1. A method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in toilet soap comprises the following implementation steps:

1) preparing a standard solution;

2) preparing a sample, dissolving the sample in hot water, mixing and extracting the sample with a solvent, and salting out to obtain an extracting solution;

3) adopting Agilent ZORBAX Eclipse XDB-C₁₈The chromatographic column is used as an analytical column, a methanol-acetonitrile-ammonium acetate ternary mobile phase is adopted, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the detection wavelength is 300nm, and the quantification is carried out by an external standard method.

2. The method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap according to claim 1, wherein the method comprises the following steps: weighing a proper amount of each standard substance in a 10mL volumetric flask, adding methanol to dissolve the standard substance and fixing the volume to a scale, and respectively preparing standard stock solutions with the mass concentration of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol being 1.0 mg/mL; preparing the standard stock solution into a mixed standard solution, and diluting the mixed standard solution step by step to respectively obtain a series of mixed standard solutions with the concentrations of 2,4, 6-trichlorophenol and pentachlorophenol being 0.25, 0.5, 1.0, 2.0, 5.0 and 10.0 mu g/mL and the concentrations of thiochlorophenol being 0.125, 0.25, 0.5, 1.0, 2.5 and 5.0 mu g/mL.

3. The method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap according to claim 1 or 2, wherein the method comprises the following steps: dispersing each sample by hot water at 60 ℃, adding acetonitrile, mixing, performing ultrasonic extraction, and finally adding sodium chloride for salting out to obtain an upper-layer extract liquor; the ultrasonic extraction time is 18-20 min.

4. The method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap according to claim 1 or 2, wherein the method comprises the following steps: in order to realize better separation effect, the influence of a mobile phase system methanol-acetonitrile-5 mmol/L ammonium acetate aqueous solution on the HPLC separation effect and the peak shape of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol is examined, and the following gradient elution program is determined through optimization experiments:

0-3 min, 30% of A, 10% of B and 60% of D; 3-35 min, 30-80% of A, 10% of B and 60-10% of D; 35-45 min, 80% of A, 10% of B and 10% of D; 45-47 min, 80-30% of A, 10% of B and 10-60% of D; 47-50 min, 30% of A, 10% of B and 60% of D; wherein, the mobile phase: a is methanol, B is acetonitrile, D is 5mmol/L ammonium acetate aqueous solution; sample introduction amount: 10 μ L.

5. The method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap according to claim 3, wherein the method comprises the following steps: in order to realize better separation effect, the influence of a mobile phase system methanol-acetonitrile-5 mmol/L ammonium acetate aqueous solution on the HPLC separation effect and the peak shape of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol is examined, and the following gradient elution program is determined through optimization experiments:

0-3 min, 30% of A, 10% of B and 60% of D; 3-35 min, 30-80% of A, 10% of B and 60-10% of D; 35-45 min, 80% of A, 10% of B and 10% of D; 45-47 min, 80-30% of A, 10% of B and 10-60% of D; 47-50 min, 30% of A, 10% of B and 60% of D; wherein, the mobile phase: a is methanol, B is acetonitrile, D is 5mmol/L ammonium acetate aqueous solution; sample introduction amount: 10 μ L.

Background

2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol have strong bactericidal action on gram-positive bacteria, so that the antiseptic bactericidal action is achieved. 2,4, 6-trichlorophenol and pentachlorophenol belong to two carcinogens issued by the international cancer research institute of the world health organization. The thiochlorophene can irritate the skin and cause contact dermatitis, which can cause damage to the skin. The national food and drug administration issues a notice (No. 268 in 2015) of cosmetic safety specifications (2015 edition) that specifies 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol as forbidden components in cosmetic compositions that should not be used.

The perfumed soap sold in the domestic market conforms to the definition of cosmetics in the cosmetics supervision and management regulations, but is not included in the cosmetics management. According to the regulations of (EC) No 1223/2009, toilet soaps are classified as cosmetics in the European Union, and 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol are definitely forbidden components in toilet soaps and cannot be used. Therefore, in the foreign trade, in order to guarantee the quality of the toilet soap exported in China, it is necessary to develop a detection and analysis method for 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in the toilet soap. At present, national standard methods suitable for detecting 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in cream, emulsion and liquid cosmetics exist in China, and the detection methods of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in cream, emulsion and liquid cosmetics include liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and capillary electrophoresis.

