CN117783396A - Method for detecting other sterols in cholesterol and application of method - Google Patents

Abstract

The invention provides a detection method and application of other sterols in cholesterol, and relates to the technical field of medicine analysis. In this assay, other sterols include 24-cholestadienol, sitosterol, cholestanol and beta-cholestanol. The detection method comprises the steps of adopting a high performance liquid chromatography combined electrospray detector to separate and measure other sterols in cholesterol, obtaining a chromatogram and calculating to obtain other sterol contents; the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of methanol and water; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid; the chromatographic column is YMC-Tiart C18 chromatographic column. The detection method has the characteristics of strong specificity, good separation degree, good precision, high accuracy, good durability, simple and quick operation and the like, and can be effectively used for controlling related substances of other sterols in the process of cholesterol production technology and the final product.

Description

Method for detecting other sterols in cholesterol and application of method

Technical Field

The invention relates to the technical field of medicine analysis, in particular to a detection method and application of other sterols in cholesterol.

Background

Cholesterol is also called cholesterol, is a special steroid compound, has wide application in various aspects such as cosmetics, emulsifying agents, medicines and the like, can be used as a multifunctional cosmetic emulsifying aid, and can improve the property of an anionic emulsifying agent and the stability of emulsion. Cholesterol, a multifunctional cosmetic emulsification aid with biological activity, has been listed in the american CTFA cosmetic raw materials handbook as a natural active substance to which cosmetics can be added. Because cholesterol has a natural steroid basic skeleton, almost all steroid hormone medicines can be prepared by modifying the cholesterol through effective chemical or microbial conversion and other means, and the cholesterol is an ideal steroid medicine precursor. The steroid ring and side chain structure of cholesterol make it have the functions of surface activity and stabilizing foam, and can be used as basic component of emulsifier to form oil-in-water or water-in-oil emulsion, so that it can be used for improving the property of anionic emulsifier and stability of emulsion. In the era of novel drug delivery systems, liposome injection is used as a novel nano drug delivery system, has the advantages of good targeting property and tissue affinity, improvement of solubility and penetrability of insoluble drugs, improvement of drug stability and the like, and is receiving more and more attention in the field of pharmacy. Cholesterol is a basic auxiliary material for manufacturing liposome, plays a role in regulating the structure and properties of the membrane, and can greatly improve the bearing capacity and stability of the liposome. Therefore, there is a great concern about the source, content, impurity level, safety, etc. of cholesterol, especially in the fields of cell therapy, gene therapy, etc. Non-cholesterol sterols such as 24-cholestadiol, sitosterol, cholestanol, beta-cholestanol in cholesterol biosynthesis and metabolism routes, which are all fat-soluble compounds similar to cholesterol in structure, are difficult to remove during the production process, so that other sterol impurities in cholesterol must be strictly controlled.

The structure and the property of other sterols in cholesterol are very similar to those of cholesterol, so that separation and analysis are difficult, the researches on sterol impurities in the prior art mostly adopt gas chromatography, the detection method has the defects of poor separation degree, poor accuracy and the like, and a great deal of researches show that even if chromatographic conditions are repeatedly optimized or chromatographic columns with different polarities are replaced, separation of the sterols from other sterols in the cholesterol is difficult to realize. Is unfavorable for the quality control of the product manufacturing process. In the prior art, various problems of poor separation degree, poor sensitivity and complex pretreatment exist in the research of sterol impurities by adopting a liquid chromatography, most sterols have no conjugated multi-part saturated structure, have no absorption in the ultraviolet absorption wavelength commonly used by the high-performance liquid chromatography, have weak absorption in the short ultraviolet wavelength (below 210 nm), have insufficient response, and have no means such as pre-column derivatization, if the impurity level in the sample injection amount allowed by effective separation is usually lower than the detection limit and cannot be detected, but the derivatization process is complex, and the derivatization reagent has great harm to human and environment, such as a derivatization agent phenyl isocyanate is a virulent reagent.

Cholesterol is a kind of carried in pharmacopoeia, and carried in both Chinese pharmacopoeia and United states pharmacopoeia, other sterol measuring items in cholesterol standard of United states pharmacopoeia USP-NF 2021 are measured by adopting a gas chromatography internal standard method, and the separation degree of beta-cholestanol from cholesterol and chain sterol peaks is extremely poor (less than 1.0), so that the content of other sterols in the cholesterol cannot be accurately measured, and the gas chromatography conditions are not improved yet. In the cholesterol standard of the current edition of Chinese pharmacopoeia, no other sterol inspection item exists, and when the other sterols in the cholesterol are measured by adopting the chromatographic conditions of the content measurement item, the cholesterol and the other sterols cannot be separated. High performance liquid chromatography has not been found in other patents and literature to simultaneously determine 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol in cholesterol. Therefore, the research of a detection method capable of accurately and rapidly detecting other sterols in cholesterol is particularly urgent for the research and development and production of medicaments for enterprises, and has great significance for controllable medicament quality.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a method for detecting other sterols in cholesterol, which is used for solving the problem that 24-cholestadienol, sitosterol, cholestanol and beta-cholestanol in the cholesterol cannot be detected simultaneously in the prior art. Another object of the present invention is to provide the use of the above detection method.

