Analysis method for detecting organic ultraviolet absorbent and metabolites thereof in urine

Abstract

The invention relates to the field of environmental chemistry and biological sample analysis, in particular to an analysis method for detecting an organic ultraviolet absorbent and metabolites thereof in urine. The method comprises the following steps: carrying out enzymolysis on a urine sample, adding an internal standard substance, extracting and purifying 24 organic ultraviolet absorbents and metabolites thereof in the sample in an extraction manner, and detecting the content of a target substance in the urine sample through a high performance liquid chromatography-mass spectrometer. The method is easy to operate and high in accuracy and reliability, eight types of 24 organic ultraviolet absorbents and metabolites thereof can be detected at the same time at a time, part of substances are reported for the first time, and the method has wide practical application value and significance for evaluating the residual conditions of the organic ultraviolet absorbents and metabolites thereof in the human body.

Description

Technical Field

[0001] The invention relates to the field of environmental chemistry and biological sample analysis, and in particular to an analytical method for detecting an organic ultraviolet absorber and its metabolites in urine. Background Art

[0002] Organic UV absorbers are a class of compounds that can absorb ultraviolet (UV) rays and are widely used in cosmetics, plastics, coatings, textiles and other fields to achieve sun protection and anti-UV aging functions. In addition, organic UV absorbers also have important applications in the industrial field, such as being added as functional additives in plastics, paints and textiles to delay the photodegradation of plastic products and the aging of polymer materials.

[0003] Organic UV absorbers have a conjugated double bond system or an aromatic system in their chemical structure, which can effectively absorb ultraviolet rays of a specific wavelength. However, with the expansion of the consumer market and the diversification of their uses, the types and complexity of organic UV absorbers are also increasing. Commonly used organic UV absorbers can be divided into several categories according to their chemical structure, mainly including p-aminobenzoic acid esters, benzophenones, camphor derivatives, cinnamic acid esters, dibenzoylmethanes, salicylates, triazinones and benzotriazoles. According to surveys, the global annual production of organic UV absorbers is about 10,000 tons.

[0004] The human body is exposed to these UV absorbers through various channels, including skin contact, inhalation, diet, etc. These substances circulate in the blood, are metabolized by the liver, and are excreted in the urine. Therefore, organic UV absorbers and their metabolites can be detected in urine samples, blood, emulsions, and other body fluids. However, exposure to organic UV absorbers may cause endocrine disruption, immune system disorders, skin irritation, and allergic reactions in the human body. This has caused concerns about the safety of organic UV absorbers.

[0005] Using organic UV absorbers as biological exposure markers and measuring their concentrations will help us gain a deeper understanding of the degree of human exposure to such substances and further evaluate their possible health effects. There are many types of organic UV absorbers, among which the basic structure of cinnamate UV absorbers (EHMC) is methoxycinnamate (methoxy and ester groups), and its metabolites include methoxycinnamic acid (methoxy and carboxyl groups) and methoxyacetophenone (methoxy and methyl ketone groups); camphor derivative UV absorbers (4-MBC and 3-BC) are mainly composed of camphor and benzene rings; aminobenzoic acid and its derivatives (DHHB) contain amino and ester functional groups in their structures; benzophenones (BP-1, BP-2, BP-3) are also used. 2, BP-3, BP-8 and 4-OHBP) are composed of two benzene rings and a carbonyl functional group; the dibenzoylmethane (AVO) structure contains two benzene rings and a methyl functional group; the salicylate (EHS and HMS) molecule contains a phenolic hydroxyl group and an ester functional group; the triazinones (BEMT and DBT) have a triazine ring and a ketone functional group; the benzotriazoles (UV-P, UV-9, UV-326, UV-328, UV-329 and UV-360) contain a benzene ring and a triazole ring functional group. Since these substances differ greatly in structure, it is very challenging to detect and analyze them simultaneously. Therefore, how to develop an analytical method with high accuracy, good stability and the ability to simultaneously detect the above 8 types of organic UV absorbers and their metabolites has become a problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and provide an analytical method for detecting organic ultraviolet absorbers and their metabolites in urine. The method can simultaneously detect 24 organic ultraviolet absorbers and their metabolites, and significantly improves the absolute recovery rate of each substance, with excellent accuracy and reliability.

[0007] In order to achieve the above-mentioned object, the present invention provides an analytical method for detecting organic ultraviolet absorbers and their metabolites in urine, which can simultaneously detect 24 organic ultraviolet absorbers and their metabolites, including EHMC, 4-MCA, 4'-MAP, 4-MBC, OC, 3-BC, AVO, BEMT, HMS, EHS, CDAA, DHHB, UV-P, UV-9, UV-326, UV-329, UV-360, UV-328, BP-1, BP-2, BP-3, BP-8, 4-OHBP and DBT, and comprises the following steps:

[0008] (1) preparing a mixed standard stock solution, a mixed internal standard stock solution and a mixed internal standard working solution, and establishing a standard curve for the above 24 organic ultraviolet absorbers and their metabolites;

[0009] (2) Sample pretreatment: adding an acidic solution, β-glucuronidase and the mixed internal standard working solution to the urine sample, performing enzymatic hydrolysis at 35-50° C. in a light-proof environment for at least 2 h to obtain an enzymatically hydrolyzed urine sample;

[0010] (3) Extraction treatment: subjecting the enzymatically hydrolyzed urine sample to at least two extraction treatments, combining the obtained organic phases, drying them, and then redissolving them to obtain the sample to be tested;

