CN114674958A - Method for measuring content of active ingredients in large soap horns - Google Patents
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Abstract
The application provides a method for determining the content of active ingredients in Chinese honeylocust fruit, which is used for rapidly detecting the content of L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, liquiritigenin, apigenin, luteolin, eriodictyol, vanilloid, fustin, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin in the Chinese honeylocust fruit by adopting ultra-high performance liquid chromatography-mass spectrometry. 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 Chinese honeylocust fruits.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicine component determination, in particular to a method for determining the content of active components in Chinese honeylocust fruit.
Background
Gleditsia sinensis (Gleditsia sinensis fruits), also known as Gleditsia sinensis Lam and Gleditsia sinensis (Gleditsia sinensis Lam.) Kuck, is a plant belonging to Gleditsia of subfamily Palmae of Leguminosae, and is one of the unique native tree species of China. Is distributed in most areas of the country, has rich resources, has the effects of dispelling wind and phlegm and removing dampness and toxicity, can reduce the toxic and side effects after being processed, and is commonly used for clinical medication. Gleditsia sinensis Lam has great medicinal value, but the quality of medicinal materials in different producing areas is uneven, and it is difficult to evaluate the quality. Therefore, a high-efficiency, rapid and high-sensitivity detection method is needed to measure the content of active ingredients in the Chinese honeylocust fruit and provide a basis for quality evaluation of the Chinese honeylocust fruit and research on the basis of medicinal substances.
Disclosure of Invention
The purpose of the present application is to provide a method for measuring the content of an active ingredient in Gleditsia sinensis Lam, which can simultaneously measure the content of 19 active ingredients in Gleditsia sinensis Lam and can be used for quality control of Gleditsia sinensis Lam. The specific technical scheme is as follows:
the application provides a method for measuring the content of active ingredients in gleditsia sinensis lam, which is characterized in that ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) is adopted to measure the content of the active ingredients in the gleditsia sinensis lam; the active ingredients comprise L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, liquiritigenin, apigenin, luteolin, eriodictyol, bergamonin, fustin, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin; the method comprises the following steps:
(1) establishing a standard curve of each active ingredient;
preparing 5-12 mixed reference substance solutions containing active ingredients with different known concentrations by taking 80-100 vol% methanol as a solvent; wherein the concentration of the L-malic acid is 40-55000 ng/mL; the concentration of the new chlorogenic acid is 2-20000 ng/mL; the concentration of the cryptochlorogenic acid is 3-10000 ng/mL; the concentration of isovitexin is 1-10000 ng/mL; the concentration of chlorogenic acid is 1.5-5000 ng/mL; the concentration of eriodictyol is 0.5-5000 ng/mL; the concentration of luteolin is 1-5000 ng/mL; the concentration of orientin is 2-3000 ng/mL; the concentration of isoorientin is 2-3000 ng/mL; the concentration of vitexin is 0.4-1000 ng/mL; protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, and fustin with concentrations of 0.5-500ng/mL respectively, and aurantiol and quercetin with concentrations of 0.1-500ng/mL respectively; the concentration of liquiritigenin is 0.25-300 ng/mL;
under the same chromatographic condition and mass spectrum condition, the volume is V1Respectively injecting the mixed reference substance solution into an ultra-high performance liquid chromatograph, determining the chromatographic peak of each active component according to the characteristic ions of each active component through mass spectrum detection, and obtaining the chromatographic peak area of each active component;
respectively establishing a standard curve of each active ingredient by taking the peak area of the chromatographic peak of each active ingredient as a vertical coordinate and the concentration of each active ingredient as a horizontal coordinate;
(2) obtaining the chromatographic peak area of each active component in the test solution;
taking a sample with mass M and volume V2Extracting with 60-80 vol% methanol, collecting supernatant, filtering to obtain sample solution, wherein M/V21mg/mL for (8-12);
taking the volume V under the same chromatographic conditions and mass spectrum conditions as in the step (1)1Injecting the test solution into an ultra-high performance liquid chromatograph, detecting by mass spectrometry, determining chromatographic peaks of the active ingredients according to characteristic ions of the active ingredients, and obtaining chromatographic peak areas of the active ingredients;
(3) determining the content of each active ingredient in a test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is respectively calculated according to the chromatographic peak area of each active ingredient in the test solution1According to the formula C ═ C1×V2and/M respectively calculating the content C of each active component in the test solution.
