Analytical method for the detection of organic uv absorbers and their metabolites in urine

Abstract

The present application relates to the field of environmental chemistry and biological sample analysis, and particularly relates to an analysis method for detecting organic ultraviolet absorbers and metabolites thereof in urine. The method comprises enzymolysis of a urine sample and addition of an internal standard, extraction and purification of 24 kinds of organic ultraviolet absorbers and metabolites thereof in the sample by extraction, and detection of the content of target substances in the urine sample by high performance liquid chromatography-mass spectrometer. The present application is simple in operation, high in accuracy and reliability, can simultaneously detect 24 kinds of organic ultraviolet absorbers and metabolites thereof in 8 categories at one time, some substances are reported for the first time in the human urine detection method, and the method has wide practical application value and significance for evaluating the residual situation of organic ultraviolet absorbers and metabolites thereof in human body.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental chemistry and biological sample analysis, and particularly relates to an analysis method for detecting organic ultraviolet absorbers and their metabolites in urine. BACKGROUND

[0002] Organic ultraviolet absorbers are a class of compounds that can absorb ultraviolet light (UV) and are widely used in cosmetics, plastics, paints, and textiles, among other fields, to achieve sun protection and anti-UV aging functions. In addition, organic ultraviolet 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, etc.

[0003] Organic ultraviolet absorbers have conjugated double bond systems or aromatic systems in their chemical structure, which can effectively absorb specific wavelengths of ultraviolet light. However, with the expansion of the consumer market and the diversification of use, the types and complexity of organic ultraviolet absorbers are also increasing. Commonly used organic ultraviolet absorbers can be divided into multiple categories according to their chemical structure, mainly including p-aminobenzoic acid esters, benzophenones, camphor derivatives, cinnamic acid esters, dibenzoylmethanes, salicylic acid esters, triazinones, and benzotriazoles, etc. According to investigations, the global annual production of organic ultraviolet absorbers is about 10,000 tons.

[0004] Humans can come into contact with these ultraviolet absorbers through various routes, including skin contact, respiratory intake, diet, etc. These substances circulate through the blood, are metabolized by the liver, and are excreted with urine. Therefore, organic ultraviolet absorbers and their metabolites can be detected in urine samples, blood, milk, and other body fluids. However, exposure to organic ultraviolet absorbers can cause endocrine disruption, immune system consistency, skin irritation, and allergic reactions, among other phenomena. This has raised concerns about the safety of organic ultraviolet absorbers.

[0005] The determination of the concentration of organic UV absorbers as biomarkers helps to understand the exposure of humans to these substances and to further assess the possible effects on health. Organic UV absorbers are numerous, among which the cinnamate UV absorbers (EHMC) have the basic structure of methoxycinnamate (methoxy and ester groups), the metabolites of which include methoxycinnamic acid (methoxy and carboxyl groups) and methoxyacetophenone (methoxy and methyl ketone groups); the camphor derivatives (4-MBC and 3-BC) mainly consist of camphor and benzene rings; the aminobenzoic acid and its derivatives (DHHB) contain amino and ester groups in their structure; the benzophenones (BP-1, BP-2, BP-3, BP-8 and 4-OHBP) consist of two benzene rings and a carbonyl group; the dibenzoylmethanes (AVO) contain two benzene rings and a methyl group in their structure; the salicylates (EHS and HMS) contain phenolic hydroxyl and ester groups in their molecules; the triazinones (BEMT and DBT) have a triazine ring and a ketone group; and the benzotriazoles (UV-P, UV-9, UV-326, UV-328, UV-329 and UV-360) contain benzene and triazole rings. Due to the great structural differences of these substances, it is very challenging to simultaneously detect and analyze these substances, and therefore, how to develop an analysis method with high accuracy and good stability for simultaneously detecting the above-mentioned 8 types of organic UV absorbers and their metabolites has become a problem to be solved. SUMMARY

[0006] The purpose of the present application is to overcome the problems existing in the prior art and provide an analysis method for detecting organic UV absorbers and their metabolites in urine. The method can simultaneously detect 24 types of organic UV absorbers and their metabolites, and significantly improve the absolute recovery rate of each substance, with excellent accuracy and reliability.

