CN113702527A - Method for measuring content of isomers of hydroxypropyl tetrahydropyrane triol by high performance liquid chromatography-differential refraction - Google Patents
Method for measuring content of isomers of hydroxypropyl tetrahydropyrane triol by high performance liquid chromatography-differential refraction Download PDFInfo
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- CN113702527A CN113702527A CN202110987123.9A CN202110987123A CN113702527A CN 113702527 A CN113702527 A CN 113702527A CN 202110987123 A CN202110987123 A CN 202110987123A CN 113702527 A CN113702527 A CN 113702527A
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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
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Abstract
The invention belongs to the field of compound analysis, and particularly relates to a method for measuring the content of each isomer of hydroxypropyl tetrahydropyran triol by high performance liquid chromatography-differential refraction. The method is realized by the following steps: firstly, preparing a standard solution and a test solution; and (3) carrying out qualitative and quantitative analysis on the solution by adopting a high performance liquid chromatography-differential refraction method. The detection method provided by the invention has good retention and separation effects on compounds with high polarity and small structural difference, can realize good separation of two isomers of hydroxypropyl tetrahydropyrane triol, improves the detection sensitivity and has low detection limit; the method has high accuracy, good precision and stable chromatographic conditions, can be used as a content determination method of various isomers of the hydroxypropyl tetrahydropyrane triol, and provides an effective detection means for ensuring the quality of the raw material and effectively screening and evaluating the skin care effect of the raw material.
Description
Technical Field
The invention belongs to the field of compound analysis, and particularly relates to a method for measuring the content of each isomer of hydroxypropyl tetrahydropyran triol by high performance liquid chromatography-differential refraction.
Background
Hydroxypropyl tetrahydropyrane triol is the main component of vitreous color factor of cosmetic raw materials, has a content of about 30%, and is included in the catalogue of used cosmetic raw materials (2021 edition). Currently, the highest historical usage in leave-on cosmetics is 10.5%. The hydroxypropyl tetrahydropyrane triol promotes the generation of mucopolysaccharide and glycosaminoglycan in the skin in an breath transfer mode, thereby filling gaps among collagen fibers, achieving the effects of increasing skin elasticity, repairing wrinkles and firming the skin, and being used as an anti-aging component in cosmetics. Currently, hydroxypropyl tetrahydropyrane triol mostly uses D-xylose as a raw material, and is firstly reacted with acetylacetone and then subjected to NaBH4Reducing to obtain a target product. The molecular structure of the compound has a plurality of chiral carbon atoms, which determine that the compound has two diastereomers which are respectively in (beta, S) and (beta, R) configurations. Research shows that the (beta, S) configuration is a dominant configuration, and the biological activity of the dominant configuration is far larger than that of a mixture of the (beta, S) and (beta, R) configurations. Therefore, how to scientifically synthesize the hydroxypropyl tetrahydropyrane triol raw material with higher (beta, S) configuration becomes the direction of efforts of researchers.
The hydroxypropyl tetrahydropyrane triol as the raw material is sold in the market at present, wherein the content of the hydroxypropyl tetrahydropyrane triol is mainly 30%, and the hydroxypropyl tetrahydropyrane triol is sold in a pure product (white powder with the content of more than 90%). The raw materials lack corresponding detection methods, the total content and the content of each configuration are difficult to detect, the disorderly phenomena of disordered use of the raw materials, poor use, difficult achievement of the effect and the like are caused, and the establishment of the corresponding detection methods is urgently needed to strengthen the management of the raw materials and ensure the quality and the effect of the raw materials.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for measuring the content of each isomer of hydroxypropyl tetrahydropyran triol by high performance liquid chromatography-differential refraction, the method can simultaneously measure the content of two isomers, and an effective detection means is provided for ensuring the quality of the raw material and effectively screening and evaluating the skin care effect of the raw material.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a method for measuring the content of each isomer of hydroxypropyl tetrahydropyrane triol by high performance liquid chromatography-differential refraction, which comprises the following steps:
(1) preparation of a standard solution: accurately weighing a hydroxypropyl tetrahydropyrane triol standard substance, placing the hydroxypropyl tetrahydropyrane triol standard substance in a volumetric flask, adding water to dissolve the hydroxypropyl tetrahydropyrane triol standard substance and fixing the volume to a scale, and shaking up to be used as a standard storage solution; accurately transferring the standard stock solution to prepare a standard series solution with the concentration of;
(2) preparing a test solution: accurately weighing a proper amount of a test sample, placing the test sample in a volumetric flask, adding water to dissolve the test sample, fixing the volume to a scale, and shaking up to obtain a test sample solution;
(3) and (3) carrying out qualitative and quantitative analysis on the solution by adopting a high performance liquid chromatography-differential refraction method.
