阅读说明：本技术 一种大鼠血浆中黄芪建中丸十二种成分的定量分析方法 (Quantitative analysis method for twelve components of astragalus mongholicus Jianzhong pills in rat plasma ) 是由 刘月涛 贾璐 秦雪梅 于 2019-10-16 设计创作，主要内容包括：本发明涉及一种大鼠血浆中黄芪建中丸十二种成分的定量分析方法,属于生物分析领域。本发明采用蛋白沉淀处理大鼠血浆样本,以山萘酚为内标采用超高效液相色谱-质谱联用(UPLC-MS/MS)方法检测大鼠血浆中黄芪建中丸十二种成分的含量。采用本发明的检测方法,灵敏度高、专属性强、稳定性高、回收率高达108.74％,既能满足回收率的要求,又可以提高检测效率。此外,本检测方法的重现性好,分析时间短,能满足黄芪建中丸在大鼠血浆中浓度的检测要求,可用于黄芪建中丸药代动力学研究和生物等效性研究。(The invention relates to a quantitative analysis method for twelve components of astragalus jianzhongwan in rat plasma, belonging to the field of biological analysis. The method adopts protein precipitation to process a rat plasma sample, and adopts an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method to detect the content of twelve components of the astragalus jianzhongwan in the rat plasma by taking kaempferol as an internal standard. The detection method has the advantages of high sensitivity, strong specificity, high stability and high recovery rate of 108.74%, and can meet the requirement of recovery rate and improve the detection efficiency. In addition, the detection method has good reproducibility and short analysis time, can meet the detection requirement of the concentration of the astragalus jianzhongwan in the plasma of rats, and can be used for pharmacokinetic research and bioequivalence research of the astragalus jianzhongwan.)

1. A quantitative analysis method for twelve components of astragalus mongholicus Jianzhong pills in rat plasma is characterized by comprising the following steps: the method comprises the following steps:

(1) processing a rat plasma sample;

(2) preparing a reference stock solution;

(3) preparing a mixed standard working solution;

(4) preparing an internal standard working solution;

(5) preparing a standard curve working solution;

(6) drawing a standard curve;

(7) the content of twelve components in rat plasma was determined by standard curve quantitation.

2. The method for quantitatively analyzing twelve components of the astragalus mongholicus jianzhong pill in the plasma of the rat according to claim 1, which is characterized in that: kaempferol was used as an internal standard.

3. The method for quantitatively analyzing twelve components of the astragalus mongholicus jianzhong pill in the plasma of the rat according to claim 1, which is characterized in that: the rat plasma sample is processed in the step (1), and the method specifically comprises the following steps: and adding 50 mu L of internal standard working solution into 200 mu L of plasma sample, adding 800 mu L of acetonitrile, vortexing for 5 minutes, centrifuging for 15 minutes at 4 ℃ and 13000rpm, taking 1000 mu L of supernatant into another 1.5mL centrifuge tube, placing into a high-speed centrifugal concentrator, preserving heat for 40 ℃, drying, redissolving 100 mu L of methanol, vortexing for 1 minute, centrifuging for 10 minutes at 4 ℃ and 13000rpm, and taking the supernatant to obtain the required sample solution.

4. The method for quantitatively analyzing twelve components of the astragalus mongholicus jianzhong pill in the plasma of the rat according to claim 1, which is characterized in that: the method for preparing the reference substance solution in the step (2) comprises the following specific steps: taking liquiritin, isoliquiritin, liquiritigenin, calycosin, formononetin, pterocarpan glycoside, calycosin, cinnamic acid, pterocarpan, formononetin, isoflavan and glycyrrhetinic acid reference substances, precisely weighing, placing into a 5mL volumetric flask, adding methanol to constant volume, and preparing into reference substance stock solutions with concentrations of 0.626mg/mL, 0.21mg/mL, 0.2mg/mL, 0.312mg/mL, 0.218mg/mL, 0.312mg/mL, 0.27mg/mL, 0.256mg/mL, 0.192mg/mL, 0.228mg/mL and 0.274mg/mL respectively.

