CN114660191A - Quasi-target metabonomics analysis method for relative quantification of various compounds in astragalus membranaceus - Google Patents

Abstract

The invention belongs to the technical field of quasi-targeted metabonomics, and particularly relates to a quasi-targeted metabonomics analysis method for relatively quantifying a plurality of compounds in astragalus. In order to solve the problems of incomplete and inaccurate quality evaluation of astragalus membranaceus, the UPLC-QTRAP-MS/MS-based quasi-targeted metabonomics analysis method specifically comprises the following steps: (1) preparing a test solution; (2) preparing an internal standard solution; (3) optimizing MRM parameters; (4) and (5) drawing a standard curve. The analysis method provided by the invention has the advantages of high coverage, good linearity and repeatability and accurate and reliable analysis, can be used for measuring the relative content of 114 compounds in the astragalus membranaceus, and provides a reference for the quality evaluation of the astragalus membranaceus.

Description

Quasi-targeting metabonomics analysis method for relative quantification of various compounds in astragalus membranaceus

Technical Field

The invention belongs to the technical field of quasi-targeted metabonomics, and particularly relates to a quasi-targeted metabonomics analysis method for relatively quantifying a plurality of compounds in astragalus.

Background

Astragalus membranaceus is a commonly used traditional Chinese medicine, has the effects of tonifying qi and invigorating yang, consolidating superficial resistance and arresting sweating, inducing diuresis to alleviate edema, and promoting fluid production and nourishing blood, and is used for treating qi deficiency and weakness, poor appetite and loose stool, sinking of middle-jiao energy and unhealing of ulceration for a long time. At present, the astragalus root collected in 'Chinese pharmacopoeia' 2020 edition is the dried root of Astragalus mongholicus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. The existing Chinese pharmacopoeia takes the content of calycosin glucoside and astragaloside as the quality evaluation indexes of astragalus, and the literature also uses high performance liquid chromatography to determine various chemical components in astragalus, and the methods select limited index components to determine, and cannot comprehensively and deeply evaluate the quality of the astragalus. A non-targeted metabonomics method of astragalus membranaceus is established in the literature, and the non-targeted metabonomics is used as a contour analysis technology, so that a large number of components can be detected, but the relative content determination accuracy of each component is not high.

The quasi-targeted metabonomics technology obtains the ion pair information of the metabolites by using high-resolution mass spectrometry when the method is established, and the abundance of the metabolites is measured by adopting a multi-reaction detection (MRM) mode when a sample is analyzed, so that the quasi-targeted metabonomics technology has the advantages of good linearity and repeatability, higher accuracy and the like compared with a non-targeted technology; compared to targeted quantification methods, coverage is high and standards are not required to define the metabolites detected. The invention aims to establish a UPLC-Q TRAP MS-based astragalus membranaceus quasi-targeted metabonomics technology and provides a new method for the quality evaluation of astragalus membranaceus.

Disclosure of Invention

In order to comprehensively, deeply and accurately evaluate the quality of the astragalus membranaceus, the invention provides a quasi-targeted metabonomics analysis method for simultaneously determining the relative quantification of various compounds in the astragalus membranaceus.

In order to achieve the purpose, the invention adopts the following technical scheme:

a pseudo-targeted metabonomics analysis method for relative quantification of a plurality of compounds in astragalus comprises the following steps:

(1) preparation of quality control sample solution: adding methanol into the sieved astragalus powder, performing ultrasonic extraction, centrifugation and standing, taking supernatant, and passing through a microporous filter membrane to obtain a sample solution, wherein all the sample solutions are accurately transferred to 100 mu L in the same container and fully mixed to prepare a quality control sample solution;

(2) preparation of internal standard solution: respectively placing hyperoside and saikosaponin A standard substances into volumetric flasks, adding methanol to a constant volume to obtain standard substance solutions, respectively sucking each standard substance solution with the same volume into a container, and fully and uniformly mixing to obtain mixed internal standard solution;