In order to solve the technical problems, the invention adopts the following technical scheme:

in a first aspect, a method is provided for detecting other sterols in cholesterol, including 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol;

the detection method comprises the steps of separating and measuring other sterols in cholesterol by adopting a high performance liquid chromatography, obtaining a chromatogram and calculating to obtain other sterol contents;

the chromatographic conditions of the high performance liquid chromatography include:

the detector of the liquid chromatograph is an electrospray detector;

the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of methanol and water; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid;

the chromatographic column is YMC-Tiart C18 chromatographic column.

In an alternative embodiment, the volume ratio of methanol to water in mobile phase a is 95:5; and/or, in the mobile phase B, the volume ratio of acetonitrile, water and glacial acetic acid is (85-95): (5-15): (0.1-0.2).

In an alternative embodiment, the volume ratio of methanol to water in mobile phase a is 95:5; in mobile phase B, the volume ratio of acetonitrile, water and glacial acetic acid is 90:10:0.1.

In an alternative embodiment, mobile phase a and mobile phase B are isocratically eluted.

In an alternative embodiment, the volume ratio of mobile phase a to mobile phase B in the isocratic elution is 80:20.

in an alternative embodiment, the column temperature of the chromatographic column is 20 to 35 ℃, preferably 30 ℃.

In an alternative embodiment, the chromatographic column is a YMC-Tiart C18 chromatographic column.

In an alternative embodiment, the column is a YMC-Tiart C18 column, having a size of 4.6mm×250mm, and a packing particle size of 3 μm.

In an alternative embodiment, the chromatographic conditions further comprise: the flow rate of the mobile phase is 1.0mL/min; and/or the sample injection amount is 20 mu L.

In an alternative embodiment, the sample to be tested comprises a test solution using ethanol as a solvent, wherein the concentration of the cholesterol sample is 4-6 mg/mL, preferably 5mg/mL.

In alternative embodiments, the external standard method is used for analysis to calculate the other sterols content.

In an alternative embodiment, the external standard method is a standard curve method.

In an alternative embodiment, the detection method includes:

(a) Preparing a test sample solution: weighing a cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, and filtering to obtain a sample solution;

preparing a reference substance solution: weighing one or more of 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol, and dissolving and diluting the solution to a known content by using absolute ethyl alcohol to obtain a test solution;

(b) Setting high performance liquid phase detection conditions: the chromatographic column is YMC-Tiart C18 chromatographic column with specification of 4.6mm×250mm and 3 μm; the detector is an electrospray detector; the column temperature is 30 ℃; the mobile phase A is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 95:5, a step of; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid, and the volume ratio of acetonitrile, water and glacial acetic acid is 90:10:0.1; the flow rate of the mobile phase is 1.0ml/min; the volume ratio of the mobile phase A to the mobile phase B is 80:20, performing isocratic elution;

(c) Respectively sucking the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, analyzing, and recording chromatograms;

(d) And analyzing and calculating the content of other sterols by adopting an external standard method.

In a second aspect, there is also provided the use of the method for the detection of other sterols in cholesterol of the first aspect in any of the following:

preparing cholesterol;

(ii) preparing a cholesterol-containing product comprising an emulsifier, a cosmetic or a pharmaceutical product.

Compared with the prior art, the invention has the following beneficial effects:

1. the method for detecting other sterols in cholesterol provided by the invention adopts a high performance liquid chromatography combined with an electrospray detector to separate and measure other sterols in cholesterol and calculate the content of other sterols, and the cholesterol and several other sterols in the detection method have good separation degree. The detection method does not need to carry out derivatization treatment on the sample to be detected, avoids the complex process of using pre-column derivatization and the harm of highly toxic derivatization reagents to human and environment in the liquid chromatography disclosed by the prior art, and is simple and convenient to operate.

2. The inventor finds that other sterols in cholesterol have very similar structures and properties to cholesterol, so that separation and analysis are difficult, and the gas chromatography disclosed in the prior art is difficult to realize effective separation of cholesterol from other sterols even if chromatographic condition parameters are repeatedly optimized or chromatographic columns with different polarities are replaced. In the liquid chromatography for analyzing sterols disclosed in the prior art, chromatographic conditions are not suitable for simultaneously measuring several other sterols in cholesterol in the study, and when the traditional C18 column is adopted for detection in the prior art, even if a mobile phase and a gradient elution program are repeatedly optimized, the separation degree between a main cholesterol peak and other sterol peaks is difficult to reach the detection requirement, and the inventor proves that a plurality of groups of research experiments prove that: the adopted YMC-Tiart C18 chromatographic column skillfully utilizes the characteristics of different viscosities and polarities of methanol, acetonitrile and water, complements glacial acetic acid, exerts different elution capacities of the four components on target components, optimizes a proper mobile phase system, and ensures that a good separation effect is achieved between a cholesterol main peak and other sterol peaks. Meanwhile, the maximum ultraviolet wavelength of cholesterol, 24-cholestadienol, sitosterol, cholestanol and beta-cholestanol is below 210nm, especially the absorption of beta-cholestanol is extremely weak, if no means such as pre-column derivatization and the like are adopted, the detection limit is above 1mg level, and the detection requirement of the limit of related substances cannot be met at all.

