Method for determining multiple sun-screening agents in serum

Abstract

The embodiment of the invention discloses a method for determining multiple sun-screening agents in serum, the method can be used for determining the sun-screening agents, and the method comprises the following steps: obtaining a to-be-detected sample according to to-be-detected serum; performing chromatography and mass spectrometry on the to-be-detected sample under a predetermined detection condition to obtain peak area data corresponding to the multiple sun-screening agents; acquiring the concentration of each sun-screening agent in the serum to be detected according to the linear equation and the peak area data of each sun-screening agent; the multiple sun-screening agents comprise at least one of cresol trozole trisiloxane and ethyl hexyl triazinone.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and more specifically to a method for determining multiple sunscreen agents in serum. Background Technology

[0002] Studies have shown that liquid chromatography-mass spectrometry (LC-MS / MS) can be used to quantitatively detect sunscreen agents using blood, plasma, or serum as a matrix, as illustrated in Table a below. Currently, there are no reports on the quantification of two relatively common sunscreen agents, cresoltrazol trisiloxane (DT) and ethylhexyl triazine (ET), using LC-MS / MS.

[0003] Table a. Studies on mass spectrometry quantitative detection of sunscreen agents using blood, plasma, or serum as a matrix.

[0004]

[0005]

[0006] Note: " / " indicates that there is no literature report.

[0007] [1] Bao Dejun, Feng Zhuangzhuang, Zhang Xu, et al. Determination of 22 organic ultraviolet absorbers in human serum by ultra-high performance liquid chromatography-tandem mass spectrometry. [J] Chromatography. 2025, 43(06): 620-9.

[0008] [2] YANG W, FENG J, LIANG W, et al. Ahigh-throughput method for the determination of 14UV-filters in human plasma by LC-MS / MS: Minimizeinterferences from proteins and phospholipids in the matrix [J]. Journal of chromatography B, Analytical technologies in the biomedical and life sciences, 2024, 1237: 124090.

[0009] [3] MATTAM K, ZUSTERZEEL R, PILLI NR, et al. Effect of sunscreen application under maximal use conditions on plasma concentration of sunscreen active ingredients: Arandomized clinical trial [J]. Jama, 2019, 321(21): 2082-91. Summary of the Invention

[0010] One objective of this application is to solve at least one of the problems mentioned in the background above and to provide corresponding beneficial effects.

[0011] Another objective of this application is to provide a method for determining multiple sunscreens in serum, which can be used to determine at least one sunscreen among cresoltrazol trisiloxane (DT) and ethylhexyl triazine ketone (ET).

[0012] The embodiments of this application mainly achieve the above objectives through the following technical solutions.

[0013] This application provides a method for determining multiple sunscreen agents in serum, including:

[0014] Obtain the test sample based on the serum to be tested;

[0015] Under predetermined detection conditions, chromatographic and mass spectrometric analyses were performed on the test samples to obtain peak area data for various sunscreen agents.

[0016] Based on the linear equations and peak area data of each sunscreen agent, the concentration of each sunscreen agent in the serum to be tested was obtained;

[0017] Various sunscreen agents include at least one of cresoltrazol trisiloxane and ethylhexyl triazine ketone.

[0018] In some technical solutions, obtaining the sample to be tested based on the serum to be tested includes:

[0019] The spiked serum solution is obtained based on the serum to be tested and the isotope internal standard solution;

[0020] The spiked serum solution was extracted with acetonitrile to obtain the first extraction solution and the extraction residue.

[0021] The spiked serum solution was extracted with ethyl acetate to obtain a second extraction solution;

[0022] The first extraction solution and the second extraction solution were mixed and dried, then redissolved in methanol to obtain the sample to be tested.

[0023] In some technical solutions, obtaining the spiked serum solution based on the serum to be tested and the isotope internal standard solution includes:

[0024] Centrifuge the serum to be tested and obtain the supernatant as the serum supernatant.

[0025] The serum supernatant and the isotope internal standard mixed solution were mixed to obtain the spiked serum solution.

[0026] In some technical solutions, multiple sunscreen agents include cresoltrazol trisiloxane and ethylhexyl triazine.

[0027] In some technical solutions, various sunscreen agents also include one or more of the following: bis(ethylhexyl)phenol methoxyphenyl triazine (BEMT), isoamyl p-methoxycinnamate (IMN), ethylhexyl salicylate (2-ES), and diethylamino hydroxybenzoyl hexyl benzoate (DHB).

[0028] In some technical solutions, various sunscreen agents also include one or more of phenylbenzimidazole sulfonic acid, octocrylene, ethylhexyl methoxycinnamate, octocrylene, ethylhexyl salicylate, and ethylhexyl methoxycinnamate.

[0029] In some technical solutions, the sample to be tested is analyzed by chromatography and mass spectrometry under predetermined detection conditions to obtain peak area data corresponding to various sunscreen agents; including:

[0030] The sample to be tested is injected into a chromatographic analysis instrument for chromatographic and mass spectrometric analysis to obtain peak area data for various sunscreen agents;

[0031] The chromatographic analysis instrument is a liquid chromatography-tandem mass spectrometer.

[0032] In some technical solutions, the liquid chromatography-tandem mass spectrometer is a liquid chromatography-tandem triple quadrupole mass spectrometer.

[0033] In some technical solutions, the method for determining the concentration of multiple sunscreen agents in serum further includes:

[0034] Obtain the linear equations for each sunscreen agent.

[0035] In some technical solutions, the linear equations for obtaining each sunscreen agent include:

[0036] Obtain multiple sunscreen mixtures of different concentrations, each containing a variety of sunscreen agents;

[0037] The corresponding samples for instrumentation were obtained based on the sunscreen mixture solution of each concentration and the corresponding blank matrix serum and isotope internal standard solution.

[0038] Under predetermined testing conditions, multiple samples were analyzed by chromatography and mass spectrometry to obtain linear equations for various sunscreen agents.

[0039] The beneficial effects of the embodiments of the present invention include:

[0040] 1. The method provided in this application embodiment is capable of detecting multiple sunscreen ingredients in the serum to be tested, including at least one of cresoltrazol trisiloxane and ethylhexyl triazine ketone.

[0041] 2. In some embodiments, the method provided in this application can detect a variety of sunscreen agents, including not only cresoltrazol trisiloxane and ethylhexyl triazine, but also one or more of bis(ethylhexylphenol) methoxyphenyl triazine (BEMT), isoamyl p-methoxycinnamate (IMN), ethylhexyl salicylate (2-ES), and diethylamino hydroxybenzoyl hexyl benzoate (DHB).

[0042] 3. In some embodiments, the method provided in this application can detect a variety of sunscreen agents, including not only cresoltrazol trisiloxane and ethylhexyl triazine, but also phenylbenzimidazole sulfonic acid, octocrylene, ethylhexyl methoxycinnamate, and octocrylene-d. 10 Ethylhexyl salicylate-d4, ethylhexyl methoxycinnamate- 13 One or more of C3.

[0043] 4. In some embodiments, the method provided in this application is capable of detecting a variety of sunscreens, including cresoltrazol trisiloxane, bis(ethylhexyloxyphenol)methoxyphenyltriazine, p-methoxycinnamate isoamyl ester, phenylbenzimidazole sulfonic acid, ethylhexyl triazine ketone, octocrylene, ethylhexyl salicylate, diethylaminohydroxybenzoyl benzoate, and ethylhexyl methoxycinnamate.

[0044] 5. In some embodiments, the method provided in this application has the advantage of a wide linear range. This means that the method of this application maintains a good linear relationship between the signal intensity (mass spectrometry response peak area or peak height) of the target compound and its actual concentration over a large concentration range. It can cover low, medium, and high concentration samples without frequent dilution or concentration; it is suitable for complex sample conditions (where the concentration of the target analyte varies greatly). Furthermore, it can reduce errors caused by concentration fluctuations.

[0045] 6. In some embodiments, the methods provided in this application have high correlation coefficients.

[0046] 7. In some embodiments, the method provided in this application has a low detection limit.

[0047] 8. In some embodiments, the method provided in this application has a low limit of quantification.

