CN108896684B - Method for determining oleanolic acid in laryngopharynx cleaning granules by using HPLC (high performance liquid chromatography) - Google Patents
Method for determining oleanolic acid in laryngopharynx cleaning granules by using HPLC (high performance liquid chromatography) Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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Abstract
The invention discloses a method for determining oleanolic acid in laryngopharynx cleaning particles by using an HPLC (high performance liquid chromatography) method, which comprises the steps of preparation of a reference substance solution, determination of chromatographic conditions and a standard curve, preparation and determination of a test substance solution and the like. The invention can more effectively control the quality of the laryngopharynx cleaning granules by using an HPLC method and ensure the curative effect.
Description
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a method for determining oleanolic acid in laryngopharynx cleaning particles by using HPLC.
Background
The granule for clearing throat and pharynx is prepared by adding sucrose and dextrin into four medicinal materials of achyranthes aspera, malan grass, plantain herb and radix angelicae pubescentis, has the effects of clearing heat and removing toxicity, relieving sore throat and relieving pain, and is used for treating sore throat, fever, thirst, constipation, tonsillitis and acute pharyngitis caused by lung and stomach excess heat. In the prior art, the quality of the laryngopharynx cleaning particles is controlled by controlling the quality of the oleanolic acid in the laryngopharynx cleaning particles through thin-layer chromatography, the stability, the accuracy and the like of the method cannot meet the prior technical requirements, and the report of controlling the quality of the laryngopharynx cleaning particles by high performance liquid chromatography is not available, so that the HPLC (high performance liquid chromatography) determination method for the oleanolic acid in the laryngopharynx cleaning particles is significant for more effectively controlling the quality of the laryngopharynx cleaning particles and ensuring the curative effect of the laryngopharynx cleaning particles.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for determining oleanolic acid in laryngopharynx cleaning particles by using HPLC.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the method for determining oleanolic acid in laryngopharynx cleaning particles by using HPLC comprises the following steps:
(1) preparation of control solutions: precisely weighing oleanolic acid reference substance, adding methanol to obtain solution containing 0.25mg of oleanolic acid per 1ml to obtain reference substance solution;
(2) determination of chromatographic conditions and standard curves: octadecylsilane chemically bonded silica is used as a filling agent; mixing acetonitrile: methanol: glacial acetic acid with volume percentage concentration of 0.5% is used as mobile phase according to volume ratio of 45:10, and the reference substance solution diluted in gradient is injected into a liquid chromatograph, and is measured under the condition of wavelength of 210 nm; taking the concentration of the diluted reference solution as an abscissa x and taking the peak area integral value as an ordinate y to obtain a standard curve: y 6064.3x +37429, R2 0.9997; wherein the unit of the abscissa x is mu g/mL, and the unit of the ordinate y is AU;
(3) preparation of a test solution: grinding laryngopharynx clearing granules, precipitating with ethanol, hydrolyzing, and extracting to obtain test solution;
(4) and (3) determination: octadecylsilane chemically bonded silica is used as a filling agent; mixing acetonitrile: methanol: 0.5% (volume percentage concentration) of glacial acetic acid is used as a mobile phase according to the volume ratio of 45:10, the sample solution is injected into a liquid chromatograph, the measurement is carried out under the condition of 210nm wavelength, and the oleanolic acid content in the laryngopharynx cleaning particles is obtained by calculation.
Preferably, in step (2), the number of theoretical plates in the control solution is determined to be not less than 4000 in terms of oleanolic acid.
Preferably, in step (3), the preparation of the test solution is specifically as follows: taking laryngopharynx cleaning particles, grinding, taking 8g, precisely weighing, placing in a beaker, adding 50ml of boiling water to dissolve, cooling, transferring to a 100ml measuring flask, washing the beaker with a small amount of absolute ethyl alcohol, merging the washing solution into the measuring flask, adding absolute ethyl alcohol to scale, shaking while adding, standing for 30min, centrifuging for 5min under the condition of 8000r/min, precisely taking 50ml of supernatant, adding 7ml of hydrochloric acid, heating and refluxing for 30min, cooling, transferring to a separating funnel, shaking and extracting for 6 times with petroleum ether at 60-90 ℃ with 25ml each time, combining the petroleum ether solutions, evaporating to dryness, adding methanol to dissolve the residue, transferring to a 10ml measuring flask, adding methanol to scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a test solution.
The invention can more effectively control the quality of the laryngopharynx cleaning granules by using an HPLC method and ensure the curative effect.
