A method for extracting phthalates and polycyclic aromatic hydrocarbons in blood

Abstract

The present application discloses a method for extracting phthalates and polycyclic aromatic hydrocarbons in blood in the field of blood detection technology, including S1, preparing standard reagents, S2, preparing stock solutions; S3, preparing internal standard solutions; S4, preparing mixed standard solutions; S5, preparing mixed standard 1 to mixed standard 6 solutions and control solutions; S6, blood pretreatment; b1, thawing; b2, standard preparation; b3, hydrolysis b4, primary extraction; b5, secondary extraction; b6, tertiary extraction, second round of extraction; S7, nitrogen blowing; S8, derivatization; S9, sample analysis. Using the monitoring method in this scheme, a total of 20 substances were detected from blood samples, of which there were ten polycyclic aromatic hydrocarbon metabolites (OH-PAHS), namely 1-OHNAP, 2-OHFLU, 3-OHPHE, 9-OHPHE, 1-OHPHE, 2-OHNAP, 4-OHPHE, 1-OHPYR, 2-OHPHE, and 9-OHFLU. There are 10 phthalate metabolites (PAEs), namely MECPP, MEOHP, MMP, MBP, MEHHP, MEP, MOP, MBZP, MEHP, and MIBP. Phthalates and polycyclic aromatic hydrocarbons in the blood were effectively detected.

Description

Technical Field

[0001] The invention relates to the technical field of blood detection, and in particular to a method for extracting phthalates and polycyclic aromatic hydrocarbons in blood. Background Art

[0002] Phthalate esters (PAEs), also known as phthalic acid esters, are a general term for esters formed by phthalic acid. When used as plasticizers, they generally refer to esters formed by phthalic acid and alcohols with 4 to 15 carbons. There are many varieties of phthalates (about 30 kinds), among which dioctyl phthalate is the most important variety. It is considered to be an environmental hormone that is ubiquitous in the living environment. Phthalate compounds are fat-soluble compounds. Packing butter and animal fat foods in plastic bags, especially hot ones, will increase the intake of the human body and is not good for health. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a preliminary list of carcinogens for reference, and di(2-ethylhexyl) phthalate was included in the list of Class 2B carcinogens.

[0003] Polycyclic aromatic hydrocarbons refer to aromatic hydrocarbons containing two or more benzene rings, referred to as PAHs. They mainly have two combinations: one is the non-condensed ring type, which includes biphenyl, biphenyls and polyphenyl aliphatic hydrocarbons; the other is the condensed ring type, that is, two carbon atoms are shared by two benzene rings. PAHs can cause a variety of harms to the human body due to their toxicity, genotoxicity, mutagenicity and carcinogenicity, such as damage to the respiratory system, circulatory system, nervous system, liver and kidneys. They are identified as major organic pollutants that affect human health.

[0004] However, current blood extraction methods usually only extract one of the substances, and lack an extraction method for the combined extraction of phthalates and PAHs. Summary of the invention

[0005] The present invention aims to provide an extraction method capable of simultaneously extracting phthalates and polycyclic aromatic hydrocarbons in blood.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a method for extracting phthalates and polycyclic aromatic hydrocarbons in blood, comprising the following steps:

[0007] S1, prepare standard reagents: prepare the following standard reagents in order;

[0008]

[0009]

[0010]

[0011] S2, prepare stock solution: take 14 10ml brown volumetric flasks, numbered 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 21, 22. Use a 1ml pipette to suck 0.5ml of acetonitrile and inject it into the reagent bottle of the standard reagent corresponding to the above serial number, then pipette and mix it and suck it into the volumetric flask of the corresponding serial number, repeat the above steps four times and then use acetonitrile to make the volume to 10ml; then take 6 2ml brown injection bottles, numbered 1, 5, 6, 18, 19, 20, use a 200μl pipette to suck 200μl acetonitrile into the packaging bottle of the standard reagent of the corresponding serial number, pipette and mix it and suck it into the injection bottle of the corresponding serial number, repeat this step four times, then add acetonitrile to make the injection bottles numbered 1, 5, 6 to 1ml, and the injection bottles numbered 18, 19, 20 to 1.25ml;

[0012]

[0013] S3, prepare internal standard solution: take 480ul of 0.25mg / ml 1-OHP-d9 stock solution and 240ul of 5mg / ml 1-OHNa-d7 stock solution, 1.2ml of 100ug / ml MEHP-c4, and 1.2ml of 100ug / ml MEHHP-c4, respectively, dilute to 24ml with acetonitrile, the mixed solution of the four is the internal standard solution, the final concentration of 1-OHNa-d7 is 50.9ug / ml, and the final concentration of 1-OHP-d9, MEHP-c4 and MEHHP-c4 is 5ug / ml;

