conditions according to the step 1.3, wherein the chromatographic column is a Waters X-Bridge C18 column, and the result is shown in figure 2. The result shows that the separation effect of each component to be measured is good (the separation degree is more than 1.5), the tailing factor is 0.96-1.17, and the theoretical plate number is more than 5000 by each chromatographic peak.

1.5. Methodology investigation

1.5.1 calibration curves and their Linear regression

And (3) performing the same chromatographic conditions as the step 1.3, taking the concentration of the standard solution of each purine reference substance as a horizontal coordinate and the chromatographic peak area as a vertical coordinate, drawing a standard curve, fitting a linear regression equation, and calculating a linear correlation coefficient (r), wherein the result is shown in table 1.

1.5.2 precision test

The test solution is prepared by the method of 1.2 for all batches of dry products of the complete tricholoma matsutake strains, the test is repeated for 6 times according to the chromatographic conditions of 1.3, the precision of the detection method is tested, and the results are shown in table 2.

1.5..3 stability test

Sampling the same sample solution at 0, 2, 4, 8, 12 and 24h, respectively, performing HPLC content determination according to the chromatographic condition of 1.3, and calculating RSD values of chromatographic peak areas of four purines respectively.

1.5.4 sample recovery test

About 0.20g of the powder of the whole strain of tricholoma matsutake (screened through a sieve four), precisely weighed, placed in a conical flask with a stopper, 0.1, 0.2 and 0.3mL of standard solutions of 0.50mg/mL control substances of four purines were added, and 5 parallel tests were performed per group under the chromatographic conditions of 1.3, and the results of the sample addition recovery rates were calculated, respectively, as shown in Table 3.

1.6. Results and analysis

1.6.1 linear equation

The results show that in the linear range of 0.05-30.00. mu.g/mL (see FIG. 3 for the standard curve), the four purines are completely separated (see FIG. 2 for the liquid chromatogram of the four purines), the linear minimum concentration signal-to-noise ratio (S/N) is greater than 10, and the detailed results of the linear relationship are shown in Table 1.

TABLE 1 Linear relationship of four purines

1.6.2 precision test

The results are shown in table 2 and show that: the same sample is tested in parallel for 6 times, the RSD% values are all less than 3.0%, and the precision is good.

TABLE 2 purine determination precision test results of complete strain of Pinus densiflora

1.6.3 stability test

The results show that: the chromatographic peak areas RSD of xanthine, guanine, adenine and hypoxanthine were 1.11%, 1.43%, 1.341%, 1.03%, 0.96% and 2.55%, respectively (n ═ 6), and the relative retention time and the relative peak area RSD of each common peak were both < 3% with respect to the retention time and peak area of adenine, indicating that the sample solution was stable within 24 hours.

1.6.4 sample recovery test

The results are shown in Table 3 and show that: the average recovery rate of the four purines is between 98.91% and 101.23%, which shows that the accuracy of the method is good.

Table 3. unit of measurement precision of purine of complete strain of tricholoma matsutake: is based on

1.7 one-test-multiple-evaluation method for determining content of four purines

1.7.1 relative correction factor (f)_s/x) Is calculated by

Precisely sucking 10 μ L of the reference solutions of xanthine, guanine, adenine and hypoxanthine with different concentrations prepared in step 1.1, performing liquid chromatography (chromatographic conditions are shown in the same manner as in step 1.3) to obtain HPLC-UV chromatogram, measuring and recording peak areas of chromatographic peaks of the purines, and calculating relative correction factors (f) of xanthine, guanine and hypoxanthine respectively by using adenine as a standard_s/x). The results showed that the average f of xanthine, guanine and hypoxanthine relative to adenine was 0.826, 0.595 and 5.213 respectively, and RSD was 1.31%, 1.21% and 1.03% respectively.

The relative correction factor (f)_s/x) The calculation formula is as follows:

in the formula: f. of_s/xRelative correction factor for purine x to be measured, A_sIs the peak area of adenine chromatographic peak, C_sIs adenine mass concentration, A_xIs the peak area of chromatographic peak of purine x to be measured, C_xThe mass concentration of purine x to be measured is shown.

1.7.2 high performance liquid chromatograph and column durability examination

The influence of two sets of chromatographic systems (Shimadzu LC-20AT, Agilent 1260) and different chromatographic columns (Waters Sunfire C18, 4.6mm X250 mm, 5 μm; Waters X-Bridge C18, 4.6mm X150 mm, 3.5 μm; Waters Atlantis T3C 18, 4.6mm X250 mm, 5 μm) on the correction factor was examined, and the results showed that the RSD was less than 3%, indicating that the relative correction factor was good in durability under different chromatographic systems and different chromatographic columns, see Table 4.

