CN113341029A - Method for detecting content of gamma-aminobutyric acid in cosmetics - Google Patents
Method for detecting content of gamma-aminobutyric acid in cosmetics Download PDFInfo
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Abstract
The invention discloses a method for detecting gamma-aminobutyric acid in cosmetics, which comprises the following steps: preparation of control: dissolving a gamma-aminobutyric acid reference substance by using hydrochloric acid, and filtering to obtain a gamma-aminobutyric acid reference substance solution; preparing a test sample: dissolving a cosmetic sample by using hydrochloric acid, and filtering to obtain a sample solution; preparation of a blank solution: filtering hydrochloric acid to obtain a blank solution; and (3) determination: and detecting the reference solution, the test solution and the blank solution by using a separation column and a post-column derivatization reaction column, thereby obtaining the content of the gamma-aminobutyric acid in the cosmetic test sample. The detection method is simple to operate, sensitive in response, high in accuracy and good in repeatability, and can be used for detecting the content of gamma-aminobutyric acid in cosmetics.
Description
Technical Field
The invention relates to the technical field of gamma-aminobutyric acid detection, and particularly relates to a method for detecting the content of gamma-aminobutyric acid in cosmetics.
Background
Gamma-aminobutyric acid (GABA) also known as aminobutyric acid, 4-aminobutyric acid, pipecolic acid, molecular formula C4H9NO2Molecular weight 103.1. Gamma-aminobutyric acid is an inhibitory neurotransmitter of the central nervous system of mammals, is widely distributed in prokaryotes and eukaryotes, and has been proved to have the effects of relieving anxiety, reducing blood pressure, improving sleep, removing wrinkles and whitening. A series of products are developed at home and abroad by utilizing the property of the gamma-aminobutyric acid, so that people who love beauty can hope, but the discomfort symptoms such as prickling, numbness, itching, burning and the like can be caused when the gamma-aminobutyric acid is contacted with the skin when the concentration of the gamma-aminobutyric acid reaches the wrinkle removing concentration or the better wrinkle removing concentration is expected to be reached, and the side effect has positive correlation with the concentration. In order to make gamma-aminobutyric acid have wider application in the field of skin care, the proper dosage of gamma-aminobutyric acid needs to be ensured.
Because gamma-aminobutyric acid has no ultraviolet absorption, the content of the gamma-aminobutyric acid is detected by using a high performance liquid chromatography, and the gamma-aminobutyric acid needs to react with a derivatization reagent firstly to generate a compound with ultraviolet absorption, but the pre-column derivatization reaction is easily interfered by the outside to form multi-stage derivatization, so that the detection result has poor repeatability and low accuracy after the separation of a chromatographic column, and the detection of the content of the gamma-aminobutyric acid is influenced. The post-column derivatization cation exchange chromatography is to separate gamma-aminobutyric acid by a cation exchange chromatography column and then perform derivatization treatment, and has the advantages of high reaction speed, good stability of a derivatization product and the like. When a common mobile phase and elution procedure for detecting and separating amino acid by an amino acid analyzer is used for detecting gamma-aminobutyric acid, the peak-off time of the gamma-aminobutyric acid is about 1.5min, the gamma-aminobutyric acid is easily influenced by a solvent peak, the peak area is small, and the detection accuracy of the gamma-aminobutyric acid is easily influenced by baseline fluctuation.
Disclosure of Invention
As described above, in order to solve the above technical problems, the present invention provides a method for detecting γ -aminobutyric acid in cosmetics, which can greatly improve the detection sensitivity while ensuring the accuracy of the detection result, and has the advantages of simple sample treatment and convenient operation.
The specific technical scheme of the invention is as follows:
1. a method for detecting gamma-aminobutyric acid in cosmetics comprises the following steps:
preparation of control: dissolving a gamma-aminobutyric acid reference substance by using hydrochloric acid, and filtering to obtain a gamma-aminobutyric acid reference substance solution;
preparing a test sample: dissolving a cosmetic sample by using hydrochloric acid, and filtering to obtain a sample solution;
preparation of a blank solution: filtering hydrochloric acid to obtain a blank solution;
and (3) determination: and detecting the reference solution, the test solution and the blank solution by using a separation column and a post-column derivatization reaction column, thereby obtaining the content of the gamma-aminobutyric acid in the cosmetic test sample.
2. The detection method according to item 1, wherein the concentration of the hydrochloric acid is 0.02mol/L to 0.06 mol/L.
3. The detection method according to item 1 or 2, wherein the dissolution is water bath dissolution, preferably, the dissolution time is 4 to 6 hours, and further preferably, the dissolution temperature is 60 to 80 ℃.
4. The detection method according to any one of claims 1 to 3, wherein the cosmetic is a cream-milky or liquid cosmetic, preferably, the cream-milky cosmetic is an emulsion, an eye cream or a facial cleanser; preferably, the liquid cosmetic is essential oil, toner or shower gel.
5. The detection method according to any one of claims 1 to 4, wherein the separation column is a cation exchange resin column, preferably a strong acid type cation exchange resin column;
preferably, the column temperature of the separation column is 50 to 57 ℃, preferably 55 ℃.