3. Other patents and literature have not found reports on methods for simultaneously measuring 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol in cholesterol by using a high performance liquid phase combined electrospray detector. The method of the invention is also strictly verified, and has the characteristics of strong specificity, good separation degree, good precision, high accuracy, good durability, simple and quick operation and the like, thereby ensuring the scientific and strict performance of the method. The method for accurately and rapidly measuring other sterols in cholesterol can be applied to the process of cholesterol production technology and the control of other sterols related substances of the final product, has strong practicability in work, and meets the urgent demands of enterprise drug research and development and production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a chromatogram of a cholesterol test solution for a specific study in example 5;

FIG. 2 is a chromatogram of a cholesterol test sample labeling solution for a specificity study in example 5;

FIG. 3 is a chromatogram of a cholesterol test solution in comparative example 1;

FIG. 4 is a chromatogram of a cholesterol test solution in comparative example 2;

FIG. 5 is a chromatogram of a cholesterol test solution in comparative example 3;

FIG. 6 is a chromatogram of a cholesterol test solution in comparative example 4.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In a first aspect, a method is provided for detecting other sterols in cholesterol, including 24-cholestadiol, sitosterol, cholestanol, and beta-cholestanol.

The detection method comprises the steps of separating and measuring other sterols in cholesterol by adopting a high performance liquid chromatography, obtaining a chromatogram and calculating to obtain other sterol contents;

the chromatographic conditions of the high performance liquid chromatography comprise the following steps:

the detector of the liquid chromatograph is an electrospray detector;

the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises a methanol and water mixed solution; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid;

the chromatographic column is YMC-Tiart C18 chromatographic column.

When the traditional C18 column is adopted for detection, even if the mobile phase and the gradient elution program are repeatedly optimized, the cholesterol and other sterols are difficult to be effectively separated, and the inventor proves that a plurality of groups of research experiments prove that: and the adopted YMC-Tiart C18 chromatographic column has a mobile phase A of a mixed solution of methanol and water, a mobile phase B of a mixed solution of acetonitrile, water and glacial acetic acid, and the mobile phase A and the mobile phase B are subjected to isocratic elution to obtain a good separation effect between a main cholesterol peak and other sterol peaks.

Because the polarity and structure of the impurities in the object detected by the invention are very similar, how to utilize the reasonable configuration of the different polarities and the composition ratio of the mobile phase system is that the chromatographic conditions of various combinations can be obtained through multiple experiments, and the innovative results can be obtained through comparison, summarization and screening. The inventor skillfully utilizes the characteristics of different viscosities and polarities of methanol, acetonitrile and water on the premise of different adsorption forces of YMC-Tiart C18 on target components, complements with glacial acetic acid, exerts different elution capacities of the four components on the target components, achieves the aim of effectively separating impurities from main components, and has the separation degree of more than 1.5. By comparing the chromatograms of the electrospray detector and the ultraviolet detector, the ultraviolet absorption of the beta-cholestanol is extremely weak, so that the sensitivity of the ultraviolet detector cannot realize the simultaneous separation and detection of cholesterol and 4 other sterols, but the detection result of the electrospray detector proves that the electrospray detector can solve the problem of poor sensitivity of the ultraviolet detector.

In an alternative embodiment, the volume ratio of methanol to water in mobile phase a is 95:5.

In an alternative embodiment, in mobile phase B, the volume ratio of acetonitrile, water and glacial acetic acid is (85-95): (5-15): (0.1-0.2), for example, but not limited to 85:15:0.1, 90:10:0.1, 95:5:0.1, 85:15:0.15, 90:10:0.15, 95:5:0.15, 85:15:0.2, 90:10:0.2, 95:5:0.2.

In an alternative embodiment, the volume ratio of methanol to water in mobile phase a is 95:5; in mobile phase B, the volume ratio of acetonitrile, water and glacial acetic acid is 90:10:0.1.

In an alternative embodiment, mobile phase a and mobile phase B are isocratically eluted.

In an alternative embodiment, the isocratic elution has a volume ratio of mobile phase a to mobile phase B of 80:20.

in an alternative embodiment, the column is a YMC-Tiart C18 column, having a size of 4.6mm×250mm, and a packing particle size of 3 μm.

In alternative embodiments, the column temperature of the chromatographic column is 20 to 35 ℃, such as, but not limited to, 20, 25, 30 or 35 ℃, preferably 30 ℃.

In an alternative embodiment, the chromatographic conditions further comprise a mobile phase flow rate of 1.0mL/min.

In an alternative embodiment, the chromatographic conditions further comprise a sample loading of 20 μl.

By adopting the further preferable chromatographic conditions of high performance liquid chromatography, better separation effect between the main cholesterol peak and other sterol peaks can be obtained.

In an alternative embodiment, the sample to be tested comprises a test solution using ethanol as a solvent, wherein the concentration of the cholesterol sample is 4-6 mg/mL, preferably 5mg/mL.

In an alternative embodiment, the method for preparing the test solution comprises the following steps: weighing a proper amount of cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, filtering, and taking the cholesterol sample as the sample solution without derivatization.

In an alternative embodiment, the sample to be tested comprises a control solution containing known amounts of one or more of 24-cholestadienol, sitosterol, cholestanol and beta-cholestanol.

In an alternative embodiment, the control solution uses ethanol as a solvent.

In an alternative embodiment, the external standard method is used for analysis and calculation of the content of other sterols;

in an alternative embodiment, the external standard method is a standard curve method.

