A quantitative and / or qualitative method for simultaneous determination of 54 hair dyes in hair dye products

Abstract

The invention discloses a method for simultaneously determining 54 kinds of hair dyes in hair dye products by high-performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry, using 70% methanol ultrasonic extraction, 25% methanol (containing and excluding D-isoascorbic acid) solvent dilution sample, and matching the quantitative method of solvent mark line, improving the stability of hair dye and the accuracy of measurement results; by selecting chromatographic column and selecting ammonium fluoride solution-acetonitrile as mobile phase, the separation of all isomers is guaranteed, and the sensitivity of negative ion monitoring components such as phenol and hydroquinone is improved; by simultaneously scanning positive and negative ion modes and data-dependent secondary ion full scanning, 54 kinds of hair dyes are accurately determined and quantified. This method solves the problems of stability, separation and specificity, difficulty in filtration and membrane adsorption in the actual detection process of hair dye, makes up for the problem that the existing literature and standard hair dyes are not fully covered, and is of great significance to the quality control of hair dye products.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics detection, and specifically relates to a quantitative and / or qualitative method for simultaneously determining 54 hair dyes in hair dye products by using high performance liquid chromatography-high resolution mass spectrometry. Background Art

[0002] Hair dye cosmetics are a type of cosmetics used to change hair color and beautify hair. With the development of the national economy, people's demand for beauty is constantly increasing, and hair dyeing has become one of the indispensable beauty methods in contemporary times. Although many hair dye products claim to be "pure plant ingredients", "natural", and "ammonia-free and mild", due to the frequent occurrence of patients with hair dye allergies, the question of whether the use of hair dye cosmetics is healthy and safe has become a focus of public concern and debate.

[0003] According to the duration of the color after dyeing, hair dye cosmetics can be divided into three categories: temporary, semi-permanent, and permanent. Most of the hair dyes currently sold on the market are permanent hair dye cosmetics, also known as oxidative hair dye products, which often contain aromatic amines and phenolic small molecule oxidative dyes, which have certain skin sensitization. Long-term use has different degrees of carcinogenicity and mutagenicity. Therefore, oxidative products need to undergo safety evaluation and obtain a special-purpose cosmetics license before they can be produced and sold. However, from the inspection results of unqualified hair dye cosmetics announced by the State Food and Drug Administration in recent years, it can be seen that there are serious problems with the ingredients of hair dyes. Many companies change the formula at will and do not produce according to the approved formula, which poses serious quality and safety risks. Most of the hair dyes contained in temporary and semi-permanent hair dye products are synthetic dyes, and long-term use also has potential hazards such as photosensitivity and reproductive toxicity.

[0004] Therefore, the key and difficulty of safety and quality control of hair dye cosmetics is whether the types of hair dyes in the products are produced according to the approved or registered formulas, and whether the content of each hair dye exceeds the limits listed in my country's "Technical Specifications for Safety of Cosmetics" (2015 edition, hereinafter referred to as the "Specifications"), that is, exclusive qualitative and accurate quantitative issues.

[0005] At present, the detection methods of hair dyes mainly include paper chromatography, liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography, gas chromatography-mass spectrometry, ion chromatography and micellar electrokinetic chromatography. Based on the properties that hair dyes are mostly multi-component aromatic polar small molecules, the most common and suitable determination methods are high performance liquid chromatography-ultraviolet / photodiode array detector (hereinafter referred to as "high performance liquid chromatography") and liquid chromatography-mass spectrometry.

[0006] The "Specification" lists a total of 75 permitted hair dyes, including 56 free radicals. Although the determination methods for 32 hair dyes such as p-phenylenediamine included in the "Specification" provide strong support for the quality and safety monitoring of hair dyes, they only include 24 permitted and 8 prohibited hair dyes. In routine inspections, it is found that there are still many hair dyes marked on the labels of hair dye products that are not among the 32 components, resulting in nearly half of the hair dye products being unable to receive effective market supervision, and there is a risk that illegal companies will take advantage of loopholes in the supervision and inspection standards. The HPLC determination included in the "Specification" method, whether using the C18 or C16 system, needs to be divided into multiple liquid phase systems to achieve complete separation of the 32 components, and the interference of the matrix cannot be completely eliminated; the accompanying LC-MS confirmation method is limited by the separation degree and sensitivity, and requires the establishment of two positive and negative detection systems.

[0007] In the actual inspection of hair dye products, it was found that the use of a single method could not meet the needs of hair dye inspection. The reasons are as follows: although after optimization and subsystem, the use of HPLC can separate dozens of hair dye components, the presence of other substances in different hair dye products cannot avoid interfering with the detection of the target hair dye; especially when the hair dye content is low, it is impossible to use the UV spectrum to determine the specificity; in addition, in order to ensure the separation degree, the HPLC method often requires a chromatographic operation time of more than 40 minutes, and the hair dye has poor stability, so large-scale inspection cannot be achieved.

[0008] Therefore, liquid chromatography-mass spectrometry has the characteristics of high specificity and high throughput (except for isomers, no complete separation is required), and has significant advantages in the inspection of hair dyes. Although there are literature reports on other LC-MS methods for the 32 hair dyes measured in the "Specifications", the following problems still exist:

[0009] (1) The types of hair dyes that can be tested simultaneously are seriously insufficient and cannot meet the needs of product quality and safety control;

[0010] (2) According to the amount of hair dye added to the actual hair dye sample, the pre-treatment methods in the literature will cause the content of common components to far exceed the upper limit of quantification (generally one to two orders of magnitude), which is easy to contaminate the instrument and form cross contamination, so it is not suitable for large-scale screening. In addition, if the sample is diluted after the screening calculation, the hair dye is likely to degrade, and the sample needs to be re-weighed and processed, which cannot really achieve rapid determination and lacks practical application value.

[0011] (3) It is not possible to take into account the sensitivity of hair dye components in both positive and negative ion modes. When the substances are completely separated in the positive ion mode, the sensitivity of the components measured in the negative ion mode is poor, far lower than that of the hair dye components measured in the positive ion mode, resulting in low overlap in the linear range of each substance, making accurate quantification difficult;

[0012] (4) Serious matrix effect: In order to accommodate components with low sensitivity, the sample dilution level is low; hair dyes are mainly polar small molecules (most hair dyes have a molecular weight below 200 Da), and co-elution is inevitable. Although some literature uses the matrix matching method to prepare the calibration line, the representativeness of the blank matrix cannot be guaranteed, and the recovery rate determination results are unreliable;

[0013] (5) There is no definite solution to the problem of hair dye component stability. In the method of the "Specification", sodium bisulfite is added as an antioxidant to prepare the stock solution of hair dye (hair dye concentration is greater than 2.5g / L), which is stored in a refrigerator at 0℃~4℃ and should be used within 48 hours; however, the standard curve solution is required to be "prepared before use". The method of Chinese patent 202010662203.2 found that most hair dyes at the concentration measured by LC-MS were significantly degraded within 20 hours. The instability of hair dyes creates serious uncertainty and uncontrollability for large-scale accurate quantification. In particular, it has been verified that for hair dyes other than the 32 types measured in the "Specification", such as 1,3-bis-(2,4-diaminophenoxy)propane, 2,6-dimethoxy-3,5-pyridinediamine hydrochloride and 2-amino-4-hydroxyethylaminoanisole, the addition of sodium bisulfite as an antioxidant cannot ensure stability within 12 hours and cannot meet the quantitative measurement requirements. Summary of the invention

[0014] In view of the above-mentioned technical problems existing in the prior art, the present invention has established an exclusive, sensitive, durable, high-throughput high-performance liquid chromatography-high-resolution mass spectrometry method, which can simultaneously detect 54 hair dye ingredients, covering both the common prohibited and restricted components in hair dye products on the market (especially oxidative hair dye products), and having practical application value. It can simultaneously realize the separation of multiple groups of isomers in the positive ion mode under the same mobile phase, and ensure the sensitivity of components such as phenol, hydroquinone, and 1-naphthol in the negative ion mode; it solves the problems of difficult filtration of hair dye product extracts and membrane adsorption, differences in the stability and matrix effect of hair dyes in different solutions and matrices, and differences in the content of hair dye components in samples, which affect the accuracy of quantitative results and the durability of the measurement method.

[0015] The detection method of the present invention can ensure the full separation of isomers under a chromatographic system, ensure the qualitative specificity, and take into account the sensitivity under negative ion conditions; the pre-treatment strategy is applicable to the application of most hair dye products, and solves the problems of matrix effect, stability, filter membrane adsorption, etc. that affect the quantitative accuracy; the proposed determination scheme can greatly improve the controllability of the results of the inspection process.

[0016] The first object of the present invention is to provide a method for simultaneously determining 54 hair dyes in hair dye products by high performance liquid chromatography-quadrupole / electrostatic field orbital trap high resolution mass spectrometry, wherein the hair dyes are 1-hydroxyethyl-4,5-diaminopyrazole sulfate (No.: A01), hydroxyethyl-p-phenylenediamine sulfate (No.: A02), tetraaminopyrimidine sulfate (No.: A03), 2,6-dihydroxyethylaminotoluene (No.: A04), 2-amino-6-chloro-4-nitrophenol (No.: A05), 2-methyl-5-hydroxyethylaminophenol (No.: A06), 3-nitro-p-hydroxyethylaminophenol (No.: A07), 4-hydroxypropylamino-3-nitrophenol (No.: A08), 5-amino-4-chloro-o-cresol (No.: A09), : A09), 5-amino-6-chloro-o-cresol (No.: A10), HC Yellow No. 2 (No.: A11), Hydroxybenzomorpholine (No.: A12), Hydroxyethyl-2-nitro-p-toluidine (No.: A13), 1,3-bis-(2,4-diaminophenoxy)propane (No.: A14), 2,6-dimethoxy-3,5-pyridinediamine hydrochloride (No.: A15), 2-amino-4-hydroxyethylaminoanisole (No.: A16), Phenol (No.: A17), N-hydroxyethyl-3,4-methylenedioxyaniline hydrochloride (No.: A18), HC Yellow No. 4 (No.: A19), HC Red No. 1 (No.: A20), HC Orange No. 1 (No.: A21), Disperse Violet (No.: A22 )、p-phenylenediamine (No.: S01), p-aminophenol (No.: S02), toluene-2,5-diamine sulfate (No.: S03), m-aminophenol (No.: S04), o-phenylenediamine (No.: S05), 2-chloro-p-phenylenediamine sulfate (No.: S06), o-aminophenol (No.: S07), resorcinol (No.: S08), 2-nitro-p-phenylenediamine (No.: S09), toluene-3,4-diamine (No.: S10), 4-amino-2-hydroxytoluene (No.: S11), 2-methylresorcinol (No.: S12), 6-amino-m-cresol (No.: S13), phenylmethylpyrazolone (No.: S14), N,N-diethyltoluene-2,5-diamine hydrochloride (No.: S15), S15), 4-amino-3-nitrophenol (S16), m-phenylenediamine (S17), 2,4-diaminophenoxyethanol hydrochloride (S18), hydroquinone (S19), 4-amino-m-cresol (S20), 2-amino-3-hydroxypyridine (S21), N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate (S22), p-methylaminophenol sulfate (S23), 4-nitro-o-phenylenediamine (S24), 2,6-diaminopyridine (S25), N,N-diethyl-p-phenylenediamine sulfate (S26), 6-hydroxyindole (S27), 4-chlororesorcinol (S28), 2,7-naphthol (No.: S29), N-phenyl-p-phenylenediamine (No.: S30), 1,5-naphthol (No.: S31), 1-naphthol (No.: S32);

