A method for detecting a plurality of liquid crystal monomers in serum

Abstract

The application provides a method for detecting multiple liquid crystal monomers in serum, and belongs to the technical field of chemical detection. The detection method provided by the application comprises the following steps: mixing serum, a mixed internal standard solution and a precipitant to obtain a mixed solution; mixing the mixed solution with n-hexane to perform first liquid-liquid extraction to obtain first supernatant and first lower layer material; mixing the lower layer material with methyl tert-butyl ether to perform second liquid-liquid extraction to obtain second supernatant and second lower layer material; combining the first supernatant and the second supernatant, and then performing solid phase extraction to obtain eluent; the eluent used in the solid phase extraction is dichloromethane; the eluent is concentrated and then redissolved to obtain a to-be-detected solution; the to-be-detected solution is detected by gas chromatography-tandem mass spectrometry to obtain the content of multiple liquid crystal monomers in serum. The application uses n-hexane and methyl tert-butyl ether as extractants, thereby improving sensitivity and accuracy.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection technology, specifically relating to a method for detecting multiple liquid crystal monomers in serum. Background Technology

[0002] Liquid crystal monomers (LCMs) are a class of artificially synthesized chemical substances that can switch between liquid and solid states under different temperature conditions. Therefore, they possess both the mechanical properties of liquids (ease of flow and deformation) and the anisotropic characteristics of crystals (birefringence), and are widely used in electronic products such as smartphones, laptops, and LCD TVs. Previous studies have found that LCMs are similar to polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), potentially exhibiting persistence and long-distance migration. Due to their potential lipophilicity, LCMs can accumulate in the human body, necessitating research into their exposure status and health risks in human populations. Therefore, accurate measurement of LCMs in human serum is a necessary prerequisite for studying their health risks in human populations.

[0003] Currently, methods for detecting liquid crystal monomers, such as gas chromatography and liquid chromatography, have relatively low sensitivity and accuracy. Therefore, improving these detection methods to enhance sensitivity and accuracy has become a pressing technical problem in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting multiple liquid crystal monomers in serum. The detection method provided by this invention has high sensitivity and high accuracy.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting multiple liquid crystal monomers in serum, comprising the following steps: (1) Mix the serum, mixed internal standard solution and precipitant to obtain a mixture; (2) The mixture obtained in step (1) is mixed with n-hexane and subjected to a first liquid-liquid extraction to obtain a first supernatant and a first lower layer substance; (3) The first lower layer substance obtained in step (2) is mixed with methyl tert-butyl ether and subjected to a second liquid-liquid extraction to obtain a second supernatant and a second lower layer substance; (4) Combine the first supernatant obtained in step (2) and the second supernatant obtained in step (3), and then perform solid phase extraction to obtain an eluent; the eluent used in the solid phase extraction is dichloromethane; (5) Concentrate the eluent obtained in step (4) and then redissolve it to obtain the test solution; (6) The test solution obtained in step (5) is subjected to gas chromatography-tandem mass spectrometry to obtain the content of various liquid crystal monomers in serum.

[0006] Preferably, the volume ratio of serum in step (1) to n-hexane in step (2) is 1:(20~30).

[0007] Preferably, the volume ratio of serum in step (1) to n-hexane in step (2) is 1:24.

[0008] Preferably, the volume ratio of serum in step (1) to methyl tert-butyl ether in step (3) is 1:(20~30).

[0009] Preferably, the volume ratio of serum in step (1) to methyl tert-butyl ether in step (3) is 1:24.

[0010] Preferably, the extraction column used for solid-phase extraction in step (4) is HyperSep. TM Silica extraction cartridges.

[0011] Preferably, the volume ratio of serum in step (1) to elution buffer in step (4) is 1:(35~45).

[0012] Preferably, the gas chromatography conditions during gas chromatography-tandem mass spectrometry detection in step (6) include: splitless injection, injection port temperature of 280~300℃, carrier gas as helium, carrier gas flow rate of 0.5~1.5mL / min, column length of 25~35m, column inner diameter of 0.15~0.30mm, column film thickness of 0.15~0.30μm, and column temperature program as follows: 90~110℃ held for 1~5min, then increased to 150~200℃ at a rate of 10~20℃ / min, then increased to 202~210℃ at a rate of 1~5℃ / min and held for 5~10min, then increased to 220~240℃ at a rate of 1~5℃ / min, and finally increased to 290~310℃ at a rate of 10~20℃ / min and held for 1~5min.

[0013] Preferably, the mass spectrometry conditions during gas chromatography-tandem mass spectrometry detection in step (6) include: the temperature of the transmission line and the electron bombardment source are independently 290~310℃, multiple reaction monitoring mode is adopted, the collision gas is argon, the pressure of argon is 50~100Psi, and the solvent delay time is 4~6min.

[0014] This invention provides a method for detecting multiple liquid crystal monomers in serum, comprising the following steps: mixing serum, a mixed internal standard solution, and a precipitant to obtain a mixture; mixing the mixture with n-hexane and performing a first liquid-liquid extraction to obtain a first supernatant and a first lower layer; mixing the first lower layer with methyl tert-butyl ether and performing a second liquid-liquid extraction to obtain a second supernatant and a second lower layer; combining the first and second supernatants and then performing solid-phase extraction to obtain an eluent; the eluent used in the solid-phase extraction is dichloromethane; concentrating the eluent and then redissolving it to obtain a test solution; and performing gas chromatography-tandem mass spectrometry on the test solution to obtain the content of multiple liquid crystal monomers in the serum. This invention employs an internal standard method for quantification, effectively reducing the interference of matrix effects on the detection results and ensuring the reliability of the detection method. Based on the principle of "like dissolves like," hexane and methyl tert-butyl ether are used sequentially as extractants for two liquid-liquid extractions, thereby improving the extraction efficiency. Dichloromethane is used as the eluent during solid-phase extraction, possessing excellent elution capabilities, which further enhances the extraction efficiency, thus further improving the sensitivity and accuracy of the detection. Experimental results show that the average recoveries of 39 LCMs in the detection method provided by this invention range from 86.87% to 134.17%, with intra-day precision of 2.45% to 8.82% and inter-day precision of 2.01% to 13.15%. Attached Figure Description

[0015] Figure 1 The figure shows the chromatogram of the mixed standard solution of 39 LCMs in Example 1. In the figure, 1 to 39 correspond to 1 to 39 in Table 2. Detailed Implementation

[0016] This invention provides a method for detecting multiple liquid crystal monomers in serum, comprising the following steps: (1) Mix the serum, mixed internal standard solution and precipitant to obtain a mixture; (2) The mixture obtained in step (1) is mixed with n-hexane and subjected to a first liquid-liquid extraction to obtain a first supernatant and a first lower layer substance; (3) The first lower layer substance obtained in step (2) is mixed with methyl tert-butyl ether and subjected to a second liquid-liquid extraction to obtain a second supernatant and a second lower layer substance; (4) Combine the first supernatant obtained in step (2) and the second supernatant obtained in step (3), and then perform solid phase extraction to obtain an eluent; the eluent used in the solid phase extraction is dichloromethane; (5) Concentrate the eluent obtained in step (4) and then redissolve it to obtain the test solution; (6) The test solution obtained in step (5) is subjected to gas chromatography-tandem mass spectrometry to obtain the content of various liquid crystal monomers in serum.

[0017] The present invention does not have any special limitations on the source of the raw materials, and commercially available products known to those skilled in the art can be used.

