CN109187780B - Detection method of compound motherwort granules - Google Patents
Detection method of compound motherwort granules Download PDFInfo
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Abstract
The invention discloses a detection method of compound motherwort granules, which comprises the following steps: step one, preparing a test solution, namely grinding, extracting and removing impurities from a compound motherwort particle sample to prepare the test solution; and step two, absorbing the test solution, and injecting the solution into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for determination. When the method is used for measuring 8 main chemical components in the compound motherwort particles, the good linear relation is realized in the measured mass concentration range, the correlation coefficients are all larger than 0.99, the average recovery rate (n is 6) range is 97.85-101.18%, and the RSD value range is 2.08-3.19%. The method can rapidly determine the content of 8 main chemical components in the compound motherwort granule, and has important significance for further perfecting the quality control of the compound motherwort granule.
Description
Technical Field
The invention relates to the field of quality control of traditional Chinese medicine preparations, in particular to a method for simultaneously measuring the content of 8 main components in compound motherwort granules.
Background
The compound motherwort particles [ Chinese medicine Z20050629] are nine kinds of new Chinese medicines produced by Jiangsu Kangyuan pharmaceutical industry limited company, are prepared from four common Chinese medicines of motherwort, angelica, Szechuan lovage rhizome and costustoot by refining, have the effects of promoting blood circulation, promoting qi circulation, removing blood stasis and relieving pain, and are mainly used for treating dysmenorrheal caused by qi stagnation and blood stasis in clinic. In the formula, the motherwort has the functions of promoting blood circulation and regulating blood stasis, and the angelica has the effects of enriching blood, regulating menstruation and stopping bleeding. The rhizoma ligustici wallichii is pungent, warm and fragrant, and can play a role in activating blood and promoting qi, and dispelling wind and relieving pain. Mu Xiang is pungent in flavor, bitter in flavor and descending, warm in nature, and fragrant in flavor, can ascend and descend, and pass qi activities of stomach, intestine and triple energizer, and is the essential herb for promoting qi circulation and relieving pain, and strengthening spleen and stomach. The combination of four herbs can not only increase the efficacy of promoting blood circulation and removing blood stasis, but also increase the force of promoting blood circulation by means of qi, so as to double the efficacy of promoting blood circulation, breaking stagnation and relieving pain.
At present, no research on the content determination of compound leonurus granules exists, and only the content of leonurine is generally determined and is determined by a Raney ammonium salt precipitation method. The method is complicated to operate, and only the content of one alkaloid in motherwort which is a medicinal material of a prescription in the compound is measured, which is not enough to comprehensively evaluate the quality of compound motherwort particles.
Disclosure of Invention
The invention aims to establish an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) method, which is used for measuring the content of 8 main components in compound motherwort particles and lays a foundation for improving the quality control of the compound motherwort particles.
In view of the above, the invention provides a qualitative or quantitative detection method for compound motherwort particles, which is characterized by comprising the following steps:
step one, preparing a test solution, namely grinding, extracting and removing impurities from a compound motherwort particle sample to prepare the test solution;
and step two, absorbing the test solution, and injecting the solution into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for determination.
Specifically, the following chromatographic conditions are used for the determination of the ultra performance liquid chromatography-triple quadrupole mass spectrometer:
a chromatographic column: agilent Zorbax Eclipse plus C18Chromatography column (50X 2.1mm, 5 μm); mobile phase: methanol (a) -water (B); gradient elution: 0-3min, 5% -40% B; 3-6min, 40% -65% B; 6-10min, 65% -95% B; 10-12min, 95% B; 12-14min, 95% -5% B; flow rate: 0.4-0.6 mL/min
Preferably, the flow rate of the chromatographic conditions is 0.5 mL-min-1(ii) a Column temperature: room temperature; sample introduction amount: 2 μ L.
