A method for the chromatographic detection of industrial lanolin alcohols

Abstract

The present application belongs to the technical field of analysis and detection, and particularly relates to a chromatographic detection method of industrial lanolin alcohol. The detection method of the present application adopts one-standard multiple-detection method, takes cholesterol as an internal reference, and detects the contents of cholesterol, lanosterol and dihydrolanosterol in industrial lanolin alcohol by high performance liquid chromatography. The present application establishes a one-standard multiple-detection method of industrial lanolin alcohol by optimizing the extraction method, chromatographic conditions and elution gradient, and only takes the cheap and easily available cholesterol as an internal reference, so as to simultaneously detect the contents of cholesterol, lanosterol and dihydrolanosterol in industrial lanolin alcohol. The detection method has good linear relationship, precision, repeatability and stability, low cost, and can more comprehensively reflect the quality of industrial lanolin alcohol, and can provide a basis for further improving the quality standard of industrial lanolin alcohol.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a chromatographic detection method for industrial lanol, and more specifically to a method for simultaneously detecting the content of cholesterol, lanosterol, and dihydrolanosterol in industrial lanol. Background Technology

[0002] Cholesterol, lanosterol, and dihydrolanosterol are important chemical components in industrial lanol, and their combined content generally exceeds 50% of the total weight. Cholesterol has the molecular formula C6H2O. 27 H 46 O, with a relative molecular mass of 386.65, is a natural steroid resource in the pharmaceutical industry due to its steroid nucleus structure. Lanosterol, with the molecular formula C... 30 H 50 O, with a relative molecular mass of 426.73, lanosterol is an important sterol with significant physiological functions and is a crucial raw material in the cosmetics, pharmaceutical, and chemical industries. Dihydrolanosterol, with the molecular formula C... 30 H 52 O, with a relative molecular mass of 428.73, dihydrolanosterol is one of the substrates of CYP51 and has the physiological activity of inhibiting cholesterol synthesis. The structural formulas of the three are shown in Formulas I, II, and III below, respectively.

[0003]

[0004] To enhance quality control and utilization efficiency of industrial lanol, manufacturers need to quantitatively analyze its chemical composition. Lanols with different chemical compositions can be used to process various downstream products. For example, industrial lanol with a high lanosterol content can be used to further extract high-purity lanosterol, while industrial lanol with a high cholesterol content can be processed through molecular distillation to extract cholesterol again.

[0005] However, there is currently a lack of reported corresponding liquid chromatography (LC) analysis methods. One reason for this is the high price of lanosterol reference standards. Therefore, it is necessary to develop LC analysis methods that can reduce the cost of reference standards. Multiple assays using a single standard are a common analytical technique in the field of traditional Chinese medicine. Using a single inexpensive reference standard to detect multiple components is expected to significantly reduce the cost of purchasing reference standards, providing a basis for controlling the quality of industrial lanosterol in production and determining its subsequent uses. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a chromatographic detection method for industrial lanosterol. This method overcomes the drawbacks of existing analytical methods, such as high detection costs and numerous steps, and also fills the gap in the analysis of intermediate components during the production of lanosterol and dihydrolanosterol. It plays an important role in controlling the quality of industrial lanosterol.

[0007] This invention provides a chromatographic detection method for industrial lanosterol, which employs a single standard for multiple determinations method, using cholesterol as an internal reference, and high performance liquid chromatography to detect the content of cholesterol, lanosterol, and dihydrolanosterol in industrial lanosterol.

[0008] This invention develops a single-standard, multiple-analysis method for industrial lanosterol based on existing industrial lanosterol detection technologies. Only three reference standards—cholesterol, lanosterol, and dihydrolanosterol—are needed initially for establishing the method. Once established, only cholesterol is required to simultaneously detect the contents of cholesterol, lanosterol, and dihydrolanosterol in industrial lanosterol. This effectively addresses the shortcomings of existing technologies, offering not only low cost but also providing a basis and method for controlling the quality of industrial lanosterol in production and determining its subsequent applications.

[0009] Preferably, in the above technical solution, the chromatographic column of the high performance liquid chromatography is a SunShell C18 column, with a model of 4.6mm×150mm and a packing particle size of 2.6μm.

[0010] Preferably, in the above technical solution, the mobile phase of the high performance liquid chromatography uses acetonitrile as phase B and water as phase A, and gradient elution is performed.

