CN113533584A - Simultaneous quantitative detection method and application of 2 blood-entering components in trichosanthes, allium macrostemon and pinellia ternate preparation - Google Patents
Simultaneous quantitative detection method and application of 2 blood-entering components in trichosanthes, allium macrostemon and pinellia ternate preparation Download PDFInfo
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Abstract
The invention provides a simultaneous quantitative detection method and application of 2 blood-entering components in a trichosanthes kirilowii and allium macrostemon pinellia preparation, wherein the detection method comprises the following steps: detecting the content of guanosine and adenosine in a plasma sample to be detected by using UPLC-MS/MS; chromatographic conditions are as follows: a reversed phase chromatographic column with silica gel particles as packing; mobile phase A: containing 0.1% formic acid water solution, and the mobile phase B is acetonitrile; gradient elution. The method can simultaneously determine the content of 2 blood-entering components of guanosine and adenosine in the trichosanthes and allium macrostemon pinellia preparation by limiting the parameters in the UPLC-MS/MS method and matching the parameters with each other, and is simple, rapid, accurate and reliable, high in sensitivity, good in specificity and good in stability.
Description
Technical Field
The invention relates to the technical field of in vivo metabolism of medicaments, in particular to a method for simultaneously and quantitatively detecting 2 blood-entering components in a trichosanthes kirilowii bulb, allium macrostemon and pinellia ternate preparation and application thereof.
Background
The classic famous formula trichosanthes kirilowii, allium macrostemon and pinellia ternate decoction is from the 'jin Kui Yao L ü e', is prepared by decocting trichosanthes kirilowii, allium macrostemon, pinellia ternate and rice wine, and is widely used for clinically treating angina pectoris coronary heart disease, rheumatic heart disease, ventricular tachycardia, intercostal neuralgia, mammary gland hyperplasia, chronic obstructive pulmonary disease, traumatic pneumothorax, senile cough and asthma, chronic bronchopneumonia, chronic cholecystitis and the like.
At present, researches on the snakegourd fruit, allium macrostemon and pinellia ternate decoction mainly focus on researches on chemical components, the pharmacodynamic substance basis of the snakegourd fruit, allium macrostemon and pinellia ternate decoction for playing the effects is not clear, and the detection means of effective components in blood is blank.
As is well known, the effective components of the traditional Chinese medicine can only exert curative effect after entering the blood of a human body, and how to quantitatively detect the trace effective components under the complex matrix condition becomes a difficult problem, so that in order to further deeply research the change situation of the trichosanthes and allium macrostemon pinellia preparation and the effective component change situation thereof in the blood and provide scientific basis for the effective substance basis of the efficacy in the trichosanthes and allium macrostemon pinellia preparation, the development of an accurate and reliable quantitative detection method for the blood-entering components of the trichosanthes and allium macrostemon pinellia preparation becomes a technical problem to be solved urgently at present.
Disclosure of Invention
Aiming at the defect that no quantitative detection method for blood-entering components of the trichosanthes and allium macrostemon pinellia preparation exists at present, the invention provides a simultaneous quantitative detection method for 2 blood-entering components in the trichosanthes and allium macrostemon pinellia preparation and application thereof, and the quantitative detection method can provide a basis for further screening quality markers and pharmacodynamic substance bases of the classical famous formula trichosanthes and allium macrostemon pinellia preparation.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a simultaneous quantitative detection method for 2 blood-entering components in a trichosanthes kirilowii maxim, allium macrostemon and pinellia ternate preparation comprises the following steps:
detecting the content of guanosine and adenosine in a plasma sample to be detected by using UPLC-MS/MS;
wherein, the chromatographic conditions are as follows: a reversed phase chromatographic column with silica gel particles as packing; mobile phase A: containing 0.1% formic acid water solution, and the mobile phase B is acetonitrile; the procedure for gradient elution was as follows:
0-1.5min, 95% mobile phase A, 5% mobile phase B;
1.5-2.0min, 95% → 80% mobile phase a, 5% -20% mobile phase B;
2.0-2.5min, 80% → 50% mobile phase a, 20% -50% mobile phase B;
2.5-3.5min, 50-95% of mobile phase A and 50-5% of mobile phase B;
3.5-5.0min, 95% mobile phase A, 5% mobile phase B;
the above percentages are volume percentages.
The method can simultaneously determine the content of 2 blood-entering components of guanosine and adenosine in the trichosanthes and allium macrostemon pinellia preparation by limiting the analysis parameters in the UPLC-MS/MS method and mutually matching the parameters, and the method for simultaneously and quantitatively detecting the 2 blood-entering components in the trichosanthes and allium macrostemon pinellia preparation is simple, rapid, reliable, high in sensitivity, good in specificity, good in accuracy, good in stability, lower in detection limit and quantitative limit.
The preparation comprises fructus Trichosanthis, Bulbus Allii Macrostemi and rhizoma Pinelliae decoction, fructus Trichosanthis, Bulbus Allii Macrostemi and rhizoma Pinelliae lyophilized powder, and fructus Trichosanthis, Bulbus Allii Macrostemi and rhizoma Pinelliae ointment.
