CN117723679B - Detection method of R-/S-hydroxypropyl tetrahydropyran triol - Google Patents
Detection method of R-/S-hydroxypropyl tetrahydropyran triol Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 27
- MTZYZFYTYOADPI-UHFFFAOYSA-N 3-(oxan-2-yl)propan-1-ol Chemical compound OCCCC1CCCCO1 MTZYZFYTYOADPI-UHFFFAOYSA-N 0.000 claims abstract description 26
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 18
- -1 aromatic acyl chloride Chemical class 0.000 claims abstract description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 12
- 238000010829 isocratic elution Methods 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 22
- MXMOTZIXVICDSD-UHFFFAOYSA-N anisoyl chloride Chemical compound COC1=CC=C(C(Cl)=O)C=C1 MXMOTZIXVICDSD-UHFFFAOYSA-N 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 17
- 238000001212 derivatisation Methods 0.000 claims description 16
- 239000000126 substance Substances 0.000 claims description 16
- 239000012488 sample solution Substances 0.000 claims description 10
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 229920002160 Celluloid Polymers 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000004033 plastic Substances 0.000 claims description 4
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 4
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- JCQPONUUPNAEGZ-UHFFFAOYSA-N 4-aminobenzoyl chloride Chemical compound NC1=CC=C(C(Cl)=O)C=C1 JCQPONUUPNAEGZ-UHFFFAOYSA-N 0.000 claims description 2
- DENKGPBHLYFNGK-UHFFFAOYSA-N 4-bromobenzoyl chloride Chemical compound ClC(=O)C1=CC=C(Br)C=C1 DENKGPBHLYFNGK-UHFFFAOYSA-N 0.000 claims description 2
- RKIDDEGICSMIJA-UHFFFAOYSA-N 4-chlorobenzoyl chloride Chemical compound ClC(=O)C1=CC=C(Cl)C=C1 RKIDDEGICSMIJA-UHFFFAOYSA-N 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 238000003260 vortexing Methods 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 7
- 239000002537 cosmetic Substances 0.000 abstract description 4
- KOGFZZYPPGQZFZ-QVAPDBTGSA-N (2s,3r,4s,5r)-2-(2-hydroxypropyl)oxane-3,4,5-triol Chemical compound CC(O)C[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O KOGFZZYPPGQZFZ-QVAPDBTGSA-N 0.000 description 47
- 239000000243 solution Substances 0.000 description 27
- 238000002474 experimental method Methods 0.000 description 13
- 239000012086 standard solution Substances 0.000 description 9
- 239000003513 alkali Substances 0.000 description 5
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical class ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- SKDHHIUENRGTHK-UHFFFAOYSA-N 4-nitrobenzoyl chloride Chemical compound [O-][N+](=O)C1=CC=C(C(Cl)=O)C=C1 SKDHHIUENRGTHK-UHFFFAOYSA-N 0.000 description 4
- 238000010812 external standard method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 229920002683 Glycosaminoglycan Polymers 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000005711 Benzoic acid Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- AEQDJSLRWYMAQI-UHFFFAOYSA-N Tetrahydropalmatine Natural products C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 description 2
- DHXVGJBLRPWPCS-UHFFFAOYSA-N Tetrahydropyran Chemical compound C1CCOCC1 DHXVGJBLRPWPCS-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 235000010233 benzoic acid Nutrition 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 239000007810 chemical reaction solvent Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
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- 238000000105 evaporative light scattering detection Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
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- 238000000926 separation method Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- AEQDJSLRWYMAQI-KRWDZBQOSA-N tetrahydropalmatine Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3C[C@H]2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-KRWDZBQOSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
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- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- QDHDQJXBEOUAGE-UHFFFAOYSA-N oxane-2,3,4-triol Chemical compound OC1CCOC(O)C1O QDHDQJXBEOUAGE-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 238000011069 regeneration method Methods 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical class ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 229910000404 tripotassium phosphate Inorganic materials 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a detection method of R-/S-hydroxypropyl tetrahydropyran triol, and belongs to the technical field of cosmetic detection. The detection method comprises the following steps: A. derivatizing hydroxypropyl tetrahydropyran in a sample to be tested by using aromatic acyl chloride; B. detecting the hydroxypropyl tetrahydropyran derivative by using high performance liquid chromatography, wherein the high performance liquid chromatography condition is CHIRALP IC columns, the detector is an ultraviolet detector, methanol or isopropanol is used as a mobile phase, isocratic elution is carried out, the flow rate is 0.5-1.5mL/min, the detection wavelength is 200 nm-280 nm, and the column temperature is 30-40 ℃. The method of the invention does not need to use special analysis and detection instruments which cannot be popularized, and has the characteristics of good universality, rapidness and accuracy.
