Method for extracting p-phenylenediamine antioxidant in liquid sample and application thereof

Abstract

The invention discloses a method for extracting a p-phenylenediamine antioxidant in a liquid sample and application of the p-phenylenediamine antioxidant. The p-phenylenediamine antioxidant comprises N-(1, 2, 4-trimethyl-1, 3-pentanediol) the organic solvent is at least one of N, N '-diphenyl-1, 3-dimethyl butyl)-N'-phenyl p-phenylenediamine, N-cyclohexyl-N '-phenyl p-phenylenediamine, N-isopropyl-N'-phenyl-p-phenylenediamine, N, N '-diphenyl-1, 4-phenylenediamine, N, N'-di-2-naphthyl p-phenylenediamine and 2-((4-methylpentane-2-yl) amino)-5-(phenyl amino) cyclohex-2, 5-diene-1, 4-diketone. According to the method, liquid-liquid extraction which is simple and efficient to operate, low in cost and good in repeatability is adopted as a sample pretreatment method, and is combined with an isotope label method and an ultra-high performance liquid chromatography-tandem mass spectrometry technology, so that separation and qualitative and quantitative detection of six p-phenylenediamine antioxidants and quinone derivatives in a sample are realized; meanwhile, the sensitivity is high, the quantity of isotope internal standards is small, the solvent loss is small, and the actual detection requirement is met.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for extracting p-phenylenediamine antioxidants from a liquid sample and an application thereof. Background Art

[0002] Paraphenylenediamine antioxidants have strong oxidizing properties. Rubber will oxidize during use, and paraphenylenediamine antioxidants prevent rubber oxidation by competing with rubber for free radicals. They are often used as rubber anti-degradants, effectively resisting the oxidation reaction of rubber products in harsh environments such as high temperature and humidity, maintaining the stability of the performance of rubber products, and are widely used in the production and manufacturing of rubber industrial products such as tires, tapes, and hoses. N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), its quinone derivative 6PPD-Q, and paraphenylenediamine antioxidants with similar structures to 6PPD have been proven to be released into the environment during use and can enter the human body through various pathways (such as breathing, diet, and skin contact). They have potential neurotoxicity, hepatotoxicity, developmental toxicity, and reproductive toxicity, and the safety management and monitoring of such compounds need to be strengthened.

[0003] At present, the main focus of the p-phenylenediamine antioxidants and quinone derivatives in the human body is on typical substances such as 6PPD and 6PPD-Q, while there is no corresponding detection method for other p-phenylenediamine antioxidants. Therefore, it is urgent to establish a method for simultaneously detecting multiple p-phenylenediamine antioxidants and quinone derivatives in human samples to expand the monitoring scope. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a method for extracting p-phenylenediamine antioxidants from a liquid sample and application thereof.

[0005] The first object of the present invention is to provide a method for extracting p-phenylenediamine antioxidants.

[0006] The second object of the present invention is to provide application of the method in detecting p-phenylenediamine antioxidants.

[0007] The third object of the present invention is to provide a method for detecting p-phenylenediamine antioxidants.

[0008] In order to achieve the above object, the present invention is implemented by the following scheme:

[0009] Paraphenylenediamine antioxidants and quinone derivatives have structures such as amino groups (-NH2) and benzene rings. Such compounds have certain polarity and also contain non-polar parts. Methyl tert-butyl ether is a non-polar solvent with good ability to dissolve non-polar compounds; dichloromethane is a medium polar solvent that can dissolve some polar and medium polar substances; n-hexane is a relatively non-polar solvent that can dissolve some weakly polar compounds. The present invention utilizes a combination of these three reagents to efficiently extract multiple paraphenylenediamine antioxidants from the aqueous phase into the organic phase by utilizing the polarity differences between the solvents.

[0010] A method for extracting a p-phenylenediamine antioxidant in a liquid sample, wherein the p-phenylenediamine compound comprises at least one of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, N-cyclohexyl-N'-phenyl-p-phenylenediamine, N-isopropyl-N'-phenyl-p-phenylenediamine, N,N'-diphenyl-1,4-phenylenediamine, N,N'-di-2-naphthyl-p-phenylenediamine and 2-((4-methylpentane-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione;

[0011] The method comprises the following steps:

[0012] S1. Fully mixing the liquid sample and the antioxidant to obtain a sample mixture;

[0013] S2. The methyl tert-butyl ether and the liquid sample mixture are thoroughly mixed and separated into layers to obtain an organic phase 1 and an aqueous phase;

[0014] S3. The aqueous phase obtained in step S2 is thoroughly mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, and the layers are separated to obtain an organic phase 2;

[0015] S4. Combining the organic phase 1 obtained in step S2 and the organic phase 2 obtained in step S3 to obtain an extract containing the p-phenylenediamine compound.

[0016] Preferably, the volume ratio of the sample mixture, methyl tert-butyl ether, saturated sodium chloride solution and dichloromethane-n-hexane solution is 0.25:(5-7.5):0.4:2.5.

