CN107300592B - Measure the LC-MS method of Paeoniflorin metabolin in enteral bacterium - Google Patents

Measure the LC-MS method of Paeoniflorin metabolin in enteral bacterium Download PDF

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CN107300592B
CN107300592B CN201710545755.3A CN201710545755A CN107300592B CN 107300592 B CN107300592 B CN 107300592B CN 201710545755 A CN201710545755 A CN 201710545755A CN 107300592 B CN107300592 B CN 107300592B
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paeoniflorin
bacterium
enteral
metabolin
enteral bacterium
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CN107300592A (en
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刘玉峰
孙珊珊
朱丽君
胡延喜
徐亮
桑育黎
卢晓丹
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Liaoning University
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

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Abstract

The present invention relates to the LC-MS methods of Paeoniflorin metabolin in measurement enteral bacterium.The technical solution adopted is that: the preparation of enteral bacterium；The enteral bacterium of Paeoniflorin converts；Sample treatment and metabolite analysis carry out Paeoniflorin metabolite structures identification in enteral bacterium by LC-MS method.The present invention has the advantages that easy to operate, sample treatment is simple, analysis speed is fast, high sensitivity.This method can detect more metabolites of Paeoniflorin simultaneously, and it can more, the more acurrate structure for deducing metabolite with the analysis method, important reference is provided for the inside and outside metabolism of Paeoniflorin, is of great significance to further investigation activated product and its mechanism of action.

Description

Measure the LC-MS method of Paeoniflorin metabolin in enteral bacterium
Technical field
The invention belongs to medical detection technique fields, and in particular to a kind of liquid matter for measuring Paeoniflorin metabolin in enteral bacterium Method for combined use.
Background technique
Radix paeoniae rubra is the traditional Chinese medicine in China, has the effect of clearing heat and cooling blood, removing blood stasis and acesodyne, can be used for febrile virulent maculae, fall Flutter damage, liver depression costalgia, the diseases such as amenorrhea and algomenorrhea and too fat to move sore.Chemical component in radix paeoniae rubra is more complex, but the monoterpene in radix paeoniae rubra Methods of glycosides is considered as the main active of radix paeoniae rubra, mainly there is Paeoniflorin etc., and major part has end of the bridge oxygen β-glucose With highly oxidized caged pinane skeleton structure, modern pharmacology shows that Paeoniflorin has expansion blood vessel, antalgic and sedative, anti-inflammatory A variety of effects such as antiulcer, antipyretic spasmolysis, diuresis.
For oral drugs, alimentary canal is one of most important drug metabolism place.Contain a large amount of intestines in enteron aisle Road flora and organized enzyme, when drug passes through will necessarily recurring structure change, and the enzyme generated in alimentary canal flora is largely It decomposes or reductase, the enzyme in kind of analogy liver is more, therefore, it is also increasingly important to study influence of the intestinal bacterium to drug. To some degree, intestinal flora can regard the supplement to liver metabolism as to the metabolism of drug.Research in recent years shows Chinese herbaceous peony Mainly with original shape from renal excretion after medicine glycosides intravenously administrable, replication in vitro is few in hepatic metabolism as the result is shown, and Paeoniflorin is in intestinal wall, liver Dirty and lung can hardly be metabolized conversion, therefore, may be mainly through intestinal bacteria metabolism.Research drug metabolism in vivo product remains In dosage limited some drawbacks and the limitation such as low with blood concentration, and there is many for the method for in vitro study drug metabolism Advantage, the conversion simulation human body enteral drug metabolism of vitro Drug enteral bacterium, the metabolite of generation is more stable, is more advantageous to it It is detected and is prepared, and is true close to drug metabolism in vivo.For inquiring into drug metabolism in vivo product, further investigation drug is living Property product and its mechanism of action be of great significance, for Scientific Usage of Drugs have directive function.So being surveyed by the conversion of enteral bacterium Determining Paeoniflorin in-vitro metabolic product becomes a kind of a kind of important method for inquiring into Paeoniflorin cylinder metabolism-ure.