Drawings
FIG. 1 is a chromatogram of an oleanolic acid control solution;
fig. 2 is a mobile phase: chromatogram of acetonitrile-methanol-0.5% glacial acetic acid (45: 10);
fig. 3 is a mobile phase: chromatogram of acetonitrile-methanol-0.5% ammonium acetate solution (67: 12: 21);
FIG. 4 is a standard graph of oleanolic acid;
FIG. 5 is a chromatogram of an oleanolic acid control;
FIG. 6 is a chromatogram of laryngopharynx clearing granules (batch number: 20170203);
FIG. 7 is a negative control chromatogram.
Detailed Description
1 Instrument and reagent
The instrument comprises the following steps: waters e2695 HPLC chromatograph 2998PDA
A chromatographic column: MG II C18(250 mm. times.4.6 mm. times.5 μm) column temperature: 35 deg.C
Oleanolic acid (batch: 110709 201607, purity: 91.7%, manufacturer: China institute for testing food and drug)
The methanol and the acetonitrile are chromatographically pure, and other reagents are analytically pure.
1.1 preparation of control solutions
(1) Precisely weighing 11.14mg of oleanolic acid reference substance, placing in a 10ml measuring flask, adding an appropriate amount of methanol for dissolving, adding methanol for diluting to scale, and shaking uniformly to obtain a reference substance solution I (1.114 mg/ml);
(2) accurately weighing oleanolic acid reference substance 12.29mg, placing in a 25ml measuring flask, adding appropriate amount of methanol for dissolving, adding methanol for diluting to scale, shaking to obtain reference substance solution 2 (0.4916 mg/ml);
(3) precisely weighing 29.46mg of oleanolic acid reference substance, placing in a 50ml measuring flask, adding appropriate amount of anhydrous ethanol for dissolving, adding anhydrous ethanol for diluting to scale, shaking to obtain reference substance solution (0.5892mg/ml), and performing recovery experiment;
1.2 selection of chromatographic conditions
1.2.1 selection of measurement wavelength
A control solution is taken, an ultraviolet-visible spectrophotometry (four parts of general rules 0401 of Chinese pharmacopoeia 2015 edition) is adopted for sample injection, the sample is measured, a spectrogram in the wavelength range of 190-300 nm is collected, and as a result, oleanolic acid has the maximum absorption at the wavelength of 200nm (see figure 1), and the detection wavelength of the product is selected to be 210nm according to the measurement wavelength of oleanolic acid under the content measurement item of loquat leaves in Chinese pharmacopoeia 2015 edition.
1.2.2 selection of Mobile phase
The investigation is carried out by referring to the mobile phase adopted in the content determination method under the item of loquat leaf in the oral liquid for laryngopharynx clearing and the section of Chinese pharmacopoeia 2015 edition I. Two mobile phases of acetonitrile-methanol-0.5% glacial acetic acid (45: 10) and mobile phase of acetonitrile-methanol-0.5% ammonium acetate solution (67: 12: 21) are respectively adopted, 10 mu l of reference solution is precisely absorbed and injected into a liquid chromatograph for determination. The chromatogram thereof is shown in FIGS. 2 and 3. The parameters of the chromatographic peaks are shown in Table 1.
Table 1 flow comparison table
| Mobile phase | Retention time (min) | Number of theoretical plates |
| Acetonitrile-methanol-0.5% glacial acetic acid (45: 10) | 16.105 | 15742 |
| Acetonitrile-methanol-0.5% ammonium acetate solution (67: 12: 21) | 14.904 | 10676 |
As can be seen from fig. 2, fig. 3 and table 1, chromatograms of the first mobile phase and the second mobile phase can achieve baseline separation, the peak pattern is good, the difference of the number of the tower plates is small, but the ammonium acetate mobile phase is easy to cause the blockage of the instrument pipeline, so the mobile phase is selected: further experiments were carried out with acetonitrile-methanol-0.5% glacial acetic acid (45: 10).
In summary, the chromatographic conditions were selected as follows:
detection wavelength: 210nm mobile phase: acetonitrile-methanol-0.5% glacial acetic acid solution (45:45: 10).
1.3 Linear relationship investigation
Precisely sucking appropriate amount of control solutions (see Table 4) and diluting, precisely taking 10 μ l of filtrate, injecting into liquid chromatograph, measuring, and drawing standard curve with peak area integral value as ordinate and diluted concentration (μ g/ml) as abscissa. The regression equation is: 6064.3x +37429, R20.9997 (fig. 4). The results show that: the oleanolic acid control has good linear relation in the range of 39.32-1114 μ g/ml, and the results are shown in Table 2.