Further, the ultra performance liquid chromatography-triple quadrupole mass spectrometer uses the following mass spectrum conditions:
performing multi-reaction monitoring by adopting a triple quadrupole mass spectrometer in an electrospray positive and negative ion mode, wherein an ion source is an electrospray ion source (ESI source); the ion source parameters are CAD: 8; c M R: 20; TEM: 500 ℃; ion SprayVoltage (IS): 5500V; atomizing Gas (GS)1): 40; auxiliary Gas (GS)2): 60, adding a solvent to the mixture; the scanning mode is multi-reaction monitoring (MRM), positive ion mode; the detected ion pairs used for the quantitative analysis are shown in table 1.
TABLE 1 detection ion pairs for quantitative analysis
Preferably, the flow rate is 0.5 mL/min.
Specifically, in the first step, compound motherwort particles are taken, ground, precisely weighed to be 0.1-0.3 g, added with 20-25 ml of 30-70% ethanol, weighed, ultrasonically extracted for 40-50 min, taken out, cooled, weighed again, added with the corresponding solvent to complement the weight, absorbed supernatant, filtered by a microporous membrane, transferred to be placed in a volumetric flask, diluted to scale by the corresponding solvent, and shaken uniformly, thus obtaining the compound motherwort tablet. Preferably, 0.2g of the ground compound motherwort particles are precisely weighed and placed in a 50mL conical flask with a plug, 25mL of 50% ethanol is precisely transferred, weighed, extracted by ultrasonic (working frequency 40kHz and power 500W) for 45min, placed to room temperature, weighed again, added with 50% ethanol to make up for the lost weight, shaken up, filtered by a microporous filter membrane (0.45 mu m), the subsequent filtrate is taken for standby, 0.2mL of the subsequent filtrate is sucked and placed in a 10mL volumetric flask, diluted to the scale by 50% ethanol and shaken up, thus obtaining the compound motherwort tablet.
Preferably, the extraction solvent in step one is selected from 50% ethanol.
Further, the method includes preparation and measurement of a control solution.
Preferably, the control of the control solution is selected from leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, angelolide A, costunolide and dehydrocostunolide.
Specifically, the control solution is prepared by: respectively precisely weighing appropriate amount of control substances of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levisticolide A, costunolide and dehydrocostunolide, and adding 30-70% methanol to obtain the following concentrations: 0.015-0.057, 0.12-0.20, 0.003-0.010, 0.250-0.300, 0.05-0.09, 0.01-0.04, 0.02-0.04, 0.01-0.04 mg/mL of mixed reference solution. Preferably, methanol is added at a concentration of 70%The solution is prepared into the concentrations of 20.80, 137.50, 5.00, 280.00, 78.00, 20.00, 30.60 and 22.40 mu g/mL respectively-1Mixing the reference solutions, and storing in a refrigerator at 4 deg.C. Filtering with 0.22 μm microporous membrane, and shaking. Placing in a refrigerator at 4 deg.C for use.
Further, in the detection method of the present invention, in the positive ion detection mode, the mixed control solution and the test solution are respectively extracted precisely, and injected into the liquid chromatograph to mix the control solution m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.2, 193.3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, 231.4 → 185.1 ion pairs to locate leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide a, angelolide a, costunolide and dehydrocostunolide color spectrum peaks, and the corresponding peak areas of the mixed control solution and the test solution m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.193, 3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, and 185.1 ion pairs are extracted. The signal-to-noise ratio of the chromatographic peak is more than 10: 1.
Calculating with external standard method to obtain the content of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levisticide A, costunolide and dehydrocostuslactone in the test solution.
The results of mass spectral parameters of 8 tested components examined in the present invention show that all compounds respond better in positive ion mode, with the best quantitative ion pair being m/Z312.3 → 181.0 (leonurine), 144.1 → 58.2 (stachydrine), 225.3 → 207.5 (senkyunolide H), 191.3 → 173.2 (Z-ligustilide), 193.3 → 147.3 (senkyunolide A), 381.4 → 191.2 (angelicinolactone A), 233.2 → 187.3 (costunolide), 231.4 → 185.1 (dehydrocostunolide).
Compared with the original determination method, the ultra-high performance liquid phase-triple quadrupole mass spectrometry combined technology established by the invention is used for determining 8 main chemical components in the following table of the compound leonurus particle, is simple, convenient and quick (only 14min of operation time is needed), increases the content determination of 7 main components in the compound leonurus particle, and lays a foundation for improving the quality control of the compound leonurus particle.