5. The method for quantitatively analyzing twelve components of the astragalus mongholicus jianzhong pill in the plasma of the rat according to claim 1, which is characterized in that: the preparation of the mixed reference solution in the step (3) specifically comprises the following steps: respectively sucking appropriate volumes of twelve control solutions into a 5mL volumetric flask, and performing constant volume to obtain mixed control solutions with final concentrations of liquiritin, isoliquiritin, liquiritigenin, calycosin, formononetin, pterocarpan glycoside, calycosin, cinnamic acid, pterocarpan, formononetin, isoflavan and glycyrrhetinic acid of 5.008 μ g/mL, 1.68 μ g/mL, 1.6 μ g/mL, 28 μ g/mL, 6.54 μ g/mL, 24.96 μ g/mL, 21.6 μ g/mL, 54.8 μ g/mL, 0.512 μ g/mL, 0.768 μ g/mL, 0.456 μ g/mL and 3.24 μ g/mL.

6. The method for quantitatively analyzing twelve components of the astragalus mongholicus jianzhong pill in the plasma of the rat according to claim 1, which is characterized in that: the internal standard working solution prepared in the step (4) specifically comprises the following steps: precisely weighing kaempferol reference substances, placing the kaempferol reference substances into a 5mL volumetric flask, and adding methanol to a constant volume, wherein the concentration is 50 mu g/mL.

7. The method for quantitatively analyzing twelve components of the astragalus mongholicus jianzhong pill in the plasma of the rat according to claim 1, which is characterized in that: the step (5) of preparing a standard working solution specifically comprises the following steps: the mixed standard solutions were diluted to a range of concentrations of 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, and 200-fold of the final concentration.

8. The method for quantitatively analyzing twelve components of the astragalus mongholicus jianzhong pill in the plasma of the rat according to claim 1, which is characterized in that: drawing a standard curve in the step (6), specifically: taking 200 mu L of blank plasma, adding 50 mu L of standard working solution and 20 mu L of internal standard solution, 800 mu L of acetonitrile, vortexing for 5 minutes, centrifuging for 15 minutes at 4 ℃ and 13000rpm, sucking 1000 mu L of supernatant into a 1.5mL centrifuge tube, placing the centrifuge tube in a high-speed centrifugal concentrator, preserving heat for 40 ℃, drying, adding 100 mu L of methanol for redissolving, vortexing for 1 minute, centrifuging for 10 minutes at 4 ℃ and 13000rpm, sucking the supernatant to obtain a blank solution, detecting by adopting ultra-high phase liquid chromatography tandem mass spectrometry, and drawing a standard curve.

9. The method of claim 8 for quantitatively analyzing twelve components of Huangqijianzhongwan in rat plasma, which is characterized in that:

the liquid chromatography conditions are specifically as follows: waters acquisition UPLC HSS T3 liquid chromatography column, specification: 2.1mm × 100mm, 1.8 μm; column temperature: 40 ℃; the sample injection amount is 5 mu L, and the liquid phase flow rate is 0.25 mL/min; mobile phase A/B: 0.1% aqueous formic acid/acetonitrile solution; the mobile phase gradient elution procedure was: 0-5min, 5% B; 5-10min, 20% B; 10-15min, 25% B; 15-18min, 30% B; 18-20min, 40% B; 20-23min, 60% B; 23-25min, 99% B, analysis time 25 min.

10. The method of claim 8 for quantitatively analyzing twelve components of Huangqijianzhongwan in rat plasma, which is characterized in that:

the mass spectrum detection conditions are specifically as follows: an MRM multi-reaction monitoring mode and an ESI ion source are adopted, the temperature of the ion source is 400 ℃, and an ionization mode is as follows: pneumatically assisted electrospray ionization; CAD: medium; and (4) CUR: 20 psi; GS 1: 45 psi; GS 2: 35 psi; TEM: 400 ℃; IS: 5500 v.

Technical Field

The invention relates to the technical field of biological sample traditional Chinese medicine component detection, and particularly relates to a quantitative analysis method for twelve components of astragalus mongholicus jianzhongwan in rat plasma.