[0017] The method comprises the following steps:

[0018] S1 Preparation of reference solution

[0019] S1-1 Preparation of mixed standard qualitative solution

[0020] Dissolve 54 hair dye reference substances in 10% to 30% methanol solution to prepare a mixed standard qualitative solution I with a mass concentration of hydroquinone (S19) of 400 to 4000 ng / mL and a mass concentration of other hair dyes of 100 to 1000 ng / mL, which is used for the qualitative analysis of o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24);

[0021] 54 hair dye reference substances were dissolved in 10% to 30% methanol solution containing antioxidants to prepare a mixed standard qualitative solution II with a mass concentration of hydroquinone (S19) of 400 to 4000 ng / mL and a mass concentration of other hair dyes of 100 to 1000 ng / mL, which was used for the qualitative analysis of the other 51 hair dyes;

[0022] Preparation of S1-2 series mixed standard curve solution

[0023] Dissolve 54 hair dye reference substances in 10% to 30% methanol solution, and dilute them serially to obtain a series of mixed standard curve solutions I with concentrations ranging from 32 to 8000 ng / mL for hydroquinone (S19) and 8 to 2000 ng / mL for other hair dyes, which are used for qualitative and / or quantitative determination of o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24);

[0024] Dissolve 54 hair dye reference substances in 10% to 30% methanol solution containing antioxidants, and dilute them continuously to obtain a series of mixed standard curve solutions II with a hydroquinone concentration range of 32 to 8000 ng / mL and other hair dye concentrations range of 8 to 2000 ng / mL, which are used for qualitative and / or quantitative determination of 51 components except o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24);

[0025] Preparation of S2 sample solution

[0026] S2-1 Preparation of sample stock solution:

[0027] Weigh the sample, dilute the alcohol solution to no more than 20 mg / mL, extract in an ice bath with ultrasonic waves for 5 to 20 min, shake well, filter through an organic filter membrane, and take the filtrate for later use to obtain the sample stock solution;

[0028] Preferably, the sample is weighed, diluted with an alcohol solution to 10-20 mg / mL, extracted with an ice bath ultrasonic wave for 5-20 min, shaken well, filtered through an organic filter membrane, and the filtrate is taken for later use to obtain a sample stock solution;

[0029] The alcohol solution is 50% to 80% methanol or 50% to 80% ethanol;

[0030] S2-2 Prepare sample solution I and sample solution II:

[0031] The sample stock solution is diluted with 10% to 30% methanol solution to prepare sample solution Ⅰ-1 and sample solution Ⅰ-2, wherein the concentration of the sample stock solution in the sample solution Ⅰ-1 is 20 μL / mL, and the concentration of the sample stock solution in the sample solution Ⅰ-2 is 2 μL / mL; used for accurate characterization and / or quantification of o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24);

[0032] The sample stock solution was diluted with a 10% to 30% methanol solution containing an antioxidant to prepare sample solution II-1 and sample solution II-2, wherein the sample stock solution concentration in the sample solution II-1 was 20 μL / mL and the sample stock solution concentration in the sample solution II-2 was 2 μL / mL; used for accurate qualitative and / or quantitative determination of the other 51 components;

[0033] S3 Chromatography-Mass Spectrometry Detection Conditions

[0034] Chromatographic conditions:

[0035] Chromatographic column: C18 column, column temperature: 40℃; mobile phase: phase A is 0.5~5mmol / L ammonium fluoride aqueous solution; phase B is methanol or acetonitrile, flow rate: 0.2~0.5mL / min. Gradient elution program: 0~2.0min, 10% B; 2.0~8.5min, 10% B~60% B; 8.5~10.0min, 60% B~95% B; 10.0~12.0min, 95% B; 12.0~12.1min, 95% B~10% B; 12.1~15.0min, 10% B; injection volume: 0.5~10μL; injection plate temperature: 4℃;

[0036] Mass spectrometry conditions:

[0037] Spray voltage: 1-4.5 kV in positive ion mode, -1-4 kV in negative ion mode; nebulization temperature: 300-400 °C; ion transfer tube temperature: 300-350 °C; sheath gas pressure: 50 Arb; auxiliary gas pressure: 10 Arb; full scan of primary mass spectrometry (Fullscan), scanning range (m / z): 50-300 Da; resolution: R = 60 000 or 120 000; data-dependent secondary ion full scan (Full MS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2), intensity threshold: 1.0×10 5 ; ddMS2 resolution: R = 15 000 or 30 000; In a particular embodiment, ion transfer tube temperature: 325°C.

[0038] S4 Qualitative and / or quantitative determination of samples

[0039] When both quantitative and qualitative determinations are required, perform S4a qualitative and quantitative determinations:

[0040] S4a-1 injects the mixed standard qualitative solution I prepared in S1-1 to determine the retention time of o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24), and the signal-to-noise ratio of the three chromatographic peaks should be greater than 100; injects the sample solution I-1 to determine whether o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24) are detected; if not detected, proceed to step S4a-3; if detected, proceed to steps S4a-2 and S4a-3;

[0041] S4a-2 injects the series mixed standard curve solution I prepared by S1-2, and uses the chromatographic peak areas of o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24) to perform linear regression on their concentrations, and draws a linear equation; injects sample solution I-2 and sample solution I-1 in sequence, and substitutes the peak areas into the linear equation of the series mixed standard curve solution I, and calculates the contents of o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24) by the external standard method;

[0042] S4a-3 injects the series mixed standard curve solution II prepared in S1-2, performs linear regression on the concentration of each chromatographic peak area, and draws a linear equation; injects sample solution II-2 and sample solution II-1 in sequence, substitutes the peak area into the linear equation of the series mixed standard curve solution II, and calculates the contents of 51 components except o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24) by the external standard method;

[0043] When only qualitative determination is required, perform S4b qualitative determination:

[0044] The sample solution I-1, the sample solution I-2 and the mixed standard qualitative solution I prepared by S1-1 are subjected to chromatography-mass spectrometry detection. When the chromatographic retention time of the sample solution I is consistent with that of the mixed standard qualitative solution I, and the fragment ion detection fragments of the sample solution I and the secondary mass spectrum are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of o-phenylenediamine (S05), toluene-3,4-diamine (S10), and 4-nitro-o-phenylenediamine (S24);

[0045] The sample solution II-1, the sample solution II-2 and the mixed standard qualitative solution II prepared with S1-1 are subjected to chromatography-mass spectrometry detection. When the chromatographic retention time of the sample solution II is consistent with that of the mixed standard qualitative solution II, and the fragment ion detection fragments of the sample solution II and in the secondary mass spectrum are consistent with those of the mixed standard qualitative solution II, the sample includes one or more of the 51 components other than o-phenylenediamine (S05), toluene-3,4-diamine (S10) and 4-nitro-o-phenylenediamine (S24).

[0046] Since the 54 hair dyes in the present application have isomers, in qualitative detection, the retention time and the fragment ion detection fragments in the secondary mass spectrometry need to be met at the same time.

[0047] Furthermore, the mixed standard qualitative solution described in S1-1 is prepared by the method described in (1) or (2):

[0048] (1) Prepare a mixed standard stock solution. The mixed standard stock solution is prepared by mixing 54 hair dye reference substances and dissolving them in 50% to 80% methanol solution to obtain a mixed standard stock solution. The mixed standard stock solution can be prepared directly as follows or stored at -20°C in the dark. When used:

[0049] Dilute the mixed standard stock solution with 10% to 30% methanol solution to prepare a mixed standard qualitative solution I;

[0050] The mixed standard stock solution is diluted with a 10% to 30% methanol solution containing an antioxidant to prepare a mixed standard qualitative solution II;

[0051] (2) Prepare single-label stock solutions. The preparation method of the single-label stock solutions is as follows: dissolve S05, S10, and S24 reference substances in 50% to 80% methanol solution; dissolve other hair dye reference substances in 50% to 80% methanol solution containing antioxidants; and obtain 54 single-label stock solutions respectively. The solutions can be directly prepared as follows or stored at -20°C in the dark. When used:

[0052] The single standard stock solutions of o-phenylenediamine, toluene-3,4-diamine and 4-nitro-o-phenylenediamine are mixed, and 10% to 30% methanol solution is added to prepare a mixed standard qualitative solution I;

[0053] The 54 single standard stock solutions were mixed, and a 10% to 30% methanol solution containing an antioxidant was added to prepare a mixed standard qualitative solution II.

[0054] Furthermore, the series of mixed standard curve solutions described in S1-2 are prepared by the method described in (1) or (2):

[0055] (1) preparing a mixed standard stock solution, wherein the mixed standard stock solution is prepared by taking 54 hair dye reference substances, mixing them, dissolving them in 50% to 80% methanol solution, and preparing a mixed standard stock solution; the mixed standard stock solution can be directly prepared as follows or stored at -20°C in the dark. When used:

[0056] Continuously diluting the mixed standard stock solution with 10% to 30% methanol solution to obtain a series of mixed standard curve solutions I of the several concentrations;

[0057] Continuously diluting the mixed standard stock solution with a 10% to 30% methanol solution containing an antioxidant to obtain a series of mixed standard curve solutions II of the aforementioned several concentrations;

[0058] (2) Prepare single-label stock solutions, the preparation method of the single-label stock solutions is as follows: o-phenylenediamine, toluene-3,4-diamine, 4-nitro-o-phenylenediamine reference substances are respectively dissolved in 50% to 80% methanol solution; other hair dye reference substances are respectively dissolved in 50% to 80% methanol solution containing antioxidants; 54 single-label stock solutions are obtained, which can be directly prepared as follows or stored at -20°C in the dark. When used:

[0059] The single standard stock solutions of o-phenylenediamine, toluene-3,4-diamine and 4-nitro-o-phenylenediamine are mixed, and 10% to 30% methanol solution is added to prepare a mixed standard qualitative solution I;

[0060] The 54 single standard stock solutions were mixed, and a 10% to 30% methanol solution containing an antioxidant was added to prepare a mixed standard qualitative solution II.

