CN112964796A

CN112964796A - Method for determining taurocholic acid in cosmetics

Info

Publication number: CN112964796A
Application number: CN202110162835.7A
Authority: CN
Inventors: 侯广月; 周莉莉; 杜营; 李大为; 李秀超; 张永菊; 刘倩; 孔寒; 冯光兴; 袁婷婷
Original assignee: Binzhou Center For Food And Drug Control; Shandong Institute for Product Quality Inspection
Current assignee: Binzhou Center For Food And Drug Control; Shandong Institute for Product Quality Inspection
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2021-06-15

Abstract

The invention belongs to the technical field of detection, and particularly relates to a method for determining taurocholic acid in cosmetics₁₈Separating with reversed phase chromatographic column, and measuring with high performance liquid chromatograph equipped with ultraviolet or diode array detector. Compared with a spectrophotometer method, a thin layer chromatography method, an amino acid automatic analysis method and the like, the method has the remarkable advantages of high analysis speed, high sensitivity, good accuracy and the like; the method can be applied to detection of the taurocholic acid in various cosmetics, and the method is good in universality.

Description

Method for determining taurocholic acid in cosmetics

Technical Field

The invention belongs to the technical field of detection, and particularly relates to a method for determining taurocholic acid in cosmetics.

Background

Taurine, a sulfur-containing non-protein amino acid, readily soluble in water and chemically stable, is a material that is allowed to be used in the directory of names of Chinese characters in the International cosmetic Material Standard (2015 edition) and in the notification of the State food and drug administration on the directory of names of used cosmetic materials (2014, 11). Research shows that the taurocholic acid has biological effects such as an antioxidant effect, and research proves that the taurocholic acid has the capability of activating skin cells and can continuously provide rapid and lasting energy supplement and multiple protection for young skin; meanwhile, the skin-care product also has the effects of improving the immunity of the skin and resisting the invasion of the external environment to the skin. At present, a plurality of cosmetic products sold in the market are definitely marked to be added with the taurocholic acid as an effective component.

The method for measuring the taurocholic acid mainly comprises a thin-layer chromatography method, an amino acid automatic analysis method, a gas chromatography method, a high performance liquid chromatography method and the like, wherein the high performance liquid chromatography method is widely applied to the measurement of the taurocholic acid in food and biological tissues due to the characteristics of high speed and high efficiency, high sensitivity, good reproducibility, high accuracy and the like, but the related reports about the application of the high performance liquid chromatography method to the measurement of the taurocholic acid in cosmetics are few. Therefore, the development of a rapid and high-sensitivity liquid chromatography analysis method has great significance for the detection and analysis of the taurocholic acid in the cosmetics.

Disclosure of Invention

Aiming at the current situation that a high performance liquid detection method for the taurocholic acid in the cosmetics is lacked, the invention provides a method for detecting the taurocholic acid in the cosmetics, which comprises the steps of extracting the taurocholic acid in the cosmetics by using acetonitrile aqueous solution, removing impurities by using n-hexane, taking supernate, carrying out derivatization reaction by using dansyl chloride, and carrying out C-treatment on the derivative₁₈Separating with reversed phase chromatographic column, and measuring with high performance liquid chromatograph equipped with ultraviolet or diode array detector.

The technical scheme of the invention is as follows:

a method for determining taurocholic acid in a cosmetic, comprising the steps of:

(1) extraction: weighing a proper amount of sample, adding acetonitrile water solution, and performing ultrasonic extraction; cooling to room temperature, adding acetonitrile water solution saturated n-hexane, mixing thoroughly, standing, sucking upper layer n-hexane flocculent substance with a suction pipe, and discarding; adding acetonitrile water saturated normal hexane into the lower layer solution, fully mixing, standing, and removing the upper layer normal hexane; fixing the volume, uniformly mixing, centrifuging, and reserving supernate for later use;

(2) derivatization: adding a sodium carbonate buffer solution and a dansyl chloride solution into the solution obtained in the step (1), carrying out light-proof derivatization reaction at room temperature, adding a methylamine hydrochloride solution, carrying out vortex mixing to terminate the reaction, and standing in the light-proof state until the precipitation is complete; filtering the supernatant with microporous membrane to be tested;

(3) and (3) determination: analyzing and measuring the derivative solution of the test solution and the standard working solution by adopting a high performance liquid chromatography;

(4) drawing a standard curve and quantifying by an external standard method.