Summary of the invention
The purpose of the present invention is being directed to the vacancy of existing research, a kind of liquid for measuring Paeoniflorin metabolin in enteral bacterium is provided Matter is combined detection method, and this method has easy to operate, and sample treatment is simple, analysis speed is fast, high specificity, high sensitivity Advantage.
The technical solution adopted by the present invention are as follows: the LC-MS method of Paeoniflorin metabolin in measurement enteral bacterium, including such as Lower step:
1) preparation of enteral bacterium: taking excrement, under the conditions of sterile oxygen free operation, excrement is put into one and is full of nitrogen In valve bag, excrement is made to homogenize, is then placed in the GAM culture medium that sterilizing finishes, gently shakes, set sterile anaerobism constant temperature In incubator, cultivated for 24 hours at 37 DEG C；
2) the enteral bacterium conversion of Paeoniflorin: in sterile anaerobic culture box, Paeoniflorin is added into the enteral bacterium of culture, so After be placed on 37 DEG C of constant-temperature tables and shake, speed 100r/min cultivates 2-24h, takes out, be added ethyl acetate terminate it is anti- It answers, obtains the intestinal bacteria metabolism product of Paeoniflorin；
3) sample treatment: using extract liquor extraction Paeoniflorin intestinal bacteria metabolism product three times, merge organic phase, rotation steam Dry, again with methanol is transferred in EP pipe, crosses 0.22 μm of miillpore filter, obtains metabolite sample；
4) sample measures: actual conditions are as follows:
Chromatographic condition: chromatographic column: Dikma DiamonsilTM ODS C18Chromatographic column；
Elution time: t=50min；
Sample volume: 10 μ L；
Detection wavelength: λ=230nm；
Column temperature: 30 DEG C；
Flow velocity: 0.5mL/min；
Mobile phase: water (A)+methanol (B), using gradient elution, program is as shown in table 1；
Table 1
Time (min) Flow velocity (mL/min) A (%) B (%)
0 0.5 85 15
10 0.5 70 30
20 0.5 60 40
35 0.5 0 100
50 0.5 0 100
Mass Spectrometry Conditions: first mass spectrometric: ionization mode: electrospray ionisation (ESI)；
Scan pattern: cation scanning；
Electron spray voltage: 4.0bar；
Dry gas stream speed: 10L/min；
Dry temperature degree: 200 DEG C；
Second order ms: ionization mode: electrospray ionisation (ESI)；
Scan pattern: cation scanning；
Collision gas: nitrogen；
Capillary voltage: 2.8kV；
Orifice potential: 3.0V；
Source temperature: 110 DEG C；
Desolventizing temperature: 350 DEG C；
Desolvation gas flow: 50L/Hr；
Collision energy: 15-35eV.
The LC-MS method of Paeoniflorin metabolin in above-mentioned measurement enteral bacterium, the excrement is from human body or moves Object.
The LC-MS method of Paeoniflorin metabolin in above-mentioned measurement enteral bacterium, in step 3), the extract liquor For water saturated ethyl acetate solution.
The LC-MS method of Paeoniflorin metabolin in above-mentioned measurement enteral bacterium, in step 3), the extract liquor is Water saturated methyl tertiary butyl ether(MTBE) (MTBE) solution.
The LC-MS method of Paeoniflorin metabolin, the chromatographic column, model in above-mentioned measurement enteral bacterium DiamonsilTM ODS C18Chromatography 250mm × 4.6mm, 5 μm.
