CN118130653A - Chromatographic detection method for industrial lanosterol - Google Patents
Chromatographic detection method for industrial lanosterol Download PDFInfo
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- CN118130653A CN118130653A CN202410250253.8A CN202410250253A CN118130653A CN 118130653 A CN118130653 A CN 118130653A CN 202410250253 A CN202410250253 A CN 202410250253A CN 118130653 A CN118130653 A CN 118130653A
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- QBSJHOGDIUQWTH-UHFFFAOYSA-N dihydrolanosterol Natural products CC(C)CCCC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 QBSJHOGDIUQWTH-UHFFFAOYSA-N 0.000 title claims abstract description 126
- BQPPJGMMIYJVBR-UHFFFAOYSA-N (10S)-3c-Acetoxy-4.4.10r.13c.14t-pentamethyl-17c-((R)-1.5-dimethyl-hexen-(4)-yl)-(5tH)-Delta8-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products CC12CCC(OC(C)=O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C BQPPJGMMIYJVBR-UHFFFAOYSA-N 0.000 title claims abstract description 91
- CHGIKSSZNBCNDW-UHFFFAOYSA-N (3beta,5alpha)-4,4-Dimethylcholesta-8,24-dien-3-ol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21 CHGIKSSZNBCNDW-UHFFFAOYSA-N 0.000 title claims abstract description 91
- XYTLYKGXLMKYMV-UHFFFAOYSA-N 14alpha-methylzymosterol Natural products CC12CCC(O)CC1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C XYTLYKGXLMKYMV-UHFFFAOYSA-N 0.000 title claims abstract description 91
- FPTJELQXIUUCEY-UHFFFAOYSA-N 3beta-Hydroxy-lanostan Natural products C1CC2C(C)(C)C(O)CCC2(C)C2C1C1(C)CCC(C(C)CCCC(C)C)C1(C)CC2 FPTJELQXIUUCEY-UHFFFAOYSA-N 0.000 title claims abstract description 91
- BKLIAINBCQPSOV-UHFFFAOYSA-N Gluanol Natural products CC(C)CC=CC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(O)C(C)(C)C4CC3 BKLIAINBCQPSOV-UHFFFAOYSA-N 0.000 title claims abstract description 91
- LOPKHWOTGJIQLC-UHFFFAOYSA-N Lanosterol Natural products CC(CCC=C(C)C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 LOPKHWOTGJIQLC-UHFFFAOYSA-N 0.000 title claims abstract description 91
- CAHGCLMLTWQZNJ-UHFFFAOYSA-N Nerifoliol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C CAHGCLMLTWQZNJ-UHFFFAOYSA-N 0.000 title claims abstract description 91
- CAHGCLMLTWQZNJ-RGEKOYMOSA-N lanosterol Chemical compound C([C@]12C)C[C@@H](O)C(C)(C)[C@H]1CCC1=C2CC[C@]2(C)[C@H]([C@H](CCC=C(C)C)C)CC[C@@]21C CAHGCLMLTWQZNJ-RGEKOYMOSA-N 0.000 title claims abstract description 91
- 229940058690 lanosterol Drugs 0.000 title claims abstract description 91
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 143
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 71
- MBZYKEVPFYHDOH-UHFFFAOYSA-N (10S)-3c-Hydroxy-4.4.10r.13t.14c-pentamethyl-17t-((R)-1.5-dimethyl-hexyl)-(5tH)-Delta8-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(C)CCCC(C)C)CCC21C MBZYKEVPFYHDOH-UHFFFAOYSA-N 0.000 claims abstract description 35
- MBZYKEVPFYHDOH-BQNIITSRSA-N 24,25-dihydrolanosterol Chemical compound C([C@@]12C)C[C@H](O)C(C)(C)[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@H](C)CCCC(C)C)CC[C@]21C MBZYKEVPFYHDOH-BQNIITSRSA-N 0.000 claims abstract description 35
- 239000013558 reference substance Substances 0.000 claims abstract description 34
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 18
- 238000004458 analytical method Methods 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 15
- 238000005259 measurement Methods 0.000 claims abstract description 8
- 238000000691 measurement method Methods 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 230000014759 maintenance of location Effects 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 22
- 239000000523 sample Substances 0.000 claims description 20
- 238000012937 correction Methods 0.000 claims description 17
- 239000012488 sample solution Substances 0.000 claims description 14
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 239000012085 test solution Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims 4
- 238000007813 chromatographic assay Methods 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 10
- 210000002268 wool Anatomy 0.000 abstract description 10
- 238000000605 extraction Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 239000011259 mixed solution Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 5
- 102220013334 rs368367224 Human genes 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- WHBHBVVOGNECLV-OBQKJFGGSA-N 11-deoxycortisol Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 WHBHBVVOGNECLV-OBQKJFGGSA-N 0.000 description 1
- 102100021695 Lanosterol 14-alpha demethylase Human genes 0.000 description 1
- 101710146773 Lanosterol 14-alpha demethylase Proteins 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000000199 molecular distillation Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
