Pretreatment method and analysis method of tetrabromobisphenol A and unilateral derivative thereof in sludge

Abstract

The invention provides a pretreatment method and an analysis method of tetrabromobisphenol A and a unilateral derivative thereof in sludge. According to the pretreatment method, step-by-step extraction is adopted, extraction agents are n-hexane and acetone and n-hexane and dichloromethane, synchronous extraction of multi-polar target objects is achieved, and interferents such as lipid and protein are removed through purification with a gel chromatographic column; the analysis method is based on an LC-HRMS (Liquid Chromatography-High Resolution Mass Spectrometry) combined technology, and high-sensitivity detection is realized through a gradient elution and multi-reaction monitoring mode. The method effectively solves the problem of synchronous analysis of the polybrominated phenolic compounds in the complex matrix, and has the advantages of simplicity and convenience in operation, strong anti-interference capability, stable recovery rate and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental pollutant analysis, and particularly relates to an efficient pretreatment method and an analysis method for tetrabromobisphenol A (TBBPA) and its mono-derivatives in sludge, which are particularly suitable for the simultaneous extraction, purification and detection of polybrominated phenols in sludge with complex matrixes. Background Art

[0002] Tetrabromobisphenol A (TBBPA) and its mono-derivatives are widely used brominated flame retardants and are frequently detected in the activated sludge of sewage treatment plants. However, the matrix of activated sludge is extremely complex, containing a large amount of organic matter (such as humic acid, protein), lipids, microorganisms and inorganic substances. These substances compete for adsorption or co-extract with the target substances, resulting in serious matrix effects and significantly reducing the detection sensitivity. In addition, the polarity differences of TBBPA and its derivatives are significant. For example, TBBPA is a medium-polarity compound, while the polarity range of its mono-derivatives (including hydroxylated, methoxylated products, etc.) spans a large range, making the simultaneous detection of multiple substances extremely challenging.

[0003] The existing analysis methods have obvious defects:

[0004] Low extraction efficiency: Using only a single solvent (such as acetone or dichloromethane) cannot cover the extraction requirements of multi-polarity target substances, resulting in extremely low recovery rates of some derivatives;

[0005] Insufficient purification: The solid-phase extraction column purification method has limited ability to remove lipids and proteins in sludge, and the residual interfering substances affect the life of the liquid chromatography column and the accuracy of the mass spectrometry signal;

[0006] Complicated operation: The multi-step pretreatment process is time-consuming and costly, and it is difficult to meet the high-throughput requirements of actual detection.

[0007] Therefore, there is an urgent need for a pretreatment method that can simultaneously extract multi-polarity target substances and efficiently remove the interference of complex matrixes to achieve the accurate analysis of TBBPA and its derivatives in sludge. Summary of the Invention

[0008] Aiming at the deficiencies of the existing technology, the present invention provides a pretreatment method and an analysis method for TBBPA and its mono-derivatives in sludge, and solves the technical problems of the simultaneous extraction and detection of polybrominated phenols in complex matrixes by optimizing the extraction agent combination, gel chromatography purification and LC-HRMS coupling technology.

[0009] To solve the above technical problems, the technical solution proposed in this application is:

[0010] The present invention provides a pretreatment method for tetrabromobisphenol A (TBBPA) and its mono-derivatives in sludge, including the following steps:

[0011] (1) Freeze-dry the sludge sample, grind it, and pass it through a 200-mesh sieve. Take 0.5 g of the sample and place it in a 2-mL centrifuge tube, then add small steel balls with a diameter of 4 mm.

[0012] (2) Sequentially add a mixed solution of n-hexane and acetone with a volume ratio of 8:2 and a mixed solution of n-hexane and dichloromethane with a volume ratio of 1:1 as extraction agents, and perform oscillatory extraction. After each extraction, centrifuge and collect the supernatant, repeating 3 times.

[0013] (3) Combine the supernatants after 6 extractions, purify them through a gel permeation chromatography column, and elute with a mixed solution of dichloromethane and n-hexane with a volume ratio of 1:1.

