Method for detecting blood-entering components of Niaodujiang mixture

Abstract

This invention discloses a method for detecting the blood-borne components of a uremic toxin compound, comprising the following steps: taking a plasma sample of the uremic toxin compound and a blank plasma sample, performing component analysis using ultra-high performance liquid chromatography-tandem mass spectrometry, and determining the blood-borne components of the uremic toxin compound by comparing the detection results. The detection method of this invention can simultaneously detect the blood-borne components of the uremic toxin compound, with high accuracy, short detection time, and good peak shape. It not only identifies the active ingredients of the uremic toxin compound that exert its pharmacological effect in vivo, but also provides a reference for further drug research.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting the blood components of Uremic Relief Compound. Background Technology

[0002] Uremic Relief Compound is a traditional Chinese medicine preparation independently developed by Zhongshan Municipal Hospital of Traditional Chinese Medicine. It is formulated with Astragalus membranaceus, Salvia miltiorrhiza, Carthamus tinctorius, Rheum palmatum, and Sanguisorba officinalis, and has the effects of strengthening the body's resistance, removing blood stasis, reducing turbidity, and clearing the meridians. Current research on Uremic Relief Compound mainly focuses on its preparation process and clinical application, but the active ingredients that exert its efficacy are still unclear. The components of compound preparations that enter the bloodstream are more complex than those of single herbs, as single herbs transform into different derivatives after entering the bloodstream.

[0003] Therefore, constructing a method for detecting the blood components of Uremic Kangheji can not only be used to analyze and identify the active ingredients that exert the efficacy of Uremic Kangheji, but also provide a reference for the further development and application of Uremic Kangheji. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting the blood components of Uremic Kangheji (a traditional Chinese medicine formula), so as to identify the active ingredients that exert the pharmacological effect of Uremic Kangheji in vivo, and to provide a basis for further research on drug metabolism.

[0005] According to a first aspect of the present invention, a method for detecting blood components of uremic remedy is provided, comprising the following steps: taking a plasma sample of uremic remedy and a blank plasma sample, performing component analysis using ultra-high performance liquid chromatography-tandem mass spectrometry, and determining the blood components by comparing the detection results; The liquid chromatography conditions were as follows: a C18 column; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, and the gradient elution program was as follows:

[0006] The flow rate was 0.2–0.4 mL / min, and the column temperature was 35–45 °C. The mass spectrometry conditions were as follows: positive ion mode, source voltage 4.0 kV, source temperature 320 °C; negative ion mode, source voltage 3.5 kV, source temperature 320 °C; nebulizer gas pressure 35 psi; sheath gas flow rate 11 L / min, sheath gas temperature 350 °C; declusivation potential 175 V; collision energies 10, 20, and 40 V; mass range m / z 50–1500 Da. Plasma samples also include the following pretreatment steps before analysis: (1) Mix the plasma sample with acetonitrile to obtain a mixture, and vortex the mixture for 20 to 40 seconds to allow the proteins to denature and precipitate fully; wherein the volume of acetonitrile is 4 times the volume of the plasma sample; (2) Placing the mixture at -20℃ for 30 min helps to precipitate proteins and macromolecules more completely; (3) Centrifuge the mixture, then aspirate the supernatant and mix it with 19 times the volume of pure water. Filter the mixture using a 0.22 μm syringe filter membrane. The filtrate is the plasma sample. This step is mainly to dilute the supernatant and improve the peak shape after UPLC injection.

[0007] This invention uses ultra-high performance liquid chromatography-tandem mass spectrometry to analyze plasma samples from the drug administration group and the blank group. By comparing the detection results of the two groups, the blood-entering components are determined. Using the chromatographic and mass spectrometric conditions of this invention, the blood-entering components in the plasma samples can be completely separated and detected, with good peak shape and high accuracy of detection results.

[0008] In some embodiments, plasma samples are obtained through the following steps: collecting blood samples (e.g., orbital blood), centrifuging the blood samples at 3000-3500 rpm for 10-15 min at 4°C, and obtaining the supernatant as the plasma sample. Specifically, blood samples that have been treated with the medication are processed to obtain uremic compound plasma samples, while blood samples that have not been treated are processed to obtain blank plasma samples. Preferably, the supernatant is obtained by centrifuging the blood samples at 3000 rpm for 10 min at 4°C.