TABLE 2 table of results of linear relationship test
1.4 preparation of test solutions
1.4.1 selection of test sample extraction method
The preparation method is prepared by referring to the content determination method of the oral liquid for laryngopharynx clearing, and comprises three steps of alcohol precipitation, hydrolysis and extraction, and the following preparation methods of test solution are examined for simplifying the operation steps:
an appropriate amount of the 20170203 lot was ground and subjected to the following experiment.
(1) Taking about 8g of the fine powder, placing the fine powder in a beaker, adding 50ml of boiling water to dissolve the fine powder, cooling the fine powder, transferring the fine powder in a 100ml volumetric flask, washing the beaker by using a small amount of absolute ethyl alcohol, transferring the beaker into the volumetric flask, adding absolute ethyl alcohol to a scale, shaking the beaker while adding the absolute ethyl alcohol, standing the beaker for 30 minutes, centrifuging the beaker (the rotating speed is 8000 revolutions per minute) for 5 minutes, precisely measuring 50ml of supernatant, adding 7ml of hydrochloric acid, heating and refluxing the solution for 30 minutes, cooling the beaker, transferring the beaker in a separating funnel, shaking and extracting the beaker by using petroleum ether (60-90 ℃) for 6 times, wherein 25ml of the solution is obtained each time, combining the petroleum ether solution, evaporating the residue to dryness, adding methanol to the residue to dissolve the residue, transferring the residue to a 10ml bottle, adding the methanol to the scale.
(2) Taking about 8g of the fine powder, placing the fine powder in a beaker, adding 50ml of boiling water to dissolve the fine powder, cooling the fine powder, transferring the fine powder in a 100ml volumetric flask, washing the beaker by using a small amount of absolute ethyl alcohol, transferring the beaker into the volumetric flask, adding absolute ethyl alcohol to scale, shaking the beaker while adding the absolute ethyl alcohol, standing the beaker for 30 minutes, centrifuging the beaker (the rotating speed is 8000 revolutions per minute) for 5 minutes, precisely measuring 50ml of supernate, adding 7ml of hydrochloric acid, heating and refluxing the beaker for 30 minutes, cooling the beaker, adding 50% of ethyl alcohol to complement and reduce the weight loss, shaking the beaker evenly, filtering the beaker, and taking a subsequent filtrate to obtain a sample solution.
(3) And (3) taking about 8g of the fine powder, placing the fine powder in a conical flask, adding 100ml of 5% methanol hydrochloride solution, heating and refluxing for 30 minutes, cooling, filtering, concentrating the filtrate to about 50ml, and enabling the residue to be scorched and not to be transferred to be subjected to the next experiment.
(4) Taking about 8g of the fine powder, placing the fine powder in an erlenmeyer flask, adding 50% ethanol 50ml and hydrochloric acid 7ml, heating and refluxing for 30 minutes, cooling, filtering, transferring the filtrate into a separating funnel, and shaking and extracting by using petroleum ether (60-90 ℃)50ml, wherein the emulsification is very serious and the delamination cannot be realized.
(5) The test solution is prepared by referring to the content determination method in the current quality standard of 'throat clearing granules':
taking about 6g of the fine powder, precisely weighing, adding 100ml of ethanol, placing on a water bath, heating, refluxing and extracting for 40 minutes, cooling, filtering, adding ethanol into the medicine residues, repeatedly refluxing for 1 time, filtering, washing the filter and the residues with a small amount of ethanol for multiple times, combining the washing liquor and the two filtrates, recovering ethanol to about 30ml, cooling, adding 5ml of hydrochloric acid, placing in the water bath, heating, refluxing for 1 hour, cooling, transferring into a separating funnel, shaking and extracting for 4 times with petroleum ether (60-90 ℃) and 20ml each time, combining the petroleum ether solutions, evaporating to dryness, adding methanol into the residues for dissolving, transferring into a 50ml volumetric flask, adding methanol to the scale, shaking uniformly, filtering, and taking the subsequent filtrate to obtain a sample solution.
The test solutions (i), (ii) and (iii) were measured, respectively, and the results are shown in Table 3:
table 3 extraction method selection results
| Test solution 1 | |
Solution of sample | |
| Results (mg/g) | 0.942 | 0.947 | 0.879 |
As can be seen from the above, the first and second test solutions are completely extracted, while the second test solution is simple and convenient, but the solution contains a large amount of acid, which causes great damage to the liquid chromatograph and the chromatographic column, and the first test solution is prepared by a method with few impurities after extraction.
