CN114674953A - Method for measuring chemical component content in dark plum - Google Patents
Method for measuring chemical component content in dark plum Download PDFInfo
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- CN114674953A CN114674953A CN202210297928.5A CN202210297928A CN114674953A CN 114674953 A CN114674953 A CN 114674953A CN 202210297928 A CN202210297928 A CN 202210297928A CN 114674953 A CN114674953 A CN 114674953A
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Abstract
The application provides a method for measuring the content of chemical components in dark plum, which adopts ultra-high performance liquid chromatography-mass spectrometry to measure the content of 16 chemical components in dark plum; the chemical components comprise: succinic acid, L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, D-quininic acid, citric acid, ferulic acid, syringic acid, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, amygdalin, crataegolic acid, corosolic acid and rutin. By adopting the method, the contents of 16 chemical components in the dark plum can be measured by reasonably selecting chromatographic conditions and mass spectrum conditions, and the method has the advantages of simplicity, convenience, high sensitivity, high analysis speed, strong specificity and the like, so that the method can be used for quality control of the dark plum.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicine component determination, in particular to a method for determining chemical component content in dark plum.
Background
Dark plum, a dry, nearly mature fruit of Prunus mume (plum, Sieb.) of the family rosaceae, has the effects of astringing lung, astringing intestine, promoting fluid production, and calming roundworms, and is commonly used for treating lung deficiency chronic cough, chronic diarrhea and dysentery, deficiency heat thirst, ascaris vomiting and abdominal pain. The dark plum is widely applied in the food field and the medicine field, and the quality of the dark plum can influence the application safety of the dark plum, so that a method for measuring the contents of various chemical components in the dark plum is established, and the quality of the dark plum can be controlled more reliably and comprehensively.
Disclosure of Invention
The application aims to provide a method for measuring the content of chemical components in dark plum, which can measure the content of 16 chemical components in dark plum and can be used for quality control of dark plum.
The application provides a method for measuring the content of chemical components in dark plum, which adopts ultra-high performance liquid chromatography-mass spectrometry to measure the content of the chemical components in dark plum; the chemical components comprise: succinic acid, L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, D-quininic acid, citric acid, ferulic acid, syringic acid, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, amygdalin, maslinic acid, corosolic acid and rutin; the method comprises the following steps:
(1) establishing a standard curve of each chemical component:
preparing 5-12 mixed reference substance solutions containing various chemical components with different known concentrations by taking 90-100 vol% methanol as a solvent; wherein the concentration of succinic acid is 2.7-3000ng/mL, the concentration of L-malic acid is 2.5-30000ng/mL, the concentration of protocatechuic aldehyde is 0.5-1500ng/mL, the concentration of protocatechuic acid is 2.6-3000ng/mL, the concentration of caffeic acid is 0.8-1500ng/mL, the concentration of D-quinic acid is 9.5-30000ng/mL, the concentration of citric acid is 40-60000ng/mL, the concentration of ferulic acid is 0.1-1500ng/mL, the concentration of syringic acid is 0.3-1500ng/mL, the concentration of cryptochlorogenic acid is 5-15000ng/mL, the concentration of neochlorogenic acid is 2.2-18000ng/mL, the concentration of chlorogenic acid is 1.6-15000ng/mL, the concentration of amygdalin is 2.9-25000ng/mL, the concentration of maslinic acid is 0.2-1500ng/mL, the concentration of corosolic acid is 0.2-1500ng/mL, the concentration of rutin is 0.3-1500 ng/mL;
under the same chromatographic condition and mass spectrum condition, respectively injecting each mixed reference solution with the volume of V1 into an ultra high performance liquid chromatograph, determining the chromatographic peak of each chemical component according to the characteristic ion peak of each chemical component through mass spectrum detection, and obtaining the chromatographic peak area of each chemical component;
respectively establishing a standard curve of each chemical component by taking the peak area of each chemical component chromatographic peak as a vertical coordinate and the concentration of each chemical component as a horizontal coordinate;
(2) obtaining the chromatographic peak area of each chemical component in the sample to be detected:
taking a sample to be detected with the mass of M, extracting by taking 70-90 vol% methanol with the volume of V2 as a solvent, taking supernate, and filtering to obtain a first sample solution to be detected, wherein M/V2 is (1-3) 1 mg/mL;
taking the first sample solution to be detected, and diluting N times by taking 70-90 vol% methanol as a solvent to obtain a second sample solution to be detected, wherein N is 40-60;
under the same chromatographic conditions and mass spectrum conditions as those in the step (1), injecting a first sample solution to be detected with the volume of V1 into an ultra-high performance liquid chromatograph, determining chromatographic peaks of the chemical components according to characteristic ion peaks of the chemical components through mass spectrum detection, and obtaining chromatographic peak areas of the chemical components except citric acid;
injecting a second sample solution to be detected with the volume of V1 into an ultra-high performance liquid chromatograph, determining the chromatographic peak of the citric acid according to the characteristic ion peak of the citric acid through mass spectrum detection, and obtaining the chromatographic peak area of the citric acid;
(3) determining the content of each chemical component in a sample to be detected:
according to the established standard curve of each chemical component, the concentration C1 of each chemical component is respectively obtained from the chromatographic peak area of each chemical component in the sample to be detected, the content C of each chemical component except citric acid in the sample to be detected is respectively calculated according to the formula C1 XV 2/M, and the content C 'of the citric acid in the sample to be detected is calculated according to the formula C' C1 XN XV 2/M.
