Construction method of semen oroxyli formula granule UPLC specific chromatogram and application thereof
A technology of formula granules and characteristic maps, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that there is no effective method for controlling the quality of wood butterfly formula granules, and achieve the effect of an effective and rapid evaluation method
Active Publication Date: 2019-04-19
GUANGDONG YIFANG PHARMA
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Problems solved by technology
[0003] Wood Butterfly formula granules are made of single Chinese herbal medicine pieces through water extraction, concentratio...
Abstract
The invention discloses a construction method of a semen oroxyli formula granule UPLC specific chromatogram and application thereof. The construction method includes the steps of preparing a referencesubstance solution, preparing a test substance solution, adopting ultra-performance liquid chromatography to carry out measurement, building the specific chromatogram, conducting similarity evaluation and the like. By adopting the construction method, the UPLC specific chromatogram of semen oroxyli formula granules is built, and through a liquid chromatography and mass spectrometry combination technology, six common components of the semen oroxyli formula granules are qualitatively and quantitatively analyzed rapidly. The construction method is a comprehensive, systematic and effective rapidevaluation method for quality evaluation and control of the semen oroxyli formula granules.
Application Domain
Component separation
Technology Topic
ChemistrySemen +1
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Examples
- Experimental program(1)
Example Embodiment
[0031] The present invention will be further described below through specific embodiments. The following examples are specific embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples.
[0032] 1. Instruments and reagents
[0033] 1.1 Instruments
[0034] Waters LC/MS (ACQUITY UPLC H-Class for liquid phase, Waters Xevo TQDMS for mass spectrometry); 1/1000000 balance (METTLER TOLEDO, XP26), 1/10000 balance (METTLER TOLEDO, ME204E), Agilent SB C 18 Chromatographic column (2.1×100mm, 1.8μm), electric heating constant temperature water bath (Shanghai Yiheng Technology Co., Ltd., HWS-28), CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., KQ500DE), ultrapure water system (Merck Inc., Milli-Q-Direct).
[0035] 1.2 Reference substances and reagents
[0036] Acetonitrile (merck company in Germany, chromatographic grade); ethanol (Xilong Science Co., Ltd., analytical grade); methanol (Xilong Science Co., Ltd., analytical grade); phosphoric acid (Tianjin Kemeiou Chemical Reagent Co., Ltd., chromatographic grade) ; Formic acid (Tianjin Kemeiou Chemical Reagent Co., Ltd., chromatographically pure); Water is laboratory-made water.
[0037] Chrysin (Batch No.: 111701-200501), Trichoside B (Batch No.: 111915-201603, Content: 91.9%), Baicalin (Batch No.: 110715-201720, Content: 93.5%), Baicalein (Batch No.: 111595-201607 , content: 98.5%) reference substance was purchased from China National Institute for Food and Drug Control; chrysin-7-O-β-D glucuronide (batch number: wkq17072702, content: 98%) reference substance was purchased from Weiqi, Sichuan Province Biotechnology Co., Ltd.; Pteroside A (batch number: 17021703, content: 98%) reference substance was purchased from Chengdu Pfide Biotechnology Co., Ltd.
[0038] 1.3 Medicinal materials
[0039] 16 batches of wood butterfly formula granules are produced by Guangdong Yifang Pharmaceutical Co., Ltd. The preparation method is as follows: take wood butterfly decoction pieces, use water as the extraction solvent, add water to decoct twice, add 10 times the amount of water for the first time, and decoct 1.5 hour, add the water of 8 times of the amount of feed for the second time, decoct for 1 hour, merge the filtrate, filter; the filtrate is concentrated under vacuum to a clear paste with a relative density of 1.06; get the clear paste and carry out spray drying to obtain dry dipping. Extract powder and dry extract powder are dry granulated to obtain wood butterfly formula particles with a particle size of 16 to 40 meshes.
[0040] 2. Methods and Results
[0041]2.1 Preparation of mixed reference solution: Weigh chrysin, baicalein, chrysin-7-O-β-D glucuronide, baicalin A, baicalin, and cylindroside B reference substances precisely, add methanol Dissolve the volume to 25mL to obtain chrysin, baicalein, chrysin-7-O-β-D glucuronide, xyloside A, baicalin, and xyloside B. The concentrations are 12.594 μg/mL, 69.600 μg/mL, respectively. The mixed reference stock solutions of μg/mL, 32.286 μg/mL, 128.256 μg/mL, 31.512 μg/mL and 60.276 μg/mL were obtained.