Background

The astragalus root jianzhong pill is prepared from a Zhongjing meridian formula astragalus root jianzhong decoction, and the whole formula comprises five medicines of astragalus root, cinnamon, white paeony root, honey-fried licorice root and Chinese date. The traditional Chinese medicine composition is mainly used for tonifying qi, dispelling cold, invigorating stomach and regulating stomach clinically. Modern pharmacological research shows that the traditional Chinese medicine has definite curative effect on treating gastrointestinal diseases caused by spleen deficiency; in addition, researches show that the astragalus root and jianzhong pills also have the effects of calming, relieving spasm, reducing blood sugar, regulating blood components, regulating immunologic function and the like. At present, methods for quantitative detection, pharmacokinetic research and bioequivalence test in astragalus center-building pills have not been reported. In order to accelerate the research and development of the classic prescription in China, a high-sensitivity and high-flux quantitative analysis method in the astragalus membranaceus middle-building pill body is urgently needed to be established.

In recent years, the technology of mass spectrometry-based coupling has been rapidly developed, especially ultra-high performance liquid chromatography tandem mass spectrometry. The ultra-high phase liquid chromatography tandem mass spectrum has the characteristics of high separation efficiency, high flux, high sensitivity and strong specificity, and is small in matrix interference, so that the sample pretreatment process is simplified, and the analysis speed is high.

Disclosure of Invention

The invention provides a quantitative analysis method with high sensitivity, good reproducibility, accurate analysis and reliability for the detection in the body of the astragalus center-strengthening pill, and provides a quantitative analysis method for twelve components of the astragalus center-strengthening pill in rat plasma.

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

a quantitative analysis method for twelve components of astragalus mongholicus and jianzhongwan in rat plasma comprises the following steps:

(1) processing a rat plasma sample;

(2) preparing a reference stock solution;

(3) preparing a mixed standard working solution;

(4) preparing an internal standard working solution;

(5) preparing a standard curve working solution;

(6) drawing a standard curve;

(7) the content of twelve components in rat plasma was determined by standard curve quantitation.

Furthermore, the quantitative analysis method for twelve components of the astragalus mongholicus Jianzhong pill in the rat plasma adopts kaempferol as an internal standard. Through a complete analysis method and combined with internal standard analysis, in-vivo quantification meeting pharmacopeia standards is established, and 12 components in plasma after the rats are orally administered with the astragalus mongholicus pills for strengthening the middle jiao.

Still further, the rat plasma sample is processed in the step (1), specifically: and adding 50 mu L of internal standard working solution into 200 mu L of plasma sample, adding 800 mu L of acetonitrile, vortexing for 5 minutes, centrifuging for 15 minutes at 4 ℃ and 13000rpm, taking 1000 mu L of supernatant into another 1.5mL centrifuge tube, placing into a high-speed centrifugal concentrator, preserving heat for 40 ℃, drying, redissolving 100 mu L of methanol, vortexing for 1 minute, centrifuging for 10 minutes at 4 ℃ and 13000rpm, and taking the supernatant to obtain the required sample solution. By introducing simple operations such as internal standard, acetonitrile deproteinization, freeze-drying and redissolution, the loss and system error of the components to be detected are reduced, the matrix effect caused by the matrix is reduced, and the extraction efficiency of 12 chemical components is improved.

Further, the method for preparing the reference solution in the step (2) comprises the following specific steps: taking liquiritin, isoliquiritin, liquiritigenin, calycosin, formononetin, pterocarpan glycoside, calycosin, cinnamic acid, pterocarpan, formononetin, isoflavan and glycyrrhetinic acid reference substances, precisely weighing, placing into a 5mL volumetric flask, adding methanol to constant volume, and preparing into reference substance stock solutions with concentrations of 0.626mg/mL, 0.21mg/mL, 0.2mg/mL, 0.312mg/mL, 0.218mg/mL, 0.312mg/mL, 0.27mg/mL, 0.256mg/mL, 0.192mg/mL, 0.228mg/mL and 0.274mg/mL respectively. The standard solution with set concentration is prepared, so that the waste of the standard substance of the object to be detected can be reduced, and the resources are saved.

Further, the step (3) of preparing the mixed reference solution specifically comprises: respectively sucking appropriate volumes of twelve control solutions into a 5mL volumetric flask, and performing constant volume to obtain mixed control solutions with final concentrations of liquiritin, isoliquiritin, liquiritigenin, calycosin, formononetin, pterocarpan glycoside, calycosin, cinnamic acid, pterocarpan, formononetin, isoflavan and glycyrrhetinic acid of 5.008 μ g/mL, 1.68 μ g/mL, 1.6 μ g/mL, 28 μ g/mL, 6.54 μ g/mL, 24.96 μ g/mL, 21.6 μ g/mL, 54.8 μ g/mL, 0.512 μ g/mL, 0.768 μ g/mL, 0.456 μ g/mL and 3.24 μ g/mL. The mixed reference solution can realize simultaneous analysis of multiple components by one sample injection, thereby saving time and cost. In addition, the ion suppression effect in the mass spectrometry process can be reduced, and the analysis efficiency can be improved.