[0009] In some implementations, blood samples are collected and mixed at 0.5h, 1h, 2h, 4h and 8h after drug administration.

[0010] In some embodiments, the blood-entering components of the uremic compound include: nitrosamine, salvianolic acid A, eucommia ulmoides alcohol, linoleic acid, tanshinone, bergamotol, vanillin, 1-O-acetyl inula lactone, stigmacanthoides, p-coumaric acid, L-isoleucine, phenylethanol ester of ferulic acid, dihydrocapsaicin, L-pyroglutamic acid, and L-tryptophan.

[0011] In some embodiments, the blood-bound components of the uremic compound, salvianolic acid A and tanshinone, are derived from tanshinone. Coumaric acid and linoleic acid may be derived from safflower.

[0012] This invention compares the mass spectrometry results of plasma samples from the blank group and the drug-treated group. After eliminating the influence of endogenous components in the plasma, 15 potential blood-entry components of Uremic Relief Compound were preliminarily identified. A spectral library search revealed that among the blood-entry components of Uremic Relief Compound, tanshinone A and tanshinone are derived from tanshinone, while coumaric acid and linoleic acid may be derived from safflower. The spectral library annotations for the remaining 11 components are inconsistent with the aforementioned prescription ingredients or do not specify the exact source of the traditional Chinese medicine. This is beneficial for studying the effective components of medicinal materials after preparation and absorption into the bloodstream.

[0013] In some implementations, in step (1), the volume of acetonitrile is 4 times the volume of the plasma sample.

[0014] In some embodiments, in step (2), the mixture is placed at -20°C for 30 min.

[0015] In some embodiments, in step (3), the supernatant is mixed with 19 times its volume of pure water. Further dilution of the supernatant can avoid the technical problem of inaccurate detection results due to poor peak shape.

[0016] In some embodiments, the centrifugation in step (3) is performed by centrifuging the mixture at 4°C and 11,000 to 13,000 rpm for 12 to 20 minutes. Preferably, the second centrifugation is performed by centrifuging the mixture at 4°C and 12,000 rpm for 15 minutes.

[0017] In some implementations, the flow rate is 0.3 mL / min and the column temperature is 40°C.

[0018] In some embodiments, the C18 column is a C18 column with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 2.7 μm.

[0019] In some embodiments, the following steps are also included: taking a sample of uremic toxin compound, analyzing the chemical components in the uremic toxin compound sample using ultra-high performance liquid chromatography-tandem mass spectrometry, comparing the chemical components of the uremic toxin compound with the blood-entering components of the uremic toxin compound, and determining the original blood-entering components among the blood-entering components.

[0020] In some implementations, the uremic compound sample undergoes the following pretreatment steps before injection: (1) Take 1~2 mL of the original solution of Uremic Relief Mixture, centrifuge for the first time and take out the first supernatant to remove insoluble substances such as drug residue; (2) Mix the first supernatant with acetonitrile at a volume of 3 times that of the first supernatant to obtain a mixture. Vortex the mixture for 20 to 40 seconds to allow the protein to denature and precipitate fully. (3) After placing the mixture at -20℃ for 30 min, perform a second centrifugation and collect the second supernatant; (4) Mix the second supernatant with 9 times the volume of pure water, filter through a 0.22 μm filter membrane, and the filtrate is the uremic compound sample.

[0021] In some embodiments, the original blood-entry components include salvianolic acid A, L-pyroglutamic acid, L-tryptophan, eucommia ulmoides alcohol, linoleic acid, bergamotol, vanillin, stigmacanthopanax, L-isoleucine and tanshinone, that is, these components belong to the original blood-entry components.

[0022] The beneficial effects of this invention are as follows: (1) The detection method of the present invention can simultaneously detect the blood-entering components of uremic compound, and the detection results are highly accurate, have a short detection time, and have good peak shape.