6. The detecting method according to any one of items 1 to 5, wherein the derivatizing agent used in the derivatizing reaction is o-phthalaldehyde, and preferably, the detection is performed by detecting the absorbance at a wavelength of 330-350 nm.
7. The detection method according to any one of items 1 to 6, wherein the post-column derivatization reaction column is a silica sand column, preferably, the column temperature is 30 to 50 ℃, and more preferably, 35 ℃.
8. The detection method according to any one of claims 1 to 7, wherein the mobile phase is one or more selected from the group consisting of a citric acid buffer solution, a trifluoroacetic acid solution, a sodium acetate solution, a mixture of sodium citrate and benzyl alcohol, and a mixture of ethanol and sodium hydroxide, and preferably, the post-column derivatized buffer solution is one or two or three selected from the group consisting of a methanol solution of o-phthalaldehyde, a mixture of mercaptoethanol and a boric acid buffer, and an ethanol solution.
9. The detection method according to item 8, wherein gradient elution is performed by the following procedure:
firstly, a citric acid buffer solution is used as a mobile phase, and a post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
then, using trifluoroacetic acid solution as a mobile phase, wherein the post-column derivatization buffer solution is a mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, using sodium acetate solution as a mobile phase, wherein the post-column derivatization buffer solution is a mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses a sodium acetate solution and a mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses the mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is the mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses the mixed solution of ethanol and sodium hydroxide, and the post-column derivatization buffer solution is ethanol solution; and
finally, citric acid buffer solution is used as the mobile phase, and ethanol solution is used as the post-column derivatization buffer solution.
10. The detection method according to any one of claims 1 to 9, wherein the amount of the sample is 10 to 20. mu.L, preferably 15 to 20. mu.L, and more preferably 20. mu.L.
11. The detection method according to any one of items 1 to 10, wherein the flow rate of the mobile phase is 0.35 to 0.55mL/min, and preferably, the flow rate of the post-column derivatized buffer solution is 0.2 to 0.4 mL/min.
12. The method according to any one of items 1 to 11, wherein the content of γ -aminobutyric acid in the cosmetic is measured using an external standard method.
13. The method according to any one of items 1 to 12, wherein the detection limit of gamma-aminobutyric acid is 3X 10 or more-4ppm。
ADVANTAGEOUS EFFECTS OF INVENTION
The method for detecting the content of the gamma-aminobutyric acid in the cosmetics is simple to operate, sensitive in response, high in accuracy and good in repeatability, and can be used for detecting the content of the gamma-aminobutyric acid in the cosmetics.
According to the method, 0.02mol/L hydrochloric acid is heated in a water bath kettle at 60 ℃ for 4 hours to extract the gamma-aminobutyric acid in the cosmetics, the method can fully extract the gamma-aminobutyric acid in the cosmetics, a test solution can be obtained after 0.22 mu m filtration, the sample treatment is simple, the operation is convenient, and the loss of the gamma-aminobutyric acid in the sample is reduced. The separation column can greatly improve the separation effect of the gamma-aminobutyric acid, avoid the diffusion and overlapping phenomena of sample peaks, ensure that the repeatability of the peak shape of the gamma-aminobutyric acid is good, and simultaneously improve the accuracy of the detection of the gamma-aminobutyric acid. Ortho-phthalaldehyde as post-column derivatization reagentThe detection limit is reduced to 10-4ppm, can greatly improve the detection sensitivity and accuracy.
Drawings
FIG. 1 is a chromatogram of a control sample obtained in example 1-1.
FIG. 2 is a chromatogram of a blank in example 1-1.
FIG. 3 is a chromatogram of the test sample in example 1-1.
Detailed Description
The present invention is described in detail in the following description of embodiments with reference to the figures, in which like numbers represent like features throughout the figures. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
The cosmetic is added with the gamma-aminobutyric acid, so that tight muscle nerve tissues can be relaxed, the skin can be quickly penetrated, fine lines can be reduced, the relaxation function of muscles can be enhanced, and the effect of quickly removing wrinkles can be achieved.
Therefore, the invention provides a method for detecting gamma-aminobutyric acid in cosmetics, which comprises the following steps:
preparation of control: dissolving a gamma-aminobutyric acid reference substance by using hydrochloric acid, and filtering to obtain a gamma-aminobutyric acid reference substance solution;
preparing a test sample: dissolving a cosmetic sample by using hydrochloric acid, and filtering to obtain a sample solution;
preparation of a blank solution: filtering hydrochloric acid to obtain a blank solution;
and (3) determination: and detecting the reference solution, the test solution and the blank solution by using a separation column and a post-column derivatization reaction column so as to obtain the content of the gamma-aminobutyric acid in the cosmetic test sample.
In one embodiment, the concentration of the hydrochloric acid is from 0.02mol/L to 0.06mol/L, preferably 0.02 mol/L.
For example, the hydrochloric acid may have a concentration of 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L, 0.06mol/L, or the like.