[0011] Preferably, in the above technical solution, the gradient elution program is: 0-19.0 min, 90.5% B; 19.0-25.0 min, 90.5%-100.0% B; 25.0-55 min, 100% B.

[0012] Preferably, in the above technical solution, the ultraviolet detector wavelength of the high performance liquid chromatography is 205 nm, and the running time is 55 min.

[0013] Preferably, in the above technical solution, the flow rate of the high performance liquid chromatography is 1.58±0.02mL / min.

[0014] Preferably, in the above technical solution, the column temperature of the high performance liquid chromatography is 37±1℃.

[0015] Preferably, in the above technical solution, the injection volume of the high performance liquid chromatography is 10±5μL.

[0016] Preferably, in the above technical solution, the chromatographic detection method includes the following specific steps:

[0017] (1) Preparation of cholesterol reference solution: Take an appropriate amount of cholesterol and add N-methylpyrrolidone to prepare a solution containing 1.76 mg of cholesterol per 1 mL;

[0018] (2) Preparation of test solution: Take 20 mg of industrial lanolin in a 10 mL volumetric flask, accurately weigh it, dissolve and dilute it with N-methylpyrrolidone, and then centrifuge it to obtain the test solution.

[0019] (3) High performance liquid chromatography determination: First, inject the reference solution and the test solution from step (1) into the high performance liquid chromatograph for analysis and determination. Cholesterol is used as an internal reference. The peak positions of cholesterol, lanosterol and dihydrolanosterol are determined according to the predetermined relative retention time. Then, the contents of cholesterol, lanosterol and dihydrolanosterol are calculated according to the relative correction factor.

[0020] Preferably, in the above technical solution, the relative retention time is the relative retention time relative to the elution time of the cholesterol chromatographic peak. For lanosterol, the elution time is the cholesterol retention time multiplied by 1.087, and for dihydrolanosterol, the elution time is the cholesterol retention time multiplied by 1.323. The relative correction factor is the relative correction factor relative to the peak area of ​​the cholesterol chromatographic peak. When calculating the lanosterol content, it is the relative correction factor between lanosterol and cholesterol (0.4227) multiplied by the cholesterol content multiplied by the lanosterol peak area and then divided by the cholesterol peak area. When calculating the dihydrolanosterol content, it is the relative correction factor between dihydrolanosterol and cholesterol (0.8228) multiplied by the cholesterol content multiplied by the dihydrolanosterol peak area and then divided by the cholesterol peak area.

[0021] Advantages compared to existing technologies:

[0022] This invention establishes for the first time a chromatographic detection method for industrial lanol, filling the gap in existing methods for detecting intermediates in the production process.

[0023] The single-standard, multi-analysis method established in this invention can simultaneously determine the content of cholesterol, lanosterol, and dihydrolanosterol in industrial lanosterol using only cholesterol as a reference standard. This greatly saves the amount of reference standard used, solves the problem of high reference standard prices, and reduces analytical costs.

[0024] The quality control and testing method (one standard, multiple tests) of the present invention is simple to operate, highly accurate, reproducible, and low in cost. Its test results fully meet the requirements compared with the external standard method, and can more comprehensively reflect the quality of industrial lanolin, providing a basis for further improving the quality standard of industrial lanolin and clarifying its subsequent uses. Attached Figure Description

[0025] Figure 1 This is the chromatogram of the test solution in Example 1 of the present invention;

[0026] Figure 2 This is a chromatogram of the reference standard mixture of the present invention;

[0027] Figure 3 The chromatogram of the test solution in Comparative Example 1 is shown below.

[0028] Figure 4 The chromatogram of the test solution in Comparative Example 2 is shown below.

[0029] Figure 5 The chromatogram of the test solution in Comparative Example 3 is shown below.

[0030] Figure 6 The chromatogram is of the test solution in Comparative Example 4. Detailed Implementation

[0031] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. However, the present invention is not limited to these embodiments, nor are these embodiments limited in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the reagents used in the following examples are all commercially available products and can be purchased from the market.

[0033] The present invention will be further described in detail below with reference to the illustrations and embodiments:

[0034] Experimental instruments and materials

[0035] 1. Reagents and reagents

[0036] Acetonitrile (chromatographic grade, Merck GmbH, Germany); N-methylpyrrolidone (analytical grade, Shanghai Maclean Company); cholesterol reference standard (99% purity, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: H2229748); lanosterol reference standard (100% purity, laboratory prepared); dihydrolanosterol reference standard (95% purity, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number A2303183); industrial lanol batch numbers 4301 and 4302, provided by Jiangxi Nuowei Biotechnology Co., Ltd.; deionized water was prepared by a water purification system (Milli-Q, Millipore GmbH, Germany).