In an alternative embodiment, the method for detecting other sterols in cholesterol comprises:

(a) Preparing a test sample solution: weighing a cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, and filtering to obtain a sample solution;

preparing a reference substance solution: weighing one or more of 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol, and dissolving and diluting the solution to a known content by using absolute ethyl alcohol to obtain a test solution;

(b) Setting high performance liquid phase detection conditions: the chromatographic column is YMC-Tiart C18 chromatographic column with specification of 4.6mm×250mm and 3 μm; the detector is an electrospray detector; the column temperature is 30 ℃; the mobile phase A is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 95:5, a step of; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid, and the volume ratio of acetonitrile, water and glacial acetic acid is 90:10:0.1; the flow rate of the mobile phase is 1.0ml/min; the volume ratio of the mobile phase A to the mobile phase B is 80:20, performing isocratic elution;

(c) Respectively sucking the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, analyzing, and recording chromatograms;

(d) And analyzing and calculating the content of other sterols by adopting an external standard method.

In a specific embodiment, the method for detecting other sterols in cholesterol is as follows:

chromatographic conditions and system adaptation test: the column was YMC-Tiart C18 (4.6mm. Times.250 mm,3 μm); the detector is an electrospray detector; the column temperature is 30 ℃; the mobile phase A is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 95:5, a step of; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid, and the volume ratio of acetonitrile, water and glacial acetic acid is 90:10:0.1; the flow rate of the mobile phase is 1.0ml/min; the volume ratio of the mobile phase A to the mobile phase B is 80:20, performing isocratic elution.

And dissolving and diluting the cholesterol, 24-cholestadienol, sitosterol, cholestanol and beta-cholestanol reference substances with absolute ethyl alcohol to obtain mixed solutions with the contents of 5mg/ml, 0.1mg/ml and 0.1mg/ml respectively, and taking the mixed solutions as system adaptive solutions. And precisely sucking 20 μl of the system adaptive solution, injecting into a high performance liquid chromatograph, and recording a chromatogram. The separation degree between the main cholesterol peak and other chromatographic peaks should be more than or equal to 1.5.

Preparing a test solution: weighing a proper amount of cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, and filtering to obtain the sample solution.

Preparing a reference substance solution: and weighing a proper amount of 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol reference substances, diluting the reference substances with absolute ethyl alcohol to a standard curve solution with 5 concentrations, and uniformly mixing the reference substances to obtain a reference substance solution.

The measuring method comprises the following steps: accurately sucking 20 μl of the sample solution and the reference solution, respectively, injecting into high performance liquid chromatograph, and recording chromatogram. And (3) taking the logarithm of the concentration of the reference substance as an abscissa, the logarithm of the corresponding peak area as an ordinate, making a standard curve, and calculating the content of other sterols by using a regression equation.

In view of the shortcomings of the existing detection methods, the inventor establishes a method for combining a high performance liquid chromatography with an electrospray detector and simultaneously measuring the absolute content of 24-cholestadienol, sitosterol, cholestanol and beta-cholestanol in cholesterol, and ensures separation and detection of the cholesterol and several sterols. The method of the invention is strictly verified, and the method is ensured to be scientific and strict. The detection method has the characteristics of strong specificity, good separation degree, good precision, high accuracy, good durability, simple and quick operation and the like, and can be effectively used for controlling related substances of other sterols in the process of cholesterol production technology and the final product.

In a second aspect, there is also provided the use of the method for the detection of other sterols in cholesterol of the first aspect in any of the following:

preparing cholesterol;

(ii) preparing a cholesterol-containing product comprising an emulsifier, a cosmetic or a pharmaceutical product.

In an alternative embodiment, the detection object of the method for detecting other sterols in cholesterol comprises pharmaceutic adjuvant extracted from egg yolk and used for injection.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

Example 1

Mobile phase screening:

sample adding standard solution preparation: precisely weighing a proper amount of a test product, adding a proper amount of 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol reference substances, dissolving with absolute ethyl alcohol, and diluting to obtain mixed solutions with the contents of 5mg/mL, 0.1mg/mL and 0.1mg/mL respectively, wherein the mixed solutions are used as sample adding standard solutions.

And precisely sucking 20 μl of the sample and the standard solution, injecting into a high performance liquid chromatograph, measuring according to the screening conditions of the mobile phase system in table 1, and recording the chromatogram. The number of peaks in the chromatogram was observed, and the degree of separation between the peaks was observed. The screening conditions and the results of the mobile phase system are shown in the following tables 1 and 2.

TABLE 1 screening conditions for mobile phase systems

TABLE 2 screening results for mobile phase systems

Conditions (conditions)	Number of detected components	Minimum degree of separation between peaks
			Mobile phase system I	6	＜1.0
Mobile phase system ii	7	＜1.0
			Mobile phase system III	3	1.5
Mobile phase system IV	7	1.0
			Mobile phase system V	7	1.6
Mobile phase system VI	6	1.5
			Mobile phase system VII	7	1.5
Mobile phase system VIII	7	1.5
			Mobile phase system IX	7	1.5

In order to achieve the aim of measuring other sterols, the concentration of a test sample is required to be high, the main component peak and the adjacent cholestanol peak in the reversed phase liquid chromatography are difficult to achieve baseline separation, the 24-cholestadienol and the sitosterol are also difficult to achieve baseline separation, and if the concentration of the sample is low, the probability of detecting related impurities in a missing way is increased.