The hydrochloric acid is prepared by diluting concentrated hydrochloric acid, for example, when the concentration of the prepared hydrochloric acid is 0.02mol/L, the preparation method comprises the following steps: 1.67mL of concentrated HCl was removed, added to purified water and made up to 1000 mL.
In one embodiment, the dissolution is a water bath dissolution during the preparation of the test article, preferably, the dissolution time is 4 to 6 hours, and further preferably, the dissolution temperature is 60 to 80 ℃.
For example, the dissolution time may be 4 hours, 5 hours, or 6 hours, and the dissolution temperature may be 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, or the like.
According to the invention, the hydrochloric acid is used for dissolving the cosmetics, so that the gamma-aminobutyric acid in the cosmetics can be effectively extracted, the sample treatment is simple, the operation is convenient, and the loss of the gamma-aminobutyric acid in the sample is reduced.
In one embodiment, the cosmetic is a cream-milky or liquid cosmetic, preferably, the cream-milky cosmetic is an emulsion, an eye cream, or a facial cleanser; preferably, the liquid cosmetic is essential oil, toner or shower gel.
In one embodiment, the separation column is a cation exchange resin column, preferably a strong acid type cation exchange resin column, preferably a macroporous strong acid styrene type cation exchange resin, further preferably a macroporous strong acid styrene type cation exchange resin containing benzenesulfonic acid groups, further preferably the packing particle size of the resin column is 5 μm; preferably, the column temperature of the separation column is 50 to 57 ℃, preferably 55 ℃.
The separation column can greatly improve the separation effect of the gamma-aminobutyric acid, avoid the diffusion and overlapping phenomena of sample peaks, ensure that the repeatability of the peak shape of the gamma-aminobutyric acid is good, and simultaneously improve the accuracy of the detection of the gamma-aminobutyric acid.
For example, the column temperature of the separation column may be 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃ or the like.
In one embodiment, the derivatization reagent used in the derivatization reaction is o-phthalaldehyde, and preferably, the detection is performed by detecting the absorbance at a wavelength of 330-.
For example, the detection wavelength may be 330nm, 335nm, 340nm, 345nm, 350nm, and the like.
In one embodiment, the post-column derivatization reaction column is a silica sand column, preferably, the column temperature is 30 to 50 ℃, and more preferably 35 ℃.
The post-column derivatization refers to a technique for converting a compound into a substance having a similar chemical structure by chemical transformation, and has the effect of converting a substance which is difficult to analyze into a substance having a similar chemical structure but easy to analyze, thereby facilitating quantification and separation. Chemical derivatization of amino acid analyzers refers to the chemical reaction of certain reagents (known as chemical derivatization reagents or labeling reagents) with sample components under certain conditions, the reaction products being useful for chromatographic detection.
In one embodiment, the mobile phase is selected from one or more of citric acid buffer solution, trifluoroacetic acid solution, sodium acetate solution, a mixture of citric acid and benzyl alcohol, and a mixture of ethanol and sodium hydroxide, and preferably, the post-column derivatization buffer solution is one or two or three of o-phthalaldehyde methanol solution, a mixture of mercaptoethanol and boric acid buffer solution, and an ethanol solution.
The citric acid buffer solution refers to a mixed solution of citric acid and ethanol solution, preferably a mixed solution of 20 wt% of citric acid and 11 wt% of ethanol solution;
the trifluoroacetic acid solution refers to a 2 wt% trifluoroacetic acid solution;
the sodium acetate solution refers to a 2.5 wt% sodium acetate solution;
the mixed solution of citric acid and benzyl alcohol refers to a mixed solution of 6 wt% of citric acid and 0.5 wt% of benzyl alcohol;
the mixed solution of ethanol and sodium hydroxide refers to a mixed solution of 79 wt% of ethanol and 0.8 wt% of sodium hydroxide.
The methanol solution of the o-phthalaldehyde refers to a methanol solution of the o-phthalaldehyde with the concentration of 2mg/mL, and each 1mL of the methanol solution contains 2mg of the o-phthalaldehyde;
the mixed solution of mercaptoethanol and a boric acid buffer refers to a mixed solution of 2mmol/L mercaptoethanol and 0.4mol/L boric acid buffer (pH 10-12);
the ethanol solution refers to a 50 wt% ethanol solution.
The post-column derivatization refers to derivatization performed by mixing a test solution with a derivatization reagent in the presence of a buffer.
In one embodiment, a gradient elution is performed, the elution procedure being:
firstly, a citric acid buffer solution is used as a mobile phase, and a post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
then, using trifluoroacetic acid solution as a mobile phase, wherein the post-column derivatization buffer solution is a mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, using sodium acetate solution as a mobile phase, wherein the post-column derivatization buffer solution is a mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses a sodium acetate solution and a mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses the mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is the mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses the mixed solution of ethanol and sodium hydroxide, and the post-column derivatization buffer solution is ethanol solution; and
finally, citric acid buffer solution is used as the mobile phase, and ethanol solution is used as the post-column derivatization buffer solution.
