CN114674958B - Method for measuring content of active ingredients in fructus gleditsiae - Google Patents
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Abstract
The application provides a method for measuring the content of active ingredients in large saponin, which adopts ultra-high performance liquid chromatography-mass spectrometry to rapidly detect the content of L-malic acid, protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, glycyrrhizin, apigenin, luteolin, eriodictyol, bergamot, yellow pigment, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin in the large saponin. The method provided by the application is simple to operate, high in sensitivity, high in analysis speed and strong in specificity, and can be used for quality control of the fructus gleditsiae.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicine component measurement, in particular to a method for measuring the content of active components in Chinese honeylocust fruits.
Background
The gleditsia sinensis (Gleditsiae sinensis fructus) is also called gleditsia sinensis, is a plant of gleditsia sinensis of the subfamily of Caesalpiniaceae of Leguminosae, and is one of the special species of the country tree in China. The Chinese medicinal composition is distributed in most regions of the whole country, has rich resources, has the effects of dispelling wind phlegm and removing dampness and toxic substances, can reduce toxic and side effects after being processed, and is commonly used for clinical medication. The fructus gleditsiae has larger medicinal value, but the quality of medicinal materials in different producing places is uneven, and the quality of the fructus gleditsiae is difficult to evaluate. Therefore, a high-efficiency, rapid and high-sensitivity detection method is needed to measure the content of active ingredients in the fructus gleditsiae, and provides a basis for quality evaluation of the fructus gleditsiae and research on the basis of pharmacodynamic substances.
Disclosure of Invention
The invention aims to provide a method for measuring the content of active ingredients in large Chinese honeylocust, which can simultaneously measure the content of 19 active ingredients in large Chinese honeylocust and can be used for quality control of large Chinese honeylocust. The specific technical scheme is as follows:
the application provides a method for measuring the content of active ingredients in large Chinese honeylocust, which is characterized in that ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) is adopted to measure the content of active ingredients in large Chinese honeylocust; the active ingredients comprise L-malic acid, protocatechuic acid, caffeic acid, scopoletin, glycyrrhizin, apigenin, luteolin, eriodictyol, bergamot, yellow pigment, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin; the method comprises the following steps:
(1) Establishing standard curves of the active ingredients;
preparing 5-12 mixed reference substance solutions containing active ingredients with different known concentrations by taking 80-100vol% methanol as a solvent; wherein the concentration of the L-malic acid is 40-55000ng/mL; the concentration of the chlorogenic acid is 2-20000ng/mL; the concentration of the chlorogenic acid is 3-10000ng/mL; the concentration of isovitexin is 1-10000ng/mL; the concentration of the chlorogenic acid is 1.5-5000ng/mL; eriodictyol concentration is 0.5-5000ng/mL; the concentration of luteolin is 1-5000ng/mL; the concentration of orientin is 2-3000ng/mL; the concentration of isoorientin is 2-3000ng/mL; the concentration of vitexin is 0.4-1000ng/mL; the concentrations of protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin and flavodoxin are respectively 0.5-500ng/mL, and the concentrations of bergamot and quercetin are respectively 0.1-500ng/mL; the concentration of the glycyrrhizin is 0.25-300ng/mL;
under the same chromatographic conditions and mass spectrum conditions, the volume is V 1 Respectively injecting the mixed reference substance solutions into an ultra-high performance liquid chromatograph, determining chromatographic peaks of the active ingredients according to characteristic ions of the active ingredients through mass spectrum detection, and obtaining chromatographic peak areas of the active ingredients;
establishing standard curves of the active ingredients respectively by taking the peak area of the chromatographic peak of each active ingredient as an ordinate and the concentration of each active ingredient as an abscissa;
(2) Obtaining chromatographic peak areas of each active ingredient in the sample solution;
taking a sample with mass M and volume V 2 Extracting with 60-80vol% methanol as solvent, collecting supernatant, filtering to obtain sample solution, wherein M/V 2 1mg/mL for (8-12);
taking the volume V under the same chromatographic conditions and mass spectrum conditions as in the step (1) 1 The sample solution is injected into an ultra-high performance liquid chromatograph, and the chromatographic peak of each active ingredient is determined according to the characteristic ion of each active ingredient through mass spectrum detection, and the chromatographic peak area of each active ingredient is obtained;
(3) Determining the content of each active ingredient in the test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is calculated from the chromatographic peak area of each active ingredient in the test solution 1 According to the formula c=c 1 ×V 2 And (3) respectively calculating the content C of each active ingredient in the test solution.