1.4.2 selection of sample size
5g, 8g and 10g of the fine powder were prepared by a preparation method of a test solution I, and the measurement results are shown in Table 4:
TABLE 4 sample size selection results
| Sample volume (g) | 5.0472 | 8.0896 | 10.0647 |
| Results (mg/g) | 0.938 | 0.932 | 0.841 |
As can be seen from the above, the results of the 5g and 8g samples are basically consistent, but since the baseline noise is large when the 5g samples are taken, the result is easy to cause error, and 8g is selected as the sample.
1.4.3 selection of extraction times
Taking three parts of the fine powder, 8g of each part, preparing by a preparation method of a test solution I, and respectively extracting by shaking petroleum ether (60-90 ℃) for 4 times, 6 times and 8 times, wherein the measurement results are shown in a table 5:
TABLE 5 selection of extraction times
| Number of extractions | 4 | 6 | 8 |
| Results (mg/g) | 0.892 | 0.935 | 0.939 |
As can be seen from the above, the results obtained after 6 and 8 extractions are substantially the same, and 6 extractions are selected to simplify the operation steps.
1.4.4 selection of the amount of hydrochloric acid added
About 8g of each of the three parts of the fine powder is taken, prepared by a preparation method of a test solution I, 5ml, 7ml and 9ml of hydrochloric acid are added respectively, and the measurement results are shown in Table 6:
TABLE 6 hydrochloric acid addition selection results
| Hydrochloric acid addition (ml) | 5 | 7 | 9 |
| Results (mg/g) | 0.784 | 0.941 | 0.934 |
As can be seen from the above, the results were substantially the same when 7ml and 9ml of hydrochloric acid were added, and 7ml of hydrochloric acid was selected for reducing the amount of hydrochloric acid added.
In summary, the preparation method of the test solution is determined as follows: taking the product under the items of different loading amounts, grinding, taking about 8g, placing the product in a beaker, adding 50ml of boiling water to dissolve the product, cooling the product, transferring the product to a 100ml volumetric flask, washing the beaker by using a small amount of absolute ethyl alcohol and transferring the beaker to the volumetric flask, adding absolute ethyl alcohol to the scale, shaking the beaker while adding the absolute ethyl alcohol, standing the beaker for 30 minutes, centrifuging the beaker (the rotating speed is 8000 turns per minute) for 5 minutes, precisely measuring 50ml of supernatant, adding 7ml of hydrochloric acid, heating and refluxing for 30 minutes, cooling the beaker, transferring the beaker to a separating funnel, shaking and extracting the beaker for 6 times by using petroleum ether (60-90 ℃) and 25ml each time, merging the petroleum ether liquid, evaporating the residue to dryness, adding methanol to dissolve the residue, transferring the residue to a 10ml volumetric flask, adding methanol to the scale, shaking the filtrate uniformly, and filtering the.
1.5 negative sample assay
Precisely weighing the medicinal materials except radix Achyranthis, preparing into radix Achyranthis negative sample, mixing with the sample solution to obtain radix Achyranthis negative control solution, and determining by the method.
As a result: in the chromatogram of the negative control, no chromatographic peak is present at the same retention time as the chromatographic peak of the oleanolic acid control, indicating that the negative sample is not interfering (FIGS. 5-7).
1.6 precision test
Precisely sucking the reference solution III, sampling for 6 times, and measuring by 10 μ l each time, wherein the RSD of the peak area integral value of oleanolic acid is 1.30%, and the result shows that the precision of the instrument is good, see Table 7.
TABLE 7 precision test results table
1.7 solution stability test
The same sample solution (added with 7ml of hydrochloric acid) under item 1.4.4 was taken, and 10. mu.l of the solution was precisely aspirated at 0, 4, 8, 12, 18, and 24 hours after the preparation, and injected into a liquid chromatograph, and the results of the measurement are shown in Table 8.
Table 8 table of stability test results
The results show that: the test solution was stable within 24 hours after formulation.
1.8 repeatability test
An appropriate amount of 20170203 lot sample was mixed, ground, weighed precisely, and measured in total 6 parts by the method, and the results are shown in Table 9.
TABLE 9 results of the repeatability tests
The results show that: the method has good repeatability.
1.9 recovery test
Taking 6 parts of samples of the same batch with the measured content, wherein the samples are about 4g (containing 0.932mg/g of oleanolic acid), accurately weighing the samples respectively, adding 50ml of boiling water into a beaker for dissolving, cooling the beaker, transferring the beaker into a 100ml measuring flask, washing the beaker by using a small amount of absolute ethyl alcohol, adding the washing liquid into the measuring flask, accurately adding 5ml of control solution (0.5892mg/ml) respectively, adding the absolute ethyl alcohol to the scale, and preparing the samples according to the preparation method of the test solution. The results of the measurement according to the content measurement method are shown in Table 10.
