CN109655535B - Detection method of seven-ingredient oral liquid for treating arthralgia - Google Patents

Detection method of seven-ingredient oral liquid for treating arthralgia Download PDF

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CN109655535B
CN109655535B CN201811383826.5A CN201811383826A CN109655535B CN 109655535 B CN109655535 B CN 109655535B CN 201811383826 A CN201811383826 A CN 201811383826A CN 109655535 B CN109655535 B CN 109655535B
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oral liquid
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arthralgia
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肖伟
耿婷
汤齐
高霞
黄文哲
王振中
曹亮
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Jiangsu Kanion Sunshine Pharmaceutical Co ltd
Jiangsu Kanion Pharmaceutical Co Ltd
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Abstract

The invention discloses a method for detecting seven-ingredient arthralgia-relieving oral liquid, which comprises the following steps of: the chromatographic column is Agilent ZORBAX SB-C18(4.6 x 250mm, 5 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), elution gradient: 0-10 min, 1-3% (A); 10-35 min, 3-20% (A); 35-50 min, 20-30% (A); 50-70 min, 30-55% (A); 70-75 min, 55-100% (A); 75-85 min, 100% (A); the flow rate is 0.8-1.2 mL/min; the column temperature is 25-30 ℃; the detection wavelength is 210-300 nm. The method characterizes the chemical components of the seven-ingredient arthralgia-relieving oral liquid, obtains a plurality of chemical components, and measures related component information and content information. Moreover, the invention analyzes the main blood components and evaluates the pharmacokinetic characteristics; finally, an HPLC content determination method based on main blood components is established on the basis of the research, so that the selection of index components is more reasonable, the quality standard is enriched, and the value and significance of the research are improved.

Description

Detection method of seven-ingredient oral liquid for treating arthralgia
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a quality control method of a seven-ingredient arthralgia-relieving oral liquid.
Background
The oral liquid is prepared from seven medicinal materials including ants, caulis sinomenii, caulis Spatholobi, herba Pyrolae, caulis et folium piperis, rhizoma homalomenae and radix Clematidis, and has effects of invigorating kidney, strengthening bone, dispelling pathogenic wind and eliminating arthralgia. As an oral preparation for treating RA, the traditional Chinese medicine preparation has definite clinical curative effect, is approved by doctors and patients, has an increased market share year by year, and has higher research value.
The traditional Chinese medicine compound medicine is a prescription medicine for preventing and treating diseases under the guidance of the traditional Chinese medicine theory, and as a traditional Chinese medicine prescription, the traditional Chinese medicine compound medicine has multiple medicinal material prescriptions and complex components, is often a key point and a difficulty point of research, and greatly hinders the modernization development of the traditional Chinese medicine. Therefore, the basic research of the material is necessary precondition by combining the research method and the thought of the predecessor. In addition, new research concepts and methods need to be developed and continuously explored to find the true connotation of the Chinese herbal compound better and more deeply.
At present, the literature reports about seven-ingredient arthralgia-relieving oral liquid mainly focus on production process optimization, pharmacodynamic evaluation, clinical RA treatment and the like. The chemical composition analysis, material basis, and in vivo metabolic process of the product have not been studied. Meanwhile, the existing quality standard control component of the seven-ingredient arthralgia-relieving oral liquid is single, so that the consistency among batches of products is difficult to reflect, and quality standard improvement research is necessary to better control the stability and uniformity of the products. In addition, few reports are provided on what components and forms of the seven-ingredient arthralgia-relieving oral liquid act in vivo and the action mechanism, and further understanding of the drug effect substance basis of the seven-ingredient arthralgia-relieving oral liquid is still lacking.
Disclosure of Invention
In view of the above, the invention is to use UPLC-QTOF-MS technology to research the chemical components and blood components of the preparation, and use UPLC-QQQ-MS technology to research the time course of main PK index in rat. On the basis, a quality control method based on blood components is established, and technical support is provided for effectively controlling the product quality and ensuring batch-to-batch consistency.
In order to achieve the above object, the present invention provides the following technical solutions:
a detection method of seven-ingredient arthralgia-relieving oral liquid is characterized by comprising the following steps: detecting a seven-ingredient arthralgia-relieving oral liquid sample and a reference substance, wherein the chromatographic conditions of the detection are as follows: the chromatographic column is Agilent ZORBAX SB-C18(4.6 x 250mm, 5 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), elution gradient: 0-10 min, 1-3% (A); 10-35 min, 3-20% (A); 35-50 min, 20-30% (A); 50-70 min, 30-55% (A); 70-75 min, 55-100% (A); 75-85 min, 100% (A); the flow rate is 0.8-1.2 mL/min; the column temperature is 25-30 ℃; detecting the wavelength of 210-300 nm;
and obtaining the component information or content information of the seven-ingredient arthralgia-relieving oral liquid according to the detection result.