When 8 main chemical components, namely leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, angelicin A, costunolide and dehydrocostuslactone, in the compound leonurus particle are measured, the linear relation is good in the measured mass concentration range, the correlation coefficients are all larger than 0.99, the average recovery rate (n is 6) range is 97.85-101.18%, and the RSD value range is 2.08-3.19%. Therefore, the invention has strong specificity, and good accuracy, precision and reproducibility. The method can rapidly determine the content of 8 main chemical components in the compound motherwort granule, is a simple and convenient method worthy of popularization, and has important significance for further perfecting the quality control of the compound motherwort granule.
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FIG. 1 is a diagram showing the results of examination of different extraction methods in the present invention;
FIG. 2 is a graph showing the results of examination of different extraction media according to the present invention;
fig. 2 abscissa: 1. water, 2, ethanol-water (3: 7), 3, ethanol-water (5: 5), 4, ethanol-water (7: 3), 5, ethanol-water (95: 5), 6, methanol-water (3: 7), 7, methanol-water (5: 5), 8, methanol-water (7: 3), 9, methanol-water (95: 5),
FIG. 3 is a diagram showing the results of investigation at different extraction times in the present invention;
FIG. 4 is a MRM extraction chromatogram of a mixed control in accordance with the present invention;
FIG. 5 is an MRM extraction chromatogram of the compound motherwort granule of the present invention;
in fig. 4 and 5, 1 stachydrine; 2. leonurine; 3. senkyunolide H; 4. senkyunolide A; z-ligustilide; 6. costunolide; 7. dehydrocostuslactone; 8. levistilide A.
Detailed Description
As mentioned above, the present invention aims at providing a method for detecting compound motherwort particles.
The method is carried out according to conventional conditions or conditions recommended by manufacturers, and the raw materials, reagents or instruments used by the method are conventional products which can be obtained commercially.
It is specifically noted that similar alternatives and modifications will be apparent to those skilled in the art, which are also intended to be included within the present invention. It will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately combining the methods and applications described herein without departing from the spirit, scope, and content of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention.
The following will be described specifically with reference to the experimental contents.
Systematic adaptability and methodology experiment for compound motherwort particle detection
1 Instrument and reagent
1.1 instruments
LC-20A ultra high performance liquid chromatograph (Shimadzu corporation, Japan); API 5000 mass spectrometer (AB SCIEX corporation, usa); METTLER XS 205 analytical balance (METTLER-toledo shanghai instruments ltd); KQ-500 ultrasonic cleaner (Kunshan ultrasonic Instrument Co., Ltd.); millipore Elix 5 water purifier (Michlibo China Co., Ltd.).
1.2 reagents and reagents
Compound YIMU granule (Jiangsu Kangyuan pharmaceutical Co., Ltd., lot number: 150701, 150802, 150901, 150902, 160301);
senkyunolide A (batch No. 16061406) and levistilide A (batch No. 140910) were purchased from Doppel Biotechnology Ltd; leonurine (batch number: 11823-; senkyunolide H (batch: 4220) Shanghai Shidande Biotech Co., Ltd; z-ligustilide (batch: M ST-16060801) was purchased from Dowmansite Biotech, Inc.; the purity meets the requirement of content measurement.
The methanol is chromatographically pure; the other reagents are analytically pure.
Water was self-made by Millipore Elix 5 water purifier.
2 methods and results
2.1 liquid chromatography conditions and Mass Spectrometry conditions
Liquid chromatography conditions:
a chromatographic column: agilent Zorbax Eclipse plus C18Chromatography column (50X 2.1mm, 5 μm); mobile phase: methanol (a) -water (B); gradient elution: 0-3min, 5% -40% B; 3-6min, 40% -65% B; 6-10min, 65% -95% B; 10-12min, 95% B; 12-14min, 95% -5% B; flow rate: 0.5mL/min-1(ii) a Column temperature: room temperature; sample introduction amount: 2 μ L.