The invention has the following advantages:
The present invention carries out In vitro metabolism to Paeoniflorin using enteral bacterium, by LC-MS detection method to metabolite It is measured, the results showed that, after Paeoniflorin is metabolized in bacterium in the intestine, in MTBE extract layer sample, detect 34 metabolins, 10 metabolites are identified, wherein 7 metabolins are to find in bacterium conversion in the intestine for the first time；In ethyl acetate extract layer, inspection 12 metabolins are measured, 1 metabolite is identified, are known metabolin.Compared to other methods, this method can detect simultaneously More metabolites of Paeoniflorin select the more other common solvents of MTBE that can extract more metabolites in culture solution, More, the more acurrate structure for deducing metabolite of energy has important meaning for further investigation activated product and its mechanism of action Justice has directive function for Scientific Usage of Drugs.
The present invention carries out In vitro metabolism to Paeoniflorin using enteral bacterium, can largely aids drug internal generation It thanks, while solving during inquiring into drug metabolism in vivo, the concentration of proto-drug and its metabolite is low, and internal disturbing factor is more The problems such as, to excluding the interference of endogenous material, accurately find metabolite, to and deeply be ground to identification metabolite Study carefully pharmaceutical activity product and its mechanism of action is of great significance.
The present invention is detected using Liquid Chromatography-Tandem Mass Spectrometry instrument, and detection time is short, and flux is high, and detection sensitivity is high, specifically Property is good, at low cost.The present invention, it is the advantages of integrating efficient liquid phase (HPLC) and mass spectrum (MS), both efficient, quasi- with HPLC Really, complex mixture sample can be carried out a point analysis of variance by the good feature of sensitivity, while also have mass spectrographic high sensitivity With highly selective feature, accurate molecular weight and structure feature information abundant are provided to the identification of unknown compound, it is therefore, special It is not suitable for the traditional Chinese medicine research that effective component is complicated, content is low.
The present invention furthers investigate activated product and its mechanism of action with important for inquiring into drug metabolism in vivo product Meaning has directive function for Scientific Usage of Drugs.Simultaneously to promotion radix paeoniae rubra new drug development and improving dosage form and to the side of Chinese medicine Pharmacology opinion all has significance.
Detailed description of the invention
Fig. 1 is that Paeoniflorin extracts separation process figure.
Fig. 2 is the total ion chromatogram of MTBE extract layer after Paeoniflorin converts in bacterium in the intestine.
Fig. 3 is control group 1MTBE extract layer total ion chromatogram.
Fig. 4 is control group 2MTBE extract layer total ion chromatogram.
Fig. 5 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 1 selection ion stream chromatogram.
Fig. 6 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 2 selection ion stream chromatogram.
Fig. 7 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 3 selection ion stream chromatogram.
Fig. 8 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 4 selection ion stream chromatogram.
Fig. 9 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 5 selection ion stream chromatogram.
Figure 10 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 6 selection ion stream chromatogram.
Figure 11 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 7 selection ion stream chromatogram.
Figure 12 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 8 selection ion stream chromatogram.
Figure 13 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 9 selection ion stream chromatogram.
Figure 14 be Paeoniflorin in the intestine bacterium conversion after MTBE extract layer metabolite 10 selection ion stream chromatogram.
Figure 15 is metabolic pathway in Paeoniflorin in the intestine bacterium.
Specific embodiment
In order to make those skilled in the art that the present invention may be better understood, to be made to protection scope of the present invention bright True restriction makes further details of explanation to the present invention With reference to embodiment.
Embodiment 1
The LC-MS method for measuring Paeoniflorin metabolin in enteral bacterium, includes the following steps
1) the extraction separation of Paeoniflorin
5Kg radix paeoniae rubra pharmaceutical decocting piece is crushed, 250g is taken, 70% ethyl alcohol of 10 times of amounts are added, a night is impregnated, using heating back Stream method extracts the radix paeoniae rubra of immersion, extracts three times altogether, extraction time is respectively 2h, 1h, 1h.By three layers of gauze mistake of extracting solution The dregs of a decoction are filtered, merges solution, is concentrated with Rotary Evaporators.Petroleum ether extraction is then first used, petroleum ether layer is abandoned, water intaking mutually continues to use second Ethyl acetate layer is abandoned in acetoacetic ester extraction, and water intaking is mutually further continued for using extracting n-butyl alcohol, is taken n-butanol layer, is crossed 200-300 mesh silica gel Column, chloroform-methanol elution, eluent are washed with the methanol of 10:1 and water mixed liquid again, obtain Paeoniflorin, specific steps such as Fig. 1.