[0023] (2) This invention can not only analyze the 15 blood-entering components of Uremic Kangheji through ultra-high performance liquid chromatography-tandem mass spectrometry, but also analyze and compare the common components of Uremic Kangheji after entering the blood. This provides a reference for further drug research while identifying the active ingredients of Uremic Kangheji that exert its pharmacological effect in vivo. Attached Figure Description

[0024] Figure 1 This is a photograph of the original liquid form of the uremic toxin compound of the present invention; Figure 2 This is an UPLC-QTOF-LSMS image of the blood-entering components of the uremic toxin-relieving agent of this invention. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available. In this embodiment, the word "approximately" used before numerical values, unless otherwise specified, indicates that the value allows for conventional process errors in the relevant field: for measurement parameters such as feed mass, volume, and relative density, it is ±5% of the corresponding value; for temperature parameters, it is ±2℃; for time parameters, it is ±5min. Those skilled in the art will understand that the technical effects of the present invention can be achieved within this error range.

[0026] 1. Preparation of samples of Uremic Relief Compound (NDK-H) (1) Source of Uremic Relief Compound Uremic Relief Compound is a preparation made within the Zhongshan Municipal Hospital of Traditional Chinese Medicine, with approval number Yueyaozhizi Z20070605. Figure 1 As shown. The traditional Chinese medicine used in the Uremic Relief Compound consists of Astragalus membranaceus, Rheum palmatum, Carthamus tinctorius, Salvia miltiorrhiza, and Sanguisorba officinalis. The production process of the raw liquid of the Uremic Relief Compound used in this invention is as follows: Weigh approximately 3000g of Rheum palmatum, 6000g of Sanguisorba officinalis, 9000g of Astragalus membranaceus, 6000g of Carthamus tinctorius, and 9000g of Salvia miltiorrhiza. Add approximately 10 times the amount of water to the five medicinal materials in the prescription, soak for approximately 30 minutes, and decoct twice (the first time for approximately 2 hours and the second time for approximately 1 hour). Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.10-1.15 (measured at 60℃). Add approximately 200g of sucrose, approximately 3g of sodium benzoate, and approximately 0.5g of ethylparaben to the clear extract, boil for approximately 15 minutes, cool to room temperature, add purified water to make up the volume, adjust the total volume of the liquid to 1000mL, stir well, and dispense. The production batch number is 2520250701.

[0027] (2) Preparation of samples of Uremic Relief Compound The uremic compound sample was obtained through pretreatment, including the following steps: Initial centrifugation: Take 1-2 mL of the original solution of Uremic Relief Mixture and place it in a centrifuge tube. Centrifuge at 4000 rpm for 10 minutes to remove insoluble matter such as drug residue.

[0028] Precise transfer: Precisely pipette 200 μL of supernatant and transfer it into a 1.5 mL centrifuge tube.

[0029] Protein precipitation: Add 600 μL of acetonitrile to the supernatant to obtain a mixture.

[0030] Vortex mixing: Vortex the centrifuge tube thoroughly for 30 seconds to allow the protein to denature and precipitate completely. At this point, the acetonitrile concentration in the mixture is 75%.

[0031] Low-temperature precipitation: Place the centrifuge tubes in a -20°C freezer for 30 minutes to more completely precipitate proteins and macromolecules.

[0032] High-speed centrifugation: Centrifuge the centrifuge tube at 12,000 rpm for 10 minutes at 4°C.

[0033] Supernatant dilution: After centrifugation, accurately pipette 100 μL of supernatant into another centrifuge tube, add 900 μL of pure water to the centrifuge tube, mix thoroughly, and the concentration of acetonitrile in the mixed sample is 7.5%.

[0034] Filtration: Filter the sample using a 0.22 μm syringe filter membrane (PTFE or Nylon material). The filtrate is the sample. Put the sample into a vial.

[0035] 2. Animal drug administration and plasma sample collection Laboratory animals: Male C57BL / 6 mice, 6-7 weeks old, weighing 20-22g, purchased from Guangdong Provincial Medical Laboratory Animal Center. They were housed according to the laboratory animal handling guidelines, with an ambient temperature of 20-25℃ and humidity of 50%-60%. They were fasted for 12 hours before the experiment, but had free access to water.