[0017] More preferably, the volume ratio of the sample mixture, methyl tert-butyl ether, saturated sodium chloride solution and dichloromethane-n-hexane solution is 0.25:5:0.4:2.5.

[0018] Preferably, the liquid sample comprises a body fluid sample.

[0019] More preferably, the body fluid sample includes any one of cerebrospinal fluid, serum or urine.

[0020] Preferably, the liquid sample comprises artificial serum.

[0021] Since p-phenylenediamine antioxidants have strong oxidizing properties, adding antioxidants with strong reducing properties can prevent the p-phenylenediamine antioxidants from oxidative degradation during the extraction process. Antioxidants including but not limited to glutathione, parahydroxybenzoates, butylated hydroxytoluene, etc. can achieve the purpose of extracting p-phenylenediamine antioxidants in the present invention.

[0022] Preferably, the antioxidant comprises 1-2 mmol / L glutathione solution.

[0023] More preferably, the antioxidant comprises 1 mmol / L glutathione solution.

[0024] Preferably, the volume ratio of the liquid sample to the antioxidant is (2-3):2.

[0025] More preferably, the volume ratio of the liquid sample to the antioxidant is 2.5:2.

[0026] Preferably, in step S1, the liquid sample and the antioxidant are mixed and then fully shaken to obtain a sample mixture.

[0027] More preferably, in step S1, after the liquid sample and the antioxidant are mixed, they are shaken at 25-30° C. for 20-40 min.

[0028] More preferably, in step S1, the sample is mixed with the antioxidant and then shaken at 27° C. for 30 min.

[0029] Preferably, in step S2, after methyl tert-butyl ether is mixed with the liquid sample, it is first vortexed, then ultrasonicated, and then separated by centrifugation to obtain an organic phase 1 and an aqueous phase.

[0030] Preferably, in step S3, the aqueous phase obtained in step S2 is mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, vortexed, ultrasonicated, and then separated by centrifugation to obtain an organic phase 2.

[0031] Preferably, in step S2, methyl tert-butyl ether and the sample mixture are fully mixed and separated to obtain an organic phase 1 and an aqueous phase 1; the aqueous phase 1 is fully mixed with methyl tert-butyl ether and separated to obtain an organic phase 3 and an aqueous phase 2; in step S3, the aqueous phase 2 obtained in step S2 is fully mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, separated to obtain an organic phase 4; in step S4, the organic phase 1 and the organic phase 3 obtained in step S2 and the organic phase 4 obtained in step S3 are combined to obtain an extract containing the p-phenylenediamine antioxidant.

[0032] More preferably, in step S2, after methyl tert-butyl ether and the sample mixture are mixed, they are first vortexed, then ultrasonicated, and then centrifuged to obtain organic phase 1 and aqueous phase 1; after the aqueous phase 1 is mixed with methyl tert-butyl ether, they are first vortexed, then ultrasonicated, and then centrifuged to obtain organic phase 3 and aqueous phase 2; in step S3, after the aqueous phase 2 obtained in step S2 is mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, they are first vortexed, then ultrasonicated, and then centrifuged to obtain an organic phase 4; in step S4, the organic phase 1 and organic phase 3 obtained in step S2 and the organic phase 4 obtained in step S3 are combined to obtain an extract containing the p-phenylenediamine antioxidant.

[0033] Further preferably, in step S2, the vortexing time is 8 to 10 minutes.

[0034] More preferably, in step S2, the vortexing time is 10 minutes.

[0035] Further preferably, in step S2, the ultrasonic conditions include: frequency 80-90KHz, power 700-750W, and time 12-15min.

[0036] More preferably, in step S2, the ultrasonic conditions include: frequency 80KHz, power 700W, time 15min.

[0037] Further preferably, in step S2, the centrifugal conditions include: a rotation speed of 4000-4500 rpm and a time of 4-5 min.

[0038] More preferably, in step S2, the centrifugal conditions include: rotation speed 4500 rpm, time 5 min.

[0039] Further preferably, in step S3, the vortexing time is 8 to 10 minutes.

[0040] More preferably, in step S3, the vortexing time is 10 minutes.

[0041] Further preferably, in step S3, the ultrasonic conditions include: frequency 80-90KHz, power 700-750W, and time 12-15min.

[0042] More preferably, in step S3, the ultrasonic conditions include: frequency 80KHz, power 700W, time 15min.

[0043] Further preferably, in step S3, the centrifugal conditions include: a rotation speed of 4000-4500 rpm and a time of 4-5 min.

[0044] More preferably, in step S3, the centrifugal conditions include: rotation speed 4500 rpm, time 5 min.

[0045] Preferably, in step S4, after combining all the organic phases obtained in steps S2 and S3, the organic phases are blown to near dryness with nitrogen to obtain a solid.

[0046] More preferably, step S4 further comprises: redissolving the solid with acetonitrile to obtain a redissolved solution.

[0047] Further preferably, step S4 further comprises: filtering the reconstituted solution.