[0048] 9. In some embodiments, the method provided in this application has a high recovery rate. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of a chromatogram from an experimental example in this application;

[0050] Figure 2 This is another chromatographic schematic diagram from the experimental examples in this application;

[0051] Figure 3 This is another chromatographic schematic diagram from the experimental examples in this application;

[0052] Figure 4 This is another chromatographic schematic diagram from the experimental examples in this application;

[0053] Figure 5 This is another chromatographic schematic diagram from the experimental examples in this application;

[0054] Figure 6 This is another chromatographic schematic diagram from the experimental examples in this application;

[0055] Figure 7 This is another chromatographic schematic diagram from the experimental examples in this application;

[0056] Figure 8 This is another chromatographic schematic diagram from the experimental examples in this application;

[0057] Figure 9 This is another chromatographic schematic diagram from the experimental examples in this application;

[0058] Figure 10 This is another chromatographic schematic diagram from the experimental examples in this application;

[0059] Figure 11 This is another chromatographic schematic diagram from the experimental examples in this application;

[0060] Figure 12 This is another chromatographic schematic diagram from the experimental examples in this application;

[0061] Figure 13 This is another chromatographic schematic diagram from the experimental examples in this application;

[0062] Figure 14 This is a schematic diagram of principal component analysis (PCA) in an experimental example of this application. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0064] Unless otherwise stated, the following terms in the embodiments of this application are defined as follows:

[0065] The terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first," "second," etc., may explicitly or implicitly include at least one of those features.

[0066] The terms “comprising” and “containing”, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0067] The term "blank matrix serum" refers to artificial serum blank matrix.

[0068] The term "occrylene-d" 10 "" refers to the deuterium-labeled compounds of octocrylene.

[0069] The term "ethylhexyl salicylate-d4" refers to the deuterium-labeled compound of ethylhexyl salicylate.

[0070] The term "ethylhexyl methoxycinnamate" 13 "C3" refers to ethylhexyl salicylate. 13 C isotope substitutions.

[0071] The term "Parent ion" refers to the parent ion.

[0072] The term "Daughter ion" refers to a daughter ion.

[0073] The term "DP" stands for Declustering Potential.

[0074] The term "CE" refers to Collision Energy.

[0075] The term "internal standard" refers to an internal standard.

[0076] In addition to the above, it should be emphasized that the reference to "embodiment" herein means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0077] Unless otherwise stated, the numerical range includes the stated number.

[0078] <Method for determining the concentration of sunscreen agents in serum>

[0079] This application provides a method for determining the concentration of multiple sunscreen agents in serum, including steps S120 to S140:

[0080] S120. Obtain the sample to be tested from the serum to be tested.

[0081] S130. Perform chromatographic and mass spectrometric analysis on the sample to be tested under predetermined detection conditions to obtain peak area data corresponding to various sunscreens; the predetermined detection conditions include liquid chromatography conditions and mass spectrometry conditions.

[0082] Specifically, the sample to be tested is used as the injection sample for the instrument and injected into the liquid chromatograph. Under predetermined detection conditions, the sample to be tested is analyzed by chromatography and mass spectrometry to obtain peak area data corresponding to various sunscreens.

[0083] In some embodiments, the chromatographic analysis instrument is a liquid chromatography-tandem mass spectrometer.

[0084] Optionally, the liquid chromatography-tandem mass spectrometer is a liquid chromatography-tandem triple quadrupole mass spectrometer.

[0085] S140. Based on the linear equations and peak area data of each sunscreen agent, obtain the concentration of each sunscreen agent in the serum to be tested.

[0086] A linear equation refers to the linear equation of the standard curve of a sunscreen agent. The linear equation for a sunscreen agent shows a correlation between peak area and concentration; by combining the peak area data of the sunscreen agent being tested with the linear equation, the concentration can be determined.

[0087] In some embodiments, peak area data refers to the ratio of the peak area of ​​the target ion of the sunscreen to that of the isotopic internal standard ion.

[0088] In some embodiments, the plurality of sunscreen agents include cresoltrazol trisiloxane (DT), bis(ethylhexylphenol) methoxyphenyl triazine (BEMT), isoamyl p-methoxycinnamate (IMN), ethylhexyl salicylate (2-ES), diethylamino hydroxybenzoyl hexyl benzoate (DHB), and ethylhexyl triazine ketone (ET).

[0089] The method provided in this application embodiment can accurately detect multiple sunscreen components in the serum to be tested.

[0090] In some embodiments, the multiple sunscreen agents also include one or more of phenylbenzimidazole sulfonic acid, octocrylene, ethylhexyl methoxycinnamate, octocrylene, ethylhexyl salicylate, and ethylhexyl methoxycinnamate.

[0091] In some embodiments, S120, obtaining the sample to be tested from the serum to be tested, includes steps S121 to S124:

[0092] S121. Obtain the spiked serum solution based on the serum to be tested and the isotope internal standard solution;

[0093] S122. Extract the spiked serum solution with acetonitrile to obtain the first extraction solution;

[0094] S123. Extract the spiked serum solution with ethyl acetate to obtain the second extraction solution;

[0095] S124. Mix the first extraction solution and the second extraction solution, dry them, and redissolve them with methanol to obtain the sample to be tested.

[0096] In some embodiments, S121 includes S1211 to S1212:

[0097] S1211. Centrifuge the serum to be tested and obtain the supernatant after centrifugation as the serum supernatant.

[0098] S1212. Mix the serum supernatant with the corresponding isotope internal standard solution to obtain the spiked serum solution.

[0099] In some embodiments, step S1211 includes step S12111:

[0100] S12111. Vortex the serum to be tested, centrifuge, and obtain the supernatant after centrifugation as the serum supernatant.

[0101] In some embodiments, step S12111 includes:

[0102] The serum to be tested was vortexed at 1500-2500 rpm for 3-10 min, and then centrifuged at 10000-15000 rpm for 3-10 min. The supernatant after centrifugation was obtained as the serum supernatant.

[0103] In some embodiments, step S12111 includes:

[0104] After removing 500 μL of the serum to be tested from the -80℃ freezer, thaw it step by step to room temperature; vortex at 2000 rpm for 5 min, and then centrifuge at 12000 rpm for 5 min to obtain the supernatant after centrifugation, which is used as the serum supernatant.

[0105] In some embodiments, in S1212, the volume ratio of serum supernatant to isotope internal standard solution is 200:10, or 20:1.

[0106] In some embodiments, step S122 includes:

[0107] Acetonitrile was added to the spiked serum solution, and the mixture was stirred at 1500–2500 rpm for 3–10 min. Liquid-liquid extraction was then performed at a constant temperature of 3–5 °C for 0.8–1.2 h. Afterward, the solution was centrifuged at 10000–15000 rpm for 3–10 min, and the entire supernatant was collected to obtain the first extraction solution. Acetonitrile is used as the extraction solvent.

[0108] In some embodiments, the volume ratio of the spiked serum solution to acetonitrile in step S122 is 210:990, or 7:33.

[0109] In some embodiments, step S122 includes:

[0110] Take 210 μL of spiked serum solution, add 990 μL of acetonitrile, mix at 2000 rpm for 5 min, and extract at 4℃ for 1 h. Then centrifuge at 12000 rpm for 3-10 min, and take all the supernatant after centrifugation to obtain the first extraction solution.

[0111] In some embodiments, step S123 includes:

[0112] Ethyl acetate was added to the spiked serum solution and mixed at 1500–2500 rpm for 3–10 min. Liquid-liquid extraction was then performed at 3–5 °C for 0.8–1.2 h. The solution was then centrifuged at 10000–15000 rpm for 3–10 min. The supernatant obtained after centrifugation was collected as the second extraction solution. Ethyl acetate was used as the extraction solvent.

[0113] In some embodiments, the volume ratio of the spiked serum solution to ethyl acetate in step S123 is 210:990, or 7:33.

[0114] In some embodiments, step S123 includes:

[0115] Take 210 μL of spiked serum solution, add 990 μL of ethyl acetate, mix at 2000 rpm for 5 min, extract at 4℃ for 1 h, then centrifuge at 12000 rpm for 5 min, and take all the supernatant after centrifugation to obtain the second extraction solution.

