Detection method of whitening component

Abstract

The invention discloses a detection method of whitening components. The invention provides a method for detecting whitening components including carbinomizole, 5-N-propyluracil or peptidomidol in cosmetics, and the method makes up the defect that a method for detecting carbinomizole, 5-N-propyluracil or peptidomidol in cosmetics does not exist in the prior art. The invention further provides a high performance liquid chromatography-tandem mass spectrometry detection method for nine substances including methimazole, carbinomizole, thiouracil, methylthiouracil, 5-N-propyluracil, propyl thiouracil, captopril, ethionamide and peptidomidol in a to-be-detected sample. The detection method is simple to operate, rapid in analysis, high in specificity and high in separation degree. The invention further provides a high performance liquid chromatography-tandem mass spectrometry detection method for the nine substances including methimazole, carbinomizole, thiouracil, methylthiouracil, 5-N-propyluracil, propyl thiouracil, captopril, ethionamide and peptidomidol. The method can be used as a detection method for the nine components in cosmetics, food, medicines and health care products, and provides technical support for analysis of forbidden substances of the cosmetics and other substances of related categories.

Description

Technical Field

[0001] The invention relates to the field of analysis and detection, and in particular to a method for detecting whitening ingredients. Background Art

[0002] The color of human skin mainly depends on the content and distribution of melanin. The synthesis of melanin is mainly affected by tyrosinase, dopachrome interconvertase and 5,6-dihydroxyindole 2-carboxylic acid oxidase activity and other factors such as dopa and endocrine factors (MSH). The main targets of commonly used skin whitening agents are tyrosinase, melanocytes and the transport metabolism of melanin. According to different working principles, skin whitening agents can be divided into tyrosinase activity inhibitors, melanocyte toxic agents, agents that affect melanin metabolism, sunscreens and chemical peels.

[0003] Related literature reports that thiourea-derived drugs such as 2-thiouracil, methylthiouracil, propylthiouracil, and thiamazole can reduce the production of melanin by non-competitively inhibiting the activity of tyrosinase, thereby producing a whitening effect. Ethionamide is an approved anti-tuberculosis drug used to treat multidrug-resistant tuberculosis. It has chemical similarities with phenylthiourea and has a non-competitive inhibitory effect on tyrosinase. However, methimazole, ethionamide, thiourea and their derivatives are listed as prohibited substances in the "Technical Specifications for Safety of Cosmetics" (2015 edition). Carbimazole is a thiourea amide drug used to treat hyperthyroidism, which can be metabolized into the active form of methimazole. Captopril is an angiotensin-converting enzyme inhibitor, widely used in the treatment of hypertension and heart failure. It can inhibit tyrosinase activity in a dose-dependent manner, thereby inhibiting melanin formation and achieving a whitening effect. Thiamidol, also known as isobutylamidothiazolylresorcinol in Chinese, is a resorcinol derivative and a patented ingredient of the Beiersdorf Group. This compound inhibits the activity of tyrosinase, thereby inhibiting the formation of melanin in cells, achieving a whitening effect. It is currently only used in some brand products. This raw material obtained my country's new raw material registration certificate in November 2024.

[0004] However, the current detection methods for substances such as methimazole are mostly found in food, medicine and clinical fields. There are existing standards and literature reports on the use of high performance liquid chromatography to separately determine some whitening ingredients in cosmetics, but there is no rapid quantitative detection method for ingredients in cosmetics including carbimazole, 5-N-propyluracil or peptide anmido. In addition, there is no literature report on the high performance liquid chromatography-mass spectrometry detection method for the simultaneous quantitative detection of nine substances including methimazole, methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide and peptide anmido. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting whitening ingredients.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides a method for detecting whitening ingredients, the method comprising the steps of:

[0007] Using a liquid chromatography-mass spectrometer to detect whitening ingredients in the sample to be tested, wherein the whitening ingredients include carbimazole, 5-N-propyl uracil and / or peptide anmido;

[0008] Wherein, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0009] Selecting an ion with a mass-to-charge ratio of 187 as a parent ion, and selecting an ion with a mass-to-charge ratio of 114.8 and / or 143.1 as a daughter ion to detect the carbimazole;

[0010] Selecting an ion with a mass-to-charge ratio of 155 as a parent ion, and selecting an ion with a mass-to-charge ratio of 138.1 and / or 82 as a daughter ion to detect the 5-N-propyluracil;

[0011] The ion with a mass-to-charge ratio of 279 was selected as the parent ion, and the ions with a mass-to-charge ratio of 209.1, 167.1 and / or 150.1 were selected as the daughter ions to detect the peptide Amido.