The method for measuring the content of the active ingredients in the fructus gleditsiae provided by the application can be used for measuring the content of 19 active ingredients in the fructus gleditsiae simultaneously by reasonably selecting chromatographic conditions and mass spectrum conditions, has the advantages of simplicity in operation, high sensitivity, high accuracy, high analysis speed, strong specificity and the like, can be used for quality control of the fructus gleditsiae, and provides a basis for quality evaluation of the fructus gleditsiae and research on the drug effect substance basis thereof.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will briefly introduce the drawings that are required to be used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other embodiments may also be obtained according to these drawings to those skilled in the art.
FIG. 1A is a multi-reaction ion monitoring graph (MRM graph) of the mixed control solution labeled 8 in example 1;
FIG. 1B is a MRM chart of 19 active ingredients in batch S10 of fructus Gleditsiae Abnormalis;
the reference numerals in the drawings represent: l-malic acid; 2. protocatechuic aldehyde; 3. protocatechuic acid; 4. caffeic acid; 5. scopoletin; 6. glycyrrhizin; 7. apigenin; 8. luteolin; 9. eriodictyol; 10. orange extract; 11. yellow pigment lignin; 12. chlorogenic acid of Cryptophan; 13. new chlorogenic acid; 14. chlorogenic acid; 15. vitexin; 16. isovitexin; 17. quercetin; 18. orientin; 19. isoorientin.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. Based on the embodiments herein, a person of ordinary skill in the art would be able to obtain all other embodiments based on the disclosure herein, which are within the scope of the disclosure herein.
The application provides a method for measuring the content of active ingredients in large Chinese honeylocust, which is characterized by measuring the content of active ingredients in large Chinese honeylocust by adopting UPLC-MS/MS; the active ingredients comprise L-malic acid, protocatechuic acid, caffeic acid, scopoletin, glycyrrhizin, apigenin, luteolin, eriodictyol, bergamot, yellow pigment, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin; the method comprises the following steps:
(1) Establishing standard curves of the active ingredients;
preparing 5-12 mixed reference substance solutions containing active ingredients with different known concentrations by taking 80-100vol% methanol as a solvent; wherein the concentration of the L-malic acid is 40-55000ng/mL; the concentration of the chlorogenic acid is 2-20000ng/mL; the concentration of the chlorogenic acid is 3-10000ng/mL; the concentration of isovitexin is 1-10000ng/mL; the concentration of the chlorogenic acid is 1.5-5000ng/mL; eriodictyol concentration is 0.5-5000ng/mL; the concentration of luteolin is 1-5000ng/mL; the concentration of orientin is 2-3000ng/mL; the concentration of isoorientin is 2-3000ng/mL; the concentration of vitexin is 0.4-1000ng/mL; the concentrations of protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin and flavodoxin are respectively 0.5-500ng/mL, and the concentrations of bergamot and quercetin are respectively 0.1-500ng/mL; the concentration of the glycyrrhizin is 0.25-300ng/mL;
under the same chromatographic conditions and mass spectrum conditions, the volume is V 1 Respectively injecting the mixed reference substance solutions into an ultra-high performance liquid chromatograph, determining chromatographic peaks of the active ingredients according to characteristic ions of the active ingredients through mass spectrum detection, and obtaining chromatographic peak areas of the active ingredients;
establishing standard curves of the active ingredients respectively by taking the peak area of the chromatographic peak of each active ingredient as an ordinate and the concentration of each active ingredient as an abscissa;
(2) Obtaining chromatographic peak areas of each active ingredient in the sample solution;
taking a sample with mass M and volume V 2 Extracting with 60-80vol% methanol as solvent, collecting supernatant, filtering to obtain sample solution, wherein M/V 2 1mg/mL for (8-12);
taking the volume V under the same chromatographic conditions and mass spectrum conditions as in the step (1) 1 The sample solution is injected into an ultra-high performance liquid chromatograph, and the chromatographic peak of each active ingredient is determined according to the characteristic ion of each active ingredient through mass spectrum detection, and the chromatographic peak area of each active ingredient is obtained;
(3) Determining the content of each active ingredient in the test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is calculated from the chromatographic peak area of each active ingredient in the test solution 1 According to the formula c=c 1 ×V 2 And (3) respectively calculating the content C of each active ingredient in the test solution.