[0007] To achieve the above-mentioned purpose, the present application provides an analysis method for detecting organic UV absorbers and their metabolites in urine, which can simultaneously detect 24 types of organic UV 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, comprising 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 standard curves of the above-mentioned 24 types of organic UV absorbers and their metabolites;

[0009] (2) sample pretreatment: adding an acidic solution, beta-glucuronidase and the mixed internal standard working solution into the urine sample, and carrying out enzymolysis at 35-50 DEG C in a light-proof environment for at least 2h to obtain an enzymolysis urine sample;

[0010] (3) extraction treatment: carrying out at least 2 times of extraction treatment on the enzymolysis urine sample, combining the obtained organic phase, blowing dry and redissolving to obtain a to-be-tested sample;

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

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

[0013] The application provides an analysis method of organic ultraviolet absorbers and metabolites based on liquid chromatography tandem mass spectrometry, which realizes purification and enrichment of urine samples, significantly reduces matrix effect, improves the absolute recovery rate of the to-be-tested substances, and realizes one-time simultaneous detection of 8 categories of 24 kinds of organic ultraviolet absorbers and metabolites by combining specific LC-MS / MS parameter conditions, and the operation is simple and efficient, and part of the substances are reported for the first time in the human urine detection method. The analysis method provided by the application has high accuracy and reliability, and has wide practical application value and significance for evaluating the residual situation of organic ultraviolet absorbers and metabolites in human body. DETAILED DESCRIPTION

[0014] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the properties. The endpoints of the ranges and any numerical values should be interpreted as approximately including values near the reported values. For numerical ranges, the endpoints are included in the ranges. The disclosure of ranges includes ranges having endpoints that are approximations.

[0015] The present application provides an analysis method for detecting organic ultraviolet absorbers and their metabolites in urine, which can simultaneously detect 24 kinds of 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, comprising the following steps:

[0016] (1) Prepare mixed standard stock solution, mixed internal standard stock solution and mixed internal standard working solution, and establish standard curves of the above 24 kinds of organic ultraviolet absorbers and their metabolites;

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

[0018] (3) Extraction treatment: perform at least 2 times of extraction treatment on the enzymatically digested urine sample, combine the obtained organic phase, blow dry and redissolve to obtain a sample to be tested;

[0019] (4) LC-MS / MS detection: perform LC-MS / MS detection on the sample to be tested, and quantify the organic ultraviolet absorbers and their metabolites in the sample to be tested according to the standard curves;

[0020] The conditions of the LC-MS / MS detection include: using Waters Acquity BEH C 18chromatographic column; 0.05-0.2% ammonia water solution as mobile phase A, methanol as mobile phase B; total needle time is at least 25 min; elution gradient is: 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; 10.1-22 min, 0% A, 100% B; 22-22.01 min, 0% A-70% A, 100% B-30% B; 22.01- elution end, 70% A, 30% B.

[0021] According to the present application, in step (1), before pretreatment and LC-MS / MS detection of the urine sample, mixed standard stock solution, mixed internal standard stock solution and mixed internal standard working solution need to be prepared to facilitate the subsequent steps and obtain better analysis effect.

[0022] According to the present application, the mixed standard stock solution comprises 24 kinds of 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.

[0023] wherein EHMC is ethylhexyl methoxy cinnamate; 4-MCA is 4-methoxycinnamic acid; 4'-MAP is 4'-methoxyacetophenone; 4-MBC is 4-methylbenzal camphor; OC is octocrilene; 3-BC is 3-benzal 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 diethylamino hydroxybenzoyl hexyl benzoate; UV-P is 2-(2'-hydroxy-5'-methylphenyl)benzotriazole; UV-9 is 2-hydroxy-4-methoxybenzophenone; UV-326 is 2'-(2'-hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole; UV-329 is octabenzone; 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; and DBT is diethylhexyl butamido triazone.

[0024] According to the present application, the mixed internal standard stock solution and the mixed internal standard working solution comprise 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, a total of 15 internal standard compounds.

[0025] wherein 4-MBC-d4 is 4-methylbenzal camphor-d4; EHS-d4 is 2-ethylhexyl salicylate-d4; HMS-d4 is homosalate-d4; EHMC-d3 is ethylhexyl methoxy cinnamate-d3; OC- 13 C3 is octocrilene- 13 C3; UV-329- 13 C6 is octabenzone- 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-benzotriazol-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-hydroxybenzophenone-d4.

[0026] According to the present application, in order to obtain better analysis effect, an analysis system is established, preferably, the concentration of each organic ultraviolet absorber and metabolite thereof in the mixed standard stock solution is simultaneously 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 the range between any values thereof.

