A UPLC-MS / MS method for simultaneous determination of multiple tetrabromodiphenyl ether metabolites in aquatic products

Abstract

The invention discloses a UPLC-MS / MS method for simultaneously determining a plurality of tetrabromodiphenyl ether metabolites in aquatic products, and specifically relates to the technical field of aquatic product pollution detection, comprising the following steps: step 1, sample extraction, S1: accurately weighing 5.0±0.2g of a homogenized sample, and sequentially adding 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate; S2: vortexing the sample solution treated in S1 for 25-32s, placing it in a water bath for ultrasonic-assisted extraction for 9-13min, centrifuging at 4000rpm for 5min, and taking the supernatant into a new tube; S3: adding 4-6mL of the above-mentioned acetone-n-hexane to the new tube containing the supernatant in S2, repeating the extraction once, centrifuging at 4000rpm for 4-6min, and combining the supernatants. Aiming at the matrix characteristics of aquatic products, the present invention combines mixed solvent-ultrasound-assisted liquid-liquid extraction and solid phase extraction purification methods to establish a high-throughput UPLC-MS / MS detection method for various BDE-47 metabolites in aquatic products, which is rapid, sensitive and accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic product pollution detection, and in particular to a UPLC-MS / MS method for simultaneously determining a plurality of tetrabromodiphenyl ether metabolites in aquatic products. Background Art

[0002] Persistent organic pollutants (POPs) are characterized by widespread, persistent and multi-toxicity, and can be accumulated and amplified through the food chain, causing serious harm to the human body and the ecological environment. Polybrominated diphenyl ethers (PBDEs) are currently the focus of attention of toxic halogenated flame retardants, which are widely detected in environmental media and organisms. Among them, four PBDEs and HBCD containing 4-7 bromine atoms have been included in the POPs list. PBDEs can be accumulated and amplified through the food chain, and different concentrations of PBDEs have been detected in many aquatic products such as fish and shellfish.

[0003] 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is one of the most abundant PBDEs homologues in the environment. It is widely distributed in the natural environment, especially in the marine environment. It has bioaccumulation characteristics and strong neurotoxicity, genotoxicity, immunotoxicity, etc. It is a new type of POPs. Toxicological studies have shown that the toxic effects of two types of BDE-47 derivatives, methoxylated BDE-47 (MeO-BDE47) and hydroxylated BDE-47 (HO-BDE47), are higher than the parent compound BDE-47 itself. Eating aquatic products contaminated by BDE-47 and its metabolites may cause serious health problems.

[0004] At present, there are no relevant standards for the determination of hydroxylated and methoxylated BDE-47 derivatives in aquatic products. The determination methods for hydroxylated and methoxylated BDE-47 include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (HPLC-MS), and most of them are based on the determination of single metabolite indicators, with complicated steps, time-consuming operations, and long detection cycles. For example, the GC-MS method requires the addition of silane reagents for derivatization, which has many interfering factors and cannot meet the actual needs of rapid and accurate determination of multiple samples and multiple indicators. Aquatic products are rich in interfering impurities such as oils and proteins, and the complex matrix interferes with instrument detection and analysis. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the problems existing in the existing UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to provide a UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products. The problem to be solved is that the method is mainly based on the determination of metabolite indicators, the steps are cumbersome, the operation is time-consuming, and the detection cycle is long. In addition, if silane reagent is added for derivatization, the interference factors are large, and the actual needs of rapid and accurate determination of multiple samples and multiple indicators cannot be met. Moreover, aquatic products are rich in interfering impurities such as oil, protein, etc., and the complex matrix interferes with instrument detection and analysis.

[0008] In order to solve the above technical problems, the present invention provides the following technical solution: a UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products, comprising the following steps:

[0009] Step 1: Sample extraction

[0010] S1: Accurately weigh 5.0±0.2g of the homogenized sample, and add 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate in sequence;

[0011] S2: The sample solution treated in S1 was vortexed for 25-32 seconds and then placed in a water bath for ultrasound-assisted extraction for 9-13 minutes. The solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was transferred to a new tube.

