CN107782828A - A kind of method by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and Sitosterolum - Google Patents
A kind of method by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and Sitosterolum Download PDFInfo
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- CN107782828A CN107782828A CN201711316125.5A CN201711316125A CN107782828A CN 107782828 A CN107782828 A CN 107782828A CN 201711316125 A CN201711316125 A CN 201711316125A CN 107782828 A CN107782828 A CN 107782828A
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- campesterol
- stigmasterol
- cupreol
- separating
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- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 title claims abstract description 55
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 title claims abstract description 55
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 title claims abstract description 55
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol 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)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 title claims abstract description 55
- 235000000431 campesterol Nutrition 0.000 title claims abstract description 55
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 title claims abstract description 54
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 title claims abstract description 54
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 title claims abstract description 54
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol 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)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 title claims abstract description 54
- 235000016831 stigmasterol Nutrition 0.000 title claims abstract description 54
- 229940032091 stigmasterol Drugs 0.000 title claims abstract description 54
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 19
- 229930182558 Sterol Natural products 0.000 claims abstract description 20
- 150000003432 sterols Chemical class 0.000 claims abstract description 20
- 235000003702 sterols Nutrition 0.000 claims abstract description 20
- 239000002798 polar solvent Substances 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 238000012545 processing Methods 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 51
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol 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)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 claims description 41
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 150000003431 steroids Chemical class 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims 2
- 244000046052 Phaseolus vulgaris Species 0.000 claims 2
- 238000000926 separation method Methods 0.000 abstract description 13
- 239000000178 monomer Substances 0.000 abstract description 10
- 238000011084 recovery Methods 0.000 abstract description 9
- 230000014759 maintenance of location Effects 0.000 abstract description 4
- 238000004445 quantitative analysis Methods 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 4
- 239000012071 phase Substances 0.000 description 13
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 3
- 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 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- KZJWDPNRJALLNS-VPUBHVLGSA-N (-)-beta-Sitosterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@@H](C(C)C)CC)C)CC4)CC3)CC=2)CC1 KZJWDPNRJALLNS-VPUBHVLGSA-N 0.000 description 1
- CSVWWLUMXNHWSU-UHFFFAOYSA-N (22E)-(24xi)-24-ethyl-5alpha-cholest-22-en-3beta-ol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(CC)C(C)C)C1(C)CC2 CSVWWLUMXNHWSU-UHFFFAOYSA-N 0.000 description 1
- KLEXDBGYSOIREE-UHFFFAOYSA-N 24xi-n-propylcholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CCC)C(C)C)C1(C)CC2 KLEXDBGYSOIREE-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- LPZCCMIISIBREI-MTFRKTCUSA-N Citrostadienol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@H]2C3=CC[C@H]4[C@H](C)[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)C(C)C LPZCCMIISIBREI-MTFRKTCUSA-N 0.000 description 1
- ARVGMISWLZPBCH-UHFFFAOYSA-N Dehydro-beta-sitosterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(CC)C(C)C)CCC33)C)C3=CC=C21 ARVGMISWLZPBCH-UHFFFAOYSA-N 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MJVXAPPOFPTTCA-UHFFFAOYSA-N beta-Sistosterol Natural products CCC(CCC(C)C1CCC2C3CC=C4C(C)C(O)CCC4(C)C3CCC12C)C(C)C MJVXAPPOFPTTCA-UHFFFAOYSA-N 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001877 deodorizing effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000010773 plant oil Substances 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 239000010499 rapseed oil Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000015500 sitosterol Nutrition 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 1
- CMXPERZAMAQXSF-UHFFFAOYSA-M sodium;1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate;1,8-dihydroxyanthracene-9,10-dione Chemical compound [Na+].O=C1C2=CC=CC(O)=C2C(=O)C2=C1C=CC=C2O.CCCCC(CC)COC(=O)CC(S([O-])(=O)=O)C(=O)OCC(CC)CCCC CMXPERZAMAQXSF-UHFFFAOYSA-M 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000003784 tall oil Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
Landscapes
- 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)
- Steroid Compounds (AREA)
Abstract
The invention discloses a kind of method by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and Sitosterolum, including, the preparation of standard liquid, prepared using polar solvent and contain campesterol, stigmasterol and Sitosterolum hybrid standard storing solution, after processing, then prepare the hybrid standard serial solution containing campesterol, stigmasterol and Sitosterolum respectively with polar solvent.We invents, 3 kinds of sterol monomers are not only made to reach baseline separation, substantially reduce retention time, it is 1.0mL/min that optimal chromatographic condition, which is finally determined, Detection wavelength 208nm, 35 DEG C of column temperature, and this method precision and average recovery are higher, disclosure satisfy that the testing requirements of 3 kinds of sterol content of monomer of accurate quantitative analysis.