[0027] Preferably, 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, for example, it can be 1 μg / mL, 1.5 μg / mL, 1.8 μg / mL and 2 μg / mL and the range between any values thereof.

[0028] Preferably, 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, for example, it can be 1 μg / mL, 1.5 μg / mL, 1.8 μg / mL and 2 μg / mL and the range between any values thereof.

[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 application, 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 methods:

[0031] Mixed standard stock solution: weigh a single compound into a single volumetric flask, and make up with methanol. In this way, 24 single standard stock solutions of organic ultraviolet absorbers and metabolites thereof are obtained respectively. Remove the same volume of 24 single standard stock solutions into a volumetric flask, and make up with methanol to obtain the mixed standard stock solution.

[0032] Mixed internal standard stock solution: single internal standard compound was weighed into a single volumetric flask, and methanol was added to constant volume. In this way, 15 single internal standard stock solutions were obtained. The same volume of each single internal standard stock solution was transferred into a volumetric flask, and methanol was added to constant volume to obtain the mixed internal standard stock solution.

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

[0034] According to the present application, in order to obtain a more reasonable standard curve and thus better determine each organic ultraviolet absorber and its metabolite in the urine sample, preferably, the step of establishing the standard curve of the above-mentioned 24 organic ultraviolet absorbers and their metabolites comprises: diluting the mixed standard stock solution into a series of mixed standard working solutions with different concentrations (such as at least 3 concentration values in more than 3 concentrations, for example, 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, etc.), each different concentration of the mixed standard working solution is mixed with the mixed internal standard working solution to constant 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 detection result is taken as the vertical coordinate, and the detection concentration is taken as the horizontal coordinate to obtain the standard curve.

[0035] More preferably, the step of establishing the standard curve of the above-mentioned 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, respectively, 50 μL of each mixed standard working solution is taken, 150 μL of the mixed internal standard working solution is added, and methanol is added to constant volume to 500 μL, LC-MS / MS detection is performed respectively, 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 detection result is taken as the vertical coordinate, 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 are taken as the horizontal coordinate to obtain the standard curve.

[0036] According to the present application, the specific pretreatment of the urine sample in step (2) can make the obtained sample have more suitable properties 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 effect. 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 0.5-2 mol / L, preferably 1-1.5 mol / L, for example, it can be 1 mol / L, 1.2 mol / L, 1.4 mol / L and 1.5 mol / L, and any range between any of these values. 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 any range between any of these values. Preferably, the amount of the acidic solution used is 100-400 μL, preferably 200-300 μL, for example, it can be 200 μL, 220 μL, 250 μL and 300 μL, and any range between any of these values, relative to 1 mL of the urine sample.

[0038] According to the present application, in the enzymatic treatment, the addition of β-glucuronidase can catalyze the hydrolysis reaction of glucuronide conjugates and generate the corresponding free compounds. In order to obtain better enzymatic effect, preferably, 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. Preferably, the amount of the β-glucuronidase used is 5-20 μL, preferably 10-15 μL, for example, it can be 10 μL, 12 μL, 14 μL and 15 μL, and any range between any of these values, relative to 1 mL of the urine sample. Preferably, the enzyme activity of the β-glucuronidase is 20-200 kU / mL, preferably 50-150 kU / mL, for example, it can be 50 kU / mL, 85 kU / mL, 100 kU / mL and 150 kU / mL, and any range between any of these values.

[0039] According to the present application, in the enzymatic treatment, the addition of mixed internal standard working solution can compensate for the individual differences of the sample (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, the amount of the mixed internal standard working solution used is 100-200 μL, preferably 140-160 μL, for example, it can be 140 μL, 150 μL, 155 μL and 160 μL, and any range between any of these values, relative to 1 mL of the urine sample.

