CN111812265B - Detection method for simultaneously determining 32 dyes in hair dye - Google Patents
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Abstract
The invention belongs to the technical field of cosmetic detection, and particularly relates to a method for simultaneously detecting 32 dyes in a hair dye by using a high performance liquid chromatography-tandem mass spectrometry technology. The invention establishes a simple, convenient, rapid and high-flux measuring method, can simultaneously and quantitatively measure 32 dyes in the hair dye, has the advantages of simple operation, high sensitivity, good recovery rate, good reproducibility, short detection time, low solvent consumption, high detection flux and high detection efficiency, and has very important significance for analyzing the quality safety of the hair dye.
Description
Technical Field
The invention belongs to the technical field of cosmetic detection, and particularly relates to a detection method for simultaneously detecting 32 dyes in a hair dye by using a high performance liquid chromatography-tandem mass spectrometry technology.
Background
The hair dye is a common cosmetic, belongs to cosmetics with special purposes in China, and is divided into an oxidation type hair dye and a non-oxidation type hair dye, and 80 percent of the hair dye sold in the market is the oxidation type hair dye. At present, more and more people like hair dyeing, and the consumption population of the hair dye is younger and younger, so the safety of the hair dye is concerned. The harm of the hair dye is mainly from dyes, the main components of the dyes are aniline and phenol compounds, the compounds have allergenicity and carcinogenicity, chronic harm can be caused to human health after long-term use, and in recent years, researches report that the risk of people suffering from breast cancer and bladder cancer is increased by at least 25% due to the use of the hair dye. The use of hair dye is closely related to the health of people, so the quality management of the hair dye is strengthened and the detection technology of the hair dye is perfected.
The detection methods of the dyes reported in relevant documents at home and abroad comprise a thin layer chromatography, a capillary electrophoresis method, a high performance liquid chromatography, a gas chromatography-mass spectrometry, a high performance liquid chromatography-mass spectrometry and the like. The thin-layer chromatography cannot accurately quantify, the gas chromatography and the gas chromatography-mass spectrometry are not favorable for analyzing heat-labile substances, and the hair dye sample matrix is complicated, has an unsatisfactory separation effect and is difficult to characterize. Although the high performance liquid chromatography-mass spectrometry has strong separation capability and high sensitivity, the matrix effect can be reduced, and the target compound can be analyzed quickly and accurately, the method for detecting dye components in the hair dye is not included in technical Specification for cosmetic safety (2015), and when a plurality of dyes in the hair dye are detected by the prior art, the response values of components such as hydroquinone, resorcinol, 2-methylresorcinol, 4-amino-3-nitrophenol, 4-chlororesorcinol, 1, 5-naphthalenediol, 2, 7-naphthalenediol and 1-naphthol are low, the detection result is inaccurate, and the simultaneous detection of 32 dyes in the hair dye cannot be realized. Therefore, a method for simultaneously detecting 32 dyes in the hair dye based on liquid chromatography-mass spectrometry is urgently needed to be established.
The invention establishes a simple, convenient, rapid and high-throughput high performance liquid chromatography-tandem mass spectrometry detection method, can simultaneously detect 32 dyes in a dye agent, simultaneously realizes the analysis of the stability of 32 dye components, provides a theoretical basis for the accurate quantification and quality monitoring of the dye components, and provides a technical support for the detection of the dyes in the hair dye.