Specifically, the test sample is prepared by precisely measuring 1mL of Qiwei Tongbi oral liquid, diluting with pure water by 10 times, centrifuging at 14000rpm for 10min, and collecting supernatant as test sample solution.
Preferably, in the chromatographic conditions, the flow rate is 1.0mL/min, the column temperature is 25 ℃, and the detection wavelength is 254 nm.
Further, the reference substance is selected from one or more of sinomenine, monotropein, formononetin, protocatechuic acid, valine, adenine, pyroglutamic acid, leucine, phenylalanine, 5-hydroxymethylfurfural, glutamic acid, magnoflorine and formononetin.
Specifically, the reference substance is prepared by preparing a solution containing 10-40 mug/mL of each reference substance by using 50% methanol. The standard curve of one or more reference substances can be established by using the detection conditions, so that the content of the effective components can be calculated according to the detection result of the test substance.
The invention also provides a method for analyzing the chemical components of the seven-ingredient arthralgia-relieving oral liquid, which is characterized in that the seven-ingredient arthralgia-relieving oral liquid is analyzed by UPLC-Q TOF-MS, wherein the chromatographic conditions and the mass spectrum conditions are respectively as follows:
chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18(4.6 x 250mm, 5 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), elution gradient: 0-10 min, 1-3% (A); 10-35 min, 3-20% (A); 35-50 min, 20-30% (A); 50-70 min, 30-55% (A); 70-75 min, 55-100% (A); 75-85 min, 100% (A); the flow rate is 0.8-1.2 mL/min; the column temperature is 25-30 ℃; detecting the wavelength of 210-300 nm; wherein, the flow rate is preferably 1.0 mL/min; the column temperature is preferably 25 ℃; the detection wavelength is preferably 254 nm;
mass spectrum conditions: electrospray ion source (ESI), positive and negative ion mode; the capillary voltage is 4000V and 3500V respectively; the ion scanning range m/z is 100-3000; the drying airflow rate is 10L/min; the temperature of the drying gas is 350 ℃; atomization pressure 60 psi; cleavage voltage 135V; the taper hole voltage is 65V; collision energy was 10, 20, 30, 40eV, respectively.
The invention also provides a detection method of main blood-entering components of the seven-ingredient arthralgia-relieving oral liquid, which is characterized in that a blood plasma sample and a standard blood plasma sample of the seven-ingredient arthralgia-relieving oral liquid are taken for detection, and the chromatographic conditions of the method are as follows: the chromatographic column is Agilent Eclipse plus C18(3 x 50mm, 1.8 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), gradient elution: 0-3 min, 10-45% (A); 45-65% (A) for 3-4 min; 4-4.3 min, 65-100% (A); 4.3-5 min, 100% (A); 5-5.5 min, 100-10% (A); 5.5-6 min, 10% (A); column temperature: 30-40 ℃; flow rate: 0.5-1.0 mL/min; wherein the column temperature is preferably 40 ℃ and the flow rate is preferably 0.6 mL/min.
According to the detection result, the information of the main blood-entering component (PK component) of the seven-ingredient arthralgia-relieving oral liquid is obtained.
Specifically, the preparation method of the processed seven-ingredient oral liquid blood plasma sample for treating arthralgia syndrome comprises the following steps: taking a plasma sample, adding an internal standard solution (chloramphenicol, 5 mu g/mL), adding methanol, uniformly mixing by vortex, centrifuging at 14000rpm, and taking supernatant fluid to obtain the pharmaceutical composition.
Further, the preparation of the standard plasma sample comprises taking several 90 μ L portions of blank plasma, adding 10 μ L of the mixed reference solution and 10 μ L of the internal standard solution (chloramphenicol, 5 μ g/mL) respectively, and preparing the concentrations of the sinomenine and the magnoflorine as follows: 240.5/102, 120.2/51, 60.13/25.5, 30.06/12.8, 15.03/6.4, 7.52/3.19, 3.76/1.59, 1.88/0.80, 0.63/0.27 μ g/mL of a series of standard plasma sample solutions.