[0012] S3: Add 4-6 mL of the above acetone-n-hexane to a new tube containing the supernatant in S2 and repeat the extraction once. Centrifuge at 4000 rpm for 4-6 min and combine the supernatants.

[0013] Step 2: Purification of sample solution

[0014] Take 5 mL of the combined supernatant and load it onto the activated Captiva EMR-Lipid (6 mL, 600 mg) solid phase extraction column. Keep the flow rate at about 2 mL / min, collect the effluent, blow it to near dryness with nitrogen at 38°C-42°C, and add acetonitrile to make up to 0.7-1.3 mL.

[0015] Step 3: Test the sample solution using liquid chromatography-tandem mass spectrometry

[0016] The liquid chromatography conditions are:

[0017] The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, the column temperature was 33°C-36°C, the injection volume was 5 μL, the mobile phase A was 0.1% formic acid aqueous solution (containing 2 mmol ammonium acetate), and the mobile phase B was acetonitrile. The gradient elution program was as follows: 0-1 min, A:B=70:30; 1-2 min, A:B=50:50; 2-5 min, A:B=5:95; 5-10 min, A:B=5:95; 10-11 min, A:B=70:30.

[0018] The mass spectrometry conditions are:

[0019] Drying gas temperature: 280℃-310℃; Drying gas flow rate: (9-12L) / min; Nebulizing gas pressure: 40psi; Capillary voltage: 3500V; Sheath gas temperature: 350℃; Sheath gas flow rate: 12L / min.

[0020] As a preferred embodiment of the UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products described in the present invention, wherein: in S1, 10 mL of acetone-n-hexane is added, and the v / v ratio of acetone to n-hexane is 1:1.

[0021] As a preferred embodiment of the UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products described in the present invention, wherein: in S1, the weight of the added sodium chloride is 1 g, and the added anhydrous sodium sulfate is 1 g.

[0022] As a preferred embodiment of the UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products described in the present invention, wherein: in S2, the sample solution is vortexed for 30 seconds and then placed in a water bath for ultrasonic-assisted extraction for 10 minutes.

[0023] As a preferred embodiment of the UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products described in the present invention, wherein: in S3, 5 mL of the above-mentioned acetone-n-hexane is added and the extraction is repeated once, and centrifuged at 4000 rpm for 5 min.

[0024] As a preferred embodiment of the UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products described in the present invention, the collected effluent in step 2 is blown to near dryness with nitrogen at 40° C., and acetonitrile is added to make the volume to 1 mL.

[0025] As a preferred embodiment of the UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products described in the present invention, the column temperature of the chromatographic column in the liquid chromatography condition in step three is 35°C.

[0026] As a preferred embodiment of the UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products described in the present invention, the drying gas temperature of the mass spectrometry conditions in step three is 300° C. and the drying gas flow rate is 10 L / min.

[0027] In summary, the present invention includes at least one of the following beneficial effects:

[0028] The present invention aims at the matrix characteristics of aquatic products, combines mixed solvent-ultrasound-assisted liquid-liquid extraction and solid phase extraction purification means, and establishes a high-throughput UPLC-MS / MS detection method for various BDE-47 metabolites in aquatic products, which is rapid, sensitive and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0030] Figure 1 is the MRM spectrum of the metabolite measured in the present invention; DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The embodiment of the present invention discloses a UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products.

[0033] Embodiment 1

[0034] A UPLC-MS / MS method for the simultaneous determination of multiple tetrabromodiphenyl ether metabolites in aquatic products comprises the following steps:

[0035] Step 1: Sample extraction

[0036] S1: Accurately weigh 5.0±0.2g of the homogenized sample, and add 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate in sequence;

[0037] S2: The sample solution treated in S1 was vortexed for 25-32 seconds and then placed in a water bath for ultrasound-assisted extraction for 9-13 minutes. The solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was transferred to a new tube.

