Methods for detecting skin whitening ingredients

Abstract

This application discloses a method for detecting skin-whitening ingredients. Specifically, it provides a method for detecting skin-whitening ingredients, including carbimazole, 5-N-propyluracil, or peptide-amidazoline in cosmetics, filling the gap in existing technologies that lack methods for detecting these ingredients in cosmetics. This application also provides a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for detecting nine substances—methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and peptide-amidazoline—in a sample. This method is simple to operate, rapid, highly specific, and has high resolution. It can be used as a detection method for the above nine ingredients in cosmetics, food, pharmaceuticals, and health products, providing technical support for the analysis of prohibited substances and other substances in related categories in cosmetics.

Description

Technical Field

[0001] This invention relates to the field of analytical testing, and in particular to a method for detecting skin-whitening ingredients. Background Technology

[0002] Human skin color primarily depends on the content and distribution of melanin. Melanin synthesis is mainly influenced by the activities of tyrosinase, dopachrome tautomerase, and 5,6-dihydroxyindole-2-carboxylic acid oxidase, as well as other factors such as dopa and endocrine factors (MSH). Currently, commonly used skin whitening agents primarily target tyrosinase, melanocytes, and melanin transport and metabolism. Based on different mechanisms of action, skin whitening agents can be classified into tyrosinase activity inhibitors, melanocyte cytotoxic agents, melanin metabolism modifiers, sunscreens, and chemical peeling agents.

[0003] Relevant literature reports that thiourea derivatives such as 2-thiouracil, methylthiouracil, propylthiouracil, and thiamazole can reduce melanin production by non-competitively inhibiting tyrosinase activity, thus producing a skin-whitening effect. Ethionamide, an approved anti-tuberculosis drug used to treat multidrug-resistant tuberculosis, is chemically similar to phenylthiourea and has a non-competitive inhibitory effect on tyrosinase. However, thiamazole, ethionamide, thiourea, and their derivatives are listed as prohibited substances in the "Cosmetic Safety Technical Specifications" (2015 edition). Carbimazole, a thiourea amide drug used to treat hyperthyroidism, can be metabolized into its active form, thiamazole. Captopril is an angiotensin-converting enzyme inhibitor widely used in the treatment of hypertension and heart failure. It inhibits tyrosinase activity in a dose-dependent manner, thereby suppressing melanin formation and achieving a skin-whitening effect. Thiamidol, also known as isobutyramide thiazolyl resorcinol, is a resorcinol derivative and a patented ingredient of the Beiersdorf Group. This compound inhibits tyrosinase activity, thereby suppressing melanin production in cells, achieving a skin-whitening effect. Currently, it is only used in some branded products. This ingredient obtained a new raw material registration certificate in my country in November 2024.

[0004] However, current detection methods for substances such as methimazole are mostly found in the food, pharmaceutical, and clinical fields. Standards and literature have reported the use of high-performance liquid chromatography (HPLC) to determine some whitening ingredients in cosmetics individually. However, there is no rapid quantitative detection method for ingredients in cosmetics, including carbimazole, 5-N-propyluracil, or peptide omeprazole. In addition, there are no literature reports on the simultaneous quantitative detection of nine substances, including methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and peptide omeprazole. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting skin-whitening ingredients.

[0006] To address the aforementioned technical problems, a first aspect of the present invention provides a method for detecting whitening ingredients, the method comprising the steps of:

[0007] The whitening components in the sample were detected using liquid chromatography-mass spectrometry, and the whitening components included carbimazole, 5-N-propyluracil, and / or peptide amido.

[0008] The mass spectrometry conditions of the liquid chromatography-mass spectrometry system include:

[0009] Ions with a mass-to-charge ratio of 187 were selected as the parent ion, and ions with a mass-to-charge ratio of 114.8 and / or 143.1 were selected as daughter ions for the detection of carbimazole.

[0010] An ion with a mass-to-charge ratio of 155 was selected as the parent ion, and ions with a mass-to-charge ratio of 138.1 and / or 82 were selected as daughter ions to detect the 5-N-propyluracil.

