CN115902024B - Stable isotope labeling and liquid chromatography-mass spectrometry analysis method for dye with strong reducibility and forbidden limit in hair dye - Google Patents
Stable isotope labeling and liquid chromatography-mass spectrometry analysis method for dye with strong reducibility and forbidden limit in hair dye Download PDFInfo
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- CN115902024B CN115902024B CN202211498578.5A CN202211498578A CN115902024B CN 115902024 B CN115902024 B CN 115902024B CN 202211498578 A CN202211498578 A CN 202211498578A CN 115902024 B CN115902024 B CN 115902024B
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- dye
- phenylenediamine
- derivatization
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- dansyl chloride
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- 239000000975 dye Substances 0.000 title claims abstract description 144
- 238000004458 analytical method Methods 0.000 title claims abstract description 27
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 title claims abstract description 16
- 239000000118 hair dye Substances 0.000 title claims description 25
- 238000001948 isotopic labelling Methods 0.000 title abstract description 15
- 238000001212 derivatisation Methods 0.000 claims abstract description 143
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 60
- XPDXVDYUQZHFPV-UHFFFAOYSA-N Dansyl Chloride Chemical class C1=CC=C2C(N(C)C)=CC=CC2=C1S(Cl)(=O)=O XPDXVDYUQZHFPV-UHFFFAOYSA-N 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 48
- 238000006243 chemical reaction Methods 0.000 claims abstract description 44
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 claims abstract description 43
- 239000000243 solution Substances 0.000 claims description 49
- 150000002500 ions Chemical class 0.000 claims description 46
- CWLKGDAVCFYWJK-UHFFFAOYSA-N 3-aminophenol Chemical compound NC1=CC=CC(O)=C1 CWLKGDAVCFYWJK-UHFFFAOYSA-N 0.000 claims description 45
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical compound NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 claims description 43
- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical compound NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 claims description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 42
- 239000000523 sample Substances 0.000 claims description 34
- 239000012086 standard solution Substances 0.000 claims description 30
- CBCKQZAAMUWICA-UHFFFAOYSA-N 1,4-phenylenediamine Chemical compound NC1=CC=C(N)C=C1 CBCKQZAAMUWICA-UHFFFAOYSA-N 0.000 claims description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 26
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 229940018564 m-phenylenediamine Drugs 0.000 claims description 24
- 229940018563 3-aminophenol Drugs 0.000 claims description 23
- WZCQRUWWHSTZEM-UHFFFAOYSA-N 1,3-phenylenediamine Chemical compound NC1=CC=CC(N)=C1 WZCQRUWWHSTZEM-UHFFFAOYSA-N 0.000 claims description 22
- 229910052757 nitrogen Inorganic materials 0.000 claims description 19
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 17
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 claims description 16
- 239000012488 sample solution Substances 0.000 claims description 15
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- 239000011550 stock solution Substances 0.000 claims description 14
- 238000005303 weighing Methods 0.000 claims description 14
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 12
- QGNGOGOOPUYKMC-UHFFFAOYSA-N 4-hydroxy-6-methylaniline Chemical compound CC1=CC(O)=CC=C1N QGNGOGOOPUYKMC-UHFFFAOYSA-N 0.000 claims description 9
- VHNQIURBCCNWDN-UHFFFAOYSA-N pyridine-2,6-diamine Chemical compound NC1=CC=CC(N)=N1 VHNQIURBCCNWDN-UHFFFAOYSA-N 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
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- 239000007924 injection Substances 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 7
- 210000000692 cap cell Anatomy 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 235000019253 formic acid Nutrition 0.000 claims description 7
- 238000004949 mass spectrometry Methods 0.000 claims description 7
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 6
- 239000007795 chemical reaction product Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 6
- IQXUIDYRTHQTET-UHFFFAOYSA-N 4-amino-3-nitrophenol Chemical compound NC1=CC=C(O)C=C1[N+]([O-])=O IQXUIDYRTHQTET-UHFFFAOYSA-N 0.000 claims description 5
- RLYCRLGLCUXUPO-UHFFFAOYSA-N 2,6-diaminotoluene Chemical compound CC1=C(N)C=CC=C1N RLYCRLGLCUXUPO-UHFFFAOYSA-N 0.000 claims description 4
- YWNSGSNRKLNTHJ-UHFFFAOYSA-N 2-(4-aminoanilino)ethanol;sulfuric acid Chemical compound OS(O)(=O)=O.NC1=CC=C(NCCO)C=C1 YWNSGSNRKLNTHJ-UHFFFAOYSA-N 0.000 claims description 4
- HCPJEHJGFKWRFM-UHFFFAOYSA-N 2-amino-5-methylphenol Chemical compound CC1=CC=C(N)C(O)=C1 HCPJEHJGFKWRFM-UHFFFAOYSA-N 0.000 claims description 4
- HVHNMNGARPCGGD-UHFFFAOYSA-N 2-nitro-p-phenylenediamine Chemical compound NC1=CC=C(N)C([N+]([O-])=O)=C1 HVHNMNGARPCGGD-UHFFFAOYSA-N 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 4
- ATGUVEKSASEFFO-UHFFFAOYSA-N p-aminodiphenylamine Chemical compound C1=CC(N)=CC=C1NC1=CC=CC=C1 ATGUVEKSASEFFO-UHFFFAOYSA-N 0.000 claims description 4
- 239000013558 reference substance Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- BOKGTLAJQHTOKE-UHFFFAOYSA-N 1,5-dihydroxynaphthalene Chemical compound C1=CC=C2C(O)=CC=CC2=C1O BOKGTLAJQHTOKE-UHFFFAOYSA-N 0.000 claims description 3
- BMTSZVZQNMNPCT-UHFFFAOYSA-N 2-aminopyridin-3-ol Chemical compound NC1=NC=CC=C1O BMTSZVZQNMNPCT-UHFFFAOYSA-N 0.000 claims description 3
- GQFGHCRXPLROOF-UHFFFAOYSA-N 2-chloro-1,4-phenylenediamine sulfate Chemical compound OS(O)(=O)=O.NC1=CC=C(N)C(Cl)=C1 GQFGHCRXPLROOF-UHFFFAOYSA-N 0.000 claims description 3
- ZWUBBMDHSZDNTA-UHFFFAOYSA-N 4-Chloro-meta-phenylenediamine Chemical compound NC1=CC=C(Cl)C(N)=C1 ZWUBBMDHSZDNTA-UHFFFAOYSA-N 0.000 claims description 3
- ZVNPWFOVUDMGRP-UHFFFAOYSA-N 4-methylaminophenol sulfate Chemical compound OS(O)(=O)=O.CNC1=CC=C(O)C=C1.CNC1=CC=C(O)C=C1 ZVNPWFOVUDMGRP-UHFFFAOYSA-N 0.000 claims description 3
- RAUWPNXIALNKQM-UHFFFAOYSA-N 4-nitro-1,2-phenylenediamine Chemical compound NC1=CC=C([N+]([O-])=O)C=C1N RAUWPNXIALNKQM-UHFFFAOYSA-N 0.000 claims description 3
- DBFYESDCPWWCHN-UHFFFAOYSA-N 5-amino-2-methylphenol Chemical compound CC1=CC=C(N)C=C1O DBFYESDCPWWCHN-UHFFFAOYSA-N 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000010813 internal standard method Methods 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- XAWPKHNOFIWWNZ-UHFFFAOYSA-N 1h-indol-6-ol Chemical compound OC1=CC=C2C=CNC2=C1 XAWPKHNOFIWWNZ-UHFFFAOYSA-N 0.000 claims description 2
- 229940113490 2,4-diaminophenoxyethanol hydrochloride Drugs 0.000 claims description 2
- PBVFDMZFBPZIMC-UHFFFAOYSA-N 2-(2,4-diaminophenoxy)ethanol;hydrochloride Chemical compound Cl.NC1=CC=C(OCCO)C(N)=C1 PBVFDMZFBPZIMC-UHFFFAOYSA-N 0.000 claims description 2
- 230000005764 inhibitory process Effects 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- DFQICHCWIIJABH-UHFFFAOYSA-N naphthalene-2,7-diol Chemical compound C1=CC(O)=CC2=CC(O)=CC=C21 DFQICHCWIIJABH-UHFFFAOYSA-N 0.000 claims description 2
- VOZKAJLKRJDJLL-UHFFFAOYSA-N 2,4-diaminotoluene Chemical compound CC1=CC=C(N)C=C1N VOZKAJLKRJDJLL-UHFFFAOYSA-N 0.000 claims 2
- 238000003556 assay Methods 0.000 claims 2
- 238000004445 quantitative analysis Methods 0.000 abstract description 10
- 239000002537 cosmetic Substances 0.000 abstract description 8
- 238000004451 qualitative analysis Methods 0.000 abstract description 8
- 239000000047 product Substances 0.000 description 95
- -1 p-phenylenediamine dansyl chloride derivative Chemical class 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 239000012071 phase Substances 0.000 description 12