In one embodiment, the elution procedure is:
when the time is 0min, a citric acid buffer solution is used as a mobile phase, and a post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
when the time is 0-3.5min, the mobile phase uses trifluoroacetic acid solution, and the post-column derivatization buffer solution is the mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
3.5-6.5min, using sodium acetate solution as mobile phase, and using o-phthalaldehyde methanol solution and mixed solution of mercaptoethanol and boric acid buffer solution as post-column derivatization buffer solution;
when the time is 6.5-12.8min, the mobile phase uses a sodium acetate solution and a mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
when 12.8-12.9min, the mobile phase uses the mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is the mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
when 12.9-21.0min, the mobile phase uses the mixed solution of ethanol and sodium hydroxide, and the post-column derivatization buffer solution is ethanol solution; and
at 21.0-40.0min, citric acid buffer solution is used as mobile phase, and ethanol solution is used as post-column derivatization buffer solution.
Preferably, at 0min, the mobile phase uses a sodium citrate buffer solution, the citric acid buffer solution is a 20 wt% citric acid and 11 wt% ethanol solution, the buffer solution after the post-column derivatization is a mixed solution of a phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution, the phthalaldehyde methanol solution is a 2mg/mL phthalaldehyde methanol solution, the mixed solution of the mercaptoethanol and the boric acid buffer solution refers to 2mmol/L mercaptoethanol +0.4mol/L boric acid buffer solution (pH is 10), and the volume ratios of the phthalaldehyde methanol solution and the mixed solution of the mercaptoethanol and the boric acid buffer solution are the same and are both 50%;
when the time is 0-3.5min, 2 wt% of trifluoroacetic acid solution is used as a mobile phase, the buffer solution for post-column derivatization is a mixed solution of an o-phthalaldehyde methanol solution and mercaptoethanol and a boric acid buffer solution, the o-phthalaldehyde methanol solution is 2mg/mL of the o-phthalaldehyde methanol solution, the mixed solution of the mercaptoethanol and the boric acid buffer solution refers to 2mmol/L of mercaptoethanol and 0.4mol/L of the boric acid buffer solution (pH is 10), and the volume ratios of the o-phthalaldehyde methanol solution to the mixed solution of the mercaptoethanol and the boric acid buffer solution are the same and are both 50%;
when the time is 3.5-6.5min, 2.5 wt% of sodium acetate solution is used as a mobile phase, the buffer solution for post-column derivatization is a mixed solution of phthalic dicarboxaldehyde methanol solution and mercaptoethanol and boric acid buffer solution, the phthalic dicarboxaldehyde methanol solution is 2mg/mL of phthalic dicarboxaldehyde methanol solution, the mixed solution of mercaptoethanol and boric acid buffer solution refers to 2mmol/L of mercaptoethanol and 0.4mol/L of boric acid buffer solution (pH is 10), and the volume ratios of the phthalic dicarboxaldehyde methanol solution to the mixed solution of mercaptoethanol and boric acid buffer solution are the same and are both 50%;
at 6.5-12.8min, the mobile phase uses sodium acetate solution and the mixed solution of citric acid and benzyl alcohol, wherein the sodium acetate solution is 2.5 wt% sodium acetate solution, the mixed solution of citric acid and benzyl alcohol refers to the mixed solution of 6 wt% citric acid and 0.5 wt% benzyl alcohol,
the buffer solution for post-column derivatization is a mixed solution of an o-phthalaldehyde methanol solution and mercaptoethanol and a boric acid buffer solution, the o-phthalaldehyde methanol solution is a 2mg/mL o-phthalaldehyde methanol solution, the mixed solution of mercaptoethanol and the boric acid buffer solution refers to 2mmol/L mercaptoethanol and 0.4mol/L boric acid buffer solution (pH is 10), and the volume ratios of the o-phthalaldehyde methanol solution to the mixed solution of mercaptoethanol and the boric acid buffer solution are the same and are both 50%;
wherein, at 6.5-6.6min, the mobile phase is a mixture of 23% sodium acetate solution and 77% citric acid and benzyl alcohol, and at 6.6-12.8min, the mobile phase is a mixture of 2% sodium acetate solution and 98% citric acid and benzyl alcohol;
at 12.8-12.9min, the mobile phase uses the mixture of citric acid and benzyl alcohol, the mixture of citric acid and benzyl alcohol refers to the mixture of 6 wt% citric acid and 0.5 wt% benzyl alcohol,
the buffer solution for post-column derivatization is a mixed solution of an o-phthalaldehyde methanol solution and mercaptoethanol and a boric acid buffer solution, the o-phthalaldehyde methanol solution is a 2mg/mL o-phthalaldehyde methanol solution, the mixed solution of mercaptoethanol and the boric acid buffer solution refers to 2mmol/L mercaptoethanol and 0.4mol/L boric acid buffer solution (pH is 10), and the volume ratios of the o-phthalaldehyde methanol solution to the mixed solution of mercaptoethanol and the boric acid buffer solution are the same and are both 50%;
when 12.9-21.0min, the mobile phase uses the mixture of ethanol and sodium hydroxide, the mixture of ethanol and sodium hydroxide refers to 79 wt% ethanol solution and 0.8 wt% sodium hydroxide solution;
the buffer solution for post-column derivatization is an ethanol solution, and the ethanol solution is a 50 wt% ethanol solution;
at 21.0-40.0min, citric acid buffer solution is used as the mobile phase, ethanol solution is used as the post-column derivatization buffer solution, the sodium citrate buffer solution is 20 wt% citric acid and 11 wt% ethanol solution, and the ethanol solution is 50 wt% ethanol solution.