The application provides a survey method of active ingredient content in big soap stone, adopts UPLC-MS/MS, through rationally selecting chromatogram condition and mass spectrum condition, can survey 19 active ingredient's in big soap stone content simultaneously, just the method has advantages such as easy operation, sensitivity height, degree of accuracy height, analysis speed are fast, the specificity is strong, can be used to the quality control of big soap stone, provides the basis for the quality evaluation of big soap stone and the research of pharmacodynamic material basis.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and it is also obvious for a person skilled in the art to obtain other embodiments according to the drawings.
FIG. 1A is a multiple reactive ion monitor (MRM) chart of the mixed control solution of 8-note in example 1;
FIG. 1B is a MRM chart of 19 active ingredients in batch S10 of Gleditsia sinensis Lam;
in the drawings, the numerical references respectively represent: l-malic acid; 2. protocatechualdehyde; 3. protocatechuic acid; 4. caffeic acid; 5. scopoletin lactone; 6. liquiritigenin; 7. apigenin; 8. luteolin; 9. eriodictyol; 10. a fragrant citrus fruit essence; 11. fustin; 12. cryptochlorogenic acid; 13. chlorogenic acid; 14. chlorogenic acid; 15. vitexin; 16. isovitexin; 17. quercetin; 18. orientin; 19. isoorientin.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the description herein are intended to be within the scope of the present disclosure.
The application provides a method for measuring the content of active ingredients in Chinese honeylocust, which is characterized in that UPLC-MS/MS is adopted to measure the content of the active ingredients in the Chinese honeylocust; the active ingredients comprise L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, liquiritigenin, apigenin, luteolin, eriodictyol, bergamonin, fustin, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin; the method comprises the following steps:
(1) establishing a standard curve of each active ingredient;
preparing 5-12 mixed reference substance solutions containing active ingredients with different known concentrations by taking 80-100 vol% methanol as a solvent; wherein the concentration of the L-malic acid is 40-55000 ng/mL; the concentration of the neochlorogenic acid is 2-20000 ng/mL; the concentration of the cryptochlorogenic acid is 3-10000 ng/mL; the concentration of isovitexin is 1-10000 ng/mL; the concentration of chlorogenic acid is 1.5-5000 ng/mL; the concentration of eriodictyol is 0.5-5000 ng/mL; the concentration of luteolin is 1-5000 ng/mL; the concentration of orientin is 2-3000 ng/mL; the concentration of isoorientin is 2-3000 ng/mL; the concentration of vitexin is 0.4-1000 ng/mL; protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, and fustin with concentrations of 0.5-500ng/mL respectively, and aurantiol and quercetin with concentrations of 0.1-500ng/mL respectively; the concentration of liquiritigenin is 0.25-300 ng/mL;
under the same chromatographic condition and mass spectrum condition, the volume is V1Respectively injecting the mixed reference substance solution into an ultra-high performance liquid chromatograph, determining the chromatographic peak of each active component according to the characteristic ions of each active component through mass spectrum detection, and obtaining the chromatographic peak area of each active component;
respectively establishing a standard curve of each active ingredient by taking the peak area of the chromatographic peak of each active ingredient as a vertical coordinate and the concentration of each active ingredient as a horizontal coordinate;
(2) obtaining the chromatographic peak area of each active component in the test solution;
taking a sample with mass M and volume V2Extracting with 60-80 vol% methanol, collecting supernatant, filtering to obtain sample solution, wherein M/V21mg/mL for (8-12);
taking the volume V under the same chromatographic conditions and mass spectrum conditions as in the step (1)1Injecting the test solution into an ultra-high performance liquid chromatograph, detecting by mass spectrometry, determining chromatographic peaks of the active ingredients according to characteristic ions of the active ingredients, and obtaining chromatographic peak areas of the active ingredients;
(3) determining the content of each active ingredient in a test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is respectively calculated according to the peak area of the chromatographic peak of each active ingredient in the test solution1According to the formula C ═ C1×V2and/M respectively calculating the content C of each active component in the test solution.