[0012] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0013] Selecting an ion with a mass-to-charge ratio of 187 as a parent ion, and selecting an ion with a mass-to-charge ratio of 114.8 or 143.1 as a daughter ion to detect the carbimazole;

[0014] Selecting an ion with a mass-to-charge ratio of 155 as a parent ion, and selecting an ion with a mass-to-charge ratio of 138.1 or 82 as a daughter ion to detect the 5-N-propyluracil;

[0015] The ion with a mass-to-charge ratio of 279 was selected as the parent ion, and the ion with a mass-to-charge ratio of 209.1, 167.1 or 150.1 was selected as the daughter ion to detect the peptide Amido.

[0016] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0017] An ion with a mass-to-charge ratio of 187 is selected as a parent ion, and an ion with a mass-to-charge ratio of 114.8 or 143.1 is selected as a daughter ion to detect the carbimazole, wherein the parent ion with a mass-to-charge ratio of 187 and the daughter ion with a mass-to-charge ratio of 114.8 are a quantitative ion pair;

[0018] An ion with a mass-to-charge ratio of 155 is selected as a parent ion, and an ion with a mass-to-charge ratio of 138.1 or 82 is selected as a daughter ion to detect the 5-N-propyl uracil, wherein the parent ion with a mass-to-charge ratio of 155 and the daughter ion with a mass-to-charge ratio of 138.1 are a quantitative ion pair;

[0019] The ion with a mass-to-charge ratio of 279 was selected as the parent ion, and the ion with a mass-to-charge ratio of 209.1, 167.1 or 150.1 was selected as the daughter ion to detect the peptide anamido, wherein the parent ion with a mass-to-charge ratio of 279 and the daughter ion with a mass-to-charge ratio of 150.1 were used as the quantitative ion pair.

[0020] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0021]

[0022] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0023] Ion source: Turbo Spray ion Drive;

[0024] Ion source temperature: 500°C;

[0025] Ion spray voltage: 5500V;

[0026] Scanning mode: positive ion scanning;

[0027] Detection method: multiple reaction monitoring (MRM) mode.

[0028] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0029] Ion source: Turbo Spray ion Drive;

[0030] Ion source temperature: 500°C;

[0031] Ion spray voltage: 5500V;

[0032] Collision gas (CAD): 9;

[0033] Curtain air (CUR): 35psi;

[0034] Heating gas (GS1): 50psi;

[0035] Auxiliary heating gas (GS2): 50psi;

[0036] Scanning mode: positive ion scanning;

[0037] Detection method: multiple reaction monitoring (MRM) mode.

[0038] In some embodiments, the chromatographic conditions of the liquid chromatography-mass spectrometry instrument include:

[0039] An aqueous solution containing formic acid (preferably a formic acid: water volume ratio of 0.1:99.9) is used as the aqueous phase, and methanol is used as the organic phase for gradient elution.

[0040] In some embodiments, the chromatographic conditions of the liquid chromatography-mass spectrometry instrument include:

[0041] An aqueous solution containing formic acid is used as the aqueous phase, methanol is used as the organic phase for gradient elution, the flow rate of the mobile phase is 0.2-0.4 mL / min, and the chromatographic column is a C18 column.

[0042] In some embodiments, the chromatographic conditions of the liquid chromatography-mass spectrometry are as follows:

[0043] Chromatographic column: Agilent ZORBAX Eclipse Plus-C18 column (2.1 mm × 100 mm, 3.5 μm);

[0044] Column temperature: 30°C;

[0045] Flow rate: 0.3 mL / min;

[0046] Injection volume: 2 μL;

[0047] Mobile phase: Mobile phase A is formic acid aqueous solution with a volume ratio of formic acid: water of 0.1:99.9, and mobile phase B is methanol;

[0048] Gradient elution program: 0-1.0min, 95% A; 1.0-4.0min, 95%-80% A; 4.0-8.0min, 80%-70% A; 8.0-10.0min, 70%-20% A; 10.0-11.0min, 20%-95% A; 11.0-15.0min, 95% A; see the table below for details:

[0049] time Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0.0 0.3 95 5 1.0 0.3 95 5 4.0 0.3 80 20 8.0 0.3 70 30 10.0 0.3 20 80 11.0 0.3 95 5 15.0 0.3 95 5 .

[0050] In one embodiment, the method further comprises the step of pre-treating the sample to be tested: dissolving the sample to be tested in methanol.

[0051] In one embodiment, the sample to be tested is food, medicine, health product or cosmetic.

[0052] In one embodiment, the sample to be tested is a cosmetic.

[0053] In one embodiment, the cosmetic is selected from at least one of toner, essence, face cream, facial cleanser, lotion, shower gel, sunscreen and isolation cream.

[0054] In one embodiment, the cosmetic is an ointment, cream, lotion or liquid water-based product.

[0055] In one embodiment, the cosmetic label also contains whitening, spot removal, yellowing or melanin removal.