The invention also provides a detection method for analyzing the blood-entering components of the seven-ingredient arthralgia-relieving oral liquid, which is characterized by comprising the following steps of:
analyzing the processed seven-ingredient Tongbi oral liquid plasma sample and blank plasma sample by UPLC-Q-TOF-MS, wherein the chromatographic conditions and the mass spectrum conditions are respectively as follows:
the chromatographic column is Agilent ZORBAX SB-C18(4.6 x 250mm, 5 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), elution gradient: 0-10 min, 1-3% (A); 10-35 min, 3-20% (A); 35-50 min, 20-30% (A); 50-70 min, 30-55% (A); 70-75 min, 55-100% (A); 75-85 min, 100% (A); the flow rate is 0.8-1.2 mL/min; the column temperature is 25-30 ℃; detecting the wavelength of 210-300 nm; preferably, the column temperature: 25 ℃, flow rate: 1.0 mL/min; the detection wavelength is 254 nm;
mass spectrum conditions: electrospray ion source (ESI), positive and negative ion mode; the capillary voltage is 4000V and 3500V respectively; the ion scanning range m/z is 100-3000; the drying airflow rate is 10L/min; the temperature of the drying gas is 350 ℃; atomization pressure 60 psi; cleavage voltage 135V; the taper hole voltage is 65V; collision energy was 10, 20, 30, 40eV, respectively.
Specifically, the preparation method of the blood plasma sample of the seven-ingredient arthralgia-relieving oral liquid for analyzing the blood components is as follows: taking a plasma sample, adding methanol, mixing uniformly, centrifuging at 14000rpm, drying supernatant in nitrogen at 40 ℃, adding 50% methanol for redissolving, centrifuging at 14000rpm, and taking supernatant to obtain the blood plasma.
The invention also provides a medicamentThe method for measuring the content of the blood-entering component based on the Weitongbi oral liquid is characterized by comprising the following steps: detecting a seven-ingredient arthralgia-relieving oral liquid sample and a reference substance, wherein the chromatographic conditions of the detection are as follows: agilent extended-C18(4.6 x 250mm, 5 μm); mobile phase: 0.05mol/LNaH2PO4(A) -acetonitrile (B); elution gradient: 0-15 min, 8% (B); 15-28 min, 8-12% (B); 28-31 min, 12-50% (B); 31-35 min, 50% (B); detection wavelength: 260-270 nm, column temperature: 20-30 ℃, flow rate: 0.8-1.2 mL/min;
according to the detection result, the content information of the seven-ingredient arthralgia-relieving oral liquid based on the blood-entering components is obtained.
Specifically, the test sample is prepared by precisely measuring 1mL of seven-ingredient arthromyodynia-relieving oral liquid, adding 50% methanol, performing ultrasonic centrifugation, and collecting supernatant as a test sample solution;
the reference substance is selected from sinomenine and magnoflorine;
the flow rate is 1.0mL/min, the column temperature is 25 ℃, and the detection wavelength is 265 nm.
The invention adopts UPLC-Q TOF-MS technology to characterize the chemical components of the seven-ingredient arthralgia-relieving oral liquid, conjectures the possible chemical components according to molecular weight information and literature reports, analyzes 47 compounds from the seven-ingredient arthralgia-relieving oral liquid, and measures the related component information and content information. Moreover, the invention also adopts UPLC-QQQ-MS technology to analyze the main blood components and evaluate the pharmacokinetic characteristics; finally, an HPLC method was established on the basis of the above studies to detect the content of the main component. The method has good precision, stability, repeatability, recovery rate and the like, and meets the detection requirements of samples, so that the method can be effectively applied to quality evaluation of preparations.
Drawings
FIG. 1 is a total ion flow diagram of seven-ingredient Tongbi oral liquid under positive and negative ion modes according to the present invention; wherein, A is in positive ion mode, and B is in negative ion mode.
Fig. 2 is an analysis diagram of the blood-entering components of the seven-ingredient arthralgia-relieving oral liquid after gastric lavage of rats.
FIG. 3 is a specificity profile of sinomenine, magnoflorine and internal standard in rat plasma in accordance with the present invention.
Fig. 4 is a drug time curve of sinomenine in blood plasma after oral administration of the seven-ingredient arthromyodynia-relieving oral liquid in rats.
Fig. 5 is a drug time curve of magnoflorine in blood plasma after oral administration of the seven-ingredient arthromyodynia-relieving oral liquid to rats.
FIG. 6 is the HPLC chromatogram of the seven ingredient Tongbi oral liquid and the reference substance of the invention.
Detailed Description
The invention discloses a quality control method of seven-ingredient arthralgia-relieving oral liquid, which can be realized by appropriately improving process parameters by taking the contents of the seven-ingredient arthralgia-relieving oral liquid as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention.
Except for special points, the medicines, reagents and instruments used in the technical scheme provided by the invention can be purchased from conventional channels or markets.
