Process for the chiral resolution of n-(1,3-dimethylbutyl)-n'-phenyl-p-phenylenediamine and its quinone, analytical method and use thereof

Abstract

The application discloses a chiral resolution method, an analysis method and application of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone (6PPDQ), and belongs to the technical field of separation and analysis of organic pollutants; the chiral resolution method of 6PPD and its quinone 6PPDQ is separated by using a preparative supercritical fluid liquid chromatography system, and chiral isomers of 6PPD or chiral isomers of 6PPDQ can be effectively obtained; thus, the blank in the research on the chiral isomers of 6PPD and 6PPDQ in the prior art can be overcome, and a material basis is laid for further exploring the influence of the chiral isomers of 6PPD and 6PPDQ on an ecological environment.

Description

Technical Field

[0001] This invention belongs to the field of separation and analysis technology of organic pollutants, and particularly relates to a chiral resolution method, analytical method and application of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine and its quinone. Background Technology

[0002] N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is a typical rubber antioxidant. Due to its excellent thermal stability, resistance to degradation, oxidation, and elastic cracking, it is widely used in various rubber products such as tires, pipes, and belts. Global production and usage of 6PPD are continuously increasing, reaching 2.0 × 10⁻⁶ in 2020. 5 This represents approximately 54% of the total production of rubber antioxidants. The ozonation product of 6PPD, N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPDQ), has been proven to be a toxic substance and is one of the most toxic chemicals to aquatic organisms. The widespread use of 6PPD has led to its widespread distribution in the environment, along with its ozonation product 6PPDQ. Both 6PPD and 6PPDQ have been detected in global air, water, sediment, soil, and even human bodies, confirming them as a global environmental pollutant. It is noteworthy that both 6PPD and 6PPDQ contain one chiral carbon atom (e.g., ...). Figure 1 As shown in the figure, each has a pair of enantiomers that can rotate the vibration plane of polarized light in different directions. Current research mainly focuses on the environmental behavior of the racemic mixtures of 6PPD and 6PPDQ. However, 6PPD and 6PPDQ have been shown to have significantly different toxic effects on rainbow trout. The toxicity of the racemic mixtures 6PPDQ and S-6PPDQ is 1.9 and 2.6 times that of R-6PPDQ, respectively. Therefore, whether 6PPD and 6PPDQ have selective environmental behavior is worth noting. The methods for resolving and applying the chiral isomers of 6PPD and 6PPDQ need further research. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method that can effectively separate 6PPD and 6PPDQ chiral isomers and determine their optical rotation, while also being able to detect, analyze and apply the content of 6PPD and 6PPDQ chiral isomers in environmental samples.

[0004] To achieve the above objectives, the first aspect of the present invention provides a chiral resolution method for 6PPD and its quinone 6PPDQ, the method comprising the following steps:

[0005] Racemic 6PPD or racemic 6PPDQ was separated using a preparative supercritical fluid liquid chromatography system. The corresponding components were then collected, concentrated, and dried to obtain (+)-6PPD and (-)-6PPD, or (+)-6PPDQ and (-)-6PPDQ.

[0006] This invention provides a method for separating chiral isomers of 6PPD or 6PPDQ. By using a preparative supercritical liquid chromatography system, the chiral isomers of 6PPD or 6PPDQ can be effectively obtained. This overcomes the gap in the existing technology for the study of chiral isomers of 6PPD and 6PPDQ, and lays a material basis for further exploring the impact of chiral isomers of 6PPD and 6PPDQ on the ecological environment.

[0007] As a preferred embodiment of the chiral separation method of the present invention, the preparative supercritical fluid liquid chromatography system is a Waters Prep 150 preparative liquid chromatography system.

[0008] In a preferred embodiment of the chiral separation method of the present invention, the mobile phase used in the separation is supercritical fluid carbon dioxide and ethanol.

[0009] In a preferred embodiment of the chiral separation method of the present invention, the elution method in the separation is isocratic elution.

[0010] As a preferred embodiment of the chiral separation method described in this invention, the separation chromatographic parameters for racemic 6PPD are as follows:

[0011] Chromatographic column: OJ normal phase chiral chromatographic column, specifications: 250×50mm, 10μm, Daicel;

[0012] Mobile phase: 40% supercritical carbon dioxide and 60% ethanol, wherein the ethanol contains 0.01% ammonia;

[0013] Flow rate: 120 g / min;

[0014] Wavelength: 220nm;

[0015] Column temperature: 35℃.

[0016] As a preferred embodiment of the chiral separation method described in this invention, the separation chromatographic parameters for racemic 6PPDQ are as follows:

[0017] Chromatographic column: IG chiral column, 250×30mm, 10μm, Daicel;

[0018] Mobile phase: 67% supercritical carbon dioxide and 33% ethanol;

[0019] Flow rate: 150 g / min;

[0020] Wavelength: 220nm;

[0021] Column temperature: 35℃.