The chromatograms of different mobile phase systems show that the minimum separation degree between peaks of the mobile phase system I, II cannot reach more than 1.5, and the minimum separation degree between peaks of the mobile phase system III can reach more than 1.5, but the main cholesterol peak emergence time is 146 minutes, and the 24-cholestadiol, the sitosterol and other small impurities with lower content cannot be detected because the peak form is flattened after the peak emergence delay, so that the mobile phase system cannot be applied to sample analysis and detection.

The mobile phase system VI is that glacial acetic acid is not added into the mobile phase B, and the separation degree is required but some small impurities cannot be detected.

When the mobile phase system V, VII, VIII, IX is adopted for gradient elution, the peak sequence of all sterol components is 24-cholestadiol, sitosterol, cholestanol, cholesterol and beta-cholestanol, and the minimum separation degree between the peaks is more than 1.5. The comparison result shows that if the solvent of the mobile phase in the embodiment of the invention is changed or the composition ratio of the solvent is changed, the separation degree effect can be affected, so that accurate detection cannot be realized, and the mobile phase system is innovative. The results show that the mobile phase system V, VII, VIII, IX can achieve the aim of separation and detection, the mobile phase A is a mixed solution of methanol and water, and the volume ratio is 95:5, a step of; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid, and the volume ratio is (85-95): (5-15): (0.1 to 0.2); with mobile phase system V being the most preferred condition.

Example 2

Detector contrast screening:

sample labeling solution preparation the same as in example 1, precisely sucking 20 μl of sample labeling solution, injecting into high performance liquid chromatograph, measuring according to the screening conditions of the detector of table 3, and recording chromatogram. The number of peaks in the chromatogram was observed, and the degree of separation between the peaks was observed. The detector screen is shown in table 3 below.

TABLE 3 detector screening conditions

The electrospray detector chromatogram shows that cholesterol and 4 other sterols can be detected, and the peak sequence is 24-cholestadienol, chain sterol, cholestanol, cholesterol, beta-cholestanol, and the minimum separation degree between peaks is more than 1.5. The ultraviolet detector chromatogram showed only chromatographic peaks of cholesterol and 24-cholestadiol, sitosterol, and cholestanol, and no component peak of beta-cholestanol was detected. The results show that the ultraviolet absorption of beta-cholestanol is very weak, so that the ultraviolet detector cannot realize simultaneous separation and detection of cholesterol and 4 other sterols, and the electrospray detector is selected to separate and detect the other sterols.

Example 3

And (3) comparing and screening by using a chromatographic column:

sample labeling solution preparation the same as in example 1, precisely sucking 20 μl of sample labeling solution, injecting into high performance liquid chromatograph, measuring according to the column screening conditions of table 4, and recording chromatogram. The number of peaks in the chromatogram was observed, and the degree of separation between the peaks was observed. The comparative screening conditions of the chromatographic columns are shown in Table 4, and the comparative research results of the chromatographic columns are shown in Table 5.

TABLE 4 column screening conditions

Table 5 results of column comparison study

Chromatographic column	Number of detected components	Minimum degree of separation between peaks
			Diamonsil	5	＜1.0
YMC	7	1.6
			Irinotecan	4	＜1.0

The chromatogram of column 2 (YMC-Tiart C18.6mm×250mm,3 μm) shows that cholesterol and 4 other sterols can be detected, the peak sequence is 24-cholestadiol, chain sterol, cholestanol, cholesterol, beta-cholestanol, and the minimum separation degree between peaks is above 1.5. When the other 2 traditional C18 columns are used for detection, even if the mobile phase and gradient elution procedures are repeatedly optimized, the separation degree between the main cholesterol peak and other sterol peaks is difficult to reach the detection requirement. The results show that when the special chromatographic column and the optimized mobile phase system are adopted for elution, the peaks can be well separated, and further, the content of other sterols can be accurately detected. The final column was therefore selected as column 2 (YMC-Tiart C18.6 mm. Times.250 mm,3 μm).

Example 4

Column temperature contrast screening:

sample labeling solution preparation the same as in example 1, precisely sucking 20 μl of sample labeling solution, injecting into high performance liquid chromatograph, measuring according to the column screening conditions of table 6, and recording chromatogram. The number of peaks in the chromatogram was observed, and the degree of separation between the peaks was observed. The results of the column comparison are shown in Table 7.

TABLE 6 column temperature screening conditions

TABLE 7 results of column temperature comparison study

Column temperature	Number of detected components	Minimum degree of separation between peaks
			20℃	6	1.5
30℃	7	1.6
			40℃	7	1.3

The chromatogram at 30 ℃ shows that cholesterol and 4 other sterols can be detected, the peak sequence is 24-cholestadienol, chain sterol, cholestanol, cholesterol and beta-cholestanol, and the minimum separation degree between the peaks is more than 1.5. The chromatogram at the column temperature of 40 ℃ shows that the separation degree among the peaks is affected to different degrees and the baseline separation is not achieved because the peak-out time accelerates the detection of cholesterol and 4 other sterols. The chromatogram at 20℃shows that cholesterol and 4 other sterols can be detected, but some minor impurities cannot be integrated after peak shape is bad due to the delay of peak time. Therefore, the final column temperature is preferably 30 ℃.

Example 5

The following methodological validation was performed using laboratory bench scale cholesterol samples, according to the chromatographic conditions of table 8:

TABLE 8 sample chromatographic conditions for small test cholesterol

Specificity investigation

(1) Solution preparation

Blank solution: absolute ethyl alcohol.