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

[0041] According to the present application, in step (3), the enzymatic urine sample obtained after the enzymatic treatment is subjected to extraction treatment to further purify and enrich the urine sample, extract the organic ultraviolet absorber and its metabolite in the urine sample, and reduce the influence of other impurities such as salts and proteins in the urine on the analysis results. In order to achieve better extraction effect and improve the absolute recovery rate of each detection component, preferably, the extraction treatment comprises adding an extraction solvent to the enzymatic urine sample, mixing and oscillating at 2000-3000rpm (for example, it can be 2000rpm, 2200rpm, 2500rpm and 3000rpm, and any range between any of these values) for 10-30min (for example, it can be 10min, 15min, 20min and 30min, and any range between any of these values), and then centrifuging at a centrifugal force of 3000-4000g (for example, it can be 3000g, 3500g, 3800g and 4000g, and any range between any of these values) for 10-30min (for example, it can be 10min, 15min, 20min and 30min, and any range between any of these values) 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 n-hexane-dichloromethane mixed solvent and / or ethyl acetate-acetonitrile mixed solvent, more preferably n-hexane-dichloromethane mixed solvent with a volume ratio of 1:0.5-1.5 and / or ethyl acetate-acetonitrile mixed solvent with a volume ratio of 1:0.5-1.5, for example, n-hexane-dichloromethane mixed solvent with a volume ratio of 1:1, n-hexane-dichloromethane mixed solvent with a volume ratio of 1:1.5, ethyl acetate-acetonitrile mixed solvent with a volume ratio of 1:0.5 and ethyl acetate-acetonitrile mixed solvent with a volume ratio of 1:1.

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

[0044] According to the present application, in order to obtain better analysis effect, preferably, the solvent for reconstitution 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 blow-drying is an inert 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 and 250 μL, and any range between any of these values.

[0047] According to the present application, in step (4), the LC-MS / MS detection of the sample to be tested can qualitatively and quantitatively analyze the organic ultraviolet absorber and its metabolites therein. The quantitative analysis is carried out by 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 application, in order to obtain faster detection speed, improve the stability and accuracy of detection, preferably, the conditions of the LC-MS / MS detection further include: injection volume 3-6 μL, preferably 4-5 μL (for example, it can be 4 μL, 4.5 μL, 4.7 μL and 5 μL, and any range between any of these values); column temperature 35-50℃, preferably 40-45℃ (for example, it can be 40℃, 42℃, 43℃ and 45℃, and any range between any of these values); injection temperature 5-12℃, preferably 8-10℃ (for example, it can be 8℃, 9℃, 9.5℃ and 10℃, and any range between any of these values); flow rate of mobile phase 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 and 0.3 mL / min, and any range between any of these values); ion source is ESI (electrospray); scanning mode is positive and negative ion scanning; monitoring mode is MRM (mass spectrometry multiple reaction monitoring); carrier gas is nitrogen, collision gas is helium; capillary voltage 2.3-2.6 kV, preferably 2.4-2.5 kV (for example, it can be 2.4 kV, 2.46 kV, 2.48 kV and 2.5 kV, and any range between any of these values); ion source temperature 120-180℃, preferably 140-160℃ (for example, it can be 140℃, 145℃, 150℃ and 160℃, and any range between any of these values); desolvation temperature 400-600℃, preferably 450-550℃ (for example, it can be 450℃, 480℃, 500℃ and 530℃, and any range between any of these values).

[0049] The application provides an analysis method of organic ultraviolet absorbers and metabolites thereof based on liquid chromatography tandem mass spectrometry, which realizes purification and enrichment of a urine sample, significantly reduces matrix effect, improves absolute recovery of the detected substances, and realizes simultaneous detection of 24 organic ultraviolet absorbers and metabolites thereof in 8 categories under specific LC-MS / MS parameter conditions, which is simple and efficient, and some of the substances are reported for the first time in the human urine detection method. The analysis method provided by the application has high accuracy and reliability, and has wide practical application value and significance for evaluating the residual situation of organic ultraviolet absorbers and metabolites thereof in the human body.

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

[0051] In the following examples, unless otherwise specified, the devices used are conventional experimental devices in the art, the experimental operations used are conventional operations in the art, and the raw materials, reagents, etc. used can be obtained by commercial purchase. 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] 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 by using methanol, and then transfer a certain amount of the above 24 single standard stock solutions into the same volumetric flask to prepare a mixed standard stock solution with a concentration of 1 μg / mL.