According to the method for measuring the content of the chemical components in the dark plum, an ultra-high performance liquid chromatography-mass spectrometry combined technology (UHPLC-MS/MS) is adopted, the content of 16 chemical components in the dark plum can be measured by reasonably selecting chromatographic conditions and mass spectrometry conditions, and the method has the advantages of simplicity, convenience, high sensitivity, high analysis speed, strong specificity and the like, so that the method can be used for controlling the quality of the dark plum in a credible, comprehensive and accurate manner.
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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 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 graph showing multiple reactive ion monitoring of 16 chemical components in the 6 th mixed control solution of example 1;
FIG. 1B is a diagram of multiple reactive ion monitoring of 16 chemical components in smoked plums of batch 8;
in the drawings, the numerical designations represent: 1. succinic acid; l-malic acid; 3. protocatechualdehyde; 4. protocatechuic acid; 5. caffeic acid; d-quinic acid; 7. citric acid; 8. ferulic acid; 9. syringic acid; 10. cryptochlorogenic acid; 11. chlorogenic acid; 12. chlorogenic acid; 13. amygdalin; 14. maslinic acid; 15. corosolic acid; 16. rutin is used.
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 chemical components in dark plum, which adopts ultra-high performance liquid chromatography-mass spectrometry to measure the content of the chemical components in dark plum; the chemical components comprise: succinic acid, L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, D-quininic acid, citric acid, ferulic acid, syringic acid, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, amygdalin, maslinic acid, corosolic acid and rutin; the method comprises the following steps:
(1) establishing a standard curve of each chemical component:
preparing 5-12 mixed reference substance solutions containing various chemical components with different known concentrations by taking 90-100 vol% methanol as a solvent; wherein the concentration of succinic acid is 2.7-3000ng/mL, the concentration of L-malic acid is 2.5-30000ng/mL, the concentration of protocatechuic aldehyde is 0.5-1500ng/mL, the concentration of protocatechuic acid is 2.6-3000ng/mL, the concentration of caffeic acid is 0.8-1500ng/mL, the concentration of D-quinic acid is 9.5-30000ng/mL, the concentration of citric acid is 40-60000ng/mL, the concentration of ferulic acid is 0.1-1500ng/mL, the concentration of syringic acid is 0.3-1500ng/mL, the concentration of cryptochlorogenic acid is 5-15000ng/mL, the concentration of neochlorogenic acid is 2.2-18000ng/mL, the concentration of chlorogenic acid is 1.6-15000ng/mL, the concentration of amygdalin is 2.9-25000ng/mL, the concentration of maslinic acid is 0.2-1500ng/mL, the concentration of corosolic acid is 0.2-1500ng/mL, the concentration of rutin is 0.3-1500 ng/mL;
under the same chromatographic condition and mass spectrum condition, respectively injecting each mixed reference solution with the volume of V1 into an ultra high performance liquid chromatograph, determining the chromatographic peak of each chemical component according to the characteristic ion peak of each chemical component through mass spectrum detection, and obtaining the chromatographic peak area of each chemical component;
respectively establishing a standard curve of each chemical component by taking the peak area of each chemical component chromatographic peak as a vertical coordinate and the concentration of each chemical component as a horizontal coordinate;
(2) obtaining the chromatographic peak area of each chemical component in the sample to be detected:
taking a sample to be detected with the mass of M, extracting by taking 70-90 vol% methanol with the volume of V2 as a solvent, taking supernate, and filtering to obtain a first sample solution to be detected, wherein M/V2 is (1-3) 1 mg/mL;
taking the first sample solution to be detected, and diluting by N times by taking 70-90 vol% methanol as a solvent to obtain a second sample solution to be detected, wherein N is 40-60;
under the same chromatographic conditions and mass spectrum conditions as those in the step (1), injecting a first sample solution to be detected with the volume of V1 into an ultra-high performance liquid chromatograph, determining chromatographic peaks of the chemical components according to characteristic ion peaks of the chemical components through mass spectrum detection, and obtaining chromatographic peak areas of the chemical components except citric acid;
injecting a second sample solution to be detected with the volume of V1 into an ultra-high performance liquid chromatograph, determining the chromatographic peak of the citric acid according to the characteristic ion peak of the citric acid through mass spectrum detection, and obtaining the chromatographic peak area of the citric acid;
(3) determining the content of each chemical component in a sample to be detected:
according to the established standard curve of each chemical component, the concentration C1 of each chemical component is respectively obtained from the chromatographic peak area of each chemical component in the sample to be detected, the content C of each chemical component except citric acid in the sample to be detected is respectively calculated according to the formula C1 XV 2/M, and the content C 'of the citric acid in the sample to be detected is calculated according to the formula C' C1 XN XV 2/M.