Paeoniflorin can also use commercial products.
2) preparation of enteral bacterium
It takes 8 healthy volunteers complete for the first time just, men and women each 4, keeps 10 days normal diets before taking just, do not take antibiosis Element and other drugs.
The appropriately sized valve bag full of nitrogen is taken, then squeezes out whole nitrogen, then be passed through nitrogen, so repeats to grasp Make 2 times, valve bag is full of nitrogen, sealing again.It is packed into all excrement of a defecation in valve bag, seals.It is squeezed with hand Valve bag is pressed, excrement is made to homogenize.Under the conditions of sterile oxygen free operation, 3-5g excrement is taken, another is put into and is full of nitrogen In valve bag, excrement is made to homogenize, is then placed in the GAM culture medium (250mL triangular flask) that 200mL sterilizing finishes, It gently shakes, sealing of jumping a queue is set in sterile anaerobism constant incubator, is cultivated for 24 hours at 37 DEG C, is obtained enteral bacterium bacterium solution.
The composition of GAM culture medium: tryptone 10g；Soya peptone 3g；Protein peptone 10g；Digest serum powder 13.5g；Yeast extract 5g；Beef extract 2.2g；Beef liver leaches powder 1.2g；Glucose 3g；KH2PO42.5g；NaCl 5g；Soluble starch 5g；Half Guang of L- Propylhomoserin HCl 0.5g；Sodium thioglycolate 0.3g；Distilled water adds to 1L.The dissolution of mentioned component Hybrid Heating, tune pH to 7.1~ 7.2,115 DEG C high pressure sterilization 20 minutes.
3) the enteral bacterium conversion of Paeoniflorin
Paeoniflorin standard items 100mg is accurately weighed, is placed in 10mL volumetric flask, methanol is added to scale, obtains 10mg/mL Standard solution.
In sterile anaerobic culture box, when adding enteral bacterium bacterium solution by 2 requirement of table in 7 bottles of GAM culture mediums, and pressing table 2 Between converted.
2 Paeoniflorin enteral bacterium translation table of table
Serial number Transformation time
Experimental group 1 2h
Experimental group 2 4h
Experimental group 3 8h
Experimental group 4 12h
Experimental group 5 24h
Control group 1 24h
Control group 2 24h
Experimental group: GAM culture medium+0.5mL enteral bacterium bacterium solution+0.5mL Paeoniflorin standard solution
Control group 1:GAM culture medium+0.5mL enteral bacterium bacterium solution
Control group 2:GAM culture medium+0.5mL Paeoniflorin standard solution
0.5mL Paeoniflorin standard solution is added in experiment group # 1-5 and control group 2, is slowly added dropwise, when being added dropwise Shaking, after being added dropwise, covers bottle stopper, seals bottleneck, label with sealed membrane.By experimental group and the sterile anaerobism of control group triangular flask Taken out in incubator, be placed on 37 DEG C of constant-temperature tables and shake, speed is 100r/min or so, start timing, respectively 2h, 4h, 8h, 12h, corresponding triangular flask is taken out for 24 hours.After reaction, ethyl acetate is added and terminates reaction, obtain the enteral of Paeoniflorin Bacterium metabolite.
4) sample treatment
The intestinal bacteria metabolism product of the Paeoniflorin of different experiments group is taken respectively, uses the water saturated acetic acid of 1:1 (v/v) respectively Ethyl ester solution extracts the intestinal bacteria metabolism product of Paeoniflorin three times, merges organic phase, is spin-dried for organic phase using Rotary Evaporators, It is transferred in EP pipe using methanol, then opening volatilizes, and the dissolution of 2mL methanol is added.Different experiments group methanol is taken to dissolve respectively again Each 0.1mL of product afterwards is incorporated into new EP pipe, crosses 0.22 μm of miillpore filter, and the metabolite for obtaining ethyl acetate extraction is surveyed Test agent.
