CN114034794B - Method for determining multi-component content of heat-clearing and detoxifying oral liquid - Google Patents
Method for determining multi-component content of heat-clearing and detoxifying oral liquid Download PDFInfo
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- CN114034794B CN114034794B CN202111328711.8A CN202111328711A CN114034794B CN 114034794 B CN114034794 B CN 114034794B CN 202111328711 A CN202111328711 A CN 202111328711A CN 114034794 B CN114034794 B CN 114034794B
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Abstract
The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly discloses a method for determining the content of a heat-clearing and detoxifying oral liquid. The quality detection method has the advantages of convenient operation, stability, reliability, strong specificity, high sensitivity and good reproducibility, can comprehensively and effectively control the quality of the heat-clearing and detoxifying oral liquid, and ensures the safety and effectiveness of clinical medication.
Description
Technical Field
The invention relates to a content determination method of a heat-clearing and detoxifying oral liquid, in particular to a UPLC-based multi-component content determination method of the heat-clearing and detoxifying oral liquid, and belongs to the field of analysis of traditional Chinese medicine preparations.
Background
The oral liquid for clearing heat and removing toxicity is collected in the first part of 2020 edition of Chinese pharmacopoeia, is extracted from 12 Chinese medicinal materials of gypsum, honeysuckle, figwort root, rehmannia root, weeping forsythia, cape jasmine fruit, Chinese violet, baical skullcap root, gentian root, indigowoad root, common anemarrhena rhizome and dwarf lilyturf tuber, has the efficacy of clearing heat and removing toxicity, and is used for treating fever and flushed face, dysphoria and thirst and sore throat caused by excessive heat toxin; the clinical effect is obvious for patients with influenza and upper respiratory tract infection with the symptoms.
At present, the current examination items about the heat-clearing and detoxifying preparation in the first part of the' 2020 edition of Chinese pharmacopoeia mainly comprise the thin-layer identification of chlorogenic acid, geniposide, phillyrin, sarsasapogenin and radix ophiopogonis and the content measurement of baicalin. The traditional Chinese medicine compound preparation has complex components, plays roles in multiple targets and multiple levels, only uses thin-layer chromatography to carry out qualitative identification on the components, and uses high performance liquid chromatography to carry out content measurement on the effective components of only one component of baicalin, and is not enough to comprehensively control the quality of the heat-clearing and detoxifying oral liquid. At present, the related patent publications and literature reports for determining the content of the heat-clearing and detoxifying oral liquid are less, Zhang Zhe Wei and the like, and the determination of the content of 4 components in the heat-clearing and detoxifying oral liquid by using a one-test-and-multiple-evaluation method only quantifies the effective components in ministerial drugs. At present, no patent publications and literature reports exist for the research on the content determination of more components in the heat-clearing and detoxifying oral liquid.
Ultra Performance Liquid Chromatography (UPLC) covers brand new technologies such as small particle packing, very low system volume, rapid detection means and the like by means of an HPLC principle, increases the flux of analysis and chromatographic peak capacity, has the characteristics of simplicity, accuracy, high sensitivity, good repeatability, multiple detector types and the like, and is one of important methods for constructing determination of the content of multiple components of a preparation. The invention discloses a UPLC method for simultaneously measuring multiple components of a heat-clearing and detoxifying oral liquid, which realizes accurate quantification of 12 components in a preparation and comprehensively evaluates and controls the quality of the heat-clearing and detoxifying oral liquid, thereby ensuring the stability of the product quality and the safety and effectiveness of clinical medication and providing data reference for improving the quality standard of the heat-clearing and detoxifying oral liquid.
Disclosure of Invention
The invention aims to solve the technical problem of further optimizing a preparation detection method, simplifying an operation method and improving the detection efficiency on the basis of 2020 version Chinese pharmacopoeia. The inventor successfully integrates UPLC content determination of 12 components of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythoside A, isochlorogenic acid C, baicalin, phillyrin and wogonin in the preparation into a whole through a large number of tests, and provides a new method for detecting the heat-clearing and detoxifying oral liquid more comprehensively and accurately.