- 238000010586 diagram Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000011159 matrix material Substances 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 238000011084 recovery Methods 0.000 description 7
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical class OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
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- 150000004987 o-phenylenediamines Chemical class 0.000 description 5
- 230000010355 oscillation Effects 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 5
- 150000002989 phenols Chemical class 0.000 description 5
- 230000004044 response Effects 0.000 description 5
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- 238000012360 testing method Methods 0.000 description 5
- 150000001448 anilines Chemical class 0.000 description 4
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- 230000037361 pathway Effects 0.000 description 4
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 3
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- 238000002360 preparation method Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
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- 238000000926 separation method Methods 0.000 description 3
- KZTWOUOZKZQDMN-UHFFFAOYSA-N 2,5-diaminotoluene sulfate Chemical compound OS(O)(=O)=O.CC1=CC(N)=CC=C1N KZTWOUOZKZQDMN-UHFFFAOYSA-N 0.000 description 2
- JQVAPEJNIZULEK-UHFFFAOYSA-N 4-chlorobenzene-1,3-diol Chemical compound OC1=CC=C(Cl)C(O)=C1 JQVAPEJNIZULEK-UHFFFAOYSA-N 0.000 description 2
- GCNTZFIIOFTKIY-UHFFFAOYSA-N 4-hydroxypyridine Chemical compound OC1=CC=NC=C1 GCNTZFIIOFTKIY-UHFFFAOYSA-N 0.000 description 2
- DGRGLKZMKWPMOH-UHFFFAOYSA-N 4-methylbenzene-1,2-diamine Chemical compound CC1=CC=C(N)C(N)=C1 DGRGLKZMKWPMOH-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
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- 125000003277 amino group Chemical group 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
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- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- RJCMROWGXQCQQI-UHFFFAOYSA-N 1-hydroxyindole-5,6-diol Chemical compound C1=C(O)C(O)=CC2=C1N(O)C=C2 RJCMROWGXQCQQI-UHFFFAOYSA-N 0.000 description 1
- 150000004782 1-naphthols Chemical class 0.000 description 1
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- QWTMTLGIDKWAOW-UHFFFAOYSA-N 2-[3-(2-hydroxyphenyl)-2-methylphenyl]phenol Chemical compound C1=C(C2=CC=CC=C2O)C(C)=C(C2=C(C=CC=C2)O)C=C1 QWTMTLGIDKWAOW-UHFFFAOYSA-N 0.000 description 1
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- MSJIPWGDQODKSK-UHFFFAOYSA-N 2-[4-(2-hydroxyphenyl)phenyl]phenol Chemical compound OC1=CC=CC=C1C1=CC=C(C=2C(=CC=CC=2)O)C=C1 MSJIPWGDQODKSK-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention belongs to the technical field of analysis, and particularly relates to a stable isotope labeling and liquid chromatography-mass spectrometry analysis method of a dye with strong reducibility forbidden. Firstly, the invention discovers that the stability of a derivative product obtained by the reaction of a dye component and a dansyl chloride derivative reagent is good; secondly, the dansyl chloride and the dansyl chloride-D6 are used as derivatization reagents, a qualitative and quantitative analysis method of the dye-stable isotope labeling liquid chromatography-mass spectrometry is established, the method improves the stability and the analysis accuracy of the dye, provides a new reference basis for qualitative and quantitative analysis of forbidden dyes, and provides a new idea for accurate and quantitative analysis of chemically unstable organic matters in cosmetics.
Description
Technical Field
The invention belongs to the technical field of analysis, and particularly relates to a stable isotope labeling and liquid chromatography-mass spectrometry analysis method of a dye with strong reducibility forbidden.
Background
The hair dye is a common special-purpose cosmetic, and is divided into oxidative color-changing hair dye and non-oxidative color-changing hair dye, wherein 80% of hair dye sold in the market is oxidative color-changing hair dye. At present, more and more people like to dye hair, and the consumer population of the hair dye is also younger and younger, so the safety of the hair dye is also of great concern. The harm of the hair dye mainly comes from dyes, the main components of the dyes are anilines and phenol compounds, the compounds have sensitization and cancerogenicity, chronic injury can be caused to human health after long-term use, and researches in recent years report that the use of the hair dye can increase the risk of people suffering from breast cancer and bladder cancer by at least 25 percent. The use of the hair dye is closely related to the health of people, so that the quality management of the hair dye and the perfection of the detection technology of the hair dye are imperative.
At present, detection methods of the reporter dye in relevant literature at home and abroad comprise thin layer chromatography, capillary electrophoresis, high performance liquid chromatography, gas chromatography-mass spectrometry and high performance liquid chromatography-mass spectrometry. Thin layer chromatography does not allow accurate quantification; the gas chromatography and the gas chromatography-mass spectrometry are not beneficial to analyzing the thermally unstable substances, and the hair dye sample has the defects of complex matrix and poor separation effect and difficult qualitative. The high performance liquid chromatography is not limited by the thermal stability and the volatility of the to-be-detected object, has strong separation capability and good durability, but has more isomers in 32 dyes, so 2 method systems are needed after the latest revision of the detection method in cosmetic safety technical Specification (2015 edition), the detection time of each system is long, the requirement on a chromatographic column is high, and the service life of the chromatographic column is short. The high performance liquid chromatography-mass spectrometry has strong separation capability and high sensitivity, can reduce matrix effect and can rapidly and accurately analyze target compounds, and the method is used for qualitatively and quantitatively analyzing dyes, but the dyes are extremely easy to oxidize, the analysis reproducibility is poor, the accurate quantification is affected, the quality control analysis is plagued, liu Yuling and other researches find that dye stock solution is stored in a refrigerator at 0-4 ℃ and is used for 2 days at most.
The invention breaks through the traditional thought, and the newly-built stable isotope labeling liquid chromatography-mass spectrometry analysis method improves the stability and the analysis accuracy of the analyte to be detected, and provides a new reference basis for qualitative and quantitative analysis of forbidden dyes. Dye-forbidden components are difficult to obtain due to isotopic labeling, and these components are compounds having similar molecular structures and reactivity; the invention discovers that the chemical property of the derivative product of the dye is stable within 10 days under the condition of 4 ℃ by taking dansyl chloride as a derivatization reagent; therefore, the invention selects dansyl chloride and dansyl chloride-D6 as derivatization reagents, establishes a dye stable isotope labeling liquid chromatography-mass spectrometry analysis method for qualitatively and quantitatively analyzing the dye, improves the stability and the analysis accuracy of the dye, provides a new reference basis for qualitatively and quantitatively analyzing forbidden dyes, and provides a new thought for accurately and quantitatively analyzing chemically unstable organic matters in cosmetics.