[0055] Prepare single standard stock solutions of 4-MBC-d4, EHS-d4, HMS-d4, EHMC-d3, OC-d3, C3, UV-329-d3, C6, UV-P-d3, AVO-d3, C, UV-328-d3, UV-326-d3, BP-1-d5, BP-2-d4, BP-3-d3, BP-8-d3 and 4-OHBP-d4 with a concentration of 1 mg / mL by using methanol, and then transfer a certain amount of the above 15 single standard stock solutions into the same volumetric flask to prepare a mixed internal standard stock solution with a concentration of 1 μg / mL. 13 C3, UV-329- 13 C6, UV-P-d3, AVO- 13 C, UV-328-d 12 , UV-326-d3, BP-1-d5, BP-2-d4, BP-3-d3, BP-8-d3 and 4-OHBP-d4 with a concentration of 1 mg / mL by using methanol, and then transfer a certain amount of the above 15 single standard stock solutions into 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) Establishing standard curves of 24 organic ultraviolet absorbers and metabolites thereof

[0058] The mixed standard stock solution was diluted 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, respectively, 50 μL of each mixed standard working solution was taken, 150 μL of the mixed internal standard working solution was added, and the volume was made up to 500 μL with methanol, and LC-MS / MS detection was performed, respectively, so that the ratio of the peak area of each organic ultraviolet absorber and metabolite thereof to the peak area of the corresponding internal standard compound in the detection result was taken as the vertical coordinate, 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 taken as the horizontal coordinates, to obtain the standard curve, and the linear range was calculated according to the standard curve, and when the linear coefficient (R 2 ) was greater than 0.999, it indicated that the linearity was good, and quantitative calculation was performed accordingly. The linear correlation parameters of each organic ultraviolet absorber and metabolite thereof are shown in Table 1.

[0059] Table 1

[0060]

[0061] As can be seen from Table 1, the method detection limits of the 24 organic ultraviolet absorbers and metabolites thereof in the present application are 0.001-0.009 ng / mL, the method quantification limits are 0.002-0.031 ng / mL, and the linear coefficients (R 2 ) are all higher than 0.999, and have good linear relationships.

[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 Company) and 300 μL of mixed internal standard working solution were added to 2 mL of urine sample. The obtained mixture was enzymatically degraded at 37℃ in a light-proof environment for 4 h to obtain an enzymatically degraded urine sample.

[0064] (iv) Extraction treatment

[0065] The enzymatic urine sample was extracted twice, the obtained organic phase was combined, dried under nitrogen, and redissolved 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 oscillated at 2500 pm / min for 20 min, and then centrifuged at 3600 g for 20 min to extract the organic phase.

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

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

[0068] The conditions for LC-MS / MS detection included: Waters Acquity BEH C 18 chromatographic column was used; 0.1% ammonia solution by volume was used as mobile phase A, and methanol was used as mobile phase B; the total needle time was 25 min; the elution gradient was as follows: 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; 10.1-22 min, 0% A, 100% B; 22-22.01 min, 0% A-70% A, 100% B-30% B; 22.01-25 min, 70% A, 30% B; the injection amount was 5 μL; the column temperature was 40°C; the injection temperature was 8°C; the flow rate of the mobile phase was 0.25 mL / min; the ion source was ESI; the scanning mode was positive and negative ion scanning; the monitoring mode was MRM; the carrier gas was nitrogen, and the collision gas was helium; the capillary voltage was 2.46 kV; the ion source temperature was 150°C; and the desolvation temperature was 500°C.

[0069] The retention time, monitoring ion, cone voltage and collision energy of 24 organic ultraviolet absorbers and their metabolites and 15 corresponding internal standard compounds are shown in Table 2.

[0070] Table 2

[0071]

[0072]

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

[0074] The urine sample of the same source was divided into 4 parts, 3 parts of which were added with appropriate amount of mixed standard stock solution to adjust the spiked concentration to 1 ng / mL, 5 ng / mL and 25 ng / mL respectively, and the four urine samples were treated and analyzed according to the method of steps (one) to (five), and each urine sample was subjected to 6 parallel experiments. The spiked recovery rate and relative standard deviation were calculated, wherein the spiked recovery rate (Recovery) = (spiked sample determination value-unspiked sample determination value) ÷ spiked amount × 100%; the relative standard deviation (RSD) = the deviation value of the spiked recovery rate ÷ the average value of the 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 recovery rate of the 24 organic ultraviolet absorbers and metabolites in the present application is 70.3-130%, which is at a higher level; the relative standard deviation is < 16.5%, which is at a lower level. It can be seen that the analysis method of the present application has excellent accuracy and reliability.