If the soap adopts a national standard method for detecting 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in cream, emulsion and liquid cosmetics, two main technical problems exist in direct methanol dissolution constant volume determination: 1) the methanol cannot dissolve the soap to disperse it so that the target cannot be extracted completely; 2) the soap is rich in surfactant, mainly sodium aliphatate (anionic surfactant), and a large amount of surfactant entering a liquid chromatography separation column without treatment can damage a chromatographic column and instruments, has large matrix interference, and is easy to cause false positive of detection results.

At present, no literature report of a method for detecting 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in toilet soap is found at home and abroad.

Disclosure of Invention

The invention provides an analysis method for determining the contents of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in export cosmetic soap by liquid chromatography, aiming at the defects of the prior art. The detection and analysis method is simple, rapid, economical and efficient.

The technical scheme adopted by the invention is as follows:

a method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in toilet soap comprises the following implementation steps:

1) preparing a standard solution; 2) preparing a sample, dissolving the sample in hot water, mixing and extracting the sample with a solvent, and salting out to obtain an extracting solution; 3) adopting Agilent ZORBAX Eclipse XDB-C₁₈A chromatographic column (5 mu m,4.6mm multiplied by 250mm) is used as an analytical column, a methanol-acetonitrile-ammonium acetate ternary mobile phase is adopted, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the detection wavelength is 300nm, and the quantification is carried out by an external standard method.

Weighing a proper amount of each standard substance in a 10mL volumetric flask, adding methanol to dissolve the standard substance and fixing the volume to a scale, and respectively preparing standard stock solutions with the mass concentration of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol being 1.0 mg/mL; preparing the standard stock solution into a mixed standard solution, and diluting the mixed standard solution step by step to respectively obtain a series of mixed standard solutions with the concentrations of 2,4, 6-trichlorophenol and pentachlorophenol being 0.25, 0.5, 1.0, 2.0, 5.0 and 10.0 mu g/mL and the concentrations of thiochlorophenol being 0.125, 0.25, 0.5, 1.0, 2.5 and 5.0 mu g/mL. Dispersing each sample by hot water at 60 ℃, adding acetonitrile, mixing, performing ultrasonic extraction, and finally adding sodium chloride for salting out to obtain an upper-layer extract liquor; the ultrasonic extraction time is 18-20 min.

In order to realize better separation effect, the method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap investigates the influence of a mobile phase system methanol-acetonitrile-5 mmol/L ammonium acetate aqueous solution (pH of an acetic acid adjusting solution is 3.5) on the HPLC separation effect and peak shape of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol, and determines the following gradient elution procedure through optimization experiments: 0-3 min, 30% of A, 10% of B and 60% of D; 3-35 min, 30-80% of A, 10% of B and 60-10% of D; 35-45 min, 80% of A, 10% of B and 10% of D; 45-47 min, 80-30% of A, 10% of B and 10-60% of D; 47-50 min, 30% A, 10% B, 60% D; wherein, the mobile phase: a is methanol, B is acetonitrile, D is 5mmol/L ammonium acetate water solution (pH3.5); sample introduction amount: 10 μ L.

The invention has the beneficial effects that:

1. the invention establishes an analysis method for measuring 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in export cosmetic soap by salting out split-phase extraction-high performance liquid chromatography. The method is simple, rapid, economic and accurate, and is suitable for rapid screening and quantitative analysis of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in export cosmetic soap. Fills the blank of the method for detecting the three substances of the export cosmetic soap, and provides a technical support for the quality detection of the export soap.

2. The invention relates to a method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in toilet soap, which selects the maximum absorption wavelength of each standard substance as the detection wavelength; comparing the total scanning of 190nm-500nm wavelength of methanol, 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol, and selecting 300nm as the optimal detection wavelength of the HPLC of the three substances. Effectively eliminates the interference of impurities and increases the sensitivity of detection and analysis.

3. According to the method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap, the influence of a mobile phase system methanol-acetonitrile-5 mmol/L ammonium acetate aqueous solution (the pH value of an acetic acid adjusting solution is 3.5) on the HPLC separation effect and the peak shape of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol is considered, and an optimal gradient elution program is determined through optimization experiments, so that a better separation effect is realized.