Description
Technical Field
The invention relates to the technical field of cosmetic detection, in particular to a detection method of R-/S-hydroxypropyl tetrahydropyranetriol.
Background
The vitriol (Pro-Xylane), also known as hydroxypropyl tetrahydropyran triol (CAS No. 439685-79-7), is a bioactive substance, can promote the production of glycosaminoglycan (GAG), can promote the synthesis of some cytokines and collagen regeneration through the GAG, and can effectively repair damaged skin, thereby enabling the skin to be compact and elastic. Therefore, the glass color is widely applied to the field of cosmetics and is mainly applied to high-end skin care products such as wrinkle resistance and the like.
The hydroxy group on the chiral carbon atom at position 7 has a stereochemical structure and coexists as a pair of diastereomers. In the existing detection method, the hydroxypropyl tetrahydropyran triol molecular structure has no conjugated double bond, lacks color development and fluorescent groups, and cannot be accurately detected under the common liquid chromatography condition. The existing detection methods of the hydroxypropyl tetrahydropyran triol mainly comprise differential refraction method, ion chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS), electrospray detector method (CAD), evaporative light detector method (ELSD) and the like, and all have the defect that special analysis detection instruments which cannot be popularized are needed to be used. No reported detection method can be used for measuring the content of hydroxypropyl tetrahydropyran triol and simultaneously separating and measuring the diastereoisomer proportion.
In view of the fact that the activity of the hydroxypropyl tetrahydropyran triol with the diastereomer ratio of 70/30 is far higher than that of the hydroxypropyl tetrahydropyran triol with the diastereomer ratio of 50/50, how to rapidly and accurately detect the content of R-/S-hydroxypropyl tetrahydropyran triol is of great significance for evaluating the quality control of the hydroxypropyl tetrahydropyran triol related product, the application provides a detection method of R-/S-hydroxypropyl tetrahydropyran triol.
Disclosure of Invention
Aiming at the technical problem that a special analysis and detection instrument which cannot be popularized is required to be used in the detection method of the R-/S-hydroxypropyl tetrahydropalmatine, the detection method of the R-/S-hydroxypropyl tetrahydropalmatine with good universality, rapidness and accuracy is necessary to be provided.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a detection method of R-/S-hydroxypropyl tetrahydropyran triol, which comprises the following steps:
(1) Derivatizing hydroxypropyl tetrahydropyran in a sample to be tested by using aromatic acyl chloride;
(2) The hydroxypropyl tetrahydropyran derivative was detected using high performance liquid chromatography.
Wherein, in the step (1), the mass ratio of the aromatic acyl chloride to the sample to be detected is 1 (2-20); preferably, the mass ratio of the aromatic acyl chloride to the sample to be tested is 1:10.
In the step (1), the aromatic acyl chloride is at least one selected from 4-methoxybenzoyl chloride, 4-chlorobenzoyl chloride, 4-bromobenzoyl chloride, 4-aminobenzoyl chloride and 4-nitroformyl chloride.
In the invention, the sample to be tested comprises a hydroxypropyl tetrahydropyran triol standard and a sample containing hydroxypropyl tetrahydropyran triol.
The 4-methoxybenzoyl chloride and the hydroxypropyl tetrahydropyran are subjected to a derivatization reaction, the hydroxypropyl tetrahydropyran derivative can be detected through a liquid phase, and effective separation (the separation degree is more than 1.5) of substances similar to the hydroxypropyl tetrahydropyran structure can be realized.