Further, in the step (1), the concentration of the standard substance stock solution is 5 mg/mL; the concentrations of the standard series solutions are 0.5, 1.0, 2.0, 4.0 and 5.0 mg/mL-1。
In the step (2), the preparation of the test solution specifically comprises the following steps: accurately weighing a proper amount of a test sample (equivalent to 100mg of hydroxypropyl tetrahydropyrane triol, accurate to 0.0001 g) and placing the test sample in a 25 mL volumetric flask, adding water to dissolve the test sample, fixing the volume to the scale, and shaking up to obtain a test sample solution.
Further, in the step (3), the specific parameters of the high performance liquid chromatography-differential refraction method are as follows: a chromatographic column: CAPCELL PAK ADME C184.6X 250 mm, 5 μm; column temperature: 35 ℃; mobile phase: water (100%); flow rate: 1.0 mL/min-1(ii) a Sample introduction amount: 10 mu L of the solution; a detector: a differential refractive detector.
The invention has the beneficial effects that:
(1) the detection method provided by the invention has good retention and separation effects on compounds with high polarity and small structural difference, can realize good separation of two isomers of hydroxypropyl tetrahydropyrane triol, improves the detection sensitivity and has low detection limit;
(2) the detection method provided by the invention is verified in terms of system adaptability, specificity, precision, stability, reproducibility, accuracy, detection limit, quantitative limit and the like, meets the requirements, is high in accuracy, good in precision and stable in chromatographic conditions, can be used as a content determination method for various isomers of hydroxypropyl tetrahydropyran triol, and provides an effective detection means for guaranteeing the quality of raw materials and effectively screening and evaluating the skin care effect of the raw materials.
Drawings
FIG. 1 is an adaptation map of the hydroxypropyl tetrahydropyran triol system;
wherein 1 is in the (β, S) configuration; 2 is in the (beta, R) configuration.
FIG. 2 is a blank solvent chromatogram.
FIG. 3 is a map of a sample to be tested with a designation specification of 30%;
wherein 1 is in the (β, S) configuration; 2 is in the (beta, R) configuration.
FIG. 4 is a chart of a sample to be tested with a label specification greater than 90%.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
Instruments and materials
1. Instrument for measuring the position of a moving object
Waters e2695 high performance liquid chromatograph (wawter corporation, usa); waters 2414 shows a differential refractive detector (Watts, USA); sartorius CP225D electronic balance (Sartorius scientific instruments ltd); KQ-500DE model digital control ultrasonic cleaner (ultrasonic instruments, Inc. of Kunshan).
2. Reagents and materials
Methanol, chromatographically pure, batch No. 195912, manufacturer Fisher Scientific; acetonitrile, chromatographically pure, batch No. U5NA1H, manufacturer Honeywell; the water is first-grade water specified in GB/T6682.
The standard was purchased from Target molecular corp. (Target Mol) in the united states, lot number 146522, identifying a purity of 99.63%.
Samples from 5 feedstock manufacturing plants were 6 batches, 3 of which were liquid at a content of about 30% and 3 of which were white powder at a content of more than 90%.
3. Chromatographic conditions
A chromatographic column: CAPCELL PAK ADME C18, 4.6X 250 mm, 5 μm; column temperature: 35 ℃; mobile phase: water (100%); flow rate: 1.0 mL min-1; sample introduction amount: 10 μ L. A detector: a differential refractive detector.
Example 1
(1) Preparation of Standard solutions
Precisely weighing 50mg of a vitreous chromogen standard substance, placing the vitreous chromogen standard substance in a 10 mL volumetric flask, adding water to dissolve the vitreous chromogen standard substance and fixing the volume to a scale, and shaking up to be used as a standard stock solution; accurately transferring the standard stock solution to prepare standard series solutions with the concentrations of 0.5, 1.0, 2.0, 4.0 and 5.0 mg.mL < -1 >.
(2) Preparation of test solution
Accurately weighing a proper amount of a test sample (equivalent to 100mg of hydroxypropyl tetrahydropyrane triol, accurate to 0.0001 g) and placing the test sample in a 25 mL volumetric flask, adding water to dissolve the test sample, fixing the volume to the scale, and shaking up to obtain a test sample solution.
(3) System suitability test
The standard solution (4 mg. mL) was precisely aspirated-1) 10 μ L, injected according to the above chromatographic conditions, and the chromatogram recorded, the result is shown in FIG. 1. As can be seen from fig. 1, the (β, S) and (β, R) configurations were well separated, the degree of separation was greater than 1.5, and the chromatographic conditions satisfied the assay requirements.
Effect verification
(1) specificity
Sampling blank solvent 10 μ L, injecting sample according to the above chromatographic conditions, recording chromatogram, and finding the result shown in FIG. 2. The result shows that the blank solvent has no interference at the peak emergence time of the component to be detected.