(3) use of internal standard solution: selecting hyperoside and saikosaponin A as internal standard in positive ion mode, and selecting hyperoside as internal standard in negative ion mode;

(4) optimization of MRM parameters: the optimal ion pairs, optimal DP and CE for 114 compounds and 2 internal standard compounds are introduced into the monitoring list of the predetermined MRM as shown in tables 1 and 2 below;

TABLE 1 Positive ion mode MRM parameters for the respective compounds

TABLE 2 negative ion mode MRM parameters for the respective compounds

(5) Drawing a standard curve: concentrating 2.5 times of the quality control sample solution, diluting to a series of concentrations of 100%, 80%, 60%, 50%, 40%, 20%, 8%, 2%, 0.4%, 0.08% and 0.04%, adding a mixed internal standard solution into each diluted quality control sample solution, analyzing by liquid chromatography tandem QTRAP mass spectrometry in a positive and negative ion mode, mapping peak area ratios of an analyte and the internal standard with dilution times to construct a standard curve, and calculating the relative content of 114 compounds in each astragalus sample according to the standard curve.

Further, the mass volume ratio of the astragalus powder to the methanol in the step (1) is 0.1 g: 10mL, and the volume fraction of the methanol is 50%.

Further, the astragalus powder sieved in the step (1) is astragalus powder sieved by a fourth sieve; the ultrasonic extraction time is 20 min; the rotation speed of the centrifugation is 3500r/min, and the time is 10 min.

Further, the pore diameter of the microporous filter membrane in the step (1) is 0.22 μm.

Further, the concentration of each internal standard in the mixed internal standard solution in the step (2) is 1.67 mg/mL.

Further, the volume fraction of methanol in the step (2) is 50%.

Further, the volume ratio of each diluted quality control sample solution to the mixed internal standard solution in the step (5) is 1 mL: 40 μ L.

Further, the liquid chromatography conditions in the step (5) are as follows: chromatographic column Waters Acquity UPLC HSS T3 column, 2.1mm × 150mm, 1.8 μm, column temperature 40 deg.C, mobile phase of 0.1% volume fraction formic acid water solution and acetonitrile, gradient elution for 35min, flow rate 0.3 mL/min.

The QTRAP mass spectrum condition is as follows: the electrospray ion source has a positive ion mode and a negative ion mode, wherein the spraying voltage is +5500V or-4500V, the temperature of the ion source is 550 ℃, the air curtain gas is 40psi, and the atomizing gas and the auxiliary gas are both 50 psi.

Compared with the prior art, the invention has the following advantages:

1. compared with non-targeting, the method has the advantages of good linearity and repeatability, high accuracy and high sensitivity;

2. compared with a targeted assay method, the method has wide coverage and does not need a standard substance to limit the detected metabolites.

Drawings

FIG. 1 is a total ion flow diagram for a UPLC-QTRAP-MS/MS based method of pseudo-targeted analysis in positive ion mode (FIG. 1A) and negative ion mode (FIG. 1B);

FIG. 2 is a distribution diagram of RSD distribution of a pseudo-targeted metabonomics analysis method for relative quantification of various compounds in Astragalus membranaceus under positive ion mode (FIG. 2A), repeatability (FIG. 2B), precision (FIG. 2C);

FIG. 3 is a RSD distribution diagram of a pseudo-targeted metabonomics analysis method for relative quantification of various compounds in Astragalus membranaceus under negative ion mode, including stability (FIG. 3A), repeatability (FIG. 3B) and precision (FIG. 3C);

FIG. 4 is a graph of PCA of a sample assayed in positive ion mode using the established pseudo-targeted metabolomics analysis method;

figure 5 is a graph of PCA of samples assayed in negative ion mode using established pseudo-targeted metabolomics analysis methods.

Detailed Description

The present invention will be described in more detail and fully with reference to the following examples.

The reagents and equipment used in the examples were as follows:

the instrument comprises the following steps: ExionLC^TMAD (AB SCIEX), 5600QTOF mass spectrometer (AB SCIEX), 3200QTRAP mass spectrometer (AB SCIEX), KQ5200E ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.), chromatographic column Waters acquisition UPLC HSS T3(2.1 mm. times.150 mm, 1.8 μm).