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

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

[0013] An ion with a mass-to-charge ratio of 187 was selected as the parent ion, and ions with a mass-to-charge ratio of 114.8 or 143.1 were selected as daughter ions to detect the carbimazole.

[0014] An ion with a mass-to-charge ratio of 155 was selected as the parent ion, and an ion with a mass-to-charge ratio of 138.1 or 82 was selected as the daughter ion to detect the 5-N-propyluracil.

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

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

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

[0018] An ion with a mass-to-charge ratio of 155 is selected as the parent ion, and an ion with a mass-to-charge ratio of 138.1 or 82 is selected as the daughter ion to detect the 5-N-propyluracil, 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 quantitative ion pairs.

[0019] An ion with a mass-to-charge ratio of 279 is selected as the parent ion, and ions with a mass-to-charge ratio of 209.1, 167.1, or 150.1 are selected as daughter ions to detect the peptide Ammido, 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 quantitative ion pairs.

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

[0021]

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

[0023] Ion source: Turbo Spray Ion Drive;

[0024] Ion source temperature: 500℃;

[0025] Ion spray voltage: 5500V;

[0026] Scanning method: positive ion scan;

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

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

[0029] Ion source: Turbo Spray Ion Drive;

[0030] Ion source temperature: 500℃;

[0031] Ion spray voltage: 5500V;

[0032] Collision air (CAD): 9;

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

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

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

[0036] Scanning method: positive ion scan;

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

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

[0039] A gradient elution was performed using an aqueous solution containing formic acid (preferably with a formic acid:water volume ratio of 0.1:99.9) as the aqueous phase and methanol as the organic phase.

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

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

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

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

[0044] Column temperature: 30℃;

[0045] Flow rate: 0.3 mL / min;

[0046] Injection volume: 2 μL;

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

[0048] Gradient elution program: 0–1.0 min, 95% A; 1.0–4.0 min, 95%–80% A; 4.0–8.0 min, 80%–70% A; 8.0–10.0 min, 70%–20% A; 10.0–11.0 min, 20%–95% A; 11.0–15.0 min, 95% A; see 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 includes a pretreatment step of the sample to be tested: dissolving the sample to be tested in methanol.

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

[0052] In one implementation scheme, the sample to be tested is a cosmetic product.

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

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

[0055] In one implementation, the cosmetic product's label also includes terms such as whitening, spot removal, yellowing removal, or melanin reduction.

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

[0057] The whitening ingredients in the sample were detected using liquid chromatography-mass spectrometry. The whitening ingredients included carbimazole, 5-N-propyluracil, and / or peptide amido. The whitening ingredients in the cosmetic also included 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] The mass spectrometry conditions of the liquid chromatography-mass spectrometry system include:

[0059] An ion with a mass-to-charge ratio of 187 was selected as the parent ion, and ions with a mass-to-charge ratio of 114.8 or 143.1 were selected as daughter ions to detect the carbimazole.

[0060] An ion with a mass-to-charge ratio of 155 was selected as the parent ion, and an ion with a mass-to-charge ratio of 138.1 or 82 was selected as the daughter ion to detect the 5-N-propyluracil.

[0061] An ion with a mass-to-charge ratio of 279 was selected as the parent ion, and ions with mass-to-charge ratios of 209.1, 167.1, or 150.1 were selected as daughter ions to detect the peptide Amidato.

[0062] Ions with a mass-to-charge ratio of 115 were selected as the parent ion, and ions with a mass-to-charge ratio of 88.1 or 81 were selected as the daughter ions to detect the methimazole.

[0063] An ion with a mass-to-charge ratio of 129 was selected as the parent ion, and ions with a mass-to-charge ratio of 111.9 or 70 were also selected. * The ions are used as daughter ions to detect the thiourea pyrimidine;

[0064] An ion with a mass-to-charge ratio of 143 was selected as the parent ion, and an ion with a mass-to-charge ratio of 126 was also selected. * Or 84 ions can be used as daughter ions to detect the methylthiouracil;

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

[0066] An ion with a mass-to-charge ratio of 218 was selected as the parent ion, and ions with a mass-to-charge ratio of 116 or 172 were also selected. * The ions are used as daughter ions to detect captopril;

[0067] An ion with a mass-to-charge ratio of 167 was selected as the parent ion, and further selections were made for mass-to-charge ratios of 107 or 140.1. * The ions are used as daughter ions to detect the ethionamide.