Classifications
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
-
- 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
- G01N30/74—Optical 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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention belongs to the technical field of analysis and detection, and particularly relates to a chromatographic detection method of industrial lanosterol. The detection method adopts a one-standard multi-measurement method, uses cholesterol as an internal reference substance, and detects the content of cholesterol, lanosterol and dihydrolanosterol in industrial lanosterol by high performance liquid chromatography. According to the invention, an industrial lanosterol one-mark multi-measurement detection method is established by optimizing an extraction method, chromatographic conditions, elution gradients and the like, and the contents of cholesterol, lanosterol and dihydrolanosterol in the industrial lanosterol can be detected simultaneously only by taking cheap and easily available cholesterol as an internal reference substance; the detection method has good linear relation, precision, repeatability and stability, is low in cost, can reflect the quality of industrial wool alcohol more comprehensively, and can provide basis for further improving the quality standard of industrial wool alcohol.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a chromatographic detection method of industrial lanosterol, in particular to a method for simultaneously detecting the contents of cholesterol, lanosterol and dihydrolanosterol in industrial lanosterol.
Background
Cholesterol, lanosterol, and dihydrolanosterol are important chemical components in industrial lanosterol, and the total content of the three components is generally more than 50% of the total weight. Cholesterol has a molecular formula of C 27H46 O and relative molecular mass of 386.65, and is a natural steroid resource in the pharmaceutical industry because of its steroid nucleus structure. Lanosterol has a molecular formula of C 30H50 O and a relative molecular weight of 426.73, is an important sterol, has important physiological effects, and is an important raw material in cosmetics, medicines and chemical industry. Dihydrolanosterol, which has molecular formula of C 30H52 O and relative molecular weight of 428.73, is one of substrates of CYP51, and has physiological activity of inhibiting cholesterol synthesis. The structural formulas of the three are shown as the following formulas I, II and III respectively.
In order to enhance the quality control and utilization efficiency of industrial wool alcohol, the production enterprises need to quantitatively detect the chemical composition of the industrial wool alcohol. Lanosterol of different chemical composition can subsequently be used in processing to produce different downstream products. For example, industrial lanosterol with higher lanosterol content can be considered for subsequent further extraction of lanosterol with high purity, while industrial lanosterol with higher cholesterol content can be subjected to treatment such as molecular distillation for further extraction of cholesterol.
However, the lack of a corresponding liquid chromatography method reports that one of the reasons for this phenomenon is that lanosterol controls are expensive, and therefore, it is necessary to develop a liquid chromatography method that can reduce the cost of the controls. One standard and multiple tests are common analysis techniques in the field of traditional Chinese medicines, and a cheap reference substance is adopted to detect various components, so that the cost of purchasing the reference substance is expected to be obviously reduced, and a basis is provided for controlling the quality of industrial lanosterol in production and determining subsequent use.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a chromatographic detection method for industrial lanosterol, which can overcome the defects of high detection cost and multiple steps of the existing analysis method, can fill the blank of an intermediate component analysis method in the process of producing lanosterol and dihydrolanosterol, and has an important effect on controlling the quality of industrial lanosterol.
The invention provides a chromatographic detection method of industrial lanosterol, which adopts a one-standard multi-measurement method, uses cholesterol as an internal reference substance, and detects the content of cholesterol, lanosterol and dihydrolanosterol in the industrial lanosterol by a high performance liquid chromatography.