Drawings

FIG. 1 is a graph of experimental ultrasound time effect versus recovery;

FIGS. 2A-2D are UV spectrum scanning graphs of a blank solvent and a standard solution; wherein A (methanol), B (2,4, 6-trichlorophenol), C (pentachlorophenol), D (thiochlorophenol);

FIGS. 3A-3C are chromatograms of a mixed standard solution (A), a negative sample spiking (B), and a negative sample (C);

FIGS. 4A-4C are chromatograms of standard substances of different chromatographic columns, wherein the appearance order is 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol. Wherein in FIG. 4A: agilent ZORBAX SB-C₁₈FIG. 4B: ZORBAX Eclipse XDB-C₁₈FIG. 4C: waters Symmetry C18.

Detailed Description

The technical solution of the present invention is further described in detail below by means of specific embodiments and with reference to the accompanying drawings.

Example 1

The invention discloses a method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in toilet soap, which comprises the following steps:

1) preparing a standard solution;

2) preparing a sample, dissolving the sample in hot water, mixing and extracting the sample with a solvent, and salting out to obtain an extracting solution;

3) adopting Agilent ZORBAX Eclipse XDB-C₁₈A chromatographic column (5 mu m,4.6mm multiplied by 250mm) is used as an analytical column, a methanol-acetonitrile-ammonium acetate ternary mobile phase is adopted, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the detection wavelength is 300nm, and the quantification is carried out by an external standard method.

The experimental result shows that the linear correlation coefficients of the three substances are all larger than 0.999, the three levels are added with the standard, the average recovery rate is 71.0-103.0%, and the Relative Standard Deviation (RSD) is 3.88-8.57% (n is 6).

Example 2

The difference between the method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap of the embodiment and the embodiment 1 is that: further, the process and the steps for preparing the standard solution are as follows:

weighing a proper amount of each standard substance in a 10mL volumetric flask, adding methanol to dissolve the standard substance and fixing the volume to a scale, and respectively preparing standard stock solutions with the mass concentration of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol being 1.0 mg/mL; preparing the standard stock solution into a mixed standard solution, and diluting the mixed standard solution step by step to respectively obtain a series of mixed standard solutions with the concentrations of 2,4, 6-trichlorophenol and pentachlorophenol being 0.25, 0.5, 1.0, 2.0, 5.0 and 10.0 mu g/mL and the concentrations of thiochlorophenol being 0.125, 0.25, 0.5, 1.0, 2.5 and 5.0 mu g/mL.

The method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap comprises the steps of dispersing each sample by hot water at 60 ℃ when preparing the sample, adding acetonitrile, mixing, performing ultrasonic extraction, and finally adding sodium chloride for salting out to obtain an upper-layer extraction liquid; the ultrasonic extraction time is 18-20 min.

Example 3

The method for detecting and analyzing the content of trichlorophenol, pentachlorophenol and thiochlorophenol in the soap of the embodiment is different from the method of the embodiment 1 and the embodiment 2 in that: in order to realize better separation effect, the influence of a mobile phase system methanol-acetonitrile-5 mmol/L ammonium acetate aqueous solution on the HPLC separation effect and the peak shape of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol is examined, and the following gradient elution program is determined through optimization experiments: 0-3 min, 30% of A, 10% of B and 60% of D; 3-35 min, 30-80% of A, 10% of B and 60-10% of D; 35-45 min, 80% of A, 10% of B and 10% of D; 45-47 min, 80-30% of A, 10% of B and 10-60% of D; 47-50 min, 30% of A, 10% of B and 60% of D; wherein, the mobile phase: a is methanol, B is acetonitrile, D is 5mmol/L ammonium acetate aqueous solution; sample introduction amount: 10 μ L.

Example 4

The following experiments show the content detection and analysis method of trichlorophenol, pentachlorophenol and thiochlorophenol in the toilet soap in more detail.