Mass spectrum conditions:
the ion source is an electrospray ion source (ESI source); the ion source parameters are CAD: 8; c M R: 20; TEM: 500 ℃; ion SprayVoltage (IS): 5500V; atomizing Gas (GS)1): 40; auxiliary Gas (GS)2): 60, adding a solvent to the mixture; the scanning mode is multi-reaction monitoring (MRM), positive ion mode; the detected ion pairs used for the quantitative analysis are shown in table 2.
TABLE 2 detection ion pairs for quantitative analysis
2.2 preparation of control solutions
Respectively taking appropriate amount of reference substances including leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levisticide A, costunolide and dehydrocostuslactone, precisely weighing, and adding 70% methanol to obtain a solution containing the above reference substances 20.80, 137.50, 5.00, 280.00, 78.00, 20.00, 30.60 and 22.40 μ g/mL-1Mixing the reference solutions, and storing in a refrigerator at 4 deg.C.
2.3 examination of Single factor of preparation method of test solution
2.3.1 selection of extraction mode
Taking 6 parts of the granules (batch number: 150701), grinding, taking about 0.2g of each part, precisely weighing, placing the three parts into a 50mL conical flask with a plug, precisely transferring 25mL of 50% ethanol, weighing, ultrasonically extracting (working frequency 40kHz and power 500W) for 30min, placing at room temperature, weighing, and supplementing the lost weight with 50% ethanol. Placing the other three parts into a 50mL round-bottom flask, precisely transferring 25mL 50% ethanol, weighing, reflux-extracting in water bath (70 deg.C) for 30min, standing at room temperature, weighing, adding 50% ethanol to the reduced weight, shaking, and collecting the filtrate. Sucking 0.2mL of the subsequent filtrate, placing in a 10mL volumetric flask, diluting with 50% ethanol to scale, shaking, and filtering with microporous membrane (0.45 μm) to obtain the test solution. Measuring according to determined chromatographic condition and mass spectrum condition, recording chromatogram peak area, and calculating and comparing the average content of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levistilide A, costunolide and dehydrocostuslactone in the test solution. The results show that the contents of 8 compounds are not greatly different by adopting the ultrasonic extraction method and the reflux extraction method, and the ultrasonic extraction method is determined to be ultrasonic extraction due to the convenient and simple operation of the ultrasonic extraction, and the table 3 and the figure 1 show.
TABLE 3 examination results of the extraction methods
2.3.2 examination of extraction vehicle
Grinding 9 parts of the granule (batch number: 150701), weighing 0.2g of the granule each part precisely, placing in a 50mL conical flask with a plug, weighing 25mL of distilled water, 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol, 30% methanol, 50% methanol, 70% methanol and 95% methanol respectively, extracting with ultrasound (working frequency 40kHz and power 500W) for 30min, cooling to room temperature, weighing, adding corresponding solvent to supplement the loss weight, shaking, filtering with microporous membrane (0.45 μm), and collecting the filtrate. Sucking 0.2mL of the subsequent filtrate, placing the subsequent filtrate in a 10mL volumetric flask, diluting the subsequent filtrate to the scale with the corresponding solvent, and shaking up to obtain the test solution. Measuring according to determined chromatographic condition and mass spectrum condition, recording chromatogram peak area, and calculating and comparing the average content of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levistilide A, costunolide and dehydrocostuslactone in each test solution. The results show that the components to be tested were not completely extracted when water and methanol were used as extraction solvents, and the highest content of 8 chemical components was measured when ethanol, especially 50% ethanol, was used as the solvent, so 50% ethanol was determined as the extraction solvent, see table 4 and fig. 2.