The volume ratio of acetonitrile to water in the acetonitrile water solution in the step (1) is 1: 9.

The reference conditions of the high performance liquid chromatograph in the step (3) are as follows:

a chromatographic column: c₁₈Reverse phase chromatography column, 250 mm × 4.6 mm (i, d), 5 μm;

mobile phase: acetonitrile: 20 mmol/L ammonium acetate solution =30:70 (volume ratio);

flow rate: 1.0 mL/min;

column temperature: 35 ℃;

sample introduction volume: 20 mu L of the solution;

detection wavelength: 254 nm.

The invention has the beneficial effects that:

(1) compared with a spectrophotometer method, a thin-layer chromatography method, an amino acid automatic analysis method and the like, the method for determining the taurocholic acid in the cosmetics through the liquid chromatography has the remarkable advantages of high analysis speed, high sensitivity, good accuracy and the like.

(2) The method can be applied to detection of the taurocholic acid in various cosmetics, and the method is good in universality.

(3) Adding appropriate amount of standard substance into blank matrix, detecting and analyzing according to the method established by the standard, determining the detection limit of taurocholic acid according to signal-to-noise ratio to be 50 mg/kg (figure 5 a), and determining the quantification limit to be 150 mg/kg (figure 5 b).

Drawings

FIG. 1 is a liquid chromatogram derived from a standard sample;

fig. 2 is a chromatogram under different mobile phase conditions (a: acetonitrile: water =30:70, b: acetonitrile: ammonium acetate =30: 70);

FIG. 3 is a diagram showing the ultraviolet absorption spectrum of a derivative of taurocholic acid;

FIG. 4 is a chromatogram of a standard solution of a derivative of taurocholic acid at different wavelengths (a, 216 nm, b, 254 nm, c, 331 nm);

FIG. 5 is a chromatogram of detection limit and quantitation limit in a blank matrix (a. detection limit, b. quantitation limit).

Detailed Description

The technical solution of the present invention is further explained and illustrated by the following specific examples.

Example 1 determination of liquid chromatography conditions

1. Selection of chromatography columns

High purity silica gel C₁₈The chromatographic column is the most commonly used reversed-phase chromatographic column, takes octadecylsilane chemically bonded silica as a filler, has better carbon content and hydrophobicity, has stronger adaptability to various compound molecules, has wide application range, and is the preferred chromatographic column type in high performance liquid chromatograph analysis. This standard chooses C₁₈The chromatographic column is used as a chromatographic separation column, and as a result, the derivative of the taurocholic acid is found to be in C₁₈The chromatographic column has better separation effect, the peak forms normal distribution, and C is selected finally₁₈Reverse phase chromatography column (250X 4.6 mm (i.d.), 5 μm).

2. Selection of mobile phase

In the test, acetonitrile-ammonium acetate solution, methanol-ammonium acetate solution, acetonitrile-water and methanol-water are respectively selected as mobile phases, and the influence of different mobile phases on the peak emergence time, the peak shape and the response intensity of the target object is examined. As a result, it was found that: under the condition of the same target concentration and the same mobile phase proportion, when a sample is eluted by methanol, the retention time of a target peak is greatly prolonged, the viscosity of the methanol is higher than that of acetonitrile, and the system pressure is high, so the acetonitrile is selected as a mobile phase for elution. In addition, the specific chromatogram of figure 2 shows the peak appearance time, peak shape and response value of the taurocholic acid when acetonitrile-water and acetonitrile-20 mmol/L ammonium acetate are respectively used as the mobile phase under the same mobile phase proportion. It can be seen from fig. 2 that when the mobile phase is acetonitrile-water, the peak-off time of the target peak is earlier than when the mobile phase is acetonitrile-20 mmol/L ammonium acetate, but the interference around the target peak is large, and the target peak pattern is better when the mobile phase is ammonium acetate. Acetonitrile-20 mmol/L ammonium acetate was selected as the mobile phase.

