CN116699024A - Method for analyzing lithocholic acid quality - Google Patents
Method for analyzing lithocholic acid quality Download PDFInfo
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- CN116699024A CN116699024A CN202310676313.8A CN202310676313A CN116699024A CN 116699024 A CN116699024 A CN 116699024A CN 202310676313 A CN202310676313 A CN 202310676313A CN 116699024 A CN116699024 A CN 116699024A
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- lithocholic acid
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- SMEROWZSTRWXGI-UHFFFAOYSA-N Lithocholsaeure Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 SMEROWZSTRWXGI-UHFFFAOYSA-N 0.000 title claims abstract description 57
- SMEROWZSTRWXGI-HVATVPOCSA-N lithocholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 SMEROWZSTRWXGI-HVATVPOCSA-N 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000000243 solution Substances 0.000 claims abstract description 67
- 239000012535 impurity Substances 0.000 claims abstract description 44
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 20
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 12
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 10
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000019253 formic acid Nutrition 0.000 claims abstract description 10
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000004458 analytical method Methods 0.000 claims abstract description 7
- 238000002347 injection Methods 0.000 claims abstract description 7
- 239000007924 injection Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 6
- 238000000889 atomisation Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 239000000523 sample Substances 0.000 claims description 22
- 239000002253 acid Substances 0.000 claims description 17
- 239000012488 sample solution Substances 0.000 claims description 10
- 239000013558 reference substance Substances 0.000 claims description 7
- 238000007865 diluting Methods 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 abstract description 9
- 238000000926 separation method Methods 0.000 abstract description 5
- 238000005220 pharmaceutical analysis Methods 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 16
- 238000011084 recovery Methods 0.000 description 10
- 230000035945 sensitivity Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 6
- 238000011088 calibration curve Methods 0.000 description 5
- 239000012491 analyte Substances 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 3
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000003039 volatile agent Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a method for analyzing lithocholic acid quality, and belongs to the technical field of chemical and pharmaceutical analysis. The method for analyzing lithocholic acid quality comprises the following steps: selecting a high performance liquid chromatograph as a detector, taking an octadecylsilane bonding phase as a stationary phase, selecting a trifluoroacetic acid acetonitrile solution or a formic acid acetonitrile solution as a mobile phase A, and performing gradient elution by taking the trifluoroacetic acid solution or the formic acid solution as a mobile phase B, wherein the atomization temperature of an electrospray detector is 35-38 ℃; the sample injection amount is 5-12 mu l; the flow rate is 0.9-1.2ml/min; the column temperature is 25-35 ℃. The method realizes effective separation of the impurities and the lithocholic acid, and can accurately determine the content of the impurities in the lithocholic acid, so that the quality of the lithocholic acid can be obtained by analysis.
Description
Technical Field
The invention relates to the technical field of chemical drug analysis, in particular to a method for analyzing lithocholic acid (LCA) quality.
Background
In the prior art, the methods for detecting the quality of lithocholic acid products mainly comprise the following steps: chemical titration, chromatography, and the like; the chemical titration method generally uses sodium hydroxide titration solution (0.1 mol/L) to perform acid-base neutralization titration, and the content is determined, so that the specificity is poor, the total acid content in the analyte can only be determined, and the interference of other acidic impurities is easy; the chromatography includes Thin Layer Chromatography (TLC), gas Chromatography (GC), high Performance Liquid Chromatography (HPLC), chromatography combined method, etc.; the detection control of lithocholic acid related substances is carried out by adopting a thin-layer chromatography, so that the specificity is poor, the sensitivity is low, and the lithocholic acid related substances cannot be directly quantified and have low accuracy; determining the content of lithocholic acid by a gas chromatography, wherein the sample needs to be subjected to derivatization treatment, and the treatment process is relatively complex; the quality control of lithocholic acid is carried out by adopting a high performance liquid chromatography, and because of the self structural characteristics of the lithocholic acid, the lithocholic acid only has terminal absorption near the wavelength of 200nm, so that the problems of low sensitivity and large interference exist when a common ultraviolet detector is used for sample analysis, the impurities with low content are difficult to effectively control, and a derivatization method is adopted, but the sample pretreatment process is complex and the reproducibility is slightly poor.