[0048] More preferably, the reconstituted solution is filtered using a 0.22 μm PTFE filter membrane.

[0049] The extract containing the p-phenylenediamine antioxidant obtained by the method should also be within the protection scope of the present invention.

[0050] The use of the extract in the preparation of a detection product for p-phenylenediamine antioxidants should also be within the protection scope of the present invention.

[0051] The application of the method in detecting p-phenylenediamine antioxidants should also be within the protection scope of the present invention.

[0052] A method for detecting a p-phenylenediamine antioxidant, wherein the p-phenylenediamine antioxidant comprises at least one of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, N-cyclohexyl-N'-phenyl-p-phenylenediamine, N-isopropyl-N'-phenyl-p-phenylenediamine, N,N'-diphenyl-1,4-phenylenediamine, N,N'-di-2-naphthyl-p-phenylenediamine and 2-((4-methylpentane-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione;

[0053] The method comprises the following steps: extracting the sample to be tested by using a method for extracting p-phenylenediamine antioxidants from a liquid sample to obtain an extract, and then detecting the extract by ultra-high performance liquid chromatography;

[0054] The method for extracting p-phenylenediamine antioxidants from a liquid sample comprises the following steps:

[0055] S1. Fully mixing the liquid sample and the antioxidant to obtain a sample mixture;

[0056] S2. The methyl tert-butyl ether and the liquid sample mixture are thoroughly mixed and separated into layers to obtain an organic phase 1 and an aqueous phase;

[0057] S3. The aqueous phase obtained in step S2 is thoroughly mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, and the layers are separated to obtain an organic phase 2;

[0058] S4. Combining the organic phase 1 obtained in step S2 and the organic phase 2 obtained in step S3 to obtain an extract containing the p-phenylenediamine antioxidant.

[0059] Preferably, the volume ratio of the sample mixture, methyl tert-butyl ether, saturated sodium chloride solution and dichloromethane-n-hexane solution is 0.25:(5-7.5):0.4:2.5.

[0060] More preferably, the volume ratio of the sample mixture, methyl tert-butyl ether, saturated sodium chloride solution and dichloromethane-n-hexane solution is 0.25:5:0.4:2.5.

[0061] Preferably, the antioxidant comprises 1-2 mmol / L glutathione solution.

[0062] More preferably, the antioxidant comprises 1 mmol / L glutathione solution.

[0063] Preferably, the volume ratio of the liquid sample to the antioxidant is (2-3):2.

[0064] More preferably, the volume ratio of the liquid sample to the antioxidant is 2.5:2.

[0065] Preferably, in step S1, the liquid sample and the antioxidant are mixed and then fully shaken to obtain a sample mixture.

[0066] More preferably, in step S1, after the liquid sample and the antioxidant are mixed, they are shaken at 25-30° C. for 20-40 min.

[0067] Further preferably, in step S1, the liquid sample is mixed with the antioxidant and then shaken at 27° C. for 30 min.

[0068] Preferably, in step S2, after methyl tert-butyl ether is mixed with the liquid sample, it is first vortexed, then ultrasonicated, and then separated by centrifugation to obtain an organic phase 1 and an aqueous phase.

[0069] Preferably, in step S3, the aqueous phase obtained in step S2 is mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, vortexed, ultrasonicated, and then separated by centrifugation to obtain an organic phase 2.

[0070] Preferably, in step S2, methyl tert-butyl ether and the liquid sample mixture are fully mixed and separated to obtain an organic phase 1 and an aqueous phase 1; the aqueous phase 1 is fully mixed with methyl tert-butyl ether and separated to obtain an organic phase 3 and an aqueous phase 2; in step S3, the aqueous phase 2 obtained in step S2 is fully mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, separated to obtain an organic phase 4; in step S4, the organic phase 1 and the organic phase 3 obtained in step S2 and the organic phase 4 obtained in step S3 are combined to obtain an extract containing the p-phenylenediamine antioxidant.

[0071] More preferably, in step S2, after methyl tert-butyl ether and the sample mixture are mixed, they are first vortexed, then ultrasonicated, and then centrifuged to obtain organic phase 1 and aqueous phase 1; after the aqueous phase 1 is mixed with methyl tert-butyl ether, they are first vortexed, then ultrasonicated, and then centrifuged to obtain organic phase 3 and aqueous phase 2; in step S3, after the aqueous phase 2 obtained in step S2 is mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, they are first vortexed, then ultrasonicated, and then centrifuged to obtain an organic phase 4; in step S4, the organic phase 1 and organic phase 3 obtained in step S2 and the organic phase 4 obtained in step S3 are combined to obtain an extract containing the p-phenylenediamine antioxidant.

[0072] Further preferably, in step S2, the vortexing time is 8 to 10 minutes.

[0073] More preferably, in step S2, the vortexing time is 10 minutes.

[0074] Further preferably, in step S2, the ultrasonic conditions include: frequency 80-90KHz, power 700-750W, and time 12-15min.

