CN114252544B - Chromatographic separation purification and detection analysis method for squalene and pure squalene - Google Patents
Chromatographic separation purification and detection analysis method for squalene and pure squalene Download PDFInfo
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- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 title claims abstract description 127
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 title claims abstract description 127
- 229940031439 squalene Drugs 0.000 title claims abstract description 127
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 title claims abstract description 127
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 title claims abstract description 126
- 238000001514 detection method Methods 0.000 title claims abstract description 37
- 238000004458 analytical method Methods 0.000 title claims abstract description 26
- 238000013375 chromatographic separation Methods 0.000 title claims abstract description 15
- 238000000746 purification Methods 0.000 title claims abstract description 10
- 239000012535 impurity Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000000926 separation method Methods 0.000 claims abstract description 24
- 238000011097 chromatography purification Methods 0.000 claims abstract description 11
- 239000003480 eluent Substances 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 239000012043 crude product Substances 0.000 claims description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000001228 spectrum Methods 0.000 claims description 9
- 239000003643 water by type Substances 0.000 claims description 9
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 8
- 238000010829 isocratic elution Methods 0.000 claims description 6
- 239000006227 byproduct Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000007670 refining Methods 0.000 claims description 5
- 238000000825 ultraviolet detection Methods 0.000 claims description 4
- 235000012424 soybean oil Nutrition 0.000 claims description 3
- 239000003549 soybean oil Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims 2
- 239000010499 rapseed oil Substances 0.000 claims 2
- 239000000463 material Substances 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 14
- 239000012071 phase Substances 0.000 description 14
- 239000000523 sample Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- 239000003814 drug Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 230000005526 G1 to G0 transition Effects 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 4
- 235000015112 vegetable and seed oil Nutrition 0.000 description 4
- 239000008158 vegetable oil Substances 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 101001068027 Homo sapiens Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Proteins 0.000 description 2
- 102100034464 Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Human genes 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- ZYBWTEQKHIADDQ-UHFFFAOYSA-N ethanol;methanol Chemical compound OC.CCO ZYBWTEQKHIADDQ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 241000195974 Selaginella Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- -1 polydimethylsiloxane Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000010686 shark liver oil Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 235000019871 vegetable fat Nutrition 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
- G01N30/60—Construction of the column
- G01N30/6034—Construction of the column joining multiple columns
- G01N30/6039—Construction of the column joining multiple columns in series
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- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The application discloses a chromatographic separation, purification, detection and analysis method of squalene and a pure squalene product. In the chromatographic separation and purification method of squalene, two chromatographic columns connected in series are adopted to carry out chromatographic separation and purification on crude squalene; the two chromatographic columns connected in series are a C18 chromatographic column and a C18-PFP chromatographic column; the crude squalene passes through a C18 chromatographic column and then a PFP chromatographic column, so that impurities and squalene are separated, eluent is collected in sections, and pure squalene with the purity of more than 99.0% is obtained, the purity of squalene is obviously improved, and the current situation that squalene is low in purity as a medicinal auxiliary material and difficult in impurity control is solved. In the detection and analysis method of squalene, the method of connecting the C18 chromatographic column and the C18-PFP chromatographic column in series is adopted, so that the good separation of the maximum impurity and the main component in squalene is realized, and further, the more accurate detection of squalene purity can be realized.
Description
Technical Field
The application relates to the technical field of separation and purification of squalene, in particular to a chromatographic separation and purification method of squalene, a high performance liquid chromatography detection and analysis method and a pure squalene product.
Background
The purity of the medicine affects the effect and toxic and side effects of the medicine, so related substances are always valued as key indexes for controlling the quality of raw medicines and preparations. ICH (The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, international pharmaceutical registration Condition) guidelines provide that the limits of identification of drug substance impurities be no more than 0.10% and defined be no more than 0.15%. When the drug contains unknown impurities above this limit, there is a greater risk of quality control.
Identification limit: refers to an impurity limit beyond which impurities require identification of their structure. Define a limit: referring to an impurity limit beyond which impurities need to be defined, certain data are obtained and evaluated to ensure the biosafety of the impurity.