Optionally, the preparation method of the trichosanthes and allium macrostemon pinellia ternate preparation comprises the following steps:
decocting fructus Trichosanthis, Bulbus Allii Macrostemi and rhizoma Pinelliae in rice wine, filtering, and making into decoction, unguent or lyophilized powder.
Preferably, the preparation method of the trichosanthes, allium macrostemon and pinellia ternate decoction comprises the following steps: weighing 10-15g of trichosanthes decoction pieces, 7-12g of allium macrostemon decoction pieces and 10-15g of pinellia ternate decoction pieces, adding 500mL of 300 rice wine, soaking for about 1.5-3.5h, decocting with strong fire until boiling, then decocting with slow fire for 15-25min, and filtering with 3-5 layers of gauze when the decoction pieces are hot.
More preferably, the preparation method of the trichosanthes, allium macrostemon and pinellia ternate decoction comprises the following steps: weighing 12.0g of trichosanthes decoction pieces, 9.0g of allium macrostemon decoction pieces and 12.0g of pinellia ternate decoction pieces, adding 400mL of rice wine, soaking for about 2 hours, decocting with strong fire until boiling, then decocting with slow fire for 20min, and filtering with 4 layers of gauze while hot to obtain the traditional Chinese medicine.
Optionally, the reverse phase chromatography column is an ACQUITY UPLC HSS T3 column.
Optionally, the chromatographic conditions further comprise: the column temperature is 30-40 ℃, the sample injection amount is 1.5-2.5 mu L, and the flow rate is 0.2-0.4 mL/min.
Preferably, the column temperature of the chromatographic conditions is 35 ℃, the sample injection amount is 2.0 μ L, and the flow rate is 0.3 mL/min.
Alternatively, the mass spectrometry conditions: the ion source is an ESI source, the capillary voltage is 3.2kv, and the desolvation gas is nitrogen; the temperature of the desolvation gas is 500 ℃, the flow rate of the desolvation gas is 600L/h, and the flow rate of the taper hole is 50L/h. The accuracy and stability of the quantitative detection method can be further improved by limiting mass spectrum conditions.
Optionally, the preparation method of the plasma sample to be tested comprises the following steps: and (3) placing the plasma sample into an EP tube, adding the internal standard solution, carrying out vortex mixing, adding acetonitrile, carrying out vortex mixing, centrifuging, and taking the supernatant, namely the plasma sample to be detected.
Optionally, the preparation method of the plasma sample to be detected further comprises the steps of taking blood, then placing the blood in a heparinized centrifugal tube, and centrifuging at 4000-.
Preferably, the preparation method of the blood plasma is as follows: blood is taken, and then the blood is placed in a heparinized centrifugal tube and centrifuged for 10min at 4500r/min to obtain plasma.
Optionally, the internal standard in the internal standard solution is zeatin.
Optionally, the concentration of zeatin in the internal standard solution is 3-8 μ g/mL.
Optionally, the internal standard solution is prepared by the following method:
weighing 5.00mg of zeatin in a 10mL volumetric flask, adding a methanol solution to fix the volume to a scale to obtain 0.5mg/mL internal standard stock solution, and diluting to obtain the internal standard solution.
The invention also provides application of the simultaneous quantitative detection method of 2 blood-entering components in the trichosanthes and allium macrostemon pinellia preparation in quality control research.
Such quality control studies include, but are not limited to, in vivo pharmacokinetic studies.
Compared with the prior art, the method has the advantages that the chromatographic conditions, the mass spectrum conditions, the internal standard substance selection and the sample pretreatment conditions are reasonably set, so that the detection sensitivity is improved, and the analysis time is shortened; the simultaneous quantitative analysis of two substances with similar polarities of guanosine and adenosine is realized; can be used for pharmacokinetic study of guanosine and adenosine after oral administration of the trichosanthes, allium macrostemon and pinellia ternate preparation; provides technical reference for simultaneous quantitative analysis of other components in the trichosanthes longstamen onion bulb pinellia preparation.
Drawings
FIG. 1 is a chromatogram of the proprietary experiment of example 2;
figure 2 is a plot of mean plasma adenosine and guanosine concentrations versus time for different modes of administration in the rat pharmacokinetic study of example 3.
Description of the drawings:
in the attached figure 1, A is a chromatogram of blank plasma of a rat, B is a chromatogram of the plasma of the rat containing a mixed reference solution and an internal standard substance, and C is a chromatogram of a plasma sample after gastric lavage administration of the rat;
in fig. 2, a is a blood concentration-time curve of adenosine in rats after three consecutive days of administration, B is a blood concentration-time curve of guanosine in rats after three consecutive days of administration, C is a blood concentration-time curve of adenosine in rats after a single administration, and D is a blood concentration-time curve of guanosine in rats after a single administration.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The traditional Chinese medicine is absorbed into blood after administration, chemical components with certain content in the body are possible to be potential bioactive components, although the content of some traditional Chinese medicine components in the compound is higher, the medicine can be restricted from playing the efficacy in the body due to limited absorption degree of a human body or faster component metabolism, and the factors of complex blood sample matrix and low sample content bring research and development difficulty to the detection of blood samples.