In one embodiment, the sample amount is 10 to 20. mu.L, preferably 15 to 20. mu.L, and more preferably 20. mu.L.
For example, the amount of sample may be 10. mu.L, 11. mu.L, 12. mu.L, 13. mu.L, 14. mu.L, 15. mu.L, 16. mu.L, 17. mu.L, 18. mu.L, 19. mu.L, 20. mu.L, or the like.
In one embodiment, the flow rate of the mobile phase is 0.35 to 0.55mL/min, and preferably, the flow rate of the post-column derivatized buffer solution is 0.2 to 0.4 mL/min.
For example, the flow rate of the mobile phase can be 0.35mL/min, 0.40mL/min, 0.45mL/min, 0.50mL/min, 0.55mL/min, and the like;
the flow rate of the post-column derivatized buffer solution can be 0.20mL/min, 0.25mL/min, 0.30mL/min, 0.35mL/min, 0.40mL/min, and the like.
In one embodiment, the content of gamma-aminobutyric acid in the cosmetic is determined by an external standard method.
The external standard method is a quantitative method which adds a certain amount of standard substance into a blank test material to prepare a reference sample, processes and detects the sample in parallel with an unknown test material, and calculates the concentration of a component to be detected in the unknown test material according to the functional relationship between the response value of the reference sample and the concentration of the added standard substance. The external standard method adopted in the invention is not limited, and a standard curve method can be adopted.
The detection method can more accurately detect the content of the gamma-aminobutyric acid, and has good repeatability and high accuracy.
In one embodiment, the gamma-aminobutyric acid is detected with a precision of 3X 10 or more-4ppm。
The detection method is adopted, the gamma-aminobutyric acid in the cosmetics is extracted by using the hydrochloric acid water bath, the operation is simple and convenient, the loss of the gamma-aminobutyric acid in the sample is reduced, and the detection limit can be reduced by 10 by using the chromatographic condition-4ppm, greatly improving the detection sensitivity and accuracy.
Examples
The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Examples 1 to 1
1. Instrument reagent
LA8080 amino acid analyzer analytical balance: mettlerlatidol AL104
Ultrapure water, hydrochloric acid (analytically pure) gamma-aminobutyric acid reference substance (99.9%)
2. Chromatographic conditions
Separation column packed cation exchange resin column (4.4 mm. times.60 mm, 5 μm type: 852-8534)
Reaction column: filling quartz sand (4.6mm x 40mm, model: 8L03600)
The separation flow rate is 0.45ml/min
Reaction flow rate: 0.3ml/min
Temperature of the separation column: 55 ℃;
temperature of the reaction column: 35 ℃;
sample introduction amount: 20 mu L of the solution;
detection wavelength: 336nm
Gradient elution procedure:
| time/min | A％ | B％ | C％ | D％ | E％ | R1％ | R2％ | R3％ |
| 0.0 | 100 | -- | -- | -- | -- | 50 | 50 | -- |
| 3.5 | -- | 100 | -- | -- | -- | 50 | 50 | -- |
| 3.6 | -- | -- | 100 | -- | -- | 50 | 50 | -- |
| 6.5 | -- | -- | 100 | -- | -- | 50 | 50 | -- |
| 6.6 | -- | -- | 23 | 77 | -- | 50 | 50 | -- |
| 12.8 | -- | -- | 2 | 98 | -- | 50 | 50 | -- |
| 12.9 | -- | -- | -- | 100 | -- | 50 | 50 | -- |
| 21.0 | -- | -- | -- | -- | 100 | -- | -- | 100 |
| 32.1 | 100 | -- | -- | -- | -- | -- | 100 | |
| 40.0 | 100 | -- | -- | -- | -- | 100 |
Wherein, the mobile phase: a: 20 wt% of citric acid, 11 wt% of ethanol solution, B: trifluoroacetic acid 2 wt%, C: sodium acetate solution 2.5 wt%, D: citric acid 6 wt%, 0.5 wt% benzyl alcohol, E: 79 wt% of ethanol solution and 0.8 wt% of sodium hydroxide.
Buffer for derivatization reaction: r1: 2mg/mL of an o-phthalaldehyde methanol solution; r2: 2mmol/L mercaptoethanol +0.4mol/L boric acid buffer (pH 10); r3 is 50% ethanol solution.
The content was determined by calculation using the following formula:
the content of the gamma-aminobutyric acid in the test sample is calculated by the formula:
note: pi- - -the content of gamma-aminobutyric acid in the sample, mg/g
Cr- -content of gamma-aminobutyric acid in control solution, mg/ml
Ai- -area of peak of test solution
Peak area of Ar- -control solution
Vi- -volume of test solution, ml
Mi- -weight of sample, g
Sample preparation:
control solution: accurately weighing 20mg to 100mL of gamma-aminobutyric acid reference substance in a volumetric flask, using 0.02mol/L hydrochloric acid to fix the volume to a scale, and uniformly mixing to obtain a reference substance solution.