1 materials of the experiment
1.1 Experimental instruments
Agilent 1290 definition ultra high performance liquid chromatograph (Agilent, usa); agilent6538Q-TOF mass spectrometer (Agilent, Inc., Agilent, USA); agilent 1100 liquid chromatograph (Agilent, usa); model ST16R high speed refrigerated centrifuge (semer flyer, usa); electronic analytical balance model Mettler ME303E (Mettler-toledo instruments ltd); a Mettler XS205 electronic analytical balance (Mettler-toledo instruments ltd); pacific tii 7 ultrapure water meter (saimmer flyer, usa); nitrogen blower (HSC-24A, Esper science instruments, Inc., Wuhan); KH2200B model ultrasonic cleaner (kunshan grass ultrasonic instrument ltd).
1.2 reagents and reagents
Seven-ingredient Tongbi oral liquid (batch No. 151202, 160504, 161206, 170506, 170602, 170802, 170904, 171201, 180303 and 180411) is produced by Jiangsu Kangyuan sunshine pharmaceutical Co., Ltd; sinomenine (batch No. 110774-; magnoflorine (batch number: B20882), formononetin (batch number: B20214) and leucine (batch number: S20045) reference substances are purchased from Shanghai leaf Biotech limited, and the purity of each reference substance is more than 98%; heparin sodium (BBI Life Sciences LOT: B805BA0008), physiological saline (Hebei Tian Cheng Yao Kogyo A17041302), methanol (chromatographically pure Merck, Germany), formic acid (ACS USA).
1.3 Experimental animals
The SPF-grade healthy SD rats are half male and female, have the weight of 200-250 g, are provided by a Qinglong mountain animal breeding farm in Jiangning district of Nanjing, and have the quality qualification certificate number of 1708240024.
2 methods and results
2.1 analysis of chemical Components of the preparation
2.1.1 detection conditions
Chromatographic conditions are as follows: the column was Agilent ZORBAX SB-C18(4.6 x 250mm, 5 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), elution gradient: 0-10 min, 1-3% (A); 10-35 min, 3-20% (A); 35-50 min, 20-30% (A); 50-70 min, 30-55% (A); 70-75 min, 55-100% (A); 75-85 min, 100% (A), column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength is 254 nm; sample introduction amount: 2 μ L.
Mass spectrum conditions: electrospray ion source (ESI), positive and negative ion mode; the capillary voltage is 4000V and 3500V respectively; the ion scanning range m/z is 100-3000; the drying airflow rate is 10L/min; the temperature of the drying gas is 350 ℃; atomization pressure 60 psi; cleavage voltage 135V; the taper hole voltage is 65V; collision energy was 10, 20, 30, 40eV, respectively.
2.1.2 preparation of solutions
Preparation of control solutions: taking a proper amount of sinomenine, monotropein, formononetin, protocatechuic acid, valine, adenine, pyroglutamic acid, leucine, phenylalanine, 5-hydroxymethyl furfural, glutamic acid, magnoflorine and formononetin respectively, precisely weighing, and preparing a mixed reference substance solution containing 10-40 mu g/mL of each component by using 50% methanol.
Preparing a test solution: precisely measuring 1mL of seven-ingredient oral liquid for treating arthralgia, diluting with pure water by 10 times, centrifuging at 14000rpm for 10min, and taking supernatant as a test solution.
2.1.3 identification and assignment of Compounds
Detecting the test solution and the mixed reference solution by UPLC-Q TQF-MS under the condition of '2.1.1', and qualitatively analyzing the chemical components in the Qiwei Tongbi oral liquid to obtain a total ion flow chart (TIC) in a positive and negative ion mode, wherein the result is shown in figure 1. As can be seen from the figure, the main components of the seven-ingredient arthralgia-relieving oral liquid have better response under the condition of positive ions.
Looking up relevant Chinese and English documents, collecting chemical component information of 7 prescription medicinal materials of the seven-ingredient Tongbi oral liquid, namely ants, caulis sinomenii, caulis spatholobi, pyrola, piper wallichii, homalomena rhizoma and radix clematidis, and establishing a compound database which comprises compound names, molecular formulas, accurate relative molecular masses, types and the like. Performing primary mass spectrometry on a test solution of the seven-ingredient Tongbi oral liquid, generating a compound molecular formula by using MassHunter software, selecting the molecular formula with an error within 5ppm, performing automatic database matching, and quickly identifying possible compounds; and selecting a target compound according to the primary detection result to perform secondary mass spectrum scanning to obtain fragment ion information of the compound, and identifying and speculating the target compound by combining the cracking rule of the compound and a literature report. A total of 47 compounds were identified and putative, 13 of which were confirmed by comparison with controls, and the results are shown in Table 1.