According to the invention, on the basis of the existing industrial lanosterol detection technology, a one-mark multi-measurement method of industrial lanosterol is researched, three reference substances of cholesterol, lanosterol and dihydro lanosterol are only needed when the one-mark multi-measurement method is established in the initial stage, after the method is established, the content of cholesterol, lanosterol and dihydro lanosterol in the industrial lanosterol can be detected at one time by only using one reference substance of cholesterol, so that the defects of the prior art are effectively overcome, the cost is low, and the basis and the method can be provided for controlling the quality of the industrial lanosterol in production and determining the subsequent use.
Preferably, in the above technical solution, the chromatographic column of the high performance liquid chromatography is SunShell C chromatographic column, the model is 4.6mm×150mm, and the filler particle size is 2.6 μm.
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 performs gradient elution.
Preferably, in the above technical solution, the gradient elution procedure is as follows: 0 to 19.0min,90.5 percent of B;19.0 to 25.0min,90.5 to 100.0 percent of B,25.0 to 55min and 100 percent of B.
Preferably, in the above technical solution, the wavelength of the ultraviolet detector of the high performance liquid chromatography is 205nm, and the running time is 55min.
Preferably, in the above technical solution, the flow rate of the high performance liquid chromatography is 1.58±0.02mL/min.
Preferably, in the above technical scheme, the column temperature of the high performance liquid chromatography is 37±1 ℃.
Preferably, in the above technical solution, the sample injection amount of the high performance liquid chromatography is 10±5 μl.
Preferably, in the above technical solution, the chromatographic detection method includes the following specific steps:
(1) Preparing a cholesterol control solution: adding N-methyl pyrrolidone into proper amount of cholesterol to prepare 1.76mg of solution containing cholesterol per 1 mL;
(2) Preparing a test solution: taking industrial lanosterol 20mg in a 10mL volumetric flask, precisely weighing, adding N-methyl pyrrolidone for dissolution, diluting, and centrifuging to obtain a sample solution;
(3) High performance liquid chromatography assay: firstly, injecting the reference substance mixed solution and the sample solution in the step (1) into a high performance liquid chromatograph for analysis and measurement, taking cholesterol as an internal reference substance, and determining peak positions of cholesterol, lanosterol and dihydrolanosterol according to a predetermined relative retention time; and then calculating the content of cholesterol, lanosterol and dihydrolanosterol according to the relative correction factors.
Preferably, in the above technical solution, the relative retention time is a relative retention time relative to a cholesterol chromatographic peak-to-peak time, which is a cholesterol retention time multiplied by 1.087 for lanosterol chromatographic peak-to-peak time and a cholesterol retention time multiplied by 1.323 for dihydrolanosterol chromatographic peak-to-peak time; the relative correction factor is relative correction factor relative to cholesterol chromatographic peak area, which is calculated by multiplying lanosterol and cholesterol relative correction factor 0.4227 by lanosterol content and dividing it by cholesterol peak area when lanosterol content is calculated, and by multiplying lanosterol and cholesterol relative correction factor 0.8228 by cholesterol content and dividing it by cholesterol peak area when dihydrolanosterol content is calculated.
Compared with the prior art, the beneficial effects are that:
The invention establishes the chromatographic detection method of industrial lanosterol for the first time, and fills the blank of the prior method for detecting the intermediate in the production process.
The one-standard multi-measurement analysis method established by the invention can simultaneously measure the contents of three components of cholesterol, lanosterol and dihydrolanosterol in industrial lanosterol by using only one reference substance of cholesterol, thereby greatly saving the consumption of the reference substance, solving the problem of high price of the reference substance and reducing the analysis cost.
The quality control detection method (one-mark multi-measurement) has the advantages of simple operation, high accuracy, good reproducibility and low cost; compared with an external standard method, the detection result completely meets the requirements, can reflect the quality of the industrial wool alcohol more comprehensively, and provides a basis for further improving the quality standard of the industrial wool alcohol and defining the subsequent use of the industrial wool alcohol.
Drawings
FIG. 1 is a chromatogram of a sample solution of example 1 of the present invention;
FIG. 2 is a chromatogram of a control solution mixture of the present invention;
FIG. 3 is a chromatogram of the sample solution of comparative example 1;
FIG. 4 is a chromatogram of the sample solution of comparative example 2;
FIG. 5 is a chromatogram of the sample solution of comparative example 3;
FIG. 6 is a chromatogram of the test solution of comparative example 4.