The invention aims to provide a content determination method of a heat-clearing and detoxifying oral liquid, which is extracted from twelve traditional Chinese medicines of gypsum, honeysuckle, figwort root, rehmannia root, weeping forsythia, gardenia, Chinese violet, scutellaria, gentiana, isatis root, anemarrhena rhizome and dwarf lilyturf tuber. Wherein the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the isochlorogenic acid A, the isochlorogenic acid B and the isochlorogenic acid C belong to the components in honeysuckle, the gentiopicroside is the component in gentian, the geniposide is the component in gardenia, the forsythiaside A and the forsythin are the components in forsythia, and the baicalin and the wogonoside are the components in scutellaria baicalensis. The invention adopts UPLC method to simultaneously determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, gentiopicroside, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythoside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside in the preparation, realizes the comprehensive evaluation and control of the quality of the heat-clearing and detoxifying oral liquid, and thus provides accurate basis for the true and false identification and the internal quality detection of the heat-clearing and detoxifying oral liquid. The method specifically comprises the following steps:
A. preparation of a test solution: precisely measuring 1ml of the heat-clearing and detoxifying oral liquid, placing the heat-clearing and detoxifying oral liquid in a 50ml volumetric flask, fixing the volume to a scale with 50% -90% methanol, shaking up, and filtering to obtain a sample solution;
B. preparation of control solutions: precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside as reference substances, placing in a 20ml volumetric flask, adding 50-90% methanol to dissolve, fixing the volume to the scale, and shaking uniformly to obtain the final product;
C. UPLC chromatographic conditions: waters CORTECS T3C 18 Chromatography column, purified in acetonitrile: taking methanol as a mobile phase A and a glacial acetic acid solution as a mobile phase B, and performing gradient elution; detection wavelength: 230-250 nm; flow rate: 0.2-0.4 ml/min; column temperature: 30-40 ℃;
D. the determination method comprises the following steps: and D, precisely sucking 2 mu l of the reference substance solution and the test solution respectively, injecting into an ultra-high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.
Preferably, step a is performed by adding 70% methanol to the volume to the mark and shaking up.
Preferably, in the step B, a proper amount of chlorogenic acid, cryptochlorogenic acid, gentiopicroside, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside reference substances are precisely weighed, placed in a volumetric flask, dissolved and diluted to a scale by adding 70% of methanol, and shaken up.
Preferably, the size of the chromatographic column in the step C is 2.1X 100mm, and the particle size is 1.6 μm.
Preferably, step C the mobile phase a acetonitrile: the ratio of methanol was 3: 1.
Preferably, the gradient elution conditions in step C are:
preferably, the detection wavelength in step C is 240nm, the flow rate is 0.3ml/min, and the column temperature is 35 ℃.
Compared with the prior art, the invention has the following beneficial technical effects:
1) according to the invention, the content determination of 12 components in the heat-clearing and detoxifying oral liquid cannot be realized through the prior art, and a method for detecting multiple evaluations and simultaneously detecting 12 components in the heat-clearing and detoxifying oral liquid is established through a large number of experiments, so that the operation method is simplified, the detection efficiency is improved, consumables and labor cost are saved, and therefore, the detection method which is more rapid, convenient, energy-saving and environment-friendly is provided.
2) The invention establishes a method for measuring the content of multiple components of the heat-clearing and detoxifying oral liquid, and adopts UPLC to quantitatively measure the content of 12 components of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, gentiopicroside, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythoside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside in the preparation, so that the quality of the heat-clearing and detoxifying oral liquid can be further effectively controlled, and the quality of the preparation can be comprehensively evaluated.
3) Compared with the existing method for measuring the content of the heat-clearing and detoxifying oral liquid, the method has the advantages of rapidness, sensitivity, good repeatability and the like, can accurately measure 12 components in the Xintong oral liquid, has strong specificity and good reproducibility, provides a new method for more comprehensively and accurately detecting and evaluating the stability of the heat-clearing and detoxifying oral liquid, is favorable for stabilizing the quality of a product, and ensures the safety and the effectiveness of clinical medication.
Drawings
FIG. 1 is a UPLC chromatogram of a sample
Wherein, the No. 1 peak is new chlorogenic acid, the No. 2 peak is chlorogenic acid, the No. 3 peak is cryptochlorogenic acid, the No. 4 peak is gentiopicrin, the No. 5 peak is geniposide, the No. 6 peak is isochlorogenic acid B, the No. 7 peak is isochlorogenic acid A, the No. 8 peak is forsythiaside A, the No. 9 peak is isochlorogenic acid C, the No. 10 peak is baicalin, the No. 11 peak is forsythin, and the No. 12 peak is wogonoside.