[0014] S4, prepare mixed standard solution: take a 10 ml centrifuge tube, add 1 ml acetonitrile and mark it as mixed standard 7, use 20 μl and 50 μl pipettes to transfer the stock solution of each standard reagent (volume see the table below) into the 10 ml centrifuge tube, then add acetonitrile to make up to 5 ml, mix on a vortexer to prepare the mixed standard solution;

[0015]

[0016] S5, preparation of mixed standard 1 to mixed standard 6 solutions and control solution:

[0017] a1. Prepare instruments: take 7 5 ml centrifuge tubes and label them as Mixed Standard 1, Mixed Standard 2, Mixed Standard 3, Mixed Standard 4, Mixed Standard 5, Mixed Standard 6, and Control 0;

[0018] a2, preparation of mixed standard 6: take 1.5 ml of mixed standard 7 solution in step S4 and 1.5 ml of acetonitrile, add to mixed standard 6 tube, mix on a vortexer for 1 min, and prepare mixed standard 6;

[0019] a3, preparation of mixed standard 5: take 1 ml of mixed standard 7 solution in step S4 and 4 ml of acetonitrile, add to mixed standard 5 tube, mix on a vortexer for 15 s, 3 times, to prepare mixed standard 5;

[0020] a4, preparation of mixed standard 4: take 2 ml of mixed standard 5 and 2 ml of acetonitrile and add them to mixed standard 4 tube, mix on a vortexer for 15 seconds, 3 times, to prepare mixed standard 4;

[0021] a5, preparation of mixed standard 3: take 1 ml of mixed standard 4 and 4 ml of acetonitrile and add them to mixed standard 3 tube, mix on a vortexer for 15 seconds, 3 times, to prepare mixed standard 3;

[0022] a6, Preparation of Mixed Standard 2: Take 2 ml of Mixed Standard 3 and 2 ml of acetonitrile and add them to Mixed Standard 2 tube, mix on a vortexer for 15 seconds, 3 times, to prepare Mixed Standard 2;

[0023] a7, Preparation of Mixed Standard 1: Take 1.5 ml of Mixed Standard 2 and 1.5 ml of acetonitrile and add them to Mixed Standard 1 tube, mix on a vortexer for 15 seconds, 3 times, to prepare Mixed Standard 1;

[0024] a8, Preparation of control 0 solution: Take 3 ml of acetonitrile and add it to control 0 tube as blank control;

[0025] S6, blood pretreatment:

[0026] b1, thawing: take the blood out of the refrigerator, thaw it, shake it well and let it stand;

[0027] b2, preparation of standard: take 8 10ml round bottom plastic centrifuge tubes, numbered B0 to B7. Add 1ml pure water and 20μl of the above mixed standard solution to each tube of B0 to B7 (B0 tube adds control O, B1 tube adds mixed standard 1, B2 tube adds mixed standard 2, B3 tube adds mixed standard 3, B4 tube adds mixed standard 4, B5 tube adds mixed standard 5, B6 tube adds mixed standard 6, B7 tube adds mixed standard 7);

[0028] b3, hydrolysis: take 1 ml of blood with a 1 ml pipette and add it to a 10 ml round-bottom plastic centrifuge tube, add 20 ul of internal standard solution to both the blood sample and the standard, add 2 ml of MgSO4·7H20, and shake on a multi-tube vortex mixer for 10 min at 3500 r / min until there is no precipitation;

[0029] b4, primary extraction: add 1 ml of ethyl acetate one by one, centrifuge for 10 min (4000 rpm), and take the supernatant into a new 5 ml centrifuge tube;

[0030] b5, secondary extraction: add 1 ml of the above n-hexane and ether (volume ratio 4:1) into the 10 ml centrifuge tube again, mix in a multi-tube vortex for 15 seconds, centrifuge for 10 minutes (4000 rpm), and remove the supernatant into a 5 ml round-bottom centrifuge tube;

[0031] b6, three extractions: use 1 ml of the above n-hexane and ether (volume ratio 4:1) respectively, mix in a multi-tube vortex instrument for 15 seconds, centrifuge for 10 minutes (speed as above), take out the supernatant (about 1 ml, the supernatant cannot be taken out completely, and 2 mm of supernatant must be left, otherwise crystals are easily precipitated) into a 5 ml round-bottom centrifuge tube;

[0032] b7. After completing the extraction process three times, the organic phase stored in the 5 ml centrifuge tube was placed on a nitrogen blower and blown dry with high-purity nitrogen. After drying, 1 ml of sodium acetate buffer and 50 ul of β-glucuronidase / sulphatase enzyme (100 μl pipette, white tip, blow to clean the tip residue) were added, the lid was tightly closed, the tube was placed in a vortexer to mix for 1 min, and then placed in a 37°C water bath for 90 min;