In the application, the 90-100 vol% methanol refers to methanol water solution or pure methanol solution with volume fraction of more than or equal to 90%; the 70-90 vol% methanol refers to 70-90 vol% methanol aqueous solution.
According to the method for measuring the content of the chemical components in the dark plum, the content of 16 chemical components in the dark plum can be measured by adopting an ultra-high performance liquid chromatography-mass spectrometry technology and reasonably selecting chromatographic conditions and mass spectrometry conditions.
In some embodiments of the present application, the mixed control solution contains 5-2500ng/mL of succinic acid and protocatechuic acid, 50-25000ng/mL of L-malic acid and D-quinic acid, 2-1000ng/mL of protocatechuic aldehyde, caffeic acid, ferulic acid, syringic acid, maslinic acid, corosolic acid and rutin, 100-50000ng/mL of citric acid, 20-10000ng/mL of cryptochlorogenic acid and chlorogenic acid, 25-12500ng/mL of neochlorogenic acid and 40-20000ng/mL of amygdalin.
The preparation method of the mixed reference 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 reference stock solution may be prepared in which the concentration of each component is equal to or greater than the concentration of each chemical component in the mixed reference solution, and then the mixed reference solution may be obtained by dilution.
In some embodiments of the present application, in step (1), 90-100 vol% methanol is used as a solvent to prepare a mixed control stock solution containing each chemical component, wherein the concentrations of succinic acid and protocatechuic acid in the mixed control stock solution are 2500-3000ng/mL, the concentrations of L-malic acid and D-quinic acid are 25000-30000ng/mL, the concentrations of protocatechuic aldehyde, caffeic acid, ferulic acid, syringic acid, maslinic acid, corosolic acid and rutin are 1000-1500ng/mL, the concentration of citric acid is 50000-60000ng/mL, the concentrations of cryptochlorogenic acid and chlorogenic acid are 10000-15000ng/mL, the concentration of neochlorogenic acid is 12500-18000ng/mL, and the concentration of amygdalin is 20000-25000 ng/mL;
and diluting the mixed reference substance stock solution by using 90-100 vol% methanol as a solvent to obtain the 5-12 mixed reference substance solutions containing different known concentrations of each chemical component.
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 invention can be achieved, and the stock solution of each chemical component may be prepared, for example, by preparing the stock solution of each chemical component separately and then taking the stock solutions of each chemical component 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 min.
By adopting the preparation method of the sample solution to be detected, the sample solution to be detected comprising 16 chemical components is obtained, so that the detection result of the content of the chemical components in the dark plum is more comprehensive, accurate and credible; wherein, a first sample solution to be detected containing 16 chemical components is obtained by ultrasonic extraction and is used for detecting the content of each chemical component except citric acid; based on the high content of citric acid in the dark plum, the first sample solution to be detected is diluted to obtain a second sample solution to be detected, and the second sample solution to be detected is used for determining the content of the citric acid, so that the detection result of the content of the citric acid 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 17-90% of phase A and 10-83% of phase B in volume fraction; flow rate: 0.1-0.5 mL/min; column temperature: 15-25 ℃; sample volume V1: 1-5 μ L.
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; flow rate: 0.2-0.4 mL/min; column temperature: 18-22 ℃; sample volume V1: 1-3 μ L.