According to the method for measuring the content of the 19 active ingredients in the saponin, UPLC-MS/MS is adopted, and the content of the 19 active ingredients in the saponin 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 is 62.5-50000ng/mL and the concentration of neochlorogenic acid is 20-16000ng/mL in each of the mixed control solutions; the concentrations of cryptochlorogenic acid and isovitexin are respectively 10-8000 ng/mL; the concentrations of chlorogenic acid, eriodictyol and luteolin are 5-4000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2.5-2000 ng/mL; vitexin is 1-800 ng/mL; the concentrations of protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, fructus Citri Junoris essence, fustin and quercetin are 0.5-400ng/mL respectively; the concentration of liquiritigenin is 0.25-200 ng/mL.
The preparation method of the mixed control solution is not particularly limited in the present application as long as the object of the present application can be achieved, and for example, a mixed control stock solution may be prepared in which the concentration of each component is equal to or higher than the concentration of each active component in the mixed control solution, and then the mixed control solution may be obtained by dilution.
In some embodiments of the present application, in step (1), 80-100 vol% methanol is used as a solvent to prepare a mixed control stock solution containing each active ingredient, 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 16000-20000 ng/mL; the concentrations of the cryptochlorogenic acid and the isovitexin are 8000-10000ng/mL respectively; the concentrations of chlorogenic acid, eriodictyol and luteolin are 4000 and 5000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2000ng/mL and 3000 ng/mL; vitexin is 800-1000 ng/mL; the concentrations of protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, aurantiol, fustin and quercitrin are respectively 400-500 ng/mL; the concentration of the liquiritigenin is 200-300 ng/mL;
and diluting the mixed reference substance stock solution by using 80-100 vol% methanol as a solvent to obtain the 5-12 mixed reference substance solutions containing the 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 may 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, and 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 reference solution used to establish the standard curve may comprise the mixed reference stock solution; the preparation of the stock solution of the mixed control is not particularly limited as long as the object of the present application can be achieved, and the stock solution of each active ingredient may be prepared, for example, by preparing the stock solution of each active ingredient separately and then taking the stock solutions of each active ingredient separately.
In some embodiments of the present application, in step (2), the extraction is ultrasonic extraction, the extraction power is 100-300W, the extraction frequency is 30-50kHz, and the extraction time is 20-60 minutes.
By adopting the preparation method of the test solution, the test solution containing 19 active ingredients is obtained, so that the detection result of the content of the active ingredients in the Chinese honeylocust is more comprehensive, accurate and reliable; wherein, a test solution containing 19 active ingredients is obtained by ultrasonic extraction and is used for measuring the content of each active ingredient, so that the detection result of the content of each active ingredient is more accurate.
In some embodiments of the present application, the chromatographic conditions comprise: a 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 column temperature is 20-30 ℃; the flow rate is 0.1-0.5 mL/min; sample introduction volume V1：1-5μL。
Preferably, 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: 20-28 ℃; flow rate: 0.2-0.4 mL/min; sample introduction volume V1：1-3μL。
The inventors have found in their studies that better separation of 19 active ingredients from the Gleditsia sinensis is achieved with the gradient elution of the present application, preferably, in some embodiments of the present application, in particular: 10 to 25 percent of phase B in 0 to 1.0 minute; 1.0-5.0 min, 25-31% of phase B; 5.0-7.5 minutes, 31-31% of phase B, 7.5-8.0 minutes, 31-50% of phase B; 8.0-9.0 minutes, 50% -95% 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, a 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 result 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 a multi-reaction ion monitoring and negative ion scanning mode; temperature of the drying gas: 250 ℃ to 350 ℃; flow rate of drying gas: 5-10L/min; atomizer pressure: 30-40 psi; temperature of sheath gas: 300 ℃ and 400 ℃; flow rate of sheath gas: 8-15L/min; capillary voltage: 3000-4000V.