FIG. 2 is a UPLC chromatogram of a mixed control
Wherein, the No. 1 peak is new chlorogenic acid, the No. 2 peak is chlorogenic acid, the No. 3 peak is cryptochlorogenic acid, the No. 4 peak is gentiopicrin, the No. 5 peak is geniposide, the No. 6 peak is isochlorogenic acid B, the No. 7 peak is isochlorogenic acid A, the No. 8 peak is forsythiaside A, the No. 9 peak is isochlorogenic acid C, the No. 10 peak is baicalin, the No. 11 peak is forsythin, and the No. 12 peak is wogonoside.
FIG. 3 is a UPLC chromatogram of negative control lacking honeysuckle
Wherein, the No. 4 peak is gentiopicrin, the No. 5 peak is jasminoidin, the No. 8 peak is forsythoside A, the No. 10 peak is baicalin, the No. 11 peak is forsythin, and the No. 12 peak is wogonoside.
FIG. 4 shows a UPLC chromatogram of Gentiana scabra negative control
Wherein, the peak 1 is new chlorogenic acid, the peak 2 is chlorogenic acid, the peak 3 is cryptochlorogenic acid, the peak 5 is geniposide, the peak 6 is isochlorogenic acid B, the peak 7 is isochlorogenic acid A, the peak 8 is forsythiaside A, the peak 9 is isochlorogenic acid C, the peak 10 is baicalin, the peak 11 is forsythin, and the peak 12 is wogonoside.
FIG. 5 is a UPLC chromatogram of negative control lacking Gardenia
Wherein the peak 1 is neochlorogenic acid, the peak 2 is chlorogenic acid, the peak 3 is cryptochlorogenic acid, the peak 4 is gentiopicrin, the peak 6 is isochlorogenic acid B, the peak 7 is isochlorogenic acid A, the peak 8 is forsythiaside A, the peak 9 is isochlorogenic acid C, the peak 10 is baicalin, the peak 11 is forsythin, and the peak 12 is wogonoside.
FIG. 6 is a UPLC chromatogram of negative control lacking forsythia
Wherein, the No. 1 peak is neochlorogenic acid, the No. 2 peak is chlorogenic acid, the No. 3 peak is cryptochlorogenic acid, the No. 4 peak is gentiopicroside, the No. 5 peak is geniposide, the No. 6 peak is isochlorogenic acid B, the No. 7 peak is isochlorogenic acid A, the No. 9 peak is isochlorogenic acid C, the No. 10 peak is baicalin, and the No. 12 peak is wogonoside.
FIG. 7 is UPLC chromatogram of negative control lacking Scutellariae radix
Wherein, the No. 1 peak is new chlorogenic acid, the No. 2 peak is chlorogenic acid, the No. 3 peak is cryptochlorogenic acid, the No. 4 peak is gentiopicrin, the No. 5 peak is geniposide, the No. 6 peak is isochlorogenic acid B, the No. 7 peak is isochlorogenic acid A, the No. 8 peak is forsythiaside A, the No. 9 peak is isochlorogenic acid C, and the No. 11 peak is forsythin.
Detailed Description
The present invention will be further described below in the context of exemplary embodiments for a more complete understanding of its practice. Meanwhile, since the above embodiments are only preferred embodiments of the present invention, the description thereof is more specific and detailed, but it should not be understood that the scope of the present invention is limited thereby. It should be noted that various modifications and improvements can be made by those skilled in the art without departing from the principle and spirit of the invention, and these modifications and improvements should also fall into the scope of the invention.
EXAMPLE 1 UPLC assay methodology Studies of 12 components of Heat-clearing and detoxifying oral liquid
1 instrument and reagent:
1.1 Instrument: WatersAcquity Arc high performance liquid chromatograph (USA), Waters 2998 PDA detector, electronic analytical balance (01193-YP601N, METTLER), KQ-50 model ultrasonic cleaner (Kunshan ultrasonic Instrument Co., Ltd.).
1.2 reagent: neochlorogenic acid (batch: DX001503), cryptochlorogenic acid (batch: 210427-035), isochlorogenic acid B (batch: DY003702), isochlorogenic acid A (batch: DY003602) and isochlorogenic acid C (batch: DY003802) as reference substances, which are purchased from Wenyang detected Biotechnology Limited; chlorogenic acid (batch: 110753-202018), gentiopicroside (batch: 110770-201918), geniposide (batch: 110749-201919), forsythoside A (batch: 111810-202108), baicalin (batch: 110715-202122), forsythin (batch: 110821-202117) and wogonin (batch: 112002-201702) were purchased from China food and drug assay institute. Methanol was Fisher corporation (chromatographic grade), water was ultrapure water, and all other reagents were analytical grade.