Disclosure of Invention
In view of the above technical problems, an object of the present invention is to provide a method for simultaneously measuring 32 dyes in a hair dye, wherein the 32 dyes are 2-amino-3-hydroxypyridine, p-phenylenediamine, p-aminophenol, m-phenylenediamine, 2, 6-diaminopyridine, m-aminophenol, toluene-2, 5-diamine sulfate, 2, 4-diaminophenoxyethanol hydrochloride, 4-amino-m-cresol, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, o-phenylenediamine, o-aminophenol, 2-chloro-p-phenylenediamine sulfate, phenylmethylpyrazolone, p-methylaminophenol sulfate, 2-nitro-p-phenylenediamine, 4-amino-2-hydroxytoluene, 4-nitro-o-phenylenediamine, toluene-3, 4-diamine, 6-amino-m-cresol, 6-hydroxyindole, N-diethyl-p-phenylenediamine sulfate, N-diethyl-toluene-2, 5-diamine hydrochloride, N-phenyl-p-phenylenediamine, hydroquinone, resorcinol, 2-methylresorcinol, 4-amino-3-nitrophenol, 4-chlororesorcinol, 1, 5-naphthalenediol, 2, 7-naphthalenediol, 1-naphthol; the method comprises the following steps:
(1) preparing a mixed standard solution;
(2) preparing a substrate labeling working solution:
adding a blank hair dye matrix into the mixed standard solution in the step (1) to obtain matrix standard-added working solutions with different concentrations, wherein the blank hair dye matrix is a hair dye without the 32 dyes in the claim 1;
(3) preparing a sample solution;
(4) respectively detecting and analyzing the standard solution and the sample solution by using a high performance liquid chromatography-tandem mass spectrometer:
wherein the chromatographic analysis conditions are as follows: a Waters Atlantis T3 chromatography column was used, column temperature: 40 ℃; the mobile phase A is 10mmol/L ammonium formate aqueous solution, and the pH value is 8; the mobile phase B is acetonitrile-methanol solution with the volume ratio of 1: 1; sample volume 2.0 μ L, flow rate: 0.5 mL/min; a gradient elution procedure was used: 0-0.5 min, 98% A; 0.5-1.3 min, 98-75% of A; 1.3-5.0 min, 75-30% of A; 5.0-6.0 min, 30-5% A; 6.0-7.0 min, 5% A; 7.0-7.1 min, 5-98% of A; 7.1-10 min, 98% A;
the mass spectrum conditions are as follows: mixing standard solution A with positive ion (ESI) of electrospray ion source+) Mode, ionization voltage is 3500V, and negative ion (ESI) is adopted in mixed standard solution B-) Mode, ionization voltage is-3500V; the heating Temperatures (TEM) were all: 500 ℃; the air curtain gas (CUR), the collision gas (CAD), the spray gas (GS1) and the auxiliary heating gas (GS2) are respectively as follows: 35psi, 8psi, 65psi, 50 psi; scanning mode: multiple Reaction Monitoring (MRM);
(5) calculation of the dye content in the sample:
and (3) carrying out regression analysis on the chromatographic peak area of the target substance detected by the matrix and standard working solution corresponding to the corresponding concentration of the target substance to obtain a standard curve linear equation, and then substituting the chromatographic peak area of the target substance detected by the sample solution into the standard curve linear equation corresponding to the target substance respectively to calculate the content of the 32 dye target substances in the sample.
Preferably, the preparation method of the mixed standard solution in the step (1) comprises the following steps:
2-amino-3-hydroxypyridine, p-phenylenediamine, p-aminophenol, m-phenylenediamine, 2, 6-diaminopyridine, m-aminophenol, toluene-2, 5-diamine sulfate, 2, 4-diaminophenoxyethanol hydrochloride, 4-amino-m-cresol, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, o-phenylenediamine, o-aminophenol, 2-chloro-p-phenylenediamine sulfate, phenylmethylpyrazolone, p-methylaminophenol sulfate, 2-nitro-p-phenylenediamine, 4-amino-2-hydroxytoluene, 4-nitro-o-phenylenediamine, toluene-3, 4-diamine, 6-amino-m-cresol, 6-hydroxyindole, N-diethyl-p-phenylenediamine sulfate, m-aminophenol, p-phenylenediamine sulfate, m-aminophenol, m-, Dissolving dye standard substances of N, N-diethyltoluene-2, 5-diamine hydrochloride and N-phenyl-p-phenylenediamine in a methanol-2 g/L sodium bisulfite water solution with the volume ratio of 1:1 to obtain a mixed standard solution A;
dissolving dye standard substances of hydroquinone, resorcinol, 2-methylresorcinol, 4-amino-3-nitrophenol, 4-chlororesorcinol, 1, 5-naphthalenediol, 2, 7-naphthalenediol and 1-naphthol by using a methanol-2 g/L sodium bisulfite water solution with the volume ratio of 1:1 to obtain a mixed standard solution B.
Preferably, the concentration of the mixed standard solution A is 600 mug/mL, and the concentration of the mixed standard solution B is 2000 mug/mL.
Preferably, the step (4) of mixing the standard solution A adopts electrospray ion source positive ion (ESI)+) Mode, ionization voltage is 3500V; the mixed standard solution B adopts negative ions (ESI)-) Mode, the ionization voltage was-3500V.