[0011] (4) LC-MS / MS detection: performing LC-MS / MS detection on the sample to be tested, and quantifying the organic ultraviolet absorber and its metabolites in the sample to be tested according to the standard curve;

[0012] The LC-MS / MS detection conditions include: using Waters Acquity BEH C 18 Chromatographic column; using ammonia solution with a volume fraction of 0.05-0.2% as mobile phase A, and methanol as mobile phase B; the full injection time is at least 25 minutes; the elution gradient is: 0-2 minutes, 70% A, 30% B; 2-5 minutes, 70% A-40% A, 30% B-60% B; 5-5.1 minutes, 40% A-20% A, 60% B-80% B; 5.1-10 minutes, 20% A, 80% B; 10-10.1 minutes, 20% A-0% A, 80% B-100% B; 10.1-22 minutes, 0% A, 100% B; 22-22.01 minutes, 0% A-70% A, 100% B-30% B; 22.01-end of elution, 70% A, 30% B.

[0013] The present invention provides an analytical method for organic ultraviolet absorbers and their metabolites based on liquid chromatography tandem mass spectrometry. The method achieves purification and enrichment of urine samples by performing enzymatic hydrolysis and extraction under specific reagents and conditions, significantly reduces matrix effects, improves the absolute recovery rate of the substance to be tested, and combines specific LC-MS / MS parameter conditions to achieve one-time simultaneous detection of 8 categories of 24 organic ultraviolet absorbers and their metabolites. The operation is simple and efficient, and some substances are reported for the first time for their human urine detection methods. The analytical method provided by the present invention has high accuracy and reliability, and has a wide range of practical application value and significance for evaluating the residual conditions of organic ultraviolet absorbers and their metabolites in the human body. DETAILED DESCRIPTION

[0014] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0015] The present invention provides an analytical method for detecting organic ultraviolet absorbers and metabolites thereof in urine. The method can simultaneously detect 24 organic ultraviolet absorbers and metabolites thereof, including EHMC, 4-MCA, 4'-MAP, 4-MBC, OC, 3-BC, AVO, BEMT, HMS, EHS, CDAA, DHHB, UV-P, UV-9, UV-326, UV-329, UV-360, UV-328, BP-1, BP-2, BP-3, BP-8, 4-OHBP and DBT, and comprises the following steps:

[0016] (1) preparing a mixed standard stock solution, a mixed internal standard stock solution and a mixed internal standard working solution, and establishing a standard curve for the above 24 organic ultraviolet absorbers and their metabolites;

[0017] (2) Sample pretreatment: adding an acidic solution, β-glucuronidase and the mixed internal standard working solution to the urine sample, performing enzymatic hydrolysis at 35-50° C. in a light-proof environment for at least 2 h to obtain an enzymatically hydrolyzed urine sample;

[0018] (3) Extraction treatment: subjecting the enzymatically hydrolyzed urine sample to at least two extraction treatments, combining the obtained organic phases, drying them, and then redissolving them to obtain the sample to be tested;

[0019] (4) LC-MS / MS detection: performing LC-MS / MS detection on the sample to be tested, and quantifying the organic ultraviolet absorber and its metabolites in the sample to be tested according to the standard curve;

[0020] The LC-MS / MS detection conditions include: using Waters Acquity BEH C 18Chromatographic column; using ammonia solution with a volume fraction of 0.05-0.2% as mobile phase A, and methanol as mobile phase B; the full injection time is at least 25 minutes; the elution gradient is: 0-2 minutes, 70% A, 30% B; 2-5 minutes, 70% A-40% A, 30% B-60% B; 5-5.1 minutes, 40% A-20% A, 60% B-80% B; 5.1-10 minutes, 20% A, 80% B; 10-10.1 minutes, 20% A-0% A, 80% B-100% B; 10.1-22 minutes, 0% A, 100% B; 22-22.01 minutes, 0% A-70% A, 100% B-30% B; 22.01-end of elution, 70% A, 30% B.

[0021] According to the present invention, in step (1), before the urine sample is pre-treated and LC-MS / MS detected, it is necessary to prepare a mixed standard stock solution, a mixed internal standard stock solution and a mixed internal standard working solution to facilitate the subsequent steps and obtain better analysis results.

[0022] According to the present invention, the mixed standard stock solution includes 24 organic ultraviolet absorbers and their metabolites, including EHMC, 4-MCA, 4'-MAP, 4-MBC, OC, 3-BC, AVO, BEMT, HMS, EHS, CDAA, DHHB, UV-P, UV-9, UV-326, UV-329, UV-360, UV-328, BP-1, BP-2, BP-3, BP-8, 4-OHBP and DBT.