Reagent: mass spec grade methanol, formic acid and acetonitrile (Fisher corporation), ultra pure water.

The test solution in each example was prepared as follows:

taking about 0.1g of radix astragali powder (sieved by a sieve of four numbers) to be put in a glass centrifuge tube, adding 10mL of methanol with volume fraction of 50%, ultrasonically extracting for 20min, centrifuging for 10min at 3500r/min, standing, taking supernatant, and filtering with a microfiltration membrane of 0.22 mu m for later use. Each sample solution was precisely measured out to 1mL and thoroughly mixed as a Quality Control (QC) sample. 25mL of qc sample was concentrated to dryness and reconstituted with 10mL volume fraction 50% methanol as a Concentrated Quality Control (CQC) sample for method validation analysis.

The internal standard solutions in the examples were prepared as follows:

taking about 10mg of each of hyperoside and saikosaponin A standard substances, respectively placing into 2mL volumetric flasks, and adding 50% methanol to constant volume. Respectively sucking the standard solutions with the same volume, and fully and uniformly mixing in a container to obtain mixed internal standard solutions with the internal standard concentrations of about 1.667 mg/mL.

The liquid chromatography conditions described in the examples are as follows:

a chromatographic column Waters Acquity UPLC HSS T3 column (2.1mm multiplied by 150mm, 1.8 mu m), the column temperature is 40 ℃, the mobile phase is 0.1 percent formic acid-water (A) and acetonitrile (B), the gradient elution is 0-5 min, and the gradient elution is 5-20 percent B; 5-10 min, 20% -30% of B; 30-43% of B for 10-15 min; 15-20 min, 43% -60% of B; 20-30 min, 60-100% of B, 30-30.5 min, 100-5% of B, 30.5-35 min, 5-5% of B, and the flow rate is 0.3 mL/min.

The ion source parameters of the mass spectrometer described in the examples were set as follows:

electrospray ion source (ESI) in positive and negative ion mode, and spray voltage (IS) of +5500V and-4500V; ion source Temperature (TEMP) 550 ℃; air curtain air (CUR) 40 psi; both the atomizing (GS1) and assist (GS2) gases were at 50 psi.

Example 1 establishment of a pseudo-targeting method

(1) Optimization of test Compound parameters

And (3) acquiring secondary mass spectrum data of different Collision Energies (CE) of the astragalus membranaceus sample by using a 5600QTOF mass spectrometer in an IDA (information association acquisition) mode, selecting 1-3 fragment ions with high responsivity as candidate ions for the characterized compound, constructing the candidate ions together with parent ions into candidate ion pairs, and selecting the optimal CE for each ion pair according to the response intensity.

Using a QTRAP mass spectrometer connected with UPLC in series, a multi-reaction monitoring (MRM) MS mode is adopted, the DP of each candidate ion pair is respectively set to be 20, 40, 60 and 80V, and according to the peak intensity of MRM ion transition, the ion pair corresponding to the ion channel with the highest response of each compound and the CE and DP values are determined as the final ion pair and related parameters of the compound. The liquid chromatography conditions and ion source parameters were the same as above, and the detection mode was MRM (multiple reaction monitoring mode).

(3) Determination of internal standard Compounds

Selection of alternative internal standard compounds: selecting rutin, quercitrin, hyperoside and hesperidin as flavonoid alternative internal standard, and ginsenoside Rg1, saikosaponin A and saikosaponin D as triterpene saponin alternative internal standard.

Optimizing MRM parameters of various alternative internal standards: and preparing a mixed standard solution containing all the alternative internal standards by using methanol with the volume fraction of 50%, wherein the concentration of each internal standard is 500-1000 ng/mL. And (3) using a needle pump sample injection mode, firstly optimizing Q1 to determine parent ions, then optimizing Q3 to determine daughter ions, and finally optimizing ion pair parameters (CE and DP) to obtain MRM parameters of each alternative internal standard.