Preferably, the configuration of the sample solution in the step (3) is as follows: accurately weighing 0.5g of sample, adding 15mL of 2:8 methanol-2 g/L sodium bisulfite aqueous solution, mixing uniformly, centrifuging, taking supernatant, filtering with 0.22 μm filter membrane, and taking filtrate as sample solution for later use.
Preferably, the construction process of the standard curve linear equation in the step (5) is as follows: precisely measuring the mixed standard solution into a volumetric flask, preparing blank matrix labeling solutions with different concentrations, carrying out high performance liquid chromatography-tandem mass spectrometer analysis according to the analysis conditions in the step (4), and carrying out regression analysis according to the chromatographic peak area of the target object detected by the matrix labeling working solution corresponding to the corresponding concentration to obtain a standard curve linear equation.
The invention has the beneficial effects that: the invention establishes a simple, convenient, rapid and high-flux measuring method, and provides technical support for the detection of 32 dyes in the hair dye; the method has the advantages of simple operation, high sensitivity, good recovery rate, good reproducibility, short detection time, low solvent consumption, high detection flux and high detection efficiency, and has very important significance for analyzing the quality condition of the hair dye.
Drawings
FIG. 1 is a chromatogram of total ion current of a dye under detection conditions in positive ion mode;
FIG. 2 is a chromatogram of total ion current of a dye under detection conditions in negative ion mode;
FIG. 3 is a graph showing the peak area of the dye component with time in the positive ion detection mode;
FIG. 4 is a graph showing the peak area of the dye component as a function of time in the negative ion detection mode.
Detailed Description
The present invention is further described with reference to the following examples and accompanying drawings, but the present invention is not limited in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The instruments and reagents used in the present invention are as follows:
triple Quad 3500 hplc-tandem mass spectrometer (SCIEX corporation, usa); atlantis T3 column (100 mm. times.2.1 mm, 3 μm, Waters Corp., USA); Milli-Q IQ 7000 ultra pure water machine (Millipore corporation, USA); vortex blenders (Changzhou guohua appliances, Inc.); electronic balance (mettler, switzerland); centrifuge (xiang instrument centrifuge instruments ltd).
And (3) a hair dye standard product: p-phenylenediamine, p-aminophenol, m-aminophenol, 4-amino-m-cresol, 2, 5-diaminotoluene sulfate, 2, 6-diaminopyridine, 2-nitro-p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, N-diethyl-p-phenylenediamine sulfate, N-diethyl-toluene-2, 5-diamine hydrochloride, toluene-3, 4-diamine, o-aminophenol, 1-naphthol, 4-chlororesorcinol, hydroquinone, resorcinol (Dr. Ehrenston. Co., Germany, purity is not less than 98.0%), 4-amino-2-hydroxytoluene, 2-amino-3-hydroxypyridine, 2-chloro-p-phenylenediamine sulfate, 6-amino-m-cresol, p-methylaminophenol sulfate, N-phenyl-p-phenylenediamine, p-cresol, p-phenylenediamine, p-toluene-2, 4-amino-3-hydroxy-pyridine, p-cresol, 4-nitrophthalenediamine, 2, 7-naphthalenediol, 1, 5-naphthalenediol, 4-amino-3-nitrophenol, 2-methylresorcinol (no less than 98.0% purity, available from Beijing Manhage Biotech Co., Ltd.), N, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, phenylmethylpyrazolone, 6-hydroxyindole (no less than 98.0% purity, available from Beijing BePure Co., Ltd.), 2, 4-diaminophenoxyethanol hydrochloride (no less than 98.0% purity, available from Canada TRC Co., Ltd.); methanol, acetonitrile (chromatographically pure, Merck, germany); ammonium formate (chromatographically pure, Optima, germany); sodium bisulfite (analytical grade, chemical reagents of national drug group, ltd.); microfiltration (0.22 μm, organic phase type); the experimental water was first grade water.