[0075] More preferably, in step S2, the ultrasonic conditions include: frequency 80KHz, power 700W, time 15min.

[0076] Further preferably, in step S2, the centrifugal conditions include: a rotation speed of 4000-4500 rpm and a time of 4-5 min.

[0077] More preferably, in step S2, the centrifugal conditions include: rotation speed 4500 rpm, time 5 min.

[0078] Further preferably, in step S3, the vortexing time is 8 to 10 minutes.

[0079] More preferably, in step S3, the vortexing time is 10 minutes.

[0080] Further preferably, in step S3, the ultrasonic conditions include: frequency 80-90KHz, power 700-750W, and time 12-15min.

[0081] More preferably, in step S3, the ultrasonic conditions include: frequency 80KHz, power 700W, time 15min.

[0082] Further preferably, in step S3, the centrifugal conditions include: a rotation speed of 4000-4500 rpm and a time of 4-5 min.

[0083] More preferably, in step S3, the centrifugal conditions include: rotation speed 4500 rpm, time 5 min.

[0084] Preferably, in step S4, after combining all the organic phases obtained in steps S2 and S3, the organic phases are blown to near dryness with nitrogen to obtain a solid.

[0085] More preferably, step S4 further comprises: redissolving the solid with acetonitrile to obtain a redissolved solution.

[0086] Further preferably, step S4 further comprises: filtering the reconstituted solution.

[0087] More preferably, the reconstituted solution is filtered using a 0.22 μm PTFE filter membrane.

[0088] Preferably, in step S1 of the method for extracting p-phenylenediamine antioxidants, the liquid sample is fully mixed with the internal standard and the antioxidant to obtain a sample mixture.

[0089] More preferably, the internal standard is isotope-labeled 2-((4-methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione.

[0090] Further preferably, the isotope is a stable isotope.

[0091] More preferably, the stable isotope is deuterium.

[0092] Still further preferably, the internal standard is D5-6PPD-quinone.

[0093] The sample to be tested is a liquid sample or a liquid sample obtained by pre-treating a solid sample, and is suitable for the method for detecting p-phenylenediamine antioxidants provided by the present invention.

[0094] Preferably, the liquid sample comprises a body fluid sample.

[0095] More preferably, the body fluid sample includes any one of cerebrospinal fluid, serum or urine.

[0096] Preferably, the liquid sample comprises artificial serum.

[0097] Further preferably, the mass volume ratio of the internal standard to the liquid sample is (0.5-1) ng: (200-250) μL.

[0098] Still further preferably, the mass volume ratio of the internal standard to the liquid sample is 0.625 ng:250 μL.

[0099] Preferably, the chromatographic conditions of the ultra-high performance liquid chromatography include: using a C18 chromatographic column; using 0.1 v / v% formic acid aqueous solution as mobile phase A, using methanol as mobile phase B, and in the gradient elution program, the volume percentage of mobile phase B changes as follows: 0-1.5 min, mobile phase B is 10%; 1.5-4 min, mobile phase B is 10%-95%; 4-7 min, mobile phase B is 95%-100%; 7-8 min, mobile phase B is 100%-10%; 8-12 min, mobile phase B is 10%.

[0100] More preferably, the C18 chromatographic column is Thermo Betasil TM The C18 high performance liquid chromatography column has a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 5 μm.

[0101] More preferably, the column temperature of the C18 chromatographic column is 38-40°C.

[0102] More preferably, the column temperature of the C18 chromatographic column is 40°C.

[0103] More preferably, the chromatographic conditions of the ultra-high performance liquid chromatography also include: an injection volume of 4 to 5 μL; a flow rate of 0.4 to 0.5 mL / min.

[0104] Further preferably, the chromatographic conditions of the ultra-high performance liquid chromatography also include: injection volume of 5 μL; flow rate of 0.4 mL / min.

[0105] Preferably, the detection conditions of the mass spectrometry include: drying gas temperature 250°C, drying gas flow rate 5.0L / min, nebulizing gas pressure 35psi, sheath gas temperature 350°C, sheath gas flow rate 11L / min, capillary voltage 3500V, and nozzle voltage 500V.

[0106] Compared with the prior art, the present invention has the following beneficial effects:

[0107] The present invention adopts liquid-liquid extraction as a sample pretreatment method with simple and efficient operation, low cost and good repeatability, and combines it with isotope labeling method and ultra-performance liquid chromatography tandem mass spectrometry technology to achieve the separation and qualitative and quantitative detection of 6 kinds of p-phenylenediamine antioxidants and quinone derivatives in the sample. At the same time, it has the advantages of high sensitivity, small number of isotope internal standards and small solvent loss, and meets the actual detection requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 This is the TIC diagram when mobile phase A is 0.1 v / v% formic acid aqueous solution and mobile phase B is methanol.

[0109] Figure 2 This is the TIC diagram of mobile phase A being 0.1 v / v% ammonium acetate aqueous solution and mobile phase B being methanol.