Preferably, the mass spectrometry conditions comprise: temperature of the drying gas: 280 ℃ and 320 ℃; flow rate of drying gas: 6-8L/min; atomizer pressure: 32-38 psi; temperature of sheath gas: 330 ℃ to 370 ℃; flow rate of sheath gas: 10-12L/min; capillary voltage: 3300-.
In some embodiments of the present application, the characteristic ions of each active ingredient include:
the instruments and reagents required for this application are described below.
Instrument for measuring the position of a moving object
Agilent 6470 triple quadrupole mass spectrometer (Agilent corporation, usa); agilent 1290 ultra high performance liquid chromatograph (Agilent corporation, usa); agilent MassHunter analysis software (Agilent Corp., USA); a one hundred thousand balance model R2 (Radwag, polish, inc.) a 60/220; Milli-Q IQ 7005 ultrapure water preparation instrument (Millipore Corp.); model 5453 high speed centrifuge (Eppendorf, Germany); vortex-2 Vortex mixer (Shanghai province Co., Ltd.); ZZ-L6DT ultrasonic cleaning tank (Technology Co., Ltd. of Tianjin).
Reagent
L-malic acid (batch number: DST201124-038), protocatechualdehyde (batch number: DST200628-080), protocatechuic acid (batch number: DSTDY008101), caffeic acid (batch number: DST191030-013), scopoletin (batch number: DSTDD006401), glycyrrhizin (batch number: DST200326-010), apigenin (batch number: DST200809-026), luteolin (batch number: DST200910-032), eriodictyol (batch number: DSTDS004201), and coumarine (batch number: DSTDX009401), fustin (batch: DST201019-147), cryptochlorogenic acid (batch: DSTDY003501), neochlorogenic acid (batch: DSTDX001501), chlorogenic acid (batch: DSTDL002101), vitexin (batch: DST200325-034), isovitexin (batch: DST200519-054), quercetin (batch: DST180506-006), orientin (batch: DSTDH004901) and isoorientin (batch: DST200815-121) are all purchased from Doudsite biotechnology Limited.
Methanol (chromatographically pure) was obtained from Fisher, usa and formic acid (chromatographically pure) from ROE.
Material
The Gleditsia sinensis Lam is obtained from Hebei, Anhui, Henan, Shanxi, Shandong, Yunnan, Guizhou, etc. The source of the medicinal materials is shown in table 1.
TABLE 1 sources of different batches of Gleditsia sinensis
The reagents and medicinal materials mentioned in the following examples can be obtained commercially or according to methods known in the art, unless otherwise specified.
Example 1
(1) Establishing a standard curve of each active ingredient
Precisely weighing L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, liquiritigenin, apigenin, luteolin, eriodictyol, citronellin, fustin, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin reference substances, dissolving with methanol respectively to obtain respective reference stock solutions with concentration of 1mg/mL, and storing in a refrigerator at 4 deg.C for use.
Accurately measuring appropriate amount of each reference substance stock solution, preparing into mixed reference substance stock solution with methanol, and marking as mark 1, wherein the concentration of L-malic acid is 50000ng/mL, the concentration of neochlorogenic acid is 16000ng/mL, the concentrations of cryptochlorogenic acid and isovitexin are 8000ng/mL, the concentrations of chlorogenic acid, eriodictyol and luteolin are 4000ng/mL, the concentrations of orientin, isoorientin are 2000ng/mL, vitexin is 800ng/mL, protocatechuic aldehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, bergamonin, xanthophyllin and quercitrin are 400ng/mL, and the concentration of liquiritigenin is 200 ng/mL; diluting the mixed reference stock solution with methanol for 2, 2.5, 2, 4, and 2 times in sequence to obtain 9 mixed reference solutions containing active ingredients with different known concentrations, including the mixed reference stock solution, and sequentially marking as mark 1-mark 9.