[0042] 2.2 Preparation of the test solution: take the formula granules of Mu Butterfly, grind them finely, take 0.1 g, accurately weigh them, put them in a conical flask, accurately add 50 mL of 70% ethanol, weigh them, and ultrasonically treat them for 30 minutes with a power of 300 W. , the frequency is 40KHz, take it out, let it cool, use 70% ethanol to make up the lost weight, shake well, and filter through a filter membrane.
[0043] 2.3 Determination by ultra-high phase liquid chromatography
[0044] Precisely draw 1 μl of the reference solution and the test solution, respectively, inject 1 μl into the ultra-high performance liquid chromatograph, measure and record the chromatogram, wherein the chromatographic conditions are:
[0045] Use octadecylsilane-bonded silica gel as filler; use acetonitrile as mobile phase A, use 0.1% phosphoric acid as mobile phase B, and carry out gradient elution as specified in the table; the detection wavelength is 276 nm, and the flow rate is 0.28 mL/ min, the injection volume was 1 μL, and the column temperature was 30 °C;
[0046]
[0047] 3. Investigation on the preparation method of the test solution
[0048] 3.1 Investigation of extraction solvent
[0049] In this experiment, the influence of different extraction solvents on the characteristic spectrum of Mu Butterfly formula granules was investigated respectively. Methanol, 70% ethanol and ethanol were selected as extraction solvents, and the characteristic spectrum of the sample solution of different extraction solvents was determined to determine the optimal extraction solvent.
[0050] Take wood butterfly formula granules, grind them finely, take 0.1g, a total of 6 parts, accurately weigh them, put them in a conical flask, accurately add methanol, 70% ethanol, and 50ml of ethanol, weigh them, and ultrasonically treat them for 30 minutes with a power of 300W. , the frequency is 40KHz, take it out, let it cool, make up the lost weight with methanol, 70% ethanol and ethanol, shake well, and filter it with a 0.22 μm filter membrane to get it.
[0051] By comparing the chromatograms of the three different extraction solvents, it can be found that the number of chromatographic peaks in the chromatograms of the three extraction solvents is consistent with the peak shape of the chromatographic peaks, and the order of the total peak area size is: 70% ethanol>methanol>ethanol; considering each solvent comprehensively Finally, 70% ethanol was used as the extraction solvent.
[0052] 3.2 Investigation of extraction solvent method
[0053] In this experiment, the influence of different extraction methods on the characteristic map of Mu Butterfly formula granules was investigated, and two methods, heating reflux and ultrasonic treatment, were selected. The characteristic spectra of sample solutions of different extraction methods were determined to determine the best extraction method.
[0054] Take wood butterfly formula granules, grind them finely, take 0.1 g, 2 parts in total, accurately weigh them, put them in a conical flask, accurately add 50 ml of 70% ethanol, weigh them, and heat them under reflux and ultrasonic treatment respectively (power is 300W, frequency 40KHz) for 30 minutes, take it out, let it cool, make up the lost weight with 70% ethanol, shake well, and filter with a 0.22 μm filter membrane to get it.
[0055] The two extraction methods were compared, and the number of chromatographic peaks in different extraction methods was consistent with the peak shape of chromatographic peaks. Considering the simplicity of experimental operation, ultrasonic treatment was selected as the extraction method.
[0056] 3.3 Extraction time investigation
[0057] In this experiment, the effects of different extraction times on the characteristic map of Mu Butterfly formula granules were investigated, and the extraction times of ultrasonic treatment were selected as: 15 minutes, 30 minutes, and 60 minutes, respectively. The characteristic spectrum of the sample solution at different extraction times was determined to determine the optimal extraction time.
[0058] Take wood butterfly formula granules, grind them finely, take 0.1 g, a total of 3 parts, accurately weigh them, put them in a conical flask, accurately add 50 ml of 70% ethanol, weigh them, and ultrasonically treat them for 15 minutes, 30 minutes, and 60 minutes respectively. Take it out, let it cool, make up the lost weight with 70% ethanol, shake well, and filter through a 0.22 μm filter membrane.
[0059] By comparing the chromatograms of Mu Butterfly formula granules at different extraction times, it can be found that with the increase of ultrasonic treatment time, the number of chromatographic peaks in the chromatogram is consistent with the peak shape of the chromatographic peaks, and the total area of the chromatogram is basically the same as the ratio of the weighing sample, indicating that the ultrasonic treatment It can be extracted completely in 15 minutes. To ensure the durability of the method, the extraction time is selected to be 30 minutes.