[0056] A second aspect of the present invention provides a method for simultaneously detecting multiple whitening ingredients, the method comprising the steps of:

[0057] Using a liquid chromatography-mass spectrometer to detect the whitening ingredients in the sample to be tested, the whitening ingredients include carbimazole, 5-N-propyl uracil and / or peptide anmido, and the whitening ingredients in the cosmetics also include at least one of methimazole, thiouracil, methylthiouracil, propylthiouracil, captopril and ethionamide (preferably at least two, preferably at least three, preferably at least four, preferably at least five, and more preferably all six);

[0058] Wherein, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0059] Selecting an ion with a mass-to-charge ratio of 187 as a parent ion, and selecting an ion with a mass-to-charge ratio of 114.8 or 143.1 as a daughter ion to detect the carbimazole;

[0060] Selecting an ion with a mass-to-charge ratio of 155 as a parent ion, and selecting an ion with a mass-to-charge ratio of 138.1 or 82 as a daughter ion to detect the 5-N-propyluracil;

[0061] An ion with a mass-to-charge ratio of 279 is selected as a parent ion, and an ion with a mass-to-charge ratio of 209.1, 167.1 or 150.1 is selected as a daughter ion to detect the peptide Anmido;

[0062] Selecting an ion with a mass-to-charge ratio of 115 as a parent ion, and selecting an ion with a mass-to-charge ratio of 88.1 or 81 as a daughter ion to detect the methimazole;

[0063] The ion with a mass-to-charge ratio of 129 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 111.9 or 70 * ions as daughter ions to detect the thiouracil;

[0064] The ion with a mass-to-charge ratio of 143 was selected as the precursor ion, and the ion with a mass-to-charge ratio of 126 was selected as the precursor ion. * or 84 as daughter ions to detect the methylthiouracil;

[0065] The ion with a mass-to-charge ratio of 171 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 153.9 or 112 were selected as the precursor ions. * The ions of are used as daughter ions to detect the propylthiouracil;

[0066] Select the ion with a mass-to-charge ratio of 218 as the precursor ion, and select the ion with a mass-to-charge ratio of 116 or 172 * ions are used as product ions to detect the captopril;

[0067] The ion with a mass-to-charge ratio of 167 was selected as the precursor ion, and the ion with a mass-to-charge ratio of 107 or 140.1 was selected as the precursor ion. * The ion of is used as a daughter ion to detect the ethionamide.

[0068] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometer include:

[0069] An ion with a mass-to-charge ratio of 187 is selected as a parent ion, and an ion with a mass-to-charge ratio of 114.8 and / or 143.1 is selected as a daughter ion to detect the carbimazole, wherein the parent ion with a mass-to-charge ratio of 187 and the daughter ion with a mass-to-charge ratio of 114.8 are a quantitative ion pair;

[0070] An ion with a mass-to-charge ratio of 155 is selected as a parent ion, and an ion with a mass-to-charge ratio of 138.1 and / or 82 is selected as a daughter ion to detect the 5-N-propyl uracil, wherein the parent ion with a mass-to-charge ratio of 155 and the daughter ion with a mass-to-charge ratio of 138.1 are a quantitative ion pair;

[0071] An ion with a mass-to-charge ratio of 279 is selected as a parent ion, and an ion with a mass-to-charge ratio of 209.1, 167.1 and / or 150.1 is selected as a daughter ion to detect the peptide Anmido, wherein the parent ion with a mass-to-charge ratio of 279 and the daughter ion with a mass-to-charge ratio of 150.1 are used as a quantitative ion pair;

[0072] An ion with a mass-to-charge ratio of 115 is selected as a parent ion, and an ion with a mass-to-charge ratio of 88.1 and / or 81 is selected as a daughter ion to detect the methimazole, wherein the parent ion with a mass-to-charge ratio of 115 and the daughter ion with a mass-to-charge ratio of 88.1 are a quantitative ion pair;

[0073] The ion with a mass-to-charge ratio of 129 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 111.9 and / or 70 * The ion of is used as a daughter ion to detect the thiouracil, wherein the parent ion with a mass-to-charge ratio of 129 and the daughter ion with a mass-to-charge ratio of 70 are a quantitative ion pair;

[0074] The ion with a mass-to-charge ratio of 143 was selected as the precursor ion, and the ion with a mass-to-charge ratio of 126 was selected as the precursor ion. * and / or 84 as daughter ions to detect the methylthiouracil, wherein the parent ion with a mass-to-charge ratio of 143 and the parent ion with a mass-to-charge ratio of 126 * The daughter ion is the quantitative ion pair;

[0075] The ion with a mass-to-charge ratio of 171 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 153.9 and / or 112 were selected as the precursor ions. * The ions of propylthiouracil are used as product ions to detect the propylthiouracil, wherein the parent ion has a mass-to-charge ratio of 171 and the * The daughter ion is the quantitative ion pair;

[0076] The ion with a mass-to-charge ratio of 218 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 116 and / or 172 were selected as the precursor ions. * The captopril was detected as a daughter ion with a mass-to-charge ratio of 218 for the parent ion and 172 for the daughter ion. * The daughter ion is the quantitative ion pair;

[0077] The ion with a mass-to-charge ratio of 167 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 107 and / or 140.1 were selected as the precursor ions. * The ions of ethionamide were used as product ions to detect the ethionamide, wherein the parent ion had a mass-to-charge ratio of 167 and the * The product ion is the quantitative ion pair.