Test solution: weighing 10g to 10mL of eye cream (Huaxi biology, lot number: 210327) test sample in a volumetric flask, adding 9mL of 0.02mol/L hydrochloric acid, extracting at 60 deg.C for 4h, fixing volume, and filtering to obtain test sample solution.
Blank solution: 0.02mol/L hydrochloric acid was filtered to prepare a blank solution.
4. Measurement of
And respectively sampling blank solution, reference solution and solution to be detected, detecting according to the chromatographic conditions, and calculating the content of the gamma-aminobutyric acid in the test solution according to the peak area of an external standard method.
5. Results
The results are shown in Table 1, the chromatograms are shown in FIGS. 1-3, respectively, and the detection limit is 3X 10-4ppm。
TABLE 1 detection results in example 1-1
Examples 1 to 2
The sample to be tested in example 1 was replaced with the batch number: 210309 measurement of the content of gamma-aminobutyric acid in the cosmetic under the same conditions as in example 1, the detection limit is 3X 10 as shown in Table 2-4ppm。
TABLE 2 examination results of examples 1 to 2
Examples 1 to 3
The detection wavelength in example 1 was changed to 350nm, and the other conditions were the same as in example 1, and the content of gamma-aminobutyric acid in the cosmetic was as shown in Table 3, with the detection limit of 7X 10-4ppm。
TABLE 3 test results of examples 1 to 3
Examples 1 to 4
The content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1 except that the flow rate of the mobile phase in example 1 was changed to 0.55ml/min, and the results are shown in Table 4, in which the detection limit was 9X 10-4ppm。
TABLE 4 test results of examples 1 to 4
Examples 1 to 5
The flow rate of the post-column derivatization buffer solution in example 1 was changed to 0.4ml/min, and the content of γ -aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 5, in which the detection limit was 6.6X 10-4ppm。
TABLE 5 results of chromatography analysis of samples to be tested of examples 1 to 5
Examples 1 to 6
The flow rate of the mobile phase in example 1 was changed to 0.35ml/min, othersThe content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 6, wherein the detection limit was 10X 10-4ppm。
TABLE 6 test results of examples 1 to 6
Examples 1 to 7
The flow rate of the post-column derivatization buffer solution in example 1 was changed to 0.2ml/min, and the content of γ -aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 7, in which the detection limit was 13.5X 10-4ppm。
TABLE 7 examination results of examples 1 to 7
Examples 1 to 8
The sample injection volume in example 1 was changed to 10. mu.L, and the content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 8, in which the detection limit was 5.4X 10-4ppm。
TABLE 8 test results of examples 1 to 8
Examples 1 to 9
The sample injection volume in example 1 was changed to 15. mu.L, and the content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 9, in which the detection limit was 7.8X 10-4ppm。
TABLE 9 test results of examples 1 to 9
Examples 1 to 10
The dissolution time for the preparation of the sample in example 1 was changed to 6 hours, the content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 10, wherein the detection limit is 3.2X 10-4ppm。
TABLE 10 test results of examples 1 to 10
Examples 1 to 11
The dissolution temperature for the sample preparation in example 1 was changed to 80 ℃ and the content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 11, wherein the detection limit is 3.1X 10-4ppm。
TABLE 11 test results of examples 1 to 11
Examples 1 to 12
The column temperature of the separation column in example 1 was changed to 50 ℃ and the content of γ -aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in table 12, in which the detection limit is 4.3 × 10-4ppm。
TABLE 12 test results of examples 1 to 12
Examples 1 to 13
The column temperature of the separation column in example 1 was changed to 57 ℃, and the content of γ -aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in table 13, with a detection limit of 5.8×10-4ppm。
TABLE 13 test results of examples 1 to 13
Examples 1 to 14
The measurement of the content of gamma-aminobutyric acid in the cosmetic was carried out under the same conditions as in example 1 except that the detection wavelength in example 1 was changed to 330nm, and the results are shown in Table 14, wherein the detection limit was 3.9X 10-4ppm。
TABLE 14 test results of examples 1 to 14
TABLE 15 chromatographic conditions, extraction conditions and limits of detection used in the examples
EXAMPLE 2-1 determination of reproducibility
The same lot number as in example 1-1 was sampled in the same manner as in example 1-1, and 6 portions were prepared in parallel, and the control solutions and the measurement conditions were the same as in example 1-1, and the measurement results are shown in Table 16.
TABLE 16 results of the content of gamma-aminobutyric acid of example 2-1
Example 2-2 measurement of daytime precision
6 portions of the same lot of samples as in example 1-1 were prepared in parallel at different times in the same manner as in example 1-1 for the sample treatment, and the results of the measurement were shown in Table 17 using the same control solutions and the same measurement conditions as in example 1-1.