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

[0069] An ion with a mass-to-charge ratio of 187 is selected as the precursor ion, and ions with a mass-to-charge ratio of 114.8 and / or 143.1 are selected as daughter ions for the detection of carbimazole, wherein the precursor ion with a mass-to-charge ratio of 187 and the daughter ion with a mass-to-charge ratio of 114.8 are quantitative ion pairs.

[0070] An ion with a mass-to-charge ratio of 155 is selected as the parent ion, and an ion with a mass-to-charge ratio of 138.1 and / or 82 is selected as the daughter ion to detect the 5-N-propyluracil, 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 quantitative ion pairs.

[0071] An ion with a mass-to-charge ratio of 279 was selected as the parent ion, and ions with mass-to-charge ratios of 209.1, 167.1 and / or 150.1 were selected as daughter ions to detect the peptide Ammido, 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 quantitative ion pairs.

[0072] An ion with a mass-to-charge ratio of 115 is selected as the parent ion, and an ion with a mass-to-charge ratio of 88.1 and / or 81 is selected as the daughter ion for the detection of 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 quantitative ion pairs.

[0073] Ions with a mass-to-charge ratio of 129 were selected as the parent ion, and those with a mass-to-charge ratio of 111.9 and / or 70 were also selected. * The ions are used as daughter ions to detect the thiourea pyrimidine, 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 quantitative ion pairs.

[0074] An ion with a mass-to-charge ratio of 143 was selected as the parent ion, and an ion with a mass-to-charge ratio of 126 was also selected. * The methylthiouracil is detected using ions of 143 and / or 84 as daughter ions, wherein the parent ion has a mass-to-charge ratio of 143 and 126. * The daughter ions are quantitative ion pairs;

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

[0076] Ions with a mass-to-charge ratio of 218 were selected as the parent ion, and ions with a mass-to-charge ratio of 116 and / or 172 were also selected. * The captopril was detected using ions as daughter ions, wherein the parent ion had a mass-to-charge ratio of 218 and a daughter ion of 172. * The daughter ions are quantitative ion pairs;

[0077] An ion with a mass-to-charge ratio of 167 was selected as the parent ion, and ions with a mass-to-charge ratio of 107 and / or 140.1 were also selected. * The ions were used as daughter ions to detect the ethionamide, wherein the parent ion had a mass-to-charge ratio of 167 and the daughter ion had a mass-to-charge ratio of 140.1. * The daughter ions are quantitative ion pairs.

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

[0079]

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

[0081] Ion source: Turbo Spray Ion Drive;

[0082] Ion source temperature: 500℃;

[0083] Ion spray voltage: 5500V;

[0084] Scanning method: positive ion scan;

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

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

[0087] Ion source: Turbo Spray Ion Drive;

[0088] Ion source temperature: 500℃;

[0089] Ion spray voltage: 5500 V;

[0090] Collision air (CAD): 9;

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

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

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

[0094] Scanning method: positive ion scan;

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

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

[0097] A gradient elution was performed using an aqueous solution containing formic acid (preferably with a formic acid:water volume ratio of 0.1:99.9) as the aqueous phase and methanol as the organic phase.

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

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

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

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

[0102] Column temperature: 30℃;

[0103] Flow rate: 0.3 mL / min;

[0104] Injection volume: 2 μL;

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

[0106] Gradient elution program: 0–1.0 min, 95% A; 1.0–4.0 min, 95%–80% A; 4.0–8.0 min, 80%–70% A; 8.0–10.0 min, 70%–20% A; 10.0–11.0 min, 20%–95% A; 11.0–15.0 min, 95% A; see 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 includes a pretreatment step of the sample to be tested: dissolving the sample to be tested in methanol.

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

[0110] In one implementation plan, 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-propyluracil or peptide omega in a sample (preferably a cosmetic), which makes up for the lack of a method in the prior art for detecting carbimazole, 5-N-propyluracil or peptide omega in cosmetics.