[0022] It should be noted that after obtaining the separated chiral isomers, a polarimeter was used to test the separated chiral isomers.

[0023] The polarimeter in question is an Anton Parr MCP500 polarimeter.

[0024] The optical rotation test procedure is as follows: 1.0 mg of the separated chiral isomer is weighed and dissolved in 1 mL of methanol to obtain the test solution; then the test solution is filled into the sample tube of the polarimeter, and the optical rotation of each chiral isomer is calculated according to the specific rotation value displayed by the polarimeter.

[0025] A second aspect of the present invention provides an analytical method for the chiral isomers of 6PPD and 6PPDQ, the analytical method comprising the following steps:

[0026] The chiral isomers of 6PPD and 6PPDQ were detected by supercritical liquid chromatography (SCLC). The parameters of the SCLC are as follows:

[0027] Supercritical liquid chromatography system: Waters ACQUITY UPC2 system;

[0028] Chromatographic column: OD-3 chiral column, 150×3.0mm, 3.0μm, Daicel;

[0029] Mobile phases: Phase A is supercritical carbon dioxide, and Phase B is methanol;

[0030] Elution program: 0-10 min, 98% phase A, 2% phase B; 10-10.1 min, 98-95% phase A, 2-5% phase B; 10.1-16 min, 95% phase A, 5% phase B; 16-16.1 min, 95-98% phase A, 5-2% phase B; equilibration 3.9 min;

[0031] Injection volume: 1-5 μL;

[0032] Column temperature: 40-50℃;

[0033] Flow rate: 1.0-2.5 mL / min.

[0034] The analytical method for chiral isomers of 6PPD and 6PPDQ provided by this invention, by selecting appropriate parameters for a supercritical liquid chromatograph, can effectively separate not only 6PPD and 6PPDQ, but also their chiral isomers, thus enabling the efficient detection of both. The analytical method provided by this invention yields chiral isomers of 6PPD and 6PPDQ with good peak shapes and high resolution; it enables the separation of 6PPD and 6PPDQ, as well as their chiral isomers, under the same analytical conditions, thereby improving the analytical efficiency and accuracy in subsequent applications.

[0035] As a preferred embodiment of the analytical method described in this invention, the parameters of the supercritical liquid chromatograph are as follows:

[0036] Injection volume: 3 μL;

[0037] Column temperature: 45℃;

[0038] Flow rate: 1.5 mL / min.

[0039] The present invention has found that when the parameters of the supercritical liquid chromatograph are further selected to the above values, the separation degree of the chiral isomers of 6PPD and 6PPDQ is higher and the peak shape is better.

[0040] A third aspect of the invention provides the application of the analytical method for the chiral isomers of 6PPD and 6PPDQ in determining the content of 6PPD and 6PPDQ chiral isomers in environmental samples.

[0041] As a preferred embodiment of the application described in this invention, the environmental sample includes any one of water sample, sediment sample, and organism sample.

[0042] Preferably, the organism sample includes any one of crabs, fish, or birds.

[0043] For example, the crabs include the rectangular crab; the fish include the gudgeon, mullet, eel, catfish, conger eel, filamentous catfish, and trevally; and the birds include the white-breasted waterhen and the brown-winged cuckoo.

[0044] As a preferred embodiment of the application described in this invention, the measurement method includes the following steps:

[0045] (1) After dissolving or insoluble in an organic solvent, the environmental sample is filtered, the filtrate is collected and concentrated, and then enriched and purified by solid phase extraction column to obtain the solution to be tested.

[0046] (2) The solution to be tested is analyzed by a triple quadrupole mass spectrometer in series using the analytical method described in this invention. The qualitative analysis is based on the retention time, and the quantitative analysis is performed by calibrating the standard curve using the multi-point external standard method. The contents of the chiral isomers of 6PPD and 6PPDQ in the environmental sample are calculated.

[0047] This invention has found that by using the above-mentioned measurement process, the contents of 6PPD and 6PPDQ chiral isomers in environmental samples can be effectively analyzed simultaneously, providing technical support for studying their environmental behavior. It also has certain technical application prospects for studying their toxic effects at the enantiomeric level and accurately assessing their ecological risks.

[0048] In a preferred embodiment of the application described in this invention, when the environmental sample is a water sample, the environmental sample is filtered, the filtrate is collected and enriched and purified by solid-phase extraction column to obtain the solution to be tested.

[0049] In a preferred embodiment of the application described in this invention, when the environmental sample is a sediment sample, the organic solvent includes acetonitrile, dichloromethane, and n-hexane.