The squalene can be used as pharmaceutical adjuvant, and is prepared from shark liver or vegetable oil and fat by reduced pressure distillation or supercritical CO 2 Extracting, and purifying by column chromatography. The highest squalene purity of about 95% can be obtained in the market, and squalene with a purity of more than 99% cannot be obtained. The low purity of squalene is unfavorable for quality control of medicines.
Disclosure of Invention
Aiming at the limitations of the prior art, the application provides a chromatographic separation and purification method of squalene, a high performance liquid chromatography detection and analysis method and a pure squalene product. Regarding the chromatographic separation and purification method of squalene, the application innovatively connects the C18 preparative chromatographic column and the C18-PFP (pentafiuorophenyl) preparative chromatographic column in series, and by optimizing the operation conditions of the serial chromatography proposed by the innovativeness, the application comprises the control of the type of mobile phase, the proportion of each component in the mobile phase and the column temperature, thereby successfully realizing the good separation of the maximum impurity in the squalene crude product from the squalene as a main component, and by collecting the squalene samples with the purity of more than 99.0 percent by sections. Regarding the high performance liquid chromatography detection and analysis method of squalene, the application innovatively connects the C18 chromatographic column and the C18-PFP (pentafiuorophenyl) chromatographic column in series, and successfully realizes the good separation of the maximum impurity in the squalene crude product and the squalene as a main component by optimizing the operation conditions of the serial chromatography proposed by the innovativeness, including the control of the type of the mobile phase, the proportion of each component in the mobile phase and the column temperature, and further successfully realizes the more accurate detection of the squalene purity by ultraviolet detection and quantitative analysis.
The application provides a chromatographic separation and purification method of squalene, which adopts two preparation chromatographic columns connected in series to carry out chromatographic separation and purification on a crude product of squalene, wherein the two preparation chromatographic columns connected in series are a C18 chromatographic column and a C18-PFP chromatographic column.
The C18 chromatographic column is the most commonly used chromatographic column in high performance liquid chromatography, and the stationary phase is obtained by bonding octadecyl on a silica gel matrix, has higher carbon content and better hydrophobicity, and is suitable for most compounds including nonpolar and polar small molecules and some polypeptides and proteins. The types of C18 columns are also increasing as each column manufacturer develops.
The C18-PFP chromatographic column is a chromatographic column with pentafluorophenyl embedded in C18 as stationary phase, and has separation principle greater than that of common C18 column by pi-pi acting force and certain selectivity to conjugated structure.
In embodiments of the application, separation of conjugated and unconjugated structural materials may be enhanced using a common C18 column and C18-PFP column tandem analysis. In the present document, a "C18-PFP column" is also referred to as a "PFP (chromatography) column". That is, in the present document, "C18-PFP column", "PFP column" have the same meaning.
In an embodiment of the application, the crude squalene is passed through a C18 chromatographic column and then through a PFP chromatographic column, so that the separation of impurities from squalene is realized, and then, by collecting eluent in a sectional manner, pure squalene with a purity of 99.0% or more can be obtained, which is significantly higher than the general squalene available in the market (the purity of squalene obtained in the market through a conventional process is usually 95%).
In an embodiment of the application, the mobile phase in the C18 chromatography column and the C18-PFP chromatography column is acetonitrile-water. Preferably, the volume ratio of acetonitrile to water is 80-92:20-8; preferably, the volume ratio of acetonitrile to water is 84:16; preferably, the volume ratio of acetonitrile to water is 88:12; preferably, the volume ratio of acetonitrile to water is 92:8.
In an embodiment of the application, the column temperature of the C18 chromatographic column and the C18-PFP chromatographic column is 15-25 ℃. Preferably, the column temperature of the C18 chromatographic column and the C18-PFP chromatographic column is 20 ℃.
In an embodiment of the application, the detection wavelength is selected to be 205-215nm; preferably, the detection wavelength is chosen to be 210nm.
In an embodiment of the application, the elution mode employed in the C18 chromatography column and the C18-PFP chromatography column is isocratic.
In an embodiment of the application, the flow rate of the mobile phase in the C18 and C18-PFP columns is 1.2-1.6ml/min, preferably the flow rate of the mobile phase is 1.4ml/min.