Table 4 comparison of extraction vehicles
2.3.3 selection of extraction time
Grinding 5 parts of the granule (batch number: 150701), weighing about 0.2g each part, precisely weighing, placing in 50mL conical flask with plug, precisely transferring 25mL 50% ethanol, weighing, ultrasonic extracting (working frequency 40kHz, power 500W) for 15min, 30min, 45min, 60min, 90min, standing at room temperature, weighing, adding 50% ethanol to supplement lost weight, shaking, filtering with microporous membrane (0.45 μm), and collecting filtrate. Sucking 0.2mL of the subsequent filtrate, placing the subsequent filtrate in a 10mL volumetric flask, diluting the subsequent filtrate to the scale with the corresponding solvent, and shaking up to obtain the test solution. Measuring according to determined chromatographic condition and mass spectrum condition, recording chromatogram peak area, and calculating and comparing the average content of leonurine, stachydrine senkyunolide H, Z-ligustilide, senkyunolide A, levistilide A, costunolide and dehydrocostuslactone in each test solution. The results show that the sample is not completely extracted in 15min of ultrasound, the extraction effect is highest in 45min of ultrasound, and the extraction time is determined to be 45min by comprehensive consideration, which is shown in table 5 and fig. 3.
TABLE 5 comparison of extraction times
2.3.4 optimal preparation method of test solution
Grinding the granules (batch number: 150701), weighing about 0.2g, precisely weighing, placing in 50mL conical flask with plug, precisely transferring 25mL 50% ethanol, weighing, ultrasonically extracting (working frequency 40kHz, power 500W) for 45min, cooling to room temperature, weighing again, adding 50% ethanol to make up for the lost weight, shaking, filtering with microporous membrane (0.45 μm), and collecting the filtrate. Sucking 0.2mL of the subsequent filtrate, placing the subsequent filtrate in a 10mL volumetric flask, diluting the subsequent filtrate to the scale with 50% ethanol, and shaking up to obtain the test solution.
2.4 methodological considerations
2.4.1 Linear relationship, detection Limit and quantitation Limit
Precisely sucking 0.03 mL, 0.13 mL, 0.26 mL, 0.52 mL, 1.04 mL and 2.5mL of the mixed reference substance, placing in a 10mL volumetric flask, diluting with 50% ethanol to scale, and shaking up to obtain mixed standard substance solutions with series concentrations. Precisely sucking 2 mu L of the mixed reference solution, measuring according to determined chromatographic conditions and mass spectrum conditions, and recording chromatogram peak area. And (4) performing regression analysis by taking the mass concentration as an abscissa and taking the peak area as an ordinate, and drawing a standard curve.
Taking the reference substance solution under the linear range determination item, diluting, determining according to the determined detection condition, and determining the lowest detection limit and the quantification limit when the signal to noise ratio is 3 and 10.
The linear equations of dehydrocostuslactone, costunolide, leonurine, Z-ligustilide, senkyunolide A, senkyunolide H, stachydrine and levistilide A are respectively as follows: 2043X +85424, 2287.9X +142921, 4866.7X +819194, 273.43X +374940, 1437X +409632, 1773X +23612, 660.82X +805486, and 4580.5X +317153, and correlation coefficient R2Respectively as follows: 0.9978, 0.9984, 0.9927, 0.9936, 09973, 0.9991, 0.9960 and 0.9960, 8 substances are respectively 70.00-2240 ng.mL-1、 95.63-3060ng·mL-1、65.00-2080ng·mL-1、1400-28000ng·mL-1、487.5 -7800ng·mL-1、15.63-500ng·mL-1、429.69-13750ng·mL-1、125-2000 ng·mL-1With a good linear relationship in the range. The detection limits are respectively: 2.8 ng/mL-1、2.9 ng·mL-1、3.2ng·mL-1、0.8ng·mL-1、2.0ng·mL-1、3.5ng·mL-1、3.7 ng·mL-1And 3.5 ng. mL-1The quantitative limit is 11.4 ng/mL respectively-1、9.6ng·mL-1、13.8 ng·mL-1、11.6ng·mL-1、10.4ng·mL-1、10.3ng·mL-1、13.5ng·mL-1And 13.5 ng. mL-1. The results are shown in Table 6.
TABLE 6 Linear relationship, detection limit and quantitation limit investigation results
2.4.2 precision test
And continuously feeding the same mixed reference substance solution for 6 times, recording the peak area of the chromatogram, and calculating the RSD of the peak area of each component to be measured.