[0110] Figure 3 This is the TIC diagram of mobile phase A being 0.1 v / v% formic acid aqueous solution and mobile phase B being acetonitrile.

[0111] Figure 4 This is the TIC diagram of mobile phase gradient condition 1 in Example 4.

[0112] Figure 5 This is the TIC diagram of mobile phase gradient condition 2 in Example 4.

[0113] Figure 6 This is the TIC diagram of mobile phase gradient condition 3 in Example 4. DETAILED DESCRIPTION

[0114] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0115] Example 1 Method for detecting six p-phenylenediamine antioxidants and quinone derivatives in a liquid sample Table 1 Information on p-phenylenediamine antioxidants and quinone derivatives

[0116]

[0117] The method provided in this embodiment detects the six p-phenylenediamine antioxidants and quinone derivatives shown in Table 1, and the specific steps are as follows:

[0118] 1. Reagents and internal standards

[0119] (1) Mixed standard solution

[0120] Acetonitrile was used to dissolve 6PPD standard, CPPD standard, IPPD standard, DPPD standard, DNPD standard and 6PPD-Q standard to obtain a mixed standard stock solution, wherein the final concentration of each compound standard was 1 μg / mL.

[0121] The mixed standard stock solution was then gradiently diluted with acetonitrile to obtain a mixed standard working solution with a final concentration of each compound standard of 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL or 100 ng / mL.

[0122] (2) Isotope-labeled internal standard

[0123] D5-6PPD-quinone (6PPD-Q-D5, CAS No.: 2750119-14-1) was used as an internal standard for detecting the six p-phenylenediamine antioxidants and quinone derivatives.

[0124] (3) Treatment reagents

[0125] Antioxidant solution: 1 mmol / L glutathione solution, used to prevent the oxidation of p-phenylenediamine antioxidants. When used, the volume ratio of the antioxidant solution to the liquid sample is 4:5.

[0126] Extraction solvent A: methyl tert-butyl ether.

[0127] Extraction solvent B: dichloromethane-n-hexane solution, obtained by thoroughly mixing dichloromethane and n-hexane in a volume ratio of 1:1.

[0128] 2. Pretreatment of liquid samples

[0129] Take 250 μL of the liquid sample to be tested, add 0.625 ng of the internal standard, mix with 200 μL of the antioxidant solution, and shake at 27° C. for half an hour to ensure full balance to obtain a mixed solution to be tested.

[0130] 250 μL of the mixed solution to be tested was mixed with 2.5 mL of extraction solvent A, vortexed for 10 min, ultrasonicated (frequency 80 KHz, power 700 W) for 15 min, and centrifuged at 4500 rpm for 5 min. After centrifugation, the solution was separated into two layers, and the upper layer solution was collected to obtain organic phase 1, and the lower layer solution was collected to obtain aqueous phase 1; aqueous phase 1 was mixed with 2.5 mL of extraction solvent A, vortexed for 10 min, ultrasonicated (frequency 80 KHz, power 700 W) for 15 min, and centrifuged at 4500 rpm for 5 min. After centrifugation, the solution was separated into two layers, and the upper layer solution was collected. The solution was obtained by mixing the organic phase 2, and the lower layer solution was collected to obtain the aqueous phase 2; 400 μL of saturated sodium chloride solution was then added to the aqueous phase 2 (used to cause the "salting-out effect" to promote stratification), and then mixed with 2.5 mL of extraction solvent B, vortexed for 10 minutes, ultrasonicated (frequency 80 KHz, power 700 W) for 15 minutes, and centrifuged at 4500 rpm for 5 minutes. After centrifugation, the solution was separated into two layers, and the upper layer solution was collected to obtain the organic phase 3; the organic phases 1 to 3 were combined, mixed, and blown to near dryness with nitrogen, and finally 125 μL of acetonitrile was added to dissolve, and the sample to be tested was obtained after filtering through a 0.22 μm PTFE filter membrane for standby use.

[0131] 3. Ultra-high performance liquid chromatography tandem mass spectrometry detection

[0132] (1) Instruments and equipment

[0133] The liquid chromatography instrument was an Agilent 1290 Infnity II ultra-high performance liquid chromatography system, and the mass spectrometry instrument was an Agilent G6470B electrospray triple quadrupole mass spectrometer.

[0134] (2) Liquid phase detection conditions

[0135] Thermo Betasil TM The C18 HPLC column had a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 5 μm. The gradient elution was performed with 0.1 v / v% formic acid aqueous solution (mobile phase A) and methanol (mobile phase B). The column temperature was 40°C, the injection volume was 5 μL, the flow rate was 0.4 mL / min, and the gradient elution program was shown in Table 2.

[0136] Table 2 Gradient elution program

[0137] Time (min) Formic acid water (v / v%) Methanol (v / v%) 0 90 10 1.5 90 10 4 5 95 7 0 100 8 90 10 12 90 10

[0138] (3) Mass spectrometry conditions

[0139] The data were collected in positive ion mode. The tandem mass spectrometry conditions used electrospray ionization. The detection conditions were: drying gas temperature 250°C, drying gas flow rate 5.0 L / min, nebulizing gas pressure 35 psi, sheath gas temperature 350°C, sheath gas flow rate 11 L / min, capillary voltage 3500 V, nozzle voltage 500 V. The specific multiple reaction mass spectrometry parameters are shown in Table 3.