Preparation of a single sterol positioning solution: and respectively taking a proper amount of 24-cholestadiol, sitosterol, cholestanol, cholesterol and beta-cholestanol reference substances, dissolving the reference substances with absolute ethyl alcohol and diluting the reference substances to 0.2mg/mL of single standard solution to be used as single sterol positioning solution.

Preparation of sample solution: accurately weighing a proper amount of the test sample, dissolving the test sample in absolute ethyl alcohol, and diluting the solution to a concentration of 5mg/mL to obtain a test sample solution.

Sample labeling solution (exclusive solution) preparation: precisely weighing a proper amount of a test product, adding a proper amount of 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol reference substances, dissolving with absolute ethyl alcohol, and diluting to obtain mixed solutions with the contents of 5mg/mL, 0.1mg/mL and 0.1mg/mL respectively, and taking the mixed solutions as sample standard adding solutions.

(2) And (3) measuring: taking blank solution, single sterol positioning solution, sample solution and specific solution, respectively injecting 20 μl, detecting according to the chromatographic conditions of table 8, recording the chromatogram, wherein the chromatogram of sample solution is shown in figure 1, and the chromatogram of cholesterol sample adding standard solution is shown in figure 2. The results of the blank solution, the single sterol positioning solution, the test solution, and the specific solution are shown in Table 9.

TABLE 9 specific isolation results

As shown in the results of Table 9, the absolute ethyl alcohol of the blank solution has no interference to the measurement, the retention time of the single sterol positioning solution is consistent with the retention time of other sterols existing in the sample solution and the specific solution, the separation degree between each peak of the sample and the sample adding standard solution is more than 1.5, and the method has good specificity and can be used for measuring other sterols in cholesterol.

(II) limit of detection and limit of quantification test

Taking a single sterol reference substance solution in specificity investigation, adding absolute ethyl alcohol to gradually dilute the solution to a low-concentration solution, respectively injecting 20 mul according to chromatographic conditions in the invention, and recording a chromatogram. When the signal to noise ratio (S/N) is more than or equal to 3, the detection limit concentration of 24-cholestanol, sitosterol, cholestanol and beta-cholestanol is 0.002119mg/mL, 0.001745mg/mL, 0.0008948mg/mL and 0.001175mg/mL respectively; when the signal to noise ratio (S/N) is more than or equal to 10, the quantitative limiting concentrations of 24-cholestadienol, sitosterol, cholestanol and beta-cholestanol are 0.005298mg/mL, 0.005235mg/mL, 0.002684mg/mL and 0.003525mg/mL respectively.

(III) Linear test

Precisely weighing the reference substances of 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol, dissolving the reference substances with absolute ethyl alcohol and diluting the reference substances to 5 reference substance solutions with different concentrations, respectively injecting 20 mu l according to the chromatographic conditions in the invention, and recording a chromatogram. And taking the logarithm of the concentration of the reference substance as an abscissa and the logarithm of the corresponding peak area as an ordinate, and making a standard curve, wherein the calculation result of the standard curve is shown in tables 10-13.

TABLE 10 24 Linear test results of cholestadienol

TABLE 11 results of chain sterol linearity test

TABLE 12 results of linear test of alkene cholestanol

Table 13 results of linear test of beta-cholestanol

The results show that 24-cholestadienol has good linear relationship in the range of 0.008475 mg/mL-0.03390 mg/mL, the chain sterol has good linear relationship in the range of 0.006980 mg/mL-0.02792 mg/mL, the cholestanol has good linear relationship in the range of 0.03580 mg/mL-01432 mg/mL, and the beta-cholestanol has good linear relationship in the range of 0.05875 mg-0.2350 mg/mL.

(IV) repeatability test, intermediate precision test

6 parts of cholesterol samples of the same batch are weighed, the relative standard deviation of other sterol contents is calculated according to the detection method of the embodiment, and RSD is less than or equal to 3%. Taking 1 batch of cholesterol samples by the same method, determining by different personnel at different dates according to the method, recording chromatograms, and calculating and determining the relative standard deviation of other sterol contents, wherein RSD is less than or equal to 3%. Compared with the repeated experimental result, the RSD of the obtained 12 data is less than or equal to 3%. The results of the precision test are shown in Table 14.

TABLE 14 precision test results

As shown in Table 14, in the repeatability test, the other sterol repeatability results of 6 samples were all less than or equal to 3% RSD, indicating good repeatability of the method. In the intermediate precision test, the total impurity content result RSD of 12 samples is less than or equal to 3%, which indicates that the precision of the method is good.

(fifth) durability test

(1) Solution stability test

1 batch of cholesterol samples are taken, 1 part of test solution is prepared according to the detection method of the invention, the chromatographic conditions of the detection method of the invention are respectively measured once in 0, 2, 4, 6, 8, 12 and 24 hours, the relative standard deviation of the peak areas of other sterols is calculated, RSD is less than or equal to 3 percent, and the result is shown in the following table 15, and the result shows that the test solution is stable in 24 hours.