[0033] b8, take out the fully enzymatically hydrolyzed sample (then set the temperature of the water bath to 90°C for standby use) and perform a second round of extraction;

[0034] b9, second round of extraction: 1st extraction, add 1ml ethyl acetate one by one (add sample in fume hood), shake on a multi-tube vortex mixer for 10min, 3500r / min, until there is no precipitation, centrifuge for 10min (speed 4000rpm), and take the supernatant to a new 5ml centrifuge tube; 2nd extraction, add the above 1ml n-hexane and ether (volume ratio 4:1) one by one to the 10ml centrifuge tube again, mix on a multi-tube vortex instrument for 15S, centrifuge for 10min (speed 4000rpm), and take the supernatant to a 5ml round-bottom centrifuge tube; 3rd extraction, use the above 1ml n-hexane and ether (volume ratio 4:1) respectively, vortex for 15s, centrifuge for 10min (speed 4000rpm), and take the supernatant to a 5ml round-bottom centrifuge tube;

[0035] S7, nitrogen blowing: After completing two rounds of extraction, the organic phase stored in the 5 ml centrifuge tube was placed on a nitrogen blower and concentrated to about 250 ul using high-purity nitrogen. The organic phase was then transferred to the inner tubes one by one, transferred to the nitrogen blower again and dried using high-purity nitrogen.

[0036] S8, derivatization: add an equal amount (100ul) of silanization reagent to each inner tube, cover the bottle cap tightly, and then place it in a water bath (temperature set at 90°C, 45min) to fully derivatize it;

[0037] S9, sample analysis: After cooling at room temperature (about 30 min) after the water bath, perform gas chromatography-mass spectrometry analysis.

[0038] Furthermore, in step S6, when thawing the blood sample during blood pretreatment, the blood sample is thawed in a water bath at room temperature or at 37° C. for more than 3 hours. After thawing, the blood is shaken and allowed to stand until the precipitate sinks to the bottom of the tube.

[0039] Furthermore, when 1 ml of ethyl acetate is added one by one in the first extraction of the two rounds of step S6, the samples need to be added in a fume hood.

[0040] Furthermore, when taking the supernatant during the first extraction of the two rounds in step S6, about 0.3 ml of the supernatant was taken, and a 2 mm height of the supernatant was left in the centrifuge tube to avoid precipitation of crystals.

[0041] Furthermore, when taking the supernatant during the two rounds of the second extraction in step S6, about 1 ml of the supernatant was taken, and a 2 mm height of the supernatant was left in the centrifuge tube to avoid precipitation of crystals.

[0042] Furthermore, when taking the supernatant during the two rounds of the third extraction in step S6, about 1 ml of the supernatant was taken, and a 2 mm height of the supernatant was left in the centrifuge tube to avoid precipitation of crystals.

[0043] Beneficial effects of the present invention: A total of 20 substances were detected from blood samples using the monitoring method in the present scheme, of which ten were polycyclic aromatic hydrocarbon metabolites (OH-PAHS), namely 1-OHNAP, 2-OHFLU, 3-OHPHE, 9-OHPHE, 1-OHPHE, 2-OHNAP, 4-OHPHE, 1-OHPYR, 2-OHPHE, and 9-OHFLU. Ten were phthalate metabolites (PAEs), namely MECPP, MEOHP, MMP, MBP, MEHHP, MEP, MOP, MBZP, MEHP, and MIBP. Phthalates and polycyclic aromatic hydrocarbons in blood were effectively detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a chromatogram of blood sample detection in an embodiment of the present invention. DETAILED DESCRIPTION

[0045] A method for extracting phthalates and polycyclic aromatic hydrocarbons in blood comprises the following steps:

[0046] S1, prepare standard reagents: prepare the following standard reagents in order;

[0047]

[0048]

[0049] S2, prepare stock solution: take 14 10ml brown volumetric flasks, numbered 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 21, 22. Use a 1ml pipette to suck 0.5ml of acetonitrile and inject it into the reagent bottle of the standard reagent corresponding to the above serial number, then pipette and mix it and suck it into the volumetric flask of the corresponding serial number, repeat the above steps four times and then use acetonitrile to make the volume to 10ml; then take 6 2ml brown injection bottles, numbered 1, 5, 6, 18, 19, 20, use a 200μl pipette to suck 200μl acetonitrile into the packaging bottle of the standard reagent of the corresponding serial number, pipette and mix it and suck it into the injection bottle of the corresponding serial number, repeat this step four times, then add acetonitrile to make the injection bottles numbered 1, 5, 6 to 1ml, and the injection bottles numbered 18, 19, 20 to 1.25ml;