[0060] 3.4 Investigation of the amount of extraction solvent
[0061] In this experiment, the effects of different extraction solvent dosages on the characteristic map of Mu Butterfly formula granules were investigated, and the extraction solvent dosages were selected as: 15ml, 25ml, 50ml and 100ml. The characteristic spectrum of the sample solution with different extraction solvent dosages was measured to determine the optimal extraction solvent dosage.
[0062] Take wood butterfly formula granules, grind them finely, take 0.1g, 4 parts in total, accurately weigh them, put them in a conical flask, add 15ml, 25ml, 50ml, 100ml of 70% ethanol precisely, weigh them, and ultrasonically treat them (power 300W, the frequency is 40KHz) for 30 minutes, take out, let it cool, supplement the lost weight with 70% ethanol, shake well, and filter with a 0.22 μm filter membrane, that is, it is obtained.
[0063] By comparing the chromatograms of wood butterfly formula granules with different amounts of extraction solvent, it can be found that the number of chromatographic peaks in the chromatogram is consistent with the peak shape of the chromatographic peaks, and the total area of the chromatogram is basically the same as the ratio of the weighing sample, indicating that the amount of extraction solvent is 15ml has been extracted completely, in order to ensure the durability of the method and select the appropriate injection concentration, the amount of extraction solvent is 50ml.
[0064] According to the above experimental results, the pre-processing method of the samples of Mu Butterfly formula granules can be determined as follows: take Mu Butterfly formula granules, grind them finely, take 0.1 g, accurately weigh them, put them in a conical flask, accurately add 50 mL of 70% ethanol, and weigh them. Determine the weight, ultrasonically treat for 30 minutes, take it out, let it cool, make up the lost weight with 70% ethanol, shake well, and filter through a filter membrane.
[0065] 4. Optimization of chromatographic conditions
[0066] 4.1 Selection of chromatographic columns
[0067] After comparison, it was found that the Agilent SB C18 column (2.1×100mm, 1.8μm) had the best separation effect and better peak shape. Therefore, the Agilent SB C18 column (2.1×100mm, 1.8μm) was selected as the chromatographic column.
[0068] 4.2 Selection of detection wavelength
[0069] A diode array detector was used to scan the sample at full wavelength, and it was found that the baseline was stable at 276 nm, and the peak area of each peak was relatively high. Therefore, the detection wavelength was set at 276 nm.
[0070] 4.3 Selection of mobile phase and elution conditions
[0071] The selection of mobile phase and elution conditions of the present invention adopts the following method to compare:
[0072] Mobile Phase Condition Optimization 1:
[0073] Use Agilent SB C18 column (2.1×100mm, 1.8μm) as the chromatographic column; use acetonitrile as mobile phase A, use 0.1% phosphoric acid as mobile phase B, and carry out gradient elution as specified in the table; the detection wavelength is 276nm, The flow rate was 0.4 mL/min, the injection volume was 1 μL, and the column temperature was 30 °C.
[0074]
[0075] Mobile Phase Condition Optimization 2:
[0076] Use Agilent SB C18 column (2.1×100mm, 1.8μm) as the chromatographic column; use acetonitrile as mobile phase A, use 0.1% phosphoric acid as mobile phase B, and carry out gradient elution as specified in the table; the detection wavelength is 276nm, The flow rate was 0.35 mL/min, the injection volume was 1 μL, and the column temperature was 30 °C.
[0077]
[0078] Mobile Phase Condition Optimization 3:
[0079] Use Agilent SB C18 column (2.1×100mm, 1.8μm) as the chromatographic column; use acetonitrile as mobile phase A, use 0.1% phosphoric acid as mobile phase B, and carry out gradient elution as specified in the table; the detection wavelength is 276nm, The flow rate was 0.28 mL/min, the injection volume was 1 μL, and the column temperature was 30 °C.
[0080]
[0081] It can be seen from the chromatogram results obtained from the three elution conditions: peak 2 in optimal condition 1 and optimal condition 2 contains other chromatographic peaks, the peak purity does not meet the requirements, and does not meet the baseline separation requirements, while optimal condition 3 Each chromatographic peak meets the baseline separation requirements, so the optimal condition 3 is selected for the experiment.
[0082] 5. Methodological investigation
[0083] 5.1 Precision test Take the test solution of wood butterfly formula granules, inject 6 times continuously under the above chromatographic conditions, measure the peak area, calculate the relative standard deviation RSD of the peak area, the result RSD<3, indicating that the precision of the instrument is good.