[0023] Among them, EHMC is ethylhexyl methoxycinnamate; 4-MCA is 4-methoxycinnamic acid; 4'-MAP is 4'-methoxyacetophenone; 4-MBC is 4-methylbenzylidene camphor; OC is octocrylene; 3-BC is 3-benzylidene camphor; AVO is avobenzone; BEMT is bis-ethylhexyloxyphenol methoxyphenyl triazine; HMS is homosalate; EHS is 2-ethylhexyl salicylate; CDAA is 2-cyano-3,3-diphenylacrylic acid; DHHB is diethylaminohydroxybenzoyl hexyl benzoate; UV-P is 2-(2'-hydroxy-5'-methylphenyl)benzotriazole; UV-9 is 2-hydroxy-4-methoxydiphenyl Ketone; UV-326 is 2'-(2'-hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole; UV-329 is oxotriazole; UV-360 is methylene bis-benzotriazolyl tetramethylbutylphenol; UV-328 is 2-(2H-benzotriazol-2-yl)-4,6-di-tert-amylphenol; BP-1 is 2,4-dihydroxybenzophenone; BP-2 is 2,2,4,4-tetrahydroxybenzophenone; BP-3 is 4-methoxy-2-hydroxybenzophenone; BP-8 is 2,2'-hydroxy-4-methoxybenzophenone; 4-OHBP is 4-hydroxy-benzophenone; DBT is diethylhexyl butyramido triazone.

[0024] According to the present invention, the mixed internal standard stock solution and the mixed internal standard working solution include 4-MBC-d4, EHS-d4, HMS-d4, EHMC-d3, OC- 13 C3, UV-329- 13 C6, UV-P-d3, AVO- 13 C,d3,UV-328-d 12 There are 15 internal standard compounds including UV-326-d3, BP-1-d5, BP-2-d4, BP-3-d3, BP-8-d3 and 4-OHBP-d4.

[0025] Among them, 4-MBC-d4 is 4-methylbenzylidene camphor-d4; EHS-d4 is salicylic acid-2-ethylhexyl ester-d4; HMS-d4 is homosalate-d4; EHMC-d3 is methoxycinnamic acid ethylhexyl ester-d3; OC- 13 C3 is octocrylene- 13 C3; UV-329- 13 C6 is oxadiazole- 13 C6; UV-P-d3 is 2-(2'-hydroxy-5'-methylphenyl)benzotriazole-d3; AVO- 13 C,d3 is avobenzone- 13 C,d3;UV-328-d 122-(2H-benzotriazole-2-yl)-4,6-di-tert-amylphenol-d 12 ; UV-326-d3 is 2'-(2'-hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole-d3; BP-1-d5 is 2,4-dihydroxybenzophenone-d5; BP-2-d4 is 2,2,4,4-tetrahydroxybenzophenone-d4; BP-3-d3 is 4-methoxy-2-hydroxybenzophenone-d3; BP-8-d3 is 2,2'-hydroxy-4-methoxybenzophenone-d3; 4-OHBP-d4 is 4-hydroxy-benzophenone-d4.

[0026] According to the present invention, in order to obtain better analysis results, an analysis system is established. Preferably, the concentration of each organic ultraviolet absorber and its metabolite in the mixed standard stock solution is 1-10 μg / mL, preferably 1-2 μg / mL, for example, it can be 1 μg / mL, 1.5 μg / mL, 1.8 μg / mL and 2 μg / mL and any value thereof.

[0027] Preferably, the concentration of each internal standard compound in the mixed internal standard stock solution is 0.5-3 μg / mL, preferably 1-2 μg / mL, for example, it can be 1 μg / mL, 1.5 μg / mL, 1.8 μg / mL and 2 μg / mL and any value in between.

[0028] Preferably, the concentration of each internal standard compound in the mixed internal standard working solution is 25-150 ng / mL, preferably 50-100 ng / mL, for example, it can be 50 ng / mL, 60 ng / mL, 80 ng / mL and 100 ng / mL and any value therebetween.

[0029] Preferably, the solvent of the mixed standard stock solution, the mixed internal standard stock solution and the mixed internal standard working solution is methanol.

[0030] According to the present invention, the mixed standard stock solution, the mixed internal standard stock solution and the mixed internal standard working solution can be obtained by conventional preparation methods in the art, for example, they can be prepared by the following method:

[0031] Mixed standard stock solution: Weigh a single compound into a single volumetric flask, dilute to volume with methanol, and obtain 24 single-standard stock solutions of organic UV absorbers and their metabolites in this way. Transfer the same volume of 24 single-standard stock solutions to a volumetric flask, dilute to volume with methanol, and obtain the mixed standard stock solution.

[0032] Mixed internal standard stock solution: Weigh a single internal standard compound into a single volumetric flask, dilute to volume with methanol, and obtain single standard stock solutions of 15 internal standard compounds in this way. Pipette the same volume of single standard stock solutions of 15 internal standard compounds into a volumetric flask, dilute to volume with methanol, and obtain the mixed internal standard stock solution.

[0033] Mixed internal standard working solution: when a mixed internal standard working solution is needed, the mixed internal standard stock solution is diluted with methanol to obtain the mixed internal standard working solution.

[0034] According to the present invention, in order to obtain a more reasonable standard curve, so as to better determine each organic ultraviolet absorber and its metabolite in the urine sample, preferably, the step of establishing the standard curve of the above 24 organic ultraviolet absorbers and their metabolites includes: diluting the mixed standard stock solution into a series of mixed standard working solutions with multiple different mass concentrations (such as more than 3 concentrations, for example, at least 3 concentration values ​​among 1ng / mL, 2.5ng / mL, 5ng / mL, 10ng / mL, 25ng / mL, 50ng / mL, 100ng / mL, 250ng / mL and 500ng / mL), each mixed standard working solution of different concentrations is mixed with the mixed internal standard working solution to a fixed volume, and LC-MS / MS detection is performed respectively, and the ratio of the peak area of ​​each organic ultraviolet absorber and its metabolite to the peak area of ​​each corresponding internal standard compound in the test result is used as the ordinate, and the detection concentration is used as the abscissa to obtain the standard curve.