(II) Linear and Linear Range
The standard series solutions prepared in example 1 were sampled according to the above chromatographic conditions, and chromatograms were recorded. The standard curve is drawn by taking the concentration of the standard series solution as the abscissa and the sum of the peak areas of the (beta, S) and (beta, R) configurations as the ordinate, and the result is shown in Table 1, wherein the linear relationship between the boscalid and the isomer is good in each range.
TABLE 1
(III) precision test
The standard solution (4 mg. mL) was precisely aspirated-1) 10 mu L of the obtained product is continuously injected for 6 times according to the chromatographic conditions, and a chromatogram is recorded. RSD of the peak areas of the (beta, S) and (beta, R) configuration peaks are respectively calculated to be 0.20% and 0.21%, which indicates that the precision of the instrument is good.
TABLE 2
(IV) repeatability test
The same sample was sampled and measured by taking 6 parts of the sample solution prepared in example 1. The RSD of the content (%) of the component to be detected is calculated, which shows that the method has good repeatability.
TABLE 3
(V) stability test
Taking a standard solution (4 mg. mL)-1) Respectively injecting 10 mu L of sample after being placed for 0, 2, 4, 8, 12 and 24 hours, and respectively injecting and measuring according to the chromatographic conditions, wherein RSD of (beta, S) and (beta, R) configuration peak areas is respectively calculated to be 0.20 percent and 0.38 percent, which shows that the stability of the component to be measured is good within 24 hours.
TABLE 4
(VI) accuracy test
Accurately weighing a proper amount of a test sample (equivalent to 50mg of hydroxypropyl tetrahydropyrane triol, accurate to 0.0001 g) and placing the test sample into a 25 mL volumetric flask, weighing 9 parts in parallel, performing high, medium and low level labeling tests, preparing a labeled test sample solution according to a preparation method of the test sample solution, measuring, and calculating the recovery rate (%). The results are shown in table 5, the recovery rate of the component to be detected is between 99.3% and 99.6%, and the RSD (n = 3) is between 0.40% and 0.76%, so that the determination requirements of the component to be detected are met.
TABLE 5
(VII) detection limit and quantification limit
Taking a standard solution (0.5 mg. mL)-1) A detected concentration meter quantitative concentration measurement sample is prepared by dilution, and is subjected to sample injection measurement according to a formulated method, wherein the signal-to-noise ratio S/N =3 is taken as a detected concentration, and S/N =10 is taken as a quantitative concentration. The detection limit of the method was calculated to be 0.13% and the quantitation limit was calculated to be 0.42% based on 0.3g of sample size.
(eight) sample measurement
Taking a sample, preparing a sample solution according to the method of example 1, injecting samples according to the chromatographic conditions for determination, calculating the content of the hydroxypropyl tetrahydropyrane triol in the sample by a standard curve method, calculating the proportion of the (beta, S) and (beta, R) configurations in the sample by a peak area normalization method, and obtaining the result shown in Table 6 and typical sample maps shown in figures 3-4.
TABLE 6
Calculating formula 1 Total Peak area
A =A(β,S)+ A(β,R)
In the formula: the sum of the peak areas of two isomers of A- (beta, S) configuration and (beta, R) configuration;
peak area of A (beta, S) -isomer of (beta, S) configuration;
peak area of A (. beta., R) - (. beta., R) configurational isomer.
Calculating formula 2: total content of
In the formula: x-total content of the component to be tested,%;
m-sample size, g;
the sum of the peak areas of two isomers of A- (beta, S) configuration and (beta, R) configuration;
v is volume of constant volume, mL;
a-standard curve slope;
b-Standard Curve intercept.
Calculating formula 3: content of each isomer
X(β,S)=X·A(β,S)/A
X(β,R)=X·A(β,R)/A
In the formula: the content of X (β, S) -isomer of (β, S) configuration,%;
content of X (β, R) — (β, R) configurational isomer,%;
x-total content of the component to be tested,%;
peak area of A (beta, S) -isomer of (beta, S) configuration;
peak area of A (. beta., R) - (beta, R) -configurational isomer;
the sum of the peak areas of two isomers of A- (beta, S) configuration and (beta, R) configuration.
Comparative example 1
(1) - (2) preparation of the standard solution and the test solution in the same manner as in example 1;
(3) a chromatographic column: NH (NH)2Columns (4.6X 250 mm, 5 μm); column temperature: 35 ℃; mobile phase: water (100%); flow rate: 1.0 mL/min-1(ii) a Sample introduction amount: 10 μ L. A detector: a differential refractive detector. Two diastereomers of (beta, S) and (beta, R) are synthesized into a chromatographic peak, and complete separation of the two isomers cannot be realized.