In an embodiment of the application, the C18 column is a Waters SunFire Prep C18 column, 150mm x 19mm,5 μm in size; the C18-PFP chromatographic column is a medium-spectrum red RD-PFP chromatographic column with the specification of 150mm multiplied by 21.5mm and 5.0 μm.
In an embodiment of the application, the crude squalene is a plant-derived squalene.
Squalene of vegetable origin is extracted from deodorized distillates (by-products of vegetable oil refining processes) of plants, particularly oil plants, and is largely enriched in deodorized distillates of vegetable oils, which are ideal raw materials for extracting plant squalene.
In an embodiment of the application, the crude squalene is a byproduct of soybean oil or vegetable oil refining.
The application provides a squalene pure product, wherein the purity of the squalene pure product is up to more than 99.0%.
The application provides a high performance liquid chromatography detection and analysis method of squalene, which adopts two chromatographic columns connected in series to carry out high performance liquid chromatography separation on a crude product of squalene, so that impurities and squalene are separated; the two chromatographic columns connected in series are a C18 chromatographic column and a C18-PFP chromatographic column; the squalene crude product firstly passes through a C18 chromatographic column and then passes through a C18-PFP chromatographic column; ultraviolet detection was performed to determine the squalene content.
In an embodiment of the present application, the conditions for the high performance liquid chromatography detection analysis are:
the mobile phase is acetonitrile-water, preferably, the volume ratio of acetonitrile to water is 80-92:20-8; preferably, the volume ratio of acetonitrile to water is 84:16; preferably, the volume ratio of acetonitrile to water is 88:12; preferably, the volume ratio of acetonitrile to water is 92:8;
the column temperature is 15-25 ℃, preferably 20 ℃;
the detection wavelength is 205-215nm, preferably 210nm;
the elution mode is isocratic elution;
the flow rate of the mobile phase is 1.2-1.6ml/min, preferably 1.4ml/min.
In an embodiment of the application, the C18 column is a Waters XTERRA RP C18 column, 150mm x 4.6mm,3.5 μm in size; the C18-PFP chromatographic column is a medium-spectrum red RD-PFP chromatographic column with the specification of 250mm multiplied by 4.0mm and 5.0 μm.
In an embodiment of the application, the chromatogram is recorded to a 2-fold retention time of the main component squalene.
Compared with the prior art, the application has at least the following advantages:
according to the squalene separation and purification method provided by the application, the double chromatographic columns are connected in series, the conventional C18 column and the C18-PFP column are combined, and the pentafluorophenyl is embedded into the C18-PFP column as a stationary phase, so that the separation principle is pi-pi acting force more than that of the conventional C18 column, the squalene separation and purification method has a certain selectivity to a conjugated structure, and can strengthen the separation of conjugated and unconjugated structural substances, further can effectively separate squalene from impurities, obtain high-purity squalene, and solve the current situations that the squalene is low in purity and difficult to control impurities as a pharmaceutical auxiliary material.
The squalene detection and analysis method provided by the application can separate the squalene crude product from impurities, so that the sensitivity and content accuracy of diagonal squalene detection are further improved. The method has the advantages of high specificity, high analysis speed, good accuracy and high repeatability, and is convenient for guaranteeing the quality detection and safety of squalene related products.
By utilizing the method for separating, purifying, detecting and analyzing the diagonal squalene, the purity of the detected crude product is 88.69 percent, the separation degree of the maximum impurity before the main squalene peak and the main squalene peak can reach 1.1, and the same condition is applied to preparing a liquid phase to obtain squalene with the purity higher than 99.0 percent.
The application optimizes the purification and detection of squalene by using a specific combination of C18 column and C18-PFP column (namely Waters SunFire Prep C column with specification of 150mm×19mm,5 μm, combined with medium-spectrum red RD-PFP preparation column with specification of 150mm×21.5mm,5.0 μm for separation and purification of squalene, and Waters XTERRA RP C column with specification of 150mm×4.6mm,3.5 μm, combined with medium-spectrum red RD-PFP column with specification of 250mm×4.0mm,5.0 μm for detection and analysis of squalene, and conducting repeated experiments on the operation conditions including type, proportion, flow rate, column temperature and the like of mobile phase.
Drawings
FIG. 1 shows the result of analysis of crude squalene by the HPLC detection and analysis method of example 1.