At present, researches on the snakegourd fruit, allium macrostemon and pinellia ternate decoction mainly focus on researches on chemical components, the basis of pharmacodynamic substances exerting efficacy in the snakegourd fruit, allium macrostemon and pinellia ternate decoction is not clear, the detection means of effective components in blood is blank, and the researches on multi-component pharmacokinetics of the snakegourd fruit, allium macrostemon and pinellia ternate decoction are not reported at home and abroad. On the basis of 84 prototype components in the currently identified trichosanthes, allium macrostemon and pinellia ternate decoction, 22 main representative components are selected, including trigonelline, uracil, thymidine, adenine, caffeic acid, uridine, guanine, guanosine, xanthine, adenosine, vanillic acid, 5-hydroxymethyl furfural, nicotinamide, rutin, quercetin, luteolin, protocatechuic acid, homoharringtonine, schaftoside, cucurbitacin B, N-p-coumaroyl tyramine and ferulic acid, so that the blood-entering prototype components of the trichosanthes, allium macrostemon and pinellia ternate decoction are determined, and the basis of drug effect substances of the trichosanthes, allium macrostemon and pinellia ternate decoction is discussed.
In order to establish a method for simultaneously and quantitatively detecting blood-entering components in a trichosanthes and allium macrostemon pinellia preparation, in the process of method development and method verification, in order to ensure multi-component synchronous detection, continuous exploration and optimization experiments are carried out on chromatographic conditions, mass spectrum conditions, a plasma sample processing mode and internal standard substance selection, an in vivo quantitative analysis method of the 22 components is researched, and the final result shows that only two components, namely adenosine and guanosine, have higher concentrations in vivo and can be accurately quantified. Specifically, the method for simultaneously and quantitatively detecting guanosine and adenosine in blood components in the trichosanthes kirilowii and allium macrostemon pinellia preparation researched and developed by the invention is as follows:
preparation of internal standard solution: weighing 5.00mg of zeatin in a 10mL volumetric flask, adding a methanol solution to fix the volume to a scale to obtain 0.5mg/mL internal standard stock solution, and diluting to obtain 4 mu g/mL internal standard solution for later use;
preparation of a plasma sample to be tested: precisely sucking 50 mu L of rat plasma sample, placing the rat plasma sample in 1.5mL of EP, precisely adding 10 mu L of the internal standard solution, uniformly mixing by swirling for 1min, then adding 140 mu L of acetonitrile solution, swirling for 5min, fully and uniformly mixing, centrifuging for 10min at 12000g, and obtaining supernatant as the plasma sample to be detected;
detecting the content of guanosine and adenosine in a plasma sample to be detected by using UPLC-MS/MS;
wherein, the mass spectrum condition is as follows: the ion source is an ESI source, the capillary voltage is 3.2kv, and the desolvation gas is nitrogen; the temperature of the desolvation gas is 500 ℃, the flow rate of the desolvation gas is 600L/h, and the flow rate of the taper hole is 50L/h; performing quantitative analysis by adopting an MRM mode;
chromatographic conditions are as follows: reversed phase chromatographic column with silica gel particles as filler, such as ACQUITY UPLC HSS T3 column; mobile phase A: formic acid water solution with the volume percentage of 0.1 percent, and the mobile phase B is acetonitrile; the elution mode is gradient elution; the column temperature is 30-40 ℃, the sample injection amount is 1.5-2.5 mu L, and the flow rate is 0.2-0.4 mL/min;
the procedure for gradient elution was as follows:
0-1.5min, 95% mobile phase A, 5% mobile phase B;
1.5-2.0min, 95% → 80% mobile phase a, 5% -20% mobile phase B;
2.0-2.5min, 80% → 50% mobile phase a, 20% -50% mobile phase B;
2.5-3.5min, 50-95% of mobile phase A and 50-5% of mobile phase B;
3.5-5.0min, 95% mobile phase A, 5% mobile phase B;
the above percentages are volume percentages.
Through a Multi-Reaction monitoring (MRM) positive ion scanning mode, combination of specific chromatographic detection conditions and selection of zeatin as an internal standard substance, the in vivo quantitative analysis method for the 22 components is researched, the blood concentrations of the 22 components in a rat body after administration are measured, pharmacokinetic parameters are calculated, the change rule of the traditional Chinese medicine in the body is known, and a basis is provided for further screening the quality markers and the pharmacodynamic substance basis of the classic famous formula trichosanthes kirilowii maxim and allium macrostemon pinellia ternate preparation.
The invention is further illustrated below in the following examples.
The experimental animals used in the following examples were provided by the laboratory animal center of Wenzhou university of medicine, SD male rats, weighing 200 + -20 g, 7-8 weeks old, and were acclimatized for one week in an environment of 22 + -2 deg.C, 55 + -10% relative humidity, and 12h light/dark cycle prior to the experiment.
The main experimental equipment and drugs used in the following examples are shown in tables 1-2 below.