The inventor finds in research that the gradient elution of the present application can achieve better separation effect of 16 chemical components in dark plum, and preferably, in some embodiments of the present application, the gradient elution is specifically as follows: 0-5min, 10-40% B; 5-5.5min, 40-80% B; 5.5-7min, 80-83% B; 7-14min, 83-83% 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 the characteristic ion peak of each chemical component after chromatographic separation so as to obtain more accurate identification result of each chemical component, 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 (MRM) mode and a 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: 34-36 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 ion peaks of each chemical component include:
the instruments and reagents required for this application are described below.
The instrument comprises the following steps: agilent 6470 triple quadrupole mass spectrometer (Agilent, usa); agilent 1290 ultra high performance liquid chromatograph (Agilent corporation, usa); agilent MassHunter analysis software (Agilent Corp., USA); one hundred thousand balances model AS 60/220.R2 (Radwag corporation, polish); Milli-Q-7005 ultrapure water preparation apparatus (Millipore Corp.); 5453 high speed centrifuge (eppendorf, Germany); s0200 vortex mixer (Labnet, USA); ZZ-L6DT ultrasonic cleaning tank (Technology Co., Ltd. of Tianjin).
Reagent: methanol (chromatographically pure) was obtained from Fisher corporation, USA, formic acid (chromatographically pure) was obtained from ROE corporation, USA, and ultrapure water was prepared by Milli-Q ultrapure water preparation apparatus.
Materials: comparison products: succinic acid (batch: DSTDH002001), L-malic acid (batch: DST201124-038), protocatechualdehyde (batch: DST200628-080), protocatechuic acid (batch: DSTDY008101), caffeic acid (batch: DST191030-013), D-quinic acid (batch: DST200822-045), citric acid (batch: DST200402-031), ferulic acid (batch: DST191112-001), syringic acid (batch: DST200927-057), cryptochlorogenic acid (batch: DSTDY003501), neochlorogenic acid (batch: DSTDX001501), chlorogenic acid (batch: DSTDL002101), maslinic acid (batch: DST200313-039), corosolic acid (batch: DST191010-005), and diterrata (batch: DST181101-017) were purchased from Texas Biotech, Inc.; amygdalin (batch number: XGZS-23HH) was purchased from the institute of food and drug testing, China. The smoked plums are purchased from Yunnan and Sichuan, and the information (production places and batch numbers) of 10 batches of smoked plums is shown in table 1.
Table 110 batch of ume information
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 chemical component:
precisely weighing 1.0mg of each of succinic acid, L-malic acid, protocatechuic acid, D-quinic acid and citric acid as reference substances, respectively adding water to dissolve, preparing into each reference substance stock solution with concentration of 1mg/mL, and storing in a refrigerator at 4 deg.C for use; precisely weighing 1.0mg of each of protocatechuic aldehyde, caffeic acid, ferulic acid, syringic acid, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, amygdalin, crataegolic acid, corosolic acid and rutin, respectively dissolving in methanol to obtain each control stock solution 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, and preparing into mixed reference substance stock solution with methanol, wherein the concentrations of citric acid is 50000ng/mL, L-malic acid and D-quinic acid are 25000ng/mL, amygdalin is 20000ng/mL, neochlorogenic acid is 12500ng/mL, cryptochlorogenic acid and chlorogenic acid are 10000ng/mL, the concentrations of succinic acid and protocatechuic acid are 2500ng/mL, and the concentrations of protocatechuic aldehyde, caffeic acid, ferulic acid, syringic acid, crataegolic acid, corosolic acid and rutin are 1000 ng/mL; the mixed control stock solution was diluted with methanol by 2, 2.5, 2, and 2 times in order to obtain 9 mixed control solutions containing each chemical component at different known concentrations, including the mixed control stock solution.
Under the same chromatographic condition and mass spectrum condition, respectively injecting 2 mu L of each mixed reference substance solution into an ultra high performance liquid chromatograph, determining chromatographic peaks of each chemical component according to characteristic ion peaks of each chemical component through mass spectrum detection, and obtaining the chromatographic peak area of each chemical component;
wherein the chromatographic conditions comprise: a chromatographic column: ACQUITY UPLC BEH C18 (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, gradient: 0-5min, 10-40% B; 5-5.5min, 40-80% B; 5.5-7min, 80-83% B; 7-14min, 83-83% B; flow rate: 0.3 mL/min; column temperature: 20 ℃; sample volume V1: 2 mu L of the solution;
the mass spectrum is a triple quadrupole mass spectrum, and the mass spectrum conditions comprise: the ion source is an electrospray ion source, and 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 ion peaks and corresponding mass spectral parameters for the 16 chemical components are shown in table 2.