[0140] Table 3 Specific parameters of multiple reaction mass spectrometry

[0141]

[0142]

[0143] 4. Results Analysis

[0144] A standard curve was established based on the peak area and concentration of the mixed standard working solution, and the peak area of ​​the sample to be tested was substituted into the standard curve to calculate the content of each compound.

[0145] Example 2: Recovery of 6 p-phenylenediamine antioxidants and quinone derivatives in cerebrospinal fluid

[0146] 1. Preparation of spiked samples

[0147] The internal standard and six standards of p-phenylenediamine antioxidants and quinone derivatives were added to 250 μL artificial cerebrospinal fluid to obtain spiked samples for determination of recovery. The spiked concentrations of the six compounds were set to 5 ng / mL and 10 ng / mL, respectively. Three parallel experiments were performed for each concentration, and an unspiked artificial cerebrospinal fluid was set as a blank control. In each gradient concentration, the spiked amount of each compound was the same, that is, the same spiked amount was added to each compound.

[0148] 2. Detection

[0149] The spiked sample prepared in this example was used as the liquid sample to be tested, and the content of each compound therein was tested according to the method of Example 1.

[0150] 3. Results

[0151] Standard curves were drawn based on the peak areas and concentrations of the standard working solutions of the six compounds, with the horizontal axis representing the relative concentration and the vertical axis representing the relative response value. Each compound was quantified using a standard curve at more than ten concentration levels. The resulting linear regression equation is shown in Table 4. It can be seen that the six target analytes had a good linear relationship in the concentration range of 0.01 to 50 ng / mL, and the correlation coefficients (all greater than 0.99) could be used for subsequent calculation of the contents of the six compounds in the spiked samples.

[0152] Table 4 Linear regression results of p-phenylenediamine antioxidants and quinone derivatives

[0153] Compound Concentration range (ng / mL) Regression equation <![CDATA[Coefficient of correlation R 2 > IPPD 0.01～50 <![CDATA[y=3.820519x 2 +10.176047x+0.001923]]> 0.9989 CPPD 0.01～50 <h2 style=";text-align:left;direction:ltr"><![CDATA[y=0.641525x <h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> +5.558927x+0.002826]]><h2 style=";text-align:left;direction:ltr"> 0.9997 DPPD 0.01～50 <![CDATA[y=0.309597x 2 +0.740176x+0.001094]]> 0.9977 DNPD 0.01～50 <![CDATA[y=-0.075855x 2 +0.198379x-0.000176]]> 0.9989 6PPD 0.01～50 <![CDATA[y=3.129596x 2 +10.697221x+0.002523]]> 0.9989 6PPD-Q 0.01～50 <![CDATA[y=0.147897x 2 +2.043164x+0.000204]]> 0.9999

[0154] The quantitative limits and spiked recoveries of the six target analytes in artificial cerebrospinal fluid are shown in Table 5. It can be seen that in the blank control, except for IPPD, the other p-phenylenediamine antioxidants and quinone derivatives had no background values. The spiked recoveries of the six target analytes at three spiked concentrations were between 72% and 109%, and the relative deviation did not exceed 15%, which met the recognized recovery standards (70% to 120%) and could accurately achieve the simultaneous extraction and determination of the six p-phenylenediamine antioxidants and quinone derivatives.

[0155] Table 5 Recovery results of p-phenylenediamine antioxidants and quinone derivatives in artificial cerebrospinal fluid

[0156]

[0157] Note: LOD stands for limit of detection.

[0158] Example 3 Detection of 6 p-phenylenediamine antioxidants and quinone derivatives in real cerebrospinal fluid

[0159] 19 cerebrospinal fluid samples from donors were collected as liquid samples to be tested, and the contents of 6 p-phenylenediamine antioxidants and quinone derivatives were detected by the method of Example 1. As shown in Table 6, in these real cerebrospinal fluids, the detection rates of IPPD, DPPD and 6PPD-Q were the highest, all 100%; followed by 6PPD, with a detection rate of 94.74%; the detection rates of CPPD and DNPD were lower, which may be due to the fact that cerebrospinal fluid samples do not necessarily contain all p-phenylenediamine antioxidants and quinone derivatives. The above results show that the method provided by the present invention can detect the presence and content of p-phenylenediamines and their quinone derivatives in cerebrospinal fluid samples.

[0160] Table 6 Detection of 6 p-phenylenediamines and their quinone derivatives in 19 cerebrospinal fluid samples

[0161]

[0162] Note: LOQ stands for limit of quantitation.

[0163] Example 4: Recovery of 6 p-phenylenediamine antioxidants and quinone derivatives in real urine

[0164] 1. Preparation of spiked samples

[0165] The spiked sample was prepared using the urine from the donor as the matrix according to the method of Example 2, and the unspiked donor urine was set as a blank control.