The intestinal bacteria metabolism product of the Paeoniflorin of different experiments group is taken respectively, uses the water saturated methyl of 1:1 (v/v) respectively Tertbutyl ether solution extracts the intestinal bacteria metabolism product of Paeoniflorin three times, merges organic phase, using Rotary Evaporators by organic phase It is spin-dried for, is transferred in EP pipe using methanol, then opening volatilizes, and the dissolution of 2mL methanol is added.Take different experiments group methanol respectively again Dissolved each 0.1mL of product is incorporated into new EP pipe, crosses 0.22 μm of miillpore filter, obtains the generation of methyl tertiary butyl ether(MTBE) extraction Thank to product test sample.
The ethyl acetate extraction that control group 1 and control group 2 are saturated with water respectively and water saturated methyl tertiary butyl ether(MTBE) (MTBE) it extracts, is tested as blank control.
5) sample measures
Enteral bacterium converted product carries out the measurement of metabolin after sample treatment, and actual conditions are as follows:
Chromatographic condition: chromatographic column: Dikma DiamonsilTM ODS C18Chromatographic column；
Elution time: t=50min；
Sample volume: 10 μ L；
Detection wavelength: λ=230nm；
Column temperature: 30 DEG C；
Flow velocity: 0.5mL/min；
Mobile phase: water (A)+methanol (B), using gradient elution, program is as shown in table 1；
Table 1
Mass Spectrometry Conditions: first mass spectrometric: ionization mode: electrospray ionisation (ESI)；
Scan pattern: cation scanning；
Electron spray voltage: 4.0bar；
Dry gas stream speed: 10L/min；
Dry temperature degree: 200 DEG C；
Second order ms: ionization mode: electrospray ionisation (ESI)；
Scan pattern: cation scanning；
Collision gas: nitrogen；
Capillary voltage: 2.8kV；
Orifice potential: 3.0V；
Source temperature: 110 DEG C；
Desolventizing temperature: 350 DEG C；
Desolvation gas flow: 50L/Hr；
Collision energy: 15-35eV.
As a result as shown in figures 1-15, Fig. 1 is that Paeoniflorin extracts separation process figure, and Paeoniflorin purity is 98.0% or more.Fig. 2 For the total ion chromatogram of the MTBE layer converted in Paeoniflorin in the intestine bacterium, Fig. 3 is control group 1MTBE extract layer total ion current Chromatogram, Fig. 4 are control group 2MTBE extract layer total ion chromatogram, are carried out by software MS-DIAL to Fig. 2, Fig. 3 and Fig. 4 Processing, can filter out the chromatographic peak newly increased, as Paeoniflorin in the intestine in bacterium conversion MTBE extract layer metabolite.Knot After fruit shows that Paeoniflorin is metabolized in bacterium in the intestine, in MTBE extract layer sample, 34 metabolins are detected.Use software MassLynx V4.1 is read out metabolite secondary ion fragment；Utilize compound database and MS2Analyzer Ver2.1 software etc. derives compound structure, and is proved using document and CFM-ID software to structure.In MTBE Extract layer identifies 10 metabolites, wherein 7 metabolins are the (metabolin in Fig. 5-14 of discovery in bacterium conversion in the intestine for the first time P2, P3, P5, P6, P8, P9, P10), same operation detects 12 metabolins, identifies 1 in ethyl acetate extract layer Metabolite is known metabolin.Compared to other methods, more metabolism that method of the invention can detect Paeoniflorin simultaneously are produced Object selects the more other solvents of MTBE that can extract more metabolites in culture solution, and with above-mentioned software and analysis method More, the more acurrate structure for deducing metabolite of energy has important meaning for further investigation activated product and its mechanism of action Justice has directive function for Scientific Usage of Drugs.Simultaneously to promotion radix paeoniae rubra new drug development and improving dosage form and to the recipe of Chinese medicine Theory all has significance.Selection ion stream chromatogram of the Fig. 5-14 for metabolite 1-10 in Paeoniflorin in the intestine bacterium, figure 15 for metabolite in the Paeoniflorin that is derived by above method in the intestine bacterium chemical structure.