FIG. 2 shows the result of analysis of crude squalene by the HPLC detection and analysis method of comparative example 1.
FIG. 3 shows the result of gas chromatography analysis of pure squalene obtained by separating and purifying crude squalene by the method of example 2;
FIG. 4 shows the results of gas chromatography analysis of crude squalene by the method of comparative example 2.
Detailed Description
The present application is described in detail below with reference to the specific drawings and examples, and it is necessary to point out that the following examples are given for further illustration of the present application only and are not to be construed as limiting the scope of the present application, since numerous insubstantial modifications and adaptations of the application to those skilled in the art will still fall within the scope of the present application.
The crude squalene products of the examples and the comparative examples are all crude squalene obtained from the same batch of plant squalene (extraction source: bean (vegetable) oil refining byproducts) purchased from the company of Selaginella tarda life sciences
The starting materials used in the examples and comparative examples were all available directly.
In the research process, the sample to be tested is firstly detected according to the method (GC) of EP10.0 (European Pharmacopeia, european pharmacopoeia 10.0), the purity of the sample (namely, the content of squalene in the sample is 87.34 percent according to the peak area) (see the following comparative example 2), an impurity is arranged before the main peak of squalene and cannot be separated from the main peak by a base line, and the impurity is proved to have a boiling point close to that of squalene, so that the impurity is difficult to purify by a rectification method.
The crude product was then analyzed by HPLC for a purity of 92.18% (calculated as peak area, see comparative example 1 below) higher than GC purity, with a significant main peak forward delay, and suspected impurities in the main peak were not separated. The C18 column is still adopted, chromatographic conditions are adjusted, the method is optimized, even the chromatographic column is replaced, an impurity peak does appear, but good separation cannot be achieved, and the polarity of the impurity is proved to be close to that of squalene, so that the impurity is difficult to separate through common column chromatography.
Then, by thin layer chromatography, it is found that there is an inseparable impurity point in the main peak spot, the impurity point can develop color under 254nm ultraviolet lamp (squalene does not develop color), according to the presumption that the polarity and boiling point of the impurity are close to the main peak, the impurity is probably a squalene double bond ectopic, forming a conjugated structure impurity, thus having a certain ultraviolet absorption. Thus, the method of the present application has been obtained through extensive research and exploration.
Example 1
A high performance liquid chromatography detection and analysis method of squalene comprises the following steps:
preparing a test solution: the crude squalene is taken, dissolved in absolute ethanol-methanol (1:9; V/V) and diluted to a solution containing about 1.0mg per 1 ml.
Chromatographic conditions and system applicability requirements: octadecylsilane chemically bonded silica is used as filler (Waters XTERRA RP C, 150mm×4.6mm,3.5 μm or equivalent in potency) and is serially connected with a mid-spectrum red RD-PFP chromatographic column (mid-spectrum technology, 250mm×4.0mm,5.0 μm or equivalent in potency); acetonitrile-water (88:12; V/V) as mobile phase; the column temperature is 20 ℃; sample volume 10. Mu.l; the detection wavelength is 210nm; the flow rate was 1.4ml per minute and isocratic elution was performed. The crude squalene is firstly passed through a C18 chromatographic column and then through a PFP chromatographic column, so that the impurity and squalene are separated, and the crude squalene is subjected to high performance liquid chromatography detection analysis. The separation degree of each impurity from the main peak meets the regulation.
Assay: 10 μl of the sample solution was taken and injected into a liquid chromatograph, and the chromatogram was recorded until the retention time of the main component was 2 times, and the specific test results are shown in FIG. 1 and Table 1.