Determination of internal standard compounds: firstly, flavonoid and triterpenoid saponin components which are not contained in the astragalus; completely separating from each component chromatographic peak; the retention time is in the middle; high response. Finally determining the internal standard as hyperin and saikosaponin A in the positive ion mode and the internal standard compound as hyperin in the negative ion mode according to the conditions.

(4) And (3) introducing the ID, MRM ion pair, retention time, optimal CE and optimal DP of each compound to be tested and the internal standard into a monitoring list of a preset MRM, and establishing a UPLC-QTRAP-MS/MS-based quasi-target analysis method. 63 compounds in positive ion mode and 71 compounds in negative ion mode, wherein 20 compounds can be detected in positive and negative ion mode simultaneously, and the MRM monitoring list is shown in tables 1 and 2.

TABLE 1 positive ion mode MRM parameters for individual compounds

TABLE 2 negative ion mode MRM parameters for the respective compounds

Example 2 Astragalus pseudo-targeted metabolomics methodology review

The QC sample solution concentrated by 2.5 times is diluted to different concentrations sequentially by 50% methanol, including 100%, 80%, 60%, 50%, 40%, 20%, 8%, 2%, 0.4%, 0.08%, 0.04%, and 1mL of each is added into 40 μ L of mixed internal standard solution for determination. A standard curve was constructed by plotting the peak area ratio of the analyte and internal standard against the dilution factor. Samples of high (80%), medium (40%), and low (2%) dilution levels were selected as quality control samples to evaluate day-to-day and day-to-day precision. In addition, a sample was selected for 6 replicate runs to evaluate the reproducibility of the test method, and the same sample was analyzed every 8 hours to evaluate the stability of the sample.

In the positive ion mode, the r values of the 63 compounds are all more than 0.995 in the concentration range of 0.04% -100%, and the linear relation is good (table 3). As shown in FIG. 2C, the results of the day precision showed that the RSD values of 58, 60 and 63 compounds were less than 15% at the low, medium and high concentration levels, respectively, and that the RSD values of 58, 59 and 59 compounds were less than 15% at the low, medium and high concentration levels, respectively. The results of the reproducibility test shown in FIG. 2B show that the RSD of 63 compounds was less than 15%. The results of the 72 hour stability test shown in figure 2A show that the RSD was less than 15% for all compounds.

In the negative ion mode, the r values of the 71 compounds are all more than 0.995 in the concentration range of 0.04% -100%, and the linear relation is good (table 4). As shown in FIG. 3C, the results of the day precision showed that the RSD values of 70, 69 and 65 compounds at the low, medium and high concentration levels were less than 15%, and the RSD values of 65, 58 and 43 compounds at the low, medium and high concentration levels were less than 15%. The results of the reproducibility test shown in FIG. 3B show that the RSD of the 69 compounds was less than 15%. The results of the 72 hour stability test shown in figure 3A show that the RSD was less than 15% for all compounds.

The conclusion shows that the established method has high precision, good reproducibility and accurate and reliable experimental results.

TABLE 3 Standard Curve, correlation coefficient and Linear Range results for each Compound in Positive ion mode

TABLE 4 Standard Curve, correlation coefficient and Linear Range results for each Compound in anion mode

Example 3 application of Astragalus membranaceus pseudo-targeted metabonomics method

The verified method is applied to the determination of 10 batches of astragalus samples (HQ 1-5 is Guangling astragalus and HQ 6-10 is turbid source astragalus), the relative content of each analyte is calculated through corresponding regression standard curves, and the results are shown in tables 5 and 6. The results of PCA analysis are shown in FIGS. 4 and 5, and there is no significant difference in the content of Astragalus membranaceus in the two origins of Guangling and muddy source.