Example 1 method for simultaneously measuring 32 dyes in hair dye
1. Preparation of standard solution
Respectively and precisely weighing 24 hair dye standard substances (2-amino-3-hydroxypyridine, p-phenylenediamine, p-aminophenol, m-phenylenediamine, 2, 6-diaminopyridine, m-aminophenol, toluene-2, 5-diamine sulfate, 2, 4-diaminophenoxyethanol hydrochloride, 4-amino-m-cresol, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, o-phenylenediamine, o-aminophenol, 2-chloro-p-phenylenediamine sulfate, phenylmethylpyrazolone, p-methylaminophenol sulfate, 2-nitro-p-phenylenediamine, 4-amino-2-hydroxytoluene, 4-nitro-o-phenylenediamine, toluene-3, 4-diamine, 6-amino-m-cresol, p-phenylenediamine, m-phenylenediamine, 6-hydroxyindole, N-diethyl-p-phenylenediamine sulfate, N-diethyl-toluene-2, 5-diamine hydrochloride and N-phenyl-p-phenylenediamine) 15mg are respectively placed in the same 25mL volumetric flask, methanol-2 g/L sodium bisulfite aqueous solution (volume ratio is 1:1) is used for constant volume to prepare positive ion mixed standard stock solution with each standard substance concentration of 600 mug/mL;
respectively and precisely weighing 50mg of 8 hair dye standard substances (hydroquinone, resorcinol, 2-methylresorcinol, 4-amino-3-nitrophenol, 4-chlororesorcinol, 1, 5-naphthalenediol, 2, 7-naphthalenediol and 1-naphthol) under the condition of negative ion mode detection in the same 25mL volumetric flask, and performing constant volume by using methanol-2 g/L sodium bisulfite aqueous solution (volume ratio of 1:1) to prepare negative ion mixed standard stock solution with the concentration of each standard substance of 2000 mu g/mL;
matrix-spiking working solution: precisely weighing the mixed standard stock solution into a volumetric flask according to the requirement, adding blank matrix (without 32 dyes), diluting and dissolving with methanol-2 g/L sodium bisulfite aqueous solution (volume ratio of 1:1) to the required concentration, and preparing the solution on site.
2. Sample pretreatment
Accurately weighing 0.5g of sample into a 25mL colorimetric tube with a plug, adding 15mL of mixed solution of methanol-2 g/L sodium bisulfite aqueous solution (volume ratio is 2:8), uniformly mixing for 1min in a vortex mode, using the mixed solution to fix the volume to 25mL, shaking up, centrifuging for 5min at 10000r/min, taking supernatant, filtering through a 0.22 mu m filter membrane, and taking filtrate as solution to be detected for later use. If necessary, the solution to be tested is diluted by a mixed solution of methanol and 2g/L sodium bisulfite aqueous solution (volume ratio is 2: 8).
3. Optimization of chromatographic and mass spectrometric conditions
Respectively adopting Atlantis T3 chromatographic column (100mm multiplied by 2.1mm, 3 mu m) and the capital Capcell PAK C1832 dyes are analyzed by a chromatographic column (150mm multiplied by 2.0mm, 3 mu m), and the result shows that the Atlantis T3 chromatographic column has good separation effect on isomers and the peak shape is more than that of C18The chromatographic column is more preferable. Meanwhile, the analysis effects of 32 dyes are compared when the pH value of 10mmol/L ammonium formate aqueous solution is 6, 7, 8 and 9, and the results show that under the optimized elution program, when the pH value of 10mmol/L ammonium formate aqueous solution is 8, the separation degree of each component is good and the response value is high. The total ion chromatogram of 24 dyes under positive ion mode detection condition and the total ion chromatogram of 8 dyes under negative ion mode detection condition are shown in FIG. 1 and FIG. 2 respectively, wherein 1 in FIG. 1 is 2-amino-3Hydroxypyridine, 2 p-phenylenediamine, 3 p-aminophenol, 4 m-phenylenediamine, 52, 6-diaminopyridine, 6 m-aminophenol, 7 toluene-2, 5-diamine sulfate, 8 2, 4-diaminophenoxyethanol hydrochloride, 9 4-amino-m-cresol, 10N, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, 11 o-phenylenediamine, 12 o-aminophenol, 13 2-chloro-p-phenylenediamine sulfate, 14 phenylmethylpyrazolone, 15 p-methylaminophenol sulfate, 16 2-nitro-p-phenylenediamine, 17 4-amino-2-hydroxytoluene, 18 4-nitro-o-phenylenediamine, 19 toluene-3, 4-diamine, 20 6-amino-m-cresol, 21 is 6-hydroxyindole, 22 is N, N-diethyl-p-phenylenediamine sulfate, 23 is N, N-diethyl-toluene-2, 5-diamine hydrochloride, and 24 is N-phenyl-p-phenylenediamine; in FIG. 2, 1 is hydroquinone, 2 is resorcinol, 3 is 2-methylresorcinol, 4 is 4-amino-3-nitrophenol, 5 is 4-chlororesorcinol, 6 is 1, 5-naphthalenediol, 7 is 2, 7-naphthalenediol, and 8 is 1-naphthol.