[0014] (4) Concentrate the eluate by nitrogen blowing, redissolve it with methanol, and make the volume up to 1 mL.

[0015] Further, in step (2), the volume of the mixed solution of n-hexane and acetone is 1 mL, the oscillatory extraction time is 5 min, and the frequency is 30 Hz.

[0016] Further, the centrifugation conditions in step (2) are: rotation speed 10000 r / min, time 10 min, and temperature 4 °C.

[0017] Further, the filler of the gel permeation chromatography column is Bio-Beads S-X3, the eluent is a mixed solution of dichloromethane and n-hexane with a volume ratio of 1:1, the elution volume is 300 mL; the eluent for rinsing is the same as the eluent, and the flow rate is 5 mL / min.

[0018] Further, after nitrogen blowing in step (4) until nearly dry, redissolve it with methanol and make the volume up to 1 mL, and transfer it to a 2-mL injection vial.

[0019] On the other hand, the present application protects an analytical method for tetrabromobisphenol A and its unilateral derivatives in sludge, including the following steps:

[0020] (a) Treat the sample using any of the aforementioned pretreatment methods;

[0021] (b) Detect and analyze the pretreated sample by liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS).

[0022] Further, the liquid chromatography conditions in step (b) are: the chromatographic column is GL-Science Inertsil ODS-4, with a specification of 3 mm × 150 mm × 2 μm, the mobile phase is gradient elution of water and methanol, the initial volume ratio is 80:20, the flow rate is 0.3 mL / min, and the column temperature is 20 °C.

[0023] Further, the mass spectrometry conditions in step (b) are as follows: electrospray ionization source, negative ion mode, capillary voltage 3500 V, drying gas flow rate 10 L / min, and temperature 300 °C.

[0024] Further, the method also includes performing quantitative analysis of the target compound using the multiple reaction monitoring mode.

[0025] Further, the method is applicable to the simultaneous analysis of TBBPA and its five unilateral derivatives in sludge, and the recovery rate of the target compound is ≥ 65%.

[0026] Compared with the prior art, the pretreatment method and analysis method of TBBPA and its unilateral derivatives in sludge of the present invention have achieved the following beneficial technical effects:

[0027] The present invention designs a stepwise extraction (a mixed solution of n-hexane + acetone and n-hexane + dichloromethane) to cover the wide polarity range of TBBPA and its unilateral derivatives, realizing the synchronous and efficient extraction of multi-polar compounds; combining the molecular sieve effect of gel permeation chromatography (Bio-Beads S-X3), selectively removing most of the macromolecular interfering substances such as lipids and proteins in sludge, significantly reducing the matrix effect; based on the LC-HRMS coupling technology, through gradient elution and multiple reaction monitoring mode (MRM), the detection limit of the target compound is as low as 0.1 ng / g, and the recovery rate is stable. Compared with the traditional method, the pretreatment process of the present invention is simplified, without complex solid-phase extraction steps, and the operation cost is reduced by more than 30%, providing a reliable solution for the accurate analysis of brominated phenolic compounds in complex matrix sludge. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is a schematic diagram of a sample pretreatment method and a mass spectrometry detection and analysis method according to an embodiment of the present application;

[0030] Figure 2 It is a schematic diagram of the detected contents of tetrabromobisphenol A and its five derivatives in a sludge sample according to an embodiment of the present application;

[0031] Figure 3 It is a schematic diagram of the retention time of the target compound in liquid chromatography during the data analysis process according to an embodiment of the present application;

[0032] Figure 4It is a schematic diagram of the recovery rate of the target substance in the data analysis process according to the embodiments of the present application. Detailed implementation manners

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] As Figures 1-4 shown, a pretreatment method for tetrabromobisphenol A (TBBPA) and its unilateral derivatives in sludge according to an embodiment of the present application includes the following steps:

[0035] (1) Freeze-dry the sludge sample, grind it through a 200-mesh sieve to eliminate the interference of moisture, take 0.5 g of the sample and place it in a 2-mL centrifuge tube, and add small steel balls with a diameter of 4 mm;