[0166] 2. Detection

[0167] The spiked sample prepared in this example was used as the liquid sample to be tested, and the content of each compound therein was tested according to the method of Example 1.

[0168] 3. Results

[0169] The quantitative limits and spiked recoveries of the six target analytes in real urine are shown in Table 7. It can be seen that in the blank control, except for IPPD, the other p-phenylenediamine antioxidants and quinone derivatives had no background values. The spiked recoveries of the six target analytes at three spiked concentrations were between 72% and 110%, and the relative deviation did not exceed 15%, which met the recognized recovery standard (70% to 120%), and could accurately achieve the simultaneous extraction and determination of the six p-phenylenediamine antioxidants and quinone derivatives.

[0170] Table 7 Spiked recovery results of p-phenylenediamine antioxidants and quinone derivatives in real urine

[0171]

[0172] Note: LOQ stands for limit of quantitation.

[0173] Example 5: Recovery of 6 p-phenylenediamine antioxidants and quinone derivatives in artificial serum

[0174] 1. Preparation of spiked samples

[0175] The spiked samples were prepared with artificial serum as the matrix according to the method of Example 2, and unspiked artificial serum was set as a blank control.

[0176] 2. Detection

[0177] The spiked sample prepared in this example was used as the liquid sample to be tested, and the content of each compound therein was tested according to the method of Example 1.

[0178] 3. Results

[0179] The quantitative limits and spiked recoveries of the six target analytes in artificial serum are shown in Table 8. It can be seen that in the blank control, except for IPPD, the other p-phenylenediamine antioxidants and quinone derivatives had no background values. The spiked recoveries of the six target analytes at three spiked concentrations were between 77% and 108%, and the relative deviation did not exceed 15%, which met the recognized recovery standards (70% to 120%) and could accurately achieve the simultaneous extraction and determination of the six p-phenylenediamine antioxidants and quinone derivatives.

[0180] Table 8 Spiked recovery results of p-phenylenediamine antioxidants and quinone derivatives in artificial serum

[0181]

[0182]

[0183] Note: LOQ stands for limit of quantitation.

[0184] Example 6 Effect of different conditions on test results

[0185] 1. Effect of extraction reagents on test results

[0186] A mixed standard working solution of 6 compound standards with a final concentration of 10 ng / mL was used as the liquid sample to be tested, and the test was performed according to the method of Example 1, with the only difference that in the step "pretreatment of cerebrospinal fluid sample", the sample to be tested was prepared with the extraction solvent shown in Table 9.

[0187] Table 9 Different extraction reagent combinations of p-phenylenediamines and their quinone derivatives

[0188]

[0189] Note: Combination 2 is the extraction reagent combination in Example 1.

[0190] As shown in Table 10, when different extraction reagent combinations were used for extraction, combination 2 had the best extraction effect, and six compounds were successfully extracted with a recovery rate of 85.4% to 108.6%, which met the recognized recovery standard (70% to 120%). Methyl tert-butyl ether is a polar solvent, while a mixture of dichloromethane and n-hexane can effectively extract polar and non-polar compounds. The synergistic effect of the three solvents further enhanced the extraction capacity, and compared with other extraction solvent combinations, combination 2 is easier to remove by evaporation after extraction, reducing residues and facilitating subsequent treatment.

[0191] Table 10 Spiked recovery of different extraction reagent combinations

[0192]

[0193] 2. The influence of different extraction times on the test results

[0194] A mixed standard working solution of 6 compound standards with a final concentration of 10 ng / mL was used as the liquid sample to be tested, and the test was performed according to the method of Example 1, with the only difference being that in the step "pretreatment of liquid sample", the sample to be tested was prepared according to the number of extractions shown in Table 11.

[0195] Table 11 Methods with different extraction times

[0196]

[0197] Note: Extraction reagent A is methyl tert-butyl ether, extraction reagent B is dichloromethane-n-hexane solution (1:1, v / v), and method 1 is the extraction times of each extraction reagent in Example 1.

[0198] As shown in Table 12, when different extraction times are used for extraction, the recovery rate of method 1 is the best overall, ranging from 85.4% to 108.6%, which meets the recognized recovery standard (70% to 120%); when the extraction times of methyl tert-butyl ether are increased alone, although the recovery rate is also within the standard range, the recovery rates of IPPD and 6PPD are relatively low; when the extraction times of methyl tert-butyl ether and the extraction times of dichloromethane-n-hexane solution are increased at the same time, the recovery rate of 6PPD is lower than the lower limit of the recovery standard. The method of increasing the number of extractions is not only time-consuming and cost-consuming, but also cannot meet the qualified spike recovery standard. Therefore, method 1 is the best extraction number selection.