TABLE 10 table of the results of the recovery test
The results show that: the method has good sample recovery rate.
1.10 comparison of measurement results of samples
The results of the assay performed on samples provided by the manufacturing companies are shown in table 11:
TABLE 11 table of the results of measuring the oleanolic acid content in laryngopharynx cleaning granules
1.12 Standard
In conclusion, the method for determining the oleanolic acid content in the laryngopharynx clearing granules by the HPLC is drawn up as follows:
[ CONTENT DETERMINATION ] is determined by high performance liquid chromatography (0512 in the four Ministry of pharmacopoeia 2015).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; eluting with acetonitrile-methanol-0.5% glacial acetic acid (45: 10); the detection wavelength was 210 nm. The number of theoretical plates is not less than 4000 calculated according to oleanolic acid.
Preparing reference solution by accurately weighing appropriate amount of oleanolic acid reference, and adding methanol to obtain solution containing 0.25mg of oleanolic acid per 1 ml.
Preparation of test solution the product under the condition of different loading amount is taken, ground, about 8g is taken, precisely weighed, placed in a beaker, added with 50ml of boiling water to dissolve, cooled, transferred to a 100ml measuring flask, the beaker is washed by a small amount of absolute ethyl alcohol, the washing solution is merged into the measuring flask, the absolute ethyl alcohol is added to the scale mark while shaking, kept stand for 30 minutes, centrifuged (the rotating speed is 8000 rpm) for 5 minutes, the supernatant is precisely taken, 7ml of hydrochloric acid is added, heated and refluxed for 30 minutes, cooled, transferred to a separating funnel, shaken and extracted for 6 times by petroleum ether (60-90 ℃) with 25ml each time, the petroleum ether solution is merged and evaporated to dryness, the residue is added with methanol to dissolve, transferred to a 10ml measuring flask, the methanol is added to the scale mark, shaken uniformly and filtered, and the subsequent filtrate is taken, thus obtaining the test solution.
The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
1.13 durability test
The results of the laboratory internal measurements are shown in table 12:
TABLE 12 comparative table of durability test results
As can be seen from the above table, the durability test results of the content measurement method are better.
Claims (2)
1. A method for determining oleanolic acid in laryngopharynx cleaning granules by using an HPLC method is characterized by comprising the following steps:
(1) preparation of control solutions: precisely weighing oleanolic acid reference substance, adding methanol to obtain solution containing 0.25mg of oleanolic acid per 1ml to obtain reference substance solution;
(2) determination of chromatographic conditions and standard curves: octadecylsilane chemically bonded silica is used as a filling agent; mixing acetonitrile: methanol: taking 0.5% glacial acetic acid as mobile phase according to volume ratio of 45:10, injecting the reference substance solution diluted in gradient into liquid chromatograph, and measuring at wavelength of 210 nm; and taking the concentration of the reference solution as an abscissa x and the peak area integral value as an ordinate y to obtain a standard curve: y 6064.3x +37429, R2 0.9997; wherein the unit of the abscissa x is mu g/mL, and the unit of the ordinate y is AU;
(3) preparation of a test solution: grinding laryngopharynx clearing granules, precipitating with ethanol, hydrolyzing, and extracting to obtain test solution;
(4) and (3) determination: octadecylsilane chemically bonded silica is used as a filling agent; mixing acetonitrile: methanol: glacial acetic acid with volume percentage concentration of 0.5% is used as mobile phase according to volume ratio of 45:10, the sample solution is injected into a liquid chromatograph, the measurement is carried out under the condition of 210nm wavelength, and the oleanolic acid content in the laryngopharynx cleaning particles is obtained by calculation;
the preparation process of the test solution comprises the following steps: taking laryngopharynx cleaning particles, grinding, taking 8g, precisely weighing, placing in a beaker, adding 50ml of boiling water to dissolve, cooling, transferring to a 100ml measuring flask, washing the beaker with a small amount of absolute ethyl alcohol, merging the washing solution into the measuring flask, adding absolute ethyl alcohol to scale, shaking while adding, standing for 30min, centrifuging for 5min under the condition of 8000r/min, precisely taking 50ml of supernatant, adding 7ml of hydrochloric acid, heating and refluxing for 30min, cooling, transferring to a separating funnel, shaking and extracting for 6 times by petroleum ether at 60-90 ℃ with shaking of 25ml each time, combining the petroleum ether solutions, evaporating to dryness, adding methanol to dissolve residues, transferring to a 10ml measuring flask, adding methanol to scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a test solution.
2. The method of claim 1, wherein in step (2), the number of theoretical plates in the determination of the control solution is not less than 4000, calculated as oleanolic acid.
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