[0116] In some embodiments, step S124 includes:

[0117] The first and second extraction solutions were mixed and vacuum evaporated, then methanol was added for redissolution. The mixture was centrifuged at 10,000–15,000 rpm for 3–10 min, and all the supernatant was collected as the sample to be tested.

[0118] In some embodiments, step S124 includes: mixing the first extraction solution and the second extraction solution, vacuum drying, adding 50 μL of methanol to reconstitute, centrifuging at 12000 rpm for 5 min, and taking all the supernatant as the sample to be tested.

[0119] In some embodiments, the method for determining the concentration of multiple sunscreen agents in serum further includes step S110:

[0120] S110. Obtain the linear equations for each sunscreen agent.

[0121] In some embodiments, S110, obtaining the linear equations for each sunscreen agent, includes steps S111 to S113:

[0122] S111. Obtain multiple sunscreen mixture solutions of different concentrations, wherein each concentration of sunscreen mixture solution contains multiple sunscreens.

[0123] S112. Based on the sunscreen mixture solution of each concentration and the corresponding blank matrix serum and isotope internal standard solution, obtain the corresponding sample for use on the instrument, and obtain multiple samples for use on the instrument.

[0124] S113. Under predetermined detection conditions, perform chromatographic and mass spectrometric analysis on multiple samples to obtain linear equations corresponding to various sunscreens. Specifically, use the samples as injection samples, inject them into a liquid chromatograph, perform chromatographic and mass spectrometric analysis, and obtain linear equations corresponding to various sunscreens.

[0125] In some embodiments, the chromatographic analysis instrument is a liquid chromatography-tandem mass spectrometer. Optionally, the liquid chromatography-tandem mass spectrometer is a liquid chromatography-tandem triple quadrupole mass spectrometer.

[0126] In some embodiments, the acquisition of the sample corresponding to each concentration of sunscreen mixture solution in step S112 includes steps S1121 to S1125:

[0127] S1121. Centrifuge the blank matrix serum to obtain the supernatant after centrifugation, which is used as matrix serum solution.

[0128] S1122. Mix the sunscreen mixture with the matrix serum and the isotope internal standard solution to obtain the spiked mixture solution;

[0129] S1123. Extract the spiked mixed solution with acetonitrile to obtain the first extraction solution;

[0130] S1124. Extract the spiked mixed solution with ethyl acetate to obtain a second extraction solution;

[0131] S1125. Mix the first extraction solution and the second extraction solution, dry them, and then redissolve them with methanol to obtain the sample for use.

[0132] In some embodiments, step S1121 includes step S11211:

[0133] S11211. Vortex the blank matrix serum, centrifuge, and obtain the supernatant after centrifugation as the matrix serum solution.

[0134] In some embodiments, step S11211 includes:

[0135] The blank matrix serum was vortexed at 1500-2500 rpm for 3-10 min, and then centrifuged at 10000-15000 rpm for 3-10 min to obtain the supernatant, which was used as the matrix serum solution.

[0136] In some embodiments, step S11211 includes:

[0137] 500 μL of blank matrix serum was taken out of the -80℃ freezer and thawed to room temperature in steps. It was then vortexed at 2000 rpm for 5 min and centrifuged at 12000 rpm for 5 min to obtain the supernatant as matrix serum solution.

[0138] In some embodiments, in step S1122, the volume ratio of the sunscreen mixture solution, the matrix serum solution, and the isotope internal standard solution is 200:200:10, i.e., 2:2:1.

[0139] In some embodiments, S1123 includes:

[0140] Add acetonitrile to the spiked mixture and mix at 1500–2500 rpm for 3–10 min. Perform liquid-liquid extraction at 3–5 °C for 0.8–1.2 h. Then centrifuge at 10000–15000 rpm for 3–10 min. Collect all the supernatant after centrifugation to obtain the first extraction solution.

[0141] In some embodiments, the volume ratio of the spiked mixed solution to acetonitrile in S1123 is 410:790, or 41:79.

[0142] In some embodiments, step S1123 includes:

[0143] Take 410 μL of the spiked mixed solution, add 790 μL of acetonitrile, mix at 2000 rpm for 5 min, and extract at 4℃ for 1 h. Then centrifuge at 12000 rpm for 3-10 min, and take all the supernatant after centrifugation to obtain the first extraction solution.

[0144] Acetonitrile is used as an extraction solvent.

[0145] In some embodiments, step S1124 includes:

[0146] Add ethyl acetate to the spiked mixture and mix at 1500–2500 rpm for 3–10 min. Perform liquid-liquid extraction at 3–5 °C for 0.8–1.2 h, followed by centrifugation at 10000–15000 rpm for 3–10 min. Collect all the supernatant after centrifugation to obtain the second extraction solution. Ethyl acetate serves as the extraction solvent.

[0147] In some embodiments, the volume ratio of the spiked mixed solution to ethyl acetate in step S1124 is 410:790, or 41:79.

[0148] In some embodiments, step S1124 includes:

[0149] Take 410 μL of the spiked mixed solution, add 790 μL of ethyl acetate, mix at 2000 rpm for 5 min, extract at 4℃ for 1 h, then centrifuge at 12000 rpm for 5 min, and take all the supernatant after centrifugation to obtain the second extraction solution.

[0150] In some embodiments, the liquid chromatography conditions are liquid chromatography conditions A, which include:

[0151] Chromatographic column: Waters CORTECS UPLC C18 column (2.1×100mm, 1.6μm);

[0152] The mobile phase system includes mobile phase A and mobile phase B. Mobile phase A is a 0.1% formic acid aqueous solution, which is composed of formic acid and water, and the volume concentration of formic acid is 0.1%. Mobile phase B is a 0.1% formic acid acetonitrile solution, which is composed of formic acid and acetonitrile, and the volume concentration of formic acid is 0.1%.

[0153] Column temperature: 35℃;

[0154] Flow rate of the mobile phase system: 0.3 mL / min;

[0155] Running time: 30 minutes;

[0156] Injection volume: 15 μL;

[0157] The program is a gradient elution program for liquid chromatography, as shown in Table 1 below;

[0158] Table 1

[0159] Running time (min) A％ B％ 0.0 90 10 2.0 90 10 5.0 20 80 14.0 0 100 28.0 0 100 29.0 90 10 30.0 90 10

[0160] In Table 1, A% refers to the volume fraction of mobile phase A in the mobile phase system, and B% refers to the volume fraction of mobile phase B in the eluent.

[0161] In addition, regarding the gradient elution program in Table 1, the volume fractions of mobile phases A and B will change over time.

[0162] The details are as follows: From the initial time (0 minutes) to the 2nd minute, the volume fraction of mobile phase A is maintained at 90%; then from the 2nd minute to the 5th minute, mobile phase A linearly decreases to 20%; from the 5th minute to the 14th minute, mobile phase A further linearly decreases to 0%; from the 14th minute to the 28th minute, the volume fraction of mobile phase A is maintained at 0%; from the 28th minute to the 29th minute, mobile phase A linearly increases to 90%; from the 29th minute to the 30th minute, the volume fraction of mobile phase A is maintained at 90%. Here, "linear increase" refers to a uniform increase, and "linear decrease" refers to a uniform decrease.

[0163] In some embodiments, the mass spectrometry conditions are mass spectrometry conditions A, which include:

[0164] Ion source: ESI positive ions;

[0165] Scanning mode: Multiple Response Monitoring (MRM) mode;

[0166] Ion source temperature: 450℃;

[0167] Collision Gas: Medium;

[0168] Curtain Gas: 35psi;

[0169] Ion spray voltage: 5000V;

[0170] Ion Source Gas1 / Gas2: 50psi;

[0171] Dwell time: 75ms;

[0172] Triple quadrupole mass spectrometer MRM parameters are shown in Table 2 below;

[0173] Table 2

[0174]

[0175]

[0176] In the Daughter ion column of Table 2, those marked with * correspond to quantitative ions, while those without * correspond to qualitative ions, used to determine the elution time of the target analyte chromatographic peak.