Comparative example 2
(1) - (2) preparation of the standard solution and the test solution in the same manner as in example 1;
(3) a chromatographic column: c18Columns (4.6X 250 mm, 5 μm); column temperature: 35 ℃; mobile phase: water (100%); flow rate: 1.0 mL/min-1(ii) a Sample introduction amount: 10 μ L. A detector: a differential refractive detector. Hydroxypropyl tetrahydropyran triol does not remain and is therefore notSuitable for the analysis of the compound.
Comparative example 3
(1) - (2) preparation of the standard solution and the test solution in the same manner as in example 1;
(3) a chromatographic column: CAPCELL PAK ADME C184.6X 250 mm, 5 μm; column temperature: 35 ℃; mobile phase as shown in mobile phase 2 or mobile phase 3; flow rate: 1.0 mL/min-1(ii) a Sample introduction amount: 10 μ L. A detector: a differential refractive detector.
Mobile phase 2: water-acetonitrile (95: 5, v/v) flow rate of 1.0 mL min-1Under the condition, the peak time of two diastereomers of (beta, S) and (beta, R) is advanced, and the diastereomers can not be separated effectively.
Mobile phase 3: water-methanol (95: 5, v/v) at a flow rate of 1.0 mL min-1Under the condition, the peak time of two diastereomers of (beta, S) and (beta, R) is advanced, and the diastereomers can not be separated effectively.
The invention respectively considers water, water-acetonitrile (95: 5, v/v) and water-methanol (95: 5, v/v) as mobile phases, and optimally selects a proper mobile phase system by taking the goal that two isomers can be completely separated. As a result, it was found that the addition of the organic phase to the mobile phase resulted in too short retention time of individual isomers of hydroxypropyl tetrahydropyran triol and an inefficient separation. The experimental result shows that the retention time is prolonged and the good separation of the isomer to be detected is realized by taking 100 percent pure water as the mobile phase.
The invention establishes an HPLC-differential refractive index content determination method of each isomer of the cosmetic raw material hydroxypropyl tetrahydropyrane triol, and optimizes a sample pretreatment method, chromatographic conditions and the like. And the method is verified methodologically in terms of system adaptability, specificity, precision, stability, reproducibility, accuracy, detection limit, quantification limit and the like of the method according to the regulations of 'inspection technical specifications of forbidden substances and limited substances in cosmetics' (No. 2010) and meets the requirements.
The method has high accuracy, good precision and stable chromatographic conditions, can be used as a content determination method of various isomers of the hydroxypropyl tetrahydropyrane triol, and provides reference for the quality standard research of the raw material.
Claims (4)
1. A method for measuring the content of each isomer of hydroxypropyl tetrahydropyrane triol by high performance liquid chromatography-differential refraction is characterized by comprising the following steps:
(1) preparation of a standard solution: accurately weighing a hydroxypropyl tetrahydropyrane triol standard substance, placing the hydroxypropyl tetrahydropyrane triol standard substance in a volumetric flask, adding water to dissolve the hydroxypropyl tetrahydropyrane triol standard substance and fixing the volume to a scale, and shaking up to be used as a standard storage solution; accurately transferring the standard stock solution to prepare a standard series solution with the concentration of;
(2) preparing a test solution: accurately weighing a proper amount of a test sample, placing the test sample in a volumetric flask, adding water to dissolve the test sample, fixing the volume to a scale, and shaking up to obtain a test sample solution;
(3) and (3) carrying out qualitative and quantitative analysis on the solution by adopting a high performance liquid chromatography-differential refraction method.
2. The method according to claim 1, wherein in step (1), the concentration of the standard stock solution is 5 mg/mL; the concentrations of the standard series solutions are 0.5, 1.0, 2.0, 4.0 and 5.0 mg/mL-1。
3. The method according to claim 1, wherein in the step (2), the sample solution is prepared by the specific process of: accurately weighing a proper amount of a test sample (equivalent to 100mg of hydroxypropyl tetrahydropyrane triol, accurate to 0.0001 g) and placing the test sample in a 25 mL volumetric flask, adding water to dissolve the test sample, fixing the volume to the scale, and shaking up to obtain a test sample solution.
4. The method according to any one of claims 1 to 3, wherein in step (3), the specific parameters of the HPLC-differential refractometry are: a chromatographic column: CAPCELL PAK ADME C184.6X 250 mm, 5 μm; column temperature: 35 ℃; mobile phase: water (100%); flow rate: 1.0 mL/min-1(ii) a Sample introduction amount: 10 mu L of the solution; a detector: a differential refractive detector.
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| CN115097052A (en) * | 2022-08-24 | 2022-09-23 | 山东君泰药业有限公司济南分公司 | Method for determining diastereoisomer ratio of hydroxypropyl tetrahydropyrane triol |
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| CN115097052A (en) * | 2022-08-24 | 2022-09-23 | 山东君泰药业有限公司济南分公司 | Method for determining diastereoisomer ratio of hydroxypropyl tetrahydropyrane triol |
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