[0078] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0079]

[0080] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0081] Ion source: Turbo Spray ion Drive;

[0082] Ion source temperature: 500°C;

[0083] Ion spray voltage: 5500V;

[0084] Scanning mode: positive ion scanning;

[0085] Detection method: multiple reaction monitoring (MRM) mode.

[0086] In one embodiment, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include:

[0087] Ion source: Turbo Spray ion Drive;

[0088] Ion source temperature: 500°C;

[0089] Ion spray voltage: 5500 V;

[0090] Collision gas (CAD): 9;

[0091] Curtain air (CUR): 35 psi;

[0092] Heating gas (GS1): 50 psi;

[0093] Auxiliary heating gas (GS2): 50 psi;

[0094] Scanning mode: positive ion scanning;

[0095] Detection method: multiple reaction monitoring (MRM) mode.

[0096] In some embodiments, the chromatographic conditions of the liquid chromatography-mass spectrometry instrument include:

[0097] An aqueous solution containing formic acid (preferably a formic acid: water volume ratio of 0.1:99.9) is used as the aqueous phase, and methanol is used as the organic phase for gradient elution.

[0098] In some embodiments, the chromatographic conditions of the liquid chromatography-mass spectrometry instrument include:

[0099] An aqueous solution containing formic acid is used as the aqueous phase, methanol is used as the organic phase for gradient elution, the flow rate of the mobile phase is 0.2-0.4 mL / min, and the chromatographic column is a C18 column.

[0100] In some embodiments, the chromatographic conditions of the liquid chromatography-mass spectrometry are as follows:

[0101] Chromatographic column: Agilent ZORBAX Eclipse Plus-C18 column (2.1 mm × 100 mm, 3.5 μm);

[0102] Column temperature: 30°C;

[0103] Flow rate: 0.3 mL / min;

[0104] Injection volume: 2 μL;

[0105] Mobile phase: Mobile phase A is formic acid aqueous solution with a volume ratio of formic acid: water of 0.1:99.9, and mobile phase B is methanol;

[0106] Gradient elution program: 0-1.0min, 95% A; 1.0-4.0min, 95%-80% A; 4.0-8.0min, 80%-70% A; 8.0-10.0min, 70%-20% A; 10.0-11.0min, 20%-95% A; 11.0-15.0min, 95% A; see the table below for details:

[0107] time Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0.0 0.3 95 5 1.0 0.3 95 5 4.0 0.3 80 20 8.0 0.3 70 30 10.0 0.3 20 80 11.0 0.3 95 5 15.0 0.3 95 5 .

[0108] In one embodiment, the method further comprises the step of pre-treating the sample to be tested: dissolving the sample to be tested in methanol.

[0109] In one embodiment, the sample to be tested is food, medicine, health product or cosmetic.

[0110] In one embodiment, the sample to be tested is food, medicine, or health product.

[0111] Compared with the prior art, the present invention has at least the following advantages:

[0112] 1) The embodiments of the present invention provide a method for detecting whitening ingredients including carbimazole, 5-N-propyl uracil or peptide anmido in a sample (preferably cosmetics), which makes up for the lack of a method for detecting carbimazole, 5-N-propyl uracil or peptide anmido in cosmetics in the prior art;

[0113] 2) The embodiment of the present invention also provides a high performance liquid chromatography tandem mass spectrometry detection method for nine substances, including methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and peptide anmito in food, medicine, health products, or cosmetics. The detection method is simple to operate, has rapid analysis, strong specificity, and high separation. It can be used as a detection method for the above nine ingredients in food, medicine, health products, or cosmetics, and provides technical support for the analysis of banned substances in cosmetics and other substances of related categories.