Other alternative detectors include differential reflectance detectors (RID), evaporative light flash detectors (ELSDs), mass Spectrometric Detectors (MSDs), electrospray detectors (CAD), and the like. The differential refraction detector (RID) has low sensitivity and is not suitable for gradient elution, so that the separation effect on complex samples is poor and the specificity is slightly poor; the HPLC-MS/MS is adopted for quantification, firstly, the analyte is ionized, the compounds which are difficult to ionize cannot be detected, the requirements on instrument reagents and the like are very high, and the corresponding cost is very high; quantification of sample boiling point using HPLC-ELSD cannot be too low, and response values are related to the amount of sample material, but are poor in linearity, sensitivity, and reproducibility; CAD (electrospray detector) is a new generation of universal detector developed in recent years, the detection principle is unique, the structure of the detector is not dependent on the analyte, the analyte does not need to be ionized, the detector can be detected as long as the substance belongs to a semi-volatile or non-volatile compound, the sensitivity can reach pg level, the reproducibility is good, and the detector is a powerful complement of the detector and is more and more favored by drug research and development institutions. The electrospray detector is a mass-sensitive detector, the detection response value of which is determined by the absolute mass of the sample injection, and neutral ions and negative ions can be detected simultaneously in one experiment, but the detector cannot realize. How to analyze the quality of lithocholic acid in combination with electrospray detectors is a problem that needs to be solved by the prior art.
Disclosure of Invention
The invention aims to overcome the technical defects, and provides a method for analyzing the quality of lithocholic acid, which solves the technical problem of how to analyze the quality of lithocholic acid by combining an electrospray detector in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention provides a method for analyzing lithocholic acid quality, which comprises the following steps: selecting a high performance liquid chromatograph as a detector, taking an octadecylsilane bonding phase as a stationary phase, selecting a trifluoroacetic acid acetonitrile solution or a formic acid acetonitrile solution as a mobile phase A, and performing gradient elution by taking the trifluoroacetic acid solution or the formic acid solution as a mobile phase B, wherein the atomization temperature of an electrospray detector is 35-38 ℃;
the sample injection amount is 5-12 mu l; the flow rate is 0.9-1.1ml/min; the column temperature is 25-35 ℃.
Further, the chromatographic column used was: the length is 100-150mm, the diameter is 2.1-4.6mm, and the particle size is 2.5-3.5 μm.
Further, the volume concentration of the trifluoroacetic acid solution or the formic acid solution is 0.1% -0.2%.
Further, the volume concentration of the trifluoroacetic acid acetonitrile solution or the formic acid acetonitrile solution is 0.1% -0.2%.
Further, the analysis time was 31-45min.
Further, the gradient elution procedure was as follows:
further, the impurities in lithocholic acid include one or more of 3α -hydroxy-5α -cholestan-24-oic acid, 3β -hydroxy-5β -cholestan-24-oic acid, 3β -hydroxy-5α -cholestan-24-oic acid, and 3-oxo-5β -cholestan-24-oic acid.
Further, the positioning solution of each impurity is prepared by the following steps: taking reference substances of various impurities, adding methanol to dissolve and dilute the reference substances to prepare a solution containing 1-1.5mg of the impurities in each 1ml, taking 1ml of the solution, placing the solution into a 100ml measuring flask, adding methanol to fix the volume to a scale, and shaking the solution uniformly to serve as a positioning solution.
Further, the sample solution is prepared by the steps of: dissolving lithocholic acid sample in methanol, and diluting to obtain solution containing 1.0-1.5mg of lithocholic acid sample per 1 ml.
Further, the control solution was prepared by the following steps: taking 1-1.5ml of the sample solution, placing into a 100ml measuring flask, adding methanol to fix volume to scale, shaking uniformly, and taking as a control solution.