[0050] Preferably, when the environmental sample is a sediment sample, acetonitrile is first added for dissolution and extraction, followed by a mixed solution of dichloromethane and n-hexane for dissolution and extraction. Finally, the filtrate after extraction with the mixed solution of dichloromethane and n-hexane is collected and enriched and purified by solid-phase extraction column.

[0051] Preferably, in the mixed solution of dichloromethane and n-hexane, the volume ratio of dichloromethane to n-hexane is 1:(0.8-1.2).

[0052] More preferably, in the mixed solution of dichloromethane and n-hexane, the volume ratio of dichloromethane to n-hexane is 1:1.

[0053] Preferably, the acetonitrile dissolution and extraction is performed once, and the mixed solution of dichloromethane and n-hexane is used for dissolution and extraction twice.

[0054] Preferably, the acetonitrile dissolution and extraction time is 4-6 min, and the dissolution and extraction time of the mixed solution of dichloromethane and n-hexane is 12-18 min.

[0055] Preferably, the mass-to-volume ratio of the sediment sample to the organic solvent is 3g:(16-24)mL.

[0056] In a preferred embodiment of the application described in this invention, when the environmental sample is a biological sample, the organic solvent includes acetonitrile and dichloromethane.

[0057] Preferably, when the environmental sample is a biological sample, acetonitrile is added first for dissolution and extraction, followed by dichloromethane for dissolution and extraction.

[0058] Preferably, the acetonitrile dissolution and extraction are performed twice, and the dichloromethane dissolution and extraction are performed twice.

[0059] Preferably, the acetonitrile extraction time is 55-65 min, and the dichloromethane extraction time is 25-35 min.

[0060] Preferably, the mass-to-volume ratio of the biological sample to the organic solvent is 1.5 g:(6-14) mL.

[0061] In a preferred embodiment of the application described in this invention, the solid-phase extraction column used for enrichment and purification is an Oasis HLB column with a specification of 6cc and 200mg.

[0062] In a preferred embodiment of the application described in this invention, the solid-phase extraction column is activated sequentially with methanol and ultrapure water before use.

[0063] In a preferred embodiment of the application described in this invention, the elution solvent in the enrichment and purification is methanol, and the amount of elution solvent used is 8-12 mL.

[0064] Preferably, the amount of elution solvent used is 10 mL.

[0065] As a preferred embodiment of the application described in this invention, when the environmental sample is a biological sample, after collecting the filtrate and before enrichment and purification by solid-phase extraction column, the process further includes adding C18 and PSA adsorbents to the filtrate for purification, followed by freezing and degreasing the purified liquid.

[0066] Preferably, 55-65 mg of C18 adsorbent and 110-130 mg of PSA adsorbent are added per 1.5 g of biological sample.

[0067] Preferably, the solvent for the cryogenic defatting is methanol, the freezing temperature is (-25)-(-15)℃, the freezing time is 115-125min, the number of freezing times is 2-3, and the solvent usage is 8-12mL per 1.5g of biological sample.

[0068] As a preferred embodiment of the application described in this invention, the parameters of the triple quadrupole mass spectrometer are as follows:

[0069] Ionization method: Electrospray ionization, positive ion mode;

[0070] Mass spectrometry scanning mode: Multiple reaction monitoring (MRM) mode;

[0071] Ion source parameters: ion source temperature is 145-155℃, desolvation gas temperature is 495-505℃, conical orifice gas flow rate is 145-155L / h, desolvation gas flow rate is 990-1010L / h, capillary voltage is 0.7-0.9kV, and conical orifice voltage is 25-35V.

[0072] Detected ions: 6PPD precursor ion 269.1, daughter ions 168.0, 184.1; 6PPDQ precursor ion 299.1, daughter ions 187.1, 215.1, 241.1.

[0073] As a preferred embodiment of the application described in this invention, the parameters of the triple quadrupole mass spectrometer are as follows:

[0074] Ion source parameters: ion source temperature is 150℃, desolvation gas temperature is 500℃, conical orifice gas flow rate is 150L / h, desolvation gas flow rate is 1000L / h, capillary voltage is 0.8kV, and conical orifice voltage is 30V.

[0075] In a preferred embodiment of the application described in this invention, the concentrations of the calibration standard curves are 0.02, 0.05, 0.5, 5, 50, and 100 ng / mL, respectively.

[0076] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0077] 1. The chiral separation method for the racemic 6PPD and 6PPDQ proposed in this invention uses an OJ normal-phase chiral column and an IG chiral column to separate the chiral isomers of 6PPD and 6PPDQ, respectively. Supercritical fluids carbon dioxide and ethanol are used as the mobile phase, and isocratic elution is employed. The corresponding components are collected simultaneously, and the pure optical isomers can be obtained by removing the solvent. The chemical purity reaches more than 99%, which fills the technical deficiencies in the existing methods for separating and preparing chiral isomers of 6PPD and 6PPDQ.