In the invention, 4-methoxybenzoyl chloride is used for derivatizing hydroxypropyl tetrahydropyran in a sample to be detected, and the reaction formula is as follows:
。
according to the method for detecting the content of the R-/S-hydroxypropyl tetrahydropyran, in the step (1), an aromatic acyl chloride solution is added into a sample solution to be detected, and the mixture is uniformly mixed at 2800 rpm for 2min in a vortex manner to carry out derivatization.
The invention completes derivatization in 5 minutes under normal temperature by vortex mixing, has mild condition, short time, quick reaction and high efficiency.
According to the method for detecting the content of the R-/S-hydroxypropyl tetrahydropyran in the specific embodiment of the invention, in the step (2), the high performance liquid chromatography condition is CHIRALP IC columns, the detector is an ultraviolet detector, methanol or isopropanol is used as a mobile phase for isocratic elution, the flow rate is 0.5-1.5mL/min, the detection wavelength is 200-280 nm, and the column temperature is 30-40 ℃.
According to the method for detecting the content of the hydroxypropyl tetrahydropyran, in the step (2), the detection wavelength is 240nm-260 nm.
According to the method for detecting the content of the hydroxypropyl tetrahydropyran, in the step (2), the detection wavelengths are 240nm, 241nm, 242nm, 243nm, 244nm, 246nm, 247nm, 248nm, 249nm, 250nm and 254nm. In some embodiments, the detection wavelength is 254nm, at which the target detection has a strong absorption.
According to the method for detecting the content of the hydroxypropyl tetrahydropyran in the specific embodiment of the invention, in the step (2), the high performance liquid chromatography condition is CHIRALP IC columns, the detector is an ultraviolet detector, methanol or isopropanol is used as a mobile phase, isocratic elution is carried out, the flow rate is 1.0mL/min, the detection wavelength is 254nm, and the column temperature is 30 ℃.
According to the method for detecting the content of the hydroxypropyl tetrahydropyran, in the step (2), a CHIRALP IC column with the thickness of 250 multiplied by 4.6mm and the thickness of 5 mu m is adopted.
According to the method for detecting the content of the hydroxypropyl tetrahydropyran, in the step (2), a chromatographic column is adopted as a large xylonite CHIRALP IC (250 multiplied by 4.6mm,5 mu m).
According to the method for detecting the content of the hydroxypropyl tetrahydropyran, in the step (1), naOH is used for catalyzing the reaction, and HCl generated by hydrolysis is neutralized, so that a solution sample to be detected is neutral, the pH is 5-7, detection and analysis are facilitated, and a chromatographic instrument is protected.
According to the method for detecting the content of the hydroxypropyl tetrahydropyran, which is an embodiment of the invention, the method comprises the following steps:
(1) Preparing a hydroxypropyl tetrahydropyran standard substance or a test substance into a standard substance solution and a test substance solution with the concentration of 1mg/mL respectively;
(2) Accurately transferring 1.0mL of the prepared standard substance solution and 1.0mL of the prepared sample solution respectively into 7.5mL of plastic centrifuge tubes, adding 1.0mL of 10mg/mL NaOH solution, and shaking uniformly. Adding 2.0mL of 4-methoxybenzoyl chloride solution with the concentration of 5mg/mL, swirling for 2min at 2800 r/min, uniformly mixing, fixing the volume to a scale mark by using ultrapure water, swirling for 15s at 2800 r/min, and filtering by using a 0.22 mu m PTFE filter membrane;
(3) Performing isocratic elution with a large xylonite CHIRALP IC (250×4.6mm,5 μm) chromatographic column with methanol or isopropanol as mobile phase, and operating for 5min at a flow rate of 1.0mL/min at a detection wavelength of 254nm at 30deg.C.
According to the method for detecting the content of the hydroxypropyl tetrahydropyran in the specific embodiment of the invention, the content of the hydroxypropyl tetrahydropyran (or the hydroxypropyl tetrahydropyran derivative) is calculated according to an external standard method by using the peak area; wherein,
Hydroxypropyl tetrahydropyran (%) = (peak area of sample to be measured/peak area of standard substance) × (concentration of standard substance/concentration of sample to be measured) ×100%.