Disclosure of Invention
Aiming at the technical problems, the invention aims to improve the stability and the analysis accuracy of the dye, provide a new reference basis for the qualitative and quantitative analysis of forbidden dyes, and particularly provide a qualitative and quantitative analysis of the dye by using a dye-stable isotope labeling liquid chromatography-mass spectrometry analysis method. The method specifically comprises the following steps:
in a first aspect, the invention provides a method for analyzing a dye component with strong reducibility and forbidden limit in a hair dye, which comprises the steps of respectively carrying out derivatization reaction on a standard substance solution of each dye component and a dye sample solution by using dansyl chloride-D6 and a dansyl chloride derivatization reagent.
Preferably, the method comprises the steps of:
(1) Preparing a derivatization mixed standard solution: preparing a mixed standard solution of each component of the dye, and adding a dansyl chloride-D6 derivatization reagent to carry out derivatization reaction to obtain a derivatization mixed standard solution;
(2) Derivatization of sample solution configuration: preparing a dye sample solution from a sample to be detected, and adding a dansyl chloride derivatization reagent to carry out derivatization reaction to obtain a derivatization sample solution;
(3) Preparing a solution to be tested: mixing the derivatization mixed standard solution in the step (1) and the derivatization test solution in the step (2) in equal volume to obtain a solution to be tested;
(4) And (3) respectively detecting and analyzing the solution to be detected by adopting a liquid chromatography-mass spectrometry (LC-MS) method:
the chromatographic conditions were: using Capcell PAK C 18 Chromatographic column, column temperature: 40 ℃; mobile phase a was 0.1% formic acid in water; mobile phase B was 0.1% acetonitrile formate; the flow rate is 0.3mL/min; sample injection amount is 1.0 mu L; gradient elution procedure was used: 0 to 10.5min,65 percent of A to 5%A;10.5 to 12.0min,5 percent of A;12.0 to 12.1min,5 to 65 percent of A;12.1 to 14 minutes, 65 percent of A;
the mass spectrometry conditions were: electrospray ion source positive ions (ESI) + ) Mode, ionization voltage is 4500V; the heating Temperature (TEM) was 550 ℃; the air curtain gas (CUR), the jet collision gas (CAD), the spray gas (GS 1) and the auxiliary heating gas (GS 2) are respectively 20 psi, 8 psi, 40psi and 40psi; the scanning mode is multi-reaction monitoring (MRM);
(5) And calculating the content of each dye component in the sample by adopting an internal standard method.
Preferably, the dye component comprises: 1, 5-naphthalenediol, 1-naphthol, 2, 4-diaminophenoxyethanol hydrochloride, 2, 6-diaminopyridine, 2, 7-naphthalenediol, 2-amino-3-hydroxypyridine, 2-chloro-p-phenylenediamine sulfate, 2-methyl-m-phenylenediamine, 2-nitro-p-phenylenediamine, 4-amino-2-hydroxytoluene, 4-amino-3-nitrophenol, 4-amino-m-cresol, 4-chloro-m-phenylenediamine, 4-nitro-o-phenylenediamine, 6-amino-m-cresol, 6-hydroxyindole, p-phenylenediamine, m-aminophenol, m-phenylenediamine, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, N-diethyl-p-phenylenediamine sulfate, N-diethyltoluene-2, 5-dihydrochloride, N-phenyl-p-phenylenediamine, o-aminophenol, p-methylphenol sulfate, p-phenylenediamine, m-phenylenediamine, toluene-2, 5-phenylenediamine, toluene-3-diamine, o-phenylenediamine, and 4-o-phenylenediamine.
Preferably, the dye component comprises: p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol and o-aminophenol.
Preferably, the configuration of the dye component mixed standard solution in the step (1) is as follows: precisely weighing reference substances p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol and o-aminophenol respectively, and fixing the volumes to 1mg/mL by methanol to obtain standard stock solutions respectively; respectively precisely measuring 1mL of p-phenylenediamine, 0.2mL of m-phenylenediamine, 1mL of o-phenylenediamine, 1mL of p-aminophenol and 0.2mL of m-aminophenol standard stock solution, placing the standard stock solutions into a volumetric flask, and fixing the volume to 10mL by using methanol to obtain the mixed standard solution of each component of the dye.
Preferably, the dye test solution in step (2) is configured to: weighing a sample, adding methanol-water solution with the volume ratio of 8:2, dissolving and mixing uniformly until the concentration is 50mg/mL, and carrying out ice bath ultrasonic extraction for 15min to obtain a dye sample solution.
Preferably, the derivatization reaction in the step (1) is: precisely measuring 50 mu L of mixed standard solution of the dye components, drying with nitrogen, adding 400 mu L of dansyl chloride-D6 derivatization reagent solution, sealing, and vibrating at 30 ℃ for 20min.
Preferably, the derivatization reaction in the step (2) is: precisely measuring 50 mu L of dye test solution, blowing with nitrogen, adding 400 mu L of dansyl chloride derivatization reagent solution, sealing, and vibrating at 30 ℃ for 20min.
Preferably, the dansyl chloride-D6 derivatizing reagent solution is configured to: dissolving 10mg of dansyl chloride-D6 with anhydrous tetrahydrofuran, adding 10 mu L of triethylamine, and fixing the volume to 10mL with the anhydrous tetrahydrofuran to obtain a dansyl chloride-D6 derivatization reagent solution with the concentration of 1 mg/mL;
preferably, the dansyl chloride derivatizing reagent solution is configured to: 10mg of dansyl chloride is dissolved by anhydrous tetrahydrofuran, 10 mu L of triethylamine is added, the volume is fixed to 10mL by the anhydrous tetrahydrofuran, and the dansyl chloride derivatization reagent solution with the concentration of 1mg/mL is obtained.
Preferably, the step (3) is: and mixing 200 mu L of the derivatization mixed standard solution and 200 mu L of the derivatization test solution respectively, adding 600 mu L of acetonitrile, and filtering by a 0.2 mu m filter membrane to obtain a filtrate which is the solution to be tested.
Preferably, the calculation formula in the step (5) is: ω= (c×v×d×100%)/M; c= (a D ×C For a pair of )/A D6 ；
Wherein ω is the dye content,%; c is the concentration of dye in the test solution, g/mL; v is the constant volume of the sample, mL; d is dilution multiple; m is the sample sampling amount, g; AD (analog to digital) converter 6 Adding dansyl chloride-D6 derivatization reagent into the standard solution to derivatize the peak area of the reaction product; AD is the peak area of the post-derivatization reaction product of the dansyl chloride derivatization reagent in the test sample; c (C) For a pair of Concentration of standard, g/mL.
The beneficial effects of the invention are as follows: firstly, the invention discovers that the dansyl chloride is used as a derivatization reagent and matched with derivatization reaction, the stability of a derivatization product obtained by the derivatization reaction of 31 dyes (except the phenylbenzimidazole sulfonic acid) is good, and the chemical property is stable within 10 days under the condition of 4 ℃; secondly, the invention discovers that the dansyl chloride and the dansyl chloride-D6 are selected as derivatization reagents, and a qualitative and quantitative analysis method of the dye-stable isotope labeling liquid chromatography-mass spectrometry is established.