[0036] (2) Sequentially add a mixed solution of n-hexane and acetone with a volume ratio of 8:2 and a mixed solution of n-hexane and dichloromethane with a volume ratio of 1:1 as extraction agents, perform oscillating extraction, centrifuge and collect the supernatant after each extraction, and repeat 3 times;

[0037] It can be seen that in the present application, the extraction is carried out in 2 steps:

[0038] First extraction: Use a mixed solution of n-hexane and acetone with a volume ratio of 8:2 to extract medium-polarity target substances (such as TBBPA);

[0039] Second extraction: Use a mixed solution of n-hexane and dichloromethane with a volume ratio of 1:1 to extract low-polarity derivatives;

[0040] (3) Combine the supernatants after 6 extractions, purify them through a gel permeation chromatography column, and elute them with a mixed solution of dichloromethane and n-hexane with a volume ratio of 1:1;

[0041] In the present application, a Bio-Beads S-X3 chromatographic column is used to further remove macromolecular interfering substances such as lipids and proteins;

[0042] (4) The eluate is concentrated by nitrogen blowing, redissolved with methanol and fixed to 1 mL.

[0043] In the present application, after the above pretreatment, an analysis method for tetrabromobisphenol A and its unilateral derivatives in sludge is also carried out, including the following steps:

[0044] (a) Process the sample by performing the pretreatment method as described above;

[0045] (b) The pretreated sample was detected and analyzed by liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS).

[0046] During the use of liquid chromatography, a gradient elution program (water / methanol) was preferably adopted to separate the target substance from interfering components. During the use of high-resolution mass spectrometry, in combination with the multiple reaction monitoring (MRM) mode, the detection sensitivity and specificity were improved.

[0047] The following further introduces the present application in conjunction with embodiments:

[0048] According to one aspect of the embodiments of the present application, an example of an analysis method for tetrabromobisphenol A and its unilateral derivatives in sludge is provided. An analysis method for tetrabromobisphenol A and its unilateral derivatives in sludge according to an embodiment of the present application includes the following steps:

[0049] Step S101, the collected sludge sample was subjected to freeze-drying treatment by a freeze dryer, ground and passed through a 200-mesh sieve, 0.5 g of the sample was taken and placed in a 2 mL centrifuge tube.

[0050] The above sample was used to detect TBBPA and its unilateral derivatives.

[0051] Step S102, a mixed solution of n-hexane and acetone (HEX / AC; 8:2, v / v); a mixed solution of n-hexane and dichloromethane (v:v = 1:1) was added to the centrifuge tube of the sample as an extractant.

[0052] Step S103, the extraction method was oscillating extraction. After the sample was extracted, the extract was centrifuged by a centrifuge, and the supernatant part in the centrifuge tube was collected.

[0053] When extracting the sample: the volume of the extractant added to the sample centrifuge tube each time was 1 mL, and the extraction time of oscillating extraction was 5 min.

[0054] Step S104, the sample supernatant obtained after repeating 6 times of oscillating extraction and centrifugation was collected.

[0055] When extracting the supernatant part in the centrifuge tube after centrifuging the extract obtained by extracting the sample by a centrifuge: the centrifugal rotation speed of the centrifuge was 10000 r / min, the centrifugal time was 10 min, and the temperature was 4 °C.

[0056] Step S105, the obtained sample supernatant was loaded onto a gel permeation chromatography column, rinsed with a mixed solution of DCM / HEX (1:1, v / v), and then eluted with a mixed solution of DCM / HEX (1:1, v / v) to collect the sample eluate.

[0057] When purifying and concentrating the sample using a gel permeation chromatography column: The packing material of the gel permeation chromatography column is Bio - Beads S - X3 (Bio - Rad Laboratories, Hercules, California, USA); the solutions used for the packing material are DCM / HEX (1:1, v / v) mixed solution; the solution used for elution is 300 mL of DCM / HEX (1:1, v / v) mixed solution (v:v = 1:1).

[0058] Step S106, use a nitrogen evaporator to evaporate the eluted liquid of the sample to near dryness with nitrogen, redissolve the sample with methanol and transfer it to a 2 - mL injection vial, and make the volume up to 1 mL with methanol.