According to the method for measuring the content of the active ingredients in the fructus gleditsiae, UPLC-MS/MS is adopted, and the content of 19 active ingredients in the fructus gleditsiae can be measured by reasonably selecting chromatographic conditions and mass spectrum conditions.
In some embodiments of the present application, the concentration of L-malic acid in each mixed control solution is 62.5-50000ng/mL, and the concentration of neochlorogenic acid is 20-16000ng/mL; the concentration of the cryptochlorogenic acid and the isovitexin is respectively 10-8000ng/mL; the concentration of chlorogenic acid, eriodictyol and luteolin is 5-4000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2.5-2000ng/mL; vitexin is 1-800ng/mL; the concentrations of protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, bergamot, flavodoxin and quercetin are respectively 0.5-400ng/mL; the concentration of the glycyrrhizin is 0.25-200ng/mL.
The method of preparing the mixed reference solution is not particularly limited as long as the object of the present application can be achieved, for example, a mixed reference stock solution may be prepared first, wherein the concentration of each component is equal to or higher than the concentration of each active component in the mixed reference solution, and then the mixed reference solution is obtained by dilution.
In some embodiments of the present application, in step (1), a mixed control stock solution containing each active ingredient is prepared by using 80-100vol% methanol as a solvent, wherein the concentration of L-malic acid in the mixed control stock solution is 50000-55000ng/mL, and the concentration of neochlorogenic acid in the mixed control stock solution is 16000-20000ng/mL; the concentration of the cryptochlorogenic acid and the isovitexin is 8000-10000ng/mL respectively; the concentration of chlorogenic acid, eriodictyol and luteolin is 4000-5000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2000-3000ng/mL; vitexin is 800-1000ng/mL; the concentrations of protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, bergamot, flavodoxin and quercetin are respectively 400-500ng/mL; the concentration of the glycyrrhizin is 200-300ng/mL;
diluting the mixed reference substance stock solution by taking 80-100vol% methanol as a solvent to obtain 5-12 mixed reference substance solutions containing active ingredients with different known concentrations.
The solvent used for preparing the mixed reference stock solution and the solvent used for diluting the mixed reference stock solution can be the same or different, preferably, the solvent used for preparing the mixed reference stock solution and the solvent used for diluting the mixed reference stock solution are the same, more preferably, the solvent used for preparing the mixed reference stock solution and the solvent used for diluting the mixed reference stock solution are methanol.
In the present application, the mixed control solution used to establish the standard curve may include the mixed control stock solution; the preparation of the mixed reference stock solution is not particularly limited as long as the object of the present application can be achieved, and the mixed reference stock solution can be prepared by preferentially and individually preparing the stock solutions of the active ingredients and then separately taking the stock solutions of the active ingredients.
In some embodiments of the present application, in step (2), the extraction is ultrasonic extraction with an extraction power of 100-300W, an extraction frequency of 30-50kHz, and an extraction time of 20-60 minutes.
By adopting the preparation method of the sample solution, the sample solution containing 19 active ingredients is obtained, so that the detection result of the content of the active ingredients in the fructus gleditsiae is more comprehensive, accurate and reliable; the method comprises the steps of obtaining a sample solution containing 19 active ingredients through ultrasonic extraction, and determining the content of each active ingredient, so that the detection result of each active ingredient content is more accurate.
In some embodiments of the present application, the chromatographic conditions include: chromatographic column: octadecylsilane chemically bonded silica chromatographic column; mobile phase: the phase A is formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the phase B is methanol; gradient elution is carried out by adopting 5-90% of phase A and 10-95% of phase B in volume fraction; column temperature is 20-30 ℃; the flow rate is 0.1-0.5 mL/min; sample injection volume V 1 ：1-5μL。
Preferably, the chromatographic conditions include: mobile phase: the phase A is formic acid aqueous solution with the volume fraction of 0.08-0.12%, and the phase B is methanol; column temperature: 20-28 ℃; flow rate: 0.2-0.4 mL/min; sample injection volume V 1 ：1-3μL。
The inventors found in the study that the gradient elution of the present application can achieve better separation effect of 19 active ingredients in the fructus gleditsiae, preferably, in some embodiments of the present application, the gradient elution is specifically: 0 to 1.0 min, 10 to 25 percent of phase B;1.0 to 5.0 minutes, 25 to 31 percent of phase B;5.0 to 7.5 minutes, 31 to 31 percent of phase B,7.5 to 8.0 minutes, and 31 to 50 percent of phase B;8.0 to 9.0 minutes, 50 to 95 percent of phase B;9.0 to 11.0 minutes, 95 to 95 percent of phase B.