Compared with the prior art, the invention has the beneficial effects that: according to the method for analyzing lithocholic acid quality, a high performance liquid chromatograph is selected as a detector, an octadecylsilane bonding phase is used as a stationary phase, a trifluoroacetic acid acetonitrile solution or a formic acid acetonitrile solution is selected as a mobile phase A, a trifluoroacetic acid solution or a formic acid solution is used as a mobile phase B for gradient elution, and the atomization temperature of an electrospray detector is 35-38 ℃; the sample injection amount is 5-12 mu l; the flow rate is 0.9-1.1ml/min; the column temperature is 25-35 ℃; the method can realize the effective separation of the impurities and the lithocholic acid, and can accurately determine the content of the impurities in the lithocholic acid, thereby analyzing and obtaining the quality of the lithocholic acid.
Drawings
FIG. 1 is a high performance liquid chromatogram of a lithocholic acid specific solution of example 1 of the present invention.
FIG. 2 is a calibration curve for impurity A of example 1 of the present invention.
FIG. 3 is a calibration curve for impurity B of example 1 of the present invention.
Fig. 4 is a calibration curve of impurity C of example 1 of the present invention.
Fig. 5 is a calibration curve of impurity D of example 1 of the present invention.
Fig. 6 is a calibration curve for LCA of example 1 of the invention.
Detailed Description
The specific embodiment provides a method for analyzing lithocholic acid quality, which comprises the following steps: selecting a high performance liquid chromatograph as a detector, taking an octadecylsilane bonding phase as a stationary phase, selecting a trifluoroacetic acid acetonitrile solution or a formic acid acetonitrile solution as a mobile phase A, and performing gradient elution by taking the trifluoroacetic acid solution or the formic acid solution as a mobile phase B, wherein the atomization temperature of an electrospray detector is 35-38 ℃; the sample injection amount is 5-12 mu l; the flow rate is 0.9-1.2ml/min; the column temperature is 25-35 ℃; the analysis time is 31-45min; the chromatographic column adopted is as follows: the length is 100-150mm, the diameter is 2.1-4.6mm, and the grain diameter is 2.5-3.5 mu m; the volume concentration of the trifluoroacetic acid solution or the formic acid solution is 0.1-0.2%; the volume concentration of the trifluoroacetic acid acetonitrile solution or the formic acid acetonitrile solution is 0.1-0.2%.
In certain embodiments, the gradient elution procedure is as follows:
in this embodiment, the impurities in lithocholic acid include one or more of 3α -hydroxy-5α -cholestan-24-oic acid, 3β -hydroxy-5β -cholestan-24-oic acid, 3β -hydroxy-5α -cholestan-24-oic acid, and 3-oxo-5β -cholestan-24-oic acid; the positioning solution of each impurity is prepared by the following steps: taking reference substances of various impurities, adding methanol to dissolve and dilute the reference substances to prepare a solution containing 1-1.5mg of the impurities in each 1ml, taking 1ml of the solution, placing the solution into a 100ml measuring flask, adding methanol to fix the volume to a scale, and shaking the solution uniformly to serve as a positioning solution.
In certain embodiments, the test solution is prepared by the steps of: dissolving lithocholic acid sample in methanol, and diluting to obtain solution containing 1.0-1.5mg of lithocholic acid sample per 1ml as sample solution; the control solution was prepared by the following steps: taking 1-1.5ml of the sample solution, placing into a 100ml measuring flask, adding methanol to fix volume to scale, shaking uniformly, and taking as a control solution.
The structural formulas of lithocholic acid and impurities are shown in table 1 below:
TABLE 1 Structure of lithocholic acid and impurities
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In the following embodiments, the corresponding components are described using the relevant codes.
Example 1
The embodiment provides a method for analyzing lithocholic acid quality, which comprises the following steps: selecting a high performance liquid chromatograph as a detector, taking an octadecylsilane bonding phase as a stationary phase, selecting acetonitrile formate as a mobile phase A, and carrying out gradient elution by taking formic acid as a mobile phase B, wherein the atomization temperature of an electrospray detector is 35 ℃; the sample injection amount is 10 μl; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the analysis time was 31min; the chromatographic column adopted is as follows: the chromatographic column is C18 with the length of 150mm, the diameter of 4.6mm and the particle diameter of 2.5 μm; the volume concentration of the formic acid is 0.1%; the volume concentration of the acetonitrile formate is 0.1%.