[0037] 2. Instruments and Equipment

[0038] High-performance liquid chromatograph (SHIMADZU LC-20AT, Shimadzu Corporation, Japan), including LC-20A infusion pump, autosampler, column oven, degasser, UV-Vis detector, and data processing software; electronic balance (00000246, Sartorius Scientific Instruments); ultrasonic instrument (KQ2200E, Kunshan Meimei Ultrasonic Instruments Co., Ltd.).

[0039] 3. Solution preparation

[0040] 3.1 Preparation of the reference standard mixed solution

[0041] Take appropriate amounts of cholesterol, lanosterol, and dihydrolanosterol reference standards, and add N-methylpyrrolidone to prepare a solution containing 1.76 mg cholesterol, 1.28 mg lanosterol, and 1.16 mg dihydrolanosterol per 1 mL.

[0042] 3.2 Preparation of cholesterol reference solution

[0043] Take an appropriate amount of cholesterol reference standard and add N-methylpyrrolidone to prepare a solution containing 1.76 mg of cholesterol per 1 mL.

[0044] 3.3 Preparation of test solution

[0045] Take approximately 20 mg of industrial lanolin sample into a 10 mL volumetric flask, accurately weigh it, dissolve it in N-methylpyrrolidone and dilute to the mark, mix thoroughly, centrifuge and collect the supernatant to obtain the product.

[0046] Example 1: A chromatographic detection method for industrial lanolin.

[0047] (1) The preparation of the test solution and the reference solution shall be carried out in accordance with the above method;

[0048] (2) The chromatographic conditions are as follows:

[0049] Chromatographic column: SunShell C18 column (4.6mm × 150mm, 2.6μm);

[0050] Mobile phase: Acetonitrile (B) - water (A) mobile phase system was used;

[0051] Flow rate: 1.58 mL / min;

[0052] Column temperature: 37℃;

[0053] Injection volume: 10 μL;

[0054] UV detector wavelength: 205nm, runtime: 55min;

[0055] Gradient elution was employed under the following conditions: 0–19.0 ​​min: 90.5% B; 19.0–25.0 min: 90.5%–100.0% B; 25.0–55 min: 100% B.

[0056] The theoretical plate number, calculated based on the cholesterol peak, is no less than 60,000.

[0057] Detection chromatogram as follows Figure 1 As shown, cholesterol, lanosterol, and dihydrolanosterol were well separated and the analysis time was short.

[0058] Example 2: Methodological Investigation

[0059] 1. Examination of linear relationships

[0060] The reference standard mixture prepared in "3.1" was diluted 3.333, 8.333, 20.83, 52.08, 130.2, and 325.5 times to obtain a series of reference standard mixtures with different concentrations. 10 μL of each mixture was injected and analyzed under the chromatographic conditions of Example 1. A standard curve was constructed with the peak area of ​​each component as the ordinate and the concentration as the abscissa to obtain the linear regression equation and analytical range.

[0061] The regression equation is: Cholesterol Y = 4.08 × 10 3 X, R 2 =0.9997; Lanosterol Y = 9.65 × 10 3 X, R 2 =1.000; dihydrolanosterol Y = 4.96 × 10 3 X, R 2 =1.000; the results indicate that cholesterol levels range from 5.42 to 529 μg / mL. -1 The linear relationship was good within the range of 3.93-384 μg·mL; lanosterol showed good linearity within this range. -1 The linear relationship was good within the range of 3.56-348 μg·mL; dihydrolanosterol showed good linearity within this range. -1 The linear relationship is good within the range.

[0062] 2. Precision test

[0063] The same sample solution from batch number 4301 was injected six times consecutively, and the RSD values ​​of the peak area and retention time of each component were calculated. The results are shown in Table 1. The RSD of the retention time of each component was less than 0.5000%, and the RSD of the peak area was less than 1.500%.

[0064] Table 1 Precision test results

[0065]

[0066]

[0067] 3. Repeatability test

[0068] Six parallel-prepared test solutions of batch number 4301 were injected and analyzed separately. The RSD values ​​of peak area and retention time for each component were calculated. The results are shown in Table 2. The RSD of retention time for each component was less than 0.5000%, and the RSD of peak area was less than 2.000%.