[0050]

[0051]

[0052] S3, prepare internal standard solution: take 480ul of 0.25mg / ml 1-OHP-d9 stock solution and 240ul of 5mg / ml 1-OHNa-d7 stock solution, 1.2ml of 100ug / ml MEHP-c4, and 1.2ml of 100ug / ml MEHHP-c4, respectively, dilute to 24ml with acetonitrile, the mixed solution of the four is the internal standard solution, the final concentration of 1-OHNa-d7 is 50.9ug / ml, and the final concentration of 1-OHP-d9, MEHP-c4 and MEHHP-c4 is 5ug / ml;

[0053] S4, prepare mixed standard solution: take a 10 ml centrifuge tube, add 1 ml acetonitrile and mark it as mixed standard 7, use 20 μl and 50 μl pipettes to transfer the stock solution of each standard reagent (volume see the table below) into the 10 ml centrifuge tube, then add acetonitrile to make up to 5 ml, mix on a vortexer to prepare the mixed standard solution;

[0054]

[0055]

[0056] S5, preparation of mixed standard 1 to mixed standard 6 solutions and control solution:

[0057] a1. Prepare instruments: take 7 5 ml centrifuge tubes and label them as Mixed Standard 1, Mixed Standard 2, Mixed Standard 3, Mixed Standard 4, Mixed Standard 5, Mixed Standard 6, and Control 0;

[0058] a2, preparation of mixed standard 6: take 1.5 ml of mixed standard 7 solution in step S4 and 1.5 ml of acetonitrile, add to mixed standard 6 tube, mix on a vortexer for 1 min, and prepare mixed standard 6;

[0059] a3, preparation of mixed standard 5: take 1 ml of mixed standard 7 solution in step S4 and 4 ml of acetonitrile, add to mixed standard 5 tube, mix on a vortexer for 15 s, 3 times, to prepare mixed standard 5;

[0060] a4, preparation of mixed standard 4: take 2 ml of mixed standard 5 and 2 ml of acetonitrile and add them to mixed standard 4 tube, mix on a vortexer for 15 seconds, 3 times, to prepare mixed standard 4;

[0061] a5, preparation of mixed standard 3: take 1 ml of mixed standard 4 and 4 ml of acetonitrile and add them to mixed standard 3 tube, mix on a vortexer for 15 seconds, 3 times, to prepare mixed standard 3;

[0062] a6, Preparation of Mixed Standard 2: Take 2 ml of Mixed Standard 3 and 2 ml of acetonitrile and add them to Mixed Standard 2 tube, mix on a vortexer for 15 seconds, 3 times, to prepare Mixed Standard 2;

[0063] a7, Preparation of Mixed Standard 1: Take 1.5 ml of Mixed Standard 2 and 1.5 ml of acetonitrile and add them to Mixed Standard 1 tube, mix on a vortexer for 15 seconds, 3 times, to prepare Mixed Standard 1;

[0064] a8, Preparation of control 0 solution: Take 3 ml of acetonitrile and add it to control 0 tube as blank control;

[0065] S6, blood pretreatment:

[0066] b1. Thawing: Take out the frozen blood sample from the -80℃ freezer, thaw it at room temperature or in a water bath (37℃) for more than 3 hours, shake well and let it stand (wait for the precipitate to sink to the bottom of the tube), and mark the number of the centrifuge tube (record and match this number with the unique number).

[0067] b2, Preparation of standard products (prepared and used immediately): Take 8 10ml round-bottom plastic centrifuge tubes, labeled B0 to B7. Add 1ml of pure water and 20μl of the above mixed standard to each tube of B0 to B7 (B0 tube adds control CO, B1 tube adds mixed standard 1, B2 tube adds mixed standard 2, B3 tube adds mixed standard 3, B4 tube adds mixed standard 4, B5 tube adds mixed standard 5, B6 tube adds mixed standard 6, B7 tube adds mixed standard 7), and prepare a series of standard concentrations, see Table 7.

[0068] b3, Hydrolysis: Use a 1ml pipette to take 1ml of blood and add it to a 10ml round-bottom plastic centrifuge tube. Add 20ul of internal standard mixture to the blood sample and standard, add 2ml of MgSO4·7H20, and shake on a multi-tube vortex mixer for 10min at 3500r / min until there is no precipitation (a very important step!).

[0069] b4, first extraction: add 1 ml of ethyl acetate one by one (add samples in a fume hood), centrifuge for 10 min (4000 rpm), take the supernatant (about 0.3 ml, the supernatant cannot be taken out completely, and a 2 mm height of supernatant must be left, otherwise crystals are easily precipitated) into a new 5 ml centrifuge tube.