Characteristic ion peaks and corresponding mass spectrum parameters of 216 chemical components in table
Taking the peak area (Y) of each chemical component chromatographic peak as a vertical coordinate, taking the concentration (X) of each chemical component as a horizontal coordinate, performing regression calculation by using a weighted least square method, wherein the weight coefficient is 1/X, and respectively establishing a standard curve of each chemical component to obtain a linear equation and a correlation coefficient r of each chemical component; the 9 th mixed control solution was diluted step by step, and the concentration of each control was taken as the lowest limit of quantitation (LLOQ) of each chemical component when the signal-to-noise ratio S/N was 10, and the results are shown in table 3, wherein, as shown in fig. 1A, a multiple reactive ion monitoring (MRM) graph of 16 chemical components in the 6 th mixed control solution shows that 16 chemical components do not interfere with each other, the chromatographic peak symmetry of each chemical component is good, and the separation degree is high.
Standard curves and minimum quantitative limits for the 316 chemical compositions in Table
(2) Obtaining the chromatographic peak area of each chemical component in the sample to be detected:
precisely weighing 50.0mg of dark plum powder (sieved by a 60-mesh sieve and with the inner diameter of a sieve pore of 0.3mm) of batch 1 in the table 1, placing the dark plum powder into a conical flask, adding 25mL of 80 vol% methanol, carrying out ultrasonic extraction for 40min, carrying out extraction power of 180W and extraction frequency of 40kHz, taking out after ultrasonic treatment, placing the dark plum powder to the room temperature, complementing the 80 vol% methanol to lose weight, taking 1mL of the solution, centrifuging the solution for 10min at 14000r/min, taking supernatant, filtering the supernatant by using a 0.22 mu m organic microporous filter membrane, and placing the subsequent filtrate, namely the first sample solution to be detected, in a refrigerator at 4 ℃ for storage and standby; and diluting the first sample solution to be detected by 50 times by using 80 vol% methanol to obtain a second sample solution to be detected. And repeatedly preparing 3 batches of the first to-be-detected sample solution and the second to-be-detected sample solution of 1 dark plum.
Under the same chromatographic conditions and mass spectrum conditions as those in the step (1), injecting 2 mu L of first sample solution to be detected into an ultra-high performance liquid chromatograph, determining chromatographic peaks of the chemical components according to characteristic ion peaks of the chemical components through mass spectrum detection, and obtaining chromatographic peak areas of the chemical components except citric acid;
under the same chromatographic condition and mass spectrum condition as those in the step (1), injecting 2 mu L of second sample solution to be detected into an ultra-high performance liquid chromatograph, determining the chromatographic peak of the citric acid according to the characteristic ion peak of the citric acid through mass spectrum detection, and obtaining the chromatographic peak area of the citric acid;
(3) determining the content of each chemical component in a sample to be detected:
according to the established standard curve of each chemical component, the concentration C1 of each chemical component is obtained from the chromatographic peak area of each chemical component in the sample to be tested, the content C of each chemical component except citric acid in the dark plum of the batch 1 is calculated according to the formula C1 × V2/M ═ C1 × 25mL/50mg, the content C 'of citric acid in the dark plum of the batch 1 is calculated according to the formula C' ═ C1 × N × V2/M ═ C1 × 50 × 25mL/50mg, and the content results of 16 chemical components in the dark plum of the batch 1 are shown in table 4, and the unit of each chemical component content in the dark plum is expressed by μ g/g based on 1ng/mg ═ 1 μ g/g.
Examples 2 to 10
The contents of 16 chemical components in the dark plums of the batch 2 to the batch 10 were obtained in the same manner as in example 1 except that the dark plums of the batch 2 to the batch 10 in table 1 were respectively used instead of the dark plums of the batch 1 in example 1, and the results are shown in table 4, wherein the multiple reactive ion monitoring diagram of the 16 chemical components in the dark plums of the batch 8 is shown in fig. 1B.
Table 410 dark plum batches with 16 chemical components (μ g/g) (n ═ 3)
Methodology validation
Precision degree
The precision in the day is as follows: precisely weighing 50.0mg of dark plum powder of batch 8, preparing a first sample solution to be detected according to the method of example 1, carrying out sample injection analysis for 6 times according to the chromatographic condition and the mass spectrum condition of example 1, and recording peak areas of chemical components except citric acid; preparing a second sample solution to be detected according to the method in the example 1, carrying out sample injection analysis according to the chromatographic condition and the mass spectrum condition in the example 1, carrying out sample injection for 6 times continuously, and recording the peak area of citric acid; the average value and the Relative Standard Deviation (RSD) value of the peak areas of the 16 chemical components are respectively calculated, the results are shown in Table 5, and the RSD values are all less than or equal to 5.0%, which shows that the method of the application has good precision within a day.