[0018] The detection method provided by this invention is applicable to the detection of liquid crystal monomers in serum, including biphenyls and analogues (BAs), fluorinated biphenyls and analogues (FBAs), and cyanobiphenyls and analogues (CBAs). More preferably, it is applicable to the detection of 39 liquid crystal monomers in serum, including 4-(trans-4-vinylcyclohexyl)benzonitrile, 1-[(trans,trans)-4'-vinyl[1,1'-bicyclohexane]-4-yl]-4-methylbenzene, 4'-ethyl-4-cyanobiphenyl, 1-ethoxy-2,3-difluoro-4-(trans-4-propylcyclohexyl)benzene, 4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl, 4-ethoxy-4'-cyanobiphenyl, 4'-n-butyl-4-cyanobiphenyl, 1-butoxy-2,3-difluoro-4-(trans-4-propylcyclohexyl)benzene, 4-ethoxy-4'-cyanobiphenyl, 4'-n-butyl-4-cyanobiphenyl, and 1-butoxy-2,3-difluoro-4-(trans-4-propylcyclohexyl)benzene. 4-Propylcyclohexylbenzene, pentylbicyclohexyltrifluoromethoxybenzene, 4-propoxy-4'-cyanobiphenyl, trans,trans-4'-ethyl-4-(3,4,5-trifluorophenyl)bicyclohexane, 4'-n-pentyl-4-cyanobiphenyl, 3,4-difluoro-4'-(4-ethylcyclohexyl)biphenyl, 4-butoxy-4'-cyanobiphenyl, trans,trans-4'-propyl-4-(4-trifluoromethoxyphenyl)bicyclohexane, trans-4-(3,4,5-trifluorophenyl)-trans-4'-propylbicyclohexane, 2',3,4,5-tetrafluoro-4'-(trans-4-propylcyclohexyl)bicyclohexane Benzene, trans, trans-4-(4-fluorophenyl)-4'-propylbicyclohexane, trans, trans-4-(3,4-difluorophenyl)-4''-propylbicyclohexane, 4'-(trans-4-propylcyclohexyl)-3,4,5-trifluorobiphenyl, 3,4-difluoro-4'-(trans-4-propylcyclohexyl)biphenyl, 4-fluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl, 4-pentoxy-4'-cyanobiphenyl, 2',3,4,5-tetrafluoro-4''-propyl-1,1':4',1”-terphenyl, 4-cyanophenyl 4-butylbenzoate, trans, trans-4-(3, 4-Difluorophenyl)-4''-butylbicyclohexane, 4-heptyl-4'-cyanobiphenyl, 4'-cyano-4-hexyloxybiphenyl, p-pentylphenol 4-propylbenzoate, trans,trans-4-(3,4-difluorophenyl)-4'-pentylbicyclohexane, 4'-n-octyl-4-cyanobiphenyl, trans-4-(4-pentylcyclohexyl)-3',4'-difluoro-1,1'-biphenyl, 4-heptyloxy-4'-cyanobiphenyl, 4-ethyl-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl, 1-ethoxy-2,3-difluoro-4-[(trans,trans)-4'-propyl[1,1'-Bicyclohexane]-4-yl]benzene, 4'-(octoxy)biphenyl-4-carboxynitrile, 1-[4-(4-butylcyclohexyl)cyclohexyl]-4-ethoxy-2,3-difluorobenzene, 4-cyano-4”-pentyl-p-terphenyl and 4-[difluoro(3,4,5-trifluorophenoxy)methyl]-2',3,5-trifluoro-4”-propyl-1,1':4',1''-terphenyl.

[0019] The present invention does not have a specific limitation on the content of liquid crystal monomers in the serum, and any sample to be tested that is well known to those skilled in the art can be used.

[0020] This invention involves mixing serum, a mixed internal standard solution, and a precipitant to obtain a mixed solution.

[0021] In this invention, the concentration of the internal standard substance in the mixed internal standard solution is preferably 0.010~0.10 μg / mL. As one embodiment, the concentration of the internal standard substance can be 0.050 μg / mL.

[0022] In this invention, the internal standard substance in the mixed internal standard solution preferably includes 13 C 12 Labeled polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). This invention does not specifically limit the particular internal standard substance used; adjustments can be made based on actual testing requirements.

[0023] As one implementation method, the internal standard substance can be 13 C 12 Labeled 2,4,4'-trichlorobiphenyl ( 13 C 12 -PCB28) 13 C 12 Labeled 2,2',5,5'-tetrachlorobiphenyl ( 13 C 12 -PCB52), 13 C 12 Labeled 2,2',4,5,5'-pentachlorobiphenyl ( 13 C 12 -PCB101) 13 C 12 Labeled 2,2',3,4,4',5'-hexachlorobiphenyl ( 13 C 12 -PCB138) 13 C 12 Labeled 2,2',3,4,4',5'-hexachlorobiphenyl ( 13 C 12 -PCB153), 13 C 12 Labeled 2,3,3',4,5'-pentachlorobiphenyl (13 C 12 -PCB108) 13 C 12 Labeled 3,3',4,4'-tetrachlorobiphenyl ( 13 C 12 -PCB77), 13 C 12 Labeled 3,4,4',5-Tetrachlorobiphenyl ( 13 C 12 -PCB81) 13 C 12 Labeled 2,3,3',4,4'-pentachlorobiphenyl ( 13 C 12 -PCB105), 13 C 12 Labeled 2,3,4,4',5-pentachlorobiphenyl ( 13 C 12 -PCB114) 13 C 12 Labeled 2,3',4,4',5-pentachlorobiphenyl ( 13 C 12 -PCB118) 13 C 12 Labeled 2',3,4,4',5-pentachlorobiphenyl ( 13 C 12 -PCB123) 13 C 12 Labeled 3,3',4,4',5-pentachlorobiphenyl ( 13 C 12 -PCB126) 13 C 12 Labeled 2,3,3',4,4',5-hexachlorobiphenyl ( 13 C 12 -PCB156), 13 C 12 Labeled 2,3,3',4,4',5'-hexachlorobiphenyl ( 13 C 12 -PCB157), 13 C 12 Labeled 2,3',4,4',5,5'-hexachlorobiphenyl ( 13 C 12 -PCB167), 13 C 12 Labeled 3,3',4,4',5,5'-hexachlorobiphenyl ( 13 C 12 -PCB169), 13 C 12Labeled 2,3,3',4,4',5,5'-heptachlorobiphenyl ( 13 C 12 -PCB189), 13 C 12 Labeled 3,3',4,4'-tetrabromodiphenyl ether ( 13 C 12 -BDE77), 13 C 12 Labeled 2,2',3,4,4',5'-hexabromodiphenyl ether ( 13 C 12 -BDE138) 13 C 12 Labeled 2,4,4'-tribromodiphenyl ether ( 13 C 12 -BDE28), 13 C 12 Labeled 2,2',4,4'-tetrabromodiphenyl ether ( 13 C 12 -BDE47), 13 C 12 Labeled 2,2',4,4',5-pentabromodiphenyl ether ( 13 C 12 -BDE99), 13 C 12 Labeled 2,2',4,4',6-pentabromodiphenyl ether ( 13 C 12 -BDE100) 13 C 12 Labeled 2,2',4,4',5,5'-hexabromodiphenyl ether ( 13 C 12 -BDE153), 13 C 12 Labeled 2,2',4,4',5,6'-hexabromodiphenyl ether ( 13 C 12 -BDE154) and 13 C 12 Labeled 2,2',3,4,4',5,6-heptabromodiphenyl ether ( 13 C 12 Several of them (-BDE183).

[0024] In this invention, the precipitant preferably comprises an acidic solution and an alcohol; the acidic solution is preferably a sulfuric acid solution; the mass concentration of the acidic solution is preferably 95-98%; the alcohol is preferably methanol; and the volume ratio of the acidic solution to the alcohol is preferably 1:(2-8). In this invention, the precipitant can precipitate proteins and remove lipids.

[0025] In one embodiment, the mass concentration of the acidic solution can be 96% or 97%; the volume ratio of the pickling solution to the alcohol can be 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:6.7 or 1:7.