[0070] b5, second extraction: add 1 ml of the above n-hexane + ether (volume ratio 4:1) into the 10 ml centrifuge tube again, place in a multi-tube vortex to mix for 15 seconds, centrifuge for 10 minutes (speed same as above), take out the supernatant (about 1 ml, the supernatant cannot be taken out completely, and 2 mm height of supernatant must be left, otherwise crystals are easy to precipitate) and put it into a 5 ml round-bottom centrifuge tube.

[0071] b6, third extraction: repeat the above steps, use 1 ml of n-hexane + ether (volume ratio 4:1) to mix in a multi-tube vortex instrument for 15 seconds, centrifuge for 10 minutes (speed is the same as above), take out the supernatant (about 1 ml, the supernatant cannot be taken out completely, and 2 mm height of supernatant must be left, otherwise crystals are easily precipitated) into a 5 ml round-bottom centrifuge tube.

[0072] b7, after completing the above three extraction processes, place the organic phase stored in the 5ml centrifuge tube on a nitrogen blower and blow dry with high-purity nitrogen. After blowing dry, add 1ml sodium acetate buffer (add sample in a fume hood) and 50ul of β-glucuronidase / sulphatase enzyme (100μl pipette, white tip, blow to clean the residue on the tip). Close the lid tightly, place in a vortexer to mix for 1min, and then place in a 37℃ water bath for 90min.

[0073] b8. Take out the fully enzymatically hydrolyzed sample (then set the temperature of the water bath to 90°C for later use) and perform a second round of extraction.

[0074] b9, first extraction: add 1 ml of ethyl acetate one by one (add samples in a fume hood), shake on a multi-tube vortex mixer for 10 minutes, 3500r / min, until there is no precipitation (a very important step!), centrifuge for 10 minutes (4000rpm), take the supernatant (about 0.3 ml, the supernatant cannot be taken out completely, and 2mm of supernatant must be left, otherwise crystals will easily precipitate) into a new 5 ml centrifuge tube.

[0075] b10, second extraction: add 1 ml of the above n-hexane + ether (volume ratio 4:1) into the 10 ml centrifuge tube again, place in a multi-tube vortex to mix for 15 seconds, centrifuge for 10 minutes (speed same as above), take out the supernatant (about 1 ml, the supernatant cannot be taken out completely, and 2 mm height of the supernatant must be left, otherwise crystals are easily precipitated) and put into a 5 ml round-bottom centrifuge tube.

[0076] b11, third extraction: repeat the above steps, use 1 ml of n-hexane + ether (volume ratio 4:1), vortex for 15 seconds, centrifuge for 10 minutes (speed same as above), take out the supernatant (about 1 ml, the supernatant cannot be taken out completely, and 2 mm height of supernatant must be left, otherwise crystals are easily precipitated) into a 5 ml round-bottom centrifuge tube.

[0077] S7, nitrogen blow: After completing the above three extraction processes, place the organic phase stored in the 5ml centrifuge tube on a nitrogen blower and use high-purity nitrogen to concentrate it to about 250ul (do not blow it completely dry! Rinse the 5ml centrifuge tube with 100ul n-hexane + ether (volume ratio 4:1)), then transfer them to the inner tube one by one, and then transfer them to the nitrogen blower again and blow them dry with high-purity nitrogen.

[0078] S8, derivatization: add an equal amount (100ul) of silanization reagent to each inner tube, cover the bottle cap tightly, and then place it in a water bath (temperature set at 90°C, 45min) to allow it to be fully derivatized.

[0079] S9, sample analysis: After cooling at room temperature (about 30 min) after the water bath, perform gas chromatography-mass spectrometry analysis.

[0080] S10, sample test:

[0081] 1) Mass spectrometry ion source: Select electron impact source (EI).

[0082] 2) The injection port was set to splitless mode, with 99.999% helium as the carrier gas and the flow rate maintained at 1.2 ml / min.

[0083] 3) Automatic sampling, each sample volume is 1μl.

[0084] 4) Heating program: The initial temperature is set at 60°C for 3 minutes, then the temperature is evenly increased to 150°C (at a rate of 10°C / min) for 3 minutes, then evenly increased to 210°C at the same rate, and finally evenly increased to 280°C at the same rate, and maintained at this temperature for 5 minutes. The whole process lasts 33 minutes. The injection port temperature is 250°C, the ion source temperature is 280°C, and the gain is 1.

[0085] 5) Qualitative analysis: When processing a single standard sample, perform qualitative analysis based on the product ion pairs of each substance and obtain the retention time of each substance. When injecting mixed standards, perform qualitative analysis based on the product ion pairs and retention time of each substance.