Precision in the daytime: precisely weighing 50.0mg of dark plum powder of batch 8, preparing a first sample solution to be detected according to the method of example 1, carrying out sample injection analysis according to the chromatographic condition and the mass spectrum condition of example 1, repeatedly carrying out sample injection for 2 times, carrying out continuous sample injection for three days, and recording peak areas of chemical components except citric acid; preparing a second sample solution to be detected according to the method of the embodiment 1, performing sample injection analysis according to the chromatographic condition and the mass spectrum condition of the embodiment 1, repeating sample injection for 2 times, performing continuous sample injection for three days, and recording the peak area of citric acid; the average value and RSD value of the peak areas of the 16 chemical components are respectively calculated, the results are shown in Table 6, and the RSD values are all less than or equal to 6.0%, which shows that the method of the application has good daytime precision.
Precision in day of 516 chemical components (n ═ 6)
Daytime precision of 616 chemical compositions (n ═ 6)
Repeatability of
Precisely weighing 6 parts of 8 batches of dark plum powder, 50.0mg of each part, preparing a first sample solution to be detected according to the method in the embodiment 1, performing sample injection analysis respectively according to the chromatographic condition and the mass spectrum condition in the embodiment 1, and recording peak areas of chemical components except citric acid; respectively preparing a second sample solution to be detected according to the method in the embodiment 1, carrying out sample injection analysis according to the chromatographic condition and the mass spectrum condition in the embodiment 1, and recording the peak area of citric acid; according to the standard curves of the chemical components in the example 1, the concentrations of the 16 chemical components are respectively calculated, and the average value and the RSD value of the concentrations of the chemical components are calculated, the result is shown in the table 7, and the RSD values are less than or equal to 5.9%, which shows that the method has good repeatability.
TABLE 716 repeatability of chemical composition (ng/mL, n ═ 6)
Stability of
Precisely weighing 50.0mg of dark plum powder of batch 8, preparing a first sample solution to be detected according to the method of example 1, performing sample injection analysis for 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 areas of chemical components except citric acid; preparing a second sample solution to be detected according to the method in the embodiment 1, performing sample injection analysis for 0 h, 2 h, 4h, 8 h, 12 h and 24h according to the chromatographic condition and the mass spectrum condition in the embodiment 1, and recording the peak area of citric acid; the average value and RSD value of the peak areas of the 16 chemical components are respectively calculated, the result is shown in table 8, the RSD values are all less than or equal to 7.1%, and the fact that the 16 chemical components in the dark plum fruit are good in stability under the condition that the dark plum fruit is placed at room temperature for 24 hours is shown.
Table 816 stability of the chemical compositions (n ═ 6)
Sample recovery rate
Precisely weighing 6 parts of dark plum powder of batch 8, wherein each part is 25.00 mg; preparing each standard solution of 16 chemical components, wherein the concentrations of the chemical components are respectively as follows: 10mg/mL of citric acid, 1mg/mL of L-malic acid, D-quinic acid, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid and amygdalin, 100 mu g/mL of succinic acid, protocatechuic acid, caffeic acid, crataegolic acid, corosolic acid and rutin, and 10 mu g/mL of protocatechuic aldehyde, ferulic acid and syringic acid; respectively taking the standard solutions: citric acid 530 mu L, L-malic acid 27 mu L, D-quinic acid 155 mu L, cryptochlorogenic acid 68 mu L, neochlorogenic acid 75 mu L, chlorogenic acid 40 mu L, amygdalin 100 mu L, succinic acid 15 mu L, protocatechuic acid 16 mu L, caffeic acid 19 mu L, maslinic acid 16 mu L, corosolic acid 36 mu L, rutin 19 mu L, protocatechualdehyde 44 mu L, ferulic acid 54 mu L and syringic acid 75 mu L, the addition values of the chemical components are shown in Table 9, the volume is determined to 25mL by 80% methanol, the first sample solution to be tested of each sample is prepared according to the method of example 1, the analysis is carried out according to the chromatographic condition and the mass spectrum condition of example 1 respectively, and the peak areas of the chemical components except for citric acid are recorded; respectively preparing a second sample solution to be detected of each standard sample according to the method of the embodiment 1, carrying out sample injection analysis according to the chromatographic condition and the mass spectrum condition of the embodiment 1, and recording the peak area of the citric acid; according to the standard curve of each chemical component in example 1, the concentrations of 16 chemical components in each standard sample are calculated respectively to obtain the content of 16 chemical components in each standard sample, the sample adding recovery rate of each standard sample is calculated according to the sample adding recovery rate (%) (measured value-original value)/added value multiplied by 100%, and the average recovery rate and RSD value of the sample adding recovery rate of each chemical component are calculated, the result is shown in table 9, the result of table 9 shows that the average recovery rate of 16 chemical components is between 88.8% and 110.4%, and the RSD value is less than or equal to 6.4%, which indicates that the method of the present application has good accuracy.