Table 1: example 1 detection and analysis of squalene crude product
| Peak number | Retention time | Area of | Height | Area percent | Degree of separation (USP) | Theoretical plate number (USP) | Tailing factor |
| 1 | 10.367 | 21230 | 1859 | 0.100 | -- | 18650 | -- |
| 2 | 11.739 | 21109 | 1107 | 0.099 | 3.425 | 8924 | -- |
| 3 | 12.782 | 18703 | 923 | 0.088 | 1.749 | 5390 | -- |
| 4 | 13.288 | 15340 | 1014 | 0.072 | 0.907 | 16082 | -- |
| 5 | 15.158 | 17979 | 816 | 0.085 | 4.260 | 17467 | -- |
| 6 | 16.403 | 93213 | 3880 | 0.439 | 2.499 | 14907 | -- |
| 7 | 17.486 | 50303 | 2623 | 0.237 | 2.083 | 16349 | -- |
| 8 | 21.287 | 16691 | 736 | 0.079 | 6.917 | 20397 | 1.064 |
| 9 | 23.192 | 19794 | 623 | 0.093 | 0.353 | 83 | -- |
| 10 | 23.782 | 59590 | 1755 | 0.280 | 0.106 | 10416 | -- |
| 11 | 24.711 | 48948 | 1049 | 0.230 | 0.869 | 6697 | -- |
| 12 | 29.765 | 12518 | 333 | 0.059 | 3.453 | 4796 | -- |
| 13 | 31.007 | 112698 | 2823 | 0.530 | 0.649 | 3460 | -- |
| 14 | 32.207 | 1197948 | 23039 | 5.639 | 0.656 | 6918 | -- |
| 15 | 33.687 | 18842324 | 434306 | 88.691 | 1.103 | 14100 | 0.960 |
| 16 | 36.375 | 31216 | 1121 | 0.147 | 1.112 | 1546 | -- |
| 17 | 37.107 | 124901 | 2587 | 0.588 | 0287 | 11109 | -- |
| 18 | 38.358 | 249340 | 3399 | 1.174 | 0.744 | 6186 | -- |
| 19 | 40.809 | 26741 | 681 | 0.126 | 1.622 | 23212 | 1.140 |
| 20 | 44.551 | 109495 | 1757 | 0.515 | 2.670 | 10589 | -- |
| 21 | 45.723 | 62210 | 1058 | 0.293 | 0.609 | 7455 | -- |
| 22 | 52.641 | 79854 | 1428 | 0.376 | 3.846 | 20252 | 1.216 |
| 23 | 55.713 | 12750 | 188 | 0.060 | 1.904 | 16264 | 1.052 |
| Totals to | 21244895 | 489104 | 100.000 |
The results of fig. 1 and table 1 correspond, and it can be seen from the above results that the crude diagonal squalene is connected in series by using double columns and under appropriate test conditions, the purity of the main peak (calculated by peak area normalization) is 88.69%, and the main peak is preceded by a large impurity peak with a 5.6% separation from the main peak of 1.1.
Comparative example 1
A high performance liquid chromatography detection and analysis method of squalene comprises the following steps:
test solution: the crude squalene was taken as in example 1, dissolved in absolute ethanol-methanol (1: 9;V/V) and diluted to give a solution containing about 1.0mg per 1 ml.
Chromatographic conditions and system applicability requirements: octadecylsilane chemically bonded silica is used as filler (Waters XTERRARP C, 250 mm. Times.4.6 mm,5 μm or equivalent in potency); acetonitrile-water (90:10; V/V) as mobile phase; the column temperature is 35 ℃; sample volume 10. Mu.l; the detection wavelength is 210nm; the flow rate was 1.4ml per minute and isocratic elution was performed. The separation degree of each impurity from the main peak meets the regulation.
Assay: 10 μl of the sample solution was taken and injected into a liquid chromatograph, and the chromatogram was recorded until the retention time of the main component was 2 times, and the specific test results are shown in fig. 2 and table 2.