Description
Technical field
The invention belongs to the separation technology field of phytosterol, and in particular to a kind of separated by high performance liquid chromatography is surveyed
Determine the method for campesterol, stigmasterol and cupreol.
Background technology
Phytosterol is a kind of natural active compound using perhydrocyclopentanophenanthrene as basic framework, and C-3 is connected with hydroxyl on position
Base, the steroid that 8~10 carbon atoms form side chain, the wherein unsaponifiable matter belonged in lipid, dish are connected with C-17 positions
Oily sterol (Campesterol), stigmasterol (Stigmasterol) and cupreol (Sitosterol) are contained in phytosterol
Highest sterol monomer is measured, its structure is shown in Fig. 1 simultaneously.Phytosterol have reduce cholesterol and risk of cardiovascular diseases, anticancer,
Anti-inflammatory and the physiological function such as anti-oxidant, are increasingly taken seriously in fields such as medicine, food, cosmetics, end the whole world in 2015
The demand of sterol is up to 1.8 ten thousand tons or so.At present, phytosterol mainly separates from plant oil deodorizing distillate and tall oil
Purifying obtains.Therefore, a kind of sensitive and accurate, fast and simple detection method is established, the content of phytosterol is determined to dividing with this
It is significant to analyse such active material.
Because only architectural difference be present on C-22 positions and C-24 positions in campesterol and stigmasterol, caused pole between the two
Property neutralizing effect campesterol and stigmasterol can be made to occur co-elute phenomenon in chromatographic separation process, research is big both at home and abroad at present
Separation determination simultaneously is carried out for one or two kinds of sterols therein more, is had no to three kinds of sterols while is carried out separation determination and grind
Study carefully.
The content of the invention
The purpose of this part is to summarize some aspects of embodiments of the invention and briefly introduce some preferably to implement
Example.It may do a little simplified or be omitted to avoid making our department in this part and the description of the present application summary and denomination of invention
Point, the purpose of specification digest and denomination of invention obscure, and this simplification or omit and cannot be used for limiting the scope of the present invention.
In view of above-mentioned and/or existing pass through high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
Method technological gap, it is proposed that the present invention.
It is therefore an object of the present invention to solves deficiency of the prior art, there is provided a kind of to pass through high performance liquid chromatography point
From the method for measure campesterol, stigmasterol and cupreol.
In order to solve the above technical problems, the invention provides following technical scheme：It is a kind of to pass through high performance liquid chromatography point
From the method for measure campesterol, stigmasterol and cupreol, including, the preparation of standard liquid, prepared and contained using polar solvent
Campesterol, stigmasterol and cupreol hybrid standard storing solution, after processing, then prepare steroid containing rape oil with polar solvent respectively
The hybrid standard serial solution of alcohol, stigmasterol and cupreol；Chromatographic condition, chromatographic column are C18 chromatographic columns, and Detection wavelength is
200~220nm, column temperature are 25~45 DEG C, and flow velocity is 0.5~1.5mL/min.
As the side of the present invention by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
A kind of preferred scheme of method, wherein：Described prepared using polar solvent contains campesterol, stigmasterol and cupreol hybrid standard
Storing solution, wherein, the polar solvent includes methanol.
As the side of the present invention by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
A kind of preferred scheme of method, wherein：It is described after processing, it is to be blown the methanol in the hybrid standard storing solution with nitrogen evaporator
It is dry.
As the side of the present invention by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
A kind of preferred scheme of method, wherein：It is described to prepare mixing containing campesterol, stigmasterol and cupreol respectively with polar solvent again
Standardization serial solution, wherein, the polar solvent includes acetonitrile.
As the side of the present invention by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
A kind of preferred scheme of method, wherein：It is described that to prepare the hybrid standard series containing campesterol, stigmasterol and cupreol respectively molten
Liquid, it is, prepares the hybrid standard serial solution of the μ g/mL containing campesterol 2.5,5,10,20,40,50, prepare containing stigmasterol 5,
10th, 20,40,80,100 μ g/mL hybrid standard serial solution, prepare the μ g/mL's containing cupreol 5,10,20,40,80,100
Hybrid standard serial solution.
As the side of the present invention by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
A kind of preferred scheme of method, wherein：The chromatographic condition, wherein, mobile phase includes 100% acetonitrile, 100% methanol, acetonitrile:Water
=85:5~99:1 (v/v) or methanol:Water=85:5~99:1(v/v).