[0035] More preferably, the step of establishing a standard curve of the above 24 organic ultraviolet absorbers and their metabolites comprises: diluting the mixed standard stock solution with methanol into a series of mixed standard working solutions with mass concentrations of 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL and 500 ng / mL, taking 50 μL of each mixed standard working solution, adding 150 μL of the mixed internal standard working solution, and then making up the volume to 500 μL with methanol, and performing LC-MS / MS detection respectively, using the ratio of the peak area of ​​each organic ultraviolet absorber and its metabolite in the test results to the peak area of ​​each corresponding internal standard compound as the ordinate, and using the detection concentrations of 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL as the abscissa to obtain the standard curve.

[0036] According to the present invention, in step (2), the specific pretreatment of the urine sample can make the obtained sample have more suitable properties, so as to ensure that the 24 organic ultraviolet absorbers and their metabolites in the sample can be fully recovered and separated from each other, thereby achieving better analysis results. Preferably, the acidic solution is selected from acetic acid-ammonium acetate buffer solution and / or acetic acid-sodium acetate buffer solution.

[0037] Preferably, the concentration of the acidic solution is 0.5-2mol / L, preferably 1-1.5mol / L, for example, it can be 1mol / L, 1.2mol / L, 1.4mol / L and 1.5mol / L and the range between any values ​​thereof. Preferably, the pH of the acidic solution is 4-6, preferably 4.5-5.5, for example, it can be 4.5, 4.8, 5 and 5.5 and the range between any values ​​thereof. Preferably, relative to 1mL of the urine sample, the amount of the acidic solution is 100-400μL, preferably 200-300μL, for example, it can be 200μL, 220μL, 250μL and 300μL and the range between any values ​​thereof.

[0038] According to the present invention, in the enzymatic treatment, the addition of β-glucuronidase can catalyze the hydrolysis reaction of glucuronic acid conjugates and generate corresponding free compounds. In order to obtain better enzymatic hydrolysis effect, preferably, the source of the β-glucuronidase is one or more of Roman snails, abalone, cattle liver and Escherichia coli, preferably Roman snails and / or abalone. Preferably, relative to 1mL of the urine sample, the dosage of the β-glucuronidase is 5-20μL, preferably 10-15μL, for example, it can be 10μL, 12μL, 14μL and 15μL and the range between any values ​​thereof. Preferably, the enzyme activity of the β-glucuronidase is 20-200kU / mL, preferably 50-150kU / mL, for example, it can be 50kU / mL, 85kU / mL, 100kU / mL and 150kU / mL and the range between any values ​​thereof.

[0039] According to the present invention, in the enzymatic hydrolysis treatment, the addition of a mixed internal standard working solution can compensate for individual differences in samples (different degrees of mass spectrometry signal suppression or enhancement caused by different urine sample matrices). In order to improve the reliability and accuracy of the final analysis results, preferably, relative to 1 mL of the urine sample, the amount of the mixed internal standard working solution is 100-200 μL, preferably 140-160 μL, for example, it can be 140 μL, 150 μL, 155 μL and 160 μL and the like and any range between the values ​​thereof.

[0040] According to the present invention, the enzymatic hydrolysis treatment needs to be carried out in a light-proof environment. Adjusting the temperature and time of the enzymatic hydrolysis can achieve better treatment effects. In order to improve the absolute recovery rate of each organic ultraviolet absorber and its metabolite, preferably, the temperature of the enzymatic hydrolysis is 40-45°C (for example, it can be 40°C, 42°C, 43°C and 45°C and the range between any values ​​thereof), and the time is 2-12h (for example, it can be 2h, 5h, 10h and 12h and the range between any values ​​thereof).

[0041] According to the present invention, in step (3), the enzymatically hydrolyzed urine sample obtained after the enzymatic hydrolysis treatment is subjected to an extraction treatment to further purify and enrich the urine sample, extract the organic ultraviolet absorber and its metabolites in the urine sample, and reduce the influence of other impurities such as salt and protein in the urine on the analysis results. In order to achieve a better extraction effect and improve the absolute recovery rate of each detection component, preferably, the extraction treatment includes adding an extraction solvent to the enzymatically hydrolyzed urine sample, and rotating the extractant at 2000-3000 rpm (for example, 2000 rpm, 2200 rpm, 2500 rpm and 3000 rpm, etc.). The mixture is mixed and shaken at 3000-4000 g (for example, it can be 3000 g, 3500 g, 3800 g, and 4000 g, and any range between them) for 10-30 min (for example, it can be 10 min, 15 min, 20 min, and 30 min, and any range between them), and then centrifuged at 3000-4000 g (for example, it can be 3000 g, 3500 g, 3800 g, and 4000 g, and any range between them) for 10-30 min (for example, it can be 10 min, 15 min, 20 min, and 30 min, and any range between them), to extract the organic phase.

[0042] Preferably, the extraction solvent is selected from one or more of ethyl acetate, dichloromethane, n-hexane and acetonitrile, preferably a n-hexane-dichloromethane mixed solvent and / or an ethyl acetate-acetonitrile mixed solvent, more preferably a n-hexane-dichloromethane mixed solvent with a volume ratio of 1:0.5-1.5 and / or an ethyl acetate-acetonitrile mixed solvent with a volume ratio of 1:0.5-1.5, for example, a n-hexane-dichloromethane mixed solvent with a volume ratio of 1:1, a n-hexane-dichloromethane mixed solvent with a volume ratio of 1:1.5, an ethyl acetate-acetonitrile mixed solvent with a volume ratio of 1:0.5 and an ethyl acetate-acetonitrile mixed solvent with a volume ratio of 1:1.