Performing primary mass spectrometry scanning on the 32 compounds respectively in a needle pump sample injection positive ion mode and a needle pump sample injection negative ion mode to obtain a parent ion (Q1) of each compound; performing secondary mass spectrometry scanning on the parent ions respectively to obtain information of fragment ions, and selecting two characteristic ions (Q3); and (3) optimizing mass spectrum parameters such as declustering voltage (DP), Collision Energy (CE), collision chamber outlet voltage (CXP) and the like of the two pairs of selected characteristic ion pairs to enable the response values of the two pairs of characteristic ion pairs to reach the best, wherein the specific parameters are shown in Table 1.
Table 132 main mass spectral parameters and retention times for the dye components
4. Optimized chromatographic and mass spectrum conditions
A chromatographic column: waters Atlantis T3 chromatography column; column temperature: 40 ℃; the sample size was 2.0. mu.L. The mobile phase a is 10mmol/L ammonium formate aqueous solution (the pH is adjusted to 8 by ammonia water), and the mobile phase B is acetonitrile-methanol solution (the volume ratio is 1: 1); flow rate: 0.5 mL/min. Gradient elution procedure: 0-0.5 min, 98% A; 0.5-1.3 min, 98-75% of A; 1.3-5.0 min, 75-30% of A; 5.0-6.0 min, 30-5% A; 6.0-7.0 min, 5% A; 7.0-7.1 min, 5-98% of A; 7.1-10 min, 98% A;
an ion source: electrospray ion source positive ion (ESI)+) Mode and negative ion (ESI-) mode; the ionization voltages (IS) are: 3500V and-3500V; the heating Temperatures (TEM) were all: 500 ℃; the air curtain gas (CUR), the collision gas (CAD), the spray gas (GS1) and the auxiliary heating gas (GS2) are respectively as follows: 35psi, 8psi, 65psi, 50 psi; scanning mode: multiple Reaction Monitoring (MRM).
5. Sample matrix effect
The Matrix Effect (ME) is the effect that causes enhancement or inhibition of the response value of the target compound:
ME (%) ═ response of blank matrix standard/response of pure solvent standard-1. times.100%
A hair dye sample without 32 dye components is used as a blank matrix, a blank matrix standard solution and a pure solvent standard solution with the same concentration are prepared, the pure solvent is methanol-2 g/L sodium bisulfite aqueous solution (the volume ratio is 1:1), and the calculated ME result is shown in Table 2. It can be seen that the matrix effect of 24 dye components in the positive ion detection mode is 21.9-44.6%, the matrix effect of 8 dye components in the negative ion detection mode is 4.1-19.5%, and the matrix effect of the dye components in the negative ion detection mode is much smaller than that of the dye components in the positive ion detection mode. The matrix effect of 24 dye components in the positive ion detection mode is relatively high, so that for accurate quantification, a matrix standard-added working solution is adopted for quantitative analysis.
Example 2 methodological validation
1. Linear range and detection limit
A sample without 32 dye components is used as a blank matrix, the peak area is used as the ordinate (Y), the corresponding substance concentration is used as the abscissa (X), and a matrix standard curve is established. The results show that the compounds have better ionisability and specificity, the components have good linear relation, and the correlation coefficient r is more than 0.995. The detection Limit (LOQ) of 24 dye components in a positive ion detection mode is 0.0052-0.43 mg/kg, and the quantification Limit (LOQ) is 0.018-1.5 mg/kg, wherein the signal-to-noise ratio (S/N) of a quantitative ion chromatographic peak is 3 and the S/N is 10; the detection limit of 8 dye components in the negative ion detection mode is 0.022-2.4 mg/kg, and the quantification limit is 0.078-7.9 mg/kg; the detection limits of 1-naphthol, 2-methyl resorcinol, hydroquinone and resorcinol are slightly higher in the negative ion detection mode. The specific results are shown in Table 2.