Under the same chromatographic condition and mass spectrum condition, respectively injecting 2 μ L of each mixed reference solution into an ultra high performance liquid chromatograph, determining the chromatographic peak of each active component according to the characteristic ions of each active component through mass spectrum detection, and obtaining the chromatographic peak area of each active component;
wherein the chromatographic conditions comprise: column ACQUITY UPLC BEH C18 column (2.1X 100mm,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: 0-1.0 minute, 10% -25% B; 1.0-5.0 minutes, 25% -31% B; 5.0-7.5 minutes, 31-31% of B, 7.5-8.0 minutes, 31-50% of B; 8.0-9.0 minutes, 50% -95% B; 9.0-11.0 minutes, 95% -95% of B, and the flow rate is 0.3 mL/minute; column temperature: 25 ℃; sample volume V1：2μL。
The mass spectrum is a triple quadrupole mass spectrum, and the mass spectrum conditions comprise: the ion source is an electrospray ion source; the detection mode is a multi-reaction ion monitoring and negative ion scanning mode; temperature of the drying gas: 300 ℃; flow rate of drying gas: 7L/min; atomizer pressure: 35 psi; temperature of sheath gas: 350 ℃; flow rate of sheath gas: 11L/min; capillary voltage: 3500V; the characteristic ions of the 19 active ingredients and the corresponding mass spectral parameters are shown in table 2.
TABLE 219 characteristic ions of active ingredients and corresponding Mass Spectrometry parameters
Taking the peak area (y) of each active ingredient chromatographic peak as a vertical coordinate, taking the concentration (x) of each active ingredient as a horizontal coordinate, performing regression calculation by using a weighted least square method, wherein the weight coefficient is 1/x, and establishing a 19-active-ingredient standard curve to obtain a linear equation and a correlation coefficient r of each active ingredient; gradually diluting the mixed reference solution of 9, and taking the concentration of each reference as the lowest limit of quantitation (LLOQ) of each active ingredient when the signal-to-noise ratio (S/N) is 10, respectively, and the results are shown in table 3; in the MRM chart of the 8 st mixed control solution shown in fig. 1A, 19 active ingredients were not interfered with each other, and the active ingredients had good symmetry of the chromatographic peak and high resolution.
TABLE 319 standard curve of active ingredients
(2) Obtaining the chromatographic peak area of each active component in the test solution:
crushing the large soap horns of the batch S1 in the table 1 (sieving with a 60-mesh sieve, and the inner diameter of a sieve pore is 0.3mm), precisely weighing 100.0mg of the crushed large soap horns powder, placing the crushed large soap horns powder into a 10mL volumetric flask, adding 70 vol% methanol to a constant volume to a scale, ultrasonically extracting for 40 minutes, wherein the extraction power is 180W, the extraction frequency is 40kHz, ultrasonically extracting, taking out, placing to room temperature, complementing the 70 vol% methanol to lose weight, taking supernatant, filtering with a 0.22-micron organic microporous filter membrane, and storing the filtrate as a sample solution in a refrigerator at 4 ℃ for later use. Injecting 2 mu L of sample solution into an ultra high performance liquid chromatograph under the same chromatographic condition and mass spectrum condition as in the step (1), determining the chromatographic peak of each active ingredient according to the characteristic ions of each active ingredient through mass spectrum detection, and obtaining the chromatographic peak area of each active ingredient;
(3) determining the content of each active ingredient in a test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is respectively calculated according to the chromatographic peak area of each active ingredient in the test solution1According to the formula C ═ C1×V2/M＝C1The content C of each active ingredient in the test solution of lot S1 was calculated by x 10mL/100mg to obtain the content results of 19 active ingredients in the test solution of lot S1, and the unit of the content of each active ingredient in macrosaponin is expressed in μ g/g based on 1ng/mg to 1 μ g/g, as shown in tables 4.1 to 4.2.