[0061] Furthermore, the mass concentration of the 54 hair dyes in the mixed standard stock solution and the single standard stock solution is not higher than 5000 μg / mL;

[0062] Preferably, the mass concentration of hydroquinone (S19) in the mixed standard stock solution and the single standard stock solution is not higher than 800 μg / mL, and the mass concentration of other hair dyes is not higher than 200 μg / mL; further preferably,

[0063] Further preferably, the mass concentration of hydroquinone (S19) in the mixed standard stock solution and the single standard stock solution is not higher than 40 μg / mL, and the mass concentration of other hair dyes is not higher than 10 μg / mL.

[0064] Furthermore, the 10% to 30% methanol solution in S1-1, S1-2, and S2-2 is a 25% methanol solution; the 10% to 30% methanol solution containing an antioxidant in S1-1, S1-2, and S2-2 is a 25% methanol solution containing an antioxidant.

[0065] Furthermore, in the 10% to 30% methanol solution containing an antioxidant in S1-1, S1-2, and S2-2, the antioxidant is D-isoascorbic acid or L-ascorbic acid; preferably, the oxidant is D-isoascorbic acid.

[0066] Furthermore, the antioxidant concentration in the 10% to 30% methanol solution containing antioxidant in S1-1, S1-2 and S2-2 is not less than 0.05%; preferably, the oxidant concentration is 0.05%.

[0067] During the research process, the present application systematically investigated the stability of each component under different antioxidants, including sodium bisulfite, D-isoascorbic acid, L-ascorbic acid, pyrogallic acid, citric acid, etc. The results show that D-isoascorbic acid and L-ascorbic acid have the best antioxidant effect, and the effect is similar, and the stability of most hair dyes is significantly improved, especially A14, A15 and A16; however, the stability of some hair dyes is reduced in the presence of antioxidants, such as A02, S03, S06 have poor stability in sodium bisulfite solution, and components containing o-phenylenediamine structure (S05, S10 and S24) will react chemically with D-isoascorbic acid (L-ascorbic acid), thereby significantly reducing stability. S21, S25, A07, A08, A09, A11, A13, A21 and A22 are all relatively stable in various solvents. In addition to affecting the stability of hair dyes, different oxidants will also produce matrix effects for components that are less retained. For example, sodium bisulfite solution will produce a matrix inhibition effect, while ascorbic acid will produce a stronger matrix enhancement effect. The intensity of the effect is positively correlated with the concentration of the antioxidant. In addition, the presence of D-isoascorbic acid, L-ascorbic acid or pyrogallic acid will significantly improve the peak shape of substances such as A02, S03, S14, S15, S22 and S26, especially S30. The concentration of D-isoascorbic acid must be greater than 0.05% to produce a matrix effect. When the concentration is greater than 0.05%, the matrix effect is stable.

[0068] Furthermore, S1 also includes S1-3 preparing a positioning solution:

[0069] Take the above single standard stock solution, add 10% to 30% methanol solution, and prepare the positioning solution I with the mass concentration of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine being 400 to 4000 ng / mL, and the mass concentration of other hair dyes being 100 to 1000 ng / mL;

[0070] Take the above-mentioned single-label stock solutions, add 10% to 30% methanol solution containing antioxidants, and prepare positioning solution II with a mass concentration of S19 of 400 to 4000 ng / mL and a mass concentration of other hair dyes of 100 to 1000 ng / mL.

[0071] During the research process of the technical solution of the present application, a single-label stock solution was used for stability investigation. When the actual operation of the detection method of the present invention was used, it was considered that there were about 8 groups of isomers in the 54 hair dyes of the present application, so a single-label stock solution was required for positioning;

[0072] Since the 54 hair dyes contain multiple groups of isomers, a single-standard positioning solution was used to determine the peak order of the isomers when establishing the method; if the chromatographic conditions of the present invention are fully adopted, relative retention can be used as a reference without the need to prepare a positioning solution.

[0073] Furthermore, the concentrations of the sample solutions I-1 and II-1 in S2-2 are 200 to 400 μg / mL; the concentrations of the sample solutions I-2 and II-2 in S2-2 are 20 to 40 μg / mL.

[0074] Furthermore, the alcohol solution in S2 is a 50% to 80% methanol solution or a 50% to 80% ethanol solution. Preferably, the alcohol solution is a 70% methanol solution, which is beneficial to the dissolution and filtration of the target substance.

[0075] Furthermore, the pore size of the organic filter membrane in S2 is 0.22 μm or 0.45 μm. Preferably, since the sample solution is difficult to filter and will be diluted in the next step, the pore size of the organic filter membrane is 0.45 μm.

[0076] Furthermore, the organic filter membrane in S2 is a polytetrafluoroethylene membrane.

[0077] During the research process of this application, water membrane and organic membrane were systematically investigated. Water membrane is easy to dissolve and has a poor recovery rate, so it is not suitable. Among organic membranes, nylon membrane and polyvinylidene fluoride filter membrane (PVDF) have large differences among different brands. Some manufacturers have serious adsorption of negative ion detection substances (such as S08, S12, S27, S29, S30 and S32). A20, A21 and A22 are easily adsorbed by the filter membrane. The adsorption of the filter membrane decreases with the decrease of the proportion of organic phase in the solvent. The recovery rate of polytetrafluoroethylene membranes from different manufacturers is relatively stable.

[0078] Further, the chromatographic conditions described in S3 are: chromatographic column: ACE Excel C18 column (150mm×2.0mm, 3μm), column temperature: 40°C; mobile phase: phase A is 1mmol / L ammonium fluoride aqueous solution; phase B is acetonitrile, flow rate: 0.3mL / min. Gradient elution program: 0-2.0min, 10% B; 2.0-8.5min, 10% B-60% B; 8.5-10.0min, 60% B-95% B; 10.0-12.0min, 95% B; 12.0-12.1min, 95% B-10% B; 12.1-15.0min, 10% B; injection volume: 1μL; injection plate temperature: 4°C.

[0079] Furthermore, the mass spectrometry conditions described in S3 are as follows: spray voltage: 1.5 kV in positive ion mode, -2.5 kV in negative ion mode; nebulization temperature: 350°C; ion transfer tube temperature: 325°C; sheath gas pressure: 50 Arb; auxiliary gas pressure: 10 Arb; primary mass spectrometry full scan (Full scan), scanning range (m / z): 50-300 Da; full scan resolution: R=60 000; data-dependent secondary ion full scan (Full MS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2), intensity threshold: 1.0×10 5 ;ddMS2 resolution: R=15 000.

[0080] Furthermore, in the Targeted Mass of the secondary ion full scan (Full MS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2) described in S3, the retention time, molecular formula, adduct ion form and collision energy of the target parent ion are set, and the mass spectrum information of the 54 hair dyes is as follows:

[0081]

[0082]

[0083]

[0084] Beneficial effects of the present invention:

[0085] (1) The method of the present invention solves the problems of stability, separation and specificity, difficulty in filtration and membrane adsorption that are inevitably encountered in the actual detection process of hair dyes, and makes up for the problem that the existing literature and standard hair dyes are not fully covered. An exclusive, sensitive, durable and high-throughput high-performance liquid chromatography-high-resolution mass spectrometry method for simultaneously detecting 54 hair dyes is established, covering the permitted and prohibited hair dyes that may be added to common hair dye products on the market, providing strong technical support for effective market supervision and preventing illegal enterprises from taking advantage of the lack of supervision and inspection standards and methods.

[0086] (2) The present invention can ensure the full separation of 8 groups of isomers under a chromatography-mass spectrometry system, ensure the qualitative specificity, and greatly improve the sensitivity of detecting hair dye components under negative ion conditions, so that the detection and quantitative range overlap of 54 hair dyes is higher, and the increase in the number of tests due to different dilution multiples is reduced, thereby truly achieving efficient screening.

[0087] (3) The present invention has carefully designed a solution for the stability of hair dyes to ensure the reliability of large-scale inspection results.

[0088] (4) The present invention dilutes the sample solution on the basis of improving sensitivity, and uses a solvent matching standard curve to calculate the content, thereby improving the accuracy of quantification.

[0089] (5) The pre-treatment strategy established by the present invention is applicable to the application of most hair dye products.

[0090] (6) The method of the present invention covers 32 hair dyes in "7.2" of the "Specifications" and adds 22 common prohibited or restricted hair dyes. Under the processing conditions of the present invention, especially the use of 70% methanol solution for ultrasonic extraction and 25% methanol solution (with and without D-isoascorbic acid) for sample dilution, and the quantitative method matching the solvent mark line, the stability of the hair dye and the accuracy of the measurement results are greatly improved; by selecting the chromatographic column and using ammonium fluoride solution-acetonitrile as the mobile phase, the separation of all isomers is guaranteed, and the sensitivity of negative ion monitoring components such as phenol and hydroquinone is greatly improved; by simultaneous scanning in positive and negative ion modes and data-dependent secondary ion full scanning (Full MS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2), accurate characterization and quantification of 54 hair dyes are achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Extracted ion chromatogram of 42 hair dyes tested in positive ion mode (mass deviation: 5ppm); among them:

[0092] The m / z of a-1 extraction from top to bottom are 109.07602, 110.06004, 110.07127, 111.05529, 123.09167, 124.07569, 141.08832 and 143.03705,

[0093] The m / z of a-2 extraction from top to bottom are 143.09274, 152.07061, 153.10224, 154.06110, 158.03672, 165.13863, 168.10191 and 169.09715,

[0094] The m / z of a-3 extraction from top to bottom are 170.09240, 175.08659, 179.15428, 182.08117, 183.07642, 183.11280, 185.10732 and 197.09207,

[0095] a-4 extracted m / z from top to bottom were 197.12845, 211.14410, 213.08698, 230.09240, 231.08642, 239.08150, 243.09755, and 289.16590;

[0096] Figure 2 Extracted ion chromatograms of 12 hair dyes tested in negative ion mode (mass deviation: 5ppm); among them:

[0097] The m / z extracted from b-1 are 93.03459, 109.02950, ​​123.04515, 132.04549 and 142.99053 from top to bottom, and the m / z extracted from b-2 are 143.05024, 153.03057, 159.04515, 186.99159 and 197.05678 from top to bottom;

[0098] Figure 3 Operation flow chart DETAILED DESCRIPTION

[0099] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0100] In the embodiment of the present invention, "qualified samples", "problem samples" and "unqualified samples" are determined in accordance with the requirements for the national cosmetics supervision and random inspection in 2021; products with hair dyes other than those marked on the label or in the approval certificate are determined to be problem samples; products with hair dye content exceeding the limit specified in the "Specifications" are unqualified samples; when the technical requirements stated on the product label are consistent with those in the approval certificate, products that do not have the aforementioned two situations are determined to be qualified samples.