[0069] Table 2 Results of Repeatability Tests

[0070]

[0071] 4. Stability test

[0072] The test solution (batch number 4301) and a mixed solution of reference standards with the same concentration were analyzed at 0, 1, 2, 4, 8, 12, and 24 hours. The RSD values ​​of peak area and retention time for each component were recorded. The results are shown in Table 3. The RSD of retention time for each component was less than 0.5000%, and the RSD of peak area was less than 2.000%.

[0073] Table 3 Stability test results

[0074]

[0075] 5. Spiking recovery test

[0076] Nine aliquots of the test solution with known concentrations were taken and divided into three groups. Three concentration levels (low, medium, and high) were established, and the ratio of the amount of reference standard added to the amount of the analyte in the test sample was controlled to be approximately 0.8:1.0, 1.0:1.0, and 1.2:1.0, respectively. Three parallel test solutions were prepared for each concentration, and the recovery rates were calculated. The results are shown in Table 4. The average recovery rate of cholesterol was 99.70%, the average recovery rate of lanosterol was 101.0%, and the average recovery rate of dihydrolanosterol was 101.8%, all meeting the requirements. Furthermore, the RSD of the recovery rates of each component was less than 3.0%.

[0077] Table 4 Results of the recovery test

[0078]

[0079]

[0080] Example 3: Screening Study of One Standard, Multiple Measurement Methods

[0081] 1. Determination of internal reference standards for multiple assays using a single standard

[0082] Internal reference standards should meet three requirements: (1) abundant content in the sample; (2) stable; and (3) easy to obtain. Cholesterol is chemically stable and abundant in the sample. In addition, cholesterol reference standards are easy to obtain and inexpensive. Therefore, cholesterol was ultimately selected as the internal reference standard in the one standard for multiple assays.

[0083] 2. Determination of Relative Retention Time (RRT) and Relative Correction Factor (f)

[0084] Accurately weigh cholesterol, lanosterol, and dihydrolanosterol reference standards separately, add N-methylpyrrolidone to prepare a mixed reference solution, inject it into the liquid chromatograph, and determine the peak area and retention time under the chromatographic conditions described above. The resulting chromatogram of the reference standards is shown in the figure below. Figure 2 As shown in Table 5, the correction factor and relative retention time were calculated using the standard curve method. The formula for calculating the correction factor is shown in Equation (1), and the formula for calculating the relative retention time is shown in Equation (2).

[0085]

[0086]

[0087] Where, f sx A is the relative correction factor between the internal reference and the analyte. s C represents the peak area of ​​the internal reference standard. s For the concentration of the internal reference standard, A x C represents the peak area of ​​a reference standard for a certain analyte. x k represents the concentration of a reference standard for a certain analyte. s k is the slope of the standard curve for the internal reference s. x R is the slope of the standard curve for the analyte x. s Retention time of internal reference, R x The retention time of the analyte.

[0088] Table 5 Relative retention time and relative correction factor

[0089]

[0090] 3. Durability assessment

[0091] The effects of column temperature (°C), flow rate (mL / min), initial acetonitrile ratio (%), first gradient end time (min), and second gradient end time (min) on relative retention time and relative correction factor were investigated using Plackett-Burman experimental design. A total of 14 experiments were conducted, including 2 center point experiments.

[0092] The experimental conditions and results are shown in Table 6. The parameters and their settings were as follows: column temperature (X1): 37.0 ± 1.0%, flow rate (X2): 1.58 ± 0.02 mL / min, B-phase ratio at 0 min (X3): 90.5 ± 0.5%, gradient 1 end time (X4): 19.0 ± 0.5 min, gradient 2 end time (X5): 25.0 ± 0.5 min, and injection volume (X6): 10.0 ± 5.0 μL. The results show that when the analytical parameters are varied within the optimized operating parameters, the relative retention times and relative correction factors of lanosterol and dihydrolanosterol, using cholesterol as an internal reference, are reproducible, with RSDs less than 3%. The three chromatographic peaks are also well separated, indicating that the established single-standard, multiple-analyte analytical method is robust.