[0079] (Seven) Matrix Effect Evaluation

[0080] Five urine samples of different sources were selected, and the method of steps (one) to (five) was used for processing, 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 methanol matrix standard curves 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 weak matrix effect; when ME is 50%-80% or 120%-150%, it is judged as moderate matrix effect; and when ME < 50% or > 150%, it is judged as strong matrix effect.

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

[0082] Table 4

[0083]

[0084]

[0085] As can be seen from Table 4, the matrix effect of the 24 organic ultraviolet absorbers and their metabolites in the application is 81.88-117.84%, which shows weak matrix effect.

[0086] (Eight) Actual sample determination

[0087] The urine samples of 128 people were collected and treated and analyzed according to the method of steps (one) to step (five), and the results showed that except that UV-9, 3-BC, AVO and DHHB were not detected, the remaining 20 substances were detected, the overall detection rate was 5.47-100%, and the detection concentration range was MDL (method detection limit) to 49.3 ng / mL. Among them, the detection rate and concentration level of 4-MAP, UV-P and BP-3 were significantly higher than those of other analytes, and the average values were 0.13, 0.16 and 0.74 ng / mL, respectively.

[0088] Example 2

[0089] According to the method of steps (one) to step (five) in Example 1, except that in step (three), the β-glucuronidase (from Roman snail, enzyme activity 85 kU / mL, purchased from Merck Company) was replaced by β-glucuronidase (from Escherichia coli, enzyme activity 80 kU / mL, purchased from Merck Company), and the enzyme hydrolysis time was 12 h, the enzyme hydrolysis efficiency decreased.

[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 low level. It can be seen that the accuracy and reliability of this method are slightly poor, and the enzyme hydrolysis effect is poor.

[0091] Comparative Example 1

[0092] According to the method of steps (one) to step (five) in Example 1, except that in step (three), the enzyme hydrolysis was not carried out in the dark environment, but in the natural light environment.

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

[0094] Comparative Example 2

[0095] According to the method of steps (one) to step (five) in Example 1, except that in step (four), the concentration method did not use nitrogen blowing, but used centrifugal concentration.

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

[0097] Comparative example 3

[0098] According to the method of steps (one) to (five) in embodiment 1, except that the acetic acid-ammonium acetate buffer solution in step (three) is replaced by potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (concentration of 0.1 mol / L, pH = 7), and the enzymatic time is 10 h, the enzymatic efficiency decreases.

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

[0100] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. An analytical method for detecting organic ultraviolet absorbers and their metabolites in urine, characterized in that, The method can simultaneously detect 24 kinds of 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, including the following steps: (1) Prepare a mixed standard stock solution, a mixed internal standard stock solution and a mixed internal standard working solution, and establish a standard curve of the above-mentioned 24 kinds of organic ultraviolet absorbers and metabolites thereof; (2) Sample pretreatment: add an acidic solution, β-glucuronidase and the mixed internal standard working solution to the urine sample, and enzymatically digest at 35-50°C in a light-proof environment for at least 2h to obtain an enzymatically digested urine sample; wherein the acidic solution is selected from acetic acid-ammonium acetate buffer solution and / or acetic acid-sodium acetate buffer solution; (3) Extraction treatment: the enzymatically digested urine sample is subjected to at least 2 times of extraction treatment, and the obtained organic phase is combined and blown dry to obtain a sample to be tested; wherein the extraction solvent used in the extraction treatment is selected from one or more of ethyl acetate, dichloromethane, n-hexane and acetonitrile; (4) LC-MS / MS detection: the sample to be tested is subjected to LC-MS / MS detection, and the organic ultraviolet absorbers and metabolites thereof in the sample to be tested are quantified according to the standard curve; The conditions of the LC-MS / MS detection include: using Waters Acquity BEH C 18 chromatographic column; using 0.05-0.2% ammonia solution as mobile phase A and methanol as mobile phase B; total needle time is at least 25 min; elution gradient is: 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; 10.1-22 min, 0% A, 100% B; 22-22.01 min, 0% A-70% A, 100% B-30% B; 22.01-end of elution, 70% A, 30% B; injection volume is 3-6 μL; column temperature is 35-50 °C; injection temperature is 5-12 °C; flow rate of mobile phase is 0.1-0.4 mL / 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 is 2.3-2.6 kV; ion source temperature is 120-180 °C; desolvation temperature is 400-600 °C.