TABLE 1 Main Experimental instruments and Equipment
| Name of instrument | Manufacturer of the product | Model number |
| Electronic balance | Beijing Sadolis Instrument systems, Inc | AB204N |
| High-speed refrigerated centrifuge | eppendorf Co Ltd | 5920r |
| UPLC-MS/MS | Waters corporation of America | |
| Freeze dryer | NINGBO SCIENTZ BIOTECHNOLOGY Co.,Ltd. | SCIENTZ-10N |
| Numerical control ultrasonic cleaner | KUNSHAN ULTRASONIC INSTRUMENTS Co.,Ltd. | HG64 |
| Vortex mixer | Jiangsu Haiman Linbel Instrument manufacturing Co., Ltd | XW-80A |
TABLE 2 drugs and reagents
| Reagent/medicinal material | Manufacturer/origin | Batch number |
| Snakegourd fruit | Henan Anyang | GL20200501 |
| Bulbus Allii Macrostemi | Shandong Xintai (a Chinese character of' Shandong | SDX20191001 |
| Pinellia ternata (Thunb.) Breit | All the year round in Hubei province | BX201807091 |
| Rice wine | Herb of Mipo | 20201202 |
| Adenosine (I) | China Institute for food and drug control | 110879-201703 |
| Guanosine | China Institute for food and drug control | 111977-201501 |
| Uridine (uridine) | China Institute for food and drug control | 140660-202002 |
| Adenine | China Institute for food and drug control | 110886-201102 |
| Xanthine | China Institute for food and drug control | 140662-200802 |
| Uracils | China Institute for food and drug control | 100469-201302 |
| Vanillic acid | China Institute for food and drug control | 110776-201503 |
| Trigonelline | China Institute for food and drug control | 110883-200502 |
| 5-hydroxymethylfurfural | Chengdu Desite Biotechnology Ltd | 111626-200906 |
| Nicotinamide | China Institute for food and drug control | 100115-202005 |
| N-P-Coumaroyl tyramine | Chengdu Desite Biotechnology Ltd | BP3309 |
| Guanine and its preparing process | China Institute for food and drug control | 140718-202003 |
| Thymidine | China Institute for food and drug control | 101215-201401 |
| Caffeic acid | China Institute for food and drug control | 110885-201703 |
| Rutin | China Institute for food and drug control | 100080-201811 |
| Quercetin | China Institute for food and drug control | 100081-201610 |
| Luteolin | China Institute for food and drug control | 111520-202006 |
| Protocatechuic acid | China Institute for food and drug control | 110809-201906 |
| Homoharringtonine | China Institute for food and drug control | 111533-201904 |
| Schaftoside | China Institute for food and drug control | 111912-201703 |
| Ferulic acid | China Institute for food and drug control | 110773-201915 |
| Cucurbitacin B | China Institute for food and drug control | 111945-201301 |
| Mass spectrum methanol | Merck Corp Ltd | |
| Mass spectrum grade acetonitrile | Merck Corp Ltd | |
| Chromatographic grade formic acid | ROE Scientific Inc | |
| Deionized water | Hangzhou Wahaha Group Co.,Ltd. |
Example 1 study on method for measuring blood-entering component content of trichosanthes, allium macrostemon and pinellia ternate preparation
1. Preparation of the solution
1.1 preparation of Mixed control solutions
Precisely weighing control substances of trigonelline, uracil, thymidine, adenine, caffeic acid, uridine, guanine, guanosine, xanthine, adenosine, vanillic acid, 5-hydroxymethyl furfural, nicotinamide, rutin, quercetin, luteolin, protocatechuic acid, homoharringtonine, schaftoside, cucurbitacin B, N-p-coumaroyl tyramine and ferulic acid, respectively placing the control substances of 5.00mg in a 10mL volumetric flask, adding methanol to a constant volume to reach a scale, and obtaining each control substance stock solution with a final concentration of 0.5 mg/mL;
precisely measuring each reference substance stock solution respectively, mixing, and diluting with methanol to obtain mixed reference substance solution; the concentrations of each control in the mixed control solution were as follows: 250 mu g/mL of trigonelline; uracil 100. mu.g/mL; thymidine 50 μ g/mL; adenine 50. mu.g/mL; caffeic acid 250 mug/mL; uridine 50 μ g/mL; guanine 50 mug/mL; guanosine 50 mu g/mL; xanthine 10 μ g/mL; adenosine 50 μ g/mL; 500 mu g/mL of vanillic acid; 250 mu g/mL of 5-hydroxymethylfurfural; nicotinamide 50 μ g/mL; rutin is 100 mu g/mL; quercetin 100 μ g/mL luteolin 500 μ g/mL; protocatechuic acid 500. mu.g/mL; homoharringtonine 100 μ g/mL; schaftoside 250 mug/mL; cucurbitacin B100 mug/mL; 20 mu g/mL of N-p-coumaroyl tyramine; ferulic acid 250. mu.g/mL.