As the side of the present invention by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
A kind of preferred scheme of method, wherein：The chromatographic condition, wherein, mobile phase includes 100% acetonitrile or acetonitrile:Water=85:5~
99:1(v/v)。
As the side of the present invention by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
A kind of preferred scheme of method, wherein：The chromatographic condition, wherein, sample size is 10~60 μ L.
Beneficial effect possessed by the present invention：
We invents, and 3 kinds of sterol monomers is reached baseline separation, substantially reduces retention time, finally determine
Optimal chromatographic condition is 1.0mL/min, Detection wavelength 208nm, 35 DEG C of column temperature, and this method precision and average recovery are equal
It is higher, it disclosure satisfy that the testing requirements of 3 kinds of sterol content of monomer of accurate quantitative analysis.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, required use in being described below to embodiment
Accompanying drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for this
For the those of ordinary skill of field, without having to pay creative labor, it can also be obtained according to these accompanying drawings other
Accompanying drawing.Wherein：
Fig. 1 is the HPLC chromatogram of 1 three kinds of sterol hybrid standard samples of embodiment, in figure 1,2,3 be respectively campesterol,
Stigmasterol and cupreol, 1,2,3 three kind of equal baseline separation of sterol, detect and are completed in 38min.
Fig. 2 is the HPLC chromatogram of 2 three kinds of sterol hybrid standard samples of embodiment.In figure 1,2,3 be respectively campesterol,
Stigmasterol and cupreol, 1,2,3 three kind of equal baseline separation of sterol, because the ratio that water is added in mobile phase improves, mobile phase
Polarity increases, and eluting power weakens, so detection time extends to 42min completions.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, with reference to specific embodiment pair
The embodiment of the present invention is described in detail.
Many details are elaborated in the following description to facilitate a thorough understanding of the present invention, still the present invention can be with
It is different from other manner described here using other to implement, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein refers to may be included at least one realization side of the present invention
Special characteristic, structure or characteristic in formula." in one embodiment " that different places occur in this manual not refers both to
Same embodiment, nor the single or selective embodiment mutually exclusive with other embodiment.
Embodiment 1
The preparation of standard liquid:
Prepared using methanol and contain campesterol, stigmasterol and cupreol hybrid standard storing solution, after nitrogen evaporator dries up,
Prepared respectively with acetonitrile again containing the μ g/mL of campesterol 2.5,5,10,20,40,50, stigmasterol and cupreol 5,10,20,40,
80th, 100 μ g/mL standard serial solution.
High-efficient liquid phase chromatogram condition is：
Chromatographic column：WaterC18 posts (4.6mm × 250mm, 5 μm)
Detector：Waters2489 ultraviolet-visible detectors
Detection wavelength：208nm
Column temperature：35℃
Mobile phase：Acetonitrile:Water=98:2(v/v)
Flow phase velocity：1.0mL/min
Sample size is 20 μ L
The measure of the rate of recovery and precision：
Homogeneous sojasterol sample is taken, is divided into 3 groups, it is molten to be separately added into the horizontal hybrid standard series of 3 various concentrations
Liquid (campesterol：10、20、40μg/mL；Stigmasterol and cupreol：20th, 40,80 μ g/mL) form high, normal, basic 3 mark-on groups
And 1 blank control group, replication 3 times, calculate the rate of recovery.
Fetch high, normal, basic 3 mark-on groups in yield experiment and repeat sample introduction 5 times interior on the same day, calculate withinday precision,
Sample introduction is repeated in continuous 3d daily 3 times, calculate day to day precision, represented with RSD.
Embodiment 2
The preparation of standard liquid:
Prepared using methanol and contain campesterol, stigmasterol and cupreol hybrid standard storing solution, after nitrogen evaporator dries up,
Prepared respectively with acetonitrile again containing the μ g/mL of campesterol 2.5,5,10,20,40,50, stigmasterol and cupreol 5,10,20,40,
80th, 100 μ g/mL standard serial solution.
High-efficient liquid phase chromatogram condition is：
Chromatographic column:WaterC18 posts (4.6mm × 250mm, 5 μm)
Detector:Waters2489 ultraviolet-visible detectors
Detection wavelength:208nm
Column temperature:30℃
Mobile phase:Acetonitrile:Water=94:6(v/v)
Flow phase velocity:1.0mL/min
Sample size is 20 μ L
The measure of the rate of recovery and precision：
Homogeneous campesterol sample is taken, is divided into 3 groups, it is molten to be separately added into the horizontal hybrid standard series of 3 various concentrations
Liquid (campesterol：10、20、40μg/mL；Stigmasterol and cupreol：20th, 40,80 μ g/mL) form high, normal, basic 3 mark-on groups
And 1 blank control group, replication 3 times, calculate the rate of recovery.