Detailed Description
The above-described features of the invention and those specifically described in the following (example embodiments) may be combined with each other to constitute new or preferred embodiments, but the invention is not limited to these embodiments, nor is they limited to them in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents described in the examples below are commercially available and are commercially available unless otherwise specified.
The invention is described in further detail below in connection with the figures and examples:
experimental apparatus and materials
1. Reagent and reagent
Acetonitrile (chromatographic purity, merck, germany); n-methylpyrrolidone (analytical grade, shanghai Michelin Co., ltd.), cholesterol control (purity 99%, shanghai Ala Di Biotechnology Co., ltd., lot number H2229748), lanosterol control (purity 100%, laboratory preparation), dihydrolanosterol control (purity 95%, shanghai Ala Di Biotechnology Co., ltd., lot number A2303183), industrial lanol lot numbers 4301, 4302, supplied by Jiangxi Norway Biotechnology Co., ltd.; deionized water was prepared by a water purification system (Milli-Q, millipore, germany).
2. Apparatus and device
High performance liquid chromatograph (SHIMADZU LC-20AT type, shimadzu corporation) including LC-20A infusion pump, autosampler, column oven, degasser, UV-Vis detector, and data processing software; an electronic balance (00000246, saidolesco instruments); ultrasonic machine (KQ 2200E, kunshanmei ultrasonic instruments Co.).
3. Solution preparation
3.1 Preparation of reference substance Mixed solution
Taking a proper amount of cholesterol, lanosterol and dihydro lanosterol reference substances, and adding N-methyl pyrrolidone to prepare a solution containing 1.76mg of cholesterol, 1.28mg of lanosterol and 1.16mg of dihydro lanosterol per 1 mL.
3.2 Preparation of cholesterol control solution
Taking a proper amount of cholesterol reference substance, adding N-methyl pyrrolidone to prepare a solution containing 1.76mg of cholesterol per 1 mL.
3.3 Preparation of sample solution
Taking about 20mg of industrial lanosterol sample in a 10mL volumetric flask, precisely weighing, adding N-methyl pyrrolidone for dissolution, fixing the volume to a scale mark, fully mixing, centrifuging and taking supernatant.
Example 1: chromatographic detection method for industrial lanosterol
(1) The preparation of the test solution and the reference solution is carried out according to the method;
(2) The chromatographic conditions are as follows:
Chromatographic column: sunShell C18A 18 column (4.6 mm. Times.150 mm,2.6 μm);
Mobile phase: using an acetonitrile (B) -water (a) mobile phase system;
Flow rate: 1.58mL/min;
Column temperature: 37 ℃;
sample injection amount: 10. Mu.L;
ultraviolet detector wavelength: 205nm, run time of 55min;
gradient elution is adopted, and the elution conditions are as follows: 0 to 19.0min:90.5% b;19.0 to 25.0min:90.5 to 100.0 percent of B;25.0 to 55min:100% b;
The theoretical plate number is not less than 60000 calculated as cholesterol peak.
The detection chromatogram is shown in figure 1, and has good separation degree of cholesterol, lanosterol and dihydrolanosterol, and short analysis time.
Example 2: methodology investigation
1. Linear relationship investigation
The control mixed solution prepared in the step of '3.1' is respectively diluted by 3.333, 8.333, 20.83, 52.08, 130.2 and 325.5 times to obtain a series of control mixed solutions with different concentrations, 10 mu L of the control mixed solutions are respectively injected, and analysis is carried out according to the chromatographic conditions of the example 1. And (3) taking the peak area measured by each component as an ordinate and the concentration as an abscissa to manufacture a standard curve, so as to obtain a linear regression equation and an analysis range.
The regression equation is: cholesterol y=4.08× 3X,R2 =0.9997; lanosterol y=9.65× 3X,R2 =1.000; lanosterol y=4.96× 3X,R2 =1.000; the result shows that the cholesterol has good linear relation within the range of 5.42-529 mug.mL -1; lanosterol has good linear relation within the range of 3.93-384 mug.mL -1; dihydrolanosterol has good linear relationship within the range of 3.56-348 mug.mL -1.
2. Precision test
The same sample solution with the batch number 4301 is continuously sampled for 6 times, and the RSD value of the peak area and the retention time of each component is calculated. As a result, the retention time of each component was less than 0.5000% and the peak area was less than 1.500% as shown in Table 1.