[0078] 2. The supercritical fluid chromatography method proposed in this invention for analyzing 6PPD and 6PPDQ, based on an OD-3 chiral column and using gradient elution, can simultaneously achieve complete separation of the chiral isomers of 6PPD and 6PPDQ, thus overcoming the technical deficiencies in the separation and detection of existing chiral isomers of 6PPD and 6PPDQ.

[0079] 3. The method for determining 6PPD and 6PPDQ chiral isomers in water samples, sediment samples, and biological samples proposed in this invention has the characteristics of good sample processing effect, high analytical efficiency, and reliable quantitative results. It can be widely used in the detection of 6PPD and 6PPDQ chiral isomers in environmental samples, providing technical support for the study of their environmental behavior, and has certain technical application prospects in studying their toxic effects at the enantiomeric level and accurately assessing their ecological risks. Attached Figure Description

[0080] Figure 1 Chemical structure diagrams of the 6PPD and 6PPDQ chiral isomers:

[0081] Chemical structure diagram of the chiral isomer of A-6PPD

[0082] Chemical structure diagram of the chiral isomer of B-6PPDQ;

[0083] Figure 2 Chromatographic separation of 6PPD and 6PPDQ chiral isomers in 20 ng / mL standard solution, mangrove sediment samples, and organism samples:

[0084] Chromatographic separation of 6PPD and 6PPDQ chiral isomers in A-20 ng / mL standard solution.

[0085] Chromatographic separation of 6PPD chiral isomers in B-mangrove sediment samples

[0086] Chromatographic separation of 6PPDQ chiral isomers in C-mangrove sediment samples

[0087] Chromatographic separation of 6PPD chiral isomers in D-organism samples

[0088] Chromatographic separation of the 6PPDQ chiral isomer in an E-organism sample. Detailed Implementation

[0089] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0090] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in the field.

[0091] Racemic 6PPD: AccuStandard, USA;

[0092] Racemic 6PPDQ: Toronto Research Chemicals, Canada.

[0093] Example 1

[0094] This invention provides a method for resolving 6PPD chiral isomers, comprising the following steps:

[0095] Take 1g of racemic 6PPD and separate it using a Waters Prep 150 preparative liquid chromatography system. Then collect the corresponding components and remove the solvent by rotary evaporation to obtain (+)-6PPD and (-)-6PPD.

[0096] The separation chromatographic parameters are as follows:

[0097] Chromatographic column: OJ normal phase chiral chromatographic column, with specifications of 250×50mm and 10μm, and the packing material is cellulose-tris(4-methylbenzoate);

[0098] Mobile phase: 40% supercritical carbon dioxide and 60% ethanol, wherein the ethanol contains 0.1% ammonia;

[0099] Flow rate: 120 g / min;

[0100] Wavelength: 220nm;

[0101] Column temperature: 35℃.

[0102] Example 2

[0103] This invention provides a method for resolving 6PPDQ chiral isomers, comprising the following steps:

[0104] Take 1g of racemic 6PPDQ and separate it using a Waters Prep 150 preparative liquid chromatography system. Then collect the corresponding components and remove the solvent by rotary evaporation to obtain (+)-6PPDQ and (-)-6PPDQ.

[0105] The separation chromatographic parameters are as follows:

[0106] Chromatographic column: IG chiral column, 250×30mm, 10μm, packing material is linear starch-tris(3-chloro-5-methylphenylcarbamate);

[0107] Mobile phase: 67% supercritical carbon dioxide and 33% ethanol;

[0108] Flow rate: 150 g / min;

[0109] Wavelength: 220nm;

[0110] Column temperature: 35℃.

[0111] Example 1

[0112] The effects of this invention were investigated using examples. Example 1 showed that the (+)-6PPD and (-)-6PPD obtained after chiral resolution, and Example 2 showed that the (+)-6PPDQ and (-)-6PPDQ obtained after chiral resolution, had a chemical purity of over 99%. Polarimetry was used for testing, including the following steps: 1.0 mg of each separated chiral isomer was weighed and dissolved in 1 mL of methanol to obtain the test solution; then, the test solution was filled into the sample tube of the polarimeter, and the optical rotation of the chiral isomers was calculated based on the specific rotation value displayed by the polarimeter. The optical rotations of (+)-6PPD and (-)-6PPD were 1.3° and -2.3°, respectively, and the optical rotations of (+)-6PPDQ and (-)-6PPDQ were 413° and -72°, respectively.