[0043] Preferably, relative to 1 mL of the enzymatically hydrolyzed urine sample, the amount of the extraction solvent used is 0.5-3 mL, preferably 1-2 mL, for example, it can be 1 mL, 1.5 mL, 1.8 mL, 2 mL, and any range therebetween.

[0044] According to the present invention, in order to obtain better analysis results, preferably, the re-dissolving solvent is selected from one or more of methanol, acetonitrile, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide, preferably methanol and / or acetonitrile.

[0045] Preferably, the gas used for the drying is an inactive gas, preferably nitrogen and / or argon.

[0046] Preferably, the volume of the sample to be tested is 100-300 μL, preferably 150-250 μL, for example, it can be 150 μL, 200 μL, 230 μL, 250 μL, and any value in between.

[0047] According to the present invention, in step (4), the sample to be tested is subjected to LC-MS / MS detection to perform qualitative and quantitative analysis on the organic ultraviolet absorber and its metabolites therein. The quantitative analysis is performed using the internal standard method, which can minimize the errors caused by the pretreatment process and instrument instability, thereby improving the accuracy of the analysis results.

[0048] According to the present invention, in order to obtain a faster detection speed and improve the stability and accuracy of the detection, preferably, the conditions of the LC-MS / MS detection also include: an injection volume of 3-6 μL, preferably 4-5 μL (for example, it can be 4 μL, 4.5 μL, 4.7 μL and 5 μL and the range between any values ​​thereof); a column temperature of 35-50°C, preferably 40-45°C (for example, it can be 40°C, 42°C, 43°C and 45°C and the range between any values ​​thereof); an injection temperature of 5-12°C, preferably 8-10°C, for example, it can be 8°C, 9°C, 9.5°C and 10°C and the range between any values ​​thereof); a mobile phase flow rate of 0.1-0.4 mL / min, preferably 0.2-0.3 mL / min (for example, it can be 0.2 mL / min, 0.25 mL / min, 0.28 mL / min in and 0.3mL / min and any range between them); the ion source is ESI (electrospray); the scanning mode is positive and negative ion scanning; the monitoring mode is MRM (mass spectrometry multiple reaction monitoring); the carrier gas is nitrogen, and the collision gas is helium; the capillary voltage is 2.3-2.6kV, preferably 2.4-2.5kV (for example, it can be 2.4kV, 2.46kV, 2.48kV and 2.5kV and any range between them); the ion source temperature is 120-180℃, preferably 140-160℃ (for example, it can be 140℃, 145℃, 150℃ and 160℃ and any range between them); the desolvation temperature is 400-600℃, preferably 450-550℃ (for example, it can be 450℃, 480℃, 500℃ and 530℃ and any range between them).

[0049] The present invention provides an analytical method for organic ultraviolet absorbers and their metabolites based on liquid chromatography tandem mass spectrometry. The method achieves purification and enrichment of urine samples by enzymatic hydrolysis and extraction under specific reagents and conditions, significantly reduces matrix effects, improves the absolute recovery rate of the substance to be tested, and achieves one-time simultaneous detection of 24 organic ultraviolet absorbers and their metabolites in 8 categories under specific LC-MS / MS parameter conditions. The operation is simple and efficient, and some substances are reported for the first time for their human urine detection methods. The analytical method provided by the present invention has high accuracy and reliability, and has a wide range of practical application value and significance for evaluating the residual conditions of organic ultraviolet absorbers and their metabolites in the human body.

[0050] The present invention will be described in detail below through examples.

[0051] In the following examples, unless otherwise specified, the devices used are all conventional experimental devices in the art, the experimental operations adopted are all conventional operations in the art, and the raw materials, reagents, etc. used can be obtained commercially. The instrument model for LC-MS / MS detection is XEVOTQXS.

[0052] Example 1

[0053] (I) Preparation of mixed standard stock solution, mixed internal standard stock solution and mixed internal standard working solution

[0054] Use methanol to prepare single standard stock solutions of EHMC, 4-MCA, 4'-MAP, 4-MBC, OC, 3-BC, AVO, BEMT, HMS, EHS, CDAA, DHHB, UV-P, UV-9, UV-326, UV-329, UV-360, UV-328, BP-1, BP-2, BP-3, BP-8, 4-OHBP and DBT with a concentration of 100 μg / L, and then transfer a certain amount of the above 24 single standard stock solutions to the same volumetric flask to prepare a mixed standard stock solution with a concentration of 1 μg / mL.

[0055] Prepare 4-MBC-d4, EHS-d4, HMS-d4, EHMC-d3, OC- 13 C3, UV-329- 13 C6, UV-P-d3, AVO- 13 C,d3,UV-328-d 12 , UV-326-d3, BP-1-d5, BP-2-d4, BP-3-d3, BP-8-d3 and 4-OHBP-d4 single standard stock solutions, and then transfer a certain amount of the above 15 single standard stock solutions to the same volumetric flask to prepare a mixed internal standard stock solution with a concentration of 1 μg / mL.