TABLE 4 determination of purine Components using different chromatography columns and chromatography instruments f_s/xValue of

1.7.3 location of purine chromatographic peaks

Referring to 1.8.1, the relative retention time of the chromatographic peaks of the xanthine, guanine and hypoxanthine components and the chromatographic peak of adenine in two sets of chromatographic systems (Shimadzu LC-20AT, Agilent 1260) and three different chromatographic columns is calculated according to the following formula, and each component to be detected is positioned. The results (see table 5) show that: the relative retention time fluctuation is small, and the RSD is less than 3 percent.

The relative retention time (t)_Rx) The calculation formula is as follows:

in the formula:representing the relative retention time of the purine x to be detected; t is t_xRepresenting the retention time of the purine x chromatographic peak to be detected; t is t_sThe retention time of the adenine chromatographic peak is shown.

TABLE 5 determination of t for purine Compounds using different chromatographic columns and chromatographic instruments_RxValue of

1.7.4 comparison of the results of the one-test and multi-test method of the invention with those of the external standard method

The contents of xanthine, guanine, adenine and hypoxanthine in the 3 samples of the tricholoma matsutake strains in the step 1.2 were measured by one-test-multiple-evaluation and external standard methods according to the chromatographic conditions shown in the 1.3, and the measurement results of the two methods are shown in the table 6.

The one-test and multi-evaluation method specifically comprises the following steps:

taking a series of adenine reference substance standard solutions with different concentrations prepared in the step 1.1 to perform liquid chromatography determination to obtain an HPLC-UV chromatogram, measuring and recording peak areas of adenine chromatographic peaks, drawing a standard curve by adopting an external standard method with the peak areas of adenine as vertical coordinates and the concentration of adenine as horizontal coordinates, and calculating to obtain a linear equation of adenine (see fig. 3 and table 1);

performing liquid chromatography determination on the sample solution prepared in the step 1.2 to obtain an HPLC-UV chromatogram, measuring and recording peak areas of chromatographic peaks of adenine, guanine, xanthine and hypoxanthine, substituting the peak area of the adenine into a linear equation of the adenine, and calculating to obtain the content of the adenine in the sample;

wherein, the content of guanine, xanthine and hypoxanthine is calculated according to the following formula:

in the formula:representing the mass concentration of the purine x to be detected in the sample of the tricholoma matsutake or the derivative thereof; c'_xRepresenting the mass concentration of the purine x to be detected in the test solution; c₀Representing the mass concentration of the tricholoma matsutake or derivative samples thereof in the test solution; a'_xShowing the peak area of chromatographic peak of purine x to be detected in the test solution.

The test results of the two methods are tested by t, and P is more than 0.05, which shows that the results calculated by the two methods have no significant difference, and the one-test-multiple-evaluation method is simpler, more convenient, easier to operate and lower in cost than an external standard method, and is feasible in the quality control of purine in the tricholoma matsutake sample, and the table 6 shows.

Table 6 results of measurement of four purines contents in different batches of tricholoma matsutake strains by two methods (n ═ 3) (%)

Note: a. external standard method; b. a multi-evaluation method; RSD is less than 2%.

Example 2

A method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake pilum based on an HPLC-UV method is as follows in the steps of example 1, except that: the sample is pileus part of Tricholoma matsutake (liquid chromatogram of pileus of Tricholoma matsutake pileus sample solution is shown in figure 4), the chromatographic conditions shown in step 1.3 are different, and the chromatographic conditions are as follows:

a chromatographic column: waters Atlantis T3C 18, 4.6mm × 250mm, 5 μm; mobile phase: 0.02mol/L KH₂PO₄- H₃PO₄A buffer solution; column temperature: 30 ℃; flow rate: 1.0 mL/min; wavelength of ultraviolet detector: 254 nm; sample introduction amount: 10 μ L.

The results are shown in table 7, which shows that the results obtained by the two methods have no significant difference, and the one-test-multiple-evaluation method is simpler, more convenient, easier to operate and lower in cost than the external standard method, and is feasible in the quality control of the multi-index components of the tricholoma matsutake.

Table 7 results of measurement of four purines in different batches of tricholoma matsutake pili by two methods (n ═ 3) (%)

Note: a. external standard method; b. a multi-evaluation method; RSD is less than 2%.

Example 3

A method for simultaneously measuring the contents of xanthine, guanine, adenine and hypoxanthine in tricholoma matsutake pilum based on an HPLC-UV method is as follows in the steps of example 2, except that: the sample is the stipe part of Tricholoma matsutake (liquid chromatogram of the sample solution is shown in FIG. 5).

Table 8 results of measurement of four purines in different batches of tricholoma matsutake stipe by two methods (n ═ 3) (%)

Note: a. external standard method; b. a multi-evaluation method; RSD is less than 2%.

The results are shown in table 8, which shows that the results obtained by the two methods have no significant difference, and the one-test-multiple-evaluation method is simpler, more convenient, easier to operate and lower in cost than the external standard method, and is feasible in the quality control of the multi-index components of the tricholoma matsutake pileus.