TABLE 15 results of solution stability test

Time (h)	0	2	4	6	8	12	24	Average value of	RSD(％)
										24-cholestadiene alcohol	0.8361	0.8316	0.8438	0.8512	0.8234	0.8512	0.8535	0.8415	1.4
Desmosterol	0.8665	0.8836	0.8923	0.8513	0.9012	0.9003	0.8956	0.8844	2.1
										Cholestanol	4.3505	4.3444	4.4512	4.3562	4.4511	4.4689	4.4565	4.4113	1.3
Beta-cholestanol	6.3117	6.3375	6.3416	6.4077	6.4142	6.4459	6.4693	6.3897	0.9

(2) System durability

According to the detection method of this example, 1 batch of cholesterol samples were taken, the other chromatographic conditions were unchanged, different column temperatures (25 ℃, 30 ℃, 35 ℃) were set, and the measurement was performed while changing the same brand of different batches of chromatographic columns (i.e., different batches of chromatographic columns of YMC-Tiart C184.6mm. Times.250 mm,3 μm) and the chromatograms were recorded, so that the relative standard deviation of other sterol contents was calculated, and RSD was not more than 3%. The test results are shown in Table 16 and Table 17.

Table 16 results of comparison of different column temperatures

TABLE 17 results of different chromatographic column tests

From the durability results, it was found that the cholesterol sample measurement had no effect on the measurement under small variations in column temperature and column chromatography, and the test sample solution had good stability within 24 hours.

In conclusion, the detection method of the embodiment has the characteristics of strong specificity, good separation degree, good precision, high accuracy, good durability and the like.

Comparative example 1

Other sterol measurement items in the cholesterol standard according to the United states pharmacopoeia USP-NF 2021 are measured by a gas chromatography internal standard method, and the specific method is as follows:

internal standard solution: a proper amount of pregnenolone isobutyrate was precisely weighed into a measuring flask, dissolved in heptane and diluted to a scale to make a solution of about 0.02mg per 1ml of pregnenolone isobutyrate.

Control stock solution: precisely weighing appropriate amount of cholesterol USP reference substance, chain sterol reference substance and cholestanol in a measuring flask, dissolving with internal standard, and diluting to scale to obtain mixed solution containing 0.1mg cholesterol, 0.2mg chain sterol and 0.1mg cholestanol per 1 ml.

System applicability solution: precisely remove 5.0mL of control stock solution in a 25mL volumetric flask, dissolve with internal standard, dilute to scale, shake well. A solution of about 0.02mg of stigmasterol, about 0.04mg of cholestanol, and about 0.02mg of cholestanol was prepared per 1ml of the USP control containing cholesterol.

Test solution: precisely weighing a proper amount of cholesterol sample in a measuring flask, and shaking uniformly by using an internal standard to reach the scale. About 2.0mg of solution was prepared per 1ml of cholesterol-containing sample. Two portions were prepared in parallel.

Chromatographic conditions: a detector: a flame ionization detector; chromatographic column: DB-1 (30 mX0.25 mm,0.25 μm; sample inlet temperature: 285 ℃, column temperature: 275 ℃, detector temperature: 300 ℃, carrier gas: helium, flow rate: 2.0mL/min; sample volume: 1.0. Mu.L; split weighing tube (4 mm X6.3 X78.5) used modified deactivated glass wool. Split ratio: 12:1; run time: 15min.

Assay: and (3) injecting the system adaptive solution and the sample solution into the liquid chromatograph. The sterols that may be present in the sample solution chromatograms are shown in the following table:

TABLE 18

Sample name	RRT
		Pregnenolone isobutyrate	1.00
Cholesterol	1.23
		Desmosterol	1.31
Cholestanol	1.34
		Beta-cholestanol	1.24
24-cholestadiene alcohol	1.42

The result is shown in figure 3, the peak time of the main cholesterol peak in the chromatogram of the sample solution in figure 3 is 9.035min, and the result shows that the detection is carried out according to comparative example 1, the pregnenolone isobutyrate in the chromatogram is the chromatographic peak of the internal standard substance added by the internal standard method, which is not the object of the research and the investigation of the invention, and the cholesterol, the beta-cholestanol and the chain sterol in the chromatogram can not reach baseline separation, so that the content of the chain sterol and the beta-cholestanol can not be accurately measured, and the reproducibility is poor.

Comparative example 2

The cholesterol content measurement item in the cholesterol standard of the 2020 edition of Chinese pharmacopoeia is measured by adopting a liquid phase condition in a liquid phase chromatography, and the specific method is as follows:

preparing a test solution: weighing a proper amount of cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, and filtering to obtain the sample solution.

The measuring method comprises the following steps: chromatographic column with octadecyl silicon bonded silica gel as filler; methanol is used as a mobile phase, 20 μl of the sample solution is precisely sucked, the sample solution is injected into a high performance liquid chromatograph, an evaporative light scattering detector is used for measurement, and a chromatogram is recorded.

The results are shown in FIG. 4, and the peak time of cholesterol in FIG. 4 is 3.648min, and the results show that the main cholesterol peak and the 4 other sterols cannot be separated according to the detection of comparative example 2, and the method for measuring the content cannot be used for measuring the content of the 4 other sterols.

Comparative example 3

Reference "high performance liquid chromatography determination of non-cholesterol sterols in serum" liquid chromatography, the following specific method:

preparing a test solution: weighing a proper amount of cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, and filtering to obtain the sample solution.

The measuring method comprises the following steps: a column was used with YMC-Tiart C18 (4.6mm.times.250 mm,3 μm); n-hexane saturated acetonitrile-water (volume ratio is 90:10) is used as a mobile phase, 20 μl of sample and standard solution are precisely sucked, the sample and standard solution are injected into a high performance liquid chromatograph, and the measurement is carried out under the wavelength of 240nm of an ultraviolet detector, and a chromatogram is recorded.