[0056] The mixed internal standard stock solution with a concentration of 1 μg / mL was diluted twenty times with methanol to obtain a mixed internal standard working solution with a concentration of 50 ng / mL.

[0057] (II) Establishment of standard curves for 24 organic UV absorbers and their metabolites

[0058] The mixed standard stock solution was diluted with methanol to a series of mixed standard working solutions with mass concentrations of 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL and 500 ng / mL, 50 μL of each mixed standard working solution was taken, and 150 μL of the mixed internal standard working solution was added, and then the volume was fixed to 500 μL with methanol, and LC-MS / MS detection was performed respectively, and the ratio of the peak area of ​​each organic ultraviolet absorber and its metabolite in the test result to the peak area of ​​each corresponding internal standard compound was used as the ordinate, and the detection concentrations of 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL were used as the abscissa to obtain the standard curve, and its linear range was calculated according to the standard curve. When the linear coefficient (R 2 )>0.999, indicating good linearity, quantitative calculation was performed accordingly. The linear correlation parameters of each organic ultraviolet absorber and its metabolite obtained are shown in Table 1.

[0059] Table 1

[0060]

[0061] As can be seen from Table 1, the detection limits of the 24 organic ultraviolet absorbers and their metabolites in the present invention are 0.001-0.009 ng / mL, the quantitative limits of the methods are 0.002-0.031 ng / mL, and the linear coefficients (R 2 ) are all higher than 0.999, showing a good linear relationship.

[0062] (III) Sample pretreatment

[0063] 500 μL of acetic acid-ammonium acetate buffer solution (concentration of 1 mol / L, pH = 5), 20 μL of β-glucuronidase (from Roman snail, enzyme activity of 85 kU / mL, purchased from Merck) and 300 μL of mixed internal standard working solution were added to 2 mL of urine sample. The obtained mixture was enzymatically hydrolyzed at 37°C in a dark environment for 4 hours to obtain an enzymatically hydrolyzed urine sample.

[0064] (IV) Extraction treatment

[0065] The enzymatic urine sample was subjected to two extraction treatments, the organic phases obtained were combined, dried with nitrogen, and re-dissolved with 200 μL of methanol to obtain the sample to be tested. The extraction process was as follows: 3 mL of ethyl acetate was added to the enzymatic urine sample, mixed and shaken at 2500 pm / min for 20 minutes, and then centrifuged at 3600 g centrifugal force for 20 minutes to extract the organic phase.

[0066] (V) LC-MS / MS detection

[0067] The samples to be tested are subjected to LC-MS / MS detection, and the organic ultraviolet absorbers and their metabolites in the samples to be tested are quantified according to the standard curve.

[0068] The conditions for LC-MS / MS detection included: using Waters Acquity BEH C 18 Chromatographic column; 0.1% ammonia aqueous solution as mobile phase A, methanol as mobile phase B; full injection time 25 min; elution gradient: 0-2 min, 70% A, 30% B; 2-5 min, 70% A-40% A, 30% B-60% B; 5-5.1 min, 40% A-20% A, 60% B-80% B; 5.1-10 min, 20% A, 80% B; 10-10.1 min, 20% A-0% A, 80% B-100% B; 10.1-2 2min, 0%A, 100%B; 22-22.01min, 0%A-70%A, 100%B-30%B; 22.01-25min, 70%A, 30%B; injection volume 5μL; column temperature 40℃; injection temperature 8℃; mobile phase flow rate 0.25mL / min; ion source is ESI; scanning mode is positive and negative ion scanning; monitoring mode is MRM; carrier gas is nitrogen, collision gas is helium; capillary voltage 2.46kV; ion source temperature is 150℃; desolvation temperature is 500℃.

[0069] The retention times, monitoring ions, cone voltages and collision energies of 24 organic UV absorbers and their metabolites and the corresponding 15 internal standard compounds are shown in Table 2.

[0070] Table 2

[0071]

[0072]

[0073] (VI) Evaluation of spike recovery and relative standard deviation

[0074] The urine samples from the same source were divided into 4 parts, 3 of which were added with appropriate amounts of mixed standard stock solution to adjust the spiked concentrations to 1 ng / mL, 5 ng / mL and 25 ng / mL, respectively. The four urine samples were processed and analyzed according to the methods of step (a) to step (v), and 6 parallel experiments were performed on each urine sample. The spiked recovery rate and relative standard deviation were calculated, where the spiked recovery rate (Recovery) = (spike sample measurement value - unspike sample measurement value) ÷ spiked amount × 100%; relative standard deviation (RSD) = deviation value of spiked recovery rate ÷ average value of spiked recovery rate × 100%. The results are shown in Table 3.

[0075] Table 3

[0076]

[0077]

[0078] As can be seen from Table 3, the average spiked recoveries of the 24 organic ultraviolet absorbers and their metabolites in the present invention are 70.3-130%, which is at a relatively high level; the relative standard deviation is <16.5%, which is at a relatively low level. It can be seen that the analytical method of the present invention has excellent accuracy and reliability.

[0079] 7. Matrix effect assessment

[0080] Five urine samples from different sources were selected and processed according to the method of step (i) to step (v), except that the organic phase obtained after extraction was used as the matrix to prepare urine matrix standard curves of 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, and 10 ng / mL, and methanol was used to prepare the methanol matrix standard curve of the same concentration. The matrix effect (ME) calculation formula is: ME = matrix calibration curve slope / solvent calibration curve slope × 100%, and the matrix effect is evaluated by the slope ratio of the urine matrix standard curve to the methanol matrix standard curve. When ME is 80%-120%, it is judged as a weak matrix effect; when ME is 50%-80% or 120%-150%, it is judged as a moderate matrix effect; when ME <50% or >150%, it is judged as a strong matrix effect.