[0177] In some embodiments, the method for preparing the isotope internal standard mixed solution includes:

[0178] Precisely transfer 10 μL of octocrylene-d 10 Stock solution, 10 μL ethylhexyl salicylate-d4 stock solution, 10 μL ethylhexyl methoxycinnamate- 13 C3 stock solution was diluted with methanol and shaken well to obtain a mixed isotope internal standard solution, in which octocrylene-d 10 The concentration of ethylhexyl salicylate-d4 is 1 μg / mL, and the concentration of ethylhexyl oxycinnamate- 13 The concentration of C3 is 1 μg / mL;

[0179] Octocrylin-D 10 The preparation method of the stock solution includes: accurately weighing 10 mg of octocrylene-d 10 To a 10 mL volumetric flask, dissolve and dilute with methanol to the mark, shake well, and obtain octocrylene-d. 10 Stock solution;

[0180] The preparation method of ethylhexyl salicylate-d4 stock solution includes: accurately weighing 10 mg of ethylhexyl salicylate-d4 into a 10 mL volumetric flask, dissolving and diluting it with methanol to the mark, shaking well to obtain ethylhexyl salicylate-d4 stock solution;

[0181] Ethylhexyl oxycinnamate - 13 The preparation method of C3 stock solution includes: accurately weighing 10 mg of ethylhexyl oxycinnamate -13 C3 was transferred to a 10 mL volumetric flask, dissolved in methanol and diluted to the mark, then shaken well to obtain ethylhexyl oxycinnamate. 13 C3 stock solution.

[0182] In some embodiments, the various sunscreen agents include cresoltrazol trisiloxane, bis(ethylhexylphenol)methoxyphenyltriazine, isoamyl p-methoxycinnamate, phenylbenzimidazole sulfonic acid, octocrylene, ethylhexyl salicylate, diethylaminohydroxybenzoylhexyl benzoate, ethylhexyl methoxycinnamate, and ethylhexyl triazine ketone. The linear equations for each sunscreen agent are shown in Table 3.

[0183] Table 3

[0184]

[0185] In Table 3, x in the linear equation represents the concentration of the sunscreen agent, and y represents the peak area ratio of the target ion to the internal standard isotope ion. The target ion for each sunscreen agent refers to the quantitative ion, i.e., the ion corresponding to the superscript * in Table 2. The internal standard isotope ion for each sunscreen agent refers to the quantitative ion of the corresponding internal standard isotope. The specific internal standard isotopes for the nine sunscreen agents to be tested are shown in Table 1. The Internal Standard section of Table 1 lists the internal standard isotopes for each sunscreen agent.

[0186] Regarding the quantitative ion, the following example is provided in Table 2: The target ion for cresoltrazol trisiloxane (DT) is the quantitative ion corresponding to 412.3, and the corresponding isotopic internal standard is OCT-d. 10 Its isotopic internal standard ion is the quantitative ion corresponding to 260.2. Similarly, the remaining eight will not be elaborated upon.

[0187] In some embodiments, the retention time, correlation coefficient, linear range, detection limit, quantitation limit and recovery rate corresponding to the linear equations of each sunscreen agent are shown in Table 3.

[0188] As can be seen from the above, the method provided in this application has the advantage of a wide linear range. This means that the method of this application maintains a good linear relationship between the signal intensity (mass spectrometry response peak area or peak height) of the target compound and its actual concentration over a large concentration range. It can cover low, medium, and high concentration samples without frequent dilution or concentration; it is suitable for complex sample conditions (where the concentration of the target analyte varies greatly). Furthermore, it can reduce errors caused by concentration fluctuations.

[0189] In addition, the method provided in this application embodiment also has advantages such as low detection limit and low quantitation limit.

[0190] As shown in Table 3, the correlation coefficients (r) of the linear equations for the nine sunscreen agents are all greater than 0.990. The method provided in this application also has the advantage of high correlation coefficients.

[0191] The recovery rate acceptance requirements are generally as follows: the recovery rate of the calibration standard should generally be within 85% to 115% of the theoretical value, and the recovery rate of the limit of quantitation concentration should be within 80% to 120%. It is also stipulated that for three repeated determinations, only >75% of the calibration standard and at least six concentration points need to meet the above calibration standard recovery criteria. Recovery rate = A / B × 100%, where A is the concentration of the target analyte obtained after processing a blank matrix with a known concentration of the target analyte, and B is the theoretical concentration of the target analyte. As shown in Table 3, the recovery rates of the nine sunscreens ranged from 80.30% to 116.17%, all within 80% to 120%, meeting the requirements. This demonstrates that the method provided in the embodiments of this application has the advantage of high recovery rate.

[0192] <Preparation of Mixed Solvents 1>

[0193] Example 1

[0194] Prepared according to the following method:

[0195] Measure 25 mL of methanol and add 25 mL of ultrapure water, mix well to obtain a 50% (volume fraction) methanol solution. Measure 10 mL of the 50% methanol solution, add 40 mL of tetrahydrofuran, mix well to obtain mixed solvent 1.

[0196] <Preparation of a stock solution of cresoltrazol trisiloxane reference standard>

[0197] Example 2

[0198] Prepared according to the following method:

[0199] Accurately weigh 10 mg of cresoltrazol trisiloxane reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with mixed solvent 1, and shake well; this is the reference standard stock solution 1 (1 mg / mL).

[0200] <Preparation of a stock solution of bis(ethylhexyloxyphenol)methoxyphenyltriazine reference standard>

[0201] Example 3

[0202] Prepared according to the following method:

[0203] Accurately weigh 10 mg of diethylhexyloxyphenol methoxyphenyl triazine reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with mixed solvent 1, and shake well; this is the reference standard stock solution 2 (1 mg / mL).

[0204] <Preparation of Stock Solution of Isoamyl p-methoxycinnamate Reference>

[0205] Example 4

[0206] Prepared according to the following method:

[0207] Accurately weigh 10 mg of isoamyl p-methoxycinnamate reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; this is the reference standard stock solution 3 (1 mg / mL).

[0208] <Preparation of a stock solution of phenylbenzimidazole sulfonic acid reference standard>

[0209] Example 5

[0210] Prepared according to the following method:

[0211] Accurately weigh 10 mg of phenylbenzimidazole sulfonic acid reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with mixed solvent 1, and shake well; this is the reference standard stock solution 4 (1 mg / mL).

[0212] <Preparation of Ethylhexyltriazinone Reference Stock Solution>

[0213] Example 6

[0214] Prepared according to the following method:

[0215] Accurately weigh 10 mg of ethylhexyltriazine ketone reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with mixed solvent 1, and shake well; this is the reference standard stock solution 5 (1 mg / mL).

[0216] <Preparation of Octocrylene Reference Stock Solution>

[0217] Example 7

[0218] Prepared according to the following method:

[0219] Accurately weigh 10 mg of octocrylene reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; this is the reference standard stock solution 6 (1 mg / mL).

[0220] <Preparation of Ethylhexyl Salicylate Reference Stock Solution>

[0221] Example 8

[0222] Prepared according to the following method:

[0223] Accurately weigh 10 mg of ethylhexyl salicylate reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; this is the reference standard stock solution 7 (1 mg / mL).

[0224] <Preparation of Diethylaminohydroxybenzoylhexylbenzoate Reference Stock Solution>

[0225] Example 9

[0226] Prepared according to the following method:

[0227] Accurately weigh 10 mg of diethylaminohydroxybenzoylhexylbenzoate reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; this is the reference standard stock solution 8 (1 mg / mL).

[0228] <Preparation of Ethylhexyl Methoxycinnamate Reference Stock Solution>

[0229] Example 10

[0230] Prepared according to the following method:

[0231] Accurately weigh 10 mg of ethylhexyl methoxycinnamate reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; this is the reference standard stock solution 9 (1 mg / mL).

[0232] <Preparation of a mixed solution of sunscreen reference standards>

[0233] Example 11

[0234] Prepared according to the following method:

[0235] Accurately transfer 100 μL of each of the following reference stock solutions to volumetric flasks: 1, 2, 3, 4, 5, and 8, and 1 mL of each of the following reference stock solutions: 6, 7, and 9, into 10 mL volumetric flasks. Dilute to the mark with methanol and shake well to obtain a mixed solution of sunscreen reference solutions, denoted as Mixed Solution 10.