1.2 internal standard stock solution and preparation of internal standard solution
Precisely weighing 5.00mg of zeatin, adding a methanol solution into a 10mL volumetric flask, fixing the volume to a scale to obtain an internal standard stock solution with the final concentration of 0.5mg/mL, and then storing in a refrigerator at 4 ℃;
and precisely measuring the internal standard stock solution, and diluting the internal standard stock solution by methanol to prepare an internal standard solution of 4 mu g/mL.
1.3 preparation of lyophilized powder of Trichosanthes and Allium macrostemon pinellia Tuber for intragastric administration
Weighing 12.0g of trichosanthes decoction pieces, 9.0g of allium macrostemon decoction pieces and 12.0g of pinellia ternate decoction pieces, adding 400mL of rice wine to soak for about 2h, decocting with strong fire until boiling, then decocting with slow fire for 20min, filtering with 4 layers of gauze while hot, freeze-drying with a freeze dryer to obtain trichosanthes and allium macrostemon and pinellia ternate freeze-dried powder, and sealing and storing in a refrigerator at the temperature of-80 ℃.
When a rat is subjected to gastric lavage, the dosage of the snakegourd fruit, allium macrostemon and pinellia ternate freeze-dried powder is 1 time per day according to the dosage of 6g/kg each time, and the mass-volume ratio of the snakegourd fruit, allium macrostemon and pinellia ternate freeze-dried powder to pure water is 1.5g:1mL each time of gastric lavage.
1.4 preparation of the plasma sample to be tested
Precisely sucking 50 μ L rat plasma sample, placing in 1.5mL EP tube, precisely adding 10 μ L internal standard solution (obtained by diluting internal standard stock solution, with concentration of 4 μ g/mL), and vortexing for 1min to mix well. And then adding 140 mu L of acetonitrile solution, vortexing for 5min, fully mixing uniformly, centrifuging for 10min at 12000g, and taking supernatant, namely the plasma sample to be detected.
2. Detection of
Precisely absorbing a rat plasma sample, preparing the plasma sample to be detected according to the method under item 1.4, and injecting the plasma sample into UPLC-MS/MS for detection.
2.1 detection conditions
Conditions of Mass Spectrometry
Waters Quattro API triple quadrupole tandem mass spectrometer (Waters corporation, usa), electrospray ion source (ESI); the capillary voltage was 3.2 kv; the desolventizing gas is nitrogen; the temperature of the solvent gas is 500 ℃; the flow rate of the desolventizing gas is 600L/h; the cone orifice flow rate was 50L/h, the samples were quantitatively analyzed in MRM mode, and the experimental data were collected and processed using MassLynx software (version 4.1, Waters Corp.).
The quantitative ion, cone hole voltage and collision voltage of each control are shown in the following table.
TABLE 3
Chromatographic conditions
A chromatographic column: ACQUITY UPLC HSS T3 column (2.1X 100mm,1.8 μm)
Mobile phase: a: water (with 0.1% formic acid), B: acetonitrile
Column temperature: 35 deg.C
Sample introduction amount: 2 μ L
Gradient elution conditions are as follows:
TABLE 4 gradient elution conditions
| Time (min) | A(％) | B(％) | Flow rate (mL/min) |
| 0.0 | 95 | 5 | 0.3 |
| 1.5 | 95 | 5 | 0.3 |
| 2.0 | 80 | 20 | 0.3 |
| 2.5 | 50 | 50 | 0.3 |
| 3.5 | 95 | 5 | 0.3 |
| 5 | 95 | 5 | 0.3 |
2.2 Final establishment of the detection conditions
The nitrogenous compound has response under the positive and negative ion detection mode of the electrospray ion source, the response under the positive ion mode is higher than that under the negative ion mode, and an obvious excimer ion peak can be detected under the positive ion mode.
The flow has great influence on the chromatographic behavior (peak symmetry and retention time) and the mass spectrum response of each object to be tested, methanol and acetonitrile are respectively used as mobile phases for investigation in the selection of the organic phase, and the result shows that acetonitrile as the organic phase has fast peak emergence, better peak shape, uniform distribution and high peak intensity, so that the acetonitrile is selected as the mobile phase. In the aspect of water phase additive selection, when no acid is added into the water phase, the tail of the chromatographic peak of the component to be detected is serious, the chromatographic behavior is obviously better after formic acid is added, the response of the object to be detected is also improved, and the result shows that the acetonitrile-0.1% formic acid water is used as the mobile phase and can obtain good ionic response.
The influence of 35 ℃ and 40 ℃ on chromatographic peaks is investigated, and the result shows that the influence of temperature on the separation of chromatographic peaks is small, and 35 ℃ is selected by combining the characteristics of the chromatographic column.
The finally determined method for measuring the content of the blood-entering components of the trichosanthes kirilowii, the allium macrostemon and the pinellia ternate preparation is the same as the detection condition under the item 2.1.