[0081] The matrix effects of different urine samples are shown in Table 4.

[0082] Table 4

[0083]

[0084]

[0085] It can be seen from Table 4 that the matrix effects of the 24 organic ultraviolet absorbers and their metabolites in the present invention are 81.88-117.84% on average, showing a weak matrix effect.

[0086] (VIII) Actual sample measurement

[0087] 128 human urine samples were collected and processed and analyzed according to the method of step (a) to step (e). The results showed that except for UV-9, 3-BC, AVO and DHHB, the remaining 20 substances were detected, with an overall detection rate of 5.47-100% and a detection concentration range of MDL (method detection limit) to 49.3 ng / mL. Among them, the detection rates and concentration levels of 4-MAP, UV-P and BP-3 were significantly higher than those of other analytes, with average values ​​of 0.13, 0.16 and 0.74 ng / mL, respectively.

[0088] Example 2

[0089] The method of step (1) to step (5) in Example 1 is different in that in step (3), β-glucuronidase (derived from Roman snail, with an enzyme activity of 85 kU / mL, purchased from Merck) is replaced with β-glucuronidase (derived from Escherichia coli, with an enzyme activity of 80 kU / mL, purchased from Merck), the enzymolysis time is 12 h, and the enzymolysis efficiency is reduced.

[0090] According to the method of step (six) in Example 1, the absolute recovery rates of HMS and EHS were only 45.22% and 41.16%, which were at a relatively low level. It can be seen that the accuracy and reliability of this method are slightly poor, and the enzymatic hydrolysis effect is poor.

[0091] Comparative Example 1

[0092] The method of step (1) to step (5) in Example 1 is different in that in step (3), the enzymatic hydrolysis is not carried out in a light-proof environment, but is carried out in a natural light environment.

[0093] According to the method of step (six) in Example 1, the average spiked recovery was 52.12-82.56%, which was at a relatively low level; the relative standard deviation was 15.82-42.32%, which was at a relatively high level. It can be seen that the accuracy and reliability of this method are not high.

[0094] Comparative Example 2

[0095] The method of step (1) to step (5) in Example 1 is different in that in step (4), the concentration method is not nitrogen blowing, but centrifugal concentration.

[0096] According to the method of step (six) in Example 1, the absolute recoveries of EHS and HMS were only 14.55% and 19.80%, which were at a relatively low level; the relative standard deviations of the absolute recoveries of BEMT and DBT were 37.34% and 43.70%, which were at a relatively high level. It can be seen that the accuracy and reliability of this method are not high.

[0097] Comparative Example 3

[0098] According to the method of step (1) to step (5) in Example 1, the difference is that in step (3), the acetic acid-ammonium acetate buffer solution is replaced with potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (concentration of 0.1 mol / L, pH=7), the enzymolysis time is 10 h, and the enzymolysis efficiency decreases.

[0099] According to the method of step (six) in Example 1, the absolute recovery rates of EHS and HMS were only 25.76% and 32.38%, which were at a relatively low level. It can be seen that the accuracy and reliability of this method are not high, and the enzymatic hydrolysis effect is poor.

[0100] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An analytical method for detecting organic ultraviolet absorbers and their metabolites in urine, characterized in that: The method can simultaneously detect 24 organic ultraviolet absorbers and their metabolites, including EHMC, 4-MCA, 4'-MAP, 4-MBC, OC, 3-BC, AVO, BEMT, HMS, EHS, CDAA, DHHB, UV-P, UV-9, UV-326, UV-329, UV-360, UV-328, BP-1, BP-2, BP-3, BP-8, 4-OHBP and DBT, and includes the following steps: (1) preparing a mixed standard stock solution, a mixed internal standard stock solution and a mixed internal standard working solution, and establishing a standard curve for the above 24 organic ultraviolet absorbers and their metabolites; (2) Sample pretreatment: adding an acidic solution, β-glucuronidase and the mixed internal standard working solution to the urine sample, performing enzymatic hydrolysis at 35-50° C. in a light-proof environment for at least 2 h to obtain an enzymatically hydrolyzed urine sample; (3) Extraction treatment: subjecting the enzymatically hydrolyzed urine sample to at least two extraction treatments, combining the obtained organic phases, drying them, and then redissolving them to obtain the sample to be tested; (4) LC-MS / MS detection: performing LC-MS / MS detection on the sample to be tested, and quantifying the organic ultraviolet absorber and its metabolites in the sample to be tested according to the standard curve; The LC-MS / MS detection conditions include: using Waters Acquity BEH C 18 Chromatographic columns; Ammonia solution with a volume fraction of 0.05-0.2% is used as mobile phase A, and methanol is used as mobile phase B; the full injection time is at least 25 minutes; the elution gradient is: 0-2 minutes, 70% A, 30% B; 2-5 minutes, 70% A-40% A, 30% B-60% B; 5-5.1 minutes, 40% A-20% A, 60% B-80% B; 5.1-10 minutes, 20% A, 80% B; 10-10.1 minutes, 20% A-0% A, 80% B-100% B; 10.1-22 minutes, 0% A, 100% B; 22-22.01 minutes, 0% A-70% A, 100% B-30% B; 22.01-end of elution, 70% A, 30% B.