[0026] In this invention, the preferred volume ratio of serum to precipitant is 1:(2~7). As one embodiment, the volume ratio of serum to precipitant can be 1:2.5, 1:2.8, 1:3, 1:3.5, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:6, or 1:6.8. Limiting the volume ratio of serum to precipitant within the above range further improves the lipid removal effect.

[0027] In this invention, the preferred ratio of serum volume to the mass of the mixed internal standard solution is (200~300) μL: (0.1~1.0) ng. As one embodiment, the ratio can be (230~270) μL: (0.2~0.8) ng, or even 250 μL: 0.5 ng.

[0028] The present invention does not have any special limitations on the operation of mixing the serum, mixed internal standard solution and precipitant, and any technical solution for preparing the mixture well known to those skilled in the art can be used.

[0029] After obtaining the mixture, the present invention mixes the mixture with n-hexane and performs a first liquid-liquid extraction to obtain a first supernatant and a first lower layer substance.

[0030] In this invention, the preferred volume ratio of serum to n-hexane is 1:(20~30). As one embodiment, the volume ratio of serum to n-hexane can be 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, or 1:29. Limiting the volume ratio of serum to n-hexane to the above range improves extraction efficiency, thereby further enhancing the sensitivity and accuracy of detection.

[0031] The present invention does not have any special limitations on the operation of the first liquid-liquid extraction; any operation known to those skilled in the art can be used.

[0032] In one embodiment, the centrifugation speed during the first liquid-liquid extraction can be 3500 r / min, and the centrifugation time can be 5 min.

[0033] After obtaining the first lower layer substance, the present invention mixes the first lower layer substance with methyl tert-butyl ether and performs a second liquid-liquid extraction to obtain a second supernatant and a second lower layer substance.

[0034] In this invention, the preferred volume ratio of serum to methyl tert-butyl ether is 1:(20-30). As one embodiment, the volume ratio of serum to methyl tert-butyl ether can be 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, or 1:29. Limiting the volume ratio of serum to methyl tert-butyl ether to the above range improves extraction efficiency, thereby further enhancing the sensitivity and accuracy of detection.

[0035] The present invention does not have any special limitations on the operation of the second liquid-liquid extraction; any operation known to those skilled in the art can be used.

[0036] In one embodiment, the centrifugation speed during the second liquid-liquid extraction can be 3500 r / min, and the centrifugation time can be 5 min.

[0037] After obtaining the first supernatant and the second lower layer, the present invention combines the first supernatant and the second supernatant, and then performs solid-phase extraction to obtain the eluent.

[0038] The present invention does not have any special limitations on the operation of combining the first supernatant and the second supernatant; any operation known to those skilled in the art can be used.

[0039] After the merging is completed, the present invention preferably concentrates the product obtained by merging; the concentration is preferably carried out by nitrogen blowing. The present invention does not have any special limitations on the nitrogen blowing operation, and any operation known to those skilled in the art can be used.

[0040] The present invention does not have a special limitation on the concentration time; it is sufficient to concentrate to 1 / 12 to 1 / 6 of the original solution volume.

[0041] In this invention, the extraction column used for solid-phase extraction is preferably HyperSep. TM Silica extraction column. The extraction column described above further improves extraction efficiency, thereby further enhancing detection sensitivity and accuracy.

[0042] In this invention, the extraction column is preferably activated before use; the activating agent used for activation is preferably methanol and n-hexane in sequence; the volume ratio of methanol to n-hexane is preferably (1~2):(1~2). This invention does not impose any special limitation on the amount of the activating agent; any amount well known to those skilled in the art can be used.

[0043] In this invention, the eluent used in the solid-phase extraction is dichloromethane; the preferred volume ratio of serum to eluent is 1:(35~45). As one embodiment, the volume ratio of serum to eluent can be 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, or 1:44. The use of dichloromethane as the eluent in solid-phase extraction further improves extraction efficiency, thereby further enhancing the sensitivity and accuracy of detection; limiting the volume ratio of serum to eluent within the above range further improves extraction efficiency, thereby further enhancing the sensitivity and accuracy of detection.

[0044] In this invention, the flow rate of the eluent is preferably 0.5 to 1.5 mL / min. As one embodiment, the flow rate of the eluent can be 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, or 1.4 mL / min.

[0045] After obtaining the eluent, the present invention concentrates the eluent and then redissolves it to obtain the test solution.

[0046] In this invention, the eluent is preferably mixed with a protective solvent before concentration. The protective solvent prevents the loss of the target compound through volatilization during concentration.

[0047] In this invention, the protective solvent is preferably decane, dodecane, or tridecane; the volume ratio of the eluent to the protective solvent is preferably (500~2000):1. As one embodiment, the volume ratio of the eluent to the protective solvent can be 1000:1 or 1500:1.

[0048] In this invention, the concentration is preferably achieved by nitrogen blowing. This invention does not impose any particular limitations on the nitrogen blowing operation; any operation well-known to those skilled in the art can be used to blow nitrogen to near dryness.

[0049] In this invention, the solvent used for resolution is preferably an organic solvent, more preferably toluene; the volume ratio of the solvent to serum is preferably 1:(10~15). As one embodiment, the volume ratio of the solvent to serum can be 1:11, 1:12, 1:12.5, 1:13 or 1:14.

[0050] After obtaining the test solution, the present invention performs gas chromatography-tandem mass spectrometry (GC-MS / MS) to detect the content of various liquid crystal monomers in the serum.

[0051] In this invention, the preferred gas chromatography conditions for gas chromatography-tandem mass spectrometry detection include: splitless injection, injection volume of 1-2 μL, injection port temperature of 280-300℃, carrier gas as helium, carrier gas flow rate of 0.5-1.5 mL / min, column length of 25-35 m, column inner diameter of 0.15-0.30 mm, column film thickness of 0.15-0.30 μm, and column temperature program as follows: 90-110℃ held for 1-5 min, then increased to 150-200℃ at a rate of 10-20℃ / min, then increased to 202-210℃ at a rate of 1-5℃ / min and held for 5-10 min, then increased to 220-240℃ at a rate of 1-5℃ / min, and finally increased to 290-310℃ at a rate of 10-20℃ / min and held for 1-5 min. By limiting the gas chromatography conditions to the above-mentioned range, this invention can further improve the sensitivity and accuracy of detection.

[0052] As one implementation method, the gas chromatography conditions for gas chromatography-tandem mass spectrometry detection can be as follows: splitless injection, injection volume of 1 μL, injection port temperature of 290°C, carrier gas of helium at a flow rate of 1.0 mL / min, column length of 30 m, column inner diameter of 0.25 mm, column film thickness of 0.25 μm, and column temperature program of 100°C for 2 min, then increasing to 180°C at a rate of 15°C / min, then increasing to 205°C at a rate of 3°C / min for 7 min, then increasing to 230°C at a rate of 3°C / min, and finally increasing to 300°C at a rate of 15°C / min for 2 min.

[0053] This invention does not specify the type of chromatographic column; any instrument or equipment well-known to those skilled in the art can be used. As one embodiment, the chromatographic column can be a TG-5SilMS.

[0054] In this invention, the preferred mass spectrometry conditions for gas chromatography-tandem mass spectrometry detection include: the temperature of the transfer line and the electron impact source (EI) are independently set to 290–310 °C; multiple reaction monitoring (MRM) mode is used; the collision gas is argon; the argon pressure is 50–100 Psi; and the solvent delay time is 4–6 min. Limiting the mass spectrometry conditions to the above range further improves the sensitivity and accuracy of the detection.

[0055] As one implementation, the mass spectrometry conditions during gas chromatography-tandem mass spectrometry detection can be as follows: the temperature of the transfer line and the electron impact source is independently 300°C, multiple reaction monitoring (MRM) mode is used, the collision gas is argon, the pressure of the argon gas is 70 Psi, and the solvent delay time is 5 min.