Examples 2 to 12
The contents of 19 active ingredients in the large gleditsia sinensis lam of the batches S2 to S12 were obtained in the same manner as in example 1 except that the large soap horns of the batches S2 to S12 in table 1 were respectively used instead of the large soap horn of the batch S1 in example 1, and the results are shown in tables 4.1 to 4.2, in which MRM patterns of 19 active ingredients in the large gleditsia sinensis lam of the batch S10 are shown in fig. 1B.
TABLE 4.1 content of 19 active ingredients in different batches of Gleditsia sinensis Lam (. mu.g/g)
TABLE 4.2 content of 19 active ingredients in different batches of Gleditsia sinensis (II) (μ g/g)
Methodology validation
Precision test
The precision in the day is as follows: precisely weighing 100.0mg of large soap powder of S12 batch, preparing a sample solution according to the method of example 1, performing sample injection analysis according to the chromatographic condition and mass spectrum condition of example 1, repeating sample injection for 6 times, and recording the peak area of each active component; the average and Relative Standard Deviation (RSD) values 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 RSD value of 19 active ingredient peak area ranged from 0.4% to 6%, and the results indicate that the method of the present application is accurate within the day.
TABLE 519 active ingredient in-day precision test results (n ═ 6)
Precision in the daytime: precisely weighing 100.0mg of large soap powder of S12 batch, preparing a sample solution according to the method of example 1, carrying out sample injection analysis according to the chromatographic condition and mass spectrum condition of example 1, repeatedly carrying out sample injection for 2 times, carrying out continuous sample injection for 3 days, and recording the peak area of each active component; the average of the peak areas of 19 active ingredients and their RSD values were calculated, respectively, and the results are shown in Table 6. As can be seen from Table 6, the RSD value of 19 active ingredient peak area is between 0.5% and 5.8%, and the results show that the method of the present application has good precision in the daytime.
TABLE 619 results of day precision test of active ingredients (n ═ 6)
Repeatability test
Precisely weighing 6 parts of large soap horns of the batch S12, each part being 100.0mg, preparing a test solution according to the method of the example 1, respectively carrying out sample injection analysis according to the chromatographic condition and the mass spectrum condition of the example 1, and recording the peak area of each active component; the concentrations of the active ingredients were calculated 19 times from the standard curve of each active ingredient in example 1, and the average value and RSD value of each active ingredient concentration were calculated, and the results are shown in table 7. As can be seen from Table 7, the RSD value of the 19 active ingredient peak area is between 2.0% and 6.6%, and the result shows that the method has good repeatability.
Table 719 repeatability tests results for active ingredients (n ═ 6, ng/mL)
Stability test
Precisely weighing 100.0mg of large soap powder of S12 batch, preparing a test solution according to the method of example 1, performing sample injection analysis at 0, 2, 4, 8, 12 and 24 hours respectively according to the chromatographic condition and mass spectrum condition of example 1, and recording the peak area of each active component; the average value of the peak area of 19 active ingredients and the RSD value were calculated, respectively, and the results are shown in Table 8. As can be seen from Table 8, the RSD value of 19 active ingredient peak area is between 0.6% and 5.6%, and the results show that the test solution of the present application has good stability under 24-hour standing at room temperature.