TABLE 5 relative content of the respective compounds in the cation mode

TABLE 6 relative content of each compound in anion mode

Claims (8)

1. A pseudo-targeted metabonomics analysis method for relatively quantifying a plurality of compounds in astragalus membranaceus is characterized by comprising the following steps:

(1) preparation of quality control sample solution: adding methanol into the sieved radix astragali powder, performing ultrasonic extraction, centrifuging, standing, collecting supernatant, and passing through microporous membrane to obtain sample solution, accurately transferring 100 μ L of all sample solutions into the same container, and mixing completely to obtain quality control sample solution;

(2) preparation of internal standard solution: respectively placing hyperoside and saikosaponin A standard substances into volumetric flasks, adding methanol to a constant volume to obtain standard substance solutions, respectively sucking each standard substance solution with the same volume into a container, and fully and uniformly mixing to obtain mixed internal standard solution;

(3) use of internal standard solution: selecting hyperoside and saikosaponin A as internal standard in positive ion mode, and selecting hyperoside as internal standard in negative ion mode;

(4) optimization of MRM parameters: the optimal ion pairs, optimal DP and CE of the 114 compounds and 2 internal standard compounds are introduced into the monitoring list of the predetermined MRM as shown in tables 1 and 2 below;

TABLE 1 Positive ion mode MRM parameters for the respective compounds

TABLE 2 negative ion mode MRM parameters of the respective compounds

(5) Drawing a standard curve: concentrating 2.5 times of the quality control sample solution, diluting to a final concentration of 100%, 80%, 60%, 50%, 40%, 20%, 8%, 2%, 0.4%, 0.08%, 0.04%, adding a mixed internal standard solution to each diluted quality control sample solution, analyzing by liquid chromatography tandem QTRAP mass spectrometry in a positive and negative ion mode, mapping the peak area ratio of the analyte and the internal standard with the dilution multiple to construct a standard curve, and calculating the relative content of 114 compounds in each astragalus sample according to the standard curve.

2. The pseudo-targeted metabonomics analysis method for relative quantification of multiple compounds in astragalus membranaceus according to claim 1, wherein the mass-to-volume ratio of the astragalus membranaceus powder to methanol in step (1) is 0.1 g: 10mL, and the volume fraction of the methanol is 50%.

3. The pseudo-targeted metabonomics analysis method for the relative quantification of various compounds in astragalus membranaceus according to claim 1, wherein the astragalus membranaceus powder sieved in step (1) is astragalus membranaceus powder sieved with a sieve IV; the ultrasonic extraction time is 20 min; the rotation speed of the centrifugation is 3500r/min, and the time is 10 min.

4. The pseudo-targeted metabonomics analysis method for relative quantification of multiple compounds in astragalus membranaceus according to claim 1, wherein the pore size of the microporous filter membrane in the step (1) is 0.22 μm.

5. The pseudo-targeted metabonomics analysis method for relative quantification of multiple compounds in astragalus according to claim 1, wherein the concentration of each internal standard in the mixed internal standard solution in the step (2) is 1.67 mg/mL.

6. The method for pseudo-targeted metabonomics analysis of relative quantification of various compounds in Astragalus membranaceus according to claim 1, wherein the volume fraction of methanol in step (2) is 50%.

7. The pseudo-targeted metabonomics analysis method for relative quantification of multiple compounds in astragalus according to claim 1, wherein the volume ratio of the diluted quality control sample solution to the mixed internal standard solution in the step (5) is 1 mL: 40 μ L.

8. The pseudo-targeted metabonomics analysis method for relative quantification of multiple compounds in astragalus according to claim 1, wherein the liquid chromatography conditions in step (5) are as follows: chromatographic column Waters Acquity UPLC HSS T3 column, 2.1mm × 150mm, 1.8 μm, column temperature 40 deg.C, mobile phase of 0.1% volume fraction formic acid water solution and acetonitrile, gradient elution for 35min, flow rate 0.3 mL/min.

The QTRAP mass spectrum condition is as follows: the electrospray ion source is in a positive ion mode and a negative ion mode, the spraying voltage is +5500V and-4500V, the temperature of the ion source is 550 ℃, the gas curtain gas is 40psi, and the atomizing gas and the auxiliary gas are both 50 psi.

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