[0059] Step S107, use liquid chromatography - tandem high - resolution mass spectrometry (LC - HRMS) to perform on - machine detection on the solution of the sample obtained after pretreatment, and obtain the instrumental analysis data of the sample.

[0060] In the embodiment of the present application, by using different extractants for the sludge sample respectively, target compounds with different polarities in the sample can be extracted, and proteins in the matrix can be removed. A gel permeation chromatography column is used to purify and concentrate the sample, and instrumental analysis is carried out based on liquid chromatography - tandem high - resolution mass spectrometry (LC - HRMS), so that information on TBBPA and its mono - side derivatives in the sample can be obtained as completely as possible. The present application solves the technical problem that the analysis methods of TBBPA and its mono - side derivatives in the related art do not have the ability to perform pretreatment and analysis on multiple derivatives and TBBPA itself simultaneously.

[0061] As an optional embodiment, the present application provides a pretreatment method for analyzing TBBPA and its mono - side derivatives in a sludge sample. Mainly according to the characteristics of the molecular size differences of TBBPA and its mono - side derivatives in the sample, different extractants are used for the sludge sample respectively to extract target compounds with different polarities in the sample, and the protein matrix in the sample is removed. A gel permeation chromatography column is used to purify and concentrate the sample, and instrumental analysis is carried out based on liquid chromatography - tandem high - resolution mass spectrometry (LC - HRMS). The specific operation steps are as follows:

[0062] As Figure 1As shown in the figure, for the sample: accurately weigh 0.5 g of the cold-dried, ground and sieved sludge sample, add 1 mL of a mixed solution of n-hexane and acetone (HEX / AC; 8:2, v / v) into a 2 mL centrifuge tube, and perform extraction by shaking 3 times. After the obtained extract is centrifuged at 10,000 r / min and 4 °C for 10 min by a centrifuge, the supernatant is extracted; add 1 mL of a mixed solution of n-hexane and dichloromethane (v:v = 1:1) into the above 2 mL centrifuge tube and repeat extraction 3 times again, and combine the supernatants. Use a gel permeation chromatography column with the packing of Bio-Beads S-X3 (Bio-Rad Laboratories, Hercules, California, USA) to purify and concentrate the sludge sample. Use 300 mL of a mixed solution of n-hexane and dichloromethane (v:v = 1:1) for elution. Collect the eluate of 120 mL - 240 mL, blow it to near dryness with nitrogen, redissolve it with methanol and transfer it to a 2 mL injection vial, and make the volume up to 1 mL with methanol, and perform instrumental analysis by liquid chromatography tandem high-resolution mass spectrometry.

[0063] This application provides a method for analyzing tetrabromobisphenol A and its unilateral derivatives in a sludge sample, and the purpose is to perform mass spectrometry detection on the sample obtained by pretreatment. The specific operation steps are as follows:

[0064] (1) Qualitative and quantitative analysis of the sample obtained by pretreatment is carried out by a combined system of an Agilent 1290 Infinity II ultra-high performance liquid chromatograph (Agilent Scientific Inc, USA) and an Agilent 6460 triple quadrupole (Agilent Scientific Inc, USA).

[0065] (2) The liquid chromatography column is selected as GL-Science Inertsil ODS-4 (3 mm × 150 mm × 2 μm), and this chromatographic column is a medium-pressure chromatographic column with a maximum pressure resistance value of 800 bar.

[0066] (3) The mobile phase of the liquid chromatography is pure water and methanol, and the gradient elution program is as follows: the initial ratio of the aqueous phase and the organic phase is 80:20, and it is maintained for 1 min; it becomes 20:80 within 4 min; the ratio gradually becomes 100% organic phase within 15 min and is maintained for 6 min; then it is restored to the initial ratio (80:20) within 1 min and kept for 1 min.