[0113] 2) Embodiments of the present invention also provide a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for the detection of nine substances—methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and peptidylcholine—in food, pharmaceuticals, health products, or cosmetics. This method is simple to operate, rapid in analysis, highly specific, and has high resolution. It can be used as a detection method for the above nine components in food, pharmaceuticals, health products, or cosmetics, providing technical support for the analysis of prohibited substances in cosmetics and other substances in related categories.

[0114] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0115] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

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

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

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

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

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

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

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

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

[0124] Figure 9 This is a liquid chromatography-mass spectrum of peptide amectoide according to an embodiment of the present invention. Detailed Implementation

[0125] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0126] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0127] Unless otherwise specified, the term “or” means the term “and / or” and is used interchangeably with the term “and / or”.

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

[0129] 1. Instruments, reagents and materials

[0130] Table 1

[0131]

[0132]

[0133] 2. Testing Process

[0134] 2.1 Preparation of Standards: Accurately weigh 5 mg each of methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and tamsulosin, and place them in separate 10 mL volumetric flasks. Dissolve each flask in methanol and dilute to the mark. Shake well. Accurately pipette 0.20 mL of each of the above solutions into 20 mL volumetric flasks, dilute to the mark with methanol, and shake well to obtain a mixed standard stock solution A with a concentration of 5.00 mg / L, consisting of methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, propylthiouracil, captopril, ethionamide, and tamsulosin. Accurately measure 500 μL of the mixed standard stock solution A into a 10 mL volumetric flask, dilute to the mark with methanol, and mix well to prepare a mixed standard intermediate solution B with a concentration of 250 ng / mL for the nine substances. Accurately measure 100 μL of the mixed standard stock solution A into a 5 mL volumetric flask, dilute to the mark with methanol, and mix well to prepare a mixed standard intermediate solution C with a concentration of 100 ng / mL for the nine substances.

[0135] 2.2 Preparation of standard solutions: Accurately pipette 20 μL of the above mixed standard intermediate solution C, and accurately pipette 20, 40, 200, 400, and 800 μL of the above 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 approximately 0.5 g of sample (accurate to 0.0001 g), place it in a 25 mL plastic centrifuge tube, add methanol to the mark, mix well, vortex for 30 s, sonicate for 20 min, centrifuge at 10000 r / min for 5 min, take an appropriate amount of supernatant, filter through a 0.22 μm filter membrane, and keep the filtrate for later use.

[0137] 2.4 Detection: The series of standard solutions in (2) and the filtrate in (3) were injected into a liquid chromatography-mass spectrometry instrument 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℃;

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

[0141] The mobile phase and elution conditions are as follows: Mobile phase A is an aqueous solution of formic acid and water with a volume ratio of 0.1:99.9; mobile phase B is 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 scan; Detection mode: Multiple reaction monitoring (MRM). Ions with high abundance and few interferences were used as quantitative ions, and the rest were used as qualitative ions (Table 3).

[0144] Table 2 MRM optimization parameters for compounds

[0145]

[0146] Note: * indicates quantitative ion pairs.

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

[0148] Table 3. Linear equations, correlation coefficients, linear ranges, and detection limits for nine substances.

[0149]

[0150]

[0151] 3. Method Optimization

[0152] 3.1 Selection of mobile phase

[0153] The composition of the mobile phase was investigated using water, 0.1% formic acid, and 0.5% formic acid as the aqueous phase, and methanol and acetonitrile as the organic phase. The results showed that when acetonitrile was used as the organic phase, methimazole, thiouracil, and methylthiouracil eluted earlier, making them susceptible to interference. Ethionamide showed significant tailing, captopril had a leading peak, and peptidyl ion response was poor, resulting in unsatisfactory peak shapes. When water was used as the mobile phase, captopril peak broadening was severe. Finally, 0.1% formic acid and methanol were selected as the mobile phase, which simultaneously improved the ethionamide peak tailing and captopril peak broadening, resulting in a stable baseline.