[0236] After the aforementioned procedures, the sunscreen reference solution contains: 10 μg / mL of cresoltrazol trisiloxane, diethylhexyloxyphenol methoxyphenyl triazine, p-methoxycinnamate isoamyl ester, phenylbenzimidazole sulfonic acid, ethylhexyl triazine ketone and diethylamino hydroxybenzoyl hexyl benzoate, and 100 μg / mL of octocrylene, ethylhexyl salicylate and ethylhexyl methoxycinnamate.

[0237] Preparation of Octocrylene-d 10 Stock solution >

[0238] Example 12

[0239] Prepared according to the following method:

[0240] Accurately weigh 10 mg of octocrylene-d 10 To a 10 mL volumetric flask, dissolve and dilute with methanol to the mark, then mix well; this is octocrylene-d. 10 Stock solution (1 mg / mL).

[0241] <Preparation of Ethylhexyl Salicylate-d4 Stock Solution>

[0242] Example 13

[0243] Prepared according to the following method:

[0244] Accurately weigh 10 mg of ethylhexyl salicylate-d4 into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; this is the stock solution of ethylhexyl salicylate-d4 (1 mg / mL).

[0245] Preparation of ethylhexyl oxycinnamate- 13 C3 stock solution>

[0246] Example 14

[0247] Prepared according to the following method:

[0248] Accurately weigh 10 mg of ethylhexyl oxycinnamate. 13 C3 was transferred to a 10 mL volumetric flask, dissolved in methanol and diluted to the mark, then shaken well to obtain ethylhexyl oxycinnamate. 13 C3 stock solution (1 mg / mL).

[0249] <Preparation of Isotope Internal Standard Mixture>

[0250] Example 15

[0251] Prepared according to the following method:

[0252] Precisely transfer 10 μL of octocrylene-d 10 Stock solution (1 mg / mL), 10 μL ethylhexyl salicylate-d4 stock solution (1 mg / mL), 10 μL ethylhexyl methoxycinnamate- 13 Add C3 stock solution (1 mg / mL) to a 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain an isotope internal standard mixed solution;

[0253] The isotope internal standard mixed solution contains: 1 μg / mL octocrylene-d 10 1 μg / mL ethylhexyl salicylate-d4 and 1 μg / mL ethylhexyl oxycinnamate- 13 C3.

[0254] <Obtaining Linear Equations>

[0255] Example 16

[0256] The linear equations for each sunscreen agent are obtained according to the methods shown in steps S101 to S103:

[0257] S101. Dilute the sunscreen reference solution (i.e., reference solution 10) with methanol to form 11 sunscreen solutions of different concentrations (see Table 4 for details).

[0258] Table 4

[0259]

[0260] S102. Based on the aforementioned 11 different concentrations of sunscreen mixed solutions and corresponding blank matrix serum and isotope internal standard mixed solutions, the corresponding samples were obtained for use on the instrument, resulting in 11 samples for use on the instrument.

[0261] In S102, the acquisition of each sample includes S1021 to S1027:

[0262] S1021. Take 500 μL of blank matrix serum (from Shenzhen Baileding Biotechnology Co., Ltd., product number BA2310X) from the -80℃ freezer and thaw it to room temperature in steps.

[0263] S1022. Vortex the thawed blank matrix serum at 2000 rpm for 5 min, then centrifuge at 12000 rpm for 5 min. After that, accurately transfer 400 μL of the supernatant as matrix serum solution, and divide it into two portions, each 200 μL. Place the first portion in a clean EP tube No. 1 and the second portion in a clean EP tube No. 2.

[0264] S1023. Add 200 μL of sunscreen mixture and 10 μL of isotope internal standard mixture to the matrix serum in tube 1, mix, and obtain the first spiked mixture.

[0265] S1024. Add 200 μL of sunscreen mixture and 10 μL of isotope internal standard mixture to the matrix serum solution in tube 2, mix, and obtain the second spiked mixture.

[0266] S1025. Add 790 μL of acetonitrile as the extraction solvent to the first spiked mixed solution in EP tube No. 1, mix at 2000 rpm for 5 min, and perform liquid-liquid extraction at 4℃ for 1 h. Then centrifuge at 12000 rpm for 5 min, and transfer all the supernatant after centrifugation to obtain the first extraction solution.

[0267] S1026. Add 790 μL of ethyl acetate as the extraction solvent to the second spiked mixed solution in tube 2. Mix at 2000 rpm for 5 min, and perform liquid-liquid extraction at 4℃ for 1 h. Then centrifuge at 12000 rpm for 5 min. Transfer all the supernatant after centrifugation to obtain the second extraction solution.

[0268] S1027. Transfer the first extraction solution and the first extraction solution to a new clean No. 3 EP tube, mix them, vacuum dry, add 50 μL of methanol to reconstitute, centrifuge at 12000 rpm for 5 min, and take all the supernatant after centrifugation to obtain the blank matrix spiked solution, which is used as the sample for instrumentation.

[0269] S103. Take 15 μL of each sample and inject it into the liquid chromatography-tandem triple quadrupole mass spectrometer for processing. Perform quantitative analysis under predetermined detection conditions. The predetermined detection conditions include chromatographic condition A and mass spectrometry condition A; the liquid chromatography-tandem triple quadrupole mass spectrometer is manufactured by AB Sciex, model Qtrap 6500+, serial number LC-MS / MS-004.

[0270] The linear equations for each sunscreen agent tested are shown in Table 3. The retention time, correlation coefficient, linear range, detection limit, quantitation limit, and recovery rate corresponding to the linear equations for each sunscreen agent are also shown in Table 3.

[0271] Selective Validation

[0272] Experimental Example 1

[0273] Experimental methods

[0274] 1. Detect methanol using the following method:

[0275] Methanol was used as the sample (injection volume of 15 μL) and fed into a liquid chromatography-tandem triple quadrupole mass spectrometer. Analysis was performed under predetermined detection conditions, and chromatograms were measured and recorded. These predetermined detection conditions included chromatographic condition A and mass spectrometry condition A. The liquid chromatography-tandem triple quadrupole mass spectrometer was manufactured by AB Sciex, model Qtrap 6500+, serial number LC-MS / MS-004.

[0276] 2. Detect blank matrix serum according to the experimental methods in steps S201-202:

[0277] S201. Obtain the corresponding samples for use based on the 6 batches of blank matrix serum, resulting in 6 samples for use.

[0278] The specific steps for obtaining the on-machine samples for each batch in S201 are as follows:

[0279] Take 500 μL of blank matrix serum from the -80℃ freezer and thaw it to room temperature in steps;

[0280] The thawed blank matrix serum was vortexed at 2000 rpm for 5 min, then centrifuged at 12000 rpm for 5 min. After that, two 200 μL portions of the supernatant were precisely transferred to serve as matrix serum solutions. One portion was placed in a clean EP tube No. 1, and the other portion was placed in a clean EP tube No. 2.

[0281] Add 200 μL of sample 1 (i.e., the sunscreen mixed solution 1 prepared in Example 16) to the matrix serum solution in EP tube No. 1, and then add 10 μL of isotope internal standard mixed solution to obtain the corresponding first spiked mixed solution.

[0282] Add 200 μL of sample 2 (i.e., the sunscreen mixed solution 1 prepared in Example 16) to the matrix serum solution in EP tube No. 2, and then add 10 μL of isotope internal standard mixed solution to obtain the corresponding second spiked mixed solution;

[0283] Add 790 μL of acetonitrile as the extraction solvent to the first spiked mixed solution in EP tube No. 1, mix at 2000 rpm for 5 min, and perform liquid-liquid extraction at 4℃ for 1 h. Then centrifuge at 12000 rpm for 5 min, and transfer all the supernatant after centrifugation to obtain the first extraction solution.

[0284] Add 790 μL of ethyl acetate as the extraction solvent to the second spiked mixed solution in EP tube No. 2, mix at 2000 rpm for 5 min, and perform liquid-liquid extraction at 4℃ for 1 h. Then centrifuge at 12000 rpm for 5 min, and transfer all the supernatant after centrifugation to obtain the second extraction solution.

[0285] The first extraction solution and the first extraction solution were transferred to a new clean No. 3 EP tube and mixed. The mixture was then vacuum-dried, and 50 μL of methanol was added to redissolve the solution. The mixture was centrifuged at 12000 rpm for 5 min. After centrifugation, all the supernatant was collected to obtain the blank matrix spiked solution, which was used as the sample for instrumentation.