In this example, the impurities in lithocholic acid include 3α -hydroxy-5α -cholestan-24-oic acid, 3β -hydroxy-5β -cholestan-24-oic acid, 3β -hydroxy-5α -cholestan-24-oic acid, and 3-oxo-5β -cholestan-24-oic acid; the positioning solution of each impurity is prepared by the following steps: dissolving reference substances of impurities in methanol, diluting to obtain solution containing 1mg of the impurities in each 1ml, placing 1ml of the solution into a 100ml measuring flask, adding methanol to a certain volume to scale, and shaking to obtain positioning solution; the sample solution is prepared by the following steps: dissolving lithocholic acid sample in methanol, and diluting to obtain solution containing 1.0mg of lithocholic acid sample per 1ml as sample solution; the control solution was prepared by the following steps: taking 1ml of the sample solution, placing the sample solution into a 100ml measuring flask, adding methanol to fix the volume to the scale, and shaking uniformly to serve as a control solution.
The procedure for the gradient elution is shown in table 2 below:
TABLE 2 gradient elution procedure
The specific substance profile and associated results are shown in fig. 1 and table 3 below, with peak sequences C, B, LCA, A and D.
TABLE 3 results of specific tests
As can be seen from fig. 1 and table 3, by this method, separation of each impurity from lithocholic acid can be achieved, and lithocholic acid and related impurities are detected.
To further verify the effective detection of impurities a-D we set up a sensitivity test with detection and quantification limits results shown in table 4.
TABLE 4 detection limit and quantitative limit detection results
| Name of the name | Code | Detection limit (ng) | Quantitative limit (ng) |
| 3 alpha-hydroxy-5 alpha-cholestane-24-carboxylic acid | A | 3.02 | 10.06 |
| 3 beta-hydroxy group-5 beta-cholestane-24-carboxylic acid | B | 2.98 | 9.94 |
| 3 beta-hydroxy-5 alpha-cholestane-24-carboxylic acid | C | 3.08 | 10.25 |
| 3-oxo-5 beta-cholestane-24-carboxylic acid | D | 3.2 | 10.66 |
To verify the stability of the test solution, we tested 24h samples using the protocol described above, the results are shown in table 5.
Table 5 test results for 24h sample
As can be seen from Table 5, the detection results at different times are closer, indicating that the components of the sample are more stable.
To further verify the accuracy of the impurities A-D, we set the addition amounts of the impurities with different contents, 5-15. Mu.g, and the relevant detection results are shown in tables 6 and 7.
TABLE 6 recovery of impurities A and B
| Impurity A | Impurity B | |||||
| Added amount of μg | Measured μg | Recovery rate | Added amount of μg | Measured μg | Recovery rate | |
| 1 | 5.0143 | 5.0356 | 100.42％ | 5.1059 | 5.0633 | 99.17％ |
| 2 | 5.0143 | 5.0939 | 101.59％ | 5.1059 | 5.0392 | 98.69％ |
| 3 | 5.0143 | 5.0647 | 101.01％ | 5.1059 | 5.0289 | 98.49％ |
| 4 | 10.0286 | 10.1693 | 101.40％ | 10.2118 | 10.2391 | 100.27％ |
| 5 | 10.0286 | 10.2941 | 102.65％ | 10.2118 | 10.1935 | 99.82％ |