Example 2 methodological verification of blood-entering component content determination method of trichosanthes kirilowii, allium macrostemon and pinellia ternata freezing agent
Detection and quantification limits (determination of blood prototype components)
Calculating the required dilution times according to the signal-to-noise ratio of each control in the mixed control solution, properly diluting each control stock solution to obtain the concentration of each control in the mixed control solution, detecting according to the detection condition under 2.1, determining the limit of quantitation by the signal-to-noise ratio (S/N) being more than 10, determining the limit of detection by the signal-to-noise ratio (S/N) being more than 3, and expressing the limit of detection and the limit of quantitation of 22 components.
TABLE 522 limits of quantitation and detection of components in rat plasma
And (2) performing intragastric administration on 12 rats for 0, 10min, 20min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h and 48h according to the condition under the item 1.3, then drawing blood, placing the blood in a heparinized centrifuge tube, centrifuging at 4500r/min for 10min to obtain blood plasma, preparing blood plasma samples to be detected according to the condition under the item 1.4 respectively, and then performing content determination on the blood concentration of 22 components in the blood plasma samples to be detected according to the detection condition under the item 2.1.
According to detection, 13 components including trigonelline, thymidine, caffeic acid, vanillic acid, 5-hydroxymethyl furfural, rutin, quercetin, luteolin, protocatechuic acid, homoharringtonine, schaftoside, cucurbitacin B and ferulic acid are not detected in blood plasma. Although 7 components, that is, nicotinamide, N-p-coumaroyltyramine, uracil, adenine, guanine, xanthine, and uridine, were detected in plasma, the blood concentration was below the minimum quantitative limit of the apparatus, and accurate quantitative analysis could not be performed. Only two components, adenosine and guanosine, are present in high amounts in the plasma, allowing accurate quantification. Therefore, two components, adenosine and guanosine, were selected for subsequent pharmacokinetic studies.
The specificity is as follows: and comparing the blank rat plasma chromatogram, the plasma chromatogram of the added mixed reference solution and the internal standard solution, and the plasma sample chromatogram after the administration by intragastric administration of the rat, and inspecting the selectivity of the method.
The preparation method of the blank rat plasma sample comprises the following steps:
precisely sucking 50 mu L of rat plasma sample, placing the rat plasma sample in a 1.5mL EP tube, then adding 150 mu L of acetonitrile solution, vortexing for 5min, fully mixing uniformly, centrifuging at 12000g for 10min, and taking supernatant fluid, namely a blank rat plasma sample.
Rat plasma samples with the control and internal standard added were prepared as follows:
adding 1mL of the internal standard solution prepared under the item 1.2 into 14mL of acetonitrile to obtain an acetonitrile solution mixed with the internal standard for later use;
centrifuging rat plasma in a low-temperature centrifuge at 4 ℃ and 4500r/min for 15min, taking 50 mu L of supernatant, adding 10 mu L of mixed control solution prepared under item 1 and 140 mu L of acetonitrile solution mixed with internal standard, centrifuging at 12000r/min for 10min after vortex for 5min, and taking supernatant, namely the rat plasma sample added with the mixed control and the internal standard.
The preparation method of the plasma sample after the intragastric administration of the rat comprises the following steps:
adding 1mL of the internal standard solution prepared under the item 1.2 into 14mL of acetonitrile to obtain an acetonitrile solution mixed with the internal standard for later use;
after the rat is subjected to intragastric administration for 1h, blood is drawn, the rat is placed in a heparinized centrifuge tube and centrifuged at 4500r/min for 10min to obtain plasma, the plasma is centrifuged at 4500r/min for 15min at 4 ℃ in a low-temperature centrifuge, 50 mu L of supernatant is added into 150 mu L of acetonitrile solution mixed with the internal standard, the mixture is centrifuged at 12000r/min for 10min after 5min of vortex, and the supernatant is obtained.
Because adenosine and guanosine are endogenous substances and have certain background concentration in a rat body, a blank biological sample cannot be obtained, the method compares the chromatogram of the blank plasma sample, the plasma sample added with a mixed reference substance and the chromatogram of the plasma sample 1h after administration when carrying out specificity research, and determines specificity by taking whether the retention time of compounds in each ion monitoring channel is interfered or not as a basis.
As shown in fig. 1: a is the chromatogram of the blank plasma of the rat, B is the chromatogram of the plasma of the rat added with the mixed reference substance and the internal standard, and C is the chromatogram of the plasma sample after the administration of the intragastric administration of the rat. As can be seen from FIG. 1, under the above chromatographic conditions, other endogenous substances and metabolites do not interfere with the measurement of the analyte in the sample.
Linearity (drawing of standard curve of plasma sample)
Precisely sucking 10 mu L of mixed reference substance solution prepared under 1.1 and 50 mu L of blank plasma, carrying out vortex mixing for 1min, then processing according to the method under 1.4 to prepare a series of standard drug-containing plasma samples with different concentrations, carrying out sample injection test analysis according to the detection conditions under 2.1, carrying out regression operation by using a weighted (1/C2) least square method and carrying out linear regression by using the concentration (C) as a horizontal coordinate and the ratio of the peak area of the substance to be detected to the peak area of the internal standard substance as a vertical coordinate. The standard curves and lower limit of quantitation (LLOQ) for guanosine and adenosine are shown in the following table,
TABLE 6 Standard Curve and lower quantitation Limit for adenosine and guanosine in plasma samples
| Name (R) | Linear equation of equations | R2 | Linear model (ng/mL) |
| Adenosine (I) | Y＝151.3x+3119.1 | 0.999 | 0.5-2500 |
| Guanosine | Y＝9.291x+1600.5 | 0.998 | 1-1500 |
As can be seen from the data in the table above, the correlation coefficient of the standard curve of each analyte is more than or equal to 0.99, which indicates that the UPLC-MS/MS analysis method has high sensitivity and good linear range.