3. Selection of detection wavelength

Since the derivative of taurocholic acid has absorption in the ultraviolet region, an ultraviolet detector is selected for detection. As can be seen from the scanning spectrogram (figure 3) of the diode array detector in the wavelength range of (210-400) nm, the taurocholic acid derivative has three characteristic absorption peaks at 216 nm, 248 nm and 331 nm, wherein the absorption is strongest at 216 nm and is 248 nm secondly. Referring to fig. 4, chromatograms of the derivatives of the taurine at different wavelengths show that if the extraction wavelength is set near 216 nm, interference peaks are many, and when the extraction wavelength is 331 nm, the signal response is weak, and finally, 254 nm is selected as the detection wavelength of the taurine.

4. Selection of column temperature

Under a certain condition, the column temperature also has an influence on the separation of the taurocholic acid derivatives, the influence of the column temperature on the taurocholic acid derivatives within the range of 25-40 ℃ is considered, and the fact that the retention time of a sample is advanced along with the rise of the column temperature is found, the peak area also changes to a certain extent, but the change is not obvious. The theoretical plate number is generally increased and the peak width is narrowed by raising the column temperature, so that a lower detection limit can be obtained, but the service life of the chromatographic column is influenced by over-high column temperature, and the column temperature is finally determined to be 35 ℃.

Example 2 determination of pretreatment method

1. Sample weighing

The cosmetic sample has complex components, and other impurities are dissolved out along with the extraction of the target substance to be detected to interfere the detection of the taurocholic acid, so that the sampling amount is not easy to be too large, and the sampling amount is determined to be 0.5 g.

2. Selection of extract and extraction mode

Taking the physicochemical properties of the taurocholic acid into consideration and combining common reagents in a laboratory and the toxicity of the taurocholic acid, a mixed solution of water, methanol and water (the volume ratio is 1: 9) and a mixed solution of acetonitrile and water (the volume ratio is 1: 9) are respectively selected as extraction solutions for experiments, and specific experimental data are shown in table 1. The results show that when the same blank was tested for additive recovery, the ratio of acetonitrile to acetonitrile in 1: 9: when water is used as an extracting solution, the recovery rate of the target substance to be detected of the taurocholic acid is higher than that of a sample obtained by mixing 1:9 methanol: the recovery rate of water and pure water as the extract.

The reaction solution is prepared by using acetonitrile: water =1:9 as the extract, and the comparison was performed by shaking and ultrasonic extraction for 25 min, respectively. Test results show that the recovery rate of ultrasonic extraction is slightly higher than that of oscillation extraction, so that ultrasonic extraction is adopted for 25 min.

TABLE 1 recovery of target under different extraction solutions

Example 3

A method for determining taurocholic acid in cosmetics by liquid chromatography comprises the following steps:

(1) a sample (0.5 g) was weighed out to the nearest 0.1 mg, placed in an Erlenmeyer flask, added about 20 mL of acetonitrile in water (acetonitrile: water =1: 9), mixed well, and extracted with ultrasound for 25 min. After cooling to room temperature, 10 mL of acetonitrile-saturated n-hexane was added, mixed well and allowed to stand, and the flocculated upper n-hexane material was carefully removed by suction. 10 mL of acetonitrile-saturated n-hexane was added to the lower layer solution, and after thorough mixing, the mixture was allowed to stand, and the upper layer n-hexane was discarded. Transfer to 25 mL volumetric flask, add acetonitrile in water (acetonitrile: water =1: 9) to volume to the mark and mix well. Centrifuging the sample solution at 6000 r/min for 5 min, and collecting the supernatant. The supernatant was stored at 4 ℃ in the dark and stabilized within 24 h.