The type of mass spectrum is not particularly limited in the present application, as long as the object of the present application can be achieved, and for example, triple quadrupole mass spectrum can be used. In order to effectively obtain characteristic ions of each active ingredient after chromatographic separation so as to obtain more accurate identification results of each active ingredient, in some embodiments of the present application, the mass spectrum is a triple quadrupole mass spectrum, and the mass spectrum conditions include: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring and anion scanning mode; drying gas temperature: 250-350 ℃; drying gas flow rate: 5-10L/min; atomizer pressure: 30-40psi; sheath temperature: 300-400 ℃; sheath air flow rate: 8-15L/min; capillary voltage: 3000-4000V.
Preferably, the mass spectrometry conditions include: drying gas temperature: 280-320 ℃; drying gas flow rate: 6-8L/min; atomizer pressure: 32-38psi; sheath temperature: 330-370 ℃; sheath air flow rate: 10-12L/min; capillary voltage: 3300-3700V.
In some embodiments of the present application, the characteristic ion of each active ingredient comprises:
the instrumentation and reagents required for this application are described below.
Instrument for measuring and controlling the intensity of light
Agilent 6470 triple quadrupole mass spectrometer (Agilent company, usa); agilent 1290 ultra-high performance liquid chromatograph (Agilent company, usa); agilent MassHunter analysis software (Agilent company, usa); A60/220.R2 one ten thousandth balance (from Radwag Corp., poland); milli-Q IQ 7005 ultra-pure water preparation apparatus (Millipore Co.); 5453 high-speed centrifuge (Eppendorf, germany); vortex mixer of Vortex-2 (Shanghai analysis Co., ltd.); ZZ-L6DT ultrasonic cleaning tank (Tianjin know technology Co., ltd.).
Reagent(s)
L-malic acid (lot number: DST 201124-038), protocatechuic acid (lot number: DST 200628-080), protocatechuic acid (lot number: DSTDY 008101), caffeic acid (lot number: DST 191030-013), scopoletin (lot number: DSTDD 006401) glycyrrhizin (lot number: DST 200326-010), apigenin (lot number: DST 200809-026), luteolin (lot number: DST 200910-032), eriodictyol (lot number: DSTDS 004201), bergamotin (lot number: DSTDX 009401), yellow lignin (lot number: DST 201019-147), cryptochlorogenic acid (lot number: DSTDY 003501), neochlorogenic acid (lot number: DSTDX 001501), chlorogenic acid (lot number: DSTDL 002101), vitexin (lot number: DST 200325-034), isovitexin (lot number: DST 200200054), quercetin (lot number: DST 200519-006), orientin (lot number: DST 004901), and isoorientin (lot number: DST 004901) from the same company.
Methanol (chromatographic purity) was purchased from Fisher, usa and formic acid (chromatographic purity) was purchased from ROE.
Material
The fructus Gleditsiae Abnormalis is purchased from Hebei, anhui, henan, shanxi, shandong, yunnan, guizhou, etc. The sources of the medicinal materials are shown in table 1.
TABLE 1 different batches of sources of fructus Gleditsiae Abnormalis
The reagents and materials referred to in the examples below may be obtained commercially or according to methods known in the art unless otherwise specified.
Example 1
(1) Establishing standard curve of each active ingredient
Precisely weighing L-malic acid, protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, glycyrrhizin, apigenin, luteolin, eriodictyol, bergamot, yellow pigment, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin reference substances respectively 10.0mg, respectively dissolving with methanol to prepare reference substance stock solutions with concentration of 1mg/mL, and storing in a refrigerator at 4deg.C for use.
Accurately measuring a proper amount of each control stock solution, preparing a mixed control stock solution by using methanol, and marking as a mark 1, wherein the concentration of L-malic acid is 50000ng/mL, the concentration of neochlorogenic acid is 16000ng/mL, the concentration of cryptochlorogenic acid and isovitexin is 8000ng/mL, the concentration of chlorogenic acid, eriodictyol and luteolin is 4000ng/mL, the concentration of orientin, the concentration of isoorientin is 2000ng/mL, the concentration of vitexin is 800ng/mL, the concentration of protocatechuic acid, scopoletin, apigenin, bergamot element, yellow pigment and quercetin is 400ng/mL, and the concentration of glycyrrhizin is 200ng/mL; and (3) sequentially diluting the mixed reference substance stock solution by methanol for 2 times, 2.5 times, 2 times, 4 times and 2 times to obtain 9 mixed reference substance solutions containing active ingredients with different known concentrations and containing the mixed reference substance stock solution, wherein the mixed reference substance solutions are sequentially marked as marks 1-9.