[0199] Table 12 Spiked recovery rates at different extraction times

[0200]

[0201] 3. The influence of different mobile phases on the test results

[0202] A mixed standard working solution with a final concentration of 10 ng / mL of each of the six compound standards was used as the liquid sample to be tested, and then the test was performed according to the method of Example 1, except that in the step "ultra-performance liquid chromatography tandem mass spectrometry detection", the mobile phase was changed to: ① 0.1 v / v% ammonium acetate aqueous solution (mobile phase A) and methanol (mobile phase B); ② 0.1 v / v% formic acid aqueous solution (phase A) and acetonitrile (phase B). A TIC (Total Ion Chromatogram) diagram was drawn based on the test results.

[0203] like Figure 1 As shown, when the detection was performed using 0.1 v / v% formic acid aqueous solution (mobile phase A) and methanol (mobile phase B) in Example 1, the peak shape of the target compound was good, the response was good, and the material separation was obvious. Figure 2 As shown in the figure, when 0.1 v / v% ammonium acetate aqueous solution is used instead of 0.1 v / v% formic acid aqueous solution as mobile phase A, the peak area of ​​the target compound is significantly reduced, the response is reduced, and the separation effect is also worse. Figure 3 As shown, when acetonitrile is used instead of methanol as mobile phase B, the peak shape is worse, the response is reduced, and miscellaneous peaks appear. Therefore, the 0.1 v / v% formic acid aqueous solution-methanol mobile phase system of Example 1 has the best response and separation effect for detecting each compound.

[0204] 4. Effect of different elution gradients on detection results

[0205] A mixed standard working solution of 6 compound standards with a final concentration of 1 ng / mL was used as the liquid sample to be tested, and the test was performed according to the method of Example 1, except that in the step "ultra-performance liquid chromatography tandem mass spectrometry detection", the test was performed according to the gradient elution program shown in Table 13. A TIC diagram was drawn based on the test results.

[0206] Table 13 Different gradient elution programs

[0207]

[0208] Note: Procedure 2 is the elution gradient condition in Example 1.

[0209] The TIC diagrams for conditions 1 to 3 are as follows: Figures 4 to 6 As shown, compared with procedure 2, the response of the target compound decreased and the separation effect became worse when using procedure 1 and procedure 3 for gradient elution. Therefore, the response and separation effect of each compound detected by the elution gradient of Example 1 are the best.

[0210] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for extracting p-phenylenediamine antioxidants from a liquid sample, characterized in that: The p-phenylenediamine compound includes at least one of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, N-cyclohexyl-N'-phenyl-p-phenylenediamine, N-isopropyl-N'-phenyl-p-phenylenediamine, N,N'-diphenyl-1,4-phenylenediamine, N,N'-di-2-naphthyl-p-phenylenediamine and 2-((4-methylpentane-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione; The method comprises the following steps: S1. Fully mixing the liquid sample and the antioxidant to obtain a sample mixture; S2. The methyl tert-butyl ether and the liquid sample mixture are thoroughly mixed and separated into layers to obtain an organic phase 1 and an aqueous phase; S3. The aqueous phase obtained in step S2 is thoroughly mixed with a saturated sodium chloride solution and a dichloromethane-n-hexane solution, and the layers are separated to obtain an organic phase 2; S4. Combining the organic phase 1 obtained in step S2 and the organic phase 2 obtained in step S3 to obtain an extract containing the p-phenylenediamine compound.

2. The method according to claim 1, characterized in that The liquid sample includes a body fluid sample.

3. The method according to claim 1, characterized in that The dichloromethane-n-hexane solution is obtained by fully mixing dichloromethane and n-hexane in a volume ratio of 1:(1-2).

4. The method according to claim 1, characterized in that: The volume ratio of the sample mixture, methyl tert-butyl ether, saturated sodium chloride solution and dichloromethane-n-hexane solution is 0.25:(5-7.5):0.4:2.

5.

5. The method according to claim 1, characterized in that The antioxidant includes 1-2 mmol / L glutathione solution.

6. Use of the method according to any one of claims 1 to 5 in detecting p-phenylenediamine compounds.

7. A method for detecting p-phenylenediamine antioxidants, characterized in that: The method comprises the following steps: extracting the sample to be tested by the method according to claim 1 to obtain an extract, and then detecting the extract by ultra-high performance liquid chromatography.

8. The method according to claim 7, characterized in that In step S1 of the method of claim 1, the sample to be tested is fully mixed with the internal standard and the antioxidant to obtain a sample mixture.

9. The method according to claim 8, characterized in that The internal standard is isotope-labeled 2-((4-methylpentane-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione.

10. The method according to any one of claims 7 to 9, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography include: using a C18 chromatographic column; using 0.1 v / v% formic acid aqueous solution as mobile phase A, using methanol as mobile phase B, and in the gradient elution program, the volume percentage of mobile phase B changes as follows: 0-1.5 min, mobile phase B is 10%; 1.5-4 min, mobile phase B is 10%-95%; 4-7 min, mobile phase B is 95%-100%; 7-8 min, mobile phase B is 100%-10%; 8-12 min, mobile phase B is 10%.

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