[0086] Metabolites and internal standards Retention time Precursor ion Product ions Collision Energy 1-OHNAP-D7 15.987 223 208 15 1-OHNAP 16.043 216 201 15 2-OHNAP 16.547 216 201 15 MMP 16.731 237 89 15 MEP 17.837 251 75 15 MIBP 17.840 223 75 15 MBP 19.785 223 75 15 9-OHFLE 20.231 254 165 20 MOP 20.426 223 73 15 MEHP 21.669 221 73 15 2-OHFLE 22.471 254 239 15 MEOHP 24.633 221 73 15 4-OHPHE 25.209 266 235 25 9-OHPHE 25.211 266 73 25 1-OHPHE 25.216 266 73 25 3-OHPHE 25.854 266 73 25 2-OHPHE 26.702 266 73 25 MBZ 27.261 179 105 20 MEHP-C4 27.619 225 73 15 MEHHP-C4 28.154 225 73 15 MEHHP 29.367 221 73 15 1-OHPYR 30.857 290 73 25 1-OHPYR-D9 31.229 299 73 25 MECPP 31.667 221 73 15

[0087] S11 injection:

[0088] 1) First, observe whether there is precipitation in the insert tube of each bottle. If there is no precipitation, the sample can be loaded. If there is precipitation, the sample cannot be loaded (to prevent the precipitation from clogging the injection needle).

[0089] 2) Open the 7010D software.

[0090] 3) Edit sequence

[0091] a) Click Edit Sequence, and after the sequence table pops up, click "New Sample" in the sequence table (the number of new rows is the number of samples loaded).

[0092] b) Fill in 1, 2, 3... in the sample bottle column

[0093] c) Method path: Find the disk, open the method file, and click OK.

[0094] d) Method file: Find the method file dMRM.M in the above folder and click OK.

[0095] e) Data path: Create a new folder in the above folder (named after the date, for example: 20200710).

[0096] f) Data file: Fill in the sample name.

[0097] g) Type: Select "Blank" for the blank control bottle, select "Calibration" for the standard series sample bottle, select "Sample" for the urine sample, and then click "OK".

[0098] h) Click the Run button under the sequence, change the save path to 20200710, and click Run again.

[0099] S12, export data:

[0100] 1) Quantitative analysis of each substance was performed using QQQ quantitative software. (The value below the limit of detection (LOD) = LOD / √2; in order to eliminate the effect of blood dilution, the concentration of PAH metabolites was corrected using urine creatinine concentration, i.e., metabolite creatinine-corrected concentration (μg / g Cr) = PAH or PAE metabolite concentration (μg / L) / [urine creatinine concentration (mmol / L) × 113.12 (g / mol) × 10-3].)

[0101] S13, Quality Control:

[0102] (1) Limit of detection, limit of quantification, recovery and precision

[0103] With 3S / N as the detection limit, this experiment adopted the standard addition method, adding three different concentrations of standard products, low (B4), medium (B5), and high (B6), to 1.5mL urine samples, with 8 tubes of each concentration in parallel, to calculate the recovery rate and precision. The results are shown in the following table

[0104]

[0105] The limit of detection (LOD) of the method was 3 times the signal-to-noise ratio, and the limit of quantification (LOQ) of the method was 10 times the signal-to-noise ratio.

[0106] LOD = 3S / N, LOQ = 10S / N, (S: signal intensity of the target compound in the sample being tested; N: noise intensity, i.e. background signal intensity).

[0107] Recovery rate (%) = ((CF-CU) / CA)*100%, (CF: concentration of target compound in spiked sample; CU: concentration of compound in unspiked sample; CA: concentration of added target compound).

[0108] Precision RSD = SD / M*100%, where SD is the standard deviation of concentration and M is the arithmetic mean of concentration.

[0109] (2) Standard curve: A calibration curve should be drawn for each sample analysis. Normally, the correlation coefficient of the calibration curve should be above 0.996.

[0110] (3) Each batch of samples has at least one experimental blank to deduct the inevitable background value in the experimental process and reagents.

[0111] (4) Internal standard: The intensity of the internal standard must be monitored in each analysis. The response value of the internal standard in the sample should be within ±30% of the response value of the calibration curve. Otherwise, it indicates that the instrument has drifted or interference has occurred. The cause should be found and the analysis should be repeated.

[0112] As attached Figure 1 As shown, this detection method detected a total of 20 substances, including ten polycyclic aromatic hydrocarbon metabolites (OH-PAHS), namely 1-OHNAP, 2-OHFLU, 3-OHPHE, 9-OHPHE, 1-OHPHE, 2-OHNAP, 4-OHPHE, 1-OHPYR, 2-OHPHE, and 9-OHFLU;

[0113] There are 10 phthalate metabolites (PAEs), namely MECPP, MEOHP, MMP, MBP, MEHHP, MEP, MOP, MBZP, MEHP, and MIBP.