TABLE 819 active stability test results (n ═ 6)
Sample application recovery test
Precisely weighing 6 parts of the large soap powder of the batch S12, 50.0mg of each part, preparing each control solution of 19 active ingredients, wherein the concentrations of the active ingredients are as follows: 1mg/mL of L-malic acid, 100 mu g/mL of luteolin, cryptochlorogenic acid, neochlorogenic acid, isovitexin, orientin, isoorientin, 5 mu g/mL of protocatechualdehyde, protocatechuic acid, eriodictyol, fustin, chlorogenic acid, caffeic acid, scopoletin, liquiritigenin, apigenin, bergamonin, vitexin and quercitrin, 1 mu g/mL of each control solution is respectively taken: 56 μ L of L-malic acid, 26 μ L of luteolin, 50 μ L of cryptochlorogenic acid, 150 μ L of neochlorogenic acid, 105 μ L of isovitexin, 27 μ L of orientin, 17 μ L of isoorientin, 24 μ L of protocatechualdehyde, 24 μ L of protocatechuic acid, 64 μ L of eriodictyol, 44 μ L of fuscin, 56 μ L of chlorogenic acid, 100 μ L of caffeic acid, 110 μ L of scopoletin, 27 μ L of glycyrrhizin, 130 μ L of apigenin, 80 μ L of sinensetin, 50 μ L of vitexin, and 60 μ L of quercetin were added to the sample to be tested, the added values of the respective active ingredients are shown in table 9, the volume is fixed to 10mL with 70 vol% methanol, the sample solutions for each of the added samples were prepared by the method of example 1, the chromatographic conditions and mass spectrometric conditions were respectively analyzed, and the peak areas of the respective active ingredients were recorded; the concentration of 19 active ingredients in each sample was calculated from the calibration curve of each active ingredient in example 1 to obtain the content of 19 active ingredients in each sample, the sample recovery rate of each sample was calculated from the sample recovery rate (%) (measured value-original value)/addition value × 100%, and the average recovery rate and RSD value of the sample recovery rate of each active ingredient were calculated, and the results are shown in table 9. The results in table 9 show that the average recovery of 19 active principles is between 88.7% and 112.9% and the RSD value is between 0.8% and 6.7%, indicating that the method of the present application is accurate.
TABLE 919 recovery test results for active ingredient loading (n ═ 6)
In summary, the method for determining the content of the active ingredients in the big saponin provided by the embodiment of the application adopts the UPLC-MS/MS method to simultaneously determine the content of L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, glycyrrhizin, apigenin, luteolin, eriodictyol, citrin, fustin, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin in the big saponin.
The above description is only for the preferred embodiment of the present application and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application are included in the protection scope of the present application.
Claims (10)
1. A method for measuring the content of active ingredients in saponin is characterized in that the content of the active ingredients in saponin is measured by adopting ultra-high performance liquid chromatography-mass spectrometry; the active ingredients comprise L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, liquiritigenin, apigenin, luteolin, eriodictyol, bergamonin, fustin, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, vitexin, isovitexin, quercetin, orientin and isoorientin; the method comprises the following steps:
(1) establishing a standard curve of each active ingredient;
preparing 5-12 mixed reference substance solutions containing active ingredients with different known concentrations by taking 80-100 vol% methanol as a solvent; wherein the concentration of the L-malic acid is 40-55000 ng/mL; the concentration of the neochlorogenic acid is 2-20000 ng/mL; the concentration of the cryptochlorogenic acid is 3-10000 ng/mL; the concentration of isovitexin is 1-10000 ng/mL; the concentration of chlorogenic acid is 1.5-5000 ng/mL; the concentration of eriodictyol is 0.5-5000 ng/mL; the concentration of luteolin is 1-5000 ng/mL; the concentration of orientin is 2-3000 ng/mL; the concentration of isoorientin is 2-3000 ng/mL; the concentration of vitexin is 0.4-1000 ng/mL; protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, and fustin with concentrations of 0.5-500ng/mL respectively, and aurantiol and quercetin with concentrations of 0.1-500ng/mL respectively; the concentration of liquiritigenin is 0.25-300 ng/mL;
under the same chromatographic condition and mass spectrum condition, the volume is V1Respectively injecting the mixed reference substance solution into an ultra-high performance liquid chromatograph, determining the chromatographic peak of each active component according to the characteristic ions of each active component through mass spectrum detection, and obtaining the chromatographic peak area of each active component;
respectively establishing a standard curve of each active ingredient by taking the peak area of the chromatographic peak of each active ingredient as a vertical coordinate and the concentration of each active ingredient as a horizontal coordinate;
(2) obtaining the chromatographic peak area of each active component in the test solution;
taking a sample with mass M and volume V2Extracting with 60-80 vol% methanol, collecting supernatant, filtering to obtain sample solution, wherein M/V21mg/mL for (8-12);
taking the volume V under the same chromatographic conditions and mass spectrum conditions as in the step (1)1Injecting the test solution into an ultra-high performance liquid chromatograph, detecting by mass spectrometry, determining chromatographic peaks of the active ingredients according to characteristic ions of the active ingredients, and obtaining chromatographic peak areas of the active ingredients;
(3) determining the content of each active ingredient in a test sample;
according to the established standard curve of each active ingredient, the concentration C of each active ingredient in the test solution is respectively calculated according to the chromatographic peak area of each active ingredient in the test solution1According to the formula C ═ C1×V2and/M respectively calculating the content C of each active component in the test solution.