Under the same chromatographic conditions and mass spectrum conditions, respectively taking 2 mu L of each mixed reference substance solution, injecting into an ultra-high performance liquid chromatograph, detecting by mass spectrum, determining chromatographic peaks of each active ingredient according to characteristic ions of each active ingredient, and obtaining chromatographic peak areas of each active ingredient;
wherein the chromatographic conditions include: column ACQUITY UPLC BEH C column (2.1X100 mm,1.7 μm, waters); mobile phase: the phase A is formic acid aqueous solution with the volume fraction of 0.1 percent, and the phase B is methanol; gradient elution, elution gradient is: 0 to 1.0 min, 10 to 25 percent of B;1.0 to 5.0 minutes, 25 to 31 percent of B;5.0 to 7.5 minutes, 31 to 31 percent of B,7.5 to 8.0 minutes, and 31 to 50 percent of B;8.0 to 9.0 minutes, 50 to 95 percent of B;9.0 to 11.0 minutes, 95 to 95 percent of B, and the flow rate is 0.3 mL/minute; column temperature: 25 ℃; sample injection amount V 1 ：2μL。
The mass spectrum is a triple quadrupole mass spectrum, and the mass spectrum conditions include: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring and anion scanning mode; drying gas temperature: 300 ℃; drying gas flow rate: 7L/min; atomizer pressure: 35psi; sheath temperature: 350 ℃; sheath air flow rate: 11L/min; capillary voltage: 3500V; the characteristic ions and corresponding mass spectral parameters of the 19 active ingredients are shown in table 2.
TABLE 2 characteristic ions of 19 active ingredients and corresponding mass spectral parameters
Carrying out regression calculation by using the peak area (y) of each active ingredient chromatographic peak as an ordinate and the concentration (x) of each active ingredient as an abscissa and using a weighted least square method, wherein the weight coefficient is 1/x, and establishing a standard curve of 19 active ingredients to obtain a linear equation and a correlation coefficient r of each active ingredient; after gradually diluting the mixed control solution of the standard 9, the concentration of each control was used as the minimum quantitative limit (LLOQ) of each active ingredient when the signal-noise ratio (S/N) =10, and the results are shown in table 3; the MRM diagram of the mixed reference substance solution of the mark 8 is shown in FIG. 1A, and the 19 active ingredients are not interfered with each other, so that the symmetry of chromatographic peaks of the active ingredients is good, and the separation degree is high.
TABLE 3 standard curves for 19 active ingredients
(2) Obtaining chromatographic peak areas of each active ingredient in the test sample solution:
crushing the large Chinese honeylocust fruits of the batch S1 in the table 1 (sieving with a 60-mesh sieve with the inner diameter of a sieve mesh of 0.3 mm), precisely weighing 100.0mg of crushed large Chinese honeylocust fruits, placing the crushed large Chinese honeylocust fruits in a 10mL volumetric flask, adding 70vol% methanol to fix the volume to a scale, performing ultrasonic extraction for 40 minutes, extracting power of 180W, extracting frequency of 40kHz, taking out the obtained product after ultrasonic extraction, putting the obtained product to room temperature, supplementing 70vol% methanol for weight loss, taking supernatant, filtering the supernatant with a 0.22 mu m organic microporous filter membrane, and storing the filtrate serving as a sample solution in a refrigerator at 4 ℃ for later use. Under the same chromatographic conditions and mass spectrum conditions as in the step (1), 2 mu L of the sample solution is taken and injected into an ultra-high performance liquid chromatograph, and the chromatographic peak of each active ingredient is determined according to the characteristic ion of each active ingredient through mass spectrum detection, and the chromatographic peak area of each active ingredient is obtained;
(3) Determining the content of each active ingredient in the test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is calculated from the chromatographic peak area of each active ingredient in the test solution 1 According to the formula c=c 1 ×V 2 /M＝C 1 The content C of each active ingredient in the sample solution of the batch S1 was calculated as X10 mL/100mg, and the results of the content of 19 active ingredients in the sample solution of the batch S1 were obtained, as shown in tables 4.1 to 4.2, based on 1 ng/mg=1. Mu.g/g, the unit of the content of each active ingredient in the fructus Gleditsiae Abnormalis was expressed in. Mu.g/g.