2, reagent testing: the 10 batches of the heat-clearing and detoxifying oral liquid are provided by the Lunan Kangpu pharmaceutical Co., Ltd, the sample batch numbers are shown in table 1, and the inspection proves that the heat-clearing and detoxifying oral liquid conforms to the provisions of all items of the heat-clearing and detoxifying oral liquid in 2020 edition 'Chinese pharmacopoeia'.
TABLE 1 sample batch number of oral liquid for clearing away Heat and toxic materials
| Sample (I) | Batch number | Sample (I) | Batch number |
| S1 | 352200012-1 | S6 | 352200022-3 |
| S2 | 352200012-2 | S7 | 352200032-1 |
| S3 | 352200012-3 | S8 | 352200032-2 |
| S4 | 352200022-1 | S9 | 352210012-1 |
| S5 | 352200022-2 | S10 | 352210012-2 |
3, chromatographic conditions: a chromatographic column: waters CORTECS T3C 18 Chromatographic column, 2.1X 100mm, particle size 1.6 μm; and (2) mixing acetonitrile: methanol is taken as a mobile phase A, glacial acetic acid solution is taken as a mobile phase B, and the detection wavelength: 240 nm; flow rate: 0.3 ml/min; column temperature: 35 ℃; gradient elution was performed as follows:
| time, min | Mobile phase, A% | Mobile phase, B% |
| 0-15 | 0％→13.5％ | 100％→86.5％ |
| 15-25 | 13.5％→20％ | 86.5％→80％ |
| 25-36 | 20％→28％ | 80％→72％ |
| 36-39 | 28％→30％ | 72％→70％ |
| 39-60 | 30％→55％ | 70％→45％ |
| 60-65 | 55％→0％ | 45％→100％ |
4 content determination methodology investigation
4.1 preparation of test solutions
Precisely measuring 1ml of the heat-clearing and detoxifying oral liquid, placing the heat-clearing and detoxifying oral liquid in a 50ml volumetric flask, fixing the volume to a scale with 70% methanol, shaking up, and filtering to obtain a test solution;
4.2 preparation of Mixed control solutions
Precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythoside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside as reference substances, and adding 70% methanol to obtain mixed reference substance solution containing 32.096 μ g, 29.294 μ g, 27.309 μ g, 39.178 μ g, 70.543 μ g, 12.080 μ g, 9.427 μ g, 10.789 μ g, 12.707 μ g, 190.674 μ g, 9.185 μ g and 19.872 μ g per 1 mL.
4.3 preparation of negative control solution
Taking a negative control containing no honeysuckle medicinal material, a negative control containing no gentian medicinal material, a negative control containing no gardenia medicinal material, a negative control containing no forsythia medicinal material and a negative control containing no scutellaria medicinal material respectively, operating according to the same method under the preparation item of the test solution to prepare a negative control solution, and observing from a chromatogram map that the positions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythoside A, isochlorogenic acid C, baicalin, phillyrin and wogonin are not interfered (see fig. 3, fig. 4, fig. 5, fig. 6 and fig. 7).
4.4 Linear relationship investigation
Accurately weighing appropriate amount of reference substance, adding 70% methanol to obtain the following concentrations, measuring under the above chromatographic conditions, respectively injecting 2 μ l sample, and measuring peak area. The results are shown in Table 2.
Table 212 component linear relationship examination results
Measuring neochlorogenic acid, taking the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, drawing standard curve to obtain regression equation y of 13778.277x-3051.154, R 2 The concentration of the new chlorogenic acid is 1.000, namely, the new chlorogenic acid has good linearity within the range of 4.012-40.12 mu g/mL.
Measuring chlorogenic acid, taking the measured peak area as ordinate and the control concentration (μ g/mL) as abscissa, drawing standard curve to obtain regression equation y of 13911.174x-2952.692, R 2 When the concentration of chlorogenic acid is 1.000, the chlorogenic acid concentration is in the range of 3.662-36.618 mu g/mL, and the linearity is good.