Fetch high, normal, basic 3 mark-on groups in yield experiment and repeat sample introduction 5 times interior on the same day, calculate withinday precision,
Sample introduction is repeated in continuous 3d daily 3 times, calculate day to day precision, represented with RSD.
As can be seen here, embodiment 1 is better, and detection time is short, and precision is high, and the rate of recovery is high, and RSD values are relatively
It is low.
It is noted that at this end during invention, due to campesterol on C-22 positions more than stigmasterol one it is double
Key, campesterol polarity is slightly weaker than stigmasterol, therefore be more easy to be eluted out by non-polar solven, but because stigmasterol is in C-24
Position methyl more than campesterol, therefore both can polarization neutralizing effect.Due to less polarity between the two
Difference, campesterol and stigmasterol often produce co-elute phenomenon, and the chromatographic condition of most of document report uses methanol
Or first alcohol and water is simultaneously bad as mobile phase, its separating effect to both, it is difficult to reaches baseline separation.Due to molten in reversed-phase column
The eluting power of agent weakens with the enhancing of sample polarity, and the polarity of methanol is better than acetonitrile, therefore uses the stronger second of eluting power
Nitrile is more conducive to the separation of stigmasterol and campesterol.But use 100% acetonitrile its polarity is relative to methanol as mobile phase
Decrease, make stigmasterol and campesterol can not be completely isolated when being separated in HPLC, so increase a certain proportion of water, increase
Add the polarity of mobile phase, so that stigmasterol and campesterol can reach baseline separation.In addition, the ratio of addition water should be strict
Control, if otherwise not under the proportioning, is just unable to reach the baseline separation of sterol monomer, stigmasterol and campesterol chromatographic peak meeting
It is inseparable.Also, we invents is tested by Optimization of mobile phase, acetonitrile and the water isocratic elution under certain proportion proportioning are found,
3 kinds of sterol monomers is reached baseline separation, and retention time was substantially reduced to 35min from 2 hours, it is final to determine
Optimal chromatographic condition is 1.0mL/min, Detection wavelength 208nm, 35 DEG C of column temperature, and this method precision and average recovery are equal
It is higher, it disclosure satisfy that the testing requirements of 3 kinds of sterol content of monomer of accurate quantitative analysis.
As can be seen here, our invention not only makes 3 kinds of sterol monomers reach baseline separation, substantially reduces retention time, most
It is 1.0mL/min, Detection wavelength 208nm, 35 DEG C of column temperature, and this method precision and sample-adding that optimal chromatographic condition is determined eventually
The rate of recovery is higher, disclosure satisfy that the testing requirements of 3 kinds of sterol content of monomer of accurate quantitative analysis.
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to preferable
The present invention is described in detail embodiment, it will be understood by those within the art that, can be to the technology of the present invention
Scheme is modified or equivalent substitution, and without departing from the spirit and scope of technical solution of the present invention, it all should cover in this hair
Among bright right.
Claims (8)
1. a kind of method by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol, its feature exists
In：Including,
The preparation of standard liquid：Prepared using polar solvent and contain campesterol, stigmasterol and cupreol hybrid standard storing solution,
After processing, then prepare the hybrid standard serial solution containing campesterol, stigmasterol and cupreol respectively with polar solvent；
Chromatographic condition：Chromatographic column is C18 chromatographic columns, and Detection wavelength is 200~220nm, and column temperature is 25~45 DEG C, flow velocity 0.5
~1.5mL/min.
2. pass through high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol according to claim 1
Method, it is characterised in that：Described prepared using polar solvent contains campesterol, stigmasterol and cupreol hybrid standard deposit
Liquid, wherein, the polar solvent includes methanol.
3. according to claim 1 or claim 2 pass through high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol
Method, it is characterised in that：It is described after processing, its be with nitrogen evaporator by the hybrid standard storing solution methanol dry up.
4. pass through high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol according to claim 1
Method, it is characterised in that：It is described to prepare the hybrid standard containing campesterol, stigmasterol and cupreol respectively with polar solvent again
Serial solution, wherein, the polar solvent includes acetonitrile.