In summary, the invention has the following beneficial technical effects:
according to the method for detecting the hydroxypropyl tetrahydropyran, disclosed by the invention, the 4-methoxybenzoyl chloride and the hydroxypropyl tetrahydropyran are subjected to derivatization reaction to produce the corresponding ultraviolet groups, the ultraviolet response characteristics are achieved, and then the content is calculated according to an external standard method. The method overcomes the defect that the accurate detection cannot be carried out under the common liquid chromatography condition, is simple and quick to operate, can be directly measured, and is proved to be accurate and reliable by the method verification of precision tests, linear relation and range tests, standard addition recovery tests and other methodologies, and the result shows that the method can be used for content analysis. Has very important significance for quality control of products containing the vitriol, and is a method for detecting the hydroxypropyl tetrahydropyran triol with good universality, rapidness and accuracy.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
In the present invention, the expressions "compound a" and "compound represented by formula a" and "formula a" mean the same compound.
In the present invention, "optional" or "optionally" means that there may or may not be; or may not be performed; the term "optionally adding a reaction solvent to the crude product obtained in the step (C)" means that the reaction solvent may be added to the crude product obtained in the step (C) or not.
Drawings
FIG. 1 is a linear relationship of hydroxypropyl tetrahydropyran triol derivative;
FIG. 2 is a chromatogram of a hydroxypropyl tetrahydropyran triol standard solution;
FIG. 3 is a chromatogram of a standard solution of 4-methoxybenzoyl chloride-derived hydroxypropyl tetrahydropyran triol;
FIG. 4 is a chromatogram of a sample solution of 4-methoxybenzoyl chloride-derived hydroxypropyl tetrahydropyran triol and performance parameters;
FIG. 5 is a chromatogram of a sample solution of 4-nitrobenzoyl chloride-derived hydroxypropyl tetrahydropyran triol and performance parameters;
FIG. 6 is a chromatogram of a benzoyl chloride derivative hydroxypropyl tetrahydropyran triol sample and performance parameters.
Detailed Description
In order to better understand the technical solution of the present invention, the following further discloses some non-limiting examples, which are further described in detail.
The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.
In the invention, h represents an hour; min represents minutes; g represents gram; mL represents milliliters.
The invention discloses a high performance liquid chromatography detection method for 4-methoxy benzoyl chloride derived hydroxypropyl tetrahydropyran through extensive research and a large number of experiments.
The experimental conditions for each example of the present invention are as follows:
Chiral chromatographic column model: CHIRALP IC, brand: large xylonite, size: 4.6X250 mm 5 μm, mobile phase: methanol or isopropanol; methanol or isopropanol is used as a mobile phase, gradient elution is carried out, the flow rate is 1.0mL/min, and the detector is used for: an ultraviolet detector with a detection wavelength of 254nm and a column temperature of 30 ℃.
Example 1
Determination of the R-/S-hydroxypropyl tetrahydropyran triol content:
(1) Sample preparation
Accurately weighing 100mg of standard substance or sample, placing into a 100mL volumetric flask, adding pure water to fix volume, and performing ultrasonic dissolution to obtain 1mg/mL hydroxypropyl tetrahydropyran triol solution.
(2) Sample derivatization
Accurately transferring 1.0mL of the prepared standard substance or sample solution into a 7.5mL plastic centrifuge tube, adding 1.0mL of 10mg/mL NaOH solution, and shaking uniformly. 2.0mL of 4-methoxybenzoyl chloride solution with the concentration of 5mg/mL is added, the mixture is uniformly mixed by vortex at 2800 r/min for 2min, the volume is fixed to a scale mark by ultrapure water, and the mixture is filtered by a PTFE filter membrane with the size of 0.22 mu m after vortex at 2800 r/min for 15 s.
(3) Blank solution preparation
1.0ML of ultrapure water was accurately removed and placed in a 7.5mL plastic centrifuge tube, and 1.0mL of 10mg/mL NaOH solution was added thereto and shaken well. Adding 2.0mL of 4-methoxybenzoyl chloride solution with the concentration of 5mg/mL, swirling for 2min at 2800 r/min, uniformly mixing, fixing the volume to a scale mark by using ultrapure water, swirling for 15s at 2800 r/min, and filtering by using a 0.22 mu m PTFE filter membrane;
And (5) respectively using set chromatographic conditions on the blank solution, the standard solution and the sample solution, and recording a chromatogram.