[0113] Example A

[0114] This invention provides an analytical method for 6PPD and 6PPDQ chiral isomers, the analytical method comprising the following steps:

[0115] Racemic 6PPD and racemic 6PPDQ were detected by supercritical liquid chromatography (SCLC). The parameters of the SCLC are as follows:

[0116] Supercritical liquid chromatography system: Waters ACQUITY UPC2 system;

[0117] Chromatographic column: OD-3 chiral column, 150×3.0mm, 3.0μm, Daicel;

[0118] Mobile phases: Phase A is supercritical carbon dioxide, and Phase B is methanol;

[0119] Elution program: 0-10 min, 98% phase A, 2% phase B; 10-10.1 min, 98-95% phase A, 2-5% phase B; 10.1-16 min, 95% phase A, 5% phase B; 16-16.1 min, 95-98% phase A, 5-2% phase B; equilibration 3.9 min;

[0120] Injection volume: 3 μL;

[0121] Column temperature: 45℃;

[0122] Flow rate: 1.5 mL / min.

[0123] Example B

[0124] This invention provides an analytical method for the chiral isomers of 6PPD and 6PPDQ. The difference between this analytical method and Example A is that the elution program is as follows: 0-6 min, 98% phase A; 6-6.01 min, 98%-92% phase A; 6.01-10 min, 92% phase A; 10-10.01 min, 92%-98% phase A; 10.01-11 min, 98% phase A.

[0125] Example C

[0126] This invention provides an analytical method for the chiral isomers of 6PPD and 6PPDQ. The difference between this analytical method and Example A is that the elution program is as follows: 0-10 min, 98% A phase; 10-10.01 min, 98%-92% A phase; 10.01-16 min, 92% A phase; 16-16.1 min, 92%-98% A phase; 16.1-17.6 min, 98% A phase.

[0127] Example D

[0128] This invention provides an analytical method for the chiral isomers of 6PPD and 6PPDQ. The analytical method differs from that in Example A in that the elution program is as follows: 0-10 min, 98% A phase; 10-10.1 min, 98%-95% A phase; 10.1-16 min, 95%-90% A phase; 16-16.1 min, 90%-98% A phase; 16.1-20 min, 98% A phase.

[0129] Example E

[0130] This invention provides an analytical method for the chiral isomers of 6PPD and 6PPDQ. The analytical method differs from that in Example A in that the elution program is as follows: 0-10 min, 98% phase A; 10-10.01 min, 98%-95% phase A; 10.01-16 min, 95% phase A; 16-16.1 min, 95%-98% phase A; 16.1-20 min, 98% phase A.

[0131] Example F

[0132] This invention provides an analytical method for 6PPD and 6PPDQ chiral isomers, the difference between the analytical method and Example A is that the flow rate is 1.0 mL / min.

[0133] Example G

[0134] This invention provides an analytical method for 6PPD and 6PPDQ chiral isomers, the difference between the analytical method and Example A is that the flow rate is 2.0 mL / min.

[0135] Example H

[0136] This invention provides an analytical method for the chiral isomers of 6PPD and 6PPDQ, the difference between the analytical method and Example A being that the flow rate is 2.5 mL / min.

[0137] Example I

[0138] This invention provides an analytical method for chiral isomers of 6PPD and 6PPDQ, the difference between the analytical method and Example A is that the injection volume is 1 μL.

[0139] Example J

[0140] This invention provides an analytical method for chiral isomers of 6PPD and 6PPDQ, the difference between the analytical method and Example A is that the injection volume is 5 μL.

[0141] Example K

[0142] This invention provides an analytical method for the chiral isomers of 6PPD and 6PPDQ, the difference between the analytical method and Example A is that the column temperature is 40°C.

[0143] Example L

[0144] This invention provides an analytical method for chiral isomers of 6PPD and 6PPDQ, the difference between the analytical method and Example A is that the column temperature is 50°C.

[0145] Example 2

[0146] The retention times of 6PPD and 6PPDQ and the separation degree of the chiral isomers of 6PPD and 6PPDQ in the analytical method of Example AL of this invention are verified. The method for calculating the separation degree Rs is as follows:

[0147]

[0148] Where T1 and T2 represent the enantiomer retention times, and W1 and W2 represent the enantiomer chromatographic peak widths;

[0149] The results are shown in Tables 1 and 2.

[0150] Table 1

[0151]

[0152]

[0153] Table 2

[0154]

[0155] As can be seen from Tables 1 and 2, compared with Example AE, using supercritical fluid carbon dioxide and methanol as the mobile phase, and with elution gradients of 0-10 min (98% A phase), 10-10.1 min (98%-95% A phase), 10.1-16 min (95% A phase), 16-16.1 min (95%-98% A phase), and 16.1-20 min (98% A phase), the resolution of the chiral isomers of 6PPD and 6PPDQ is greater than 1.5 (1.64 and 1.62, respectively), achieving simultaneous detection of the enantiomers of 6PPD and 6PPDQ with baseline separation, and the corresponding enantiomer peaks are the highest. Under other elution gradient conditions, complete baseline separation of the chiral isomers of 6PPD and 6PPDQ cannot be achieved simultaneously, with resolutions ranging from 0.54 to 1.01. Comparing Examples A and FH, it is evident that the chiral separation effect varies under different mobile phase flow rates. Example A shows significantly better separation of the 6PPD and 6PPDQ enantiomers than Example FH, indicating that the separation effect of 6PPD and 6PPDQ enantiomers is optimal at a flow rate of 1.5 mL / min. Comparing Examples A and IJ, it is evident that the enantiomer separation effect is significantly better at an injection volume of 3 μL than in Example IJ. Comparing Examples A and KL, it is evident that the separation effect of the 6PPD and 6PPDQ chiral isomers is optimal at a column temperature of 45℃.