1. Main reagent and instrument for experiment

2,4, 6-trichlorophenol (ω ═ 99.5%), thiochlorophenol (ω ═ 97.9%), standard, dr.ehrentorfer; pentachlorophenol (ω 98.0%), standard, altar ink quality testing technologies, ltd; methanol, acetonitrile, pure chromatogram, Shanghai' an spectrum experiment science and technology, Inc.; sodium chloride, acetic acid (glacial acetic acid), analytically pure, manufactured by west longa science ltd; ammonium acetate, analytical grade, national pharmaceutical group chemical reagents ltd; 0.22 μm organic microporous filter membrane, Shanghai' an spectral laboratory science and technology Co., Ltd; the laboratory water was Millipore Milli-Q ultrapure water.

Agilent 1260 high performance liquid chromatograph equipped with quaternary gradient pump, autosampler, column oven, ultraviolet detector, Agilent technologies, Inc. of America; ultra pure water machines, Millipore corporation, usa; EL204 electronic balance (precision 0.0001g), AE204-S electronic balance (precision 0.00001g), PH meter, mettler-tollido instruments (shanghai) ltd; 1-14 small bench top centrifuge, Sigma, germany; SB25-12DT ultrasonic cleaning machine, Ningbo Xinzhi Biotech GmbH; VORTEX-GENIE2 VORTEX mixer, Scientific Industries, USA.

2. Experimental methods

2.1 chromatographic conditions

Agilent ZORBAX Eclipse XDB-C₁₈Chromatography column (5 μm,4.6 mm. times.250 mm); mobile phase: a is methanol, B is acetonitrile, D is 5mmol/L ammonium acetate aqueous solution (pH 3.5). Sample introduction amount: 10 μ L. Flow rate: 1.0 mL/min; column temperature: at 30 ℃. Detection wavelength: 300 nm. Gradient elution procedure: 0-3 min, 30% of A, 10% of B and 60% of D; 3-35 min, 30-80% of A, 10% of B and 60-10% of D; 35-45 min, 80% of A, 10% of B and 10% of D; 45-47 min, 80-30% of A, 10% of B and 10-60% of D; 47-50 min, 30% A, 10% B and 60% D.

2.2 preparation of Standard solutions

Weighing a proper amount of each standard substance (2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol) in a 10mL volumetric flask, adding methanol to dissolve the standard substance and fixing the volume to a scale, and preparing standard stock solutions with the mass concentration of the 2,4, 6-trichlorophenol, the pentachlorophenol and the thiochlorophenol being 1.0mg/mL respectively. Preparing the standard stock solution into a mixed standard solution, and diluting the mixed standard solution step by step to respectively obtain 2,4, 6-trichlorophenol, wherein the concentration of pentachlorophenol is 0.25, 0.5, 1.0, 2.0, 5.0 and 10.0 mu g/mL, and the concentration of thiochlorophenol is 0.125, 0.25, 0.5, 1.0, 2.5 and 5.0 mu g/mL.

2.3 sample preparation

Weighing 0.5g of soap sample (uniform scraps) into a 10mL colorimetric tube with a plug, adding 5mL of 60 ℃ water, performing vortex for 1min for dispersion, adding 5mL of acetonitrile, performing vortex for 1min, performing ultrasonic treatment for 20min, and then adding 1g of sodium chloride, and performing vortex layering. Standing for 1 min. The supernatant is removed and centrifuged at 12000 r/min, and the supernatant is measured on a machine after passing through a 0.22 mu m organic microporous filter membrane.

3. Experimental results and discussion

3.1 selection of extraction mode

2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol have better solubility in organic solvents of methanol and acetonitrile. The solvent is directly extracted and is used for passing through a membrane and a machine, so that the interference of the matrix is large, the chromatographic column is polluted, and the service life of the chromatographic column is damaged. If saturated sodium chloride solution is adopted for dispersion, acetonitrile can only be used for extraction and layering because methanol and water have no salting-out effect. Because the two-phase extraction is insufficient, the experiment finally selects to disperse by 60 ℃ hot water, add acetonitrile to mix and then carry out ultrasonic extraction, and finally add sodium chloride to salt out to obtain the upper-layer extraction liquid.

3.2 ultrasound timing

Ultrasonic treatment is selected for 10min, 20min, 30min and 40min for extraction, and the recovery rate is changed along with the increase of extraction time. The result shows that the 10min extraction effect is not as good as 20min, 30min and 40min, and the extraction effects of 20min, 30min and 40min are not obviously different. In order to shorten the time required by the experiment and improve the recovery rate, the ultrasonic treatment is selected for 20min for extraction. The experimental ultrasound time effect and recovery are shown in figure 1.