[0056] In this invention, the retention time (RT) of the liquid crystal monomer 4-(trans-4-vinylcyclohexyl)benzonitrile (VcHCP) during mass spectrometry detection is preferably 13-14 min, more preferably 13.122 min, the quantitative ion is preferably 182.1→116.1, the qualitative ion is preferably 168.1→141.1, and the collision energy is preferably 10-15 eV.

[0057] In this invention, the retention time (RT) of the liquid crystal monomer 1-[(trans,trans)-4'-vinyl[1,1'-bicyclohexane]-4-yl]-4-methylbenzene (MPVBC) during mass spectrometry detection is preferably 14-15 min, more preferably 14.776 min, the quantitative ion is preferably 282.2→156.1, the qualitative ion is preferably 282.2→121.1, and the collision energy is preferably 10-30 eV.

[0058] In this invention, the retention time (RT) of the liquid crystal monomer 4'-ethyl-4-cyanobiphenyl (2CB) during mass spectrometry detection is preferably 14-15 min, more preferably 14.784 min, the quantitative ion is preferably 207.2→192.2, the qualitative ion is preferably 192.2→165.2, and the collision energy is preferably 10-15 eV.

[0059] In this invention, the retention time (RT) of the liquid crystal monomer 1-ethoxy-2,3-difluoro-4-(trans-4-propylcyclohexyl)benzene (EDFPrCB) during mass spectrometry detection is preferably 14-15 min, more preferably 14.800 min, the quantitative ion is preferably 169.1→121.1, the qualitative ion is preferably 282.2→169.1, and the collision energy is preferably 10-15 eV.

[0060] In this invention, the retention time (RT) of the liquid crystal monomer 4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl (EDFPrB) during mass spectrometry detection is preferably 16-17 min, more preferably 16.324 min, the quantitative ion is preferably 276.2→219.2, the qualitative ion is preferably 219.0→170.2, and the collision energy is preferably 15-20 eV.

[0061] In this invention, the retention time (RT) of the liquid crystal monomer 4-ethoxy-4'-cyanobiphenyl (2OCB) during mass spectrometry detection is preferably 17~18 min, more preferably 17.874 min, the quantitative ion is preferably 223.1→195.1, the qualitative ion is preferably 195.1→166.1, and the collision energy is preferably 8~15 eV.

[0062] In this invention, the retention time (RT) of the liquid crystal monomer 4'-n-butyl-4-cyanobiphenyl (4CB) during mass spectrometry detection is preferably 19-20 min, more preferably 19.868 min, the quantitative ion is preferably 235.1→192.1, the qualitative ion is preferably 192.1→165.1, and the collision energy is preferably 10-15 eV.

[0063] In this invention, the retention time (RT) of the liquid crystal monomer 1-butoxy-2,3-difluoro-4-(trans-4-propylcyclohexyl)benzene (BDFPrCB) during mass spectrometry detection is preferably 19-20 min, more preferably 19.896 min, the quantitative ion is preferably 156.1→127.1, the qualitative ion is preferably 310.3→156.1, and the collision energy is preferably 10-15 eV.

[0064] In this invention, the retention time (RT) of the liquid crystal monomer pentyl dicyclohexyl trifluoromethoxybenzene (EDFPCB) during mass spectrometry detection is preferably 20-21 min, more preferably 20.181 min, the quantitative ion is preferably 184.1→156.1, the qualitative ion is preferably 169.1→121.1, and the collision energy is preferably 10-15 eV.

[0065] In this invention, the retention time (RT) of the liquid crystal monomer 4-propoxy-4'-cyanobiphenyl (PBIPHCN) during mass spectrometry detection is preferably 20-21 min, more preferably 20.974 min, the quantitative ion is preferably 237.1→195.1, the qualitative ion is preferably 195.1→166.1, and the collision energy is preferably 10-15 eV.

[0066] In this invention, the retention time (RT) of the liquid crystal monomer trans,trans-4'-ethyl-4-(3,4,5-trifluorophenyl)bicyclohexane (ETFPBC) during mass spectrometry detection is preferably 22-23 min, more preferably 22.052 min, the quantitative ion is preferably 324.2→158.1, the qualitative ion is preferably 324.2→216.2, and the collision energy is preferably 10-15 eV.

[0067] In this invention, the retention time (RT) of the liquid crystal monomer 4'-n-pentyl-4-cyanobiphenyl (5CB) during mass spectrometry detection is preferably 23-24 min, more preferably 23.783 min, the quantitative ion is preferably 192.1→165.1, the qualitative ion is preferably 249.1→192.1, and the collision energy is preferably 10-15 eV.

[0068] In this invention, the retention time (RT) of the liquid crystal monomer 3,4-difluoro-4'-(4-ethylcyclohexyl)biphenyl (DFECB) during mass spectrometry detection is preferably 24~25 min, more preferably 24.873 min, the quantitative ion is preferably 229.1→214.1, the qualitative ion is preferably 300.2→229.1, and the collision energy is preferably 10~15 eV.

[0069] In this invention, the retention time (RT) of the liquid crystal monomer 4-butoxy-4'-cyanobiphenyl (4OCB) during mass spectrometry detection is preferably 25-26 min, more preferably 25.216 min, the quantitative ion is preferably 195.0→166.1, the qualitative ion is preferably 251.1→195.1, and the collision energy is preferably 10-15 eV.

[0070] In this invention, the retention time (RT) of the liquid crystal monomer trans,trans-4'-propyl-4-(4-trifluoromethoxyphenyl)bicyclohexane (PrTFMPBC) during mass spectrometry detection is preferably 25-26 min, more preferably 25.990 min, the quantitative ion is preferably 188.0→91.1, the qualitative ion is preferably 368.2→188.1, and the collision energy is preferably 10-20 eV.

[0071] In this invention, the retention time (RT) of the liquid crystal monomer trans-4-(3,4,5-trifluorophenyl)-trans-4'-propylbicyclohexane (PrTFPBC) during mass spectrometry detection is preferably 26-27 min, more preferably 26.057 min, the quantitative ion is preferably 282.2→129.1, the qualitative ion is preferably 282.2→118.1, and the collision energy is preferably 10-20 eV.

[0072] In this invention, the retention time (RT) of the liquid crystal monomer 2',3,4,5-tetrafluoro-4'-(trans-4-propylcyclohexyl)biphenyl (TeFPrCB) during mass spectrometry detection is preferably 26-27 min, more preferably 26.334 min, the quantitative ion is preferably 350.2→252.2, the qualitative ion is preferably 239.0→219.1, and the collision energy is preferably 10-15 eV.

[0073] In this invention, the retention time (RT) of the liquid crystal monomer trans,trans-4-(4-fluorophenyl)-4'-propylbicyclohexane (FPPrBC) during mass spectrometry detection is preferably 26-27 min, more preferably 26.468 min, the quantitative ion is preferably 122.1→96.1, the qualitative ion is preferably 302.3→122.1, and the collision energy is preferably 10-15 eV.

[0074] In this invention, the retention time (RT) of the liquid crystal monomer trans,trans-4-(3,4-difluorophenyl)-4”-propylbicyclohexane (DFPPrBC) during mass spectrometry detection is preferably 26~27 min, more preferably 26.906 min, the quantitative ion is preferably 320.2→140.1, the qualitative ion is preferably 320.2→127.1, and the collision energy is preferably 10~30 eV.

[0075] In this invention, the retention time (RT) of the liquid crystal monomer 4'-(trans-4-propylcyclohexyl)-3,4,5-trifluorobiphenyl (TFPrCCB) during mass spectrometry detection is preferably 27~28 min, more preferably 27.484 min, the quantitative ion is preferably 332.2→234.1, the qualitative ion is preferably 234.1→219.1, and the collision energy is preferably 10~15 eV.