TABLE 17 content results of gamma-aminobutyric acid of example 2-2
Examples 2-3 determination of accuracy
The blank solution of gamma-aminobutyric acid is taken, the blank solution is processed according to the test sample processing mode in the embodiment 1-1, the reference sample in the embodiment 1-1 is precisely weighed, and the simulation formula is prepared into test sample solutions with the concentrations of 80%, 100% and 120% of the prescription amount, and 3 parts of the test sample solutions are respectively prepared in parallel for 9 parts. The control solution and the measurement conditions were the same as in example 1-1, and the measurement results are shown in Table 18.
TABLE 18 Gamma-aminobutyric acid accuracy verification results
Comparative example 1
Table 19 shows the results of measuring the content of gamma-aminobutyric acid in the cosmetic by replacing o-phthalaldehyde in example 1 with ninhydrin under the same conditions as in example 1, with the detection limit of 500X 10-4ppm。
TABLE 19 test results of comparative example 1
As can be seen from the above table, the data parallelism is poor.
Comparative example 2
The reaction column temperature in example 1 was changed to 135 deg.C, and the content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in Table 20, wherein the detection limit was 70X 10-4ppm。
TABLE 20 test results of comparative example 2
As can be seen from the above table, increasing the temperature of the reaction column may affect the progress of the derivatization reaction, resulting in larger deviation of the results.
Comparative example 3
The extraction method of gamma-aminobutyric acid in example 1 was changed to 9ml of water, extraction was performed at 60 ℃ for 4 hours, then the volume was fixed, filtration was performed, the other conditions were the same as in example 1, and the content of gamma-aminobutyric acid in the cosmetic was measured, and the results are shown in table 21, where the detection limit was 100 × 10-4ppm。
TABLE 21 test results of comparative example 3
As can be seen from the above table, water cannot completely extract gamma-aminobutyric acid in the cosmetic, resulting in a small result.
Comparative example 4
The extraction method of gamma-aminobutyric acid in example 1 was changed to 9ml of 0.02mol/L hydrochloric acid, and after ultrasonic extraction for 4 hours, the volume was determined, the content was filtered, and the other conditions were the same as in example 1, and the content of gamma-aminobutyric acid in the cosmetic was measured, and the results are shown in Table 22, wherein the detection limit was 135X 10-4ppm。
TABLE 22 test results of comparative example 4
As can be seen from the above table, the ultrasonic extraction for 4h could not completely extract the γ -aminobutyric acid in the cosmetic, resulting in a smaller result.
Comparative example 5
The extraction mode of gamma-aminobutyric acid in the example 1 is changed as follows: dissolving a sample by petroleum ether, extracting by 0.02mol/L hydrochloric acid,the lower layer liquid was taken out and the volume was fixed, filtered, and the content of gamma-aminobutyric acid in the cosmetic was measured under the same conditions as in example 1, and the results are shown in table 23, where the detection limit is 309 × 10-4ppm。
TABLE 23 test results of comparative example 5
As can be seen from the above table, the result parallelism is poor and the extraction mode operation is complicated.
Comparative example 6
A, B, C, D, E, R1, R2 and R3 in example 1 were subjected to gradient elution with the elution ratios and times shown in Table 24:
TABLE 24 elution procedures
| Time/min | A％ | B％ | C％ | D％ | E％ | R1％ | R2％ | R3％ |
| 0.0 | 100 | -- | -- | -- | -- | 50 | 50 | -- |
| 3.0 | 100 | 100 | -- | -- | -- | 50 | 50 | -- |
| 3.1 | -- | 100 | -- | -- | -- | 50 | 50 | -- |
| 5.4 | -- | -- | 100 | -- | -- | 50 | 50 | -- |
| 5.5 | -- | -- | 100 | -- | -- | 50 | 50 | -- |
| 13.8 | -- | -- | -- | 100 | -- | 50 | 50 | -- |
| 13.9 | -- | -- | -- | 100 | -- | 50 | 50 | -- |
| 30.0 | -- | -- | -- | -- | 100 | -- | -- | -- |
| 30.1 | -- | -- | -- | -- | 100 | -- | -- | -- |
| 32.0 | -- | -- | -- | -- | 100 | -- | -- | -- |
| 32.1 | -- | -- | -- | -- | 100 | -- | -- | 100 |
| 33.0 | -- | -- | -- | -- | -- | -- | -- | 100 |
| 33.1 | -- | 100 | -- | -- | -- | -- | -- | 100 |
| 34.0 | -- | 100 | -- | -- | -- | -- | -- | 100 |
| 34.1 | 100 | -- | -- | -- | -- | -- | -- | 100 |
| 37.0 | 100 | -- | -- | -- | -- | -- | -- | 100 |
| 37.1 | 100 | -- | -- | -- | -- | 50 | 50 | -- |
| 53.0 | 100 | -- | -- | -- | -- | 50 | 50 | -- |
The remaining test conditions were the same as those in example 1, and the content of gamma-aminobutyric acid in the cosmetic was measured, and the results are shown in Table 25.
TABLE 25 test results of comparative example 6
As can be seen from the table above, the elution mode is the gradient elution condition of a common amino acid analyzer, the gamma-aminobutyric acid peak time is 1.887min, the gamma-aminobutyric acid peak time is seriously interfered by a solvent peak, the parallelism is poor, and the detection limit is high.