(2) Derivatization of a test solution:

accurately sucking 1.00 mL of the obtained supernatant into a 10 mL glass test tube with a plug, adding 1.00 mL of sodium carbonate buffer solution (pH 9.5, 80 mmol/L) and 1.00 mL of dansyl chloride solution (1.5 mg/mL), fully mixing, carrying out derivatization reaction at room temperature in the dark for 2 h (shaking for 1 time after 1 h), adding 0.10 mL of methylamine hydrochloride solution (20 mg/L), carrying out vortex mixing to terminate the reaction, and standing in the dark until the precipitate is complete. Filtering the supernatant with organic phase microporous membrane, and measuring. The derived solution can be stored for 48 h at below 4 deg.C under dark condition.

Another 1.00 mL standard working solution was taken and derivatized simultaneously with the test solution.

(3) Reference conditions of the apparatus

A chromatographic column: c₁₈Reverse phase chromatography column, 250 mm × 4.6 mm (i, d), 5 μm.

Mobile phase: acetonitrile: 20 mmol/L ammonium acetate solution =30:70 (volume ratio).

Flow rate: 1.0 mL/min.

Column temperature: 35 ℃ is carried out.

Sample introduction volume: 20 μ L.

Detection wavelength: 254 nm.

(4) Preparation of Standard Curve

And (3) measuring the derivative solution of the standard working solution according to the reference condition of the instrument, and drawing a standard curve by taking the mass concentration of the standard series working solution as an abscissa and taking the peak area as an ordinate.

(5) Measurement of sample solution

And (3) measuring the derivative solution of the sample solution according to the instrument reference condition, and quantifying by an external standard method. The response value of the tested object in the sample solution is within the linear range of the standard curve, and if the response value exceeds the linear range, the mobile phase is used for dilution and then analysis is carried out.

(6) Presentation of analytical results

The content of the taurocholic acid in the sample is calculated according to the formula (1):

.......................(1)

in the formula:

x-the amount of taurocholic acid in milligrams per kilogram (mg/kg) in the sample;

c-concentration of taurocholic acid in units of micrograms per milliliter (. mu.g/mL) in the sample solution from the standard curve;

v-volume of sample extraction solution in milliliters (mL);

n is the dilution multiple of the sample;

m is the mass of the sample in grams (g).

The results retain 3 significant digits, expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions.

(7) Precision degree

The absolute difference between two independent measurements obtained under repetitive conditions must not exceed 10% of the arithmetic mean.

(8) Detection limit and quantification limit

The detection limit of the method for the taurocholic acid is 50 mg/kg, and the quantification limit is 150 mg/kg.

The solutions used above were as follows:

sodium carbonate buffer solution (pH 9.5) (80 mmol/L): 0.424 g of anhydrous sodium carbonate was weighed. Adding 40 mL of water for dissolving, adjusting the pH to 9.5 by using 1 mol/L of hydrochloric acid solution, and adding water for metering to 50 mL. The solution was stable at room temperature for 3 months.

Dansyl chloride solution (1.5 mg/mL): 0.15 g dansyl chloride was weighed, dissolved with acetonitrile and made to volume 100 mL. It is prepared immediately before use.

Methylamine hydrochloride solution (20 mg/L): 2.0 g of methylamine hydrochloride is weighed, dissolved in water and made to 100 mL. The solution was stable for 3 months when stored at 4 ℃.

Aqueous acetonitrile (acetonitrile: water =1: 9): 100 mL of acetonitrile is weighed, 900 mL of water is added, and the mixture is fully mixed.