[0084] 5.2 Repeatability test Take the same batch of wood butterfly formula granules (batch number: 8016072), prepare 6 test solutions in parallel according to the test preparation method, inject samples according to the above chromatographic conditions, measure the peak area, and calculate the relative standard deviation RSD of the peak area. , the results RSD<3, the experimental results show that the method has good reproducibility.
[0085] 5.4 Stability test Precisely draw the same test solution, inject samples after 0, 2, 4, 6, 8, and 12 hours, measure the peak area, and calculate the relative standard deviation RSD of the peak area. The result RSD is less than 3. The test solution has good stability within 12h.
[0086] 6. Establishment of characteristic map of wood butterfly formula particles
[0087] Take 16 batches of wood butterfly formula granules samples, prepare the test solution according to the method under "2.2", inject 1 μL, and determine the chromatogram. The spectrum was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching, and the characteristic spectrum of Mu Butterfly Formula Granules was established. figure 1 , the generated common schema see figure 2.
[0088] Taking the No. 1 peak as the reference peak, the 6 common peaks in the UPLC-TUV chromatogram of the wood butterfly formula granule are marked, and the sum of the peak areas of the common peaks is greater than 90%. Calculate the RSD value of the relative retention time of each chromatographic peak and the reference peak. The results showed that the relative retention time RSD values of the common peaks in each chromatogram of the samples were all less than 3.0%; while the RSD values of the relative peak areas were quite different, see Table 1 and Table 2.
[0089] Table 1 Relative retention time of common peaks of 16 batches of wood butterfly characteristic maps
[0090]
[0091]
[0092] Table 2 Relative peak areas of common peaks in 16 batches of wood butterfly characteristic maps
[0093]
[0094] The similarity of 16 batches of wood butterfly formula granules samples were calculated respectively, as shown in Table 3. The similarity of 16 batches of wood butterfly formula particles was greater than 0.90, indicating that the consistency of chemical composition of wood butterfly formula particles was good and the quality was relatively stable.
[0095] Table 3 16 batches of wood butterfly formula particle similarity
[0096]
[0097]
[0098] 7. Characteristic spectrum peak identification:
[0099] The 6 common peak components were confirmed by UPLC-Q-TOF-MS method. The chromatographic conditions were as follows: use octadecylsilane-bonded silica gel as filler; use acetonitrile as mobile phase A, and use 0.01% formic acid as mobile phase B , carry out gradient elution as specified in the table; the detection wavelength is 276 nm, the flow rate is 0.28 mL/min, the injection volume is 1 μL, and the column temperature is 30 °C;
[0100]
[0101] Mass spectrometry conditions were: electrospray ionization (ESI + ), the spray voltage was 1.55kV; the desolvation temperature was 597°C, and the ion source temperature was 148°C; the desolvation gas flow was 997 L/hr; the nitrogen flow was 1 L/hr; Optimization of Cone parameters and Collision mass spectrometry parameters to tune each standard compound by means of peristaltic needle pump injection. The obtained parameters are shown in Table 4.
[0102] Table 4 Mass spectrometry parameters of standard compounds detected by LC-MS
[0103]
[0104] Remarks: There are "*" for quantitative ions.
[0105] 8. Content determination:
[0106] 8.1 Linear relationship Precisely measure the mixed reference solution under item "2.1", dilute it step by step according to the dilution ratio of 1, 1.2, 1.5, 2, 3, 6, and set the volume to obtain a series of mixed controls with 6 concentration gradients The sample solution was measured according to the chromatographic conditions and mass spectrometry conditions in "7", and the peak area was used to perform linear regression on the concentration of the injection volume, and a standard curve was drawn to obtain the regression equation, correlation coefficient, and linear range of each component, as shown in Table 5.
[0107] Table 5 The results of the linear relationship investigation of the 6 components in the Mu Butterfly formula granules
[0108]
[0109] 8.2 According to the preparation method of the test solution in "2.2" and the chromatographic conditions and mass spectrometry conditions in "7", calculate the chrysin, baicalein, chrysin-7-O-β- The contents of 6 components of D-glucuronide, styroside A, baicalin and styroside B, the results are shown in Table 6.
[0110] Table 6 sample content determination results (mg g -1 , n=2)
[0111]
PUM
| Property | Measurement | Unit |
| Granularity | 16.0 ~ 40.0 | mesh |
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