The RSD values (n is 6) of the peak areas of dehydrocostuslactone, costunolide, leonurine, Z-ligustilide, senkyunolide A, senkyunolide H, stachydrine and levisticide A in the mixed standard product are respectively 1.36%, 1.65%, 1.50%, 0.84%, 1.21%, 0.91%, 2.82% and 0.91%, and the RSD values are all less than 3.0%, which indicates that the precision of the instrument is good. The results are shown in Table 7.
TABLE 7 precision, repeatability and stability results
2.4.3 repeatability test
Taking compound motherwort particles (batch number: 150701), grinding, weighing about 0.2g, precisely weighing, preparing 6 parts of test solution in parallel according to the method under the item '2.3.4', taking 2 mu L for sample injection analysis, recording the peak area of a chromatogram, and calculating the RSD of the peak area of each component to be measured.
The RSD values of dehydrocostuslactone, costunolide, leonurine, Z-ligustilide, senkyunolide A, senkyunolide H, stachydrine and levisticide A in the test solution are respectively 1.65%, 2.21%, 2.49%, 1.12%, 2.11%, 1.92%, 2.28% and 1.69%, which shows that the method has good repeatability. The results are shown in Table 7.
2.4.4 stability test
Taking compound motherwort particles (batch number: 150701), grinding, taking about 0.2g, precisely weighing, preparing a test solution in parallel according to the method under the item '2.3.4', respectively carrying out sample injection analysis 0, 2, 4, 8, 12 and 24 hours after the test solution is prepared, recording the peak area of a chromatogram, and calculating the RSD of the content of each component to be detected.
The RSD values of the peak areas of dehydrocostuslactone, costunolide, leonurine, Z-ligustilide, senkyunolide A, senkyunolide H, stachydrine and levisticide A in the test solution are respectively 2.61%, 1.65%, 3.23%, 2.24%, 3.15%, 2.43%, 2.98% and 0.72%, which indicates that the test solution has good stability after being placed at room temperature for 12 hours. The results are shown in Table 7.
2.4.5 sample recovery test
Taking compound motherwort particles (batch number: 150701), grinding, taking about 0.1g, precisely weighing, respectively adding a certain amount of mixed reference substance solution according to the approximate proportion of the reference substance of the content of the test substance, carrying out parallel backup on the test substance solution according to the preparation method under the item '2.3.4', taking 2 mu L for sample injection analysis, recording the peak area of a chromatogram, and calculating the sample addition recovery rate and RSD.
The average recovery rates of dehydrocostuslactone, costunolide, leonurine, Z-ligustilide, senkyunolide A, senkyunolide H, stachydrine and levistilide A in the test solution are 97.85%, 99.95%, 100.20%, 100.78%, 101.18%, 98.18%, 99.58% and 101.07%, respectively, and the RSD values are 2.99%, 2.08%, 2.26%, 3.08%, 2.88%, 3.19%, 2.45% and 2.28%, respectively. The results show that the recovery of the process is good. The detailed results are shown in Table 8.
Table 8 recovery test results (n ═ 6)
2.4.6 sample determination
Taking 3 batches of compound motherwort particles, preparing a test solution according to the method under the item 2.3.4, measuring according to determined detection conditions, and recording the chromatographic peak area.
The content of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, angelicin A, costunolide and dehydrocostuslactone in the test solution of 3 batches was calculated by external standard method. 3 parts of each batch of samples were measured.
The results of measuring the contents of dehydrocostuslactone, costunolide, leonurine, Z-ligustilide, senkyunolide A, senkyunolide H, stachydrine, and levistilide A in 3 batches of compound leonurus granules are shown in Table 9. The MRM extraction chromatograms of the mixed standard and sample are shown in fig. 4 and 5, respectively. It is clear from Table 9 that the content values of 8 chemical components in 3 batches are stable.