Drawings
FIG. 1 is a dye-stable isotope labeling liquid chromatography-mass spectrometry analysis method;
FIG. 25 Dan sulfonyl chloride derivatization reaction Processes;
FIG. 35 ion flow diagrams of dye dansyl chloride derivative products, wherein the dye 2, 6-diaminopyridine derivative product, p-phenylenediamine derivative product, 4-amino-m-cresol derivative product, m-benzenediol derivative product and 1-naphthol derivative product are respectively from left to right;
FIG. 4 optimum time, optimum reaction temperature, optimum reaction solvent, optimum addition of triethylamine for the dansyl chloride derivatization; wherein each group of data is respectively dye 1-naphthol, 2, 6-diaminopyridine, 4-amino-m-cresol, p-phenylenediamine and m-benzenediol from left to right;
FIG. 5 is a graph of phenylmethyl pyrazolone stability;
FIG. 6 is an ion flow diagram of the 31 dye dansyl chloride derivative; wherein from left to right are dye 2, 6-diaminopyridine, N-diethyl-p-phenylenediamine sulfate, p-phenylenediamine, 2, 4-diaminophenoxy ethanol sulfate, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, 2, 5-diaminotoluene sulfate, N-diethyltoluene-2, 5-diamine hydrochloride, m-phenylenediamine, p-aminophenol, 2-amino-3-hydroxypyridine, m-aminophenol, 4-amino-m-cresol, o-phenylenediamine derivative, 2-nitro-p-phenylenediamine, o-aminophenol, 4-amino-2-hydroxytoluene, 4-nitro-o-phenylenediamine, 2-chloro-p-phenylenediamine sulfate, p-methylaminophenol sulfate, toluene-3, 4-diamine, 6-amino-m-cresol, p-phenylenediphenol, m-phenylenediphenol, 2-methyl-m-phenylenediphenol, 4-amino-3-nitrophenol, N-phenyl-p-phenylenediamine, 2, 7-naphthol, 6-hydroxy-1, 5-dihydroxyindole, 1-dihydroxynaphthalene;
FIG. 7 is a quantitative ion flow diagram of p-phenylenediamine, m-phenylenediamine, o-phenylenediamine derivative products wherein 1 is a p-phenylenediamine derivative product, 2 is an m-phenylenediamine derivative product, and 3 is an o-phenylenediamine derivative product;
FIG. 8 is a quantitative ion flow diagram of p-aminophenol, m-aminophenol, and o-aminophenol derivatization products, wherein 1 is p-aminophenol derivatization product, 2 is m-aminophenol derivatization product, and 3 is o-aminophenol derivatization product;
FIG. 9 is a quantitative ion flow diagram of dansyl chloride and dansyl chloride-D derivative products of aniline dyes; wherein 1 is p-phenylenediamine dansyl chloride derivative, 2 is m-phenylenediamine dansyl chloride derivative, 3 is o-phenylenediamine dansyl chloride derivative, 4 is p-phenylenediamine dansyl chloride-D6 derivative, 5 is m-phenylenediamine dansyl chloride-D6 derivative, and 6 is o-phenylenediamine dansyl chloride-D6 derivative;
FIG. 10 is a quantitative ion flow diagram of the dansyl chloride and dansyl chloride-D derivative products of a phenolic dye; wherein 1 is p-aminophenol dansyl chloride derivative, 2 is m-aminophenol dansyl chloride derivative, 3 is o-aminophenol dansyl chloride derivative, 4 is p-aminophenol dansyl chloride-D6 derivative, 5 is m-aminophenol dansyl chloride-D6 derivative, and 6 is o-aminophenol dansyl chloride-D6 derivative;
FIG. 11 is a graph of peak area of dye component over time in positive ion detection mode;
FIG. 12 is a graph of peak area of dye component over time in anion detection mode;
FIG. 13P-aminophenol hair dye molecule attenuation;
FIG. 14 is a graph of peak area of the 5 dye-derivatized products as a function of time.
Detailed Description
The following describes the aspects of the invention in connection with specific examples, but the aspects of the invention are not limited to the examples described below.
The apparatus described in the following examples comprises: triple Quad 3500 high performance liquid chromatography-tandem mass spectrometer (SCIEX company, usa); capcell PAK C18 column (150 mm. Times.2.1 mm,3 μm, japan senior Co.); sample concentration nitrogen blower (organization company, usa); constant temperature mixer (Miou instruments, hangzhou); milli-Q IQ7000 ultra-pure water machine (Millipore Co., U.S.A.); electronic balance (mertrehler, switzerland); ultrasonic extractors (Kunshan ultrasonic instruments Co., ltd.); vortex mixer (Changzhou national electrical Co.);
hair dye standard as described in the following examples: 1-naphthol, 2, 6-diaminopyridine, 2-nitro-p-phenylenediamine, 4-amino-m-cresol, 4-chloro-m-phenylenediamine, p-phenylenediamine, m-aminophenol, m-phenylenediamine, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, N-diethyl-p-phenylenediamine sulfate, N-diethyltoluene-2, 5-diamine hydrochloride, o-aminophenol, p-phenylenediamine, m-phenylenediamine, toluene-2, 5-diamine sulfate, toluene-3, 4-diamine, o-phenylenediamine (Germany Dr. Ehrenstorfer company, purity not less than 98.0%), 1, 5-naphthalenediol, 2-methyl-m-phenylenediamine, 4-amino-3-nitrophenol, 6-amino-m-cresol, N-phenyl-p-phenylenediamine, p-methylaminophenol sulfate (North-Aminomann Ha Ge biological limited, non-98%, purity less than 2, 7-hydroxy-3, 4-phenylenediamine, 4-hydroxypyridine, 4-p-phenylenediamine, 4-phenylene hydrochloride, 4-phenylene diamine (purity not less than 98.0% by Germany, 4-hydroxy-4-1, 5-naphthalenediol, 2-methyl-m-phenylenediamine, p-nitrophenol, p-methylamino-5-phenylenediamine sulfate, p-mann Ha Ge biological limited, p-diaminophenol sulfate, p-5-hydroxy-4, p-phenylene hydrochloride, p-hydroxypyridine, p-4, p-phenylene hydrochloride, p-hydroxy-4, p-phenylene ether hydrochloride, purity 98.0%); methanol, acetonitrile (chromatographic purity, merck, germany); benzyl bromide (purity >98.8%, shanghai milin); dansyl chloride (purity 98%, shanghai microphone Corp.); dansyl chloride-D6 (purity 98%, TRC, canada); tetrahydrofuran (99.5% pure, shanghai microphone company); n, N-dimethylformamide (purity 99.8%, shanghai milin); absolute ethanol (99.9% pure), SIGMA-ALDRICH; triethylamine (analytically pure, national drug group chemicals limited); formic acid (mass spectrum purity, merck, germany); microporous filter membrane (0.2 μm, organic phase type); the experimental water is primary water.
Example 1 optimization of dye derivatization conditions and selection of optimal derivatizing reagents
By comparing two derivatization reagents of benzyl bromide and dansyl chloride, the benzyl bromide is adopted as the derivatization reagent, the derivatization products of p-benzenediol and 4-chloro-m-benzenediol are not found, and the response value of the derivatization products of partial phenolic compounds (m-benzenediol, 2-methyl-m-benzenediol and 4-amino-3-nitrophenol) is weak; in addition, the derivatization process requires high temperature and takes a long time. The method adopts the dansyl chloride as the derivatization reagent, can react at room temperature, has high reaction speed, and the generated derivatization product has high response value and good peak type, and finally selects the dansyl chloride as the optimal derivatization reagent.
1. Method of
The 32 dyes were classified and found by statistics to be 28.1% phenol compounds, 43.8% aniline compounds, 25.0% aminophenols and 3.1% others. 5 representative dyes were selected based on a combination of classification and compound structural formula: 1-naphthol, 2, 6-diaminopyridine, 4-amino-m-cresol, m-benzenediol and p-phenylenediamine. The dansyl chloride is selected as a derivatization reagent to react with 5 representative dyes (1-naphthol, 2, 6-diaminopyridine, 4-amino-m-cresol, m-benzenediol and p-phenylenediamine) respectively, the derivatization reaction process is reasonably designed, and the derivatization reaction conditions are optimized.