[0286] S202. Six samples were fed into the liquid chromatography-tandem triple quadrupole mass spectrometer (15 μL for each sample) and analyzed under predetermined detection conditions. Chromatograms were then measured and recorded. The predetermined detection conditions included chromatographic condition A and mass spectrometry condition A. The liquid chromatography-tandem triple quadrupole mass spectrometer was manufactured by AB Sciex, model Qtrap 6500+, serial number LC-MS / MS-004.

[0287] Experimental results

[0288] The experimental results show that no interfering peaks were observed in the chromatogram of the blank methanol solution, confirming that the methanol solvent itself does not produce interfering signals. This demonstrates that the method of this embodiment can specifically detect the target analyte; see also [link to relevant documentation]. Figure 1 .

[0289] The chromatograms of the spiked solutions from all six batches of blank serum matrix showed the target peaks. Furthermore, the ratios of the baseline peak response to the detected peak area response for the nine target analytes (nine sunscreen agents) within 0.5 min before and after the retention time in the six batches ranged from 0.27% to 12.53%, all within 20%. The three internal standards (octocrylene-d) in the six batches also showed positive results. 10 Ethylhexyl salicylate-d4, ethylhexyl methoxycinnamate- 13 The ratios of the baseline peak response to the detected peak area response within 0.5 min before and after the retention time of C3) ranged from 0.41% to 5.02%, all within 5%; the aforementioned results can also be found in [reference needed]. Figures 2-13 .

[0290] The above test results all meet the requirements, indicating that the method of this application embodiment can accurately and specifically detect sunscreen agents without being interfered with by other components in the sample.

[0291] Figure 1 A chromatogram of methanol as a blank sample; Figure 2 A chromatogram of DT; Figure 3 A chromatogram of BEMT; Figure 4 A chromatogram of IMN; Figure 5 A chromatogram of PSA; Figure 6 This is a chromatogram of OCT. Figure 7 A chromatogram of 2-ES; Figure 8 A chromatogram of DHB; Figure 9 A chromatogram of 2-EM; Figure 10 A chromatogram of ET; Figure 11 For OCT-d 10 A chromatogram; Figure 12 A chromatogram of 2-ES-d4; Figure 13 For 2-EM- 13 A chromatogram of C3;

[0292] Figures 1-13 In the figure, the horizontal axis represents retention time in minutes (min); the vertical axis represents signal intensity, which expresses the relative abundance of ions in the sample (sample used in the instrument). Intensity is reflected by the mass spectrometry response value cps (counts per second), which is the number of ions detected per second, reflecting the abundance of ions.

[0293] <Evaluation of detection limit (DL) and quantitation limit (QL)>

[0294] Experimental Example 2

[0295] Experimental methods

[0296] The experimental method is as follows:

[0297] 1. Prepare the detection limit solution sample

[0298] The blank matrix spiked solution was obtained according to steps S1021 to S1027 above; wherein, the sunscreen mixture solution was selected from sunscreen mixture solution 1 in Example 16, wherein the concentrations of OCT, 2-ES and 2-EM were 2 ng / mL, and the concentrations of the other 6 sunscreen reference standards were 0.2 ng / mL;

[0299] The resulting blank matrix spiked solution is the limit of detection solution sample. Six limit of detection solution samples were prepared using the same method.

[0300] 2. Prepare a sample solution with a specified limit.

[0301] There are two types of limit-of-quantitation (LOQ) solution samples: a first blank matrix spiked solution and a second blank matrix spiked solution. The specific preparation methods are as follows:

[0302] The first blank matrix spiking solution and the second blank matrix spiking solution were obtained according to steps S1021 to S1027 above. In the preparation of the first blank matrix spiking solution, the sunscreen mixed solution was selected from sunscreen mixed solution 2 in Example 16, wherein the concentrations of OCT, 2-ES and 2-EM were 5 ng / mL, and the concentrations of the other 6 sunscreen reference standards were 0.5 ng / mL. In the preparation of the second blank matrix spiking solution, the sunscreen mixed solution was selected from sunscreen mixed solution 4 in Example 16, wherein the concentrations of OCT, 2-ES and 2-EM were 20 ng / mL, and the concentrations of the other 6 sunscreen reference standards were 2 ng / mL.

[0303] Following the above method, prepare six replicates of each limit-of-quantitation solution sample.

[0304] 3. Sample detection and analysis

[0305] The limit of detection (LOD) and limit of quantitation (LOQ) solutions were used as the sample inputs to the liquid chromatography-tandem triple quadrupole mass spectrometer (LC-MS / MS-004). Analysis was performed under predetermined detection conditions, and chromatograms were measured and recorded. The predetermined detection conditions included chromatographic condition A and mass spectrometry condition A. The LC-MS / MS-004 was manufactured by AB Sciex, model Qtrap 6500+.

[0306] It should be noted that:

[0307] The matrix mixture solution was used as the limit of quantitation solution sample and measured under the instrument operating conditions described above. S / N is the ratio of the chromatographic peak response value to the baseline signal-to-noise ratio.

[0308] The detection limit is the lowest concentration of the target analyte in a sample that can be detected, and the S / N ratio at this concentration level should not be less than 3. The formula for calculating the detection limit concentration is as follows:

[0309] C DL =C Low ×(3 / (S / N) Low )

[0310] Among them, C DL To determine the detection limit concentration, C Low To determine the actual concentration of low-concentration samples in the detection limit experiment, (S / N) Low To determine the signal-to-noise ratio of low-concentration samples in the detection limit experiment.

[0311] The limit of quantitation (LOQ) is the lowest concentration of the target analyte in a sample that can be reliably quantified. At this concentration level, the signal-to-noise ratio (S / N) of the chromatographic peak response should be no less than 10. If the lowest concentration is lower than the lowest point of the standard curve, the concentration at the lowest point of the standard curve is used as the method LOQ. The formula for calculating the LOQ concentration is as follows:

[0312] C QL =C Low ×(10 / (S / N) Low )

[0313] Among them, C QL For the limit of quantitation concentration, C Low For low sample concentrations during limit of quantitation tests, (S / N) Low This represents the signal-to-noise ratio of low-concentration samples in the limit of quantitation experiment.

[0314] Experimental results

[0315] Method validation results for detection limits: When S / N ≥ 3, the detection limit for DT is 0.2 ng / mL, and the three-needle signal-to-noise ratio (S / N) at the detection limit level is 434.72–577.46; the detection limit for BEMT is 0.2 ng / mL, and the three-needle S / N at the detection limit level is 835.40–894.35; the detection limit for IMN is 0.2 ng / mL, and the three-needle S / N at the detection limit level is 90.09–108.04; the detection limit for PSA is 0.2 ng / mL, and the three-needle S / N at the detection limit level is 378.56–459.83; the detection limit for OCT is 2 ng / mL. The detection limits for the three-injection signal-to-noise ratio (S / N) were 34.92–53.79 at the detection limit level; the detection limit for 2-ES was 5 ng / mL, and the S / N for the three-injection signal-to-noise ratio (S / N) was 76.95–107.28 at the detection limit level; the detection limit for DHB was 0.2 ng / mL, and the S / N for the three-injection signal-to-noise ratio (S / N) was 687.87–740.03 at the detection limit level; the detection limit for 2-EM was 2 ng / mL, and the S / N for the three-injection signal-to-noise ratio (S / N) was 159.92–218.39 at the detection limit level; the detection limit for ET was 0.2 ng / mL, and the S / N for the three-injection signal-to-noise ratio (S / N) was 171.52–329.47 at the detection limit level; all met the requirements.