[0114] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0116] Figure 1 is a liquid chromatography-mass spectrum of methimazole according to an embodiment of the present invention;

[0117] Figure 2 is a liquid chromatography-mass spectrum of carbimazole according to an embodiment of the present invention;

[0118] Figure 3 is a liquid chromatography-mass spectrum of thiouracil according to an embodiment of the present invention;

[0119] Figure 4 is a liquid chromatography-mass spectrum of methylthiouracil according to an embodiment of the present invention;

[0120] Figure 5 is a liquid chromatography-mass spectrum of 5-N-propylthiouracil according to an embodiment of the present invention;

[0121] Figure 6 is a liquid chromatography-mass spectrum of propylthiouracil according to an embodiment of the present invention;

[0122] Figure 7 is a liquid chromatography-mass spectrum of captopril according to an embodiment of the present invention;

[0123] Figure 8 is a liquid chromatography-mass spectrum of ethionamide according to an embodiment of the present invention;

[0124] Fig. 9 It is a liquid chromatography-mass spectrum of the peptide Anmido in the example of the present invention. DETAILED DESCRIPTION

[0125] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0126] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0127] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0128] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0129] 1. Instruments, Reagents, and Materials

[0130] Table 1

[0131]

[0132]

[0133] 2. Testing process

[0134] 2.1 Preparation of standard products: Accurately weigh 5 mg each of methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and peptide anmido standard products, place them in 10 mL volumetric flasks, dissolve them with methanol, make up to the mark, shake well, accurately pipette 0.20 mL of each of the above solutions into a 20 mL volumetric flask, make up to the mark with methanol, shake well, and obtain a mixed standard stock solution A of methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and peptide anmido with a concentration of 5.00 mg / L. Accurately measure 500 μL of mixed standard stock solution A into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare a 9-substance mixed standard intermediate solution B with a concentration of 250 ng / mL. Accurately measure 100 μL of mixed standard stock solution A into a 5 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare a 9-substance mixed standard intermediate solution C with a concentration of 100 ng / mL.

[0135] 2.2 Preparation of standard solutions: Accurately pipette 20 μL of the mixed standard intermediate solution C, and accurately pipette 20, 40, 200, 400, and 800 μL of the mixed standard intermediate solution B, respectively, add methanol to make up to 1 mL, shake well, and obtain a series of standard solutions with concentrations of 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL.

[0136] 2.3 Sample pretreatment: Accurately weigh about 0.5 g of sample (accurate to 0.0001 g), place it in a 25 mL plastic centrifuge tube, add methanol to the scale, mix, vortex for 30 seconds, ultrasonic for 20 minutes, centrifuge at 10000 r / min for 5 minutes, take an appropriate amount of supernatant, filter through a 0.22 μm filter membrane, and set the filtrate aside.

[0137] 2.4 Detection: The series of standard solutions in (2) and the filtrate in (3) are respectively injected into a liquid chromatograph for quantitative analysis.

[0138] 2.4.1 Liquid chromatography conditions

[0139] Chromatographic column: Agilent ZORBAX Eclipse Plus-C18 column (2.1 mm × 100 mm, 3.5 μm); column temperature: 30 °C;

[0140] Flow rate: 0.5 mL / min; injection volume: 2 μL.

[0141] The mobile phase and elution conditions were as follows: mobile phase A was a formic acid aqueous solution with a formic acid: water volume ratio of 0.1:99.9, and mobile phase B was methanol. Gradient elution program: 0-1 min, 95% A; 1.01-4 min, 80% A; 4.01-8 min, 70% A; 8.01-10 min, 20% A; 10.01-11 min, 95% A; 11.01-15 min, 95% A.

[0142] 2.4.2 Mass spectrometry conditions

[0143] Ion source: Turbo Spray lon Drive; ion source temperature: 500℃, ion spray voltage: 5500V; collision gas (CAD): 9; curtain gas (CUR): 35psi; heating gas (GS1): 50psi; auxiliary heating gas (GS2): 50psi. Scanning mode: positive ion scanning; detection mode: multiple reaction ion monitoring mode (MRM). The ions with relatively high abundance and less interference were used as quantitative ions, and the rest were used as qualitative ions (Table 3).

[0144] Table 2 MRM optimization parameters of compounds

[0145]

[0146] Note: * indicates quantitative ion pair

[0147] The linear equations, correlation coefficients, linear ranges and detection limits of methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide and peptide anamido are shown in Table 3.

[0148] Table 3 Linear equations, correlation coefficients, linear ranges and detection limits of nine material components

[0149]

[0150]

[0151] 3. Method Optimization

[0152] 3.1 Mobile phase selection

[0153] Water, 0.1% formic acid, and 0.5% formic acid were used as the aqueous phase, and methanol and acetonitrile were used as the organic phase for the mobile phase composition. The results showed that when acetonitrile was used as the organic phase, the peaks of methimazole, thiouracil, and methylthiouracil appeared earlier and were easily interfered. Ethionamide had obvious tailing, captopril had a front peak, and peptide anmido ion response was poor, and satisfactory chromatographic peak shapes could not be obtained. When water was used as the mobile phase, the captopril peak was severely broadened. Finally, 0.1% formic acid and methanol were selected as the mobile phase, which improved the tailing of the ethionamide peak and the broadening of the captopril peak, and the baseline was stable.