Claims

1. A method for extracting phthalates and polycyclic aromatic hydrocarbons in blood, characterized in that: The following steps are involved: S1, prepare standard reagents: prepare standard reagents in order; 1-naphthol, 2-naphthol, 2-hydroxyfluorene, 9-hydroxyfluorene, 9-hydroxyphenanthrene, 1-hydroxypyrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, monoethylhexyl phthalate, monoethyl phthalate, monomethyl phthalate, monooctyl phthalate, monobenzyl phthalate, monoisobutyl phthalate, monobutyl phthalate, mono-phthalic acid, phthalic acid monoesters and phthalic acid monoesters; S2, prepare stock solution: take 14 10ml brown volumetric flasks, numbered 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 21, 22 respectively; use a 1ml pipette to suck 0.5ml acetonitrile and inject it into the reagent bottle of the standard reagent corresponding to the above number, then mix it by blowing and suck it into the volumetric flask with the corresponding number, repeat the above steps four times and then make up the volume to 10ml with acetonitrile; then take 6 2ml brown injection bottles, numbered 1, 5, 6, 18, 19, 20 respectively, use a 200μl pipette to suck 200μl acetonitrile into the packaging bottle of the corresponding numbered standard reagent, mix it by blowing and suck it into the injection bottle with the corresponding number, repeat this step four times, and then add acetonitrile to make up the volume of the injection bottles numbered 1, 5, and 6 to 1ml, and the injection bottles numbered 18, 19, and 20 to 1.25ml; S3, prepare internal standard solution: take 480ul of 0.25 mg / ml 1-OHP-d9 stock solution, 240ul of 5mg / ml 1-OHNa-d7 stock solution, 1.2 ml of 100 ug / ml MEHP-c4, and 1.2 ml of 100 ug / ml MEHHP-c4, respectively, and dilute to 24ml with acetonitrile. The mixed solution of the four is the internal standard solution, the final concentration of 1-OHNa-d7 is 50.9 ug / ml, and the final concentrations of 1-OHP-d9, MEHP-c4 and MEHHP-c4 are 5 ug / ml; S4, prepare mixed standard solution: take a 10 ml centrifuge tube, add 1 ml acetonitrile and mark it as mixed standard 7, use 20 μl and 50 μl pipettes to transfer the stock solution of each standard reagent into the 10 ml centrifuge tube, then add acetonitrile to make up to 5 ml, mix well on a vortexer to prepare the mixed standard solution; S5, preparation of mixed standard 1 to mixed standard 6 solutions and control solution: a1. Prepare instruments: take 7 5 ml centrifuge tubes and label them as Mixed Standard 1, Mixed Standard 2, Mixed Standard 3, Mixed Standard 4, Mixed Standard 5, Mixed Standard 6, and Control 0; a2, preparation of mixed standard 6: take 1.5 ml of mixed standard 7 solution in step S4 and 1.5 ml of acetonitrile, add to mixed standard 6 tube, mix on a vortexer for 1 min, and prepare mixed standard 6; a3, preparation of mixed standard 5: take 1 ml of mixed standard 7 solution in step S4 and 4 ml of acetonitrile, add to mixed standard 5 tube, mix on a vortexer for 15 seconds, 3 times, to prepare mixed standard 5; a4, preparation of mixed standard 4: take 2 ml of mixed standard 5 and 2 ml of acetonitrile and add them to mixed standard 4 tube, mix on a vortexer for 15 seconds, 3 times, to prepare mixed standard 4; a5, preparation of mixed standard 3: take 1 ml of mixed standard 4 and 4 ml of acetonitrile and add them to mixed standard 3 tube, mix on a vortexer for 15 seconds, 3 times, to prepare mixed standard 3; a6, Preparation of Mixed Standard 2: Take 2 ml of Mixed Standard 3 and 2 ml of acetonitrile and add them to Mixed Standard 2 tube, mix on a vortexer for 15 seconds, 3 times, to prepare Mixed Standard 2; a7, Preparation of Mixed Standard 1: Take 1.5 ml of Mixed Standard 2 and 1.5 ml of acetonitrile and add them to Mixed Standard 1 tube, and mix them on a vortexer for 15 seconds, three times, to prepare Mixed Standard 1; a8, Preparation of control 0 solution: Take 3 ml of acetonitrile and add it to control 0 tube as blank control; S6, blood pretreatment: b1, thawing: take the blood out of the refrigerator, thaw it, shake it well and let it stand; b2, preparation of standard: take 8 10ml round-bottom plastic centrifuge tubes, numbered B0~B7, add 1ml pure water and 20μl of the above mixed standard solution to each tube, add control O to tube B0, add mixed standard 1 to tube B1, add mixed standard 2 to tube B2, add mixed standard 3 to tube B3, add mixed standard 4 to tube B4, add mixed standard 5 to tube B5, add mixed standard 6 to tube B6, and add mixed standard 7 to tube B7; b3, hydrolysis: use a 1 ml pipette