Determining cryptochlorogenic acid, taking the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, drawing standard curve to obtain regression equation y of 11710.276x-1944.462, R 2 When the concentration of the cryptochlorogenic acid is 1.000, the linearity is good within the range of 3.414-34.136 mu g/mL.
Determining gentiopicroside, taking the measured peak area as ordinate and the concentration of reference substance (μ g/mL) as abscissa, drawing standard curve, and obtaining regression equation y of 8972.627x-3111.615, R 2 ＝1.000, i.e., the gentiopicroside concentration is in the range of 4.897-48.972 μ g/mL with good linearity.
Determining geniposide, drawing standard curve with the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, and obtaining regression equation y of 12362.411x-6271.923, R 2 When the concentration of geniposide is 1.000, the concentration of geniposide is in the range of 8.818-88.179 mu g/mL, the linearity is good.
Determining isochlorogenic acid B, taking the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, drawing standard curve to obtain regression equation y of 14643.659x-1627.308, and R 2 When the concentration of the isochlorogenic acid B is 0.999, the linearity is good within the range of 1.510-15.100 mu g/mL.
Determining isochlorogenic acid A, taking the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, drawing standard curve, and obtaining regression equation y of 16759.996x-1605.231, R 2 When the concentration of the isochlorogenic acid A is 0.999, the linearity is good when the concentration of the isochlorogenic acid A is within the range of 1.178-11.784 mu g/mL.
Determining forsythoside A, taking the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, drawing standard curve to obtain regression equation y of 7184.001x-744.231, R 2 When the concentration is 1.000, the forsythiaside A concentration is in the range of 1.349-13.487 mu g/mL, and the linearity is good.
Determining isochlorogenic acid C, taking the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, drawing standard curve, and obtaining regression equation y of 15719.191x +545.007, R 2 When the concentration of the isochlorogenic acid C is 0.999, the linearity is good within the range of 1.588-15.884 mu g/mL.
And (3) measuring the baicalin, taking the measured peak area as an ordinate and the concentration (mu g/mL) of a reference substance as an abscissa, drawing a standard curve, and obtaining a regression equation y which is 8549.015x +35939.231, wherein R is 2 When the concentration of baicalin is 0.999, the baicalin concentration is in the range of 23.834-238.343 mu g/mL, and the linearity is good.
And (3) determining phillyrin, taking the measured peak area as an ordinate and the concentration (mu g/mL) of a reference substance as an abscissa, drawing a standard curve, and obtaining a regression equation y which is 5154.340x-551.538, wherein R is 2 When the concentration is 1.000, the phillyrin concentration is in the range of 1.148-11.481 mug/mL, and the linearity is good.
Measuring wogonoside, taking the measured peak area as ordinate and the reference substance concentration (μ g/mL) as abscissa, drawing standard curve, and obtaining regression equation y of 7599.647x-4397.692, R 2 When the concentration is 0.998, the wogonoside concentration is in the range of 2.484-24.840 mug/mL, and the linearity is good.
4.5 precision test
And precisely sucking the reference substance solution, repeatedly injecting sample for 6 times, wherein each time is 2 mu l, measuring peak areas, and the RSD of each component peak area is less than 2.0 percent, which indicates that the precision of the instrument is good. The results are shown in Table 3.
TABLE 3 results of precision test investigation
4.6 stability test
And (3) taking the same test solution, and respectively measuring the test solution for 0, 3, 6, 9, 12 and 24 hours after preparation, wherein the peak areas RSD of all components are less than 2.0%, which indicates that the test solution is stable within 24 hours. The results are shown in Table 4.
Table 4 stability test investigation results
4.7 repeatability test
Respectively measuring 6 samples of the same batch number, carrying out sample introduction according to the preparation method under the item of preparation of the test solution, respectively measuring peak areas, and calculating the content. The results are shown in Table 5.
TABLE 5 repeatability test results
4.8 recovery test
Precisely measuring 6 samples with known content, each sample with 0.5ml, placing in a 50ml volumetric flask, precisely adding 0.8024mg/ml neochlorogenic acid reference solution 1.30ml, 0.6103mg/ml chlorogenic acid reference solution 1.50ml, 0.8534mg/ml cryptochlorogenic acid reference solution 1.05ml, 0.3498mg/ml gentiopicroside reference solution 2.30ml, 0.4199mg/ml jasminoidin reference solution 5.60ml, 0.7550mg/ml isochlorogenic acid B reference solution 0.55ml, 0.7856mg/ml isochlorogenic acid A reference solution 0.30ml, 0.8991mg/ml forsythoside A reference solution 0.40ml, 0.7942mg/ml isochlorogenic acid C reference solution 0.50ml, 0.8667mg/ml baicalin reference solution 2.80ml, 0.7654mg/ml forsythoside A reference solution 0.30ml, and 0.2160mg/ml baicalin reference solution 3.20ml, the peak area was measured by the same method as that used in the preparation of the test sample solution. The results are shown in Table 6.