Precision and accuracy
In the same day, the treatment method under item 1.4 is adopted to process and prepare the plasma samples containing the medicine with low, medium and high concentrations, 6 parts are respectively made for each concentration, and the accuracy and precision (RSD) in the day are calculated.
The plasma samples containing drug at three concentrations, low, medium and high, were processed and prepared according to the 1.4 treatment method, 3 portions for each concentration, and measured over 5 consecutive days. The mass concentration of the sample is calculated according to the standard curve of the day, the structure of the quality control sample is subjected to variance analysis, the accuracy and precision (RSD) of the day are calculated, and the detection results are shown in the table.
TABLE 7 precision and accuracy of adenosine and guanosine in plasma samples
As can be seen from the experimental data in the above table, the daily precision and the daytime precision of the 2 components are both less than 15%, and the accuracy is within 83.1% -94.4%, which indicates that the precision and the accuracy of the UPLC-MS/MS analysis method are good.
Matrix Effect and extraction recovery
Precisely sucking 10 mu L of mixed reference substance solution under the item 1.1 and 50 mu L of blank plasma, vortexing for 1min, uniformly mixing, processing plasma samples under the method of the item 1.4, preparing quality control samples (QC) containing high, medium and low 3 concentrations, wherein at least 6 samples under each concentration have a peak area marked as A;
precisely absorbing blank plasma 50 μ L and acetonitrile 150 μ L, vortexing for 8min, and mixing well. Centrifuge at 12000g for 10min, take 50. mu.L of supernatant, add to EP tube containing 10. mu.L of mixed control solution and 10. mu.L of internal standard solution (concentration 50ng/mL), and vortex for 1 min. Processing the plasma samples according to the method under item 1.4 to prepare quality control samples (QC) containing high, medium and low 3 concentrations, at least 6 samples at each concentration, and the peak area is marked as B;
precisely sucking 10 μ L of the mixed control solution and 50 μ L of ultrapure water, vortexing for 1min, mixing, and processing the sample under 1.4 to obtain quality control sample (QC) with high, medium, and low concentrations, wherein at least 6 samples with peak area of C are obtained at each concentration.
The extraction recovery rate is the ratio (A/B) of chromatographic peak area A of the extracted sample to chromatographic peak area B of the unextracted sample. The matrix effect was calculated from the peak area ratio (B/C) of the two treatments at the same concentration. The extraction recovery and matrix effect results are shown in the following table.
TABLE 8 matrix Effect and extraction recovery of adenosine and guanosine in plasma samples
As can be seen from the data in the table above, the matrix effects of adenosine and guanosine are 77.7% -90.4% and 81.9% -89.9%, respectively, which indicates that the matrix effects of the quality control samples of each concentration level of the 2 components all meet the detection requirements; the extraction recovery rate range of 2 components in rat plasma under the quality control level is 67.2% -78.6%, and the extraction recovery rate range is stable, and meets the measurement requirements of biological samples.
Stability of
Precisely sucking blank plasma of 50 μ L, preparing quality control sample (QC) containing high, medium and low 3 concentrations by referring to the method operation under item 1.4, and performing stability inspection on the components to be tested. Stability studies included short-term stability studies (quality control samples were stored at room temperature for 12h), long-term stability studies (quality control samples were stored in a-80 ℃ freezer for 15 days), and freeze-thaw stability studies (quality control samples were subjected to 3 freeze-thaw cycles), with the stability results shown in table 9.
TABLE 9 stability of adenosine and guanosine in plasma samples
As can be seen from the data in the table above, the short-term stability, the long-term stability and the freeze-thaw stability RSD of adenosine and guanosine are less than 15%, and all meet the detection requirements, which indicates that the stability of the UPLC-MS/MS analysis method is good.
Example 3 in vivo pharmacokinetic Studies in rats
12 healthy male SD rats are respectively subjected to intragastric administration of snakegourd fruit, allium macrostemon and pinellia ternate freeze-dried powder (operated according to item 1.3) according to the dose of 6g/kg, blood is taken immediately after 6 of the snakegourd fruit, allium macrostemon and pinellia ternate freeze-dried powder are subjected to intragastric administration for 3 days, the rats are subjected to blood taking after the intragastric administration for 3 days, the blood taking time points are 0min, 10min, 20min, 30min, 1h, 2h, 4h, 8h and 12h respectively, then the rats are placed in heparinized centrifuge tubes and centrifuged at 4500r/min for 10min, blood plasma is sucked and then processed according to the method under item 1.4, and the blood plasma sample to be detected obtained after processing is stored at the temperature of minus 20 ℃ for analysis. The plasma concentrations at different times were measured, and the DAS software analyzed pharmacokinetic parameters (version 3.0, shanghai, china), and the results of the major pharmacokinetic parameters of adenosine and guanosine after administration in different ways are shown in the table below, and the mean plasma concentration-time curves of adenosine and guanosine after administration in different ways are shown in fig. 2.