[0316] Method validation results for limits of quantitation: When S / N ≥ 10, the limit of quantitation for DT is 0.5 ng / mL, with a signal-to-noise ratio (SNR) of 908.56–1260.86 at the limit of quantitation level; the limit of quantitation for BEMT is 0.5 ng / mL, with an SNR of 1260.41–1423.24 at the limit of quantitation level; the limit of quantitation for IMN is 0.5 ng / mL, with an SNR of 65.60–92.56 at the limit of quantitation level; the limit of quantitation for PSA is 0.5 ng / mL, with an SNR of 305.85–544.29 at the limit of quantitation level; and the limit of quantitation for OCT is 5n. The signal-to-noise ratio (SNR) at the limit of quantitation (LOQ) level is 66.82–112.65 for g / mL; the LOQ for 2-ES is 20 ng / mL, with an SNR of 86.70–136.56 at the LOQ level; the LOQ for DHB is 0.5 ng / mL, with an SNR of 1176.28–1822.74 at the LOQ level; the LOQ for 2-EM is 5 ng / mL, with an SNR of 169.83–226.68 at the LOQ level; and the LOQ for ET is 0.5 ng / mL, with an SNR of 214.62–756.8 at the LOQ level; all meet the requirements.

[0317] As explained above, the method provided in this application has advantages such as low detection limit and low quantitation limit.

[0318] <Detection of the content of multiple sunscreen agents>

[0319] Experimental Example 3

[0320] Sunscreen B actually contains the following sunscreen ingredients: cresoltrazol trisiloxane (DT), bis(ethylhexylphenol) methoxyphenyl triazine (BEMT), isoamyl p-methoxycinnamate (IMN), octocrylene (OCT), ethylhexyl salicylate (2-ES), diethylamino hydroxybenzoyl hexyl benzoate (DHB), and ethylhexyl triazine ketone (ET).

[0321] The method provided in the embodiments of this application will be applied to monitoring. The residual amount of sunscreen ingredient B in human blood. All subjects signed informed consent forms after understanding the purpose, procedure and potential risks of sample collection. This study was approved by the Medical Ethics Committee of the University of Hong Kong-Shenzhen Hospital (Approval No.: Lun

[2024] 154), Clinical Trial Registration No.: HKUSZH-CONTRACT-RTD-RH2024001.

[0322] Specifically, based on the following evaluation indicators, 10 volunteers were selected, their blood was collected and processed through centrifugation to obtain serum for testing, and the residual amount of sunscreen ingredients in human blood was detected.

[0323] (1) Key endpoint evaluation indicators for 10 volunteers

[0324] Referring to the "Cosmetic Safety Technical Specifications (2022 Edition)," blood samples were collected from 10 volunteers 10 times, resulting in a total of 100 blood samples. The detection of sunscreen substances was used as the primary endpoint evaluation indicator.

[0325] Baseline: The first blood sample taken 0 hours before the volunteer used the sunscreen product was used as the baseline.

[0326] Measurement variables: Blood samples collected from subjects on days 7 from day 2 to day 8 were used as measurement variables.

[0327] Final values: The 9th and 10th blood samples collected from the subjects were used as the final values.

[0328] Valid volunteers: Blood samples from valid volunteers who did not experience adverse reactions during the use of sunscreen products were considered valid subjects.

[0329] (2) Secondary evaluation indicators for 10 volunteers

[0330] No adverse reactions such as allergies to cosmetics were observed among the volunteers during the use of sunscreen products.

[0331] More specific testing methods are as follows:

[0332] 1. Obtain the serum to be tested

[0333] 1.1 Obtaining volunteers to provide serum for testing

[0334] Volunteers were selected according to the following criteria:

[0335] (1) Inclusion and exclusion criteria and application requirements: The test subjects were 10 people aged 18-60 years, with no skin diseases such as allergies or acne; the subjects were required not to use any sunscreen products one week before the visit.

[0336] (2) Application procedure: Apply sunscreen continuously to the face, neck, hands, and arms, covering approximately 25% of the total body surface area; apply once daily in the morning, using an amount of 2 mg / cm³. 2 .

[0337] (3) Requirements for blood collection from volunteers and method for marking blood samples: The serum sample number is BXXYY, where BXX represents the volunteer's serial number and YY represents the serial number of each volunteer's serum sample. For example, 10 volunteers are numbered B01 to B10, and blood is collected 10 times as required. The blood samples of each volunteer are numbered in the following order: Volunteer No. 1 is marked as B01, and the 10 blood samples are marked as B0101, B0102, B0103...B0110 respectively; Volunteer No. 2 is marked as B02, and the 10 blood samples are marked as B0201, B0202, B0203...B0210 respectively, and so on.

[0338] 1.2 Blood Sample Collection

[0339] Collect blood samples according to the following requirements:

[0340] Day 1: Visit in the morning, first blood collection 0 hours after application; sunscreen is applied on site, second blood collection 2 hours after sunscreen application, and third blood collection 6 hours after application.

[0341] Day 2: Apply sunscreen in the morning, and have your blood drawn for the fourth time 2 hours later;

[0342] Day 3: Apply sunscreen in the morning, and have your fifth blood draw 2 hours later;

[0343] Day 4: Apply sunscreen in the morning, and have your blood drawn for the sixth time 2 hours later;

[0344] Day 5: Apply sunscreen in the morning;

[0345] Day 6: Apply sunscreen in the morning;

[0346] Day 7: Apply sunscreen in the morning, and have your blood drawn for the seventh time 2 hours later;

[0347] Day 8: Do not use sunscreen, go directly to the site for the eighth blood draw;

[0348] Day 9: Do not use sunscreen, go directly to the site for the ninth blood draw;

[0349] Stop using sunscreen on the tenth and eleventh days;

[0350] Day 12: No sunscreen used, tenth blood draw.

[0351] Blood samples were collected on days 1, 2, 3, 4, 7, 8, 9, and 12. The blood was processed by centrifugation to obtain serum, which was stored at -80°C and used as test serum to detect the residual amount of sunscreen in the serum.

[0352] 2. Test the blood sample.

[0353] The serum samples obtained above were tested according to the following method:

[0354] After removing 500 μL of the serum to be tested from the -80℃ freezer, thaw it step by step to room temperature; then, vortex at 2000 rpm for 5 min, and centrifuge at 12000 rpm for 5 min. After that, accurately transfer 400 μL of the supernatant as the serum supernatant and divide it into two equal portions (200 μL each). The first portion of the serum supernatant was placed in a clean tube No. 1, and the second portion of the serum supernatant was placed in an EP tube No. 2.

[0355] Add 10 μL of isotope internal standard mixed solution to the serum supernatant in tube 1 to obtain the first spiked serum solution;

[0356] Add 10 μL of isotope internal standard mixed solution to the serum supernatant in tube 2 to obtain the second spiked serum solution;

[0357] Add 990 μL of acetonitrile as the extraction solvent to the first spiked serum solution in EP tube No. 1, mix at 2000 rpm for 5 min, and perform liquid-liquid extraction at 4℃ for 1 h. Then centrifuge at 12000 rpm for 5 min, and transfer all the supernatant after centrifugation to obtain the first extraction solution.

[0358] Add 990 μL of ethyl acetate as the extraction solvent to the second spiked serum solution in EP tube No. 2, mix at 2000 rpm for 5 min, and extract at 4℃ for 1 h. Then centrifuge at 12000 rpm for 5 min, and transfer all the supernatant after centrifugation to obtain the second extraction solution.

[0359] The first extraction solution and the first extraction solution were transferred to a new clean No. 3 EP tube and mixed. The mixture was then vacuum-dried, and 50 μL of methanol was added to reconstitute the solution. The mixture was centrifuged at 12000 rpm for 5 min. The supernatant was collected after centrifugation to obtain the sample to be tested, which was used as the sample for instrumentation. 15 μL of the sample was injected into a liquid chromatography-tandem triple quadrupole mass spectrometer for instrumentation. Chromatographic and mass spectrometric analysis was performed under predetermined detection conditions to obtain the peak area data corresponding to each sunscreen agent in the serum sample. The predetermined detection conditions included chromatographic condition A and mass spectrometric condition A. The liquid chromatography-tandem triple quadrupole mass spectrometer was manufactured by AB Sciex, model Qtrap 6500+.

[0360] The concentration of each sunscreen agent in the serum sample was obtained based on the peak area data and corresponding linear equations. Specifically, the concentration of sunscreen agents in the serum sample was quantitatively calculated based on the linear equations of the standard curves described in Table 3.

[0361] The specific quantitative method is the internal standard method to quantify the concentration of the target substance in the serum to be tested, calculated according to the following formula.