[0093] Table 6. Plackett-Burman Experimental Design and Results

[0094]

[0095] Note: Where Y1: relative retention time of lanosterol, Y2: relative correction factor of lanosterol, Y3: relative retention time of dihydrolanosterol, Y4: relative correction factor of dihydrolanosterol, X1: column temperature (°C), X2: flow rate, X3: proportion of phase B at 0 min (%), X4: end time of the first gradient (min), X5: end time of the second gradient (min), X6: injection volume (μL).

[0096] 4. Determination of the content of lanolin in industrial lanolin samples

[0097] The contents of cholesterol, lanosterol, and dihydrolanosterol in two batches (batch numbers 4301 and 4302) of industrial lanol were determined according to the above method, and the results are shown in Table 7. Specifically, the industrial lanol sample with batch number 4301 contained 35.49 μg / mg of cholesterol, 33.23 μg / mg of lanosterol, and 24.87 μg / mg of dihydrolanosterol. The obtained chromatogram is shown below. Figure 1 As shown, the industrial lanosterol sample with batch number 4302 contained 68.38 μg / mg cholesterol, 99.90 μg / mg lanosterol, and 73.40 μg / mg dihydrolanosterol.

[0098] Table 7 Results of quantitative component content detection in industrial lanolin samples

[0099]

[0100] Comparative Example 1: A chromatographic method for the detection of industrial lanolin.

[0101] (1) Use the same test solution and control solution as in Example 1;

[0102] (2) The chromatographic conditions are as follows:

[0103] Chromatographic column: SunShell C18 column (4.6mm × 150mm, 2.6μm);

[0104] Mobile phase: Acetonitrile (B) - water (A) mobile phase system was used;

[0105] Flow rate: 1.58 mL / min;

[0106] Column temperature: 37℃;

[0107] Injection volume: 10 μL;

[0108] UV detector wavelength: 205nm, runtime: 55min;

[0109] Gradient elution was used, with the following elution conditions: 0–19.0 ​​min: 94.0% B; 19.0–25.0 min: 94.0%–100.0% B; 25.0–55 min: 100% B.

[0110] Detection chromatogram as follows Figure 3 As shown, cholesterol merges with the preceding impurity peaks and cannot be completely separated.

[0111] Comparative Example 2: A chromatographic method for the detection of industrial lanolin.

[0112] (1) Use the same test solution and control solution as in Example 1;

[0113] (2) The chromatographic conditions are as follows:

[0114] Chromatographic column: SunShell C18 column (4.6mm × 150mm, 2.6μm);

[0115] Mobile phase: Acetonitrile (B) - water (A) mobile phase system was used;

[0116] Flow rate: 1.58 mL / min;

[0117] Column temperature: 32℃;

[0118] Injection volume: 10 μL;

[0119] UV detector wavelength: 205nm, runtime: 55min;

[0120] Gradient elution was used, with the following elution conditions: 0–19.0 ​​min: 90.5% B; 19.0–25.0 min: 90.5%–100.0% B; 25.0–55 min: 100% B.

[0121] Detection chromatogram as follows Figure 4 As shown, the peak elution time is prolonged, and the detection efficiency is reduced.

[0122] Comparative Example 3: A chromatographic method for the determination of industrial lanolin.

[0123] (1) Use the same test solution and control solution as in Example 1;

[0124] (2) The chromatographic conditions are as follows:

[0125] Chromatographic column: SunShell C18 column (4.6mm × 150mm, 2.6μm);

[0126] Mobile phase: Acetonitrile (B) - water (A) mobile phase system was used;

[0127] Flow rate: 1.58 mL / min;

[0128] Column temperature: 42℃;

[0129] Injection volume: 10 μL;

[0130] UV detector wavelength: 205nm, runtime: 55min;

[0131] Gradient elution was used, with the following elution conditions: 0–19.0 ​​min: 90.5% B; 19.0–25.0 min: 90.5%–100.0% B; 25.0–55 min: 100% B.

[0132] Detection chromatogram as follows Figure 5 As shown, the separation degree between cholesterol and the preceding impurity peaks is 1.39, which is less than 1.50 and does not meet the requirements.

[0133] Comparative Example 4: A chromatographic method for the detection of industrial lanolin.