As a result, as shown in FIG. 5, in order to avoid using the virulent derivatization reagent phenyl isocyanate, the test sample solution of the comparative test is directly dissolved in absolute ethanol and then is detected according to the chromatographic conditions of comparative example 3, and the result shows that the main cholesterol peak and the 4 other sterol peaks cannot be detected, thus proving that the chromatographic conditions of comparative example 3 cannot be used for directly measuring the content of the 4 other sterols in the cholesterol without pre-column derivatization.

Comparative example 4

Reference "development of cholesterol purity substance" liquid chromatography, the specific method is as follows:

preparing a test solution: weighing a proper amount of cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, and filtering to obtain the sample solution.

The measuring method comprises the following steps: a column was used with YMC-Tiart C18 (4.6mm.times.250 mm,3 μm); methanol-ethanol-acetonitrile (volume ratio is 75:10:15) is used as a mobile phase, 20 μl of sample and standard solution are precisely absorbed, the sample and standard solution are injected into a high performance liquid chromatograph, the measurement is carried out at the wavelength of 240nm of an ultraviolet detector, and a chromatogram is recorded.

As a result, as shown in FIG. 6, in order to avoid using the virulent derivatization reagent phenyl isocyanate, the test sample solution of the comparative test is directly dissolved in absolute ethanol and then is detected according to the chromatographic conditions of comparative example 3, and the result shows that the main cholesterol peak and the 4 other sterol peaks cannot be detected, thus proving that the chromatographic conditions of comparative example 3 cannot be used for directly measuring the content of the 4 other sterols in the cholesterol without pre-column derivatization.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

1. A method for detecting other sterols in cholesterol, wherein the other sterols comprise 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol;

the detection method comprises the steps of separating and measuring other sterols in cholesterol by adopting a high performance liquid chromatography, obtaining a chromatogram and calculating to obtain other sterol contents;

the chromatographic conditions of the high performance liquid chromatography include:

the detector of the liquid chromatograph is an electrospray detector;

the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of methanol and water; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid;

the chromatographic column is YMC-Tiart C18 chromatographic column.

2. The method according to claim 1, wherein the volume ratio of methanol to water in mobile phase a is 95:5;

and/or, in the mobile phase B, the volume ratio of acetonitrile, water and glacial acetic acid is (85-95): (5-15): (0.1 to 0.2);

preferably, in the mobile phase A, the volume ratio of methanol to water is 95:5; in mobile phase B, the volume ratio of acetonitrile, water and glacial acetic acid is 90:10:0.1.

3. The method according to claim 1, wherein mobile phase a and mobile phase B are subjected to isocratic elution;

preferably, the volume ratio of mobile phase a to mobile phase B in the isocratic elution is 80:20.

4. the method according to claim 1, wherein the column temperature of the chromatographic column is 20-35 ℃, preferably 30 ℃.

5. The method of claim 1, wherein the chromatographic column is a YMC-Ttiart C18 chromatographic column;

preferably, the column is a YMC-Tiart C18 column, with a specification of 4.6mm×250mm, and a packing particle size of 3 μm.

6. The method of detection according to claim 1, wherein the chromatographic conditions further comprise: the flow rate of the mobile phase is 1.0mL/min; and/or the sample injection amount is 20 mu L.

7. The method according to any one of claims 1 to 6, wherein the sample to be tested comprises a sample solution containing ethanol as a solvent, and wherein the concentration of the cholesterol sample is 4 to 6mg/mL, preferably 5mg/mL.

8. The method according to any one of claims 1 to 6, wherein the content of other sterols is calculated by analysis using an external standard method;

preferably, the external standard method is a standard curve method.

9. The method of detection according to claim 8, wherein the method of detection comprises:

(a) Preparing a test sample solution: weighing a cholesterol sample to be measured, dissolving and diluting the cholesterol sample to be measured into a solution with the content of 5mg/ml of the sample by using absolute ethyl alcohol, uniformly mixing, and filtering to obtain a sample solution;

preparing a reference substance solution: weighing one or more of 24-cholestadiol, sitosterol, cholestanol and beta-cholestanol, and dissolving and diluting the solution to a known content by using absolute ethyl alcohol to obtain a test solution;

(b) Setting high performance liquid phase detection conditions: the chromatographic column is YMC-Tiart C18 chromatographic column with specification of 4.6mm×250mm and 3 μm; the detector is an electrospray detector; the column temperature is 30 ℃; the mobile phase A is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 95:5, a step of; the mobile phase B is a mixed solution of acetonitrile, water and glacial acetic acid, and the volume ratio of acetonitrile, water and glacial acetic acid is 90:10:0.1; the flow rate of the mobile phase is 1.0ml/min; the volume ratio of the mobile phase A to the mobile phase B is 80:20, performing isocratic elution;

(c) Respectively sucking the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, analyzing, and recording chromatograms;

(d) And analyzing and calculating the content of other sterols by adopting an external standard method.

10. Use of the method for detecting other sterols in cholesterol according to any one of claims 1 to 9 in any one of the following:

preparing cholesterol;

(ii) preparing a cholesterol-containing product comprising an emulsifier, a cosmetic or a pharmaceutical product.