[0038] S3: Add 4-6 mL of the above acetone-n-hexane to a new tube containing the supernatant in S2 and repeat the extraction once. Centrifuge at 4000 rpm for 4-6 min and combine the supernatants.

[0039] Step 2: Purification of sample solution

[0040] Take 5 mL of the combined supernatant and load it onto the activated Captiva EMR-Lipid (6 mL, 600 mg) solid phase extraction column. Keep the flow rate at about 2 mL / min, collect the effluent, blow it to near dryness with nitrogen at 38°C-42°C, and add acetonitrile to make up to 0.7-1.3 mL.

[0041] Step 3: Test the sample solution using liquid chromatography-tandem mass spectrometry

[0042] The liquid chromatography conditions are:

[0043] The chromatographic column was a Waters ACQUITY UPLC BEHC18 column, the column temperature was 33°C-36°C, the injection volume was 5 μL, the mobile phase A was 0.1% formic acid aqueous solution (containing 2 mmol ammonium acetate), and the mobile phase B was acetonitrile. The gradient elution program was as follows: 0-1 min, A:B=70:30; 1-2 min, A:B=50:50; 2-5 min, A:B=5:95; 5-10 min, A:B=5:95; 10-11 min, A:B=70:30.

[0044] The mass spectrometry conditions are:

[0045] Drying gas temperature: 280℃-310℃; Drying gas flow rate: (9-12L) / min; Nebulizing gas pressure: 40psi; Capillary voltage: 3500V; Sheath gas temperature: 350℃; Sheath gas flow rate: 12L / min.

[0046] The specific values ​​in the above steps are as follows: in S1, 8 mL of acetone-n-hexane is added, and the v / v of acetone and n-hexane is 1:1, the weight of sodium chloride added is 0.7 g, and the weight of anhydrous sodium sulfate added is 0.8 g. In S2, the sample solution is vortexed for 25 seconds and then placed in a water bath for ultrasonic-assisted extraction for 9 minutes. In S3, 4 mL of the above acetone-n-hexane is added and the extraction is repeated once, and the mixture is centrifuged at 4000 rpm for 4 minutes. The collected effluent in step 2 is blown to near dryness with nitrogen at 38°C, and acetonitrile is added to make the volume to 0.7 mL. The column temperature of the chromatographic column under the liquid chromatography conditions in step 3 is 33°C, and the drying gas temperature under the mass spectrometry conditions in step 3 is 280°C; the drying gas flow rate is 9 L / min.

[0047] Embodiment 2

[0048] A UPLC-MS / MS method for the simultaneous determination of multiple tetrabromodiphenyl ether metabolites in aquatic products comprises the following steps:

[0049] Step 1: Sample extraction

[0050] S1: Accurately weigh 5.0±0.2g of the homogenized sample, and add 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate in sequence;

[0051] S2: The sample solution treated in S1 was vortexed for 25-32 seconds and then placed in a water bath for ultrasound-assisted extraction for 9-13 minutes. The solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was transferred to a new tube.

[0052] S3: Add 4-6 mL of the above acetone-n-hexane to a new tube containing the supernatant in S2 and repeat the extraction once. Centrifuge at 4000 rpm for 4-6 min and combine the supernatants.

[0053] Step 2: Purification of sample solution

[0054] Take 5 mL of the combined supernatant and load it onto the activated Captiva EMR-Lipid (6 mL, 600 mg) solid phase extraction column. Keep the flow rate at about 2 mL / min, collect the effluent, blow it to near dryness with nitrogen at 38°C-42°C, and add acetonitrile to make up to 0.7-1.3 mL.

[0055] Step 3: Test the sample solution using liquid chromatography-tandem mass spectrometry

[0056] The liquid chromatography conditions are:

[0057] The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, the column temperature was 33°C-36°C, the injection volume was 5 μL, the mobile phase A was 0.1% formic acid aqueous solution (containing 2 mmol ammonium acetate), and the mobile phase B was acetonitrile. The gradient elution program was as follows: 0-1 min, A:B=70:30; 1-2 min, A:B=50:50; 2-5 min, A:B=5:95; 5-10 min, A:B=5:95; 10-11 min, A:B=70:30.