[0101] 1 Instrument

[0102] Vanquish-Orbitrap Exploris 120 liquid chromatography-electrostatic field orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific, USA); XP6 one-millionth electronic balance (Mettler Toledo, Switzerland); CPA224S one-tenth electronic balance (Satorius, Germany); UA22MFD ultrasonic cleaning instrument (Wiggens, Germany); Milli-QIQ 7000 ultrapure water system (Millipore, USA).

[0103] 2. Reagents

[0104] Methanol, acetonitrile, ethanol (chromatographically pure, Merck, USA); formic acid (chromatographically pure, ACS, USA), acetic acid (Shanghai McLean Biochemical Technology Co., Ltd.), ammonium acetate (chromatographically pure, Sigma-Aldrich, Germany), ammonia water (chromatographically pure, Shanghai Aladdin Biochemical Technology Co., Ltd.); pyrogallic acid, sodium bisulfite, citric acid, sodium citrate (analytical pure, Sinopharm Chemical Reagent Co., Ltd.); D-isoascorbic acid (99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); ammonium fluoride (superior pure, Sinopharm Chemical Reagent Co., Ltd.).

[0105] 3 Reference substances (numbering rules for reference substances: S represents the hair dye test method included in the Technical Specifications "7.2 32 components including hydroquinone"; A represents the 22 hair dye components added by the present invention)

[0106] The numbers and information of the 54 hair dye reference substances are detailed in Table 1. Among them, reference substances A11, A18-22 were purchased from Tianjin Altascientific Company; S01, S02, S03, S01-05, S07-10 and S32 were purchased from Dr. Ehrenstorfer Company in Germany; S11-13, A02 and A16 were purchased from Panphy-chemicals Company in the United States; A01, A03-10, A12-14, A17, S14-31 were purchased from Stanford Analytical Chemicals Company in the United States; S06 was purchased from TRC Company in Canada; A15 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The purity of all reference substances was not less than 97%.

[0107] Table 1 Numbers and reference material information of 54 hair dyes

[0108]

[0109]

[0110]

[0111] Example 1 Method for simultaneously determining 54 hair dyes in hair dye products

[0112] This embodiment takes the determination of a batch of hair dye products labeled "a certain brand of hair dye (red)" as an example to introduce in detail the quantitative and qualitative determination methods of 54 hair dyes.

[0113] 1. Preparation of reference solution

[0114] 1.1 Preparation of mixed standard stock solution

[0115] Accurately weigh about 20 mg of hydroquinone (S19) and 5.0 mg of other reference substances, place them in the same 25 mL volumetric flask, dissolve and dilute to the scale with 70% methanol, shake well, and obtain a mixed standard solution with a mass concentration of 800 μg / mL for S19 and 200 μg / mL for other hair dyes.

[0116] Accurately measure 1 mL of the above mixed standard solution, place it in a 20 mL volumetric flask, dilute it to the mark with 70% methanol, and shake well to obtain a mixed standard stock solution with a mass concentration of 40 μg / mL for S19 and 10 μg / mL for other hair dyes.

[0117] The mixed standard solution and mixed standard stock solution were stored at -20°C away from light and were stable within one week.

[0118] 1.2 Preparation of mixed standard qualitative solution

[0119] Measure 40.0 μL of the mixed standard stock solution, put it in a sample injection vial, add 960 μL of 25% methanol solution, and shake well to obtain the mixed standard qualitative solution I (the mass concentration of S19 is 1600 ng / mL, and the mass concentration of other hair dyes is 400 ng / mL), which is used for the qualitative analysis of S05, S10, and S24.

[0120] Measure 40.0 μL of the mixed standard stock solution, put it in a sampling vial, add 960 μL of 25% methanol solution containing 0.05% D-isoascorbic acid, shake well, and obtain the mixed standard qualitative solution II (the mass concentration of S19 is 1600 ng / mL, and the mass concentration of other hair dyes is 400 ng / mL), which is used for the qualitative analysis of the other 51 hair dyes except S05, S10, and S24.

[0121] 1.3 Preparation of a series of mixed standard curves

[0122] Accurately measure an appropriate amount of the mixed standard stock solution and dilute it serially with 25% methanol solution to obtain a series of mixed standard curve solutions I;

[0123] Accurately measure an appropriate amount of the mixed standard stock solution and dilute it serially with 25% methanol solution (containing 0.05% D-isoascorbic acid) to obtain a series of mixed standard curve solutions II.

[0124] In the series of mixed standard curve solutions I and II, the concentration range of S19 is 32-8000 ng / mL, and the concentration range of other hair dyes except S19 is 8-2000 ng / mL. Prepare them before use.

[0125] In this embodiment, the concentrations of S19 in the series of mixed standard curve solutions I and the series of mixed standard curve solutions II are: 32, 80, 160, 320, 800, 1600, 3200, 8000 ng / mL; the concentrations of other hair dyes except S19 are: 8, 20, 40, 80, 200, 400, 800, 2000 ng / mL.

[0126] 1.4 Positioning solution

[0127] Single-label stock solutions were prepared. The single-label stock solutions were prepared by: dissolving o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine reference substances in 70% methanol solution; dissolving other hair dye reference substances in 70% methanol solution (containing 0.05% D-isoascorbic acid); and obtaining 54 single-label stock solutions respectively;

[0128] The single standard stock solutions of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine were diluted with 25% methanol solution to prepare the positioning solution I with a mass concentration of 400 ng / mL for the three hair dyes;

[0129] The 54 single-label stock solutions were mixed and diluted with 25% methanol solution containing an antioxidant to prepare positioning solution II with a mass concentration of 1600 ng / mL for S19 and a mass concentration of 400 ng / mL for other hair dyes.

[0130] The positioning solution is used for positioning and stability investigation.

[0131] 2 Preparation of sample solution

[0132] Accurately weigh 0.2 g of the sample, place it in a 10 mL stoppered colorimetric tube, add 1 mL of 70% methanol solution, vortex for 30 seconds, add 70% methanol solution to the scale, extract in an ice bath with ultrasound for 10 minutes, shake well to obtain the sample solution, filter through a 0.45 μm polytetrafluoroethylene membrane filter, take the filtrate to obtain the sample stock solution, and store at -20 °C in the dark.

[0133] (1) Accurately measure 200 μL of the sample stock solution, place it in a 10 mL volumetric flask, add 25% methanol to dilute to the mark, and shake well to obtain sample solution I-1. The concentration of the sample stock solution in the sample solution I-1 is 20 μL / mL.

[0134] Accurately measure 20 μL of the sample stock solution, place it in a 10 mL volumetric flask, add 25% methanol to dilute to the scale, and shake well to obtain sample solution I-2. The concentration of the sample stock solution in the sample solution I-2 is 2 μL / mL.

[0135] Sample solution I-1 and sample solution I-2 are used for accurate characterization and quantification of S05, S10, and S24.

[0136] (2) Accurately measure 200 μL of the sample stock solution, place it in a 10 mL volumetric flask, add 25% methanol (containing 0.05% isoascorbic acid) to the mark, and shake well to obtain sample solution II-1.

[0137] Accurately measure 20 μL of the sample stock solution, place it in a 10 mL volumetric flask, add 25% methanol (containing 0.05% isoascorbic acid) to the scale, shake well, and obtain sample solution II-2.

[0138] Sample solution II-1 and sample solution II-2 are used for accurate characterization and quantification of the other 51 components except S05, S10, and S24.

[0139] 3 Chromatographic-mass spectrometric conditions

[0140] 3.1 Chromatographic conditions

[0141] Chromatographic column: ACE Excel C18 column (150mm×2.0mm, 3μm), column temperature: 40℃; mobile phase: phase A is 1mM ammonium fluoride aqueous solution; phase B is acetonitrile, flow rate: 0.3mL / min. Gradient elution program: 0~2.0min, 10% B; 2.0~8.5min, 10% B~60% B; 8.5~10.0min, 60% B~95% B; 10.0~12.0min, 95% B; 12.0~12.1min, 95% B~10% B; 12.1~15.0min, 10% B. Injection volume: 1μL; injection plate temperature: 4℃.

[0142] Since the 54 hair dyes contain multiple groups of isomers, when establishing the method, positioning solutions were used for separate sampling to determine the peak order of the isomers; if the chromatographic conditions of the present invention are fully adopted, relative retention can be used as a reference without the need to prepare positioning solutions.

[0143] 3.2 Mass spectrometry conditions

[0144] Spray voltage: 1.5 kV in positive ion mode, -2.5 kV in negative ion mode; nebulization temperature: 350 °C; ion transfer tube temperature: 325 °C; sheath gas pressure: 50 Arb; auxiliary gas pressure: 10 Arb; full scan of primary mass spectrometry (Full scan), scanning range (m / z): 50-300 Da; full scan resolution: R = 60 000; data-dependent secondary ion full scan (Full MS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2), intensity threshold: 1.0 × 10 5 ddMS2 resolution: R = 15 000. In the Targeted Mass of the data-dependent secondary ion full scan (Full MS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2), the retention time, parent ion (m / z) and ion mode of the target parent ion were set.

[0145] The chromatographic-mass spectrometric information of 54 hair dyes is shown in Table 2.

[0146] Table 2 Chromatographic and mass spectrometric information of 54 hair dyes

[0147]

[0148]

[0149]

[0150]

[0151]

[0152] 4 Qualitative and quantitative determination of samples (after instrument calibration)

[0153] (1) Inject the mixed standard qualitative solution I to determine the retention time of S05, S10, and S24. The signal-to-noise ratio of the three chromatographic peaks should be greater than 100. Inject the sample solution I-1 to determine whether S05, S10, and S24 are detected. If the result is not detected, skip to step (3).

[0154] (2) Inject the series of mixed standard curve solution I, perform linear regression on the concentrations of S05, S10 and S24 using the chromatographic peak areas, and draw a linear equation. Inject sample solution I-2 and sample solution I-1 in sequence, substitute the peak areas into the linear equation of the series of mixed standard curve solution I, and calculate the contents of S05, S10 and S24 using the external standard method. (Since S05, S10 and S24 were not detected in step (1), this step (2) was not performed).

[0155] (3) Inject the series of mixed standard curve solution II, perform linear regression on the concentration of each chromatographic peak area, and draw a linear equation. Inject sample solution II-2 and sample solution II-1 in sequence, substitute the peak area into the linear equation of the series of mixed standard curve solution II, and calculate the content by external standard method. As a result, S02, S03, S11, S32 and A01 were detected, and the contents were 0.079%, 0.066%, 0.333%, 0.017% and 0.095% respectively after conversion ratio and calculation as free radical.