Examples 2 to 12
The results are shown in tables 4.1 to 4.2, except that the large saponins of the batches S2 to S12 in table 1 were taken to replace the large saponins of the batch S1 in example 1, respectively, and the contents of 19 active ingredients in the large saponins of the batches S2 to S12 were obtained, respectively, as shown in fig. 1B as an MRM chart of 19 active ingredients in the large saponins of the batch S10.
TABLE 4.1 content of 19 active ingredients in different batches of Gleditsia sinensis (one) (μg/g)
TABLE 4.2 content of 19 active ingredients in different batches of Gleditsia sinensis (two) (μg/g)
Methodological verification
Precision test
Within-day precision: precisely weighing 100.0mg of the large saponin powder of the batch S12, preparing a sample solution according to the method of the example 1, carrying out sample injection analysis according to the chromatographic conditions and the mass spectrum conditions of the example 1, repeating sample injection for 6 times, and recording the peak area of each active ingredient; the average value and the Relative Standard Deviation (RSD) value of the peak areas of the 19 active ingredients were calculated, respectively, and the results are shown in table 5. As can be seen from Table 5, the peak areas of the 19 active ingredients have RSD values ranging from 0.4% to 6%, and the result shows that the method has good daily precision.
Table 5 results of within-day precision test of 19 active ingredients (n=6)
Daytime precision: precisely weighing 100.0mg of the large saponin powder of the batch S12, preparing a sample solution according to the method of the example 1, carrying out sample injection analysis according to the chromatographic conditions and the mass spectrum conditions of the example 1, repeating sample injection for 2 times, continuously carrying out sample injection for 3 days, and recording the peak area of each active ingredient; the average value of peak areas of 19 active ingredients and RSD value thereof were calculated, respectively, and the results are shown in table 6. As can be seen from Table 6, the RSD values of the peak areas of the 19 active ingredients are between 0.5% and 5.8%, and the result shows that the daytime precision of the method is good.
Table 6 results of daytime precision test of 19 active ingredients (n=6)
Repeatability test
Precisely weighing 6 parts of fructus Gleditsiae Abnormalis of batch S12, each 100.0mg, preparing sample solution according to the method of example 1, analyzing by sample injection according to the chromatographic condition and mass spectrometry condition of example 1, and recording peak area of each active ingredient; the concentrations of 19 active ingredients were calculated from the standard curves of the respective active ingredients in example 1, and the average value and RSD value of the concentrations of the respective active ingredients were calculated, and the results are shown in table 7. As can be seen from Table 7, the RSD values of the peak areas of the 19 active ingredients are between 2.0% and 6.6%, and the result shows that the method of the application has better repeatability.
TABLE 7 repeatability test results of 19 active ingredients (n=6, ng/mL)
Stability test
Precisely weighing 100.0mg of the large saponin powder of the batch S12, preparing a sample solution according to the method of the example 1, performing sample injection analysis at 0, 2, 4, 8, 12 and 24 hours according to the chromatographic conditions and the mass spectrum conditions of the example 1, and recording the peak areas of the active components; the average value of peak areas and RSD values of the 19 active ingredients were calculated, respectively, and the results are shown in table 8. As can be seen from Table 8, the RSD values of the peak areas of the 19 active ingredients are between 0.6% and 5.6%, and the results show that the test sample solution has good stability under the condition of being placed at room temperature for 24 hours.