[0154] Chromatographic column selection and elution gradient optimization

[0155] Since the structures of methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, and propylthiouracil are similar, the retention times of the compounds are similar, and different compounds can produce the same fragments, in order to ensure the accurate identification and quantification of the compounds, it is necessary to ensure that the chromatographic peaks of each substance are completely separated. Three chromatographic columns, Waters UPLC BEHC18 column (2.1mm×100mm, 1.7μm), Waters UPLC BEH C18 column (2.1mm×150mm, 1.7μm), and Agilent ZORBAX Eclipse Plus-C18 column (2.1mm×100mm, 3.5μm), were used for separation effect experiments.

[0156] The results showed that carbimazole and methylthiouracil could not be completely separated when the first two chromatographic columns were used. When the Agilent ZORBAX Eclipse Plus-C18 column (2.1mm×100mm, 3.5μm) was used, when the organic phase ratio was higher than 30% by volume, the retention times of thiouracil and methimazole were close, and the retention times of carbimazole, methylthiouracil, 5-N-propyluracil and propylthiouracil were close. By adjusting the mobile phase gradient elution program, the nine substances had good separation and suitable retention time.

[0157] 3.2 Selection of ion pairs

[0158] All compounds were treated with [M+H] + As the parent ion. When the secondary mass spectrometry was used to determine its daughter ions, it was found that thiouracil, with 129 as the parent ion, could produce three daughter ions of 70, 71.9 and 111.9, and the response intensity was 111.9>71.9>70. However, in the actual determination, it was found that the response was poor when 71.9 was the daughter ion, and it was easily interfered by the sample matrix due to its short retention time. The response was relatively high when 70 was the daughter ion, and the recovery rate was good in liquid water matrix and cream emulsion matrix. Methylthiouracil uses 143 as the parent ion, and the relative abundance of daughter ions with mass-to-charge ratios of 71.9, 84, 87.9, 115, and 126 is high. When 87.9 and 115 are daughter ions, the recovery rate and precision are poor and the back-calibration accuracy is low. When 71.9 and 86 are daughter ions, the baseline is unstable and the signal-to-noise ratio is low. After the daughter ions are changed to 126 and 84, the recovery results in two matrices are good, ranging from 96.72-109.33% and 95.68-99.75%, respectively. When carbimazole uses 187 as the parent ion, it can produce fragments of 114.8, 143.1, and 88, and 114.8 can also produce two fragments of 88 and 81. The experiment optimized and investigated the CE values ​​of the three fragments, and found that the fragment response of 88 was unstable, so 143.1 and 114.8 were selected as daughter ions. The response of 5-N-propyluracil daughter ion 82 was good, but the response of daughter ions with molecular weight above 100 was slightly low. The parameters of 125.9, 136.9 and 138.1 were investigated and optimized, and 138.1 was finally selected as the daughter ion.

[0159] 3.3 Investigation of stability

[0160] Carbimazole is a derivative of amino formic acid. Due to the influence of the imidazole ring on the amide bond of amino formic acid, its amide bond is more easily hydrolyzed than the ester bond, generating the corresponding secondary amine form, namely methimazole. In order to further investigate the stability of the solution, carbimazole single standard solution was prepared with water, 80% methanol and methanol to 1500ng / mL and 600ng / mL, respectively. The changes of carbimazole over time when stored in a refrigerator (4℃) and room temperature (25℃) were compared: the sample was injected and analyzed at 0, 24 and 72h, and the peak area of ​​carbimazole was recorded. The peak area at 0h was 100%, and the recovery rate at each point was investigated. The results showed that when the standard solution was diluted with 80% methanol or methanol and refrigerated at 4℃, it was more conducive to the stability of carbimazole, and the peak area recovery rate at 24h was 102.79% and 92.42%, respectively. Under room temperature conditions, the carbimazole content decreased significantly at 24h, and the solution stability was worse at lower concentrations. Therefore, the reference solution and the test solution should be freshly prepared before use, stored at low temperature and away from light, and injected in time. Taking into account factors such as sample pretreatment operation and solution stability, methanol is used for standard solution dilution and sample pretreatment.

[0161] Example 1

[0162] Two commercially available cosmetic matrices were used as test objects, spiked with 0.25 μg / g, 0.50 μg / g and 2.50 μg / g, respectively, and parallel tests (n=6) were performed at the same time. The recovery rate and RSD were shown in Table 5. The results showed that the recovery rate of the nine components under high, medium and low spike concentration conditions ranged from 85.90% to 113.00%, and the relative deviation RSD (n=6) was 0.42% to 6.26%, indicating that the recovery rate of this method was good.

[0163] Table 4 Recovery results (n = 6)

[0164]

[0165]

[0166] This detection method was applied to 65 batches of commercially available creams, lotions and liquid water-based anti-freckle and whitening cosmetics (toner, essence, face cream, etc.) for testing. Results: 3 batches of samples were found to contain peptide Anmito, with a content range of 0.09%-0.20%, and the other compounds were not detected.