[0156] (4) Qualitative judgment:

[0157] The sample solution I-1, the sample solution I-2 and the mixed standard qualitative solution I prepared by S1-2 are subjected to chromatography and mass spectrometry detection: when the chromatographic retention time of the sample solution I is consistent with that of the mixed standard qualitative solution I, and when the fragment ion detection fragments of the sample solution I and the secondary mass spectrometry are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of S05, S10 and S24;

[0158] S05, S10 and S24 were not detected in the sample solution of this example.

[0159] The mixed standard qualitative solution II prepared by sample solution II-1, sample solution II-2 and S1-2 is subjected to chromatography and mass spectrometry detection; when the chromatographic retention time of sample solution II is consistent with that of the mixed standard qualitative solution II, and when the fragment ion detection fragments of sample solution I and in the secondary mass spectrometry are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of the 51 components except S05, S10, and S24.

[0160] The retention time of S02, S03, S11, S32 and A01 in the sample solution of this example and the fragments detected in the secondary mass spectrometry are consistent with those of the hair dye in the reference solution, that is, the sample of this example includes S02, S03, S11, S32 and A01, and the qualitative results correspond to the quantitative results.

[0161] (5) Result analysis: The sample label does not match the ingredients in the approval document, and is a problematic sample. The high performance liquid chromatography method included in the "Specifications" was used to determine 32 types of hair dyes such as p-phenylenediamine, but none of them were detected, and it was impossible to determine whether the ingredients were consistent with the label or the approval document. The LC-HRMS determination established by the present invention showed that the actual ingredients of the sample were consistent with the label and inconsistent with the approval document. The contents of the five hair dyes (S02, S03, S11, S32 and A01) were all calculated based on sample solution II-2; in sample solution II-1, the concentrations of the four hair dyes except S32 exceeded the linear range.

[0162] Example 2 Method for Simultaneously Determining 54 Hair Dyes in Hair Dye Products

[0163] This embodiment takes the determination of a batch of hair dye products labeled "a certain school of hair dye (natural black)" as an example to introduce in detail the qualitative and quantitative methods of 54 hair dyes.

[0164] 1. Preparation of reference solution

[0165] Same as "Example 1"

[0166] 2 Preparation of sample solution

[0167] Same as "Example 1"

[0168] 3 Chromatographic-mass spectrometric conditions

[0169] Same as "Example 1"

[0170] 4 Qualitative and quantitative determination of samples (after instrument calibration)

[0171] (1) Inject the mixed standard qualitative solution I to determine the retention time of S05, S10, and S24. The signal-to-noise ratio of the three chromatographic peaks should be greater than 100. Inject the sample solution I-1 to determine whether S05, S10, and S24 are detected.

[0172] If the result is not detected, skip to step (3).

[0173] (2) Inject the series of mixed standard curve solution I, perform linear regression on the concentrations of S05, S10 and S24 using the chromatographic peak areas, and draw a linear equation. Inject sample solution I-2 and sample solution I-1 in sequence, substitute the peak areas into the linear equation of the series of mixed standard curve solution I, and calculate the contents of S05, S10 and S24 using the external standard method. (Since S05, S10 and S24 were not detected in step (1), this step (2) was not performed)

[0174] (3) Inject the series of mixed standard curve solution II, perform linear regression on the concentration of each chromatographic peak area, and draw a linear equation. Inject sample solution II-2 and sample solution II-1 in sequence, substitute the peak area into the linear equation of the series of mixed standard curve solution II, and calculate the content by external standard method. As a result, S01, S04, S14, S25 and A02 were detected, and the contents were 1.31%, 0.302%, 0.248%, 0.066% and 0.0011% respectively according to the proportion and free radical calculation.

[0175] (4) Qualitative judgment:

[0176] The sample solution I-1, the sample solution I-2 and the mixed standard qualitative solution I prepared by S1-2 are subjected to chromatography and mass spectrometry detection: when the chromatographic retention time of the sample solution I is consistent with that of the mixed standard qualitative solution I, and the fragment ion detection fragments of the sample solution I and the secondary mass spectrometry are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of S05, S10, and S24;

[0177] S05, S10 and S24 were not detected in the sample solution of this example.

[0178] The mixed standard qualitative solution II prepared by sample solution II-1, sample solution II-2 and S1-2 is subjected to chromatography and mass spectrometry detection; when the chromatographic retention time of sample solution II is consistent with that of the mixed standard qualitative solution II, and the fragment ion detection fragments of sample solution I and in the secondary mass spectrometry are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of the 51 components except S05, S10, and S24.

[0179] The retention time of S01, S04, S14, S25 and A02 in the sample solution of this example and the hair dye in the reference solution and the fragment ion detection fragments in the secondary mass spectrometry are all consistent, that is, the sample of this example includes S01, S04, S14, S25 and A02, and the qualitative results correspond to the quantitative results.

[0180] (5) Result analysis:

[0181] This product has no label and is a problematic sample. The approval certificate contains three hair dyes: S01, S04 and S08. According to the LC-HRMS measurement established by the present invention, the actual ingredients of the sample are not completely consistent with the approval certificate, and hair dyes other than those in the approval certificate are detected: S14, S25 and A02. Among the five hair dyes detected, the contents of S01, S04, S14 and S25 are calculated using sample solution II-2, and A02 is calculated using the concentration of sample solution II-1. S08 in the approval certificate was not detected. Calculated using sample solution II-1, the detection limit of S08 in this method is 4.0 μg / g, which is 1 / 8 of the method in the "Specification".

[0182] Example 3 (Comparative Liquid Phase) Method for Simultaneously Determining 54 Hair Dyes in Hair Dye Products

[0183] This embodiment takes the determination of a batch of hair dye products labeled "a certain brand of hair dye 4G" as an example to introduce in detail the qualitative and quantitative methods of 54 hair dyes.

[0184] 1. Preparation of reference solution

[0185] Same as "Example 1"

[0186] 2 Preparation of sample solution

[0187] Same as "Example 1"

[0188] 3 Chromatographic-mass spectrometric conditions

[0189] Same as "Example 1"

[0190] 4 Qualitative and quantitative determination of samples (after instrument calibration)

[0191] (1) Inject the mixed standard qualitative solution I to determine the retention time of S05, S10, and S24. The signal-to-noise ratio of the three chromatographic peaks should be greater than 100. Inject the sample solution I-1 to determine whether S05, S10, and S24 are detected.

[0192] If the result is not detected, skip to step (3).

[0193] (2) Inject the series of mixed standard curve solution I, perform linear regression on the concentrations of S05, S10 and S24 using the chromatographic peak areas, and draw a linear equation. Inject sample solution I-2 and sample solution I-1 in sequence, substitute the peak areas into the linear equation of the series of mixed standard curve solution I, and calculate the contents of S05, S10 and S24 using the external standard method. (Since S05, S10 and S24 were not detected in step (1), this step (2) was not performed)

[0194] (3) Inject the series of mixed standard curve solution II, perform linear regression on the concentration of each chromatographic peak area, and draw a linear equation. Inject sample solution II-2 and sample solution II-1 in sequence, substitute the peak area into the linear equation of the series of mixed standard curve solution II, and calculate the content by external standard method. As a result, S03, S18 and A14 were detected, and the contents were calculated by conversion ratio to 0.0039%, 0.0013% and 0.0025% respectively.

[0195] (4) Qualitative judgment:

[0196] The sample solution I-1, the sample solution I-2 and the mixed standard qualitative solution I prepared by S1-2 are subjected to chromatography and mass spectrometry detection: when the chromatographic retention time of the sample solution I is consistent with that of the mixed standard qualitative solution I, and the fragment ion detection fragments of the sample solution I and the secondary mass spectrometry are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of S05, S10, and S24;

[0197] S05, S10 and S24 were not detected in the sample solution of this example.

[0198] The mixed standard qualitative solution II prepared by sample solution II-1, sample solution II-2 and S1-2 is subjected to chromatography and mass spectrometry detection; when the chromatographic retention time of sample solution II is consistent with that of the mixed standard qualitative solution II, and the fragment ion detection fragments of sample solution I and in the secondary mass spectrometry are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of the 51 components except S05, S10, and S24.

[0199] The retention time of S03, S18 and A01 in the sample solution of this embodiment and the fragment ion detection fragments in the secondary mass spectrometry are consistent with those of the hair dye in the reference solution, that is, the sample of this embodiment includes S03, S18 and A14, and the qualitative results correspond to the quantitative results.

[0200] (5) Result analysis:

[0201] The product approval certificate and label identification are consistent, and should contain S01, S02, S04, S08, S11 and S24. However, according to the HPLC method of the "Specification", the above 6 hair dyes were not detected, and the sample is qualified.

[0202] The LC-HRMS established by the present invention determined that the actual composition of the sample did not match the composition in the approval document (and label), and was a problematic sample. Not only were the six hair dyes marked in the approval document not detected, but hair dyes other than those marked in the approval document (and label) were also detected: S03, S18, and A14. The concentrations of the three detected hair dyes were calculated using the sample solution II-1.

[0203] Example 4 Optimization of Chromatographic Conditions

[0204] The present invention investigated 54 kinds of hair dyes with 0.1% acetic acid solution, 5mmol / L acetic acid solution, 5mmol / L ammonium acetate solution, 5mmol / L ammonium formate solution, 5mmol / L ammonium acetate solution (ammonia water adjusted to pH 7.5), 5mmol / L ammonium acetate solution (ammonia water adjusted to pH 8.0), 1mmol / L ammonium fluoride solution and 2mmol / L ammonium fluoride solution as aqueous phase, in ACE Excel 3C18 (2.1mm×150mm,3μm), WATERS BEH Shield RP18(2.1mm×150mm,2.5μm)、WATERSatlantis TM T3 (2.1mm×150mm, 3μm), Phenomenex Separation on C18(2) (2 mm×150 mm, 3 μm) and Agilent Poroshell 120PFP (2.1 mm×150 mm, 2.7 μm).

[0205] The results showed that under the acidic mobile phase, the three groups of isomers of m / z 109.07602, m / z 110.06004 and m / z 124.07569 could not be completely separated on each chromatographic column, among which the Agilent PFP column had the best separation.

[0206] The separation of ammonium formate, ammonium acetate, and ammonium fluoride solutions was similar. The higher the pH, the stronger the retention of the hair dye and the higher the sensitivity. TM The pH tolerance range of conventional C18 chromatographic columns such as T3 (2.1mm×150mm, 3μm) is 2 to 8; when a mobile phase with pH=8 is used for large-scale determination of hair dyes, the peak shape deteriorates significantly, and the mobile phase with pH=8.0 is very easy to "grow hair", which is not conducive to large-scale sample determination.