Example 2
And (3) methodological verification:
1. Quantitative limit experiment
According to the solution preparation method in example 1, a standard solution for a quantitative limit experiment, i.e., a standard solution having a concentration of 0.025 mg/mL at the lowest point of the linear solution was prepared as a quantitative limit solution), and detection analysis was performed to record a chromatogram.
According to the signal to noise ratio S/N of the quantitative limit being more than or equal to 10, the hydroxypropyl tetrahydropyran triol standard solution is diluted, and the corresponding chromatogram is shown in figure 4 (quantitative limit) according to the chromatographic condition determination of the embodiment. The signal to noise ratio was calculated automatically by software and the experimental results are shown in table 1.
TABLE 1 quantitative limit determination of hydroxypropyl tetrahydropyran triol
Experimental results show that when the hydroxypropyl tetrahydropyran triol with the concentration of 0.025 mg/mL is derivatized, the ratio of the chromatographic peak height to the baseline noise of isomer 1 and isomer 2 can still reach 65.1 or 63.2, and the sensitivity is good.
2 Linear relation and Range experiments
According to the solution preparation method in example 1, a standard stock solution of hydroxypropyltetrahydro-pyran triol derivative having a concentration of 10.0mg/mL was prepared, the appropriate stock solution was transferred to a 10mL volumetric flask, and diluted with water to a scale to prepare a linear standard solution having a concentration of hydroxypropyltetrahydro-pyran triol derivative of 0.025mg/mL (quantitative limit solution), 0.1mg/mL, 0.5mg/mL, 0.75mg/mL, 1.0mg/mL, and 1.2mg/mL, and the derivative product of the hydroxypropyltetrahydro-pyran triol solution was obtained by carrying out the derivative under the derivative conditions in example 1.
The prepared standard solution for linear experiments was tested and continuously sampled, each sample was fed into a needle, the retention time and peak area of the derivative were recorded, and the sample feeding amount (X) and peak area (Y) were subjected to data regression analysis according to the analysis method of the present invention, and the recorded results are shown in Table 2 and FIG. 1.
TABLE 2 results of Linear relationship experiments
By calculation, the linear regression equation is y=153634x+14956, r 2 =0.9995, r=0.99977. The result shows that the hydroxypropyl tetrahydropyran triol derivative has good linear relation between peak area and sample injection amount in the concentration range of 0.025mg/mL (quantitative limit concentration) -20mg/mL (150% concentration).
3 Precision experiment
According to the solution preparation method in example 1, a sample solution for precision experiments was prepared, a hydroxypropyltetrahydro-pyran triol solution having a concentration of 1.0mg/mL was prepared, and 6 parts were prepared in parallel, and derivatization was performed according to the derivatization conditions in example 1 to obtain a hydroxypropyltetrahydro-pyran triol derivative having a concentration of 0.1 mg/mL.
And detecting the prepared sample solution for precision experiments, continuously injecting samples, feeding each sample into one needle, recording the retention time and the peak area of the derivative, calculating the result by an external standard method, evaluating the result, and recording the result as follows.
TABLE 3 results of precision experiments
As shown in Table 3, the RSD value of the content measurement is 0.24%, which indicates that the accuracy and reproducibility of the content measurement of the hydroxypropyl tetrahydropyran triol by the derivatization method of the invention are high.
4, Experiment of recovery rate by adding standard
Accurately weighing a hydroxypropyl tetrahydropyran triol sample with known content, respectively adding a certain amount of hydroxypropyl tetrahydropyran triol standard solution to prepare solutions with the concentration of 0.8mg/mL (80%), 1.0mg/mL (100%) and 1.2mg/mL (120%), and preparing 3 parts of each concentration in parallel.
The recovery was calculated by the external standard method according to the procedure described in example 1, with an average recovery of 99.3% and RSD of 0.37% indicating good standard recovery.