1mg of 1-naphthol, 2, 6-diaminopyridine, 4-amino-m-cresol, m-benzenediol and p-phenylenediamine standard substances are precisely weighed respectively, and 1mL of methanol is added for dissolution. Diluting the 5 compound solutions by 10 times, putting 50 mu L of each solution into a 1.5mL centrifuge tube respectively, drying by nitrogen, adding 50 mu L of 1mg/mL derivatization reagent (oscillation time is 60min, temperature is 60 ℃, rotation speed is 1000r/min, solvents are absolute ethyl alcohol, absolute methyl alcohol, absolute tetrahydrofuran and absolute dimethylformamide respectively), sealing the centrifuge tube, keeping constant temperature (time is 60min, rotation speed is 1000r/min, temperature is 30, 40, 50 and 60 ℃) for oscillation reaction for a certain time (temperature is 60 ℃, rotation speed is 1000r/min, time is 20, 40, 60, 80, 100 and 120 min), cooling to room temperature after the reaction is finished, adding 1mL of acetonitrile, filtering by a 0.2 mu m filter membrane, and taking filtrate as a solution to be detected for standby.
Optimizing the addition amount of triethylamine: according to the operation steps, triethylamine 0, 10, 20 and 30 mu L are respectively added into the derivatization reagent, the constant-temperature oscillation time is 60min, the temperature is 60 ℃, and the rotating speed is 1000r/min.
2. Results
After derivatization of 5 representative dyes with dansyl chloride, the amino or phenolic hydroxyl groups in the dye molecules are replaced by N, N-dimethylnaphthyl groups; the derivatization products which are all monosubstituted are obtained, and the derivatization process is shown in figure 2; the ion flow diagram of the derivative product of the 5 dye dansyl chloride is shown in figure 3. In the process of the dansyl chloride derivatization reaction, the area of the extracted ion flow peak of the 5 dye derivatization products does not change obviously along with the reaction time, which indicates that the dansyl chloride derivatization reaction is fast, and the optimal reaction time is 20min; the area of the extracted ion flow peak of the 5 representative dye derivatization products is reduced along with the increase of the reaction temperature, and the optimal reaction temperature of the 5 dyes is 30 ℃; the derivatization effect of all 5 dyes in anhydrous tetrahydrofuran is the best; the optimal adding amount of triethylamine of 1-naphthol and m-dihydroxybenzene is 10 mu L, the area of the extracted ion flow peak of the derivative products of the rest compounds is reduced along with the increase of the adding amount of the triethylamine, and the optimal adding amount of the triethylamine is comprehensively considered to be 10 mu L; the above results are shown in particular in fig. 4. The above results indicate that the optimum condition for the derivatization reaction of the dansyl chloride is a reaction time of 20min, a reaction temperature of 30 ℃ and a derivatization solvent of tetrahydrofuran and an addition amount of triethylamine in the derivatization solvent of 10 μl.
Example 2 high Performance liquid chromatography-Mass Spectrometry analysis method of dye-derived products results were established
Respectively precisely weighing 1mg of 31 dyes (except phenylbenzimidazole sulfonic acid) respectively, adding 1mL of methanol for dissolution, diluting for 10 times, respectively taking 50 mu L of the solution into a centrifuge tube with the same 1.5mL, drying by nitrogen, adding 400 mu L of dansyl chloride-D6 derivatization reagent (1 mg/mL), sealing the centrifuge tube for constant-temperature oscillation reaction, cooling to room temperature after the reaction is finished, respectively adding 600 mu L of acetonitrile, mixing in an equal volume, filtering by a 0.2 mu m filter membrane, and taking the filtrate as a solution to be tested for standby.
Chromatographic conditions: chromatographic column: capcell PAK C for senior hall 18 A chromatographic column; column temperature: 40 ℃; the sample injection amount was 1.0. Mu.L. Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile; flow rate: 0.3mL/min. Gradient elution procedure: 0-2 min,90% A; 2-20 min,90% A-5%A; 20-25 min,5% A; 25-25.1 min, 5-90% A; 25.1-30 min,90% A.
Mass spectrometry conditions: ion source: electrospray ion source positive ions (ESI) + ) A mode; the ionization voltage (IS) IS: 4500V; the heating Temperature (TEM) was all: 550 ℃; the air curtain air (CUR), the jet Collision Air (CAD), the spray air (GS 1) and the auxiliary heating air (GS 2) are respectively: 20. 8, 40psi; scanning mode: multiple Reaction Monitoring (MRM).
Among the 32 dyes, phenyl methyl pyrazolone was the only molecular component containing no aniline or phenol structure, and the extracted ion peak area was stable well (Δ < 20%) within 50 hours, while demonstrating the stability of the instrument and the operating conditions (see fig. 5 for the results). In the process of the derivatization reaction of the other 31 dyes by using the dansyl chloride, all dye derivatization products obtain characteristic ions (delta 170.2 Da) of N, N-dimethylnaphthyl positive ions; the 4-chloro-m-benzenediol is derivatized to obtain a disubstituted derivatization product, the other compounds generate monosubstituted derivatization products, and the molecular structural formulas of the dye and the dansyl chloride derivatization products are shown in the table 1; scanning the dye derivative Product by adopting a Q1Multiple (Q1 MI) positive Ion mode, simultaneously scanning the compound by adopting a Product Ion (MS 2) positive Ion mode, setting different CE values, obtaining a parent Ion and two characteristic daughter ions of the dye derivative Product, and establishing an MRM method of the derivative Product, wherein the result is shown in Table 2; the ion flow diagram of the 31 dye dansyl chloride derivative product is shown in figure 6.
TABLE 1 molecular Structure of 31 dye and Danesulfonyl chloride derived products
TABLE 2 Main Mass Spectrometry parameters and Retention times for 31 Danesulfonyl chloride derived products
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Example 3 establishment of a method for dye-stable isotope labeling LC-MS analysis
1. Establishment of dye stable isotope labeling liquid chromatography-mass spectrometry analysis method
In view of the fact that chromatographic peaks of dye isomers are not easy to separate, aniline isomers (p-phenylenediamine, m-phenylenediamine and o-phenylenediamine) and phenol isomers (p-aminophenol, m-aminophenol and o-aminophenol) are selected in an experiment, after 6 dyes are respectively subjected to derivatization reaction with dansyl chloride and dansyl chloride-D6, amino groups in dye molecules are respectively substituted by N, N-dimethylnaphthyl and N, N-dimethylnaphthyl-D6, and monosubstituted derivatization products are obtained.
Derivatization reaction process: respectively precisely weighing 1mg of p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol and o-aminophenol standard substances, adding 1mL of methanol for dissolution, diluting the 6 compound solutions by 10 times, respectively taking 50 mu L of each solution into a centrifuge tube with the same 1.5mL, drying by nitrogen, adding 400 mu L of dansyl chloride-D6 derivatization reagent (1 mg/mL), sealing the centrifuge tube for constant-temperature oscillation reaction, cooling to room temperature after the reaction is finished, respectively adding 600 mu L of acetonitrile, mixing in an equal volume, filtering by a 0.2 mu m filter membrane, and taking the filtrate as a solution to be measured for standby.
Optimizing chromatographic conditions: chromatographic column: capcell PAK C for senior hall 18 A chromatographic column; column temperature: 40 ℃; the sample injection amount was 1.0. Mu.L. Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile; flow rate: 0.3mL/min; gradient elution procedure: 0 to 10.5min,65 percent of A to 5%A;10.5 to 12.0min,5 percent of A;12.0 to 12.1min,5 to 65 percent of A; 12.1-14 min,65% A.