[0154] Column selection and elution gradient optimization

[0155] Because the analytes methimazole, carbimazole, thiouracil, methylthiouracil, 5-N-propyluracil, and propylthiouracil have similar structures, their retention times are similar, and different compounds can produce the same fragments, it is necessary to ensure complete separation of the chromatographic peaks of each substance to guarantee accurate qualitative and quantitative analysis. Separation performance was tested using three columns: a Waters UPLC BEHC18 column (2.1 mm × 100 mm, 1.7 μm), a Waters UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm), and an Agilent ZORBAX Eclipse Plus-C18 column (2.1 mm × 100 mm, 3.5 μm).

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

[0157] 3.2 Selection of ion pairs

[0158] All compounds are represented by [M+H]. + As the parent ion, when determining its daughter ions using secondary mass spectrometry, it was found that thiouracil, with 129 as the parent ion, can produce three daughter ions: 70, 71.9, and 111.9, with the response intensity being 111.9 > 71.9 > 70. However, in actual measurements, it was found that the response was poor when 71.9 was used as the daughter ion, and due to the short retention time, it was easily affected by the sample matrix. The response was relatively high when 70 was used as the daughter ion, and the recovery rate was good in liquid water matrix and cream / emulsion matrix. Methylthiouracil, with 143 as the parent ion, exhibits relatively high abundance of daughter ions with mass-to-charge ratios of 71.9, 84, 87.9, 115, and 126. However, using 87.9 and 115 as daughter ions results in poor recovery and precision, along with low backcalibration accuracy. Using 71.9 and 86 as daughter ions leads to baseline instability and low signal-to-noise ratio. Changing the daughter ions to 126 and 84 yields good recoveries in two matrices, ranging from 96.72% to 109.33% and 95.68% to 99.75%, respectively. Carbimazole, with 187 as the parent ion, generates fragments of 114.8, 143.1, and 88. Furthermore, 114.8 generates two additional fragments, 88 and 81. The CE values ​​of the three fragments were optimized and investigated. The results showed that fragment 88 exhibited unstable response; therefore, 143.1 and 114.8 were selected as daughter ions. The 5-N-propyluracil daughter ion 82 showed a good response, but the daughter ions with a molecular weight of 100 or higher showed a slightly lower response. The parameters of 125.9, 136.9 and 138.1 were investigated and optimized in the experiment, and 138.1 was finally selected as the daughter ion.

[0159] 3.3 Stability Assessment

[0160] Carbimazole is a carbamic acid derivative. Due to the influence of the imidazole ring on the amide bond of carbamic acid, the amide bond is more easily hydrolyzed than the ester bond, yielding the corresponding secondary amine form, methimazole. To further investigate the stability of the solution, carbimazole single-standard solutions were prepared to concentrations of 1500 ng / mL and 600 ng / mL with water, 80% methanol, and methanol, respectively. The changes in carbimazole concentration over time were compared when stored in a refrigerator (4℃) and at room temperature (25℃). Analysis was performed at 0, 24, and 72 h, and the peak area of ​​carbimazole was recorded. The peak area at 0 h was taken as 100%, and the recovery rate at each point was examined. The results showed that dilution of the standard solution with 80% methanol or methanol and refrigeration at 4℃ was more conducive to the stability of carbimazole, with peak areas at 24 h showing recoveries of 102.79% and 92.42%, respectively. At room temperature, the carbimazole content decreased significantly after 24 h, and the solution stability was worse at lower concentrations. Therefore, the reference solution and the test solution should be freshly prepared immediately before use, stored at low temperature and protected from light, and injected promptly. Considering factors such as sample pretreatment procedures and solution stability, methanol was chosen for diluting the standard solution and for sample pretreatment.

[0161] Example 1

[0162] Two commercially available cosmetic bases were used as test subjects, spiked at concentrations of 0.25 μg / g, 0.50 μg / g, and 2.50 μg / g, respectively. Parallel experiments (n=6) were conducted, and the recoveries and RSDs were determined (Table 5). The results showed that the recoveries of the nine components ranged from 85.90% to 113.00% under high, medium, and low spiked concentrations, with relative deviations (RSDs) (n=6) ranging from 0.42% to 6.26%. This indicates that the recovery results of this method are good.

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

[0164]

[0165]

[0166] Using this detection method, 65 batches of commercially available creams, lotions, and water-based whitening and freckle-removing cosmetics (toners, serums, face creams, etc.) were tested according to the method. Results showed that 3 batches of samples contained peptide Anmido, with a content ranging from 0.09% to 0.20%, while all other compounds were not detected.