[0156] Application Example 1

[0157] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample, wherein the environmental sample is a sediment sample from the Qi'ao Island Mangrove Nature Reserve in Zhuhai. The determination method includes the following steps:

[0158] (1) Sediment sample processing: After freeze-drying the sediment sample, it was passed through an 80-mesh sieve. The sieve residue was collected, and 3.0 g of the residue was weighed into a clean Teflon centrifuge tube (washed sequentially with acetone, dichloromethane, and n-hexane). The tube was then dissolved in acetonitrile and a mixed solution of dichloromethane and n-hexane (volume ratio of dichloromethane to n-hexane was 1:1) and subjected to ultrasonic extraction. The solvent volume was 20 mL, the extraction temperature was 25 °C, the extraction time was 15 min, and the extraction was performed once with acetonitrile and twice with the mixed solution of dichloromethane and n-hexane. After extraction, the extract was concentrated by rotary evaporation, then redissolved in 0.5 mL of methanol and 50 mL of ultrapure water. The extract was then extracted using an Oasis HLB solid-phase extraction column (Waters, Oasis HLB 6cc Vac). Cartridge (200 mg) was purified and then washed twice with 6.5 wt% methanol aqueous solution and ultrapure water, respectively. The solution was then extracted under vacuum for 30 min and finally eluted with 10 mL of methanol. The eluent was collected, concentrated by rotation, dried under nitrogen, and diluted to 200 μL with acetonitrile. The solution was then filtered through a 0.22 μm filter membrane and the filtrate was collected to obtain the test solution.

[0159] The Oasis HLB solid-phase extraction column was activated with 10 mL of methanol and 10 mL of ultrapure water before use.

[0160] (2) The test solution was analyzed using a supercritical liquid chromatograph-triple quadrupole tandem mass spectrometer:

[0161] The parameters for supercritical liquid chromatography are as follows:

[0162] Supercritical liquid chromatography system: Waters ACQUITY UPC2 system;

[0163] Chromatographic column: OD-3 chiral column, 150×3.0mm, 3.0μm, Daicel;

[0164] Mobile phases: Phase A is supercritical carbon dioxide, and Phase B is methanol;

[0165] Elution program: 0-10 min, 98% phase A, 2% phase B; 10-10.1 min, 98-95% phase A, 2-5% phase B; 10.1-16 min, 95% phase A, 5% phase B; 16-16.1 min, 95-98% phase A, 5-2% phase B; equilibration 3.9 min;

[0166] Injection volume: 3 μL;

[0167] Column temperature: 45℃;

[0168] Flow rate: 1.5 mL / min;

[0169] The parameter conditions for the triple quadrupole tandem mass spectrometer are as follows:

[0170] Ionization method: Electrospray ionization, positive ion mode;

[0171] Mass spectrometry scanning mode: Multiple reaction monitoring (MRM) mode;

[0172] Ion source parameters: ion source temperature is 150℃, desolvation gas temperature is 500℃, conical gas flow rate is 150L / h, desolvation gas flow rate is 1000L / h, capillary voltage is 0.8kV, conical voltage is 30V, and the detected ions are shown in Table 3.

[0173] Table 3

[0174]

[0175] (3) Qualitative analysis was performed based on retention time, and quantitative analysis was performed using a multi-point (external standard method) calibration standard curve. The concentrations of the calibration standard curves were 0.02, 0.05, 0.5, 5, 50 and 100 ng / mL, respectively.

[0176] (4) Calculate the contents of (+)-6PPD, (-)-6PPD, (+)-6PPDQ and (-)-6PPDQ in the sediment sample.

[0177] Application Example 2

[0178] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in environmental samples. The only difference between this method and Application Example 1 is the environmental sample and its processing. The environmental sample in this application is a water sample from the Zhuhai Qi'ao Island Mangrove Nature Reserve. The water sample processing includes the following steps:

[0179] Take 1L of the collected water sample into a clean beaker, enrich and extract the sample using an Oasis HLB solid-phase extraction column, wash twice with 6.5wt% methanol aqueous solution and ultrapure water, then perform vacuum extraction for 30min, and finally elute with 10mL of methanol. Collect the eluent, then concentrate by rotation, dry with nitrogen, and make up to 200μL with acetonitrile. Filter through a 0.22μm filter membrane and collect the filtrate to obtain the solution to be tested.