TABLE 612 component recovery test results
The results of measuring the contents of 12 components in 4.910 batches of oral liquid for clearing away heat and toxic materials are as follows (see Table 7).
TABLE 710 contents of 12 components in the heat-clearing and detoxicating oral liquid
EXAMPLE 2 UPLC assay methodology Studies of 12 components of Heat-clearing and detoxifying oral liquid
A. Preparation of test solution
Precisely measuring 1ml of the heat-clearing and detoxifying oral liquid, placing the heat-clearing and detoxifying oral liquid in a 50ml volumetric flask, fixing the volume to a scale with 50% methanol, shaking up, and filtering to obtain a test solution;
B. preparation of Mixed control solutions
Precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside as reference substances, placing in a 20ml volumetric flask, adding 50% methanol for dissolving, fixing the volume to the scale, and shaking uniformly to obtain the final product;
C. UPLC chromatographic conditions
A chromatographic column: waters CORTECS T3C 18 Chromatographic column, 2.1X 100mm, particle size 1.6 μm; mixing acetonitrile: methanol ratio 3:1 as mobile phase A, glacial acetic acid solution as mobile phase B, wavelength: 250 nm; flow rate: 0.4 ml/min; column temperature: 30 ℃; gradient elution was performed as follows:
| time, min | Mobile phase, A% | Mobile phase, B% |
| 0-15 | 0％→13.5％ | 100％→86.5％ |
| 15-25 | 13.5％→20％ | 86.5％→80％ |
| 25-36 | 20％→28％ | 80％→72％ |
| 36-39 | 28％→30％ | 72％→70％ |
| 39-60 | 30％→55％ | 70％→45％ |
| 60-65 | 55％→0％ | 45％→100％ |
D. The measuring method comprises the following steps: and D, precisely sucking 2 mu l of the reference solution and the test solution respectively, injecting into an ultra-high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.
EXAMPLE 3 UPLC assay methodology Studies of 12 components of Heat-clearing and detoxifying oral liquid
A. Preparation of test solution
Precisely measuring 1ml of the heat-clearing and detoxifying oral liquid, placing the heat-clearing and detoxifying oral liquid in a 50ml volumetric flask, fixing the volume to a scale with 90% methanol, shaking up, and filtering to obtain a test solution;
B. preparation of Mixed control solutions
Precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside as reference substances, placing in a 20ml volumetric flask, adding 90% methanol for dissolving, fixing the volume to the scale, and shaking uniformly to obtain the final product;
C. UPLC chromatographic conditions
A chromatographic column: waters CORTECS T3C 18 Chromatographic column, 2.1X 100mm, particle size 1.6 μm; mixing acetonitrile: methanol ratio 3:1 as mobile phase A, glacial acetic acid solution as mobile phase B, wavelength: 230 nm; flow rate: 0.2 ml/min; column temperature: at 40 ℃; gradient elution was performed as follows:
D. the measuring method comprises the following steps: and D, precisely sucking 2 mu l of the reference solution and the test solution respectively, injecting into an ultra-high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.