[0076] In this invention, the retention time (RT) of the liquid crystal monomer 3,4-difluoro-4'-(trans-4-propylcyclohexyl)biphenyl (DFPrB) during mass spectrometry detection is preferably 28~29 min, more preferably 28.686 min, the quantitative ion is preferably 229.1→214.1, the qualitative ion is preferably 314.2→229.1, and the collision energy is preferably 10~15 eV.

[0077] In this invention, the retention time (RT) of the liquid crystal monomer 4-fluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl (FPrCB) during mass spectrometry detection is preferably 28-29 min, more preferably 28.690 min, the quantitative ion is preferably 211.1→196.1, the qualitative ion is preferably 296.2→211.2, and the collision energy is preferably 10-15 eV.

[0078] In this invention, the retention time (RT) of the liquid crystal monomer 4-pentoxy-4'-cyanobiphenyl (5OCB) during mass spectrometry detection is preferably 29~30 min, more preferably 29.238 min, the quantitative ion is preferably 195.0→166.1, the qualitative ion is preferably 265.1→195.1, and the collision energy is preferably 10~15 eV.

[0079] In this invention, the retention time (RT) of the liquid crystal monomer 2',3,4,5-tetrafluoro-4''-propyl-1,1':4',1''-terphenyl (TeFPrT) during mass spectrometry detection is preferably 29-30 min, more preferably 29.300 min, the quantitative ion is preferably 344.1→315.1, the qualitative ion is preferably 315.1→275.1, and the collision energy is preferably 10-20 eV.

[0080] In this invention, the retention time (RT) of the liquid crystal monomer 4-butylbenzoic acid 4-cyanophenyl ester (4BzoCP) during mass spectrometry detection is preferably 29~30 min, more preferably 29.530 min, the quantitative ion is preferably 161.1→91.1, the qualitative ion is preferably 161.1→65.1, and the collision energy is preferably 15~30 eV.

[0081] In this invention, the retention time (RT) of the liquid crystal monomer trans,trans-4-(3,4-difluorophenyl)-4”-butylbicyclohexane (BDFPBC) during mass spectrometry detection is preferably 30~31 min, more preferably 30.851 min, the quantitative ion is preferably 140.0→114.1, the qualitative ion is preferably 334.2→140.1, and the collision energy is preferably 10~15 eV.

[0082] In this invention, the retention time (RT) of the liquid crystal monomer 4-heptyl-4'-cyanobiphenyl (7CB) during mass spectrometry detection is preferably 31~32 min, more preferably 31.529 min, the quantitative ion is preferably 192.0→190.1, the qualitative ion is preferably 277.2→192.1, and the collision energy is preferably 10~15 eV.

[0083] In this invention, the retention time (RT) of the liquid crystal monomer 4'-cyano-4-hexyloxybiphenyl (6OCB) during mass spectrometry detection is preferably 32-33 min, more preferably 32.420 min, the quantitative ion is preferably 195.0→166.1, the qualitative ion is preferably 279.2→195.1, and the collision energy is preferably 10-15 eV.

[0084] In this invention, the retention time (RT) of the liquid crystal monomer 4-propylbenzoic acid p-pentylphenol ester (3Bzo5P) during mass spectrometry detection is preferably 32-33 min, more preferably 32.632 min, the quantitative ion is preferably 147.1→91.1, the qualitative ion is preferably 310.2→147.1, and the collision energy is preferably 8-15 eV.

[0085] In this invention, the retention time (RT) of the liquid crystal monomer trans,trans-4-(3,4-difluorophenyl)-4'-pentylbicyclohexane (DFPBC) during mass spectrometry detection is preferably 33~34 min, more preferably 33.300 min, the quantitative ion is preferably 140.0→114.1, the qualitative ion is preferably 348.3→140.1, and the collision energy is preferably 10~15 eV.

[0086] In this invention, the retention time (RT) of the liquid crystal monomer 4'-n-octyl-4-cyanobiphenyl (8CB) during mass spectrometry detection is preferably 33~34 min, more preferably 33.560 min, the quantitative ion is preferably 192.1→165.1, the qualitative ion is preferably 291.2→192.1, and the collision energy is preferably 10~15 eV.

[0087] In this invention, the retention time (RT) of the liquid crystal monomer trans-4-(4-pentylcyclohexyl)-3',4'-difluoro-1,1'-biphenyl (DFPCB) during mass spectrometry detection is preferably 34~35 min, more preferably 34.059 min, the quantitative ion is preferably 342.2→216.1, the qualitative ion is preferably 342.2→229.1, and the collision energy is preferably 10~15 eV.

[0088] In this invention, the retention time (RT) of the liquid crystal monomer 4-heptoxy-4'-cyanobiphenyl (7OCB) during mass spectrometry detection is preferably 34~35 min, more preferably 34.132 min, the quantitative ion is preferably 195.1→166.1, the qualitative ion is preferably 293.2→195.1, and the collision energy is preferably 10~15 eV.

[0089] In this invention, the retention time (RT) of the liquid crystal monomer 4-ethyl-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl (EPrCPB) during mass spectrometry detection is preferably 34~35 min, more preferably 34.280 min, the quantitative ion is preferably 306.2→178.1, the qualitative ion is preferably 306.2→221.2, and the collision energy is preferably 10~30 eV.

[0090] In this invention, the retention time (RT) of the liquid crystal monomer 1-ethoxy-2,3-difluoro-4-[(trans,trans)-4'-propyl[1,1'-bicyclohexane]-4-yl]benzene (EDFPBB) during mass spectrometry detection is preferably 34~35 min, more preferably 34.740 min, the quantitative ion is preferably 364.2→156.1, the qualitative ion is preferably 364.2→169.1, and the collision energy is preferably 15~20 eV.

[0091] In this invention, the retention time (RT) of the liquid crystal monomer 4'-(octoxy)biphenyl-4-carboxynitrile (8OCB) during mass spectrometry detection is preferably 35~36 min, more preferably 35.283 min, the quantitative ion is preferably 195.0→166.1, the qualitative ion is preferably 307.2→195.1, and the collision energy is preferably 10~15 eV.

[0092] In this invention, the retention time (RT) of the liquid crystal monomer 1-[4-(4-butylcyclohexyl)cyclohexyl]-4-ethoxy-2,3-difluorobenzene (BCEDB) during mass spectrometry detection is preferably 35-36 min, more preferably 35.800 min, the quantitative ion is preferably 184.1→156.1, the qualitative ion is preferably 378.2→156.1, and the collision energy is preferably 10-15 eV.

[0093] In this invention, the retention time (RT) of the liquid crystal monomer 4-cyano-4''-pentyl-p-terphenyl (5CT) during mass spectrometry detection is preferably 38~39 min, more preferably 38.890 min, the quantitative ion is preferably 325.2→268.1, the qualitative ion is preferably 268.1→165.1, and the collision energy is preferably 10~20 eV.

[0094] In this invention, the retention time (RT) of the liquid crystal monomer 4-[difluoro(3,4,5-trifluorophenoxy)methyl]-2',3,5-trifluoro-4''-propyl-1,1':4',1''-terphenyl (PDTFMTFT) during mass spectrometry detection is preferably 39~40 min, more preferably 39.160 min, the quantitative ion is preferably 346.1→275.1, the qualitative ion is preferably 375.2→346.1, and the collision energy is preferably 20~30 eV.