TABLE 1 precision test results
3. Repeatability test
Six parallel prepared sample solutions with batch number 4301 are taken, sample analysis is performed respectively, and RSD values of peak areas and retention times of the components are calculated. As a result, the retention time of each component was less than 0.5000% and the peak area was less than 2.000% as shown in Table 2.
TABLE 2 repeatability test results
4. Stability test
Sample solutions of the sample with the batch number 4301 and the reference substance mixed solution with the same concentration are taken, and sample analysis is carried out at 0,1,2,4,8, 12 and 24 hours, wherein the peak area and the RSD value of the retention time of each component are obtained. As a result, the retention time of each component was less than 0.5000% and the peak area was less than 2.000% as shown in Table 3.
TABLE 3 stability test results
5. Sample addition recovery test
9 Parts of sample solutions with known contents are respectively taken and divided into 3 groups. The ratio of the addition amount of the reference substance to the component to be detected in the test sample is controlled to be about 0.8:1.0,1.0:1.0 and 1.2:1.0 by setting the three concentration levels of low, medium and high, 3 parts of test sample solutions are prepared in parallel for each concentration to measure, and the recovery rate is calculated, and the result is shown in table 4, wherein the average recovery rate of cholesterol is 99.70%, the average recovery rate of lanosterol is 101.0, the average recovery rate of dihydrolanosterol is 101.8%, the average recovery rate of dihydrolanosterol meets the requirements, and the RSD of each component recovery rate is lower than 3.0%.
TABLE 4 sample recovery test results
Example 3: screening research of one-mark multi-test detection method
1. Determination of one-standard-multiple-measurement reference substance
The reference substance should satisfy three requirements: (1) the content in the sample is rich; (2) stabilization; (3) easy access. The cholesterol has stable chemical property, is rich in the sample, and the cholesterol reference substance is easy to obtain and low in price, so that the cholesterol is finally determined to be used as an internal reference substance in one standard and multiple tests.
2. Determination of Relative Retention Time (RRT) and relative correction factor (f)
Respectively precisely weighing cholesterol, lanosterol, and dihydro lanosterol reference substances, adding N-methyl pyrrolidone to obtain mixed reference substance solution, and injecting into liquid chromatograph, and determining peak area and retention time according to the above chromatographic conditions, wherein the chromatogram of the obtained reference substance is shown in figure 2. The calibration factor and the relative retention time are calculated by a standard curve method, the calculation formula of the calibration factor is shown in a formula (1), the calculation formula of the relative retention time is shown in a formula (2), and the result is shown in a table 5.
Wherein f sx is the relative correction factor of the internal reference substance and the component to be detected, A s is the peak area of the reference substance, C s is the concentration of the reference substance, A x is the peak area of the reference substance of a certain component to be detected, C x is the concentration of a reference substance of a component to be detected, k s is the slope of a standard curve of an internal reference substance s, k x is the slope of a standard curve of a component x to be detected, R s is the retention time of the internal reference substance, and R x is the retention time of the component to be detected.
TABLE 5 relative retention time and relative correction factor
3. Durability inspection
The effect of column temperature (. Degree.C.), flow (mL/min), initial acetonitrile fraction (%), first gradient end time (min), second gradient end time (min) on relative retention time and relative correction factor was examined by the Plackett-Burman test design for a total of 14 experiments, with a center point of 2.
The test conditions and results are shown in table 6, and the respective parameters and setting ranges are: column temperature (X 1): 37.0±1.0%, flow rate (X 2): 1.58+ -0.02 mL/min, phase B ratio at 0min (X 3): 90.5±0.5%, gradient 1 end time (X 4): 19.0±0.5min, gradient 2 end time (X 5): 25.0+ -0.5 min, sample injection amount (X 6): 10.0+ -5.0 μL. The result shows that when the analysis parameters are changed within the optimization range of the operation parameters, cholesterol is used as an internal reference, the relative retention time and the relative correction factor repeatability of lanosterol and dihydrolanosterol are good, RSD is less than 3%, and three chromatographic peaks are fully separated, so that the robustness of the established one-standard multi-measurement analysis method is good.