5 Stability test
Samples of hydroxypropyl tetrahydropyran triol were prepared by derivatization according to the method of example 1, and after standing at room temperature for 0h, 12h, 24h and 48h, the samples were tested by the method of example 1 to calculate the relative standard deviation of the peak areas of the hydroxypropyl tetrahydropyran triol derivatives.
After the samples of the derivatized hydroxypropyltetrahydropyran triol were left at room temperature for 0h, 12h, 24h and 48h, the RSD of the peak area measured for each sample was 0.47%, indicating that the solution was stable within 48h after the hydroxypropyltetrahydropyran triol derivatization.
Example 3
Comparing other aromatic acid chlorides with sulfonyl chloride derivatives:
Aromatic acyl chloride is selected to be derivatised with hydroxypropyl tetrahydropyran triol in alkaline solution, such as common alkali like NaOH, KOH, etc., or alkali weak acid salt, such as phosphate (such as Na 3PO4、K3PO4, etc.), hydrogen phosphate (such as Na 2HPO4、K2HPO4, etc.), sodium tetraborate alkali weak acid salt, or mixed solution of alkali weak acid salt and alkali, aiming at introducing aromatic ring group with ultraviolet absorption, so that the hydroxypropyl tetrahydropyran triol can be detected by common high performance liquid chromatography.
In the invention, 4-methoxybenzoyl chloride, 4-nitrobenzoyl chloride and benzoyl chloride aromatic acyl chloride are taken as examples, and are derived from hydroxypropyl tetrahydropyran triol standard. The solution after the derivatization is analyzed by a high performance liquid chromatography mass spectrometer, and the 4-methoxybenzoyl chloride, the 4-nitrobasic benzoyl chloride and the benzoyl chloride can be completely derivatized.
In FIG. 4, the chromatogram of the 4-methoxybenzoyl chloride derivative solution shows that the main peaks of the hydroxypropyl tetrahydropyran triol derivative are shown at the positions of 8.038min and 9.041 after the retention time is confirmed by LCMS, and the characteristic peaks have good response and are completely derived from the hydroxypropyl tetrahydropyran triol.
In FIG. 5, the chromatogram of the solution of the 4-nitrobenzoyl chloride derivative shows that the main peak of the hydroxypropyl tetrahydropyran triol derivative is at the positions of 5.706min and 10.790min in retention time, the benzoic acid peak is at the position of 1.749min, the response of the characteristic peak is weaker than that of the 4-methoxybenzoyl chloride derivative, and the proportion deviation of the two peaks is larger.
In fig. 6, the chromatograms of the benzoyl chloride derivative solutions are shown, and LCMS confirms that the main peak of the hydroxypropyl tetrahydropyran triol derivative is located at the positions of 11.729min and 14.458min, the benzoic acid peak is located at the position of 4.958min, the characteristic peak response of the hydroxypropyl tetrahydropyran triol derivative is weaker than that of the 4-methoxybenzoyl chloride derivative, and the proportion deviation of the two peaks is larger.
In experiments, when 4-methoxyphenylacyl chloride, 4-nitrobenzoyl chloride and benzoyl chloride are respectively used for derivatizing hydroxypropyl tetrahydropyran triol, peaks can be obtained under the chromatographic conditions of the invention, but the response of the two peaks is weaker, the deviation between the ratio of the two main peaks and the actual hydroxypropyl tetrahydropyran triol is larger, and the ratio of the main peaks before derivatization can be better reflected by the 4-methoxyphenylacyl chloride, so that the 4-methoxybenzoyl chloride is preferably selected as a derivatizing agent.
Example 4
The method of the invention is compared with other methods:
The existing detection method of the hydroxypropyl tetrahydropyran triol comprises a group standard T/SDSBME 001-2022 raw material hydroxypropyl tetrahydropyran triol for cosmetics, and a high performance liquid chromatography method is adopted according to the fourth section rule 0512 of the Chinese pharmacopoeia 2015. However, no specific detection method is disclosed, according to the method in the document CN112697933a, which requires a XAminde chromatographic column and simultaneously an ELSD detector, the invention performs detection on three batches of samples from different manufacturers, and the samples from each manufacturer are measured according to the corresponding chromatographic conditions, 2 parts of the samples are measured in parallel, and the content is calculated as an average value; peak areas of diastereomers were recorded simultaneously, and the percent peak areas were calculated, with the comparison results shown below.