[0095] This invention preferably uses toluene as a solvent to prepare a series of standard solutions containing internal standards, which are then detected by gas chromatography-tandem mass spectrometry. The ratio of the quantitative ion peak area of ​​the target analyte (liquid crystal monomer) to the quantitative ion peak area of ​​the corresponding internal standard (A) is used. i / A s A standard curve is plotted with the ordinate (y-axis) and the concentration (C) of the standard solution containing the internal standard (x-axis) as the abscissa, as shown in Equation I: A i / A s =a+b×C i / C s Formula I; In the formula, A i Quantify the ion peak area of ​​the target analyte; A s 1. Quantitative peak area of ​​the internal standard; a. Standard curve intercept; b. Standard curve slope; C. i The concentration of the target analyte standard solution is expressed in μg / L; C s The concentration of the internal standard solution is expressed in μg / L.

[0096] The concentration of the target analyte (liquid crystal monomer) in serum was automatically calculated by the data processing system using the internal standard method, as shown in Equation II: ρ=C'×V1 / V Equation II; In the formula, ρ is the actual sample concentration in μg / L; C' is the sample concentration calculated from the standard curve in μg / L; V1 is the fixed volume in μL; and V is the sample volume in μL.

[0097] The present invention does not impose any special limitations on the operation of preparing a series of standard solutions containing internal standard substances using toluene as a solvent; any technical solution known to those skilled in the art can be used.

[0098] This invention employs an internal standard method for quantification, effectively reducing the interference of matrix effects on the detection results and ensuring the reliability of the detection method. It uses n-hexane and methyl tert-butyl ether as extractants for two liquid-liquid extractions, thereby improving extraction efficiency. The use of dichloromethane as an eluent during solid-phase extraction further enhances extraction efficiency, thus further improving the sensitivity and accuracy of the detection.

[0099] The detection method provided by this invention has a wide range of detection types, including the three most widely used LCMs: diphenyl compounds, fluorinated diphenyl compounds, and cyanodiphenyl compounds, and can simultaneously detect 39 compounds.

[0100] The detection method provided by this invention has the advantage of high throughput, using an automated biological sample pretreatment platform that can process 64 samples at a time; the detection method provided by this invention has the advantage of high sensitivity, using an electron impact ion source to simultaneously detect the content of 39 LCMs in a small volume (250 μL) of serum from both general and occupational populations; the detection method provided by this invention has the advantage of high accuracy, as the lack of commercially available LCM isotope-labeled standard materials currently affects the accuracy of quantification. This invention expands the range of selectable isotope internal standards by using methods such as molecular lipophilic descriptor (LogP, LogD) matching, and evaluates the accuracy of results using relative error, effectively improving the confidence of measurement results.

[0101] The present invention also provides a detection kit comprising: a mixed internal standard solution, a precipitant, n-hexane, methyl tert-butyl ether, and dichloromethane.

[0102] In this invention, the detection kit is used to detect 39 liquid crystal monomers in serum.

[0103] In this invention, the mixed internal standard solution and precipitant are preferably the same as those described above, and will not be repeated here.

[0104] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0105] The instruments and equipment used in each embodiment and comparative example were a Trace 1310-TSQ 9000 gas chromatograph-triple quadrupole tandem mass spectrometer (GC-MS / MS) (Thermo Fisher Scientific, USA), equipped with a TG-5SilMS column (Thermo Fisher Scientific, USA); an FV64 nitrogen blower (Guangzhou Detai Instrument Technology Co., Ltd.), a DT5-4B centrifuge (Hebei Yizhong Medical Instrument Co., Ltd.), and a Fotector-08HT high-throughput fully automated solid-phase extraction system (Ruike Group Co., Ltd.).

[0106] The reagents and materials used in each embodiment and comparative example are as follows: Standards: 39 LCMs standards were purchased from Tianjin Alta Technology Co., Ltd., of which 38 were 100 μg / mL solutions, and PDTFMTFT was a 10.09 mg solid dissolved in toluene to a concentration of 100 μg / mL. Of the 39 LCMs standards, 29 were mixed standards, and the remaining 10 were single standards (including EDPprB, BDFPrCB, EDFPCB, BCEDB, PDTFMTFT, DFECB, TeFPrCB, TeFPrT, FPPrBC, and FPrCB). Specific information is shown in Table 1.

[0107] Internal standard: using 13 C 12 Stable isotope-labeled polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were used as quantitative internal standards. P48-M-ES (1 μg / mL, soluble in toluene) was one of six internal standards. 13 C 12 -PCB-28, 13 C 12 -PCB-52, 13 C 12 -PCB-101, 13 C 12 -PCB-138, 13 C 12 -PCB-153, 13 C 12 -PCB-108); WP-LCS (1 μg / mL, soluble in toluene, containing 12 internal standards including...). 13 C 12-PCB-77, 13 C 12 -PCB-81, 13 C 12 -PCB-105, 13 C 12 -PCB-114, 13 C 12 -PCB-118, 13 C 12 -PCB-123, 13 C 12 -PCB-126, 13 C 12 -PCB-156, 13 C 12 -PCB-157, 13 C 12 -PCB-167, 13 C 12 -PCB-169, 13 C 12 -PCB-189); MBDE-MXFR (2 μg / mL, soluble in toluene and n-nonane, two internal standards including...). 13 C 12 -BDE-77, 13 C 12 - BDE-138); MBDE-MXFS (2 μg / mL, soluble in toluene, containing 7 internal standards including...) 13 C 12 -BDE-28, 13 C 12 -BDE-47, 13 C 12 -BDE-99, 13 C 12 -BDE-100, 13 C 12 -BDE-153, 13 C 12 -BDE-154, 13 C 12 -BDE-183), all of which were purchased from Wellington Laboratories in the United States.

[0108] Other reagents and materials: The solid-phase extraction column used was a 6mL HyperSep column from Thermo Fisher Scientific, USA. TMSilica extraction columns; methanol (MeOH), dichloromethane (DCM), and acetone (ACE) were all chromatographically pure and purchased from Thermo Fisher Scientific, USA; n-hexane (HEX, pesticide residue grade, Thermo Fisher Scientific, USA); toluene (TOL, chromatographically pure, Tedia Scientific, USA); n-dodecane (Dodecane, chromatographically pure, Acros Scientific, Belgium); methyl tert-butyl ether (MTBE, pesticide residue grade, Shanghai Anpu Experimental Technology Co., Ltd.); other organic reagents used were analytical grade reagents with a purity greater than 98%.

[0109] Serum samples were obtained from recruited volunteers who signed written informed consent forms. The study was approved by the Ethics Committee of the Institute of Environmental and Health Related Product Safety, Chinese Center for Disease Control and Prevention. Serum was obtained by collecting fasting blood samples from the subjects and stored at -80°C.

[0110] Table 1. Basic information on 39 types of LCMs

[0111] The solution is prepared as follows: 39 LCMs mixed standard solutions: Take 4 mL of a 100 μg / mL mixed standard stock solution of 29 LCMs and dilute to 40 mL with toluene to prepare a 10 μg / mL 29 LCMs mixed standard stock solution. Take another 4 mL of the 10 μg / mL 29 LCMs mixed standard stock solution and dilute to 40 mL with toluene to prepare a 1 μg / mL 29 LCMs mixed standard stock solution. Take 10 μL of a 100 μg / mL single standard stock solution of 10 LCMs and dilute to 1 mL with toluene to prepare a 1 μg / mL 10 LCMs mixed standard stock solution. In preparation for use, take 100 μL of the 1 μg / mL mixed standard stock solution of 29 LCMs and 10 LCMs and dilute to 500 μL with toluene to prepare a 0.2 μg / mL mixed standard solution of 39 LCMs-1; take 90 μL of the 39 LCMs mixed standard solution-1 and dilute to 720 μL with toluene to prepare a 0.025 μg / mL mixed standard solution of 39 LCMs-2.

[0112] Mixed internal standard solution: Take 50 μL of 2 μg / mL MBDE-MXFR and MBDE-MXFS stock solutions and dilute to 100 μL with toluene to prepare 1 μg / mL mixed standard stock solutions of MBDE-MXFR and MBDE-MXFS, respectively. Take 25 μL of 1 μg / mL P48-M-ES, WP-LCS, MBDE-MXFR and MBDE-MXFS mixed standard stock solutions and dilute to 500 μL with toluene to prepare 0.050 μg / mL mixed internal standard solutions.