[0067] (4) The injection volume is 5 μL, the column temperature is 20 °C, and the mobile phase flow rate is 0.3 mL·min -1 。

[0068] (5) The mass spectrometry ion source is an electrospray ionization (ESI) source (electron spray ionization, ESI), with the temperature set at 300 °C, the drying gas flow rate at 10 L / min, the protective gas temperature at 325 °C, and the flow rate at 11 L / min.

[0069] (6) The determination is carried out in multiple reaction monitoring (MRM) mode and negative ion mode (negative), with Delta EMV at -600 V, and the capillary voltage is set at 3500 V.

[0070] As Figure 2 shown, this application provides the content of the target substance detected in the sample; as Figure 3 shown, it provides the retention time of the target substance in the mass spectrometry data; as Figure 4 shown, it provides the recovery rate of the target substance.

[0071] Compared with the prior art, the advantages and positive effects of this method are: by using different extractants for sludge samples respectively, target compounds with different polarities in the samples can be extracted, and a gel permeation chromatography purification column is used to purify and concentrate the samples. Based on liquid chromatography tandem high-resolution mass spectrometry for instrumental analysis, information on tetrabromobisphenol A and its derivatives in the samples can be obtained as completely as possible.

[0072] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pretreatment method for tetrabromobisphenol A (TBBPA) and its unilateral derivatives in sludge, characterized in that, It includes the following steps: (1) After freeze-drying and grinding the sludge sample through a 200-mesh sieve, take 0.5 g of the sample and place it in a 2 mL centrifuge tube, and add small steel beads with a diameter of 4 mm; (2) Sequentially add a mixed solution of n-hexane and acetone with a volume ratio of 8:2 and a mixed solution of n-hexane and dichloromethane with a volume ratio of 1:1 as the extractant, perform oscillating extraction, centrifuge and collect the supernatant after each extraction, and repeat 3 times; (3) Combine the supernatants after 6 extractions, purify them through a gel permeation chromatography column, and elute with a mixed solution of dichloromethane and n-hexane with a volume ratio of 1:1; (4) After concentrating the eluate by nitrogen blowing, redissolve it with methanol and make the volume constant to 1 mL.

2. The pre-treatment method according to claim 1, characterized in that, In step (2), the volume of the mixed solution of n-hexane and acetone is 1 mL, the oscillating extraction time is 5 min, and the frequency is 30 Hz.

3. The preprocessing method according to claim 1, wherein The centrifugation conditions in step (2) are: rotation speed 10000 r / min, time 10 min, temperature 4 °C.

4. The pre-treatment method according to claim 1, characterized in that, The packing of the gel permeation chromatography column is Bio-Beads S-X3, the eluent is a mixed solution of dichloromethane and n-hexane with a volume ratio of 1:1, the elution volume is 300 mL; the eluent for rinsing is the same as the eluent, and the flow rate is 5 mL / min.

5. The pre-treatment method according to claim 1, wherein In step (4), after concentrating by nitrogen blowing until nearly dry, redissolve it with methanol and make the volume constant to 1 mL, and transfer it to a 2 mL injection vial.

6. An analytical method for tetrabromobisphenol A and its unilateral derivatives in sludge, characterized in that, It includes the following steps: (a) Treat the sample by using the pretreatment method described in any one of claims 1-5; (b) Detect and analyze the pretreated sample by liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS).

7. The analysis method according to claim 6, characterized in that, The liquid chromatography conditions in step (b) are: the chromatographic column is GL-Science Inertsil ODS-4, with a specification of 3 mm×150 mm×2 μm, the mobile phase is gradient elution of water and methanol, the initial volume ratio is 80:20, the flow rate is 0.3 mL / min, and the column temperature is 20 °C.

8. The analysis method according to claim 6, characterized in that, The mass spectrometry conditions in step (b) are: electrospray ionization source, negative ion mode, capillary voltage 3500 V, drying gas flow rate 10 L / min, temperature 300 °C.

9. The analysis method according to claim 6, characterized in that The method also includes using the multiple reaction monitoring mode for quantitative analysis of the target substance.

10. The analysis method according to claim 6, characterized in that The method is applicable to the simultaneous analysis of TBBPA and its five unilateral derivatives in sludge, and the recovery rate of the target substance is ≥65%.

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