[0167] The foregoing description illustrates and illustrates 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 to the above embodiments, which are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made in accordance with the scope of the patent and the description should still fall within the scope of the present invention. The scope of protection of this invention is defined by the appended claims and their equivalents.

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

Claims

1. A method for detecting a whitening ingredient, characterized by, The method includes the following steps: The skin-whitening components in the sample were detected using liquid chromatography-mass spectrometry. The skin-whitening components included carbimazole, 5-N-propyluracil, and peptide omeprazole. The mass spectrometry conditions of the liquid chromatography-mass spectrometry system include: Ions with a mass-to-charge ratio of 187 were selected as the parent ion, and ions with a mass-to-charge ratio of 114.8 and / or 143.1 were selected as daughter ions for the detection of carbimazole. An ion with a mass-to-charge ratio of 155 was selected as the parent ion, and ions with a mass-to-charge ratio of 138.1 and / or 82 were selected as daughter ions to detect the 5-N-propyluracil. An ion with a mass-to-charge ratio of 279 was selected as the parent ion, and ions with mass-to-charge ratios of 209.1, 167.1, and / or 150.1 were selected as daughter ions to detect the peptide Amidato. The mass spectrometry conditions of the liquid chromatography-mass spectrometry system include: Ion source: Turbo Spray Ion Drive; Ion source temperature: 500℃; Ion spray voltage: 5500V; Scanning method: positive ion scan; Detection method: Multiple reaction monitoring (MRM) mode; The chromatographic conditions for the liquid chromatography-mass spectrometry (LC-MS) instrument are as follows: Column: Agilent ZORBAX Eclipse Plus-C18 column; Column temperature: 30℃; Flow rate: 0.3 mL / min; Injection volume: 2 μL; Mobile phase: Mobile phase A is an aqueous solution of formic acid 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; 8.0–10.0 min, 70%–20% A; 10.0–11.0 min, 20%–95% A; 11.0–15.0 min, 95% A.

2. The detection method according to claim 1, characterized in that, The mass spectrometry conditions of the liquid chromatography-mass spectrometry system include: An ion with a mass-to-charge ratio of 187 is selected as the precursor ion, and an ion with a mass-to-charge ratio of 114.8 or 143.1 is selected as the daughter ion to detect the carbimazole, wherein the precursor ion with a mass-to-charge ratio of 187 and the daughter ion with a mass-to-charge ratio of 114.8 are quantitative ion pairs. An ion with a mass-to-charge ratio of 155 is selected as the parent ion, and an ion with a mass-to-charge ratio of 138.1 or 82 is selected as the daughter ion to detect the 5-N-propyluracil, 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 quantitative ion pairs. An ion with a mass-to-charge ratio of 279 is selected as the parent ion, and ions with a mass-to-charge ratio of 209.1, 167.1, or 150.1 are selected as daughter ions to detect the peptide Ammido, 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 quantitative ion pairs.

3. The method of claim 1, wherein, The mass spectrometry conditions of the liquid chromatography-mass spectrometry system include: In the table, * indicates quantitative ion pairs.