[0180] Application Example 3

[0181] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in environmental samples. The only difference between this method and Application Example 1 is the environmental sample and its processing. The environmental sample in this application is a biological sample from the Zhuhai Qi'ao Island Mangrove Nature Reserve, specifically a gudgeon. The processing of the biological sample includes the following steps:

[0182] After cleaning and dissecting the biological sample, muscle tissue was freeze-dried, ground, and passed through an 80-mesh sieve. The sieve residue was collected, and 1.5g of the residue was mixed with 500mg of anhydrous sodium sulfate. The mixture was kept dry and placed in a Teflon centrifuge tube. The tissue was then extracted sequentially with acetonitrile and dichloromethane, followed by ultrasonic extraction. The solvent volume was 10mL, the extraction temperature was 40℃ for acetonitrile and 30℃ for dichloromethane, the extraction time was 60min for acetonitrile and 30min for dichloromethane, and the extraction was performed twice with acetonitrile and twice with dichloromethane. After extraction, 60mg of C18 and 120mg of sodium sulfate were added to the extract. PSA was purified with 120 mg of anhydrous magnesium sulfate. After vortexing for 1 min, the mixture was centrifuged, and the supernatant was collected and concentrated by rotary evaporation. The supernatant was redissolved in 10 mL of methanol and transferred to a glass test tube. The tube was then frozen at -20 °C for 120 min. This freezing operation was repeated twice, and the supernatant was collected. The supernatant was concentrated by rotary evaporation, dried under nitrogen, and brought to a final volume of 200 μL with acetonitrile. The solution was then filtered through a 0.22 μm filter membrane, and the filtrate was collected to obtain the solution to be tested.

[0183] Application Example 4

[0184] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is a long-legged crab.

[0185] Application Example 5

[0186] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is mullet.

[0187] Application Example 6

[0188] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is a tarpon.

[0189] Application Example 7

[0190] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is catfish.

[0191] Application Example 8

[0192] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is a moray eel.

[0193] Application Example 9

[0194] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is a filamentous catfish.

[0195] Application Example 10

[0196] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is a scorpion.

[0197] Application Example 11

[0198] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is a white-breasted waterhen.

[0199] Application Example 12

[0200] This invention provides a method for determining the content of 6PPD and 6PPDQ chiral isomers in an environmental sample. The only difference between this method and Application Example 3 is that the biological sample is a brown-winged cuckoo.

[0201] Example 3

[0202] The separation effect and corresponding content of 6PPD and 6PPDQ chiral isomers in environmental samples were investigated in Examples 1-12 of this invention. The results are shown in Tables 4-5.

[0203] Table 4

[0204]

[0205]

[0206] Table 5

[0207]

[0208] As can be seen from Tables 4-5, the resolution of the enantiomers of 6PPD and 6PPDQ was greater than 1.5 in all samples, achieving complete separation. In addition, within the mass concentration ranges of 0.02, 0.05, 0.5, 5, 50 and 100 ng / mL, the chromatographic peak area and mass concentration of the chiral isomers of 6PPD and 6PPDQ showed good linearity, with correlation coefficients greater than 0.999.

[0209] In addition, during the testing process, the chromatograms of the separation of 6PPD and 6PPDQ chiral isomers in the 20 ng / mL standard solution, sediment sample, and biological sample (Application Example 3) are shown in the figure. Figure 2As shown; from Figure 2 As can be seen from the above, the technical solution provided by the present invention can effectively separate and analyze the chiral isomers of 6PPD and 6PPDQ in environmental samples.

[0210] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A chiral resolution method for 6PPD and its quinone 6PPDQ, characterized in that, The method includes the following steps: Racemic 6PPD or racemic 6PPDQ was separated using a preparative supercritical fluid liquid chromatography system. The corresponding components were then collected, concentrated, and dried to obtain (+)-6PPD and (-)-6PPD, or (+)-6PPDQ and (-)-6PPDQ.

2. The chiral decomposition method according to claim 1, characterized in that, The preparative supercritical fluid liquid chromatography system is a Waters Prep 150 preparative liquid chromatography system; And / or, the mobile phase used in the separation is supercritical fluid carbon dioxide and ethanol; And / or, the elution method in the separation is isocratic elution.

3. The chiral separation method according to claim 2, characterized in that, The separation chromatographic parameters for racemic 6PPD are as follows: Chromatographic column: OJ normal phase chiral chromatographic column, specifications: 250×50mm, 10μm, Daicel; Mobile phase: 40% supercritical carbon dioxide and 60% ethanol, wherein the ethanol contains 0.01% ammonia; Flow rate: 120 g / min; Wavelength: 220nm; Column temperature: 35℃.