TABLE 9 measurement results of contents (mg. g)-1,n＝3)
Method for measuring content of 8 main chemical components in compound motherwort particles
A method for measuring 8 main components in compound motherwort particles by using an ultra-fast liquid chromatography-tandem triple quadrupole mass spectrometry comprises the following steps:
(1) preparation of mixed control solution: accurately weighing leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levisticolide A, costunolide and dehydrocostunolide as reference substancesAdding 70% methanol solution to obtain solutions with concentrations of 20.80, 137.50, 5.00, 280.00, 78.00, 20.00, 30.60 and 22.40 μ g/mL-1Mixed control solution of (4). That is, every 1mL of control solution contains 20.80, 137.50, 5.00, 280.00, 78.00, 20.00, 30.60 and 22.40 μ g of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levigatone A, costunolide and dehydrocostunolide respectively, the solvent is 50% ethanol, and the solution is filtered by 0.22 μm microporous membrane;
(2) preparation of a test solution: precisely weighing 0.2g of the ground compound motherwort particles, placing in a 50mL conical flask with a plug, precisely transferring 25mL of 50% ethanol, weighing, ultrasonically extracting for 45min at the working frequency of 40kHz and the power of 500W, placing at room temperature, weighing again, adding 50% ethanol to supplement the loss weight, shaking up, filtering through a microporous filter membrane (0.45 mu m), and taking the subsequent filtrate for later use. Sucking 0.2mL of the subsequent filtrate, placing the subsequent filtrate in a 10mL volumetric flask, diluting the subsequent filtrate to a scale with 50% ethanol, and shaking up to obtain the final product;
(3) chromatographic condition and system adaptability test: chromatographic condition and system adaptability test: a chromatographic column: an AgilentZorbax Eclipse plus C18 chromatographic column, taking methanol solution as a mobile phase A and water as a mobile phase B, and adopting a gradient elution mode: 0min → 3min → 6min → 10min → 12min → 14min, mobile phase a: 5% → 40% → 65% → 95% → 95% → 5%, mobile phase B: 95% → 60% → 35% → 5% → 5% → 95%; the flow rate is 0.5 mL/min; adopting a triple quadrupole mass spectrometry detector, carrying out electrospray positive ion mode, carrying out multi-reaction monitoring, and selecting m/Z312.3 → 181.0 (leonurine), 144.1 → 58.2 (stachydrine), 225.3 → 207.5 (senkyunolide H), 191.3 → 173.2 (Z-ligustilide), 193.3 → 147.3 (senkyunolide A), 381.4 → 191.2 (angelica lactone A), 233.2 → 187.3 (costunolide), 231.4 → 185.1 (dehydrocostunolide) as a detection ion pair; when the sample amount is 2 mu L, the chromatographic peak signal-to-noise ratio of the mixed control solution m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.2, 193.3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, 231.4 → 185.1 in the ion flow graph is greater than 10: 1;
(4) the determination method comprises the following steps: precisely absorbing 2 μ L of each of the mixed control solution and the test solution respectively in positive ion detection mode, injecting into liquid chromatograph, aligning leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, angelolide A, costunolide and dehydrocostuslactone chromatographic peak with mixed control solution m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.2, 193.3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, 231.4 → 185.1 ions, measuring m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.2, 193.3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, and sample solution m/z H, Z, respectively, and calculating target area of senkyunolide, thus obtaining target solution and senkyunolide by corresponding to the ion extraction method, and calculating target area of the target solution, Contents of senkyunolide A, levistilide A, costunolide and dehydrocostuslactone.
In the above examples, all compounds had a good linear relationship in the measured mass concentration range, the correlation coefficient was greater than 0.99, and the average recovery (n ═ 6) was less than 3.08%. The method provided by the invention has the advantages of good repeatability, good instrument precision and stable components in the sample solution within 24 hours. The research result provides a useful reference for qualitative and quantitative research of trace components in complex components or complex matrixes of traditional Chinese medicines, and also provides data support for clinical safety and reasonable medication.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (9)
1. A detection method of compound motherwort granules is characterized by comprising the following steps:
step one, preparing a test solution, namely grinding, extracting and removing impurities from a compound motherwort particle sample to prepare the test solution;
absorbing the test solution, and injecting the test solution into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for measurement;
the chromatographic conditions were as follows: the chromatographic column is an Agilent Zorbax Eclipse plus C18 chromatographic column, methanol is used as a mobile phase A, water is used as a mobile phase B, and a gradient elution mode is adopted: 0min → 3min → 6min → 10min → 12min → 14min, mobile phase a: 5% → 40% → 65% → 95% → 95% → 5%, mobile phase B: 95% → 60% → 35% → 5% → 5% → 95%; the flow rate is 0.4-0.6 mL/min;
the substances detected are dehydrocostuslactone, costunolide, leonurine, Z-ligustilide, senkyunolide A, senkyunolide H, stachydrine and levistilide A.