[0167] The above description shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be within the scope of the present invention. The scope of protection required by the present invention is defined by the attached claims and their equivalents.

[0168] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A method for detecting whitening ingredients, characterized in that: The method comprises the steps of: Using a liquid chromatography-mass spectrometer to detect whitening ingredients in the sample to be tested, wherein the whitening ingredients include carbimazole, 5-N-propyl uracil and / or peptide anmido; Wherein, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include: Selecting an ion with a mass-to-charge ratio of 187 as a parent ion, and selecting an ion with a mass-to-charge ratio of 114.8 and / or 143.1 as a daughter ion to detect the carbimazole; Selecting an ion with a mass-to-charge ratio of 155 as a parent ion, and selecting an ion with a mass-to-charge ratio of 138.1 and / or 82 as a daughter ion to detect the 5-N-propyluracil; The ion with a mass-to-charge ratio of 279 was selected as the parent ion, and the ions with a mass-to-charge ratio of 209.1, 167.1 and / or 150.1 were selected as the daughter ions to detect the peptide Amido.

2. The detection method according to claim 1, characterized in that: The mass spectrometry conditions of the liquid chromatography-mass spectrometer include: An ion with a mass-to-charge ratio of 187 is selected as a parent ion, and an ion with a mass-to-charge ratio of 114.8 or 143.1 is selected as a daughter ion to detect the carbimazole, wherein the parent ion with a mass-to-charge ratio of 187 and the daughter ion with a mass-to-charge ratio of 114.8 are a quantitative ion pair; An ion with a mass-to-charge ratio of 155 is selected as a parent ion, and an ion with a mass-to-charge ratio of 138.1 or 82 is selected as a daughter ion to detect the 5-N-propyl uracil, wherein the parent ion with a mass-to-charge ratio of 155 and the daughter ion with a mass-to-charge ratio of 138.1 are a quantitative ion pair; The ion with a mass-to-charge ratio of 279 was selected as the parent ion, and the ion with a mass-to-charge ratio of 209.1, 167.1 or 150.1 was selected as the daughter ion to detect the peptide anamido, wherein the parent ion with a mass-to-charge ratio of 279 and the daughter ion with a mass-to-charge ratio of 150.1 were used as the quantitative ion pair.

3. The detection method according to claim 1, characterized in that: The mass spectrometry conditions of the liquid chromatography-mass spectrometer include: 。 4. A method for simultaneously detecting multiple whitening ingredients, characterized in that: The method comprises the steps of: using a liquid chromatography-mass spectrometer to detect whitening ingredients in a sample to be tested, wherein the whitening ingredients include carbimazole, 5-N-propyl uracil and / or peptide anmido, and the whitening ingredients in the cosmetics further include at least one of methimazole, thiouracil, methylthiouracil, propylthiouracil, captopril and ethionamide (preferably at least two, preferably at least three, preferably at least four, preferably at least five, and more preferably all six); Wherein, the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument include: Selecting an ion with a mass-to-charge ratio of 187 as a parent ion, and selecting an ion with a mass-to-charge ratio of 114.8 or 143.1 as a daughter ion to detect the carbimazole; Selecting an ion with a mass-to-charge ratio of 155 as a parent ion, and selecting an ion with a mass-to-charge ratio of 138.1 or 82 as a daughter ion to detect the 5-N-propyluracil; An ion with a mass-to-charge ratio of 279 is selected as a parent ion, and an ion with a mass-to-charge ratio of 209.1, 167.1 or 150.1 is selected as a daughter ion to detect the peptide Anmido; Selecting an ion with a mass-to-charge ratio of 115 as a parent ion, and selecting an ion with a mass-to-charge ratio of 88.1 or 81 as a daughter ion to detect the methimazole; The ion with a mass-to-charge ratio of 129 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 111.9 or 70 * ions as daughter ions to detect the thiouracil; The ion with a mass-to-charge ratio of 143 was selected as the precursor ion, and the ion with a mass-to-charge ratio of 126 was selected as the precursor ion. * or 84 as daughter ions to detect the methylthiouracil; The ion with a mass-to-charge ratio of 171 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 153.9 or 112 were selected as the precursor ions. * The ions of are used as daughter ions to detect the propylthiouracil; Select the ion with a mass-to-charge ratio of 218 as the precursor ion, and select the ion with a mass-to-charge ratio of 116 or 172 * ions are used as product ions to detect the captopril; The ion with a mass-to-charge ratio of 167 was selected as the precursor ion, and the ion with a mass-to-charge ratio of 107 or 140.1 was selected as the precursor ion. * The ion of is used as a daughter ion to detect the ethionamide.