to take 1 ml of blood and add it to a 10 ml round-bottom plastic centrifuge tube. Add 20 ul of internal standard solution to both the blood sample and the standard, add 2 ml of MgSO4•7H20, and shake on a multi-tube vortex mixer for 10 min at 3500 r / min until there is no precipitation. b4, primary extraction: add 1 ml of ethyl acetate one by one, centrifuge for 10 min at 4000 rpm, and take the supernatant into a new 5 ml centrifuge tube; b5, secondary extraction: add 1 ml of n-hexane and ether to a 10 ml centrifuge tube again, with a volume ratio of 4:1, place in a multi-tube vortex to mix for 15 s, centrifuge for 10 min at 4000 rpm, and remove the supernatant to a 5 ml round-bottom centrifuge tube; b6, three extractions: use 1 ml of the above n-hexane and ether, respectively, with a volume ratio of 4:1, place in a multi-tube vortex to mix for 15 s, centrifuge for 10 min at 4000 rpm, and remove the supernatant into a 5 ml round-bottom centrifuge tube; b7. After completing the extraction process three times, the organic phase stored in the 5 ml centrifuge tube was placed on a nitrogen blower and blown dry with high-purity nitrogen. After drying, 1 ml of sodium acetate buffer and 50 ul of β-glucuronidase / sulphatase enzyme were added, a 100 μl pipette, and a white tip. The tip was blown to clean the residue, the lid was tightly closed, and the mixture was mixed in a vortexer for 1 min, and then placed in a 37°C water bath for 90 min. b8, take out the fully enzymatically hydrolyzed sample, then set the temperature of the water bath to 90°C and set aside for the second round of extraction; b9, second round of extraction: 1st extraction, add 1 ml of ethyl acetate one by one, add samples in a fume hood, shake on a multi-tube vortex mixer for 10 min, 3500r / min, until there is no precipitation, centrifuge for 10 min at 4000 rpm, and take the supernatant to a new 5 ml centrifuge tube; 2nd extraction, add the above 1 ml of n-hexane and ether to the 10 ml centrifuge tube one by one again, with a volume ratio of 4:1, put on a multi-tube vortex instrument to mix for 15 s, centrifuge for 10 min at 4000 rpm, and take the supernatant to a 5 ml round-bottom centrifuge tube; 3rd extraction, use the above 1 ml of n-hexane and ether respectively, with a volume ratio of 4:1, vortex for 15 s, centrifuge for 10 min at 4000 rpm, and take the supernatant to a 5 ml round-bottom centrifuge tube; S7, nitrogen blowing: After completing two rounds of extraction, the organic phase in the 5 ml centrifuge tube was placed on a nitrogen blower and concentrated to about 250 ul using high-purity nitrogen. The organic phase was then transferred to the inner tubes one by one, transferred to the nitrogen blower again and dried using high-purity nitrogen. S8, derivatization: add 100 ul of silanization reagent to each inner tube, cover the bottle tightly, and then place it in a water bath at 90°C for 45 min to fully derivatize it; S9, sample analysis: After the water bath is finished, cool to room temperature and then perform gas chromatography-mass spectrometry analysis.

2. The method for extracting phthalates and polycyclic aromatic hydrocarbons in blood according to claim 1, characterized in that: Step S6: When thawing the blood sample during blood pretreatment, the blood sample is thawed in a water bath at room temperature or at 37°C for more than 3 hours. After thawing, the blood is shaken and allowed to stand until the precipitate sinks to the bottom of the tube.

3. The method for extracting phthalates and polycyclic aromatic hydrocarbons in blood according to claim 2, characterized in that: When 1 ml of ethyl acetate is added one by one in the first extraction of the two rounds of step S6, the samples need to be added in a fume hood.

4. The method for extracting phthalates and polycyclic aromatic hydrocarbons in blood according to claim 3, characterized in that: When taking the supernatant during the first extraction of the two rounds in step S6, take about 0.3 ml of the supernatant and leave 2 mm of the supernatant in the centrifuge tube to avoid crystallization; When taking the supernatant during the two rounds of second extraction in step S6, take about 1 ml of the supernatant and leave 2 mm of the supernatant in the centrifuge tube to avoid crystallization; When taking the supernatant during the two rounds of the third extraction in step S6, take about 1 ml of the supernatant and leave 2 mm of the supernatant in the centrifuge tube to avoid precipitation of crystals.

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