TABLE 10 pharmacokinetic parameters of adenosine and guanosine in rats after administration (n ═ 6)
As can be seen from the data in the above table in conjunction with FIG. 2, the C of adenosine and guanosine after a single administrationmaxValues of 875.46 + -439.12 ng/mL and 1137.51 + -407.19 ng/mL, respectively, and C of adenosine and guanosine after three consecutive days of administrationmaxThe values are 709.69 +/-343.75 ng/mL and 351.26 +/-85.94 ng/mL respectively, the blood concentration is relatively high, the medicine effect can be exerted only by chemical components with certain content in the body after the medicine is absorbed into the blood, and therefore adenosine and guanosine can be the main active components in the trichosanthes macrostemon pinellia preparation. In addition, the time to peak (T) of adenosine and guanosine following a single administrationmax) The values are 0.63 +/-0.77 h and 0.61 +/-0.70 h respectively, which shows that the trichosanthes and allium macrostemon pinellia preparation is absorbed quickly after entering a body and can quickly enter blood circulation to play a curative effect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A simultaneous quantitative detection method for 2 blood-entering components in a trichosanthes kirilowii maxim, allium macrostemon and pinellia ternate preparation is characterized by comprising the following steps:
detecting the content of guanosine and adenosine in a plasma sample to be detected by using UPLC-MS/MS;
wherein, the chromatographic conditions are as follows: a reversed phase chromatographic column with silica gel particles as packing; mobile phase A: containing 0.1% formic acid water solution, and the mobile phase B is acetonitrile; the elution procedure was as follows:
0-1.5min, 95% mobile phase A, 5% mobile phase B;
1.5-2.0min, 95% → 80% mobile phase a, 5% -20% mobile phase B;
2.0-2.5min, 80% → 50% mobile phase a, 20% -50% mobile phase B;
2.5-3.5min, 50-95% of mobile phase A and 50-5% of mobile phase B;
3.5-5.0min, 95% mobile phase A, 5% mobile phase B;
the above percentages are volume percentages.
2. The method for simultaneously and quantitatively detecting 2 blood-entering components in the trichosanthes kirilowii bulb and pinellia ternate preparation according to claim 1, wherein the reversed phase chromatographic column is an ACQUITY UPLC HSST 3 column.
3. The method for simultaneously and quantitatively detecting 2 blood-entering components in the trichosanthes kirilowii maxim and allium macrostemon pinellia ternate preparation according to claim 1, wherein the chromatographic conditions further comprise: the column temperature is 30-40 ℃, the sample injection amount is 1.5-2.5 mu L, and the flow rate is 0.2-0.4 mL/min.
4. The method for simultaneously and quantitatively detecting 2 blood-entering components in the trichosanthes kirilowii maxim, allium macrostemon and pinellia ternate preparation according to any one of claims 1 to 3, is characterized in that the mass spectrum conditions are as follows: the ion source is an ESI source, the capillary voltage is 3.2kv, and the desolvation gas is nitrogen; the temperature of the desolvation gas is 500 ℃, the flow rate of the desolvation gas is 600L/h, and the flow rate of the taper hole is 50L/h.
5. The method for simultaneously and quantitatively detecting 2 blood-entering components in the trichosanthes kirilowii and allium macrostemon pinellia ternate preparation according to claim 1, wherein the preparation method of the blood plasma sample to be detected comprises the following steps: and (3) placing the plasma sample into an EP tube, adding the internal standard solution, carrying out vortex mixing, adding acetonitrile, carrying out vortex mixing, centrifuging, and taking the supernatant to obtain the plasma sample to be detected.
6. The method for simultaneously and quantitatively detecting 2 blood-entering components in the trichosanthes kirilowii bulb and allium macrostemon pinellia ternate preparation according to claim 5, wherein an internal standard substance in an internal standard solution is zeatin.
7. The method for simultaneously and quantitatively detecting 2 blood-entering components in the trichosanthes kirilowii bulb and pinellia ternate preparation according to claim 6, wherein the concentration of the zeatin in the internal standard solution is 3-8 μ g/mL.
8. The method for simultaneously and quantitatively detecting 2 blood-entering components in the trichosanthes kirilowii maxim, allium macrostemon and pinellia ternate preparation according to any one of claims 5 to 7, wherein the preparation method of the internal standard solution is as follows:
weighing 5.00mg of zeatin in a 10mL volumetric flask, adding a methanol solution to fix the volume to a scale to obtain 0.5mg/mL internal standard stock solution, and diluting to obtain the internal standard solution.
9. The use of the simultaneous quantitative determination method of 2 blood-entering components in the preparation of any one of claims 1-8 in the quality control research of the preparation.
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