[0207] In positive ion mode, the peak symmetry of 5mmol / L ammonium acetate solution is the best; however, in negative ion mode, the responses of various substances, especially S19 (hydroquinone) and A17 (phenol), in ammonium fluoride solution are significantly stronger than those in ammonium acetate and ammonium formate solutions, with a sensitivity difference of more than one order of magnitude. The peak shape of 2mmol / L ammonium fluoride is better than that of 1mmol / L ammonium fluoride solution, but the sensitivity is slightly worse. When ammonium formate, ammonium acetate, and ammonium fluoride solutions are used as the aqueous phase, the peak shape of the Agilent PFP column is the worst, especially for S15, S21, and S26. The isomers S29 and S31 in negative ion mode are significantly different in ACE Excel 3C18 and WATERS The chromatographic column can easily separate the and Waters Atlantis TM T3 needs to optimize the gradient conditions. Agilent PFP cannot be separated under the conditions investigated in this experiment. The peak shapes of A05 and A15 in ACE Excel 3C18 are significantly better than those in WATERS. and Waters Atlantis TM T3.

[0208] Therefore, 1mmol / L ammonium fluoride solution was finally selected as the aqueous phase and ACE Excel 3C18 as the stationary phase. The 54 hair dyes were strongly retained in methanol, but the peak shapes of components such as S15 and S26 were significantly worse than those of acetonitrile; both methanol and acetonitrile can separate isomers on the ACE Excel3C18 column, and acetonitrile is preferred as the organic phase in the present invention. The 54 hair dyes after optimization can be separated and determined within 15 minutes (such as Figure 1 and Figure 2 shown).

[0209] Example 5 Optimization of mass spectrometry parameters

[0210] The monitoring modes of each substance were investigated, and the results are shown in Table 2. 12 hair dyes including A05 were monitored in negative ion mode, and the remaining 42 components were measured in positive ion mode. Among them, S27 was measured in positive ion mode in the "Specification", and negative ion mode was selected for measurement under the conditions of this paper. The ion source parameters such as spray voltage, atomization temperature and ion transfer tube temperature were optimized. Almost all hair dyes have the highest response when the atomization temperature is 350℃ and the ion transfer tube temperature is 325℃, but there are differences in the optimal spray voltage. The response of most hair dyes monitored by positive ions increases with the decrease of spray voltage. Finally, 1.5kV was selected as the positive ion spray voltage. S19 has the lowest sensitivity in negative ion mode, and its optimal voltage -2.5kV was selected as the negative ion spray voltage.

[0211] Table 2 lists the theoretical accurate mass numbers and retention times of the parent ions of 54 target substances. The full scan mode of positive and negative ion switching is adopted, and the mass tolerance is set to 5.0ppm through the integration of the primary mass spectrometry extracted ion flow chart to meet the specificity and quantitative requirements. The collision energy of the target compound is optimized to obtain richer fragment information. As shown in Table 2, when the parent ion in the list is found during the primary mass spectrometry scan and the intensity reaches the set threshold, the secondary mass spectrometry scan is automatically performed. When measuring the sample, the retention time, the precise mass of the parent ion and the precise mass of the secondary fragment ion are compared for qualitative confirmation.

[0212] Example 6 Optimization of sample pretreatment

[0213] (1) Selection of extraction solvent

[0214] Hair dye cosmetics are characterized by the fact that they often contain a variety of hair dyes with varying contents. The solubility of the 54 hair dyes in aqueous solutions and organic solvents varies greatly. The main factors considered in sample pretreatment optimization are the solubility and stability of the hair dye components in the extraction solution, as well as the convenience and recovery rate of filtration. First, by optimizing the preparation of the single-standard stock solution of the reference substance, a 70% methanol solution was selected to extract the sample, which can make each hair dye have better solubility and be easier to filter.

[0215] Experimental method: The filtration, storage (-20°C) and stability of 54 reference solutions extracted with 50% to 100% methanol, 50% to 100% ethanol, 50% to 100% methanol (containing 0.05% D-isoascorbic acid) and 50% to 80% methanol (containing 1g / L sodium bisulfite) solutions were investigated. The comparison was set up in a gradient of 10% within the above concentration range.

[0216] The results showed that the extraction conditions of methanol and ethanol were similar, but the 70% methanol solution could make the hair dyes have better solubility and easier to filter. The sample solutions extracted with 50% methanol and 50% ethanol would freeze when stored at -20℃; the sample solution extracted with 1g / L sodium bisulfite had a higher viscosity and did not significantly improve the stability of the components with poor stability; the sample extracted with the solution containing D-isoascorbic acid, although the room temperature stability of A14, A15 and A16 increased, but the room temperature stability of S05, S10 and S24 decreased significantly.

[0217] Therefore, the present invention selects 70% methanol solution to extract the sample, mainly considering that the extracted sample is immediately stored at -20°C after filtration to extend the stability time of the hair dye.

[0218] (2) Selection of filter membrane

[0219] Using 50% methanol as the initial condition, the mixed reference solution was diluted and the adsorption of hair dye by water membranes (polyethersulfone) and organic filter membranes (nylon membrane, vinylidene fluoride membrane, polytetrafluoroethylene membrane) of different brands and materials was investigated.

[0220] Experimental method: accurately measure 0.4mL of mixed standard stock solution, dilute to 10mL with 50%, 60%, 70% and 80% methanol respectively, shake well, and obtain the membrane inspection reference solution. Use different brands of filter membranes (specifications are 25mm, 0.45μm), filter about 0.5mL of the membrane inspection reference solution in sequence (do not discard the initial filtrate), place in a 2mL injection bottle, shake well, and inject directly.

[0221] The results showed that water membranes are easy to dissolve and have poor recovery rates, so they are not applicable. Among organic membranes, nylon membranes and polyvinylidene fluoride membranes (PVDF) vary greatly from brand to brand. Nylon membranes and polyvinylidene fluoride membranes from some manufacturers have serious adsorption of phenolic compounds (such as S08, S12, S27, S29, S30 and S32) for negative ion monitoring, which has a great impact on the recovery rate; A20, A21 and A22 are easily adsorbed by the membrane, and the membrane adsorption decreases with the decrease of the proportion of organic phase in the solvent; the recovery rates of polyvinylidene fluoride membranes of different brands vary greatly. The recovery rate of polytetrafluoroethylene membranes is relatively stable.

[0222] (3) Selection of dilution solvent

[0223] The sensitivity of hair dye determination by LC-Orbitrap-MS is very high. Since these components are present in high concentrations in hair dye products and their concentrations vary greatly, appropriate dilution is required. After dilution, the matrix effect is significantly reduced, but the stability of the components is also significantly reduced. Therefore, it is necessary to select a suitable solvent to enhance the stability of the components.

[0224] The stability of each component under different antioxidants, including sodium bisulfite, isoascorbic acid, L-ascorbic acid, pyrogallic acid, citric acid, etc., was systematically investigated.

[0225] Experimental method: 40 μL of the mixed standard stock solution was accurately measured and placed in a sample injection bottle. 960 μL of 25% methanol and 25% methanol (containing 1g / L sodium bisulfite, 0.01%-0.1% isoascorbic acid, 0.05% L-ascorbic acid, 0.05% pyrogallic acid, and 0.005 mol / L citric acid) were added respectively. The solution was shaken well to obtain the stability test solution of different solvents. The solution was placed in a 4°C sample injection tray and injected at 0, 2, 6, 12, 24, 48, and 72 hours to test the stability.

[0226] The results showed that D-isoascorbic acid and L-ascorbic acid had the best antioxidant effect and similar effects, and significantly improved the stability of most hair dyes, especially A14, A15 and A16; however, the stability of some hair dyes decreased in the presence of antioxidants, such as A02, S03, and S06, which had poor stability in sodium bisulfite solution. Components containing o-phenylenediamine structure (S05, S10 and S24) would react chemically with D-isoascorbic acid (L-ascorbic acid), thus significantly reducing their stability. S21, S25, A07, A08, A09, A11, A13, A21 and A22 were all stable in various solvents. In addition to affecting the stability of hair dyes, different oxidants also produced matrix effects for components with weaker retention, such as sodium bisulfite solution produced matrix inhibition effects, while ascorbic acid produced a stronger matrix enhancement effect, and the intensity of the effect was positively correlated with the concentration of antioxidants. In addition, the presence of D-isoascorbic acid, L-ascorbic acid or pyrogallic acid significantly improved the peak shapes of substances such as A02, S03, S14, S15, S22 and S26, especially S30.

[0227] In the injection solution, a high proportion of organic solvents will cause solvent effects, resulting in chromatographic peak fronts. Therefore, 25% methanol and 25% methanol (containing 0.05% D-isoascorbic acid) solutions were finally selected to dilute the sample stock solution, and the corresponding solvents were used to prepare standard curves and other reference solutions. Among the 54 hair dyes, S05, S10 and S24 were qualitatively and quantitatively analyzed using reference substances and sample solutions diluted with 25% methanol solution; the other 51 components were qualitatively and quantitatively analyzed using reference substances and sample solutions containing D-isoascorbic acid to ensure qualitative and quantitative accuracy.

[0228] Example 7 Methodological Investigation

[0229] 1. Preparation of reference solution

[0230] Same as "Example 1"

[0231] 2 Preparation of sample solution

[0232] Same as "Example 1"

[0233] 3 Chromatographic-mass spectrometric conditions

[0234] Same as "Example 1"

[0235] 4 Methodological investigation

[0236] 4.1 Linear range, detection limit and quantification limit

[0237] Two standard curves were prepared according to the method described in "Example 1, 1.3 Preparation of a series of mixed standard curves". The concentrations of the 54 hair dye standards were 8.00, 20.0, 40.0, 80.0, 200, 400, 800, and 2000 ng / mL, and the determination was carried out according to the "3" chromatography-mass spectrometry conditions. Linear regression was performed with the concentration (x, ng / mL) as the horizontal coordinate and the peak area (y) of the extracted ion current (extracted ion m / z see Table 2, Masstolerance: 5.0ppm) as the vertical coordinate. The content of the target substance when the chromatographic peak signal-to-noise ratio (S / N) was ≥3 and 10 was used as the detection limit and quantification limit of the method, and the detection concentration (LOD, ng / g) and quantitative concentration (LOQ, ng / g) were calculated based on the dilution multiples of sample solution I-1 and sample solution II-1 (0.2g sample diluted to 500mL).