TABLE 6Plackett-Burman test design and results Table
Remarks: wherein, Y 1: lanosterol relative retention time, Y 2: lanosterol relative correction factor, Y 3: relative retention time of dihydrolanosterol, Y 4: relative correction factor of dihydro lanosterol, X 1: column temperature (. Degree. C.), X 2: flow, X 3: phase B ratio (%), X 4 at 0 min: first gradient end time (min), X 5: second gradient end time (min), X 6: sample introduction amount (μl).
4. Determination of the content of Industrial wool alcohol samples
The cholesterol, lanosterol, and dihydrolanosterol contents of 2 batches (batches 4301 and 4302) of industrial lanosterol were measured as described above, and the results are shown in Table 7. Wherein, the cholesterol content in the industrial lanosterol sample with batch number 4301 is 35.49 mug/mg, the lanosterol content is 33.23 mug/mg, and the dihydrolanosterol content is 24.87 mug/mg, and the obtained chromatogram is shown in figure 1; the commercial lanosterol sample with batch number 4302 had a cholesterol content of 68.38 μg/mg, lanosterol content of 99.90 μg/mg and dihydrolanosterol content of 73.40 μg/mg.
TABLE 7 quantitative ingredient content detection results of Industrial lanonol samples
Comparative example 1: chromatographic detection method for industrial lanosterol
(1) The same test solution and control solution as in example 1 were used;
(2) The chromatographic conditions are as follows:
Chromatographic column: sunShell C18A 18 column (4.6 mm. Times.150 mm,2.6 μm);
Mobile phase: using an acetonitrile (B) -water (a) mobile phase system;
Flow rate: 1.58mL/min;
Column temperature: 37 ℃;
sample injection amount: 10. Mu.L;
ultraviolet detector wavelength: 205nm, run time of 55min;
Gradient elution is adopted, and the elution conditions are as follows: 0 to 19.0min:94.0% b;19.0 to 25.0min:94.0 to 100.0 percent of B;25.0 to 55min:100% B.
As shown in FIG. 3, cholesterol was combined with the previous impurity peak, and could not be completely separated.
Comparative example 2: chromatographic detection method for industrial lanosterol
(1) The same test solution and control solution as in example 1 were used;
(2) The chromatographic conditions are as follows:
Chromatographic column: sunShell C18A 18 column (4.6 mm. Times.150 mm,2.6 μm);
Mobile phase: using an acetonitrile (B) -water (a) mobile phase system;
Flow rate: 1.58mL/min;
Column temperature: 32 ℃;
sample injection amount: 10. Mu.L;
ultraviolet detector wavelength: 205nm, run time of 55min;
Gradient elution is adopted, and the elution conditions are as follows: 0 to 19.0min:90.5% b;19.0 to 25.0min:90.5 to 100.0 percent of B;25.0 to 55min:100% B.
The detection chromatogram is shown in FIG. 4, and shows that the peak time is prolonged and the detection efficiency is reduced.
Comparative example 3: chromatographic detection method for industrial lanosterol
(1) The same test solution and control solution as in example 1 were used;
(2) The chromatographic conditions are as follows:
Chromatographic column: sunShell C18A 18 column (4.6 mm. Times.150 mm,2.6 μm);
Mobile phase: using an acetonitrile (B) -water (a) mobile phase system;
Flow rate: 1.58mL/min;
column temperature: 42 ℃;
sample injection amount: 10. Mu.L;
ultraviolet detector wavelength: 205nm, run time of 55min;
Gradient elution is adopted, and the elution conditions are as follows: 0 to 19.0min:90.5% b;19.0 to 25.0min:90.5 to 100.0 percent of B;25.0 to 55min:100% B.
As shown in FIG. 5, the separation degree of cholesterol from the previous impurity peak is 1.39, less than 1.50, which is not satisfactory.
Comparative example 4: chromatographic detection method for industrial lanosterol
(1) The same test solution and control solution as in example 1 were used;
(2) The chromatographic conditions are as follows:
Chromatographic column: sunShell C18A 18 column (4.6 mm. Times.150 mm,2.6 μm);
Mobile phase: using an acetonitrile (B) -water (a) mobile phase system;
Flow rate: 1.2mL/min;
Column temperature: 37 ℃;
sample injection amount: 10. Mu.L;
ultraviolet detector wavelength: 205nm, run time of 55min;
Gradient elution is adopted, and the elution conditions are as follows: 0 to 19.0min:90.5% b;19.0 to 25.0min:90.5 to 100.0 percent of B;25.0 to 55min:100% B.