After the derivatization reaction of the 6 representative dyes with dansyl chloride-D6, the amino groups in the dye molecules are replaced by N, N-dimethylnaphthyl-D6 to obtain monosubstituted derivatization products. The p-phenylenediamine derivative product, m-phenylenediamine derivative product and o-phenylenediamine derivative product are isomers, the p-aminophenol derivative product, the derivative product and the o-aminophenol derivative product are isomers, and as can be obtained from fig. 7 and 8, the isomers of the two groups of derivative products are well separated, and accurate qualitative and quantitative analysis can be realized. In qualitative and quantitative analysis of the 6 dyes, isomers are difficult to separate, a T3 chromatographic column is needed, and a 10mmol/L ammonium formate aqueous solution with pH of 8 is needed as a mobile phase, so that the isomers are easier to separate after derivatization compared with the non-derivatization.
And (3) optimizing mass spectrum conditions: ion source: electrospray ion source positive ions (ESI) + ) A mode; the ionization voltage (IS) IS: 4500V; the heating Temperature (TEM) was all: 550 ℃; the air curtain air (CUR), the jet Collision Air (CAD), the spray air (GS 1) and the auxiliary heating air (GS 2) are respectively: 20. 8, 40psi; scanning mode: multiple Reaction Monitoring (MRM).
The method comprises the steps of scanning 6 dye derivatization products respectively by adopting a Q1Multiple (Q1 MI) positive Ion mode, simultaneously scanning the 6 compounds respectively by adopting a Product Ion (MS 2) positive Ion mode, setting different CE values, obtaining parent ions and two characteristic ions of the 6 dye derivatization products, respectively optimizing DP and CE values in the 6 dye derivatization products MRM method by adopting a needle pump sample injection, and establishing the 6 dye derivatization products MRM method (specific parameters are shown in Table 3). Since N, N-dimethylnaphthyl-D6 is very easy to leave in the ion fragmentation process, the characteristic ion (delta 176.2) of N, N-dimethylnaphthyl-D6 is obtained from all 6 dye derivatization products.
Table 3 6 main mass spectrum parameters and retention times of the derivatized products
Note that: the derivatizing agent of the p-phenylenediamine derivatization product, the m-phenylenediamine derivatization product, the o-phenylenediamine derivatization product, the p-aminophenol derivatization product, the m-aminophenol derivatization product and the o-aminophenol derivatization product is dansyl chloride; p-phenylenediamine derivative products * M-phenylenediamine derivative products * O-phenylenediamine derivative products * P-aminophenol derivative products * M-aminophenol derivative products * And o-aminophenol derivative products * The derivatizing agent of (2) is dansyl chloride-D6.
2. Dye dansyl chloride derivatization pathway and dansyl chloride-D6 derivatization pathway comparison
6, preparing a dye mixed standard solution: respectively precisely weighing reference substances of p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol and o-aminophenol 10mg, fixing the volume to 10mL by using methanol to obtain the 6 dye standard stock solutions, respectively precisely weighing 1mL of the p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol standard stock solution and 0.2mL of the o-aminophenol standard stock solution, and uniformly shaking the solution in a 10mL volumetric flask by using methanol to obtain the 6 dye mixed standard solution.
Preparation of derivatizing reagent: and respectively precisely weighing 10mg of dansyl chloride-D6 derivatization reagent and 10mg of dansyl chloride derivatization reagent in a 10mL volumetric flask, adding anhydrous tetrahydrofuran for dissolution, fixing the volume, adding 20 mu L of triethylamine, and shaking uniformly to obtain a dansyl chloride-D6 derivatization reagent solution and a dansyl chloride derivatization reagent solution with the concentration of 1 mg/mL.
Derivatization reaction process: precisely measuring 2 parts of 6 dye mixed standard solutions, respectively placing the two mixed standard solutions in 1.5mL centrifuge tubes, drying by nitrogen, adding 400 mu L of dansyl chloride-D6 derivatization reagent solution into one centrifuge tube, adding 400 mu L of dansyl chloride derivatization reagent solution into the other centrifuge tube, sealing the centrifuge tubes, vibrating the centrifuge tubes at constant temperature for reaction, cooling to room temperature after the reaction is finished, precisely measuring 200 mu L of each solution in the two test tubes, mixing, adding 600 mu L of acetonitrile, filtering by a 0.2 mu m filter membrane, and taking the filtrate as a solution to be measured for standby. Analysis of the derivatised product using established liquid-based methods revealed that the dansyl chloride derivatised product of the aniline dye (p-phenylenediamine, m-phenylenediamine, o-phenylenediamine) and the dansyl chloride-D6 derivatised product had the same retention time (see figure 9 for results) and a peak area RSD of 2.2% to 12%) (see table 4 for results); the retention time of the dansyl chloride derivative products of the phenolic dyes (p-aminophenol, m-aminophenol, o-aminophenol) and the dansyl chloride-D6 derivative products were the same (see FIG. 10 for the results) and the peak area RSD was 4.1% to 7.5% (see Table 4 for the results). In summary, the dye was identical to the dansyl chloride and dansyl chloride-D6 derivatization reaction pathways (derivatization pathways are shown in FIG. 1).
Table 4 6 RSD (%) of peak area of dansyl chloride derivative and dansyl chloride-D6 derivative of dye (n=6)
Example 4 methodological validation of dye-stable isotope labeling liquid chromatography-mass spectrometry
The optimal derivatization reagents dansyl chloride and dansyl chloride-D6, aniline isomers (p-phenylenediamine, m-phenylenediamine and o-phenylenediamine) and phenol isomers (p-aminophenol, m-aminophenol and o-aminophenol) are selected as target compounds, and the 6 dye isotope labeling liquid mass spectrometry analysis methods are established.
The chromatographic conditions are as follows: chromatographic column: capcell PAK C for senior hall 18 A chromatographic column; column temperature: 40 ℃; the sample injection amount was 1.0. Mu.L. Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile; flow rate: 0.3mL/min; gradient elution procedure: 0 to 10.5min,65 percent of A to 5%A;10.5 to 12.0min,5 percent of A;12.0 to 12.1min,5 to 65 percent of A;12.1 to 14 minutes, 65 percent of A;
the mass spectrometry conditions were ion source: electrospray ion source positive ions (ESI) + ) A mode; the ionization voltage (IS) IS: 4500V; the heating Temperature (TEM) was all: 550 ℃; the air curtain air (CUR), the jet Collision Air (CAD), the spray air (GS 1) and the auxiliary heating air (GS 2) are respectively: 20. 8, 40psi; scanning mode: multiple Reaction Monitoring (MRM), the main mass spectrum parameters and retention times of the derivatized products are shown in table 3.
1. Concentration range, recovery rate and precision
And (5) performing standard recovery rate and precision measurement by adopting a hair dye negative sample. Adding 6 dye single standard stock solutions (1 mg/mL) into a hair dye negative sample, precisely measuring 50 mu L of the treated 6 dye standard solutions in a 1.5mL centrifuge tube, taking 6 parts (A1, A2, A3, B1, B2 and B3) in parallel, drying by nitrogen, adding 400 mu L of dansyl chloride derivatization reagent (1 mg/mL) into A1, A2 and A3 respectively, adding 400 mu L of dansyl chloride-D6 derivatization reagent (1 mg/mL) into B1, B2 and B3 respectively, sealing the centrifuge tube, vibrating at constant temperature for reaction, cooling to room temperature after the reaction is finished, adding 0.6mL of acetonitrile, mixing A1 with B1 (V: V=1:1) is C1, mixing A2 with B2 (V: V=1:6) is C2, mixing A3 with B3 (V: 1:1/10) is C3, and the mixed solution is C1, C2 and C3. The C1, C2 and C3 are respectively measured in parallel for 6 times, 3 rounds of measurement are carried out simultaneously, the peak areas of the dansyl chloride derivative products and the dansyl chloride-D6 derivative products are measured (main mass spectrum parameters are shown in table 3), and the recovery rate of the dansyl chloride derivative products and the precision of the peak areas are calculated (the calculation formula is shown in (1)). Calculating the concentration range of 6 dye derivative products to be 0.45-4.3 mug/mL; as can be obtained from table 5, the average recovery of the 6 dye-derived products is 83.7-114.3%, and the recovery of C2 is generally higher because of the exchange of hydrogen and deuterium during the derivatization process using the dansyl chloride-D6 derivatization reagent; the Relative Standard Deviation (RSD) is 2.3-8.9%, and the result shows that the method has better precision.