TABLE 916 sample recovery for chemical components (n ═ 6)
Dilution effect
Precisely sucking each control stock solution with the concentration of 1mg/mL in the embodiment 1 to prepare a mixed control stock solution with the concentrations of citric acid of 500 mug/mL, L-malic acid and D-quinic acid of 250 mug/mL, amygdalin of 200 mug/mL, neochlorogenic acid of 125 mug/mL, cryptochlorogenic acid and chlorogenic acid of 100 mug/mL, succinic acid and protocatechuic acid of 25 mug/mL, protocatechuic aldehyde, caffeic acid, ferulic acid, syringic acid, crataegolic acid, corosolic acid and rutin of 10 mug/mL; the mixed reference stock solution is diluted by 20, 50 and 100 times respectively, 6 parts of the mixed reference stock solution are diluted by each time, sample injection analysis is performed according to the chromatographic condition and the mass spectrum condition of example 1, the peak areas of the chemical components are recorded, the concentrations of the 16 chemical components under each dilution time are calculated respectively according to the standard curve of the chemical components in example 1, the average value and the RSD value of the concentrations are calculated, the RE value is calculated according to the relative error (RE,%) (average value-measured value)/measured value multiplied by 100 percent, the result is shown in Table 10, the absolute value of RE is less than or equal to 7.3 percent according to Table 10, and the RSD value is less than or equal to 6.9 percent, thereby further indicating that the method has better accuracy.
TABLE 1016 dilution Effect of chemical compositions (n ═ 6)
In conclusion, the UHPLC-MS/MS analysis method is established in the application, the contents of 16 chemical components including succinic acid, L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, D-quinic acid, citric acid, ferulic acid, syringic acid, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, amygdalin, crataegolic acid, corosolic acid and rutin in dark plums under different processing methods can be measured, and the method is simple, convenient, high in sensitivity, high in analysis speed and strong in specificity.
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 determining chemical component content in mume fructus adopts ultra-high performance liquid chromatography-mass spectrometry to determine chemical component content in mume fructus; the chemical components comprise: succinic acid, L-malic acid, protocatechualdehyde, protocatechuic acid, caffeic acid, D-quininic acid, citric acid, ferulic acid, syringic acid, cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, amygdalin, maslinic acid, corosolic acid and rutin; the method comprises the following steps:
(1) establishing a standard curve of each chemical component:
preparing 5-12 mixed reference substance solutions containing various chemical components with different known concentrations by taking 90-100 vol% methanol as a solvent; wherein the concentration of succinic acid is 2.7-3000ng/mL, the concentration of L-malic acid is 2.5-30000ng/mL, the concentration of protocatechuic aldehyde is 0.5-1500ng/mL, the concentration of protocatechuic acid is 2.6-3000ng/mL, the concentration of caffeic acid is 0.8-1500ng/mL, the concentration of D-quinic acid is 9.5-30000ng/mL, the concentration of citric acid is 40-60000ng/mL, the concentration of ferulic acid is 0.1-1500ng/mL, the concentration of syringic acid is 0.3-1500ng/mL, the concentration of cryptochlorogenic acid is 5-15000ng/mL, the concentration of neochlorogenic acid is 2.2-18000ng/mL, the concentration of chlorogenic acid is 1.6-15000ng/mL, the concentration of amygdalin is 2.9-25000ng/mL, the concentration of maslinic acid is 0.2-1500ng/mL, the concentration of corosolic acid is 0.2-1500ng/mL, the concentration of rutin is 0.3-1500 ng/mL;
respectively injecting each mixed reference solution with the volume of V1 into an ultra high performance liquid chromatograph under the same chromatographic condition and mass spectrum condition, determining the chromatographic peak of each chemical component according to the characteristic ion peak of each chemical component through mass spectrum detection, and obtaining the chromatographic peak area of each chemical component;
respectively establishing a standard curve of each chemical component by taking the peak area of each chemical component chromatographic peak as a vertical coordinate and the concentration of each chemical component as a horizontal coordinate;
(2) obtaining the chromatographic peak area of each chemical component in the sample to be detected:
taking a sample to be detected with the mass of M, performing ultrasonic extraction by taking 70-90 vol% methanol with the volume of V2 as a solvent, taking supernate, and filtering to obtain a first sample solution to be detected, wherein M/V2 is (1-3) 1 mg/mL;
taking the first sample solution to be detected, and diluting by N times by taking 70-90 vol% methanol as a solvent to obtain a second sample solution to be detected, wherein N is 40-60;
under the same chromatographic conditions and mass spectrum conditions as those in the step (1), injecting a first sample solution to be detected with the volume of V1 into an ultra-high performance liquid chromatograph, determining chromatographic peaks of the chemical components according to characteristic ion peaks of the chemical components through mass spectrum detection, and obtaining chromatographic peak areas of the chemical components except citric acid;
injecting a second sample solution to be detected with the volume of V1 into an ultra-high performance liquid chromatograph, determining the chromatographic peak of the citric acid according to the characteristic ion peak of the citric acid through mass spectrum detection, and obtaining the chromatographic peak area of the citric acid;
(3) determining the content of each chemical component in a sample to be detected:
according to the established standard curve of each chemical component, the concentration C1 of each chemical component is respectively obtained from the chromatographic peak area of each chemical component in the sample to be detected, the content C of each chemical component except citric acid in the sample to be detected is respectively calculated according to the formula C1 XV 2/M, and the content C 'of the citric acid in the sample to be detected is calculated according to the formula C' C1 XN XV 2/M.
2. The method according to claim 1, wherein the concentration of succinic acid and protocatechuic acid in the mixed control solution is 5-2500ng/mL, the concentration of L-malic acid and D-quinic acid is 50-25000ng/mL, the concentration of protocatechuic aldehyde, caffeic acid, ferulic acid, syringic acid, maslinic acid, corosolic acid and rutin is 2-1000ng/mL, the concentration of citric acid is 100-50000ng/mL, the concentration of cryptochlorogenic acid and chlorogenic acid is 20-10000ng/mL, the concentration of neochlorogenic acid is 25-12500ng/mL, and the concentration of amygdalin is 40-20000 ng/mL.
3. The method as claimed in claim 1, wherein in step (1), 90-100 vol% methanol is used as solvent to prepare a mixed control stock solution containing each chemical component, wherein the concentrations of succinic acid and protocatechuic acid in the mixed control stock solution are 2500-3000ng/mL respectively, the concentrations of L-malic acid and D-quinic acid are 25000-30000ng/mL respectively, the concentrations of protocatechuic aldehyde, caffeic acid, ferulic acid, syringic acid, crataegolic acid, corosolic acid and rutin are 1000-1500ng/mL respectively, the concentration of citric acid is 50000-60000ng/mL, the concentrations of cryptochlorogenic acid and chlorogenic acid are 10000-15000ng/mL respectively, the concentration of new chlorogenic acid is 12500-18000ng/mL, and the concentration of amygdalin is 20000-25000 ng/mL;
and diluting the mixed reference substance stock solution by using 90-100 vol% methanol as a solvent to obtain the 5-12 mixed reference substance solutions containing different known concentrations of each chemical component.
4. The method as claimed in claim 1, wherein 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 min.
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 17-90% of phase A and 10-83% of phase B in volume fraction; flow rate: 0.1-0.5 mL/min; column temperature: 15-25 ℃; sample volume V1: 1-5 μ L.
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; flow rate: 0.2-0.4 mL/min; column temperature: 18-22 ℃; sample volume V1: 1-3 μ L.
7. The method according to claim 5, wherein the gradient elution is in particular: 0-5min, 10-40% B; 5-5.5min, 40-80% B; 5.5-7min, 80-83% B; 7-14min, 83-83% B.
8. The method of any one of claims 1-4, wherein the mass spectrometry is triple quadrupole mass spectrometry, the mass spectrometry conditions comprising: the ion source is an electrospray ion source, and the detection mode is a multi-reaction ion monitoring and negative ion scanning mode; temperature of the drying gas: 250 ℃ and 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: 34-36 psi; temperature of sheath gas: 330 ℃ to 370 ℃; flow rate of sheath gas: 10-12L/min; capillary voltage: 3300-.
10. The method of any of claims 1-4, wherein the characteristic ion peak of each chemical component comprises:
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