[0058] The mass spectrometry conditions are:

[0059] Drying gas temperature: 280℃-310℃; Drying gas flow rate: (9-12L) / min; Nebulizing gas pressure: 40psi; Capillary voltage: 3500V; Sheath gas temperature: 350℃; Sheath gas flow rate: 12L / min.

[0060] The specific values ​​in the above steps are as follows: in S1, 10 mL of acetone-n-hexane is added, and the v / v of acetone and n-hexane is 1:1, the weight of sodium chloride added is 1 g, and the weight of anhydrous sodium sulfate added is 1 g. In S2, the sample solution is vortexed for 30 seconds and then placed in a water bath for ultrasonic-assisted extraction for 10 minutes. In S3, 5 mL of the above acetone-n-hexane is added and the extraction is repeated once, and the mixture is centrifuged at 4000 rpm for 5 minutes. The collected effluent in step 2 is blown to near dryness with nitrogen at 40°C, and acetonitrile is added to make the volume 1 mL. The column temperature of the chromatographic column under the liquid chromatography conditions in step 3 is 35°C, and the drying gas temperature under the mass spectrometry conditions in step 3 is 300°C; the drying gas flow rate is 10 L / min.

[0061] Embodiment 3

[0062] A UPLC-MS / MS method for the simultaneous determination of multiple tetrabromodiphenyl ether metabolites in aquatic products comprises the following steps:

[0063] Step 1: Sample extraction

[0064] S1: Accurately weigh 5.0±0.2g of the homogenized sample, and add 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate in sequence;

[0065] S2: The sample solution treated in S1 was vortexed for 25-32 seconds and then placed in a water bath for ultrasound-assisted extraction for 9-13 minutes. The solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was transferred to a new tube.

[0066] S3: Add 4-6 mL of the above acetone-n-hexane to a new tube containing the supernatant in S2 and repeat the extraction once. Centrifuge at 4000 rpm for 4-6 min and combine the supernatants.

[0067] Step 2: Purification of sample solution

[0068] Take 5 mL of the combined supernatant and load it onto the activated Captiva EMR-Lipid (6 mL, 600 mg) solid phase extraction column. Keep the flow rate at about 2 mL / min, collect the effluent, blow it to near dryness with nitrogen at 38°C-42°C, and add acetonitrile to make up to 0.7-1.3 mL.

[0069] Step 3: Test the sample solution using liquid chromatography-tandem mass spectrometry

[0070] The liquid chromatography conditions are:

[0071] The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, the column temperature was 33°C-36°C, the injection volume was 5 μL, the mobile phase A was 0.1% formic acid aqueous solution (containing 2 mmol ammonium acetate), and the mobile phase B was acetonitrile. The gradient elution program was as follows: 0-1 min, A:B=70:30; 1-2 min, A:B=50:50; 2-5 min, A:B=5:95; 5-10 min, A:B=5:95; 10-11 min, A:B=70:30.

[0072] The mass spectrometry conditions are:

[0073] Drying gas temperature: 280℃-310℃; Drying gas flow rate: (9-12L) / min; Nebulizing gas pressure: 40psi; Capillary voltage: 3500V; Sheath gas temperature: 350℃; Sheath gas flow rate: 12L / min.

[0074] The specific values ​​in the above steps are as follows: in S1, 11 mL of acetone-n-hexane is added, and the v / v of acetone and n-hexane is 1:1, the weight of sodium chloride added is 1.3 g, and the weight of anhydrous sodium sulfate added is 1.1 g. In S2, the sample solution is vortexed for 32 seconds and then placed in a water bath for ultrasonic-assisted extraction for 13 minutes. In S3, 6 mL of the above acetone-n-hexane is added and the extraction is repeated once, and the mixture is centrifuged at 4000 rpm for 6 minutes. The collected effluent in step 2 is blown to near dryness with nitrogen at 42°C, and acetonitrile is added to make the volume to 1.3 mL. The column temperature of the chromatographic column under the liquid chromatography conditions in step 3 is 36°C, and the drying gas temperature under the mass spectrometry conditions in step 3 is 310°C; the drying gas flow rate is 12 L / min.