Comparative example 7
In example 1, the B and C values in the mobile phase used were changed to B: citric acid solution 22 wt%, C: the content of gamma-aminobutyric acid in the cosmetic composition was measured in the same manner as in example 1 except that the content of the sodium citrate solution was 1.3 wt%, and the measurement limit was 240X 10 as shown in Table 26-4ppm。
TABLE 26 test results of comparative example 7
As can be seen from the table above, the citric acid and sodium citrate solution has poor elution capability on gamma-aminobutyric acid, so that the peak area of the gamma-aminobutyric acid is low, the result is small, and the detection limit is high.
Comparative example 8
The derivatization reagent buffer solution mercaptoethanol in the example 1 is changed into mercaptopropionic acid, other conditions are the same as those in the example 1, the detection result shows that no peak of gamma-aminobutyric acid in the test sample is found, and the detection limit is 0.
Comparative example 9
The detection wavelength in the example 1 is changed to 570nm, other conditions are the same as the example 1, no peak of gamma-aminobutyric acid in the test sample is found in the detection result, and the detection limit is 0.
TABLE 27 chromatographic conditions, extraction conditions and limits of detection for comparative examples
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for detecting gamma-aminobutyric acid in cosmetics comprises the following steps:
preparation of control: dissolving a gamma-aminobutyric acid reference substance by using hydrochloric acid, and filtering to obtain a gamma-aminobutyric acid reference substance solution;
preparing a test sample: dissolving a cosmetic sample by using hydrochloric acid, and filtering to obtain a sample solution;
preparation of a blank solution: filtering hydrochloric acid to obtain a blank solution;
and (3) determination: and detecting the reference solution, the test solution and the blank solution by using a separation column and a post-column derivatization reaction column, thereby obtaining the content of the gamma-aminobutyric acid in the cosmetic test sample.
2. The detection method according to claim 1, wherein the concentration of the hydrochloric acid is 0.02mol/L to 0.06 mol/L;
preferably, the dissolution is water bath dissolution, preferably, the dissolution time is 4-6h, and further preferably, the dissolution temperature is 60-80 ℃.
3. The detection method according to any one of claims 1 to 2, wherein the cosmetic is a cream-milky or liquid cosmetic, preferably, the cream-milky cosmetic is an emulsion, an eye cream, or a facial cleanser; preferably, the liquid cosmetic is essential oil, toner or shower gel.
4. The detection method according to any one of claims 1 to 3, wherein the separation column is a cation exchange resin column, preferably a strong acid type cation exchange resin column;
preferably, the column temperature of the separation column is 50-57 ℃, and is preferably 55 ℃;
preferably, the derivatization reagent used in the derivatization reaction is o-phthalaldehyde, and preferably, the detection is to detect the absorbance with the wavelength of 330-350 nm;
preferably, the post-column derivatization reaction column is a quartz sand column, and the column temperature is preferably 30-50 ℃, and more preferably 35 ℃.
5. The detection method according to any one of claims 1 to 4, wherein the mobile phase is one or more selected from the group consisting of a citric acid buffer solution, a trifluoroacetic acid solution, a sodium acetate solution, a mixture of sodium citrate and benzyl alcohol, and a mixture of ethanol and sodium hydroxide, and preferably, the post-column derivatized buffer solution is one or two or three selected from the group consisting of a methanol solution of o-phthalaldehyde, a mixture of mercaptoethanol and a boric acid buffer, and an ethanol solution.
6. The detection method according to claim 5, wherein a gradient elution is performed, and the elution procedure is:
firstly, a citric acid buffer solution is used as a mobile phase, and a post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
then, using trifluoroacetic acid solution as a mobile phase, wherein the post-column derivatization buffer solution is a mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, using sodium acetate solution as a mobile phase, wherein the post-column derivatization buffer solution is a mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses a sodium acetate solution and a mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is a mixed solution of an o-phthalaldehyde methanol solution and a mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses the mixed solution of citric acid and benzyl alcohol, and the post-column derivatization buffer solution is the mixed solution of o-phthalaldehyde methanol solution and mercaptoethanol and boric acid buffer solution;
then, the mobile phase uses the mixed solution of ethanol and sodium hydroxide, and the post-column derivatization buffer solution is ethanol solution; and
finally, citric acid buffer solution is used as the mobile phase, and ethanol solution is used as the post-column derivatization buffer solution.
7. The detection method according to any one of claims 1 to 6, wherein the amount of the sample is 10 to 20. mu.L, preferably 15 to 20. mu.L, and more preferably 20. mu.L.
8. The detection method according to any one of claims 1 to 7, wherein the flow rate of the mobile phase is 0.35 to 0.55mL/min, and preferably, the flow rate of the post-column derivatized buffer solution is 0.2 to 0.4 mL/min.
9. The method according to any one of claims 1 to 8, wherein the content of γ -aminobutyric acid in the cosmetic is measured by an external standard method.
10. The method according to any one of claims 1 to 9, wherein the detection limit of gamma-aminobutyric acid is 3 x 10 or more-4ppm。
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