As shown in FIG. 6, the detection chromatogram has prolonged peak time and reduced detection efficiency, and the dihydrolanosterol is combined with the previous impurity peak and cannot be completely separated.
In summary, the invention establishes an industrial lanosterol one-standard multi-measurement detection method by adopting a high performance liquid chromatography technology, namely, the content of cholesterol, lanosterol and dihydrolanosterol in the industrial lanosterol can be detected simultaneously by taking cheap and easily available cholesterol as an internal reference substance; the detection method has good linear relation, precision, repeatability and stability, is low in cost, can reflect the quality of industrial wool alcohol more comprehensively, and provides a basis for further improving the quality standard of industrial wool alcohol.
Finally, it should be emphasized that the foregoing description is merely illustrative of the preferred embodiments of the invention, and that various changes and modifications can be made by those skilled in the art without departing from the spirit and principles of the invention, and any such modifications, equivalents, improvements, etc. are intended to be included within the scope of the invention.
Claims (10)
1. A chromatographic detection method for industrial lanosterol is characterized in that a one-standard multi-measurement method is adopted, cholesterol is used as an internal reference, and the content of cholesterol, lanosterol and dihydrolanosterol in the industrial lanosterol is detected by a high performance liquid chromatography.
2. The method for detecting industrial lanosterol by chromatography according to claim 1, wherein the chromatographic column of the high performance liquid chromatography is SunShell C chromatographic column, the model is 4.6mm×150mm, and the filler particle size is 2.6 μm.
3. The method for detecting industrial lanosterol by chromatography according to claim 1, wherein the mobile phase of the high performance liquid chromatography uses acetonitrile as phase B and water as phase a, and gradient elution is performed.
4. A chromatographic assay method of industrial lanosterol in accordance with claim 3 wherein the gradient elution procedure is: 0 to 19.0min,90.5 percent of B;19.0 to 25.0min,90.5 to 100.0 percent of B,25.0 to 55min and 100 percent of B.
5. The method for chromatographic detection of industrial lanosterol according to claim 1, wherein the ultraviolet detector wavelength of the high performance liquid chromatography is 205nm, and the running time is 55min.
6. The method for detecting industrial lanosterol by chromatography according to claim 1, wherein the flow rate of the high performance liquid chromatography is 1.58+ -0.02 mL/min.
7. The method for chromatographic detection of industrial lanosterol according to claim 1, wherein the column temperature of the high performance liquid chromatography is 37±1 ℃.
8. The method for detecting industrial lanosterol by chromatography according to claim 1, wherein the sample injection amount of the high performance liquid chromatography is 10+ -5 μl.
9. The method for chromatographic detection of industrial lanosterol according to any one of claims 1 to 8, comprising the specific steps of:
(1) Preparing a cholesterol control solution: adding N-methyl pyrrolidone into proper amount of cholesterol to prepare 1.76mg of solution containing cholesterol per 1 mL;
(2) Preparing a test solution: taking industrial lanosterol 20mg in a 10mL volumetric flask, precisely weighing, adding N-methyl pyrrolidone for dissolution, diluting, and centrifuging to obtain a sample solution;
(3) High performance liquid chromatography assay: firstly, injecting the reference substance solution and the sample solution in the step (1) into a high performance liquid chromatograph for analysis and measurement, taking cholesterol as an internal reference substance, and determining peak positions of cholesterol, lanosterol and dihydrolanosterol according to a predetermined relative retention time; and then calculating the content of cholesterol, lanosterol and dihydrolanosterol according to the relative correction factors.
10. The method for chromatographic detection of industrial lanosterol according to claim 9, wherein the relative retention time is relative retention time relative to cholesterol chromatographic peak-to-peak time, which is cholesterol retention time times 1.087 for lanosterol chromatographic peak-to-peak times 1.323 for dihydrolanosterol chromatographic peak-to-peak times; the relative correction factor is relative correction factor relative to cholesterol chromatographic peak area, which is calculated by multiplying lanosterol and cholesterol relative correction factor 0.4227 by lanosterol content and dividing it by cholesterol peak area when lanosterol content is calculated, and by multiplying lanosterol and cholesterol relative correction factor 0.8228 by cholesterol content and dividing it by cholesterol peak area when dihydrolanosterol content is calculated.
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