Table 2 linear equations, linear ranges, correlation coefficients, detection limits, quantitation limits, matrix effects for 32 dyes in a sample (n ═ 3)
2. Recovery and precision
And (5) performing the standard adding recovery rate and precision measurement by adopting a hair dye negative sample. The mixed standard solution with the concentration of 0.2, 2 and 10mg/kg is added into the hair dye negative sample, each concentration is parallelly measured for 6 times, 3 times of measurement are carried out, the average recovery rate of 24 dye components in a positive ion detection mode is 80.2-109.3%, and the average recovery rate of 8 dye components in a negative ion detection mode is 90.8-106.2%, and can be obtained from table 3. The matrix standard-added mixed solution with three concentrations is subjected to parallel measurement for 6 times, the total measurement is carried out for 3 rounds, the precision of the peak area is calculated, the precision can be obtained from the table 3, the Relative Standard Deviation (RSD) of the 24 dye components in the positive ion detection mode is 1.4-9.8%, the Relative Standard Deviation (RSD) of the 8 dye components in the negative ion detection mode is 0.8-8.5%, and the result shows that the method has better precision.
Table 3 recovery, precision (n ═ 6) of 32 dyes in the sample
Example 3 dye stability analysis
Because aniline and phenol molecules in 32 dyes are easy to oxidize, stability analysis is carried out. And (3) continuously injecting a mixed standard solution with the same concentration (the concentration of 24 dye components is 5mg/L in a positive ion detection mode, and the concentration of 8 dye components is 20mg/L in a negative ion detection mode) for 48 hours, wherein the phenyl methyl pyrazolone is the only one of 32 dyes and does not contain aniline and phenol compounds, and the chemical property is stable. Taking p-phenylenediamine, p-aminophenol, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, m-aminophenol and 4-amino-2-hydroxytoluene as examples, a time-dependent change diagram of the dye component peak area in a positive ion detection mode is shown in fig. 3, the dye component peak area in the positive ion detection mode increases with the increase of time, the 10-hour peak area increases by 11.7% -67.3%, and the average increase is 30.3%. Taking 2, 7-naphthalenediol, 1, 5-naphthalenediol, 4-amino-3-nitrophenol, 1-naphthol and 4-chlororesorcinol as examples, the peak area of the dye component in the negative ion detection mode changes with time as shown in fig. 4, the peak area of the dye component in the negative ion detection mode decreases with time, the peak area of most compounds decreases significantly at 20 hours, and the peak area decreases by 29.6% on average.
The above embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications made based on the structure, characteristics and principles of the invention should be included in the claims of the present invention.
Claims (3)
1. A method for simultaneously measuring 32 dyes in a hair dye, wherein the 32 dyes are 2-amino-3-hydroxypyridine, p-phenylenediamine, p-aminophenol, m-phenylenediamine, 2, 6-diaminopyridine, m-aminophenol, toluene-2, 5-diamine sulfate, 2, 4-diaminophenoxyethanol hydrochloride, 4-amino-m-cresol, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, o-phenylenediamine, o-aminophenol, 2-chloro-p-phenylenediamine sulfate, phenylmethylpyrazolone, p-methylaminophenol sulfate, 2-nitro-p-phenylenediamine, 4-amino-2-hydroxytoluene, 4-nitro-o-phenylenediamine, toluene-3, 4-diamine, 6-amino-m-cresol, p-phenylenediamine, p, 6-hydroxyindole, N-diethyl-p-phenylenediamine sulfate, N-diethyl-toluene-2, 5-diamine hydrochloride, N-phenyl-p-phenylenediamine, hydroquinone, resorcinol, 2-methyl resorcinol, 4-amino-3-nitrophenol, 4-chlororesorcinol, 1, 5-naphthalenediol, 2, 7-naphthalenediol, 1-naphthol; characterized in that the method comprises the following steps:
(1) preparation of mixed standard solution:
2-amino-3-hydroxypyridine, p-phenylenediamine, p-aminophenol, m-phenylenediamine, 2, 6-diaminopyridine, m-aminophenol, toluene-2, 5-diamine sulfate, 2, 4-diaminophenoxyethanol hydrochloride, 4-amino-m-cresol, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, o-phenylenediamine, o-aminophenol, 2-chloro-p-phenylenediamine sulfate, phenylmethylpyrazolone, p-methylaminophenol sulfate, 2-nitro-p-phenylenediamine, 4-amino-2-hydroxytoluene, 4-nitro-o-phenylenediamine, toluene-3, 4-diamine, 6-amino-m-cresol, 6-hydroxyindole, N-diethyl-p-phenylenediamine sulfate, m-aminophenol, p-phenylenediamine sulfate, m-aminophenol, m-, Dissolving dye standard substances of N, N-diethyltoluene-2, 5-diamine hydrochloride and N-phenyl-p-phenylenediamine in a methanol-2 g/L sodium bisulfite water solution with the volume ratio of 1:1 to obtain a mixed standard solution A;
dissolving dye standard substances of hydroquinone, resorcinol, 2-methylresorcinol, 4-amino-3-nitrophenol, 4-chlororesorcinol, 1, 5-naphthalenediol, 2, 7-naphthalenediol and 1-naphthol by using a methanol-2 g/L sodium bisulfite water solution with the volume ratio of 1:1 to obtain a mixed standard solution B;
(2) preparing a substrate labeling working solution:
adding a blank hair dye substrate into the mixed standard solution in the step (1) to obtain substrate standard-added working solutions with different concentrations, wherein the blank hair dye substrate is a hair dye without the 32 dyes;
(3) preparation of sample solution:
accurately weighing 0.5g of sample, adding 15mL of sodium bisulfite aqueous solution with the volume ratio of 2:8 methanol to 2g/L, uniformly mixing, centrifuging, taking supernatant, filtering with a 0.22 mu m filter membrane, and taking filtrate as sample solution for later use;
(4) respectively detecting and analyzing the standard solution and the sample solution by using a high performance liquid chromatography-tandem mass spectrometer:
wherein the chromatographic analysis conditions are as follows: a Waters Atlantis T3 chromatography column was used, column temperature: 40 ℃; the mobile phase A is 10mmol/L ammonium formate aqueous solution, and the pH value is 8; the mobile phase B is acetonitrile-methanol solution with the volume ratio of 1: 1; sample volume 2.0 μ L, flow rate: 0.5 mL/min; a gradient elution procedure was used: 0-0.5 min, 98% A; 0.5-1.3 min, 98-75% of A; 1.3-5.0 min, 75-30% of A; 5.0-6.0 min, 30-5% A; 6.0-7.0 min, 5% A; 7.0-7.1 min, 5-98% of A; 7.1-10 min, 98% A;
the mass spectrum conditions are as follows: electrospray ion source positive ion (ESI)+) Mode, ionization voltage is 3500V; electrospray ion source negative ion (ESI)-) Mode, ionization voltage is-3500V; the heating Temperatures (TEM) were all: 500 ℃; the air curtain gas (CUR), the collision gas (CAD), the spray gas (GS1) and the auxiliary heating gas (GS2) are respectively as follows: 35psi, 8psi, 65psi, 50 psi; scanning mode: multiple Reaction Monitoring (MRM);
the mixed standard solution A adopts electrospray ion source positive ion (ESI)+) Mode, ionization voltage is 3500V; the mixed standard solution B adopts negative ions (ESI)-) Mode, ionization voltage is-3500V;
(5) calculation of the dye content in the sample:
and (3) carrying out regression analysis on the chromatographic peak area of the target substance detected by the matrix and standard working solution corresponding to the corresponding concentration of the target substance to obtain a standard curve linear equation, and then substituting the chromatographic peak area of the target substance detected by the sample solution into the standard curve linear equation corresponding to the target substance respectively to calculate the content of the 32 dye target substances in the sample.
2. The method of claim 1, wherein the concentration of mixed standard solution a is 600 μ g/mL and the concentration of mixed standard solution B is 2000 μ g/mL.
3. The method of claim 1, wherein the standard curve linear equation in the step (5) is constructed by: precisely measuring the mixed standard solution into a volumetric flask, preparing blank matrix labeling solutions with different concentrations, carrying out high performance liquid chromatography-tandem mass spectrometer analysis according to the analysis conditions in the step (4), and carrying out regression analysis according to the chromatographic peak area of the target object detected by the matrix labeling working solution corresponding to the corresponding concentration to obtain a standard curve linear equation.
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