5. pass through high efficiency liquid chromatography for separating and determining campesterol, stigmasterol according to any one of claim 1,2 or 4
With the method for cupreol, it is characterised in that：It is described to prepare the hybrid standard containing campesterol, stigmasterol and cupreol respectively
Serial solution, it is, prepares the hybrid standard serial solution of the μ g/mL containing campesterol 2.5,5,10,20,40,50, and preparation contains beans
The μ g/mL of sterol 5,10,20,40,80,100 hybrid standard serial solution, prepare the μ containing cupreol 5,10,20,40,80,100
G/mL hybrid standard serial solution.
6. pass through high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol according to claim 5
Method, it is characterised in that：The chromatographic condition, wherein, mobile phase includes 100% acetonitrile, 100% methanol, acetonitrile:Water=85:5
~99:1 (v/v) or methanol:Water=85:5~99:1(v/v).
7. pass through high efficiency liquid chromatography for separating and determining campesterol, beans steroid according to any one of claim 1,2,4 or 6
The method of alcohol and cupreol, it is characterised in that：The chromatographic condition, wherein, mobile phase includes 100% acetonitrile or acetonitrile:Water
=85:5~99:1(v/v).
8. pass through high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and cupreol according to claim 7
Method, it is characterised in that：The chromatographic condition, wherein, sample size is 10~60 μ L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109738553A (en) * | 2019-03-08 | 2019-05-10 | 云南中烟工业有限责任公司 | A kind of pre-treating method for the measurement simultaneously of free, reference state sterol in tobacco |
| CN110333300A (en) * | 2019-06-12 | 2019-10-15 | 舟山新诺佳生物工程有限责任公司 | Unsaponifiable matter and stearic type detection method in a kind of fish oil grease |
| CN111122732A (en) * | 2019-12-27 | 2020-05-08 | 贵州景峰注射剂有限公司 | Quality detection method for caulis mahoniae medicine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011067144A1 (en) * | 2009-12-03 | 2011-06-09 | Dsm Ip Assets B.V. | Production of non-yeast sterols by yeast |
| CN102565217A (en) * | 2011-12-16 | 2012-07-11 | 中国农业科学院农产品加工研究所 | Method for simultaneously determining phytosterol and squalene in vegetable oil |
| CN103018389A (en) * | 2011-09-23 | 2013-04-03 | 北大方正集团有限公司 | High performance liquid chromatography method and application thereof |
| CN103091406A (en) * | 2011-11-04 | 2013-05-08 | 上海医药工业研究院 | Detection method of fatty acid compounds and/or sterol compounds in rape bee pollen |
| CN104297377A (en) * | 2014-10-24 | 2015-01-21 | 曲阜师范大学 | Detection and analysis method of phytosterol |
-
2017
- 2017-12-12 CN CN201711316125.5A patent/CN107782828A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011067144A1 (en) * | 2009-12-03 | 2011-06-09 | Dsm Ip Assets B.V. | Production of non-yeast sterols by yeast |
| CN103018389A (en) * | 2011-09-23 | 2013-04-03 | 北大方正集团有限公司 | High performance liquid chromatography method and application thereof |
| CN103091406A (en) * | 2011-11-04 | 2013-05-08 | 上海医药工业研究院 | Detection method of fatty acid compounds and/or sterol compounds in rape bee pollen |
| CN102565217A (en) * | 2011-12-16 | 2012-07-11 | 中国农业科学院农产品加工研究所 | Method for simultaneously determining phytosterol and squalene in vegetable oil |
| CN104297377A (en) * | 2014-10-24 | 2015-01-21 | 曲阜师范大学 | Detection and analysis method of phytosterol |
Non-Patent Citations (7)
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109738553A (en) * | 2019-03-08 | 2019-05-10 | 云南中烟工业有限责任公司 | A kind of pre-treating method for the measurement simultaneously of free, reference state sterol in tobacco |
| CN110333300A (en) * | 2019-06-12 | 2019-10-15 | 舟山新诺佳生物工程有限责任公司 | Unsaponifiable matter and stearic type detection method in a kind of fish oil grease |
| CN110333300B (en) * | 2019-06-12 | 2022-02-22 | 舟山新诺佳生物工程有限责任公司 | Method for detecting types of unsaponifiable matters and oil esters in fish oil and fat |
| CN111122732A (en) * | 2019-12-27 | 2020-05-08 | 贵州景峰注射剂有限公司 | Quality detection method for caulis mahoniae medicine |
| CN111122732B (en) * | 2019-12-27 | 2022-09-09 | 贵州景峰注射剂有限公司 | Quality detection method for caulis mahoniae medicine |
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