4. The chiral separation method according to claim 2, characterized in that, The separation chromatographic parameters for racemic 6PPDQ are as follows: Chromatographic column: IG chiral column, 250×30mm, 10μm, Daicel; Mobile phase: 67% supercritical carbon dioxide and 33% ethanol; Flow rate: 150 g / min; Wavelength: 220nm; Column temperature: 35℃.

5. An analytical method for 6PPD and 6PPDQ chiral isomers, characterized in that, The analytical method includes the following steps: The chiral isomers of 6PPD and 6PPDQ were detected by supercritical liquid chromatography (SCLC). The parameters of the SCLC are as follows: Supercritical liquid chromatography system: Waters ACQUITY UPC2 system; Chromatographic column: OD-3 chiral column, 150×3.0mm, 3.0μm, Daicel; Mobile phases: Phase A is supercritical carbon dioxide, and Phase B is methanol; Elution program: 0-10 min, 98% phase A, 2% phase B; 10-10.1 min, 98-95% phase A, 2-5% phase B; 10.1-16 min, 95% phase A, 5% phase B; 16-16.1 min, 95-98% phase A, 5-2% phase B; equilibration 3.9 min; Injection volume: 1-5 μL; Column temperature: 40-50℃; Flow rate: 1.0-2.5 mL / min.

6. The analytical method according to claim 5, characterized in that, The parameters of the supercritical liquid chromatograph are as follows: Injection volume: 3 μL; Column temperature: 45℃; Flow rate: 1.5 mL / min.

7. The application of the analytical method according to any one of claims 5-6 in determining the content of 6PPD and 6PPDQ chiral isomers in environmental samples.

8. The application according to claim 7, characterized in that, The environmental samples include any one of water samples, sediment samples, and biological samples.

9. The application according to claim 7, characterized in that, The determination method includes the following steps: (1) After dissolving or insoluble in an organic solvent, the environmental sample is filtered, the filtrate is collected and concentrated, and then enriched and purified by solid phase extraction column to obtain the solution to be tested. (2) The solution to be tested is analyzed by a triple quadrupole mass spectrometer in series with the analytical method described in any one of claims 5-6, and qualitative analysis is performed based on retention time. The standard curve is calibrated by multi-point external standard method for quantitative analysis, and the contents of 6PPD and 6PPDQ chiral isomers in the environmental sample are calculated.

10. The application according to claim 9, characterized in that, When the environmental sample is a water sample, the environmental sample is filtered, the filtrate is collected and enriched and purified by solid phase extraction column to obtain the solution to be tested; And / or, when the environmental sample is a sediment sample, the organic solvent includes acetonitrile, dichloromethane, and n-hexane; And / or, when the environmental sample is a biological sample, the organic solvent includes acetonitrile and dichloromethane.

11. The application according to claim 9, characterized in that, In the solid-phase extraction column enrichment and purification process, the solid-phase extraction column is an Oasis HLB column with a specification of 6cc and 200mg. And / or, the solid-phase extraction column is used after being activated sequentially with methanol and ultrapure water; And / or, the elution solvent in the enrichment and purification is methanol, and the amount of elution solvent used is 8-12 mL.

12. The application according to claim 9, characterized in that, When the environmental sample is a biological sample, after collecting the filtrate and before enrichment and purification by solid phase extraction column, the process includes adding C18 and PSA adsorbents to the filtrate for purification, followed by freezing and degreasing the purified liquid.

13. The application according to claim 9, characterized in that, The parameters of the triple quadrupole mass spectrometer are as follows: Ionization method: Electrospray ionization, positive ion mode; Mass spectrometry scanning mode: Multiple reaction monitoring (MRM) mode; Ion source parameters: ion source temperature is 145-155℃, desolvation gas temperature is 495-505℃, conical orifice gas flow rate is 145-155L / h, desolvation gas flow rate is 990-1010L / h, capillary voltage is 0.7-0.9kV, and conical orifice voltage is 25-35V. Detected ions: 6PPD precursor ion 269.1, daughter ions 168.0, 184.1; 6PPDQ precursor ion 299.1, daughter ions 187.1, 215.1, 241.

1.

14. The application according to claim 9, characterized in that, The parameters of the triple quadrupole mass spectrometer are as follows: Ion source parameters: ion source temperature is 150℃, desolvation gas temperature is 500℃, conical orifice gas flow rate is 150L / h, desolvation gas flow rate is 1000L / h, capillary voltage is 0.8kV, and conical orifice voltage is 30V.

15. The application according to claim 9, characterized in that, The concentrations of the calibration standard curves were 0.02, 0.05, 0.5, 5, 50, and 100 ng / mL, respectively.

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