[0075] By analyzing the difference in the values ​​in different steps of the three embodiments, it is concluded that the values ​​in Embodiment 2 are the best values. The quantitative and qualitative ion pairs in Embodiment 2 are shown in the following table, where * indicates quantitative ions.

[0076]

[0077] In addition, the linear correlation coefficients (R 2 ) is 0.996-0.999, with good linear correlation. The detection limit is 0.1-0.3μg / kg, and the quantification limit is 0.3-0.9μg / kg. The method has high sensitivity, as shown in the following table.

[0078]

[0079] Finally, a few points should be explained: First, in the description of this application, it should be noted that, unless otherwise specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, which may refer to mechanical connection or electrical connection, or internal communication between two components, or direct connection. "upper", "lower", "left", "right", etc. are only used to indicate relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship may change;

[0080] Secondly: In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other;

[0081] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products, characterized in that: The following steps are involved: Step 1: Sample extraction S1: Accurately weigh 5.0±0.2g of the homogenized sample, and add 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate in sequence; S2: The sample solution treated in S1 was vortexed for 25-32 seconds and then placed in a water bath for ultrasound-assisted extraction for 9-13 minutes. The solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was transferred to a new tube. S3: Add 4-6 mL of the above acetone-n-hexane to the new tube containing the supernatant in S2 and repeat the extraction once, centrifuge at 4000 rpm for 4-6 min, and combine the supernatants; Step 2: Purification of sample solution Take 5 mL of the combined supernatant and load it onto the activated Captiva EMR-Lipid, 6 mL, 600 mg solid phase extraction column. Keep the flow rate at about 2 mL / min, collect the effluent, blow it to near dryness with nitrogen at 38°C-42°C, and add acetonitrile to make up to 0.7-1.3 mL. Step 3: Test the sample solution using liquid chromatography-tandem mass spectrometry The liquid chromatography conditions are: The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, the column temperature was 33°C-36°C, the injection volume was 5 μL, the mobile phase A was 0.1% formic acid aqueous solution containing 2 mmol ammonium acetate, and the mobile phase B was acetonitrile; the gradient elution program was as follows: 0-1 min, A:B=70:30; 1-2 min, A:B=50:50; 2-5 min, A:B=5:95; 5-10 min, A:B=5:95; 10-11 min, A:B=70:30; The mass spectrometry conditions are: Drying gas temperature: 280℃-310℃; Drying gas flow rate: 9-12L / min; Nebulizing gas pressure: 40psi; Capillary voltage: 3500V; Sheath gas temperature: 350℃; Sheath gas flow rate: 12L / min; Among them, the six metabolites are 3-HO-BDE47, 5-HO-BDE47, 6-HO-BDE47, 3-McO-BDE47, 5-MeO-BDE47 and 6-MeO-BDE47.

2. The UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that: In S1, 10 mL of acetone-n-hexane was added, and the v / v ratio of acetone to n-hexane was 1:

1.

3. The UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that: In S1, the weight of the added sodium chloride is 1 g, and the weight of the added anhydrous sodium sulfate is 1 g.

4. The UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that: In S2, the sample solution was vortexed for 30 seconds and then placed in a water bath for ultrasound-assisted extraction for 10 minutes.

5. The UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that: In S3, 5 mL of the above acetone-n-hexane was added and the extraction was repeated once, and centrifuged at 4000 rpm for 5 min.

6. The UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that: The collected effluent in step 2 was blown to near dryness with nitrogen at 40°C, and acetonitrile was added to make up to 1 mL.

7. The UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that: The column temperature of the chromatographic column in the liquid chromatography condition in step 3 is 35°C.

8. The UPLC-MS / MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that: The drying gas temperature of the mass spectrometry conditions in step 3 is 300° C. and the drying gas flow rate is 10 L / min.

Images