Table 8 results of stability test of 19 active ingredients (n=6)
Sample recovery test
Precisely weighing 6 parts of the large Chinese honeylocust fruit powder in precisely weighed batch S12, and preparing each reference substance solution of 19 active ingredients with the concentration of each active ingredient of 50.0 mg: l-malic acid 1mg/mL, luteolin, cryptochlorogenic acid, neochlorogenic acid, isovitexin, orientin, isoorientin 100 μg/mL, protocatechuic aldehyde, protocatechuic acid, eriodictyol, xanthosine, chlorogenic acid 5 μg/mL, caffeic acid, scopoletin, glycyrrhizin, apigenin, bergamot, vitexin, quercetin 1 μg/mL, and taking each control solution: 56 mu L of L-malic acid, 26 mu L of luteolin, 50 mu L of cryptochlorogenic acid, 150 mu L of neochlorogenic acid, 105 mu L of isovitexin, 27 mu L of orientin, 17 mu L of isoorientin, 24 mu L of protocatechuic acid, 24 mu L of eriodictyol, 44 mu L of yellow lignin, 56 mu L of chlorogenic acid, 100 mu L of caffeic acid, 110 mu L of scopoletin, 27 mu L of glycyrrhizin, 130 mu L of apigenin, 80 mu L of bergamot, 50 mu L of vitexin and 60 mu L of quercetin are added to samples to be tested, the addition values of the active ingredients are shown in table 9, 70vol% methanol is used for constant volume to 10mL, sample solutions of the samples to be marked are prepared according to the method of example 1, and peak areas of the active ingredients are recorded by sample injection analysis according to the chromatographic conditions and mass spectrum conditions of example 1; the concentrations of 19 active ingredients in each of the labeled samples were calculated according to the standard curves of the active ingredients in example 1, respectively, to obtain the contents of 19 active ingredients in each of the labeled samples, the sample recovery rate of each of the labeled samples was calculated according to the sample recovery rate (%) = (measured value-original value)/added value×100%, and the average recovery rate and RSD value of the sample recovery rate of each of the active ingredients were calculated, and the results are shown in table 9. The results in Table 9 show that the average recovery rate of 19 active ingredients is between 88.7% and 112.9%, the RSD value is between 0.8% and 6.7%, and the results show that the method has good accuracy.
Table 9 results of sample recovery of 19 active ingredients (n=6)
In summary, the method for measuring the content of the active ingredient in the fructus gleditsiae provided by the embodiment of the application adopts a UPLC-MS/MS method, and simultaneously measures the content of L-malic acid, protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, glycyrrhizin, apigenin, luteolin, eriodictyol, bergamot, yellow lignin, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercitrin, orientin and isoorientin in the fructus gleditsiae, and the method is simple to operate, high in sensitivity, high in accuracy and high in analysis speed, and can provide a basis for quality evaluation of the fructus gleditsiae and research on the drug effect substance basis.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the present application. Any modifications, equivalent substitutions, improvements, etc. that are within the spirit and principles of the present application are intended to be included within the scope of the present application.
Claims (9)
1. A method for measuring the content of active ingredients in fructus gleditsiae is characterized in that ultra-high performance liquid chromatography-mass spectrometry is adopted for combination to measure the content of active ingredients in fructus gleditsiae; the active ingredients comprise L-malic acid, protocatechuic acid, caffeic acid, scopoletin, glycyrrhizin, apigenin, luteolin, eriodictyol, bergamot, yellow pigment, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin; the method comprises the following steps:
(1) Establishing standard curves of the active ingredients;
preparing 5-12 mixed reference substance solutions containing active ingredients with different known concentrations by taking 80-100vol% methanol as a solvent; wherein the concentration of the L-malic acid is 40-55000ng/mL; the concentration of the chlorogenic acid is 2-20000ng/mL; the concentration of the chlorogenic acid is 3-10000ng/mL; the concentration of isovitexin is 1-10000ng/mL; the concentration of the chlorogenic acid is 1.5-5000ng/mL; eriodictyol concentration is 0.5-5000ng/mL; the concentration of luteolin is 1-5000ng/mL; the concentration of orientin is 2-3000ng/mL; the concentration of isoorientin is 2-3000ng/mL; the concentration of vitexin is 0.4-1000ng/mL; the concentrations of protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin and flavodoxin are respectively 0.5-500ng/mL, and the concentrations of bergamot and quercetin are respectively 0.1-500ng/mL; the concentration of the glycyrrhizin is 0.25-300ng/mL;
under the same chromatographic conditions and mass spectrum conditions, the volume is V 1 The mixed reference substance solutions of (2) are respectively injected into an ultra-high performance liquid chromatograph, detected by mass spectrum and according to the activitiesThe characteristic ion of the component determines the chromatographic peak of each active component and obtains the chromatographic peak area of each active component;
establishing standard curves of the active ingredients respectively by taking the peak area of the chromatographic peak of each active ingredient as an ordinate and the concentration of each active ingredient as an abscissa;
wherein the chromatographic conditions include: chromatographic column: octadecylsilane chemically bonded silica chromatographic column; mobile phase: the phase A is formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the phase B is methanol; gradient elution is carried out by adopting 5-90% of phase A and 10-95% of phase B in volume fraction; the gradient elution specifically comprises the following steps: 0 to 1.0 min, 10 to 25 percent of phase B;1.0 to 5.0 minutes, 25 to 31 percent of phase B;5.0 to 7.5 minutes, 31 to 31 percent of phase B,7.5 to 8.0 minutes, and 31 to 50 percent of phase B;8.0 to 9.0 minutes, 50 to 95 percent of phase B;9.0 to 11.0 minutes, 95 to 95 percent of phase B;
(2) Obtaining chromatographic peak areas of each active ingredient in the sample solution;
taking a sample with mass M and volume V 2 Extracting with 60-80vol% methanol as solvent, collecting supernatant, filtering to obtain sample solution, wherein M/V 2 1mg/mL for (8-12);
taking the volume V under the same chromatographic conditions and mass spectrum conditions as in the step (1) 1 The sample solution is injected into an ultra-high performance liquid chromatograph, and the chromatographic peak of each active ingredient is determined according to the characteristic ion of each active ingredient through mass spectrum detection, and the chromatographic peak area of each active ingredient is obtained;
(3) Determining the content of each active ingredient in the test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is calculated from the chromatographic peak area of each active ingredient in the test solution 1 According to the formula c=c 1 ×V 2 And (3) respectively calculating the content C of each active ingredient in the test sample.