TABLE 1 UPLC-Q-TOF-MS/MS identification and medicinal material attribution of seven-ingredient oral liquid for relieving arthralgia
Figure BDA0001872512160000081
Figure BDA0001872512160000091
Figure BDA0001872512160000101
Figure BDA0001872512160000111
Figure BDA0001872512160000121
Figure BDA0001872512160000131
The label [ a ] is the compound which has been aligned with the reference
The detection method obtained according to the conditions has good precision, the stability of the reference substance solution and the sample solution at room temperature in each sample and within 24 hours of the reference is good, different batches of solutions are prepared by the same method, and the detection result shows that the method has good repeatability.
2.2 study of the blood-entering Components and pharmacokinetic characteristics of the preparations
2.2.1 sample Collection
100mL of seven-ingredient oral liquid for relieving arthralgia is taken and concentrated to 20 mL. 18 healthy SD rats were randomly divided into 3 groups and administered by gavage at doses 4 times (2.16mL/kg), 8 times (4.32mL/kg) and 12 times (6.48mL/kg) the clinical dose. Fasting was performed for 12 hours before administration and water was freely available. Collecting blood of 0.3mL in orbital vein before administration and 10min, 20min, 30min, 45min, 1h, 2h, 4h, 6h, 8h, 10h and 24h after administration, placing in 1.5mL heparinized EP tube, centrifuging at 4000rpm for 10min, collecting supernatant plasma, and storing at-80 deg.C.
2.2.2 sample treatment
And (3) placing 500 mu L of plasma sample in an EP tube, adding 1.5mL of methanol, uniformly mixing in a vortex mode, centrifuging for 10min at 14000rpm, drying supernatant in nitrogen at 40 ℃, adding 100 mu L of 50% methanol for redissolving, centrifuging for 10min at 14000rpm, taking supernatant for sample injection, and analyzing possible blood components and metabolites.
100 mu L of plasma sample is taken and placed in a 1mL EP tube, 10 mu L of internal standard is added, 300 mu L of methanol is added, vortex mixing is carried out, centrifugation is carried out for 10min at 14000rpm, and the sample injection of supernatant is taken for analyzing the time course of main components in rat body.
2.2.3 detection conditions
2.2.3.1UPLC-Q TQF-MS conditions
The detection conditions were the same as those under the item "2.1.1".
2.2.3.2UPLC-QQQ-MS Condition
Chromatographic conditions are as follows: the column was Agilent Eclipse plus C18(3 x 50mm, 1.8 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), gradient elution: 0-3 min, 10-45% (A); 45-65% (A) for 3-4 min; 4-4.3 min, 65-100% (A); 4.3-5 min, 100% (A); 5-5.5 min, 100-10% (A); 5.5-6 min, 10% (A); column temperature: 40 ℃; flow rate: 0.6 mL/min.
Mass spectrum conditions: ESI ion source; a positive ion mode; ion source Temperature (TEM): 500 ℃; auxiliary gas 1(GS1, N2)40psi, auxiliary gas 2(GS2, N2)50 psi; air curtain air (CUR)35 psi; collision gas (CAD)8 psi; spray voltage (IS) 5500V; the scanning mode is multiple ion reaction detection (MRM); the mass spectrometric detection parameters of the target components are shown in table 2.
TABLE 2 Mass spectrometric detection parameters for sinomenine, magnoflorine and internal standards
Figure BDA0001872512160000151
2.2.4 analysis of blood constituents
Based on the detection result of chemical components, the possible blood components and metabolites thereof of the seven-ingredient arthromyodynia-relieving oral liquid after oral administration of rats are analyzed by adopting the detection condition under the item '2.2.3.1'. Because the response of each component in the negative ion mode is not good, the detection result in the positive ion mode is mainly analyzed. By comparing with blank plasma, 8 possible blood components are detected in plasma samples after administration, and 6 of the blood components are prototype components and 2 are metabolites by comparing with chemical components of the preparation and combining with the secondary mass spectrum cracking rule of target components. The results are shown in FIG. 2 and Table 3.
TABLE 3 UPLC-Q-TOF-MS/MS identification of blood-entering components of seven-ingredient Tongbi oral liquid
Figure BDA0001872512160000152
Figure BDA0001872512160000161
The label [ a ] is the compound which has been aligned with the reference
2.2.5 study of the time course of the principal Components
(1) Specialization inspection
Respectively taking blank rat plasma, adding 10 μ L of mixed standard plasma samples (final concentrations of sinomenine and magnoflorine are respectively 30.1 μ g/mL and 12.8 μ g/mL) and plasma samples after administration for 20min, processing according to the method under the item '2.2.2', and injecting sample for analysis. The result shows that the retention time of the sinomenine, the magnoflorine and the internal standard in the plasma sample is respectively 1.55min, 2.13min and 3.35min, the determination of the target component is not interfered by the endogenous component, and the specificity is better. The results are shown in FIG. 3.