Table 2: comparative example 1 detection and analysis result of squalene crude product
| Peak number | Retention time | Area of | Height | Area percent | Degree of separation (USP) | Theoretical plate number (USP) | Tailing factor |
| 1 | 5.924 | 41168 | 5066 | 0.164 | -- | 11115 | -- |
| 2 | 6.390 | 143143 | 13541 | 0.569 | 1.951 | 10164 | -- |
| 3 | 6.675 | 35488 | 3718 | 0.141 | 0.927 | 5500 | -- |
| 4 | 7.040 | 34382 | 2909 | 0.137 | 1.143 | 10200 | -- |
| 5 | 7.692 | 19808 | 1847 | 0.079 | 2.390 | 13253 | -- |
| 6 | 8.039 | 71283 | 6941 | 0.284 | 1.294 | 14349 | 1.233 |
| 7 | 8.697 | 89943 | 7376 | 0.358 | 2.221 | 11545 | 0.994 |
| 8 | 10.209 | 136143 | 6836 | 0.542 | 3.382 | 5125 | -- |
| 9 | 10.452 | 49272 | 4437 | 0.196 | 0.357 | 2786 | -- |
| 10 | 10.743 | 33008 | 2789 | 0.131 | 0.486 | 11278 | -- |
| 11 | 11.303 | 17105 | 802 | 0.068 | 1.168 | 6632 | 1.070 |
| 12 | 13.102 | 23173255 | 1413918 | 92.177 | 3.727 | 16322 | 0.804 |
| 13 | 14.010 | 220574 | 10405 | 0.877 | 1.750 | 7997 | -- |
| 14 | 14.723 | 576535 | 18950 | 2.293 | 1.060 | 6711 | -- |
| 15 | 15.307 | 71761 | 2987 | 0.285 | 0.638 | 3042 | -- |
| 16 | 18.109 | 234603 | 8477 | 0933 | 3.108 | 10916 | 0.822 |
| 17 | 20.054 | 113441 | 6185 | 0.451 | 3.262 | 25799 | 1.029 |
| 18 | 20.880 | 61013 | 2826 | 0.243 | 1.513 | 19930 | 0.991 |
| 19 | 23.154 | 18067 | 778 | 0.072 | 3.741 | 22009 | 1.054 |
| Totals to | 2513992 | 1520788 | 100.000 |
From the results shown in fig. 2 and table 2, it can be seen that the same batch of crude squalene is used in comparative example 1 and example 1, but the purity of the main peak 92.18% (calculated by peak area normalization method) in the detection result is significantly higher than that in example 1 because the impurity overlaps the main peak before the main peak, and good separation is not achieved, thus resulting in the detection purity of comparative example 1 being higher than that in example 1 of the present application. Therefore, the method for separating the squalene crude product is better, and the detection data is more accurate.
Example 2
A chromatographic separation and purification method of squalene adopts two preparation chromatographic columns connected in series to carry out chromatographic separation and purification on crude squalene; the method comprises the following steps:
preparation of chromatographic separation conditions: octadecylsilane chemically bonded silica was used as a filler (Waters SunFire Prep C18150 mm. Times.19 mm,5 μm) in series with a medium-spectrum red RD-PFP preparative chromatographic column (150 mm. Times.21.5 mm,5.0 μm); acetonitrile-water (88:12; V/V) as mobile phase; the column temperature is 20 ℃; the detection wavelength is 210nm; the flow rate was 1.4ml per minute and isocratic elution was performed. The crude squalene is firstly passed through a C18 chromatographic column and then through a PFP chromatographic column, so that the impurity and squalene are separated, and the eluent is collected in sections to obtain pure squalene.
After the above-mentioned collected eluate was concentrated, the yield was 79.8%.
The pure squalene obtained after the concentration was tested according to the test method for squalene in EP10.0 (European Pharmacopeia, european pharmacopoeia 10.0) (GC) under the following conditions.
A detector: FID carrier gas: n (N) 2
Capillary column: agilent DB-1 (30 m 0.32mm 1.0 μm); 100% polydimethylsiloxane as stationary phase
Flow rate: 1.7ml/min; sample injection amount: 1.0 μl; column temperature: 290 ℃;
sample inlet temperature: 275 deg.c; detector temperature: 300 DEG C
Split ratio: 12:1
Sample injection mode: a direct sample injection method; mode: sampling according to constant flow mode
Run time: 25min
The detection results of GC are shown in fig. 3 and table 3.
Table 3: GC analysis results of squalene pure product prepared in example 2
The data in FIG. 3 and Table 3 correspond, and it can be seen from the data in FIG. 3 and Table 3 that in the pure squalene obtained by the method of the present application, the impurity peak before the main peak has been separated and removed, and the purity of the main peak is as high as 99.22%. From this, it was found that a squalene sample of higher purity was obtained by the separation and purification method of the present application.
Comparative example 2
The purity of the crude squalene was measured under the same GC measurement conditions as in example 2, and the measurement results are shown in FIG. 4 and Table 4.