TABLE 4 results of experiments with different detection methods
The method of CN112697933a in the literature, both column and detector are unconventional and expensive. The derivatization method uses a conventional chromatographic column, the column is stable and durable, the retention time is stable, the repeatability is good, the result is accurate and reliable, and the equipment price is relatively low. The derivatization method disclosed by the invention is simple to operate, short in time, quick in derivatization, and good in accuracy and specificity.
While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and combinations of the methods and applications described herein can be made and applied within the spirit and scope of the invention. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention.
Claims (9)
1. The detection method of the R-/S-hydroxypropyl tetrahydropyran triol is characterized by comprising the following steps of:
(1) Derivatizing hydroxypropyl tetrahydropyran in a sample to be tested by using aromatic acyl chloride;
(2) Detecting the hydroxypropyl tetrahydropyran derivative by using high performance liquid chromatography;
wherein in the step (1), the aromatic acyl chloride is at least one selected from 4-methoxybenzoyl chloride, 4-chlorobenzoyl chloride, 4-bromobenzoyl chloride, 4-aminobenzoyl chloride and 4-nitroformyl chloride;
In the step (2), the high performance liquid chromatography condition is CHIRALP IC columns, the detector is an ultraviolet detector, methanol or isopropanol is used as a mobile phase, isocratic elution is carried out, the flow rate is 0.5-1.5mL/min, the detection wavelength is 200 nm-280 nm, and the column temperature is 30-40 ℃.
2. The method according to claim 1, wherein in the step (1), the mass ratio of the aromatic acid chloride to the sample to be measured is 1 (2-20).
3. The method according to claim 1, wherein in the step (1), the aromatic acid chloride is 4-methoxybenzoyl chloride.
4. The method according to claim 1, wherein in the step (1), the aromatic acid chloride solution is added into the sample solution to be measured, and the mixture is uniformly mixed by vortexing at 2800 rpm for 2min, and the derivatization is performed.
5. The method according to claim 1, wherein in the step (2), the detection wavelength is 240nm to 260nm.
6. The method according to claim 1, wherein in the step (2), the conditions of high performance liquid chromatography are CHIRALP IC columns, the detector is an ultraviolet detector, methanol or isopropanol is used as a mobile phase, isocratic elution is performed, the flow rate is 1.0mL/min, the detection wavelength is 254nm, and the column temperature is 30 ℃.
7. The method according to claim 1, wherein in the step (2), a chiral chromatographic column model is used: CHIRALP IC, brand: large xylonite, size: 4.6 x 250mm, 5 μm.
8. The method according to claim 1, wherein in step (1), naOH is used to catalyze the reaction to neutralize HCl produced by the hydrolysis, so that the sample of the solution to be detected is neutral and has a pH of 5 to 7.
9. The method of detection according to claim 1, comprising the steps of:
(1) Preparing a hydroxypropyl tetrahydropyran standard substance or a test substance into a standard substance solution and a test substance solution with the concentration of 1mg/mL respectively;
(2) Accurately transferring 1.0mL of the prepared standard substance solution and 1.0mL of the prepared sample solution respectively into 7.5mL of plastic centrifuge tubes, adding 1.0mL of 10mg/mL of NaOH solution, and shaking uniformly; adding 2.0mL of 4-methoxybenzoyl chloride solution with the concentration of 5mg/mL, swirling for 2min at 2800 r/min, uniformly mixing, fixing the volume to a scale mark by using ultrapure water, swirling for 15s at 2800 r/min, and filtering by using a 0.22 mu m PTFE filter membrane;
(3) The chiral chromatographic column model is adopted: CHIRALP IC, brand: large xylonite, size: 4.6X250 mm, 5 μm, methanol or isopropanol as mobile phase, and then running for 5min at a flow rate of 1.0mL/min at a detection wavelength of 254nm and column temperature of 30deg.C.
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