[0036] Mice were divided into a blank control group (n=1) and a drug treatment group (n=3), for a total of 20 mice.

[0037] Dosage: NDK-H 26 mL / kg.

[0038] After drug administration, blood samples (500-1000 μL each time) were collected at 0.5, 1, 2, 4, and 8 hours. The specific experimental procedure was as follows: Mice were anesthetized with isoflurane inhalation. After the righting reflex disappeared, they were fixed in place, and the sides of the neck were moderately compressed to cause the eyeballs to protrude. Blood was collected from the orbital venous plexus using the enucleation method. Immediately after blood collection, cervical dislocation was performed for euthanasia. Blood samples from different sampling time points were mixed and added to anticoagulant tubes. The blood samples were centrifuged at 3000 rpm for 10 min at 4°C to obtain plasma. After centrifugation, the plasma was stored at -80°C or directly pretreated.

[0039] Preprocessing includes the following steps: (1) Precise transfer: Precisely aspirate 200 μL of supernatant and place the supernatant into a 1.5 mL centrifuge tube.

[0040] (2) Protein precipitation: Add 800 μL of acetonitrile to the supernatant to obtain a mixture.

[0041] (3) Vortex mixing: Vortex the centrifuge tube thoroughly for 30 seconds to allow the protein to denature and precipitate completely. At this point, the acetonitrile concentration in the mixture is 80%.

[0042] (4) Low temperature precipitation: Place the centrifuge tube in a -20℃ refrigerator for 30 minutes to more completely precipitate proteins and macromolecules.

[0043] (5) High-speed centrifugation: Centrifuge the centrifuge tube at 12,000 rpm for 15 minutes at 4°C.

[0044] (6) Dilution of supernatant: After centrifugation, accurately pipette 100 μL of supernatant into another centrifuge tube, add 1900 μL of pure water to the centrifuge tube, mix thoroughly, and the concentration of acetonitrile in the mixed sample is 4%.

[0045] (7) Filtration: Use a 0.22μm syringe filter membrane (PTFE or Nylon material) to filter the sample. The filtrate is the sample. Put the sample into a vial.

[0046] It should be noted that different pretreatment methods were used in this invention because the sample matrices of the uremic toxin compound solution and plasma are different. The uremic toxin compound solution is an aqueous traditional Chinese medicine preparation. It was first centrifuged at 4000 rpm to remove insoluble substances such as dregs. Since the protein and lipid content is relatively low, the final acetonitrile concentration was set to 75%, and centrifugation was performed at 12000 rpm for 10 minutes. Plasma, on the other hand, contains a large amount of protein, lipids, and endogenous interfering substances. Therefore, the final acetonitrile concentration was set to 80%, and the high-speed centrifugation time was extended to 15 minutes to improve protein precipitation and matrix purification. Subsequently, 900 μL and 1900 μL of water were added respectively to reduce the acetonitrile concentration in the uremic toxin compound solution sample and the plasma sample to 7.5% and 4%, respectively. This was to reduce the strength of the injection solvent and improve the peak shape in C18 reversed-phase chromatography. Therefore, the difference in the two sets of parameters represents adjustments to the processing conditions for different sample matrices.

[0047] 3. Ultra-high performance liquid chromatography-tandem mass spectrometry analysis The mass spectrometry instrument used was: ultra-high performance liquid chromatography (1290 II, Agilent Technologies, USA)-quadrupole time-of-flight mass spectrometry (6546, Agilent Technologies, USA).

[0048] (1) Liquid chromatography conditions Chromatographic column: C18 column, Agilent Poroshell 120 EC-C18 (2.1x150 mm, 2.7 μm); mobile phase: phase A (aqueous phase) was 0.1% formic acid aqueous solution, phase B (organic phase) was acetonitrile; flow rate: 0.3 mL / min; column temperature: 40℃; injection volume: 5 μL; elution program is shown in Table 1.