[0238] The results showed that the 54 hair dyes had a good linear relationship within the range shown in Table 3, and the correlation coefficients (r) were all greater than 0.995. This method has high sensitivity and can meet the requirements of actual measurement. The linear range, LOD and LOQ and other related data are shown in Table 3.

[0239] Table 3 Linear range, linear equation, correlation coefficient, detection limit and quantification limit of 54 hair dyes

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] y: peak area; x: mass concentration (ng / mL)

[0246] *: Calculation is based on the dilution factor of sample solution I. LOD / LOQ (μg / g) = LOD / LOQ (ng / mL) × 500 / 0.2 (g)

[0247] 4.2 Recovery, repeatability and injection precision

[0248] A batch of 54 samples with low hair dye content (sample solution Ⅰ-1 and sample solution Ⅱ-1 were prepared according to "Example 1, 2", containing a small amount of S03, S17 and A14, and the concentration was less than 50 ng / mL) were screened out as the spike recovery matrix for method validation.

[0249] Weigh 11 mixed matrix samples, add mixed standard solutions of low, medium and high concentration levels shown in Table 4 respectively, and perform pretreatment according to "Example 1, 2 Sample Solution I-1 and Sample Solution II-1", 3 samples for each level, calculate the spiked recovery and repeatability; one of each level is repeated 6 times, calculate the relative standard deviation (RSD), and obtain the injection precision. The results are shown in Table 4. The average recovery of 54 hair dyes is 60.0% to 118.4%, the repeatability is 4.0% to 14.9%, and the RSD is 0.6% to 10.0%.

[0250] Table 4 Background concentration, added concentration, recovery, repeatability (n=9) and relative standard deviation (RSD, n=6) of 54 hair dye samples

[0251]

[0252]

[0253]

[0254] *Recovery rate = (measured concentration - background concentration) / spiked concentration × 100%.

[0255] 4.3 Stability investigation

[0256] Take the "mixed standard stock solution" prepared in Example 1, 1.1, and dilute it with 25% methanol and 25% methanol (containing 0.05% D-isoascorbic acid) to make 400 ng / mL mixed standard qualitative solution I and mixed standard qualitative solution II, respectively, place them in a 4°C injector, and measure them at 0, 6, 12, 24 and 48 h. The stability is determined by 85% of the peak area at 0 h.

[0257] The results showed that in 25% methanol (containing 0.05% D-isoascorbic acid), A03 was stable for 12 hours, A14 was stable for 24 hours, and the remaining 49 hair dyes (except S05, S10 and S24) were stable for 48 hours; in 25% methanol, S05, S10 and S24 were stable for 48 hours.

[0258] Example 8 20 batches of measurement results

[0259] 1. Preparation of reference solution

[0260] Same as "Example 1"

[0261] 2 Preparation of sample solution

[0262] Same as "Example 1"

[0263] 3 Chromatographic-mass spectrometric conditions

[0264] Same as "Example 1"

[0265] 4. Testing of actual samples

[0266] The operation is the same as in Example 1. The qualitative results are shown in Table 5.

[0267] The method established by the present invention was used to measure 135 batches of hair dye products on the market, and a total of 27 hair dyes were detected (Table 5), of which 9 were hair dyes other than the "7.2 p-phenylenediamine and other 32 components" in the "Specifications". A total of 131 batches were detected with hair dyes numbered S, accounting for 97.0% of the total batches; 40 batches were detected with hair dyes numbered A, accounting for 29.6% of the total batches, of which 2 batches only contained hair dyes numbered A. 23 batches were detected with hair dyes other than those on the labels or batches, which were problematic samples: 10 batches were detected with hair dyes numbered A other than those on the labels or batches; 15 batches were detected with hair dyes numbered S other than those on the labels or batches. Among the hair dyes numbered A, A01, A04, A10 and A11 had the highest detection rates, and they were also the hair dyes other than those on the labels or batches that were often detected in problematic samples, and they require special attention. In addition, the retention time of A02 should be 1.52 min. In the chromatograms of some samples, there are interfering chromatographic peaks near 1.65-1.70 min, but their secondary mass spectra are different, so it is determined that A02 is not detected. This shows the importance of secondary mass spectrometry in assisting judgment when the retention times are close.

[0268] Table 5 Detection rates of target hair dye ingredients in 135 batches of hair dye products

[0269] serial number Hair dye name Number of batches checked Detection rate (%) S08 Resorcinol 91 67.4 S01 p-phenylenediamine 87 64.4 S04 m-Aminophenol 77 57.0 S02 p-Aminophenol 46 34.1 S03 Toluene-2,5-diamine sulfate 40 29.6 S18 2,4-Diaminophenoxyethanol hydrochloride 36 26.7 S11 4-Amino-2-hydroxytoluene 34 25.2 S22 N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate 30 22.2 S14 Phenylmethylpyrazolone 22 16.3 A01 1-Hydroxyethyl-4,5-diaminopyrazole sulfate 16 11.9 S12 2-Methylresorcinol 16 11.9 S32 1-Naphthol 15 11.1 A16 2-Amino-4-hydroxyethylaminoanisole 12 8.9 A04 2,6-Dihydroxyethylaminotoluene 7 5.2 S20 4-Amino-m-cresol 7 5.2 S28 4-Chlororesorcinol 6 4.4 A02 Hydroxyethyl p-phenylenediamine sulfate 5 3.7 A10 5-Amino-6-chloro-o-cresol 5 3.7 A11 HC Yellow No. 2 5 3.7 S21 2-Amino-3-hydroxypyridine 4 3.0 A05 2-Amino-6-chloro-4-nitrophenol 3 2.2 A06 2-Methyl-5-hydroxyethylaminophenol 3 2.2 A12 Hydroxybenzone 2 1.5 S25 2,6-Diaminopyridine 2 1.5 S10 Toluene-3,4-diamine 1 0.7 S13 6-Amino-m-cresol 1 0.7 S23 p-Methylaminophenol sulfate 1 0.7

Claims

1. A method for simultaneously determining 54 hair dyes in hair dye products by high performance liquid chromatography-quadrupole / electrostatic field orbital trap high resolution mass spectrometry, wherein the hair dyes are 1-hydroxyethyl-4,5-diaminopyrazole sulfate, hydroxyethyl-p-phenylenediamine sulfate, tetraaminopyrimidine sulfate, 2,6-dihydroxyethylaminotoluene, 2-amino-6-chloro-4-nitrophenol, 2-methyl-5-hydroxyethylaminophenol, 3-nitro-p-hydroxyethylaminophenol, 4-hydroxypropylamino- 3-Nitrophenol, 5-amino-4-chloro-o-cresol, 5-amino-6-chloro-o-cresol, HC Yellow No. 2, Hydroxybenzomorpholine, Hydroxyethyl-2-nitro-p-toluidine, 1,3-bis-(2,4-diaminophenoxy)propane, 2,6-dimethoxy-3,5-pyridinediamine hydrochloride, 2-amino-4-hydroxyethylaminoanisole, Phenol, N-hydroxyethyl-3,4-methylenedioxyaniline hydrochloride, HC Yellow No. 4, HC Red No. 1, HC Orange No. 1, Disperse Violet, p-phenylenediamine, p-aminophenol, toluene-2,5-diamine sulfate, m-aminophenol, o-phenylenediamine, 2-chloro-p-phenylenediamine sulfate, o-aminophenol, resorcinol, 2-nitro-p-phenylenediamine, toluene-3,4-diamine, 4-amino-2-hydroxytoluene, 2-methylresorcinol, 6-amino-m-cresol, phenylmethylpyrazolone, N,N-diethyltoluene-2,5-diamine hydrochloride, 4-amino-3-nitrophenol, m- Phenylenediamine, 2,4-diaminophenoxyethanol hydrochloride, hydroquinone, 4-amino-m-cresol, 2-amino-3-hydroxypyridine, N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate, p-methylaminophenol sulfate, 4-nitro-o-phenylenediamine, 2,6-diaminopyridine, N,N-diethyl-p-phenylenediamine sulfate, 6-hydroxyindole, 4-chlororesorcinol, 2,7-naphthalenediol, N-phenyl-p-phenylenediamine, 1,5-naphthalenediol, 1-naphthol; It is characterized in that The method comprises the following steps: S1 Preparation of reference solution S1-1 Preparation of mixed standard qualitative solution Dissolve 54 hair dye reference substances in 10% to 30% methanol solution to prepare a mixed standard qualitative solution I with a mass concentration of hydroquinone of 400 to 4000 ng / mL and a mass concentration of other hair dyes of 100 to 1000 ng / mL, which is used for the qualitative determination of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine; 54 hair dye reference substances were dissolved in 10% to 30% methanol solution containing antioxidants to prepare mixed standard qualitative solution II with a mass concentration of hydroquinone of 400 to 4000 ng / mL and a mass concentration of other hair dyes of 100 to 1000 ng / mL, which was used for the qualitative analysis of the other 51 hair dyes; Preparation of S1-2 series mixed standard curve solution Dissolve 54 hair dye reference substances in 10% to 30% methanol solution, and dilute them continuously to obtain a series of mixed standard curve solutions I with hydroquinone concentrations ranging from 32 to 8000 ng / mL and other hair dye concentrations ranging from 8 to 2000 ng / mL, which are used for qualitative and / or quantitative determination of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine; Dissolve 54 hair dye reference substances in 10% to 30% methanol solution containing antioxidants, and dilute them continuously to obtain a series of mixed standard curve solutions II with a hydroquinone concentration range of 32 to 8000 ng / mL and other hair dye concentrations range of 8 to 2000 ng / mL, which are used for qualitative and / or quantitative determination of 51 components except o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine; Preparation of S2 sample solution S2-1 Preparation of sample stock solution: Weigh the sample, dilute the alcohol solution to no more than 20 mg / mL, extract in an ice bath with ultrasonic waves for 5 to 20 min, shake well, filter through an organic filter membrane, and take the filtrate for later use to obtain the sample stock solution; The alcohol solution is 50% to 80% methanol or 50% to 80% ethanol; S2-2 Prepare sample solution I and sample solution II: The sample stock solution is diluted with 10% to 30% methanol solution to prepare sample solution Ⅰ-1 and sample solution Ⅰ-2, wherein the concentration of the sample stock solution in the sample solution Ⅰ-1 is 20 μL / mL, and the concentration of the sample stock solution in the sample solution Ⅰ-2 is 2 μL / mL; used for accurate qualitative and / or quantitative determination of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine; The sample stock solution was diluted with a 10% to 30% methanol solution containing an antioxidant to prepare sample solution II-1 and sample solution II-2, wherein the sample stock solution concentration in the sample solution II-1 was 20 μL / mL and the sample stock solution concentration in the sample solution II-2 was 2 μL / mL; used for accurate qualitative and / or quantitative determination of the other 51 components; S3 Chromatography-Mass Spectrometry Detection Conditions Chromatographic conditions: Chromatographic column: C18 column, column temperature: 40°C; mobile phase: phase A is 0.5-5mmol / L ammonium fluoride aqueous solution; phase B is methanol or acetonitrile, flow rate: 0.2-0.5mL / min; gradient elution program: 0-2.0min, 10%B; 2.0-8.5min, 10%B-60%B; 8.5-10.0min, 60%B-95%B; 10.0-12.0min, 95%B; 12.0-12.1min, 95%B-10%B; 12.1-15.0min, 10%B; injection volume: 0.5-10μL; injection plate temperature: 4°C; Mass spectrometry conditions: Spray voltage: 1-4.5 kV in positive ion mode, -1-4 kV in negative ion mode; atomization temperature: 300-400 °C; ion transfer tube temperature: 300-350 °C; sheath gas pressure: 50 Arb; auxiliary gas pressure: 10 Arb; primary mass spectrometry full scan, scanning range m / z: 50-300 Da; resolution: R = 60 000 or 120 000; data-dependent secondary ion full scan FullMS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2, intensity threshold: 1.0×10 5 ;ddMS2 resolution: R = 15 000 or 30 000; S4 Qualitative and / or quantitative determination of samples S4a qualitative and quantitative determination: S4a-1 injects the mixed standard qualitative solution I prepared in S1-1 to determine the retention time of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine, and the signal-to-noise ratio of the three chromatographic peaks should be greater than 100; injects the sample solution I-1 to determine whether o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine are detected; if not detected, proceed to step S4a-3; if detected, proceed to steps S4a-2 and S4a-3; S4a-2 injects the series mixed standard curve solution I prepared in S1-2, and uses the chromatographic peak areas of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine to perform linear regression on their concentrations, and draws a linear equation; injects sample solution I-2 and sample solution I-1 in sequence, and substitutes the peak areas into the linear equation of the series mixed standard curve solution I, and calculates the contents of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine by the external standard method; S4a-3 injects the series of mixed standard curve solutions II prepared in S1-2, performs linear regression on the concentration of each chromatographic peak area, and draws a linear equation; injects sample solution II-2 and sample solution II-1 in sequence, substitutes the peak area into the linear equation of the series of mixed standard working solutions II, and calculates the contents of 51 components except o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine by the external standard method; S4b Qualitative judgment: The sample solution I-1, the sample solution I-2 and the mixed standard qualitative solution I prepared by S1-1 are subjected to chromatography-mass spectrometry detection. When the chromatographic retention time of the sample solution I is consistent with that of the mixed standard qualitative solution I, and the fragment ion detection fragments of the sample solution I and the secondary mass spectrometry are consistent with those of the mixed standard qualitative solution I, the sample includes one or more of o-phenylenediamine, toluene-3,4-diamine and 4-nitro-o-phenylenediamine; The sample solution II-1, the sample solution II-2 and the mixed standard qualitative solution II prepared by S1-1 are subjected to chromatography-mass spectrometry detection. When the chromatographic retention time of the sample solution II is consistent with that of the mixed standard qualitative solution II, and the fragment ion detection fragments of the sample solution II and in the secondary mass spectrum are consistent with those of the mixed standard qualitative solution II, the sample includes one or more of the 51 components other than o-phenylenediamine, toluene-3,4-diamine and 4-nitro-o-phenylenediamine.