(2) Standard curve and lower limit of quantitation
Taking a proper amount of sinomenine and magnoflorine reference substances, and preparing mixed standard substance solutions with the concentrations of 60.13 mu g/mL and 25.5 mu g/mL respectively by using methanol. Then diluted by methanol into a series of mixed standard working solutions with the concentrations of 2405/1020, 1202/510, 601.3/255, 300.6/128, 150.3/64, 75.2/31.9, 37.6/15.9, 18.8/8.0 and 6.3/2.7ng/mL respectively.
Taking several parts of 90 μ L blank plasma, respectively adding 10 μ L mixed reference solution and 10 μ L internal standard solution (chloramphenicol, 5 μ g/mL), and preparing into sinomenine and magnoflorine with concentrations of 240.5/102, 120.2/51, 60.13/25.5, 30.06/12.8, and 15.03/6, respectively, by the method under item "2.2.2".4. 7.52/3.19, 3.76/1.59, 1.88/0.80, 0.63/0.27ng/mL of the series of standard plasma sample solutions, analyzing by sample injection, using the concentration (C, ng/mL) of the analyte as an independent variable (x), the peak area ratio of the analyte to the internal standard as a dependent variable (y), and adopting weighting (W is 1/C)2) Performing regression operation by using a least square method to obtain a linear regression equation of the sinomenine: y is 0.00978x +0.00155 (concentration range is 0.63-240.5 ng/mL, LLOQ is 0.63ng/mL, r is 0.9996); magnoflorine: y is 0.0773x +0.00402 (the concentration range is 0.27-102 ng/mL, LLOQ is 0.27ng/mL, r is 0.9989), and all meet the detection requirements of the biological sample.
(3) Method verification
The method has the advantages that the verification result meets the requirements of biological sample detection on accuracy, precision, selectivity, sensitivity, extraction recovery rate, dilution reliability and stability, and can be used for detecting sinomenine and magnoflorine in a rat plasma sample and evaluating pharmacokinetic characteristics.
(4) Data analysis
Adopting the detection conditions under the item of '2.2.3', measuring the concentrations of the sinomenine and the magnoflorine in the plasma of the rat at different time points, and drawing a blood concentration-time curve; calculation of pharmacokinetic parameters was performed in a non-compartmental model using DAS 3.0 software. The results are shown in FIGS. 4 and 5, and Table 4. TABLE 4 pharmacokinetic parameters of sinomenine and magnoflorine in plasma of different doses of Qiwei Tongbi oral liquid after oral administration in rat (Mean + -SD)
Figure BDA0001872512160000171
The results show that the dynamics of sinomenine in rats are basically consistent under different administration doses, TmaxWithin 1h, absorption is rapid, t1/2The time is 1.65-2.48 h. AUC over the dose range administered0-∞The dosage of the composition is obviously increased along with the increase of the dosage, wherein the dosage of C is low or mediummaxIncrease with increasing dosage, increase at high dosage is not obvious or even slightly reduced compared with the medium dosage, and may be related to the interaction of multiple components of complex Chinese medicine, orThe difference between medium and high doses is not much related. Meanwhile, the dynamics behaviors of magnoflorine in rats are basically consistent under different administration doses, and T ismaxWithin 0.21-0.26 h, the absorption is fast, t1/2The time is 1.65-2.48 h. AUC over the dose range administered0-∞And CmaxAll show obvious increasing trend along with the increase of the administration dosage.
2.3 quality control Studies based on blood admission composition
2.3.1 detection method
A chromatographic column: agilent extended-C18(4.6 x 250mm, 5 μm); mobile phase: 0.05mol/LNaH2PO4(A) -acetonitrile (B); elution gradient: 0-15 min, 8% (B); 15-28 min, 8-12% (B); 28-31 min, 12-50% (B); 31-35 min, 50% (B); detection wavelength: 265nm, column temperature: 25 ℃, flow rate: 1.0mL/min, and a sample size of 10. mu.L.
2.3.2 preparation of solutions
Preparation of control solutions: taking appropriate amount of sinomenine and magnoflorine reference substances respectively, precisely weighing, and preparing into mixed reference substance stock solutions with concentrations of 3.05 and 1.52mg/mL respectively with 50% methanol.