Claims (5)

1. measuring the LC-MS method of Paeoniflorin metabolin in enteral bacterium, it is characterised in that include the following steps:
1) preparation of enteral bacterium: taking excrement, under the conditions of sterile oxygen free operation, excrement is put into one and is full of the self-styled of nitrogen In bag, excrement is made to homogenize, is then placed in the GAM culture medium that sterilizing finishes, gently shakes, set sterile anaerobism constant temperature incubation In case, cultivated for 24 hours at 37 DEG C；
2) the enteral bacterium conversion of Paeoniflorin: in sterile anaerobic culture box, Paeoniflorin is added into the enteral bacterium of culture, then puts It sets and is shaken on 37 DEG C of constant-temperature tables, speed 100r/min cultivates 2-24h, takes out, and ethyl acetate is added and terminates reaction, obtains The intestinal bacteria metabolism product of Paeoniflorin；
3) sample treatment: using extract liquor extraction Paeoniflorin intestinal bacteria metabolism product three times, merge organic phase, rotation is evaporated, Again with methanol is transferred in EP pipe, crosses 0.22 μm of miillpore filter, obtains metabolite sample；
4) sample measures: actual conditions are as follows:
Chromatographic condition: chromatographic column: Dikma DiamonsilTMODS C18Chromatographic column；
Elution time: t=50min；
Sample volume: 10 μ L；
Detection wavelength: λ=230nm；
Column temperature: 30 DEG C；
Flow velocity: 0.5mL/min；
Mobile phase: water+methanol, using gradient elution, program is as shown in table 1；
Table 1
Time min Flow velocity mL/min Water % Methanol % 0 0.5 85 15 10 0.5 70 30 20 0.5 60 40 35 0.5 0 100 50 0.5 0 100
Mass Spectrometry Conditions: first mass spectrometric: ionization mode: electrospray ionisation ESI；
Scan pattern: cation scanning；
Electron spray voltage: 4.0bar；
Dry gas stream speed: 10L/min；
Dry temperature degree: 200 DEG C；
Second order ms: ionization mode: electrospray ionisation ESI；
Scan pattern: cation scanning；
Collision gas: nitrogen；
Capillary voltage: 2.8kV；
Orifice potential: 3.0V；
Source temperature: 110 DEG C；
Desolventizing temperature: 350 DEG C；
Desolvation gas flow: 50L/Hr；
Collision energy: 15-35eV.
2. the LC-MS method of Paeoniflorin metabolin in measurement enteral bacterium according to claim 1, which is characterized in that institute The excrement stated derives from human body or animal body.
3. the LC-MS method of Paeoniflorin metabolin in measurement enteral bacterium according to claim 1, which is characterized in that step It is rapid 3) in, the extract liquor be water saturated ethyl acetate solution.
4. the LC-MS method of Paeoniflorin metabolin in measurement enteral bacterium according to claim 1, which is characterized in that step It is rapid 3) in, the extract liquor be water saturated t-butyl methyl ether solution.
5. the LC-MS method of Paeoniflorin metabolin in measurement enteral bacterium according to claim 1, which is characterized in that institute The chromatographic column stated, model DiamonsilTMODS C18Chromatography 250mm × 4.6mm, 5 μm.
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CN101181373A (en) * 2007-12-07 2008-05-21 戴敏 Cortex moutan valid target pharmaceutical combination, preparation method and application thereof

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Determination of paeoniflorin in rat hippocampus by high-performance liquid chromatography after intravenous administration of Paeoniae Radix extract;Xihui He et al.;《Journal of Chromatography B》;20041231;277–281 *
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