2. The method according to claim 1, wherein: The mixed standard stock solution includes 24 organic ultraviolet absorbers and their metabolites, including EHMC, 4-MCA, 4'-MAP, 4-MBC, OC, 3-BC, AVO, BEMT, HMS, EHS, CDAA, DHHB, UV-P, UV-9, UV-326, UV-329, UV-360, UV-328, BP-1, BP-2, BP-3, BP-8, 4-OHBP and DBT; And / or, the mixed internal standard stock solution and the mixed internal standard working solution include 4-MBC-d4, EHS-d4, HMS-d4, EHMC-d3, OC- 13 C3, UV-329- 13 C6, UV-P-d3, AVO- 13 C,d3,UV-328-d 12 There are 15 internal standard compounds including UV-326-d3, BP-1-d5, BP-2-d4, BP-3-d3, BP-8-d3 and 4-OHBP-d4.

3. The method according to claim 1 or 2, wherein: The concentration of each organic ultraviolet absorber and its metabolite in the mixed standard stock solution is 1-10 μg / mL, preferably 1-2 μg / mL; And / or, the concentration of each internal standard compound in the mixed internal standard stock solution is simultaneously 0.5-3 μg / mL, preferably 1-2 μg / mL; And / or, the concentration of each internal standard compound in the mixed internal standard working solution is simultaneously 25-150 ng / mL, preferably 50-100 ng / mL; And / or, the solvent of the mixed standard stock solution, the mixed internal standard stock solution and the mixed internal standard working solution is methanol.

4. The method according to any one of claims 1 to 3, wherein: The acidic solution is selected from acetic acid-ammonium acetate buffer solution and / or acetic acid-sodium acetate buffer solution; and / or, the concentration of the acidic solution is 0.5-2 mol / L, preferably 1-1.5 mol / L; and / or, the pH of the acidic solution is 4-6, preferably 4.5-5.5; And / or, the source of the β-glucuronidase is one or more of Roman snail, abalone, bovine liver and Escherichia coli, preferably Roman snail and / or abalone; And / or, the enzymatic activity of the β-glucuronidase is 20-200 kU / mL, preferably 50-150 kU / mL.

5. The method according to any one of claims 1 to 4, wherein: Relative to 1 mL of the urine sample, the amount of the acidic solution is 100-400 μL, preferably 200-300 μL; and / or, relative to 1 mL of the urine sample, the amount of the β-glucuronidase is 5-20 μL, preferably 10-15 μL; And / or, relative to 1 mL of the urine sample, the amount of the mixed internal standard working solution is 100-200 μL, preferably 140-160 μL.

6. The method according to any one of claims 1 to 5, wherein: The temperature of the enzymolysis is 40-45°C and the time is 2-12h.

7. The method according to any one of claims 1 to 6, wherein: The extraction treatment comprises adding an extraction solvent to the enzymatically hydrolyzed urine sample, mixing and oscillating at 2000-3000 rpm for 10-30 minutes, and then centrifuging at a centrifugal force of 3000-4000 g for 10-30 minutes to extract an organic phase; Preferably, the extraction solvent is selected from one or more of ethyl acetate, dichloromethane, n-hexane and acetonitrile, preferably a n-hexane-dichloromethane mixed solvent and / or an ethyl acetate-acetonitrile mixed solvent, more preferably a n-hexane-dichloromethane mixed solvent with a volume ratio of 1:0.5-1.5 and / or an ethyl acetate-acetonitrile mixed solvent with a volume ratio of 1:0.5-1.5; Preferably, relative to 1 mL of the enzymatically hydrolyzed urine sample, the amount of the extraction solvent used is 0.5-3 mL, preferably 1-2 mL.

8. The method according to any one of claims 1 to 7, wherein: The re-dissolving solvent is selected from one or more of methanol, acetonitrile, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide, preferably methanol and / or acetonitrile; And / or, the volume of the sample to be tested is 100-300 μL, preferably 150-250 μL.

9. The method according to any one of claims 1 to 8, wherein: The step of establishing the standard curve of the above-mentioned 24 organic ultraviolet absorbers and their metabolites includes: diluting the mixed standard stock solution into a series of mixed standard working solutions with multiple different mass concentrations, mixing each mixed standard working solution of different concentrations with the mixed internal standard working solution to a fixed volume, and performing LC-MS / MS detection respectively, using the ratio of the peak area of ​​each organic ultraviolet absorber and its metabolite in the detection results to the peak area of ​​each corresponding internal standard compound as the vertical coordinate, and using the detection concentration as the horizontal coordinate to obtain the standard curve.

10. The method according to any one of claims 1 to 9, wherein: The conditions for the LC-MS / MS detection also include: injection volume 3-6 μL, preferably 4-5 μL; column temperature 35-50°C, preferably 40-45°C; injection temperature 5-12°C, preferably 8-10°C; mobile phase flow rate 0.1-0.4 mL / min, preferably 0.2-0.3 mL / min; ion source ESI; scanning mode positive and negative ion scanning; monitoring mode MRM; carrier gas nitrogen, collision gas helium; capillary voltage 2.3-2.6 kV, preferably 2.4-2.5 kV; ion source temperature 120-180°C, preferably 140-160°C; desolvation temperature 400-600°C, preferably 450-550°C.

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