Preparation of a test solution: precisely measuring 1mL of seven-ingredient oral liquid for treating arthralgia syndrome, placing into a 10mL measuring flask, diluting with 50% methanol, fixing volume to scale, performing ultrasonic treatment for 10min, centrifuging, and collecting supernatant.
2.3.3 Linear relationship investigation
Taking a proper amount of the mixed reference stock solution under the item 2.3.2, obtaining mixed reference solutions with series concentrations by adopting a stepwise dilution method, carrying out sample injection analysis according to the detection method under the item 2.3.1, recording peak areas of various spectral peaks, drawing a standard curve by taking the sample concentration (mg/mL) as a horizontal coordinate (x) and the peak areas as a vertical coordinate (y), and calculating a regression equation. The results are shown in Table 5. The results show that the linear relationship between sinomenine and magnoflorine in the selected concentration range is good.
TABLE 5 Linear regression equation, correlation coefficient and Linear Range investigation
Figure BDA0001872512160000191
2.3.4 precision test
Taking the mixed reference substance solution (the concentrations of the sinomenine and the magnoflorine are 0.152 mg/mL and 0.076mg/mL respectively), continuously injecting a sample for 6 needles, recording the peak area of each chromatographic peak, and calculating the RSD value. The result shows that the peak areas RSD of the two components are less than 1 percent, which indicates that the precision of the instrument is good.
2.3.5 stability test
Sample solutions (batch: 160504) to be tested are taken, sample injection is carried out for 0, 2, 4, 8, 12 and 24 hours after preparation, peak areas of components to be tested are recorded, and RSD values are calculated. The result shows that the peak area RSD of the sinomenine in the test solution is 0.25%, and the peak area RSD of the magnoflorine is 0.15%, which shows that the test solution has good stability within 24 hours.
2.3.6 repeatability test
Taking the same batch of seven-ingredient oral liquid (batch: 160504) for dredging the arthralgia, preparing a test solution according to the method under the item '2.3.2', preparing 6 parts in parallel, measuring according to the detection condition under the item '2.3.1', recording the peak area of each component to be measured, and calculating the RSD value of the content. The result shows that the RSD of the sinomenine content is 0.33 percent, and the RSD of the magnoflorine content is 0.62 percent, which indicates that the method has good repeatability.
2.3.7 sample recovery test
6 parts of seven-ingredient Tongbi oral liquid (batch number: 160504, the contents of the sinomenine and the magnoflorine are respectively 1.45mg/mL and 0.44mg/mL) with known content are taken, 0.5mL of each part is added with a proper amount of mixed reference substance solution according to the proportion of 1:1, the operation is carried out according to the proposed content determination method, and the recovery rate and RSD of each component are calculated according to the method determination. The result shows that the recovery rate of the sinomenine is between 100.9 and 101.9 percent, the RSD is 0.34 percent, the recovery rate of the magnoflorine is between 100.3 and 101.5 percent, and the RSD is 0.44 percent, which shows that the method has good accuracy. The results are shown in Table 6.
TABLE 6 sample recovery examination
Figure BDA0001872512160000201
2.3.8 Multi-batch sample determination
Taking 10 batches of the seven-ingredient oral liquid for dredging the arthralgia, preparing a test solution according to the method under the item 2.3.2, and determining the contents of the sinomenine and the magnoflorine according to the chromatographic conditions under the item 2.3.1. Simultaneously, the 10 batches of preparation are taken, and the content of the sinomenine is determined by an HPLC method in the current standard. The results are shown in Table 7. The content of the sinomenine is 1.30-1.53 mg/mL, the content of the magnoflorine is 0.45-0.93 mg/mL, and the content of the sinomenine is basically consistent with the detection result of the current standard, so that the method is stable and reliable and can be effectively applied to the quality control of products. HPLC chromatograms of the seven-ingredient arthralgia-relieving oral liquid and the reference substance obtained by the method are shown in FIG. 6.