[0049] Table 1 Elution Procedure

[0050] (2) Mass spectrometry conditions Acquisition modes: positive and negative ions; positive ion mode: source voltage 4.0 kV, source temperature 320 °C; negative ion mode: source voltage 3.5 kV, source temperature 320 °C; drying gas flow rate 8 L / min, nebulizer gas pressure 35 psig, sheath gas temperature 350 °C, sheath gas flow rate 11 L / min; Fragmentor voltage 175 V, Skimmer 1 voltage (first-stage skimmer voltage) 65 V, Octopole RF Peak voltage 750 V. The mass range of the first-stage mass spectrometry scan was 100–1500 Da; the second-stage mass spectrometry used Auto MS / MS mode, with a scan range of m / z 50–1500, an acquisition rate of 2.5 spectra / s, and fixed collision energies of 10, 20, and 40 V. All acquired data were processed using MassHunter Qualitative Analysis 12.0 software (Agilent Technologies, USA). The parameters of the electrospray ionization source are shown in Table 2.

[0051] Table 2 Electrospray ionization source parameters

[0052] 4. Analysis Results Mass spectrometry analysis results of plasma components from the blank control group and the drug-treated group were compared to eliminate the influence of endogenous components in the plasma, thus determining the components of the uremic compound that entered the bloodstream. The results of the blood component analysis of the uremic compound are shown in Table 3.

[0053] Mass spectrometry analysis of plasma components from the control group and the treatment group was performed. After eliminating the influence of endogenous components in the plasma, 15 potential blood-entering components of the Uremic Relief Compound were preliminarily identified. Among them, tanshinone A and tanshinone are derived from tanshinone, while p-coumaric acid and linoleic acid may be derived from safflower. No components explicitly identified in the spectral library as originating from astragalus, safflower, rhubarb, or sanguisorba were detected. The spectral library sources of the remaining 11 components were inconsistent with the above-mentioned prescription ingredients or did not specify the specific Chinese medicine source. This indicates that tanshinone A and tanshinone are relatively well-defined characteristic blood-entering components of tanshinone, while p-coumaric acid and linoleic acid may be characteristic blood-entering components of safflower.

[0054] Table 3. Results of Blood Component Detection of Uremic Relief Compound

[0055] The mass spectrometry results of the chemical components of the Uremic Relief Compound were compared with the spectral library identification results of the administered plasma samples. The common components are listed in Table 4. Table 4 revealed 10 components with identical names and molecular formulas that showed a spectral library match, including tanshinone A, L-pyroglutamic acid, L-tryptophan, eucommia ulmoides alcohol, linoleic acid, tanshinone, bergamotol, vanillin, schizophyllum commune, and L-leucine. These can be tentatively listed as potential precursor components entering the bloodstream. However, the retention times of some components differed significantly between the undiluted Uremic Relief Compound and the plasma samples, requiring further confirmation based on the retention times of reference standards and MS / MS characteristic fragments.

[0056] Coumaric acid, phenylethanol ferulic acid, dihydrocapsaicin, 1-O-acetylspirin lactone, and nisose were detected only in plasma samples of the uremic remedy, which does not preclude the determination that these components are in vivo metabolites. These components may be original blood-borne components of the uremic remedy below the detection limit of the original solution, in vivo metabolic or hydrolysis products, or may involve isomer transformation, library mis-injection, or plasma background interference. Specifically, p-coumaric acid glucoside was detected in the original uremic remedy solution, suggesting that p-coumaric acid may be formed through in vivo hydrolysis. The sources and metabolic relationships of other components require further verification through secondary mass spectrometry fragmentation, metabolic transformation relationships, and reference standards.