[0113] Preparation of standard curve: Take an appropriate amount of 39 LCMs mixed standard solution-1 and mixed internal standard solution and dilute them with toluene in a gradient to prepare a series of standard solutions S1-S8 with a concentration range of 0.5~100 μg / L, namely 0.5, 1, 2, 5, 10, 20, 50 and 100 μg / L. The concentration of the internal standard solution in each gradient is 25 μg / L.

[0114] Example 1 A method for detecting multiple liquid crystal monomers in serum comprises the following steps: (1) Take 250 μL of fetal bovine serum and place it in a clean 12 mL glass centrifuge tube. Add 0.5 ng of mixed internal standard solution, then add 200 μL of 98% concentrated sulfuric acid and 1 mL of methanol in sequence. Vortex to mix and obtain a mixture. (2) The mixture obtained in step (1) is subjected to a first liquid-liquid extraction with 6 mL of n-hexane and centrifuged at 3500 r / min for 5 min to obtain the first supernatant and the first lower layer. (3) The first lower layer substance obtained in step (2) is mixed with 6 mL of methyl tert-butyl ether, and a second liquid-liquid extraction is performed. The mixture is then centrifuged at 3500 r / min for 5 min to obtain the second supernatant and the second lower layer substance. (4) Combine the first supernatant obtained in step (2) and the second supernatant obtained in step (3) into the same 12 mL glass centrifuge tube, concentrate it to 1 / 6 of the original solution volume by nitrogen blowing, and then perform HyperSep TM The Silica extraction column was activated sequentially with 2 mL of methanol and 2 mL of n-hexane, then loaded with the sample for solid-phase extraction. The target analyte was eluted with 10 mL of dichloromethane to obtain the eluent. The flow rate of dichloromethane was 1.0 mL / min. (5) Add dodecane to the eluent obtained in step (4) and blow it with nitrogen until it is almost dry, then redissolve it with 20 μL of toluene to obtain the test solution; (6) Prepare a standard solution, and then transfer the test solution obtained in step (5) to a brown injection bottle for injection. The injection volume is 1 μL. Gas chromatography-tandem mass spectrometry is used for detection. A standard curve is plotted with the ratio of the quantitative ion peak area of ​​the liquid crystal monomer to the quantitative ion peak area of ​​the corresponding internal standard (Ai / As) as the ordinate (y-axis) and the concentration of the standard solution containing the internal standard (C) as the abscissa (x-axis) to obtain the content of various liquid crystal monomers in serum. The chromatographic conditions are: chromatographic column TG-5SilMS (30m×0.25mm×0.25μm), splitless injection, and injection port temperature. The temperature was 290℃, the carrier gas was helium, and the flow rate was 1.0 mL / min. The column temperature program was 100℃ for 2 min, then increased to 180℃ at 15℃ / min, then increased to 205℃ at 3℃ / min for 7 min, then increased to 230℃ at 3℃ / min, then increased to 300℃ at 15℃ / min for 2 min. The mass spectrometry conditions were: transfer line and electron impact source temperature were both 300℃; multiple reaction monitoring mode was used, argon was used as the collision gas at a pressure of 70 Psi, and the solvent delay was 5 min. The detection conditions for liquid crystal monomers and their internal standards during mass spectrometry detection are shown in Table 2. Table 2. Mass spectrometry parameters and detection conditions for internal standards of 39 LCMs

[0115] In Table 2, the first value in the collision energy (CE) corresponds to quantitative ions, and the second value corresponds to qualitative ions.

[0116] The chromatogram of the mixed standard solution of 39 LCMs in Example 1 is shown below. Figure 1 As shown, Figure 1 1 to 39 in the middle correspond to 1 to 39 in Table 2.

[0117] Comparative Example 1 Based on Example 1, n-hexane in step (2) was replaced with methyl tert-butyl ether, while other conditions remained unchanged.

[0118] Comparative Example 2 Based on Example 1, the methyl tert-butyl ether in step (3) was replaced with n-hexane, while other conditions remained unchanged.

[0119] Comparative Example 3 Based on Example 1, the methyl tert-butyl ether in step (3) was replaced with dichloromethane, while other conditions remained unchanged.

[0120] In Example 1, the extraction efficiency of 39 substances in two liquid-liquid extractions ranged from 48.97% to 73.30%. The extraction efficiency was more concentrated among the 39 LCMs and the standard deviation was generally lower.

[0121] In Comparative Example 1, the extraction efficiency ranged from 2.21% to 131.53% when 39 substances were extracted twice using liquid-liquid extraction, and the extraction efficiency of 20 target substances was greater than 100%.

[0122] In Comparative Example 2, the extraction efficiency of 39 substances in two liquid-liquid extractions ranged from 27.80% to 96.65%.

[0123] After two liquid-liquid extractions in Comparative Example 3, a large amount of red precipitate appeared in the solution during the first nitrogen purging. This may be because dichloromethane disrupted the transferrin in serum, releasing Fe. 3+ During nitrogen blowing concentration, it hydrolyzes to form ferric hydroxide colloid.

[0124] Based on the above results, using n-hexane and methyl tert-butyl ether as extractants for two-stage liquid-liquid extraction is optimal.

[0125] Comparative Example 4 Based on Example 1, the eluent was changed to n-hexane and dichloromethane, with a volume ratio of n-hexane to dichloromethane of 1:1, while other conditions remained unchanged.

[0126] Comparative Example 5 Based on Comparative Example 4, the extraction column was changed to Florisil PR (60~100 mesh), while other conditions remained unchanged.

[0127] Comparative Example 6 Based on Comparative Example 4, the extraction column was changed to Alumina-A acidic alumina, while other conditions remained unchanged.

[0128] Comparative Example 7 Based on Comparative Example 4, the extraction column was changed to HLB, while other conditions remained the same.

[0129] The extraction efficiencies of the extraction columns in Comparative Examples 4-7 for extracting 39 substances were 50.90%–65.41%, 45.47%–61.30%, 43.22%–60.67%, and 35.31%–57.37%, respectively; among them, HyperSep… TM The Silica extraction column had the highest extraction efficiency and performed relatively best among 39 LCMs.

[0130] Comparative Example 8 Based on Example 1, the eluent was changed to n-hexane, while other conditions remained unchanged.

[0131] The extraction efficiencies of the elution solvents in Examples 1, 4, and 8 ranged from 48.97% to 73.30%, 50.90% to 65.41%, and 38.56% to 53.34%, respectively. The extraction results with n-hexane were relatively low. Although the lowest extraction efficiency with n-hexane and dichloromethane as eluents was higher than that with dichloromethane as eluent, the extraction efficiency of 35 out of 39 LCMs was higher with dichloromethane as eluent than with n-hexane and dichloromethane as eluents.

[0132] Comparative Example 9 Based on Example 1, the external standard method was used, with other conditions remaining unchanged.

[0133] Human serum matrix components are complex. Even after purification through liquid-liquid extraction and solid-phase extraction, co-extracted impurities may still interfere with the detection of target analytes, easily leading to ion inhibition / enhancement effects. Therefore, the matrix effect (ME) of the detection method needs to be evaluated. Using selected target analyte isotope-labeled internal standards, the absolute matrix effect (AME) and relative matrix effect (RME) were evaluated using both external and internal standard methods, and calculated according to the formula: ME (%) = (slope of matrix standard curve / slope of solvent standard curve) × 100%. When calculating the absolute matrix effect, Y in the standard curve Y = aX + b represents Ai (the area of ​​the quantitative ion peak of the target analyte), and X represents Ci (the concentration of the target analyte corresponding to the standard curve). When calculating the relative matrix effect, Y in the standard curve Y = aX + b represents Ai / As (the area of ​​the quantitative ion peak of the target analyte / the area of ​​the quantitative ion peak of the internal standard), and X represents Ci / Cs (the concentration of the target analyte corresponding to the standard curve / the concentration of the internal standard corresponding to the standard curve).