2. The method according to claim 1, characterized in that The data described in S3 rely on the Targeted Mass of the secondary ion full scan Full MS / Intensity / Dynamic Exclusion / Targeted Mass / ddMS2, in which the retention time, m / z and polarity of the target precursor ion are set. The mass spectrometry information of the 54 dyes is as follows:

3. The method according to claim 1, characterized in that The mixed standard qualitative solution described in S1-1 is prepared by the method described in (1) or (2): (1) preparing a mixed standard stock solution, wherein the mixed standard stock solution is prepared by taking 54 hair dye reference substances, mixing them, and dissolving them in a 50% to 80% methanol solution to prepare a mixed standard stock solution; Dilute the mixed standard stock solution with 10% to 30% methanol solution to prepare a mixed standard qualitative solution I; The mixed standard stock solution is diluted with a 10% to 30% methanol solution containing an antioxidant to prepare a mixed standard qualitative solution II; (2) preparing single-label stock solutions, wherein the single-label stock solutions are prepared by dissolving o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine reference substances in 50% to 80% methanol solution, respectively; and dissolving other hair dye reference substances in 50% to 80% methanol solution containing an antioxidant, respectively; and obtaining 54 single-label stock solutions; The single standard stock solutions of o-phenylenediamine, toluene-3,4-diamine and 4-nitro-o-phenylenediamine are mixed, and 10% to 30% methanol solution is added to prepare a mixed standard qualitative solution I; The 54 single standard stock solutions were mixed, and a 10% to 30% methanol solution containing an antioxidant was added to prepare a mixed standard qualitative solution II.

4. The method according to claim 1, characterized in that: The series of mixed standard curve solutions described in S1-2 are prepared by the method described in (1) or (2): (1) preparing a mixed standard stock solution, wherein the mixed standard stock solution is prepared by taking 54 hair dye reference substances, mixing them, and dissolving them in a 50% to 80% methanol solution to prepare a mixed standard stock solution; Continuously diluting the mixed standard stock solution with 10% to 30% methanol solution to obtain a series of mixed standard curve solutions I of the several concentrations; Continuously diluting the mixed standard stock solution with a 10% to 30% methanol solution containing an antioxidant to obtain a series of mixed standard curve solutions II of the aforementioned several concentrations; (2) preparing single-label stock solutions, wherein the single-label stock solutions are prepared by dissolving o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine reference substances in 50% to 80% methanol solution, respectively; and dissolving other hair dye reference substances in 50% to 80% methanol solution containing an antioxidant, respectively; and obtaining 54 single-label stock solutions; The single standard stock solutions of o-phenylenediamine, toluene-3,4-diamine and 4-nitro-o-phenylenediamine are mixed, and 10% to 30% methanol solution is added to prepare a mixed standard qualitative solution I; The 54 single standard stock solutions were mixed, and a 10% to 30% methanol solution containing an antioxidant was added to prepare a mixed standard qualitative solution II.

5. The method according to claim 3 or 4, characterized in that: The mass concentrations of the 54 hair dyes in the mixed standard stock solution and the single standard stock solution are no higher than 5000 μg / mL.

6. The method according to claim 5, characterized in that The mass concentration of hydroquinone in the mixed standard stock solution and the single standard stock solution is not higher than 800 μg / mL, and the mass concentration of other hair dyes is not higher than 200 μg / mL.

7. The method according to claim 6, characterized in that The mass concentration of hydroquinone in the mixed standard stock solution and the single standard stock solution is not higher than 40 μg / mL, and the mass concentration of other hair dyes is not higher than 10 μg / mL.

8. The method according to claim 1, characterized in that The 10% to 30% methanol solution in S1-1, S1-2 and S2-2 is a 25% methanol solution; the 10% to 30% methanol solution containing an antioxidant in S1-1, S1-2 and S2-2 is a 25% methanol solution containing an antioxidant.

9. The method according to claim 1, characterized in that: In the 10% to 30% methanol solution containing an antioxidant in S1-1, S1-2 and S2-2, the antioxidant is D-isoascorbic acid or L-ascorbic acid.

10. The method according to claim 9, characterized in that In the 10% to 30% methanol solution containing an antioxidant in S1-1, S1-2 and S2-2, the antioxidant is D-isoascorbic acid.

11. The method according to claim 1, characterized in that: In the 10% to 30% methanol solution containing antioxidants, the concentration of the antioxidants in S1-1, S1-2 and S2-2 is not less than 0.05%.

12. The method according to claim 10, characterized in that In the 10% to 30% methanol solution containing antioxidants in S1-1, S1-2 and S2-2, the concentration of antioxidants is 0.05%.

13. The method according to claim 3 or 4, characterized in that: S1 also includes S1-3 preparation of positioning solution: Take the single-label stock solution, add 10% to 30% methanol solution respectively, and prepare the positioning solution I with the mass concentration of o-phenylenediamine, toluene-3,4-diamine, and 4-nitro-o-phenylenediamine being 100 to 1000 ng / mL; Take the single-label stock solution, add 10% to 30% methanol solution containing antioxidants, and prepare positioning solution II with a mass concentration of hydroquinone of 400 to 4000 ng / mL and a mass concentration of other hair dyes of 100 to 1000 ng / mL.

14. The method according to claim 1, characterized in that Weigh the sample in S2-1 and dilute it to 10-20 mg / mL with alcohol solution.

15. The method according to claim 1, characterized in that The concentrations of the sample solutions Ⅰ-1 and Ⅱ-1 in S2-2 are 200 to 400 μg / mL; the concentrations of the sample solutions Ⅰ-2 and Ⅱ-2 in S2-2 are 20 to 40 μg / mL.

16. The method according to claim 1, characterized in that The alcohol solution in S2 is a 70% methanol solution.

17. The method according to claim 1, characterized in that The pore size of the organic filter membrane in S2 is 0.22 μm or 0.45 μm.

18. The method according to claim 17, characterized in that The pore size of the organic filter membrane in S2 is 0.45 μm.

19. The method according to claim 18, characterized in that The organic filter membrane in S2 is a polytetrafluoroethylene membrane.

20. The method according to claim 1, characterized in that S3 described chromatographic conditions: chromatographic column: ACE Excel C18 column 150mm×2.0mm, 3μm, column temperature: 40°C; mobile phase: phase A is 1mmol / L ammonium fluoride aqueous solution; phase B is acetonitrile, flow rate: 0.3mL / min; gradient elution program: 0~2.0min, 10%B; 2.0~8.5min, 10%B~60%B; 8.5~10.0min, 60%B~95%B; 10.0~12.0min, 95%B; 12.0~12.1min, 95%B~10%B; 12.1~15.0min, 10%B; injection volume: 1μL; injection plate temperature: 4℃.

21. The method according to claim 1, characterized in that The mass spectrometry conditions described in S3 were as follows: spray voltage: 1.5 kV in positive ion mode, -2.5 kV in negative ion mode; nebulization temperature: 350 °C; ion transfer tube temperature: 325 °C; sheath gas pressure: 50 Arb; auxiliary gas pressure: 10 Arb; primary mass spectrometry full scan, scanning range m / z: 50-300 Da; full scan resolution: R=60000; data-dependent secondary ion full scan Full MS / Intensity / Dynamic Exclusion / TargetedMass / ddMS2, intensity threshold: 1.0×10 5 ;ddMS2 resolution: R=15 000.

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