Comparative example 1 UPLC determination of 12 Components of Heat-clearing and detoxicating oral liquid Mobile phase study
The UPLC is used for simultaneously measuring 12 components of the heat-clearing and detoxifying oral liquid by the prior art, and the specific technical scheme is as follows:
A. preparation of test solution
Precisely measuring 1ml of the heat-clearing and detoxifying oral liquid, placing the heat-clearing and detoxifying oral liquid in a 50ml volumetric flask, fixing the volume to a scale with 70% methanol, shaking up, and filtering to obtain a test solution;
B. preparation of Mixed control solutions
Precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside as reference substances, placing in a 20ml volumetric flask, adding 70% methanol for dissolving, fixing the volume to scale, and shaking uniformly to obtain the final product;
C. UPLC chromatographic conditions
A chromatographic column: waters CORTECS T3C 18 Chromatographic column, 2.1X 100mm, particle size 1.6 μm; the mobile phase A is methanol, the mobile phase B is glacial acetic acid solution, and the wavelength is as follows: 230 nm; flow rate: 0.3 ml/min; column temperature: 35 ℃; gradient elution was performed as follows:
| time, min | Mobile phase, A% | Mobile phase, B% |
| 0-15 | 0％→13.5％ | 100％→86.5％ |
| 15-25 | 13.5％→20％ | 86.5％→80％ |
| 25-36 | 20％→28％ | 80％→72％ |
| 36-39 | 28％→30％ | 72％→70％ |
| 39-60 | 30％→55％ | 70％→45％ |
| 60-65 | 55％→0％ | 45％→100％ |
D. The determination method comprises the following steps: and D, precisely sucking 2 mu l of the reference solution and the test solution respectively, injecting into an ultra-high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.
The UPLC method results show that the separation degree is not good, and the content measurement of 12 components cannot be realized.
The invention establishes a method for measuring the content of 12 components of the heat-clearing and detoxifying oral liquid through a large number of experiments, wherein the mobile phase A in the step C is methanol, acetonitrile and acetonitrile respectively: methanol (9:1, 6:1, 3:1, 1:3, 1:6, 1:9), and the mobile phase B is glacial acetic acid.
The test results show that: mobile phase a was acetonitrile: the UPLC chromatogram obtained when methanol (3:1) and mobile phase B are glacial acetic acid is stable and reliable, has strong specificity and good reproducibility, and can realize content determination of 12 components in the heat-clearing and detoxifying oral liquid.
Claims (5)
1. A method for determining contents of heat-clearing and detoxifying oral liquid is characterized in that the method adopts ultra-high performance liquid chromatography to determine contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside in a preparation, and comprises the following steps:
A. preparation of a test solution: precisely measuring 1ml of the heat-clearing and detoxifying oral liquid, placing the heat-clearing and detoxifying oral liquid in a 50ml volumetric flask, fixing the volume to a scale with 50% -90% methanol, shaking up, and filtering to obtain a sample solution;
B. preparation of control solutions: precisely weighing appropriate amount of reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, gentiopicrin, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside, placing in a 20ml volumetric flask, adding 50-90% methanol for dissolving, fixing the volume to scale, and shaking uniformly to obtain the final product;
C. UPLC chromatographic conditions: waters CORTECS T3C 18 Chromatography column, purified in acetonitrile: methanol =3:1 is a mobile phase A, glacial acetic acid solution is a mobile phase B, gradient elution is adopted, and the conditions of gradient content elution of the mobile phase are as follows:
detection wavelength: 230-250 nm; flow rate: 0.2-0.4 ml/min; column temperature: 30-40 ℃;
D. the determination method comprises the following steps: and D, precisely sucking 2 mu l of the reference substance solution and the sample solution respectively, injecting the reference substance solution and the sample solution into an ultra-high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the sample solution.
2. The content determination method according to claim 1, wherein the volume is determined to the scale by adding 70% methanol in step A, and shaking up.
3. The content determination method of claim 1, wherein in the step B, a proper amount of chlorogenic acid, cryptochlorogenic acid, gentiopicroside, geniposide, isochlorogenic acid B, isochlorogenic acid A, forsythiaside A, isochlorogenic acid C, baicalin, phillyrin and wogonoside reference substance are precisely weighed, placed in a volumetric flask, dissolved and diluted to scale by adding 70% methanol, and shaken well.
4. The method according to claim 1, wherein the column of step C has a size of 2.1X 100mm and a particle diameter of 1.6. mu.m.
5. The method according to claim 1, wherein the detection wavelength in step C is 240nm, the flow rate is 0.3ml/min, and the column temperature is 35 ℃.
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| CN110187044A (en) * | 2019-01-17 | 2019-08-30 | 山东省农业科学院家禽研究所 | A kind of quality determining method of Yinqiao Jiedu oral liquid |
| CN113009023A (en) * | 2021-02-26 | 2021-06-22 | 河南福森药业有限公司 | Method for measuring chemical component content in traditional Chinese medicine compound preparation |
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| CN110187044A (en) * | 2019-01-17 | 2019-08-30 | 山东省农业科学院家禽研究所 | A kind of quality determining method of Yinqiao Jiedu oral liquid |
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