3.3 selection of chromatographic conditions

3.3.1 selection of detection wavelength

To increase sensitivity to exclude interference from impurities, the wavelength of maximum absorption of each standard substance is generally selected as the detection wavelength. Comparing the wavelength full scans of 190nm-500nm of the solvents methanol, 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol, see figure 2. The maximum absorption wavelength of the 2,4, 6-trichlorophenol is 210nm-220nm, the maximum absorption wavelength of the pentachlorophenol is 210-250nm, the maximum absorption wavelength of the thiochlorophenol is 210-270nm, the solvent effect of the methanol is present near 190nm-260nm, the solvent methanol near 300nm has no absorption value, and the 2,4, 6-trichlorophenol, the pentachlorophenol and the thiochlorophenol have strong absorption at 300nm and have no other interference. Therefore, 300nm is selected as the optimum detection wavelength of the HPLC for the three substances.

3.3.2 Mobile phase selection

In order to realize better separation effect, three different mobile phase systems, namely methanol-acetonitrile-water, methanol-acetonitrile-0.1% formic acid aqueous solution, and methanol-acetonitrile-5 mmol/L ammonium acetate aqueous solution (the pH value of acetic acid regulating solution is 3.5), are considered to influence the HPLC separation effect and the peak shape of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol, and through optimization experiments, the gradient elution program in '1.2.1' is finally determined, so that the separation can be effectively realized, the peak shape is sharp, and the sensitivity is high. The high performance liquid chromatogram of the mixed standard solution of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol, the liquid chromatogram of the negative sample with the label and the liquid chromatogram of the negative sample are shown in FIGS. 3A-3C.

3.3.3 selection of chromatography columns

Gradient wash in "1.2.1Agilent ZORBAX SB-C was compared under the program-off condition₁₈、ZORBAX Eclipse XDB-C₁₈And Waters Symmetry C₁₈Three liquid chromatography columns, all 5 μm,4.6mm × 250mm in specification. Referring to FIG. 4, the order of appearance of peaks is 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol, and the comparison of the number of plates and the shape of the peaks shows that all three peaks in FIG. 4(A) have a certain tailing degree, and the tail of the thiochlorophenol in FIG. 4(C) is severe, resulting in a decrease in peak height. Therefore, ZORBAX Eclipse XDB-C is selected₁₈Is a chromatographic column detected by the method.

3.4 Linear relationship and quantitative Limit

The test is prepared by mixing a series of standard working solutions for chromatographic determination and quantifying by an external standard method. The CAS numbers, linear ranges, linear equations, correlation coefficients, and quantitative limits for 2,4, 6-trichlorophenol, pentachlorophenol, and thiochlorophenol are shown in Table 1.

TABLE 1 CAS number, Linear Range, Linear equation, correlation coefficient, quantitation Limit for three materials

3.5 recovery, precision

According to the detection of the chromatographic conditions and the pretreatment method, the standard addition recovery experiment is carried out on the mixed standard solution with the addition level of 3 levels, namely the quantitative limit, the double quantitative limit and the quadruple quantitative limit, in the negative sample, 6 parallel samples are prepared at each standard addition concentration level, and the addition recovery rate and the relative standard deviation of the negative sample are shown in table 2. The result shows that the average recovery rate of the method is 71.0-103.0%, the relative standard deviation is 3.88-8.57%, and the method has accurate measurement result and can be used for quantitative analysis of export cosmetic soap 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol.

Table 2 negative sample addition recovery and relative standard deviation (n ═ 6)

3.6 determination of the actual sample

The established method is adopted to detect 8 batches of export cosmetic soap, forbidden components of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol are not detected in the result, and the interference of other components is not found.

And (4) conclusion: the research establishes an analysis method for measuring 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in the export cosmetic soap by salting out split-phase extraction-high performance liquid chromatography. The method is simple, rapid, economic and accurate, and is suitable for rapid screening and quantitative analysis of 2,4, 6-trichlorophenol, pentachlorophenol and thiochlorophenol in export cosmetic soap. Fills the blank of the method for detecting the three substances of the export cosmetic soap, and provides a technical support for the quality detection of the export soap.