TABLE 7 determination results of sinomenine and magnoflorine contents in multi-batch seven-ingredient oral liquid for treating arthralgia
Figure BDA0001872512160000202
Figure BDA0001872512160000211
In the above embodiments, the present invention first systematically studies the chemical component spectrum and the blood component spectrum of the preparation, and defines the main blood components, thereby providing a strong support for the basic study of the drug-effective substances. Then, a UPLC-QQQ-MS detection method of sinomenine and magnoflorine in rat plasma is established and is effectively applied to pharmacokinetic research in a rat body, and the result shows that the time course of the target component in the body is well characterized. Based on the method, an HPLC content determination method of main blood-entering components of sinomenine and magnoflorine of the seven-ingredient arthromyodynia-relieving oral liquid is established through experiments, so that the selection of index components is more reasonable, and the value and significance of quality standard improvement research are enriched.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (7)

1. A detection method of seven-ingredient arthralgia-relieving oral liquid is characterized by comprising the following steps: detecting a seven-ingredient arthralgia-relieving oral liquid sample and a reference substance, wherein the chromatographic conditions of the detection are as follows: the chromatographic column is Agilent ZORBAX SB-C184.6 × 250mm, 5 μm; mobile phase: methanol is used as A, 0.1% formic acid aqueous solution is used as B, and elution gradient is as follows: 0-10 min, 1-3% A; 10-35 min, 3-20% A; 35-50 min, 20-30% A; 30-55% of A for 50-70 min; 70-75 min, 55-100% A; 75-85 min, 100% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-30 ℃; detecting the wavelength of 210-300 nm;
mass spectrum conditions: electrospray ion source ESI, positive and negative ion mode; the capillary voltage is 4000V and 3500V respectively; the ion scanning range m/z is 100-3000; the flow rate of the drying gas is 10L/min; the temperature of the drying gas is 350 ℃; atomization pressure 60 psi; cleavage voltage 135V; the taper hole voltage is 65V; collision energy is respectively 10 eV, 20 eV, 30 eV and 40 eV;
and obtaining the component information or content information of the seven-ingredient arthralgia-relieving oral liquid according to the detection result.
2. The detection method according to claim 1, wherein the sample is prepared by precisely measuring 1ml of Qiwei Tongbi oral liquid, diluting with pure water by 10 times, centrifuging at 14000rpm for 10min, and collecting the supernatant as a sample solution.
3. The detection method according to claim 1, wherein the flow rate is 1.0mL/min, the column temperature is 25 ℃, and the detection wavelength is 254 nm.
4. The assay of claim 1, wherein the control is selected from one or more of sinomenine, monotropein, formononetin, protocatechuic acid, valine, adenine, pyroglutamic acid, leucine, phenylalanine, 5-hydroxymethylfurfural, glutamic acid, magnoflorine, and formononetin.
5. A detection method for analyzing blood-entering components of a seven-ingredient arthralgia-relieving oral liquid is characterized by comprising the following steps:
analyzing the processed seven-ingredient Tongbi oral liquid plasma sample and blank plasma sample by UPLC-Q-TOF-MS, wherein the chromatographic conditions and the mass spectrum conditions are respectively as follows:
the chromatographic column is Agilent ZORBAX SB-C184.6 × 250mm, 5 μm; mobile phase: methanol is used as A, 0.1% formic acid aqueous solution is used as B, and elution gradient is as follows: 0-10 min, 1-3% A; 10-35 min, 3-20% A; 35-50 min, 20-30% A; 30-55% of A for 50-70 min; 70-75 min, 55-100% A; 75-85 min, 100% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-30 ℃; detecting the wavelength of 210-300 nm;
mass spectrum conditions: electrospray ion source ESI, positive and negative ion mode; the capillary voltage is 4000V and 3500V respectively; the ion scanning range m/z is 100-3000; the flow rate of the drying gas is 10L/min; the temperature of the drying gas is 350 ℃; atomization pressure 60 psi; cleavage voltage 135V; the taper hole voltage is 65V; collision energy was 10, 20, 30, 40eV, respectively.
6. A content determination method of a seven-ingredient arthralgia-relieving oral liquid based on blood components is characterized by comprising the following steps: detecting a seven-ingredient arthralgia-relieving oral liquid sample and a reference substance, wherein the chromatographic conditions of the detection are as follows: agilent extended-C184.6 x 250mm, 5 μm; mobile phase: a: 0.05mol/LNaH2PO4And B: acetonitrile; elution gradient: 0-15 min, 8% B; 15-28 min, 8-12% of B; 28-31 min, 12-50% B; 31-35 min, 50% B; the flow rate is 0.8-1.2 mL/min; the column temperature is 20-30 ℃; detecting the wavelength of 260-270 nm;
the reference substance is selected from sinomenine and magnoflorine;
according to the detection result, the content information of the seven-ingredient arthralgia-relieving oral liquid based on the blood-entering components is obtained.
7. The detection method according to claim 6,
the preparation of the test sample comprises precisely measuring 1mL of seven-ingredient oral liquid for relieving arthralgia, adding 50% methanol, ultrasonically centrifuging, and collecting supernatant as test sample solution;
the flow rate is 1.0mL/min, the column temperature is 25 ℃, and the detection wavelength is 265 nm.
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