2. The method of claim 1, wherein, The mixed standard stock solution comprises 24 kinds of 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 / 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 , UV-326-d3, BP-1-d5, BP-2-d4, BP-3-d3, BP-8-d3 and 4-OHBP-d4, a total of 15 internal standard compounds.

3. The method of claim 1, wherein, The concentration of each organic ultraviolet absorber and metabolite in the mixed standard stock solution is 1-10μg / mL at the same time; And / or, the concentration of each internal standard compound in the mixed internal standard stock solution is 0.5-3μg / mL at the same time; And / or, the concentration of each internal standard compound in the mixed internal standard working solution is 25-150ng / mL at the same time; 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 of claim 3, wherein, The concentration of each organic ultraviolet absorber and metabolite in the mixed standard stock solution is 1-2μg / mL at the same time; And / or, the concentration of each internal standard compound in the mixed internal standard stock solution is 1-2μg / mL at the same time; And / or, the concentration of each internal standard compound in the mixed internal standard working solution is 50-100ng / mL at the same time.

5. The method of claim 1, wherein, The concentration of the acidic solution is 0.5-2mol / L; And / or, the pH of the acidic solution is 4-6; And / or, the source of the β-glucuronidase is one or more of Roman snail, abalone, bovine liver and Escherichia coli. And / or, the enzyme activity of the beta-glucuronidase is 20-200 kU / mL.

6. The method of claim 5, wherein, The concentration of the acidic solution is 1-1.5 mol / L; And / or, the pH of the acidic solution is 4.5-5.5; And / or, the source of the beta-glucuronidase is Roman snail and / or abalone; And / or, the enzyme activity of the beta-glucuronidase is 50-150 kU / mL.

7. The method of claim 1, wherein, The amount of the acidic solution is 100-400 μL relative to 1 mL of the urine sample; And / or, the amount of the beta-glucuronidase is 5-20 μL relative to 1 mL of the urine sample; And / or, the amount of the mixed internal standard working solution is 100-200 μL relative to 1 mL of the urine sample.

8. The method of claim 7, wherein, The amount of the acidic solution is 200-300 μL relative to 1 mL of the urine sample; And / or, the amount of the beta-glucuronidase is 10-15 μL relative to 1 mL of the urine sample; And / or, the amount of the mixed internal standard working solution is 140-160 μL relative to 1 mL of the urine sample.

9. The method of claim 1, wherein, The temperature of the enzymolysis is 40-45℃, and the time is 2-12 h.

10. The method of claim 1, wherein, The extraction treatment comprises adding the extraction solvent to the enzymolyzed urine sample, mixing and oscillating at 2000-3000 rpm for 10-30 min, and then centrifuging at a centrifugal force of 3000-4000 g for 10-30 min to extract the organic phase.

11. The method of claim 1, wherein, The extraction solvent is a n-hexane-dichloromethane mixed solvent and / or an ethyl acetate-acetonitrile mixed solvent.

12. The method of claim 11, wherein, The extraction solvent is 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.

13. The method of claim 1, wherein, The amount of the extraction solvent is 0.5-3 mL relative to 1 mL of the enzymolyzed urine sample.

14. The method of claim 13, wherein, The amount of the extraction solvent is 1-2 mL relative to 1 mL of the enzymolyzed urine sample.

15. The method of claim 1, wherein, The resolubilizing solvent is selected from one or more of methanol, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide; And / or, the volume of the to-be-tested sample is 100-300 μL.

16. The method of claim 15, wherein, The resolubilizing solvent is methanol and / or acetonitrile; And / or, the volume of the to-be-tested sample is 150-250 μL.

17. The method of claim 1, wherein, The step of establishing the standard curve of the 24 organic ultraviolet absorbers and metabolites comprises: diluting the mixed standard stock solution into a series of mixed standard working solutions with different concentrations, mixing each different concentration of the mixed standard working solution with the mixed internal standard working solution to constant volume, and respectively performing LC-MS / MS detection, so that the ratio of the peak area of each organic ultraviolet absorber and metabolite to the peak area of each corresponding internal standard compound in the detection result is taken as the vertical coordinate, and the detection concentration is taken as the horizontal coordinate, to obtain the standard curve.

18. The method of claim 1, wherein, The conditions of the LC-MS / MS detection further include: injection volume 4-5 μL; column temperature 40-45℃; injection temperature 8-10℃; flow rate of mobile phase 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 2.4-2.5 kV; ion source temperature 140-160℃; desolvation temperature 450-550℃.

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