| 6 | 10.0286 | 9.9775 | 99.49％ | 10.2118 | 10.0494 | 98.41％ |
| 7 | 15.0429 | 15.0937 | 100.34％ | 15.3177 | 15.4206 | 100.67％ |
| 8 | 15.0429 | 15.3442 | 102.00％ | 15.3177 | 15.4981 | 101.18％ |
| 9 | 15.0429 | 15.4396 | 102.64％ | 15.3177 | 15.5309 | 101.39％ |
| Average value of | / | / | 101.28％ | / | / | 99.79％ |
| Standard deviation SD | / | / | 1.07％ | / | / | 1.16％ |
| RSD | / | / | 1.06％ | / | / | 1.16％ |
TABLE 7 recovery of impurities C and D
| Impurity C | Impurity D | |||||
| Added amount of μg | Measured quantityμg | Recovery rate | Added amount of μg | Measured μg | Recovery rate | |
| 1 | 5.0084 | 5.0576 | 100.98％ | 5.0375 | 5.0298 | 99.85％ |
| 2 | 5.0084 | 5.0893 | 101.62％ | 5.0375 | 4.9735 | 98.73％ |
| 3 | 5.0084 | 4.9572 | 98.98％ | 5.0375 | 5.0502 | 100.25％ |
| 4 | 10.0168 | 10.1349 | 101.18％ | 10.075 | 10.3329 | 102.56％ |
| 5 | 10.0168 | 10.3107 | 102.93％ | 10.075 | 10.2196 | 101.44％ |
| 6 | 10.0168 | 10.2646 | 102.47％ | 10.075 | 9.9906 | 99.16％ |
| 7 | 15.0252 | 15.0013 | 99.84％ | 15.1125 | 15.2158 | 100.68％ |
| 8 | 15.0252 | 14.7961 | 98.48％ | 15.1125 | 15.3533 | 101.59％ |
| 9 | 15.0252 | 15.0632 | 100.25％ | 15.1125 | 14.9427 | 98.88％ |
| Average value of | / | / | 100.75％ | / | / | 100.35％ |
| Standard deviation SD | / | / | 1.50％ | / | / | 1.33％ |
| RSD | / | / | 1.49％ | / | / | 1.33％ |
It can be seen from tables 6 and 7 that different concentrations of impurities a-D all have a higher accuracy.
To further verify the accuracy of detecting the impurity A-D, we set the addition amount of 10. Mu.g of the impurity, and conducted a repeatability test, the results of which are shown in tables 8 and 9.
TABLE 8 repeatability test results of impurities A and B
| Impurity A | Impurity B | |||||
| Added amount of μg | Measured μg | Recovery rate | Added amount of μg | Measured μg | Recovery rate | |
| 1 | 10.0286 | 10.1693 | 101.40％ | 10.2118 | 10.2391 | 100.27％ |
| 2 | 10.0286 | 10.2941 | 102.65％ | 10.2118 | 10.1935 | 99.82％ |
| 3 | 10.0286 | 9.9775 | 99.49％ | 10.2118 | 10.0494 | 98.41％ |
| 4 | 10.0286 | 10.0095 | 99.81％ | 10.2118 | 10.3054 | 100.92％ |
| 5 | 10.0286 | 10.1947 | 101.66％ | 10.2118 | 10.2217 | 100.10％ |
| 6 | 10.0286 | 10.2022 | 101.73％ | 10.2118 | 10.1853 | 99.74％ |
| Average value of | / | / | 101.12％ | / | / | 99.88％ |
| RSD | / | / | 1.21％ | / | / | 0.83％ |
TABLE 9 repeatability test results of impurities C and D
| Impurity C | Impurity D | |||||
| Added amount of μg | Measured μg | Recovery rate | Added amount of μg | Measured μg | Recovery rate | |
| 1 | 10.0168 | 10.3549 | 103.38％ | 10.075 | 10.3329 | 102.56％ |
| 2 | 10.0168 | 10.3107 | 102.93％ | 10.075 | 10.2196 | 101.44％ |
| 3 | 10.0168 | 10.2646 | 102.47％ | 10.075 | 9.9906 | 99.16％ |
| 4 | 10.0168 | 10.0003 | 99.84％ | 10.075 | 9.9372 | 98.63％ |
| 5 | 10.0168 | 10.0543 | 100.37％ | 10.075 | 9.9846 | 99.10％ |
| 6 | 10.0168 | 10.1941 | 101.77％ | 10.075 | 10.1149 | 100.40％ |
| Average value of | / | / | 101.79％ | / | / | 100.21％ |
| RSD | / | / | 1.40％ | / | / | 1.54％ |
As can be seen from tables 8 and 9, the detection of impurities A-D was found to have good reproducibility.