Acetonitrile water saturated n-hexane: and (3) adding 200 mL of n-hexane into a 250 mL separating funnel, adding a proper amount of 10% acetonitrile aqueous solution, violently oscillating, standing for layering, and removing the lower acetonitrile-water layer to obtain the acetonitrile-water layer.

Ammonium acetate solution (0.01 mol/L): 0.77 g of ammonium acetate is weighed, dissolved by adding a proper amount of water, and is added with water to be constant volume to 1000 mL, and the mixture is filtered by a 0.22 mu m water phase microporous membrane for later use.

Standard stock solution of taurocholic acid (1.0 mg/mL): accurately weighing appropriate amount of cholic acid standard (accurate to 0.1 mg), and dissolving with water to obtain standard stock solution with concentration of 1.0 mg/mL. Storing at 2-8 ℃ for 7 days.

Standard working solution of taurocholic acid: the standard stock solution of the taurocholic acid is diluted by water into a series of standard solutions of 0. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL, 5.0. mu.g/mL, 10.0. mu.g/mL, 20.0. mu.g/mL and 50.0. mu.g/mL in sequence. Is prepared before use.

Example 4

Examination of the Linear relationship of example 3

Standard stock solutions were formulated into a range of concentrations of standard working solutions: after derivatization treatment, the sample injection analysis is carried out according to standard chromatographic conditions to determine peak areas, and the peak areas of chromatographic peaks are taken as ordinate and the corresponding solution concentrations are taken as abscissa to draw a standard curve, thereby obtaining a linear equation and a correlation coefficient (Table 2). The result shows that the linear relation of the taurocholic acid is good in the concentration range of 0.50-50.00 mu g/mL.

TABLE 2 Linear relationship of taurocholic acid

Example 5

Precision examination of example 3

The low, medium and high concentration of the taurocholic acid are selected and added into the blank substrate, the precision in the day and the precision in the day are examined, and the results are shown in table 3.

Precision of the method of Table 3: (n=6）

Example 6

Example 3 recovery test

According to the standard, the standard solutions of the taurocholic acid with the concentration levels of 150 mg/kg, 300 mg/kg and 500 mg/kg are respectively added into the liquid cosmetics, the milk cosmetics and the cream cosmetics, and the standard solutions are processed, analyzed and detected according to the method established by the standard, and the recovery rate is calculated. The results in Table 4 show that the recovery rate of the taurocholic acid in the liquid, milk and cream cosmetics is good, and the recovery rate is between 82.7 and 97.7 percent.

TABLE 4 recovery of taurocholic acid in cosmetic products (n = 6)

Example 7

Examination of detection limits and quantitation limits in example 3

Adding appropriate amount of standard substance into blank matrix, detecting and analyzing according to the method established by the standard, determining the detection limit of taurocholic acid according to signal-to-noise ratio to be 50 mg/kg (figure 5 a), and determining the quantification limit to be 150 mg/kg (figure 5 b).

Claims

1. A method for determining taurocholic acid in cosmetics is characterized by comprising the following steps:

(4) drawing a standard curve and quantifying by an external standard method.

2. The method for determining taurocholic acid in a cosmetic according to claim 1, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution in step (1) is 1: 9.

3. The method for determining taurocholic acid in a cosmetic according to claim 1, wherein the reference conditions of the HPLC in step (3) are as follows:

mobile phase: acetonitrile: the volume ratio of the 20 mmol/L ammonium acetate solution is 30: 70;

flow rate: 1.0 mL/min;

column temperature: 35 ℃;

sample introduction volume: 20 mu L of the solution;

detection wavelength: 254 nm.

4. The method for detecting the taurocholic acid in the cosmetic according to claim 1, wherein the acetonitrile water saturated n-hexane preparation process of step (1) is as follows: and (3) putting n-hexane into a separating funnel, adding a proper amount of 10% acetonitrile aqueous solution, violently oscillating, standing for layering, and removing the lower acetonitrile-water layer to obtain the acetonitrile-water layer.