C 1 : content of dansyl chloride derivative product, C 2 : content of Danesulfonyl chloride-D6 derivative product, A 1 : peak area of dansyl chloride derivative product, A 2 ：
Peak area of dansyl chloride-D6 derivative product
Table 56 recovery of dansyl chloride-D6 derivative products, precision (n=6)
2. Detecting the concentration
The signal to noise ratio (S/N) =3 of the quantitative ion chromatographic peak of the dansyl chloride-D6 derivative product is taken as a detection Limit (LOD), and S/N=10 is taken as a quantitative Limit (LOQ), so that the detection limit of the 6 dye derivative products is 0.011-0.056 mg/kg (see table 6). The 6 dyes are not subjected to derivatization treatment, the detection limit of detection by adopting a liquid method is 0.16-1.4 mg/kg, and the result shows that the sensitivity of the 6 dyes after derivatization is obviously improved.
Table 6 6 quantitative limits and detection limits for the Danesulfonyl chloride-D6 derived products
And carrying out methodology verification experiments on the established stable isotope labeling liquid chromatography-mass spectrometry analysis method of 6 strongly-reducibility forbidden dyes, wherein the concentration range of 6 dye derivatization products is 0.45-4.3 mug/mL, the average recovery rate is 83.7-114.3%, and the Relative Standard Deviation (RSD) is 2.3-8.9%. The method not only solves the problem of unstable dye, but also can exclude the substrate effect from accurately and quantitatively analyzing the dye, and the detection concentration of the dye is improved by 100 times compared with that before derivatization.
3. Dye-derived product stability analysis
Stability analysis before dye derivatization: according to the requirements of cosmetic safety technical Specification (2015 edition), 31 dye mixed standard solutions are prepared, sodium bisulphite (with the concentration of 2 g/L) is added to prevent standard substances from being oxidized, continuous sample injection is carried out for 50 hours, and the change of peak areas of ion flow diagrams extracted from each molecular component is observed. The results show that even if sodium sulfite as an antioxidant is added according to the specification, most of the aniline components are denatured within 10 hours, and the peak area rises by more than 20%. It is possible that hydrophobic oxidation products are formed, producing the same MRM ion pair causing interference (see fig. 11 for results). Most of the peak areas of the molecular components of the phenol-based hair dye decayed to 80% or less of the initial peak area within 2 hours (see fig. 12 for the results). As shown in fig. 13, p-aminophenol starts to deform after more than 7 hours in the sample pan (4 ℃) and after 20 hours a significant disturbance of impurities is generated, which impurities have a higher chromatographic retention time under reversed phase chromatographic conditions, indicating that the oxidation process produces a hydrophobic product with a higher ionization efficiency resulting in a higher chromatographic peak area than the true value.
Dye derivatization product stability analysis: and (3) selecting an optimal derivatization reagent to react with 5 representative dyes, and continuously injecting the dye derivatization products at the temperature of 4 ℃ for 10 days. The dansyl chloride is a derivatization reagent, and the chemical property of the derivatization product after the derivatization reaction is stable (delta < 20%) within 10 days at the temperature of 4 ℃ (see figure 14), which shows that after the derivatization reaction of the nucleophilic substitution reagent (dansyl chloride) with the amino or phenolic hydroxyl of the dye, the stability of the dye is improved.
Example 5 determination of dye content in samples
1. Sample matrix effect
The Matrix Effect (ME) is the effect that results in an enhancement or inhibition of the response value of the target compound:
response value of ME (%) = |blank matrix standard-response value of pure solvent standard-1×100%
And taking a hair dye sample which does not contain 6 representative dyes as a blank matrix, preparing a blank matrix adding standard solution and a pure solvent adding standard solution with the same concentration, and calculating to obtain ME. The results show that the matrix effect of the 6 dyes is 29.4-44.6%, which indicates that the dye components are complex and the matrix effect is high, and in addition, the quality control samples of the hair dye cannot be purchased in the market, so that the quantitative analysis by an internal standard method is adopted for accurate quantification.
2. Sample content determination
Preparing a dye mixed standard solution: respectively precisely weighing reference substances of p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol and o-aminophenol 10mg, fixing the volume to 10mL by using methanol to obtain the 6 dye standard stock solutions, respectively precisely weighing 1mL of the p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol standard stock solution and 0.2mL of the o-aminophenol standard stock solution, and uniformly shaking the solution in a 10mL volumetric flask by using methanol to obtain the 6 dye mixed standard solution.
Sample solution preparation: accurately weighing 0.5g of the sample in a 10mL colorimetric tube with a plug, adding a mixed solution of methanol-water solution (volume ratio is 8:2), mixing for 1min by vortex, fixing the volume of the mixed solution to 10mL, performing ultrasonic extraction for 15min in ice bath, and obtaining a sample solution which can be diluted if necessary.
Preparation of derivatizing reagent: precisely weighing 10mg of dansyl chloride-D6 derivatization reagent in a 10mL volumetric flask, adding anhydrous tetrahydrofuran for dissolution and volume fixing, adding 20 mu L of triethylamine, and shaking uniformly to obtain a dansyl chloride-D6 derivatization reagent solution with the concentration of 1 mg/mL; precisely weighing 10mg of dansyl chloride derivative reagent in a 10mL volumetric flask, adding anhydrous tetrahydrofuran for dissolution and constant volume, adding 20 mu L of triethylamine, and shaking uniformly to obtain a dansyl chloride derivative reagent solution with the concentration of 1 mg/mL.
Derivatization reaction process: precisely measuring 50 mu L of each of 6 dye mixed standard solutions and test sample solutions, respectively placing the solutions into a 1.5mL centrifuge tube, drying the solutions by nitrogen, adding 400 mu L of dansyl chloride-D6 derivatization reagent solution into the centrifuge tube of the dye mixed standard solutions dried by nitrogen, adding 400 mu L of dansyl chloride derivatization reagent solution into the centrifuge tube of the dye test sample solutions dried by nitrogen, sealing the centrifuge tube, vibrating the centrifuge tube at constant temperature for reaction, precisely measuring 200 mu L of each of the solutions in the two centrifuge tubes for mixing after the reaction is finished and cooling to room temperature, adding 600 mu L of acetonitrile, filtering the solution by a 0.2 mu m filter membrane, and taking the filtrate as the solution to be measured for standby.
Determination of the derivatization products: chromatographic conditions: chromatographic column: capcell PAK C for senior hall 18 A chromatographic column; column temperature: 40 ℃; the sample injection amount was 1.0. Mu.L. Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile; flow rate: 0.3mL/min. Gradient elution procedure: 0 to 10.5min,65 percent of A to 5%A;10.5 to 12.0min,5 percent of A;12.0 to 12.1min,5 to 65 percent of A; 12.1-14 min,65% A. Mass spectrometry conditions: ion source: electrospray ion source positive ions (ESI) + ) A mode; the ionization voltage (IS) IS: 4500V; the heating Temperature (TEM) was all: 550 ℃; the air curtain air (CUR), the jet Collision Air (CAD), the spray air (GS 1) and the auxiliary heating air (GS 2) are respectively: 20. 8, 40psi; scanning mode: multiple Reaction Monitoring (MRM). Specific parameters of the MRM method of the 6 dye derivatization products are shown in Table 3; the whole process of the content measurement of the sample is shown in figure 1.