2. The method of claim 1, wherein the concentration of L-malic acid in the mixed control solution is 62.5-50000ng/mL and the concentration of neochlorogenic acid is 20-16000ng/mL; the concentration of the cryptochlorogenic acid and the isovitexin is respectively 10-8000ng/mL; the concentration of chlorogenic acid, eriodictyol and luteolin is 5-4000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2.5-2000ng/mL; vitexin is 1-800ng/mL; the concentrations of protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, bergamot, flavodoxin and quercetin are respectively 0.5-400ng/mL; the concentration of the glycyrrhizin is 0.25-200ng/mL.
3. The method according to claim 1, wherein in the step (1), 80-100vol% of methanol is used as a solvent to prepare a mixed reference stock solution containing each active ingredient, wherein the concentration of L-malic acid in the mixed reference stock solution is 50000-55000ng/mL and the concentration of neochlorogenic acid in the mixed reference stock solution is 16000-20000ng/mL; the concentration of the cryptochlorogenic acid and the isovitexin is 8000-10000ng/mL respectively; the concentration of chlorogenic acid, eriodictyol and luteolin is 4000-5000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2000-3000ng/mL; vitexin is 800-1000ng/mL; the concentrations of protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, bergamot, flavodoxin and quercetin are respectively 400-500ng/mL; the concentration of the glycyrrhizin is 200-300ng/mL;
diluting the mixed reference substance stock solution by taking 80-100vol% methanol as a solvent to obtain 5-12 mixed reference substance solutions containing active ingredients with different known concentrations.
4. The method according to claim 1, wherein in the step (2), the extraction is ultrasonic extraction with an extraction power of 100-300W, an extraction frequency of 30-50kHz, and an extraction time of 20-60 minutes.
5. The method of any one of claims 1-4, wherein the chromatographic conditions further comprise:
column temperature is 20-30 ℃; the flow rate is 0.1-0.5 mL/min; sample injection volume V 1 ：1-5μL。
6. According to the weightsThe method of claim 5, wherein the chromatographic conditions comprise: mobile phase: the phase A is formic acid aqueous solution with the volume fraction of 0.08-0.12%, and the phase B is methanol; column temperature: 22-28 ℃; flow rate: 0.2-0.4 mL/min; sample injection volume V 1 ：1-3μL。
7. The method of any one of claims 1-4, wherein the mass spectrum is a triple quadrupole mass spectrum, the mass spectrum conditions comprising: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring and anion scanning mode; drying gas temperature: 250-350 ℃; drying gas flow rate: 5-10L/min; atomizer pressure: 30-40psi; sheath temperature: 300-400 ℃; sheath air flow rate: 8-15L/min; capillary voltage: 3000-4000V.
8. The method of claim 7, wherein the mass spectrometry conditions comprise: drying gas temperature: 280-320 ℃; drying gas flow rate: 6-8L/min; atomizer pressure: 32-38psi; sheath temperature: 330-370 ℃; sheath air flow rate: 10-12L/min; capillary voltage: 3300-3700V.
9. The method according to any one of claims 1 to 4, wherein the characteristic ions of each active ingredient comprise:
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