2. The detection method according to claim 1, wherein the ultra performance liquid chromatography-triple quadrupole mass spectrometer uses the following mass spectrometry conditions:
the multi-reaction monitoring is carried out by adopting a triple quadrupole mass spectrometer in an electrospray positive and negative ion mode, and m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.2, 193.3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, 231.4 → 185.1 are selected as detection ion pairs.
3. The detection method according to claim 1, wherein the flow rate is 0.5 mL/min.
4. The detection method according to any one of claims 1 to 3, characterized in that: in the first step, taking compound motherwort particles, grinding, precisely weighing 0.1-0.3 g, adding 20-25 ml of 30-70% ethanol, weighing, performing ultrasonic extraction for 40-50 min, taking out, cooling, weighing again, adding a corresponding solvent for complementing weight, sucking supernatant, filtering with a microporous filter membrane, transferring filtrate, placing in a volumetric flask, diluting to a scale with the corresponding solvent, and shaking up to obtain the compound motherwort tablet.
5. The detection method according to any one of claims 1 to 3, characterized in that: in the first step, taking compound motherwort particles, grinding, precisely weighing 0.2g of the ground compound motherwort particles, placing the particles in a 50mL conical flask with a plug, precisely transferring 25mL of 50% ethanol, weighing, performing ultrasonic treatment, performing extraction for 45min at the working frequency of 40kHz and the power of 500W, placing the solution at room temperature, weighing again, adding 50% ethanol to complement the lost weight, shaking up the solution, filtering the solution through a 0.45 mu m microporous membrane, taking the filtrate for later use, sucking 0.2mL of the subsequent filtrate, placing the subsequent filtrate in a 10mL volumetric flask, diluting the subsequent filtrate to the scale with 50% ethanol, and shaking up the subsequent to obtain the compound motherwort particles.
6. The detection method according to any one of claims 1 to 3, characterized in that: the method comprises the preparation and measurement of a control solution.
7. The detection method according to claim 6, characterized in that: the reference substance of the reference substance solution is selected from leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levistilide A, costunolide and dehydrocostunolide.
8. The detection method according to claim 7, characterized in that: the control solution was prepared as follows: respectively precisely weighing control substances of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, levisticolide A, costunolide and dehydrocostuslactone, and adding 30-70% methanol to obtain the following components: 0.015-0.057, 0.12-0.20, 0.003-0.010, 0.250-0.300, 0.05-0.09, 0.01-0.04, 0.02-0.04, 0.01-0.04 mg/mL of mixed reference solution.
9. The detection method according to claim 8, characterized in that:
precisely sucking the mixed reference solution and the test solution respectively in a positive ion detection mode, injecting the solution into a liquid chromatograph, and aligning the m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.2, 193.3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, 231.4 → 185.1 ion pairs to determine the chromatographic peaks of leonurine, stachydrine, senkyunolide H, Z-ligustilide, senkyunolide A, angelolide A, costunolide and dehydrocostunolide, and determining the chromatographic peaks of m/z 312.3 → 181.0, 144.1 → 58.2, 225.3 → 207.5, 191.3 → 173.2, 193.3 → 147.3, 381.4 → 191.2, 233.2 → 187.3, 231.4 → 185.1 ion flow diagram of the mixed reference solution and the test solution, and calculating the peak areas of leonurine, stachydrine, and senkyunolide by a corresponding method except for extracting ions in the test solution, Z-ligustilide, senkyunolide A, levistilide A, costunolide and dehydrocostuslactone.
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