[0362]

[0363] in,

[0364] C SPL : The concentration of the target substance (i.e., sunscreen agent) in the serum to be tested;

[0365] A SPL : The ratio of the peak area of ​​the target ion to the peak area of ​​the isotopic internal standard ion in the serum to be tested;

[0366] k: The slope in the linear equation;

[0367] b: The intercept in the linear equation.

[0368] Understandably, a linear equation can generally be expressed as y = kx + b, where y is the peak area ratio of the target ion to the isotopic internal standard ion, x is the concentration, k is the slope, and b is the intercept. The slope and intercept are definite constants that can be obtained from the linear equation.

[0369] 3. Experimental Results

[0370] The specific test results are shown in Table 5.

[0371] Table 5. Calculation results of linear equations for sunscreen components in 100 serum samples.

[0372]

[0373]

[0374]

[0375] In the table, "<" means less than.

[0376] The modified z-score (Median Absolute Deviation, MAD) method and principal component analysis (PCA) were used to remove outliers and perform data analysis on the data in Table 5. The results are shown in Table 6.

[0377] (*Modified zscore method (Median Absolute)) Deviation (MAD) is a median-based outlier detection method that uses the median and absolute median difference of data to determine whether the data is an outlier. The outlier screening in this project includes the following steps: (1) The corrected zscore (mad_z) is greater than 2.5; (2) The detection value is greater than the detection limit; (3) Handling method: Outliers are those whose detection results are far beyond the standard value and whose corrected zscore (mad_z) value is greater than 2.5. Analyzing the occasional occurrence of outliers in one or several items in 10 tests of the same volunteer, it is considered that the sample may be contaminated, or that the volunteer applied other cosmetics or came into contact with other chemical agents on the day of the test, which is considered to have no clinical significance. (4) Further principal component analysis (PCA) dimensionality reduction analysis was performed on the longitudinal multi-index data of the sample group testers. First, each tester was measured at 10 time points from the data dimension, and each time point contained 8 indicators, so each tester had a total of 80 features (10×8). The 80 features were reduced in dimensionality by PCA to find outliers.

[0378] Table 6. Detection results of 10 volunteers after outlier removal.

[0379]

[0380]

[0381]

[0382]

[0383] In Table 6, NA indicates that the detection concentration of the target analyte does not exceed the corresponding method detection limit.

[0384] The results of principal component analysis (PCA) of the 10 subjects are as follows: Figure 14 As shown. Figure 14In the diagram, the horizontal axis PC1 represents the first principal component and its corresponding contribution rate; the first principal component has the highest contribution rate. The vertical axis PC2 represents the second principal component and its corresponding contribution rate; the second principal component has the second highest contribution rate. It should be noted that the contribution rate is the proportion of the variance of the corresponding principal component in the overall variance; a higher proportion indicates a higher weight in the overall data.

[0385] The experimental results showed that test subject B03 significantly deviated from other individuals in the PCA space, exhibiting a clear outlier phenomenon, suggesting a possible anomaly in the sample. Therefore, it is considered that the sample may have been contaminated, or that the volunteer may have applied other cosmetics or been exposed to other chemical agents on the day of testing, which is deemed clinically insignificant.

[0386] The experimental results showed that the detection results of cresoltrazol trisiloxane (DT), bis(ethylhexylphenol) methoxyphenyl triazine (BEMT), isoamyl p-methoxycinnamate (IMN), ethylhexyl salicylate (2-ES), diethylaminohydroxybenzoylhexyl benzoate (DHB), and ethylhexyl triazine ketone (ET) were all <0.5 ng / mL; the results of octocrylene (OCT) were all "NA", which did not exceed the detection limit (5 ng / mL).

[0387] In addition, the FDA recommends a plasma concentration threshold of 0.5 ng / mL for sunscreens. In 100 serum samples, the mean values ​​of seven sunscreen ingredients—cresoltrazol trisiloxane (DT), bis(ethylhexylphenol) methoxyphenyl triazine (BEMT), isoamyl p-methoxycinnamate (IMN), ethylhexyl salicylate (2-ES), diethylamino hydroxybenzoyl hexyl benzoate (DHB), and ethylhexyl triazine ketone (ET)—were all within the method detection limit and less than 0.5 ng / mL, which complies with the FDA's recommendations regarding the amount of sunscreen entering the bloodstream. Octocrylene (OCT) may have a small amount of residue entering the bloodstream, but this did not exceed the method detection limit (5 ng / mL).

[0388] The above experiments demonstrate that the method provided in the embodiments of this application can be used to detect various sunscreen ingredients in blood.

[0389] The above detailed embodiments have described the present invention in detail, but these are not intended to limit the invention. The scope of protection of the present invention is not limited to the above embodiments; any equivalent modifications or variations made by those skilled in the art based on the disclosure of the present invention should be included within the scope of protection set forth in the claims.

Claims

1. A method for determining a plurality of sunscreens in serum, characterized in that, The method comprises the following steps: obtaining a test sample from the test serum; performing chromatographic and mass spectrometric analysis on the test sample under predetermined detection conditions to obtain peak area data corresponding to the plurality of sunscreen agents; obtaining the concentrations of the plurality of sunscreen agents in the test serum according to the linear equations of the sunscreen agents and the peak area data; the plurality of sunscreen agents include at least one of methylphenyl triazone and ethylhexyl triazone.

2. The method for determining a plurality of sunscreen agents in serum according to claim 1, wherein: the test sample is obtained from the test serum by the following steps: obtaining a spiked serum solution from the test serum and an isotopic internal standard solution; extracting the spiked serum solution with acetonitrile to obtain a first extraction solution; extracting the spiked serum solution with ethyl acetate to obtain a second extraction solution; mixing the first extraction solution and the second extraction solution, drying, and then redissolving with methanol to obtain the test sample.

3. The method for determining a plurality of sunscreen agents in serum according to claim 1, wherein: the spiked serum solution is obtained from the test serum and the isotopic internal standard solution by the following steps: centrifuging the test serum to obtain a supernatant as a serum supernatant; mixing the serum supernatant and the isotopic internal standard mixed solution to obtain the spiked serum solution.

4. The method for determining a plurality of sunscreen agents in serum according to claim 1, wherein: the plurality of sunscreen agents include methylphenyl triazone and ethylhexyl triazone.

5. The method for determining a plurality of sunscreen agents in serum according to claim 4, wherein: the plurality of sunscreen agents further include one or more of bis-ethylhexyl phenol methoxyphenyl triazine, isoamyl p-methoxycinnamate, ethylhexyl salicylate, and hexyl diaminohydroxybenzo-glycolate.

6. The method for determining a plurality of sunscreen agents in serum according to claim 4, wherein: the plurality of sunscreen agents further include one or more of phenyl benzimidazole sulfonic acid, octocrylene, ethylhexyl methoxy cinnamate, octocrylene, ethylhexyl salicylate, and ethylhexyl methoxy cinnamate.

7. The method for determining a plurality of sunscreen agents in serum according to claim 6, wherein: the peak area data corresponding to the plurality of sunscreen agents are obtained by performing chromatographic and mass spectrometric analysis on the test sample under predetermined detection conditions, which comprises: injecting the test sample into a chromatographic analysis instrument to perform chromatographic and mass spectrometric analysis and obtain the peak area data corresponding to the plurality of sunscreen agents; wherein the chromatographic analysis instrument is a liquid chromatograph tandem mass spectrometer.

8. The method for determining a plurality of sunscreen agents in serum according to claim 7, wherein: the liquid chromatograph tandem mass spectrometer is a liquid chromatograph tandem triple quadrupole mass spectrometer.

9. The method for determining a plurality of sunscreen agents in serum according to claim 1, characterized by, The method further comprises the following steps: obtaining linear equations of the plurality of sunscreen agents.

10. The method for determining a plurality of sunscreen agents in serum according to claim 9, characterized by, The step of obtaining linear equations of the plurality of sunscreen agents comprises: obtaining a plurality of sunscreen agent mixed solutions with different concentrations, each of which contains the plurality of sunscreen agents; obtaining corresponding samples for analysis from each concentration of the sunscreen agent mixed solution and the corresponding blank matrix serum and isotopic internal standard solution; performing chromatographic and mass spectrometric analysis on the plurality of samples for analysis under predetermined detection conditions to obtain linear equations corresponding to the plurality of sunscreen agents.

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