[0134] (1) Use the same test solution and control solution as in Example 1;

[0135] (2) The chromatographic conditions are as follows:

[0136] Chromatographic column: SunShell C18 column (4.6mm × 150mm, 2.6μm);

[0137] Mobile phase: Acetonitrile (B) - water (A) mobile phase system was used;

[0138] Flow rate: 1.2 mL / min;

[0139] Column temperature: 37℃;

[0140] Injection volume: 10 μL;

[0141] UV detector wavelength: 205nm, runtime: 55min;

[0142] Gradient elution was used, with the following elution conditions: 0–19.0 ​​min: 90.5% B; 19.0–25.0 min: 90.5%–100.0% B; 25.0–55 min: 100% B.

[0143] Detection chromatogram as follows Figure 6 As shown, the peak elution time is prolonged, the detection efficiency is reduced, and dihydrolanosterol merges with the preceding impurity peaks, making complete separation impossible.

[0144] In summary, this invention establishes a single-standard, multi-analysis method for industrial lanosterol using high-performance liquid chromatography (HPLC). This method uses readily available and inexpensive cholesterol as an internal reference to simultaneously detect the contents of cholesterol, lanosterol, and dihydrolanosterol in industrial lanosterol. The method exhibits good linearity, precision, repeatability, and stability, and is low-cost. It provides a more comprehensive reflection of the quality of industrial lanosterol, offering a basis for further improving the quality standards of industrial lanosterol.

[0145] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A chromatographic detection method for industrial lanolin, characterized in that, The method of one standard and multiple determinations was adopted, with cholesterol as an internal reference, and the contents of cholesterol, lanosterol and dihydrolanosterol in industrial lanol were determined by high performance liquid chromatography. The chromatographic column used in the high-performance liquid chromatography method is a SunShell C18 column; The mobile phase of the high-performance liquid chromatography method is acetonitrile as phase B and water as phase A, and gradient elution is performed. The program is: 0-19.0 min, 90.5% B. 19.0~25.0 min, 90.5%~100.0%B, 25.0~55 min, 100%B; The high-performance liquid chromatography (HPLC) method uses an ultraviolet detector with a wavelength of 205 nm, a running time of 55 min, a flow rate of 1.58 ± 0.02 mL / min, and a column temperature of 37 ± 1 °C. The manufacturer of the industrial lanol is Jiangxi Nuowei Biotechnology Co., Ltd. The preparation method of the test solution is as follows: take 20 mg of industrial lanol into a 10 mL volumetric flask, accurately weigh it, add N-methylpyrrolidone to dissolve and dilute it, and then centrifuge to obtain the test solution.

2. The chromatographic detection method for industrial lanolin according to claim 1, characterized in that, The SunShell C18 column is 4.6 mm × 150 mm in size and has a particle size of 2.6 μm.

3. The chromatographic detection method for industrial lanolin according to claim 1, characterized in that, The injection volume for the high-performance liquid chromatography method is 10±5 μL.

4. A chromatographic detection method for industrial lanolin according to any one of claims 1-3, characterized in that, The specific steps include the following: (1) Preparation of cholesterol reference solution: Take an appropriate amount of cholesterol and add N-methylpyrrolidone to prepare a solution containing 1.76 mg of cholesterol per 1 mL; (2) Preparation of test solution: Take 20 mg of industrial lanolin into a 10 mL volumetric flask, weigh accurately, dissolve and dilute with N-methylpyrrolidone, and centrifuge to obtain the test solution; (3) High performance liquid chromatography determination: First, inject the reference solution in step (1) and the test solution in step (2) into the high performance liquid chromatograph for analysis and determination. Cholesterol is used as an internal reference. The peak positions of cholesterol, lanosterol and dihydrolanosterol are determined according to the predetermined relative retention time. Then, the contents of cholesterol, lanosterol and dihydrolanosterol are calculated according to the relative correction factor.

5. The chromatographic detection method for industrial lanolin according to claim 4, characterized in that, The relative retention time is relative to the elution time of the cholesterol chromatographic peak. For lanosterol, the elution time is the cholesterol retention time multiplied by 1.087; for dihydrolanosterol, the elution time is the cholesterol retention time multiplied by 1.

323. The relative correction factor is relative to the peak area of ​​the cholesterol chromatographic peak. When calculating the lanosterol content, it is calculated as the relative correction factor between lanosterol and cholesterol (0.4227) multiplied by the cholesterol content multiplied by the lanosterol peak area, then divided by the cholesterol peak area. When calculating the dihydrolanosterol content, it is calculated as the relative correction factor between dihydrolanosterol and cholesterol (0.8228) multiplied by the cholesterol content multiplied by the dihydrolanosterol peak area, then divided by the cholesterol peak area.

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