2. The method of claim 1, wherein the concentration of L-malic acid is 62.5-50000ng/mL and the concentration of neochlorogenic acid is 20-16000ng/mL in the mixed control solution; the concentrations of cryptochlorogenic acid and isovitexin are respectively 10-8000 ng/mL; the concentrations of chlorogenic acid, eriodictyol and luteolin are 5-4000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2.5-2000 ng/mL; vitexin is 1-800 ng/mL; the concentrations of protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, fructus Citri Junoris essence, fustin and quercetin are 0.5-400ng/mL respectively; the concentration of liquiritigenin is 0.25-200 ng/mL.
3. The method as claimed in claim 1, wherein in step (1), 80-100 vol% methanol is used as solvent to prepare a mixed control stock solution containing each active ingredient, 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 16000-20000 ng/mL; the concentrations of the cryptochlorogenic acid and the isovitexin are 8000-10000ng/mL respectively; the concentrations of chlorogenic acid, eriodictyol and luteolin are 4000 and 5000ng/mL respectively; the concentrations of orientin and isoorientin are respectively 2000-3000 ng/mL; vitexin is 800-1000 ng/mL; the concentrations of protocatechualdehyde, protocatechuic acid, caffeic acid, scopoletin, apigenin, aurantiol, fustin and quercitrin are respectively 400-500 ng/mL; the concentration of the liquiritigenin is 200-300 ng/mL;
and diluting the mixed reference substance stock solution by using 80-100 vol% methanol as a solvent to obtain the 5-12 mixed reference substance solutions containing the active ingredients with different known concentrations.
4. The method as claimed in claim 1, wherein in step (2), the extraction is ultrasonic extraction, the extraction power is 100-.
5. The method of any one of claims 1-4, wherein the chromatographic conditions comprise:
a 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 column temperature is 20-30 ℃; the flow rate is 0.1-0.5 mL/min; sample introduction volume V1：1-5μL。
6. The 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 introduction volume V1：1-3μL。
7. The method according to claim 5, the gradient elution being in particular: 10 to 25 percent of phase B in 0 to 1.0 minute; 1.0-5.0 min, 25-31% of phase B; 5.0-7.5 minutes, 31-31% of phase B, 7.5-8.0 minutes, 31-50% of phase B; 8.0-9.0 minutes, 50% -95% of phase B; 9.0 to 11.0 minutes, 95 to 95 percent of phase B.
8. The method of any one of claims 1-4, wherein the mass spectrometry is triple quadrupole mass spectrometry, and wherein the mass spectrometry conditions comprise: the ion source is an electrospray ion source; the detection mode is a multi-reaction ion monitoring and negative ion scanning mode; temperature of the drying gas: 250 ℃ to 350 ℃; flow rate of drying gas: 5-10L/min; atomizer pressure: 30-40 psi; temperature of sheath gas: 300 ℃ and 400 ℃; flow rate of sheath gas: 8-15L/min; capillary voltage: 3000-4000V.
9. The method of claim 8, wherein the mass spectrometry conditions comprise: temperature of the drying gas: 280 ℃ and 320 ℃; flow rate of drying gas: 6-8L/min; atomizer pressure: 32-38 psi; temperature of sheath gas: 330 ℃ to 370 ℃; flow rate of sheath gas: 10-12L/min; capillary voltage: 3300-.
10. The method according to any one of claims 1 to 4, wherein the characteristic ions of each active ingredient comprise:
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