[0057] Table 4. Results of Detection of Prototype Blood Components of Uremic Relief Compound

[0058] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for detecting the blood components of uremic rehydration solution, characterized in that, The process includes the following steps: taking plasma samples of the uremic rehydration solution and blank plasma samples, performing component analysis using ultra-high performance liquid chromatography-tandem mass spectrometry, and determining the blood-entering components of the uremic rehydration solution by comparing the detection results; the plasma samples also include the following pretreatment steps before analysis: (1) Mix the plasma sample with acetonitrile to obtain a mixture; wherein the volume of acetonitrile is 4 times the volume of the plasma sample; (2) Place the mixture at -20℃ for 30 minutes; (3) Centrifuge the mixture, then remove the supernatant, mix the supernatant with 19 times the volume of pure water solution, filter, and the filtrate is the plasma sample; The liquid chromatography conditions were as follows: C18 column, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, flow rate was 0.2~0.4 mL / min, column temperature was 35~45℃; gradient elution program was as follows: The mass spectrometry conditions were as follows: positive ion mode, source voltage 4.0 kV, source temperature 320 °C; negative ion mode, source voltage 3.5 kV, source temperature 320 °C; nebulizer gas pressure 35 psi; sheath gas flow rate 11 L / min, sheath gas temperature 350 °C; declustering potential 175 V; collision energies 10, 20, and 40 V; and mass range m / z 50–1500 Da.

2. The method for detecting the blood components of the uremic toxin compound according to claim 1, characterized in that, The plasma sample is obtained through the following steps: blood sample is collected, and the supernatant obtained by centrifuging the blood sample at 3000~3500 rpm for 10~15 min at 4℃ is the plasma sample.

3. The method for detecting the blood components of the uremic toxin compound according to claim 1 or 2, characterized in that, The blood samples were collected and mixed at 0.5h, 1h, 2h, 4h and 8h after drug administration.

4. The method for detecting the blood components of the uremic toxin compound according to claim 3, characterized in that, The blood-entering components of Uremic Relief Compound include: naesin, salvianolic acid A, eucommia ulmoides alcohol, linoleic acid, tanshinone, bergamotol, vanillin, 1-O-acetyl inula lactone, stigmacanthoides, p-coumaric acid, L-isoleucine, phenylethanol ester of ferulic acid, dihydrocapsaicin, L-pyroglutamic acid, and L-tryptophan.

5. The method for detecting the blood components of the uremic toxin compound according to claim 3, characterized in that, Among the blood-entering components of Uremic Relief Compound, Tanshinone A and Tanshinone are derived from Tanshinone.

6. The method for detecting the blood components of the uremic toxin compound according to claim 1, characterized in that, The conditions used for centrifugation in step (3) are to centrifuge the mixture at 4°C and 11,000 to 13,000 rpm for 12 to 20 minutes.

7. The method for detecting the blood components of the uremic toxin compound according to claim 1, characterized in that, In step (1), the volume of acetonitrile is 4 times the volume of the plasma sample; in step (3), the volume of pure aqueous solution is 19 times the volume of the supernatant; the flow rate is 0.3 mL / min, and the column temperature is 40℃.

8. The method for detecting the blood components of the uremic toxin compound according to claim 1, characterized in that, It also includes the following steps: Samples of the uremic toxin compound were taken, and the chemical components in the samples were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. The chemical components of the uremic toxin compound were compared with the blood-entering components of the uremic toxin compound to determine the original blood-entering components.

9. The method for detecting the blood components of the uremic toxin compound according to claim 8, characterized in that, The following pretreatment steps were taken for the uremic compound sample before injection: (1) Take the original solution of Uremic Relief Mixture, centrifuge it for the first time and aspirate the first supernatant; (2) Mix the first supernatant with acetonitrile at a volume of 3 times that of the first supernatant to obtain a mixture. Vortex the mixture for 20 to 40 seconds to allow the protein to denature and precipitate fully. (3) After placing the mixture at -20℃ for 30 min, perform a second centrifugation and collect the second supernatant; (4) Mix the second supernatant with 9 times the volume of pure water, filter through a 0.22 μm filter membrane, and the filtrate is the uremic compound sample.

10. The method for detecting the blood components of the uremic toxin compound according to claim 8, characterized in that, The prototype blood-entering components include salvianolic acid A, L-pyroglutamic acid, L-tryptophan, eucommia ulmoides alcohol, linoleic acid, tanshinone, bergamotol, vanillin, stigmacanoidin and L-isoleucine.

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