Table 4: comparative example 2 GC analysis results on crude squalene
The data in FIG. 4 and Table 4 correspond, and it can be seen from FIG. 4 and Table 4 that the crude squalene has a main peak purity of 87.34% and a 5.42% impurity content, which is close to the boiling point of squalene and is more difficult to purify by rectification. Comparative example 2 corresponds to the detection result of example 1, but the gas-phase FID is a destructive detector and cannot be used for production. Comparative example 2 in comparison with example 2, the squalene sample purified in example 2 was significantly more pure than the crude sample under the same assay.
In conclusion, the application adopts a method of connecting C18 column and PFP column in series, optimizes chromatographic conditions, controls column temperature, can realize good separation of maximum impurities and main components in squalene, can effectively detect the purity of a crude squalene product, provides a more effective detection method, can collect eluent in liquid phase elution in sections to obtain a squalene sample with the purity of more than 99.0%, obviously improves squalene purity, and solves the current situations that squalene is used as a pharmaceutical adjuvant and has low purity and difficult impurity control.
It is to be understood that the above embodiments are merely illustrative of the application of the principles of the present application, but not in limitation thereof. Various modifications and improvements may occur to those skilled in the art without departing from the spirit and the substance of the application, and such modifications and improvements are considered to be within the scope of the application.
Claims (2)
1. A chromatographic separation and purification method of squalene is characterized in that:
the purification method adopts two preparation chromatographic columns connected in series to carry out chromatographic separation and purification on the crude product of the diagonal squalene;
the two preparation chromatographic columns connected in series are a C18 chromatographic column and a C18-PFP chromatographic column;
the squalene crude product firstly passes through a C18 chromatographic column, then passes through a C18-PFP chromatographic column, so that impurities and squalene are separated, and eluent is collected in sections to obtain pure squalene;
wherein:
in the C18 chromatographic column and the C18-PFP chromatographic column, the adopted mobile phase is acetonitrile-water, and the volume ratio of acetonitrile to water is 88:12;
the column temperature of the C18 chromatographic column and the C18-PFP chromatographic column is 20 ℃;
the elution mode in the C18 chromatographic column and the C18-PFP chromatographic column is isocratic elution;
the flow rate of the mobile phase is 1.2-1.6ml/min;
the C18 chromatographic column is a Waters SunFire Prep C chromatographic column with the specification of 150mm multiplied by 19mm and 5 mu m;
the C18-PFP chromatographic column is a medium-spectrum red RD-PFP preparation chromatographic column with the specification of 150mm multiplied by 21.5mm and 5.0 μm;
the squalene crude product is a byproduct of refining soybean oil or rape oil.
2. A high performance liquid chromatography detection and analysis method of squalene is characterized in that:
performing high performance liquid chromatography separation on the crude squalene by adopting two chromatographic columns connected in series to separate impurities from squalene, wherein the two chromatographic columns connected in series are a C18 chromatographic column and a C18-PFP chromatographic column;
the squalene crude product firstly passes through a C18 chromatographic column and then passes through a C18-PFP chromatographic column;
performing ultraviolet detection to determine the content of squalene;
wherein:
the C18 chromatographic column is Waters XTERRA RP C18 chromatographic column with the specification of 150mm multiplied by 4.6mm and 3.5 μm;
the C18-PFP chromatographic column is a medium-spectrum red RD-PFP chromatographic column with the specification of 250mm multiplied by 4.0mm and 5.0 μm;
in the high performance liquid chromatography detection analysis, the adopted mobile phase is acetonitrile-water mixed solution, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 88:12;
the column temperature is 20 ℃;
the ultraviolet detection wavelength is 205-215nm;
the elution mode is isocratic elution;
the flow rate of the mobile phase is 1.2-1.6ml/min;
the squalene crude product is a byproduct of refining soybean oil or rape oil.
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Denomination of invention: A chromatographic separation, purification, detection and analysis method for squalene and its pure product Granted publication date: 20231110 Pledgee: Hunan Bank Co.,Ltd. Jinshi Branch Pledgor: Hunan Jiujian Pharmaceutical Technology Co.,Ltd. Registration number: Y2024980020309 |