4. A method for simultaneously detecting a plurality of whitening ingredients, characterized by, The method comprises the steps of: using a liquid chromatograph mass spectrometer to detect whitening ingredients in a to-be-detected sample, the whitening ingredients comprising carbamazide, 5-N-propyl uracil and peptide amido, and the whitening ingredients further comprising at least one of methimazole, thiouracil, methyl thiouracil, propyl thiouracil, captopril and ethionamide; The mass spectrometry conditions of the liquid chromatograph mass spectrometer comprise: 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 carbamazide; 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; An ion with a mass-to-charge ratio of 279 is selected as a parent ion, and ions with mass-to-charge ratios of 209.1, 167.1 or 150.1 are selected as daughter ions to detect the peptide amido; An ion with a mass-to-charge ratio of 115 is selected as a parent ion, and ions with mass-to-charge ratios of 88.1 or 81 are selected as daughter ions to detect the methimazole; The thiouracil is detected by selecting ions having a mass-to-charge ratio of 129 as parent ions and ions having a mass-to-charge ratio of 111.9 or 70 * as daughter ions. The ion having a mass-to-charge ratio of 143 is selected as a parent ion, and the ion having a mass-to-charge ratio of 126 * or 84 is selected as a daughter ion to detect the methylthiouracil. The propyl thioureido pyrimidine is detected by selecting ions having a mass-to-charge ratio of 171 as parent ions and ions having a mass-to-charge ratio of 153.9 or 112 * as daughter ions. ions having a mass-to-charge ratio of 218 are selected as parent ions, and ions having a mass-to-charge ratio of 116 or 172 are selected as daughter ions to detect the captopril; * ; ions having a mass-to-charge ratio of 167 are selected as parent ions, and ions having a mass-to-charge ratio of 107 or 140.1 * are selected as daughter ions to detect the ethionamide; The mass spectrometry conditions of the liquid chromatograph mass spectrometer comprise: Ion source: Turbo Spray lon Drive; Ion source temperature: 500 DEG C; Ion spray voltage: 5500 V; Scan mode: positive ion scan; Detection mode: multiple reaction ion monitoring mode; The chromatography conditions of the liquid chromatograph mass spectrometer are as follows: Chromatography column: Agilent ZORBAX Eclipse Plus-C18 column; Column temperature: 30 DEG C; Flow rate: 0.3 mL / min; Injection volume: 2 mu L; Mobile phase: mobile phase A is a formic acid aqueous solution with a formic acid: water volume ratio 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; 8.0-10.0 min, 70%-20% A; 10.0-11.0 min, 20%-95% A; 11.0-15.0 min, 95% A.

5. The method of claim 4, wherein, The mass spectrometry conditions of the liquid chromatograph mass spectrometer comprise: An ion with a mass-to-charge ratio of 187 is selected as a parent ion, and ions with mass-to-charge ratios of 114.8 and 143.1 are selected as daughter ions to detect the carbamazide, 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 ions with mass-to-charge ratios of 138.1 and 82 are selected as daughter ions 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 ions with mass-to-charge ratios of 209.1, 167.1 and 150.1 are selected as daughter ions to detect the peptide amido, 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 a quantitative ion pair; The ion with a mass-to-charge ratio of 115 is selected as a parent ion, and the ion with a mass-to-charge ratio of 88.1 and 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; ions having a mass to charge ratio of 129 are selected as parent ions and ions having a mass to charge ratio of 111.9 and 70 are selected as daughter ions to detect the thiouracil, wherein the parent ion having a mass to charge ratio of 129 and the daughter ion of 70 are the quantitation ion pair * ​ ions having a mass-to-charge ratio of 143 are selected as parent ions, and ions having a mass-to-charge ratio of 126 * and 84 are selected as daughter ions to detect the methylthiouracil, wherein the parent ions having a mass-to-charge ratio of 143 and the daughter ions having a mass-to-charge ratio of 126 * are the quantitation ion pairs; ions having a mass-to-charge ratio of 171 were selected as parent ions, and ions having a mass-to-charge ratio of 153.9 and 112 * were selected as daughter ions to detect the propyl thioureido pyrimidine, wherein the parent ion having a mass-to-charge ratio of 171 and the daughter ion having a mass-to-charge ratio of 112 * were a quantitative ion pair; ions having a mass-to-charge ratio of 218 are selected as parent ions, and ions having a mass-to-charge ratio of 116 and 172 are selected as daughter ions to detect the captopril, wherein the parent ions having a mass-to-charge ratio of 218 and the daughter ions having a mass-to-charge ratio of 172 are quantitative ion pairs * * ;​ ions having a mass to charge ratio of 107 and 140.1 * The parent ion having a mass to charge ratio of 167 and the daughter ion having a mass to charge ratio of 140.1 * are the quantifier ion pairs.

6. The method of claim 5, wherein, The mass spectrometry conditions of the liquid chromatograph-mass spectrometer include: In the table, * indicates a quantitative ion pair.

7. The method according to any one of claims 1 to 6, characterized in that, The sample to be detected is a food, a medicine, a health product or a cosmetic.

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