The method is used for detecting the commercial 13 hair dye samples and calculating the content of 6 representative dyes in the samples (the calculation formula is shown as (2)), and meanwhile, the method of '32 components such as 7.2 p-phenylenediamine and the like' in the 'safety technical regulations for cosmetics' (2015) is adopted for detecting the samples, and the result shows that the two methods detect the consistent dye components in the samples, and the measured value by the method is slightly different from the measured value by the reference 'safety technical regulations for cosmetics' (2015), but the content is in the same order of magnitude, and the phenomenon is generated because the quantitative linear range is inconsistent by adopting a liquid phase method and a liquid quality method.
Wherein the dye content,%; c-concentration of dye in the sample solution, g/mL; v-sample constant volume, mL; d-dilution factor; m-sample sampling amount, g; a is that D6 -peak area of dye control added with dansyl chloride-D6 derivatization reagent reaction product; a is that D -adding dansyl chloride derivatization reagent reaction product peak area to the sample; c (C) For a pair of Concentration of standard, g/mL.
The absolute difference between the results of two independent tests obtained under the same conditions must not exceed 15% of the arithmetic mean.
The measurement results of the 6 dyes in the sample are shown in Table 7.
TABLE 7 determination of 6 dyes in samples
Note that: (1) for the measurement results of the method of reference cosmetic safety technology rules (2015 edition); (2) the results of the measurement according to the method of the present invention.
Claims (6)
1. A method for analyzing a dye component for a strong reducing inhibition in a hair dye, the dye component comprising: 1, 5-naphthalenediol, 1-naphthol, 2, 4-diaminophenoxyethanol hydrochloride, 2, 6-diaminopyridine, 2, 7-naphthalenediol, 2-amino-3-hydroxypyridine, 2-chloro-p-phenylenediamine sulfate, 2-methyl-m-phenylenediamine, 2-nitro-p-phenylenediamine, 4-amino-2-hydroxytoluene, 4-amino-3-nitrophenol, 4-amino-m-cresol, 4-chloro-m-phenylenediamine, 4-nitro-o-phenylenediamine, 6-amino-m-cresol, 6-hydroxyindole, p-phenylenediamine, m-aminophenol, m-phenylenediamine, N-bis (2-hydroxyethyl) -p-phenylenediamine sulfate, N-diethyl-p-phenylenediamine sulfate, N-diethyltoluene-2, 5-dihydrochloride, N-phenyl-p-phenylenediamine, o-aminophenol, p-methylaminophenol sulfate, p-phenylenediamine, m-toluenediamine, 4-toluenediamine, 3-toluenediamine, m-toluenediamine, 4-toluenediamine, 3-o-phenylenediamine sulfate; the method comprises the following steps:
(1) Preparing a derivatization mixed standard solution: preparing a mixed standard solution of each component of the dye by using methanol, and adding a dansyl chloride-D6 derivatization reagent to carry out a derivatization reaction to obtain a derivatization mixed standard solution;
(2) Derivatization of sample solution configuration: preparing a dye sample solution from a sample to be detected, and adding a dansyl chloride derivatization reagent to carry out derivatization reaction to obtain a derivatization sample solution;
(3) Preparing a solution to be tested: mixing the derivatization mixed standard solution in the step (1) and the derivatization test solution in the step (2) in equal volume to obtain a solution to be tested;
(4) And (3) respectively detecting and analyzing the solution to be detected by adopting a liquid chromatography-mass spectrometry (LC-MS) method:
the chromatographic conditions were: using Capcell PAK C 18 Chromatographic column, column temperature: 40 ℃; mobile phase a was 0.1% formic acid in water; mobile phase B was 0.1% acetonitrile formate; the flow rate is 0.3mL/min; sample injection amount is 1.0 mu L; gradient elution procedure was used: 0 to 10.5min,65 percent of A to 5%A;10.5 to 12.0min,5 percent of A;12.0 to 12.1min,5 to 65 percent of A;12.1 to 14 minutes, 65 percent of A;
the mass spectrometry conditions were: electrospray ion source positive ions (ESI) + ) Mode, ionization voltage is 4500V; the heating Temperature (TEM) was 550 ℃; the air curtain gas (CUR), the jet collision gas (CAD), the spray gas (GS 1) and the auxiliary heating gas (GS 2) are respectively 20 psi, 8 psi, 40psi and 40psi; the scanning mode is multi-reaction monitoring (MRM);
(5) Calculating the content of each component of the dye in the sample by adopting an internal standard method;
wherein the dansyl chloride-D6 derivatizing reagent solution or dansyl chloride derivatizing reagent solution is configured to: dissolving 10mg of dansyl chloride-D6 or dansyl chloride with anhydrous tetrahydrofuran, adding 10 mu L of triethylamine, and fixing the volume to 10mL with the anhydrous tetrahydrofuran to obtain a dansyl chloride-D6 derivatization reagent solution or a dansyl chloride derivatization reagent solution with the concentration of 1 mg/mL;
the derivatization reaction is as follows: precisely measuring 50 mu L of dye component mixed standard solution or sample solution, drying with nitrogen, adding 400 mu L of dansyl chloride-D6 derivatization reagent solution or dansyl chloride derivatization reagent solution, sealing, and vibrating at 30 ℃ for 20min.
2. The assay of claim 1, wherein the dye component comprises: p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol and o-aminophenol.
3. The analytical method according to claim 2, wherein the dye component-mixed standard solution in step (1) is configured such that: precisely weighing reference substances p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, p-aminophenol, m-aminophenol and o-aminophenol respectively, and fixing the volumes to 1mg/mL by methanol to obtain standard stock solutions respectively; respectively precisely measuring 1mL of p-phenylenediamine, 0.2mL of m-phenylenediamine, 1mL of o-phenylenediamine, 1mL of p-aminophenol and 0.2mL of m-aminophenol standard stock solution, placing the standard stock solutions into a volumetric flask, and fixing the volume to 10mL by using methanol to obtain the mixed standard solution of each component of the dye.
4. The assay of claim 2, wherein the dye test solution of step (2) is configured to: weighing a sample, adding methanol-water solution with the volume ratio of 8:2, dissolving and mixing uniformly until the concentration is 50mg/mL, and carrying out ice bath ultrasonic extraction for 15min to obtain a dye sample solution.
5. The method of analysis according to claim 2, wherein the step (3) is: and mixing 200 mu L of the derivatization mixed standard solution and 200 mu L of the derivatization test solution respectively, adding 600 mu L of acetonitrile, and filtering by a 0.2 mu m filter membrane to obtain a filtrate which is the solution to be tested.
6. The analytical method according to claim 1, wherein the calculation formula in the step (5) is:
ω＝(C×V×D×100％)/M；C＝(A D ×C for a pair of )/A D6 ；
Wherein ω is the dye content,%;
c is the concentration of dye in the test solution, g/mL;
v is the constant volume of the sample, mL;
d is dilution multiple;
m is the sample sampling amount, g;
A D6 adding dansyl chloride-D6 derivatization reagent into the standard solution to derivatize the peak area of the reaction product;
A D adding dansyl chloride derivatization reagent into the sample to obtain the peak area of the reaction product;
C for a pair of Concentration of standard, g/mL.
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| 离子对高效液相色谱法同时测定氧化型染发剂中5种染料;李莉，徐海娥，吉文亮.;《环境监测管理与技术》;第25卷(第4期);第31-35页 * |
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