[0134] Experimental results show that the AME range is 106.32%~333.20%, and after internal standard correction, the RME range decreases to 76.20%~127.76%. According to industry consensus, when the RME value is between 80% and 120%, the matrix effect can be considered insignificant. Therefore, this invention uses the internal standard method for quantification, effectively reducing the interference of the matrix effect on the detection results and ensuring the reliability of the detection method. The specific matrix effects of the target analytes are shown in Table 3.

[0135] In Example 1, the 39 LCMs showed good linearity in the range of 0.5–100 μg / L, with a coefficient of determination R0. 2All values ​​were greater than 0.999. Based on relevant FDA documents, the method limit of detection was estimated using a signal-to-noise ratio of 3, and a low-concentration spike of 5 times was used. Calculations were performed on calf serum samples, and after 7 repeated assays, the method limit of detection was calculated to be 0.045–0.272 μg / L using 3 times the standard deviation of the experimental results. The method limit of quantitation was calculated to be 0.150–0.908 μg / L using 10 times the standard deviation. The specific limits of detection and quantitation for the target analytes are shown in Table 3.

[0136] Table 3. Linear range and coefficient of determination (R²) of 39 LCMs 2 Limit of detection (MDL), limit of quantitation (LOQ), and matrix effect

[0137] Test case Fetal bovine serum was used as the matrix for spiking at three concentration levels: 1 μg / L, 2 μg / L, and 5 μg / L. Six replicates were performed for each spiking level, and the assays were repeated over six consecutive days. The spiked recovery, intra-day precision (RSD), and inter-day RSD of the detection method were evaluated, as shown in Table 4. Table 4. Spike recoveries and precision of 39 LCMs

[0138] As shown in Table 4, the average recoveries of the 39 LCMs were 86.87–134.17%, with intra-day RSDs of 2.45–8.82% and inter-day RSDs of 2.01–13.15%. Therefore, the detection recovery and precision of this invention are both good.

[0139] Example 2 The amount of concentrated sulfuric acid added in step (1) of Example 1 was replaced with 250 μL, and all other parameters were the same as in Example 1.

[0140] The method of Example 2 was used to test 10 serum samples collected from workers in the liquid crystal display manufacturing industry. The results are shown in Table 5 (<MDL in the table indicates that it cannot be qualitatively determined, the same below).

[0141] Table 5. Detection results of 39 LCMs in serum samples

[0142] As shown in Table 5, a total of 32 LCMs were detected in all samples. Among them, 23 had a detection rate of ≥50%, and 14 had a detection rate of ≥80%. 7CB had the highest median mass concentration. The median mass concentration range of all detected LCMs was 0.105~20.179 μg / L.

[0143] Example 3 The amount of concentrated sulfuric acid added in step (1) of Example 1 was replaced with 150 μL, and all other parameters were the same as in Example 1.

[0144] The method described in Example 3 was used to test serum samples from 58 ordinary adults in a region of East China. The results are shown in Table 6.

[0145] Table 6. Detection results of 39 LCMs in serum samples

[0146] As shown in Table 6, all 38 LCMs were detected, with 13 having a detection rate ≥50% and 9 having a detection rate ≥80%. BCEDB had the highest median mass concentration. The median mass concentration range of all detected LCMs was 0.109~6.012 μg / L.

[0147] Example 4 Replace the amount of methanol added in step (1) of Example 1 with 500 μL, and keep all other parameters the same as in Example 1.

[0148] The method described in Example 4 was used to test 64 serum samples from ordinary adults in Beijing. The results are shown in Table 7.

[0149] Table 7. Detection results of 39 LCMs in serum samples

[0150] As shown in Table 7, a total of 20 LCMs were detected in all samples. Among them, 11 had a detection rate of ≥50%, and 8 had a detection rate of ≥80%. BCEDB had the highest median mass concentration. The median mass concentration range of all detected LCMs was 0.102~5.715 μg / L.

[0151] Example 5 Replace the amount of methanol added in step (1) of Example 1 with 1.5 mL, and keep all other parameters the same as in Example 1.

[0152] The method described in Example 5 was used to test 61 serum samples from ordinary adults in Guangdong Province. The results are shown in Table 8.

[0153] Table 8. Detection results of 39 LCMs in serum samples

[0154] As shown in Table 8, a total of 21 LCMs were detected in all samples. Among them, 11 had a detection rate of ≥50%, and 7 had a detection rate of ≥80%. BCEDB had the highest median mass concentration. The median mass concentration range of all detected LCMs was 0.113~3.170 μg / L.

[0155] As can be seen from the examples and comparative examples, the detection method provided by the present invention has high sensitivity and high accuracy.

[0156] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting multiple liquid crystal monomers in serum, characterized in that, Includes the following steps: (1) The serum, mixed internal standard solution and precipitant are mixed to obtain a mixture; the precipitant is an acidic solution and an alcohol; the acidic solution is a sulfuric acid solution; the mass concentration of the acidic solution is 98%; the alcohol is methanol; the volume ratio of the acidic solution to the alcohol is 1:(2~8); the volume ratio of the serum to the precipitant is 1:(2~7). (2) The mixture obtained in step (1) is mixed with n-hexane and subjected to a first liquid-liquid extraction to obtain a first supernatant and a first lower layer substance; (3) The first lower layer substance obtained in step (2) is mixed with methyl tert-butyl ether and subjected to a second liquid-liquid extraction to obtain a second supernatant and a second lower layer substance; (4) Combine the first supernatant obtained in step (2) and the second supernatant obtained in step (3), and then perform solid phase extraction to obtain an eluent; the eluent used in the solid phase extraction is dichloromethane; (5) Concentrate the eluent obtained in step (4) and then redissolve it to obtain the test solution; (6) The test solution obtained in step (5) is subjected to gas chromatography-tandem mass spectrometry to obtain the content of various liquid crystal monomers in serum; The extraction column used for solid-phase extraction in step (4) is HyperSep. TM Silica extraction cartridges; The chromatographic column was a TG-5SilMS, with a length of 30m, an inner diameter of 0.25mm, and a film thickness of 0.25μm. The temperature program for the column was as follows: 100℃ held for 2 min, then increased to 180℃ at a rate of 15℃ / min, followed by 205℃ at a rate of 3℃ / min and held for 7 min, then increased to 230℃ at a rate of 3℃ / min, and finally increased to 300℃ at a rate of 15℃ / min and held for 2 min.

2. The detection method according to claim 1, characterized in that, The volume ratio of serum in step (1) to n-hexane in step (2) is 1:(20~30).

3. The detection method according to claim 1 or 2, characterized in that, The volume ratio of serum in step (1) to n-hexane in step (2) is 1:

24.

4. The detection method according to claim 1, characterized in that, The volume ratio of serum in step (1) to methyl tert-butyl ether in step (3) is 1:(20~30).

5. The detection method according to claim 1 or 4, characterized in that, The volume ratio of serum in step (1) to methyl tert-butyl ether in step (3) is 1:

24.

6. The detection method according to claim 1, characterized in that, The volume ratio of serum in step (1) to elution buffer in step (4) is 1:(35~45).

7. The detection method according to claim 1, characterized in that, In step (6), the mass spectrometry conditions during gas chromatography-tandem mass spectrometry detection include: the temperature of the transmission line and the electron bombardment source are independently 290~310℃, multiple reaction monitoring mode is used, the collision gas is argon, the pressure of argon is 50~100Psi, and the solvent delay time is 4~6min.

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