Further, the preparation of the internal standard working solution in the step (4) specifically comprises: precisely weighing kaempferol reference substances, placing the kaempferol reference substances into a 5mL volumetric flask, and adding methanol to a constant volume, wherein the concentration is 50 mu g/mL. The kaempferol is a common flavonoid ingredient mother core, has a structure similar to that of an ingredient to be detected, can reduce interference caused by a plasma biological matrix, and can be accurately and quantitatively analyzed.

Further, in the step (5), a standard working solution is prepared, specifically: the mixed standard solutions were diluted to a range of concentrations of 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, and 200-fold of the final concentration. The standard solutions with the series of concentrations are configured, so that the concentration curve of the substance to be detected in the plasma can be simulated, and the dynamic concentration range of the drug in the plasma can be met.

Furthermore, the step (6) draws a standard curve, specifically: taking 200 mu L of blank plasma, adding 50 mu L of standard working solution and 20 mu L of internal standard solution, 800 mu L of acetonitrile, vortexing for 5 minutes, centrifuging for 15 minutes at 4 ℃ and 13000rpm, sucking 1000 mu L of supernatant into a 1.5mL centrifuge tube, placing the centrifuge tube in a high-speed centrifugal concentrator, preserving heat for 40 ℃, drying, adding 100 mu L of methanol for redissolving, vortexing for 1 minute, centrifuging for 10 minutes at 4 ℃ and 13000rpm, sucking the supernatant to obtain a blank solution, detecting by adopting ultra-high phase liquid chromatography tandem mass spectrometry, and drawing a standard curve. Introducing a blank matrix (blank plasma), and simulating the interference of the biological matrix to the object to be detected through parallel experimental operation, thereby accurately determining the quality and the quantity.

Further, the liquid chromatography conditions are specifically that the chromatographic column: waters acquisition UPLC HSS T3 liquid chromatography column, specification: 2.1mm × 100mm, 1.8 μm; column temperature: 40 ℃; the sample injection amount is 5 mu L, and the liquid phase flow rate is 0.25 mL/min; mobile phase A/B: 0.1% aqueous formic acid/acetonitrile solution; the mobile phase gradient elution procedure was: 0-5min, 5% B; 5-10min, 20% B; 10-15min, 25% B; 15-18min, 30% B; 18-20min, 40% B; 20-23min, 60% B; 23-25min, 99% B, analysis time 25 min; and (3) obtaining better separation degree in a short time through chromatographic gradient separation, and quickly preparing qualitative and quantitative analysis on the object to be detected.

Further, the mass spectrometry detection conditions are specifically as follows: an MRM multi-reaction monitoring mode and an ESI ion source are adopted, the temperature of the ion source is 400 ℃, and an ionization mode is as follows: pneumatically assisted electrospray ionization; CAD: medium; and (4) CUR: 20 psi; GS 1: 45 psi; GS 2: 35 psi; TEM: 400 ℃; IS: 5500 v. And (3) constructing an MRM quantitative mode suitable for different components to be measured, and realizing accurate and sensitive quantification.

Preparing a QC solution: and (4) selecting the standard working solution with the low, medium and high concentrations in the step (4) as the QC solution.

And (3) carrying out methodology investigation on the established quantitative analysis method of the twelve components of the astragalus mongholicus Jianzhong pill in the rat plasma by using a QC solution. The accuracy and the robustness of the analysis method are ensured, and the accuracy of a quantitative result is ensured.

Compared with the prior art, the invention has the following beneficial effects: 1. the analysis time is short; 2. the separation degree is better; 3. has good reproducibility and can accurately measure the content of the twelve components.

The method can be used for pharmacokinetics research of the radix astragali Jianzhong pill.

Drawings

FIG. 1 IS a mass spectrometric total ion flow diagram of a sample solution (IS: internal standard).

Detailed Description

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