To further determine whether there is a linear relationship between concentration and peak area, we performed measurements of different concentrations, the results being shown in FIGS. 2-6 and Table 10.
TABLE 10 lithocholic acid related substance method validation results
From the summary, it can be seen from fig. 2-6 and table 10 that the method has good sensitivity, high accuracy and good repeatability; the R value of the regression equation of the impurity A, B, C and D is 0.9990-0.9997 in the range of about 0.5-20 mug/ml, and the correlation between the concentration of the sample and the peak area is good; the test solution was stable over 24 hours.
The method provided by the invention has better selectivity, accuracy and sensitivity for measuring the lithocholic acid content, and is simple and convenient to operate, and quick and easy to measure.
Further, we changed one of the conditions for the relevant detection, set the column temperature to 25℃or 35℃and the flow rate to 0.9ml or 1.1ml, and exchanged the chromatographic column to Agilent eclipse XDB-C18 length 150mm, diameter 4.6mm, particle size 3.5 μm, i.e. chromatographic column 2, while the other conditions were unchanged to develop five new groups of detection, the detection results are shown in Table 11.
TABLE 11 detection results under different conditions
It can be seen from table 11 that changing a certain condition can still achieve effective separation of the individual components without affecting detection.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made in accordance with the technical idea of the present invention shall be included in the scope of the claims of the present invention.
Claims (10)
1. A method for analysing the quality of lithocholic acid, comprising the steps of: selecting a high performance liquid chromatograph as a detector, taking an octadecylsilane bonding phase as a stationary phase, selecting a trifluoroacetic acid acetonitrile solution or a formic acid acetonitrile solution as a mobile phase A, and performing gradient elution by taking the trifluoroacetic acid solution or the formic acid solution as a mobile phase B, wherein the atomization temperature of an electrospray detector is 35-38 ℃;
the sample injection amount is 5-12 mu l; the flow rate is 0.9-1.1ml/min; the column temperature is 25-35 ℃.
2. The method for analyzing lithocholic acid quality according to claim 1, wherein the chromatographic column used is: the length is 100-150mm, the diameter is 2.1-4.6mm, and the particle size is 2.5-3.5 μm.
3. The method for analysing the quality of lithocholic acid according to claim 1, characterized in that the formic acid has a volume concentration of 0.1% to 0.2%.
4. The method for analyzing lithocholic acid quality according to claim 1, wherein the volume concentration of the trifluoroacetic acid solution, the trifluoroacetic acid acetonitrile solution or the formic acid acetonitrile is 0.1% -0.2%.
5. The method for analyzing the quality of lithocholic acid according to claim 1, wherein the analysis time is 31-45min.
6. The method for analysing lithocholic acid mass according to claim 1, characterized in that the gradient elution procedure is as follows:
7. the method of analyzing the quality of lithocholic acid according to claim 1, wherein the impurities in lithocholic acid include one or more of 3α -hydroxy-5α -cholestan-24-oic acid, 3β -hydroxy-5β -cholestan-24-oic acid, 3β -hydroxy-5α -cholestan-24-oic acid, and 3-oxo-5β -cholestan-24-oic acid.
8. The method for analyzing lithocholic acid quality according to claim 7, wherein the positioning solution of each impurity is prepared by the steps of: taking reference substances of various impurities, adding methanol to dissolve and dilute the reference substances to prepare a solution containing 1-1.5mg of the impurities in each 1ml, taking 1ml of the solution, placing the solution into a 100ml measuring flask, adding methanol to fix the volume to a scale, and shaking the solution uniformly to serve as a positioning solution.
9. The method for analyzing lithocholic acid quality according to claim 1, wherein the sample solution is prepared by the steps of: dissolving lithocholic acid sample in methanol, and diluting to obtain solution containing 1.0-1.5mg of lithocholic acid sample per 1 ml.
10. The method for analyzing lithocholic acid quality according to claim 9, wherein the control solution is prepared by: taking 1-1.5ml of the sample solution, placing into a 100ml measuring flask, adding methanol to fix volume to scale, shaking uniformly, and taking as a control solution.
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