5. The detection method according to claim 4, characterized in that: The mass spectrometry conditions of the liquid chromatography-mass spectrometer include: An ion with a mass-to-charge ratio of 187 is selected as a parent ion, and an ion with a mass-to-charge ratio of 114.8 and / or 143.1 is selected as a daughter ion to detect the carbimazole, wherein the parent ion with a mass-to-charge ratio of 187 and the daughter ion with a mass-to-charge ratio of 114.8 are a quantitative ion pair; An ion with a mass-to-charge ratio of 155 is selected as a parent ion, and an ion with a mass-to-charge ratio of 138.1 and / or 82 is selected as a daughter ion to detect the 5-N-propyl uracil, wherein the parent ion with a mass-to-charge ratio of 155 and the daughter ion with a mass-to-charge ratio of 138.1 are a quantitative ion pair; An ion with a mass-to-charge ratio of 279 is selected as a parent ion, and an ion with a mass-to-charge ratio of 209.1, 167.1 and / or 150.1 is selected as a daughter ion to detect the peptide Anmido, wherein the parent ion with a mass-to-charge ratio of 279 and the daughter ion with a mass-to-charge ratio of 150.1 are used as a quantitative ion pair; An ion with a mass-to-charge ratio of 115 is selected as a parent ion, and an ion with a mass-to-charge ratio of 88.1 and / or 81 is selected as a daughter ion to detect the methimazole, wherein the parent ion with a mass-to-charge ratio of 115 and the daughter ion with a mass-to-charge ratio of 88.1 are a quantitative ion pair; The ion with a mass-to-charge ratio of 129 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 111.9 and / or 70 * The ion of is used as a daughter ion to detect the thiouracil, wherein the parent ion with a mass-to-charge ratio of 129 and the daughter ion with a mass-to-charge ratio of 70 are a quantitative ion pair; The ion with a mass-to-charge ratio of 143 was selected as the precursor ion, and the ion with a mass-to-charge ratio of 126 was selected as the precursor ion. * and / or 84 as daughter ions to detect the methylthiouracil, wherein the parent ion with a mass-to-charge ratio of 143 and the parent ion with a mass-to-charge ratio of 126 * The daughter ion is the quantitative ion pair; The ion with a mass-to-charge ratio of 171 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 153.9 and / or 112 were selected as the precursor ions. * The ions of propylthiouracil are used as product ions to detect the propylthiouracil, wherein the parent ion has a mass-to-charge ratio of 171 and the * The daughter ion is the quantitative ion pair; The ion with a mass-to-charge ratio of 218 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 116 and / or 172 were selected as the precursor ions. * The captopril was detected as a daughter ion with a mass-to-charge ratio of 218 for the parent ion and 172 for the daughter ion. * The daughter ion is the quantitative ion pair; The ion with a mass-to-charge ratio of 167 was selected as the precursor ion, and the ions with a mass-to-charge ratio of 107 and / or 140.1 were selected as the precursor ions. * The ions of ethionamide were used as product ions to detect the ethionamide, wherein the parent ion had a mass-to-charge ratio of 167 and the * The product ion is the quantitative ion pair.

6. The detection method according to claim 5, characterized in that: The mass spectrometry conditions of the liquid chromatography-mass spectrometer include: 。 7. The detection method according to any one of claims 1 to 7, characterized in that: The mass spectrometry conditions of the liquid chromatography-mass spectrometer include: Ion source: Turbo Spray ion Drive; Ion source temperature: 500°C; Ion spray voltage: 5500V; Scanning mode: positive ion scanning; Detection method: multiple reaction monitoring (MRM) mode.

8. The detection method according to any one of claims 1 to 7, characterized in that: The chromatographic conditions of the liquid chromatography-mass spectrometry instrument include: using an aqueous solution containing formic acid (preferably a formic acid: water volume ratio of 0.1:99.9) as the aqueous phase, and using methanol as the organic phase for gradient elution.

9. The detection method according to any one of claims 1 to 7, characterized in that: The chromatographic conditions of the liquid chromatography-mass spectrometer are as follows: Chromatographic column: Agilent ZORBAX Eclipse Plus-C18 column (2.1 mm × 100 mm, 3.5 μm); Column temperature: 30°C; Flow rate: 0.3 mL / min; Injection volume: 2 μL; Mobile phase: Mobile phase A is formic acid aqueous solution with a volume ratio of formic acid: water of 0.1:99.9, and mobile phase B is methanol; Gradient elution program: 0-1.0 min, 95% A; 1.0-4.0 min, 95%-80% A; 4.0-8.0 min, 80%-70% A; 8.0~10.0min,70%~20%A; 10.0~11.0min,20%~95%A; 11.0~15.0min,95%A.

10. The detection method according to any one of claims 1 to 7, characterized in that: The samples to be tested are food, medicine, health products or cosmetics.

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