Quantitative method for determining quorum sensing signal molecules through ultrasonic extraction and liquid-liquid extraction

Abstract

A quantitative method for determining quorum sensing signal molecules through ultrasonic extraction and liquid-liquid extraction comprises the following steps: (1) centrifuging a to-be-detected sludge sample, taking lower-layer sludge solids, and freeze-drying the lower-layer sludge solids; (2) performing ultrasonic extraction, and adding internal standard C7-HSL, C9-HSL and C11-HSL before ultrasonic extraction to obtain an ultrasonic extraction concentrated solution; (3) performing liquid-liquid extraction on the ultrasonic extraction concentrated solution through dichloromethane to obtain a solution to be detected; and (4) quantitatively analyzing the AHLs in the to-be-detected solution by liquid chromatography-tandem mass spectrometry, measuring the peak area of the spectrogram of the AHLs in the to-be-detected solution, and calculating the content of the AHLs in the to-be-detected sample by combining the standard curve. The method has good applicability to sludge samples of various sewage biological treatment systems, 13 AHLs in the sludge samples can be effectively and accurately determined, and a reliable quantitative method is provided for research on AHLs signal molecule distribution in the sewage biological treatment systems.

Description

Technical Field

[0001] The invention relates to a method for quantitatively detecting quorum sensing signal molecules (N-acyl homoserine lactones, AHLs for short) in solid phase samples of sewage treatment systems, belonging to the technical field of signal molecule detection. Background Art

[0002] N-acyl-homoserine lactones (AHLs) are a key signaling molecule for microbial quorum sensing (QS). The latest research results show that QS is involved in a variety of wastewater biological treatment processes. For example, in membrane bioreactors (MBRs), AHLs affect the formation of membrane fouling. In addition, AHLs also play an important role in moving bed biofilm reactor (MBBR) systems, which can enhance wastewater treatment efficiency by promoting the formation of biofilms on the surface of fillers. In activated sludge systems (AGS), AHLs also play an important role and play a key role in the formation of granular sludge. In addition, AHLs are also involved in multiple steps in the nitrogen conversion process, including nitrification, denitrification, and anaerobic ammonium oxidation. It is worth noting that AHLs not only play a role in the above-mentioned wastewater treatment processes, but also play a regulatory role in the degradation of special organic matter.

[0003] There are at least 70 kinds of quorum sensing microorganisms mediated by AHLs in sewage biological treatment systems, which can secrete 11 kinds of AHLs. And due to the diversity of microorganisms, the distribution and concentration of AHLs in sewage biological treatment systems vary with different sewage treatment processes, operating parameters and sewage characteristics. Therefore, understanding the distribution of quorum sensing signal molecules among microorganisms in sewage treatment systems is of great significance for improving the efficiency of sewage biological treatment systems.

[0004] At present, the most widely used method for detecting signal molecules AHLs in solid phase samples is ultrasonic extraction (UE) + HPLC-MS / MS. It realizes the transfer and enrichment and concentration of AHLs in solid phase samples by ultrasonic extraction. In this process, the recovery rate of AHLs has an important influence on the accuracy of the detection results. However, most existing detection methods lack the analysis and verification of the recovery rate of AHLs, resulting in low reliability of their quantitative results. Secondly, compared with traditional biological indicator bacteria methods and high performance liquid chromatography (HPLC), HPLC-MS / MS, as a commonly used chemical substance analysis method, combines the separation ability of high performance liquid chromatography with the high specificity and high sensitivity of triple quadrupole mass spectrometry. The mass analysis capability has the advantages of high efficiency, high sensitivity and high precision. For example, HPLC-MS / MS can provide molecular ion peaks and fragment ion peaks for qualitative and quantitative analysis. In addition, this method has been widely used in environmental testing, drug analysis and other fields.

[0005] Nevertheless, existing research still needs to pay further attention to the recovery rate of AHLs in solid phase samples to improve the accuracy and reliability of the detection results. Some studies have shown that the recovery rate of AHLs can be significantly improved by optimizing ultrasonic extraction conditions and using different organic extraction solvents. For example, by controlling the ultrasonic extraction temperature to less than 40°C and using an appropriate solvent combination, the extraction efficiency can be effectively improved. These improvement measures help to ensure the reliability and repeatability of the test results. In addition, for complex sludge samples, during the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection process, non-target products may co-elute with the target analytes, thereby interfering with their ionization efficiency, causing the signal to become higher or enhanced, thereby affecting the accuracy of the results. However, most of the existing methods for detecting AHLs based on mass spectrometry often lack the evaluation of the accuracy of the test results due to the matrix effect when dealing with such complex samples, which significantly affects the accuracy and reliability of the test results. Summary of the invention

[0006] In view of the shortcomings of the existing AHLs detection technology in sludge, the present invention proposes a more accurate and efficient quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction based on the combination of ultrasonic extraction and liquid-liquid extraction.

[0007] The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction of the present invention comprises the following steps:

[0008] (1) collecting the sludge-water mixture in the sewage treatment system, centrifuging and freeze-drying the sludge solid to obtain a sludge sample;

[0009] (2) performing ultrasonic extraction (UE), adding ethyl acetate and internal standard to the sludge sample, and extracting by water bath ultrasound; the above extraction process is repeated three times, and the ethyl acetate obtained three times is combined to obtain an ultrasonic extract;

[0010] (3) evaporating the ultrasonic extraction liquid obtained in step (2) with nitrogen, then re-dissolving it with methanol, and adding ultrapure water to change its physical and chemical properties, thereby obtaining an ultrasonic extraction concentrate;

[0011] (4) liquid-liquid extraction (LLE) is performed on the ultrasonic extraction concentrate obtained in step (3) using dichloromethane, and after mixing, the mixture is shaken and centrifuged, and the lower layer solution (dichloromethane layer) is transferred to a centrifuge tube and evaporated to dryness by nitrogen blowing, and then re-dissolved by vortexing with methanol, and then centrifuged after re-dissolution, and the upper clear liquid is taken as the test solution;

[0012] (5) The AHLs in the test solution are quantitatively analyzed by liquid chromatography-tandem mass spectrometry, the peak area of ​​the spectrum of AHLs in the test solution is measured, and the content of AHLs in the test sample is calculated based on the standard curve.

[0013] The rotation speed of the centrifugal treatment in step (1) is 10000 rpm and the centrifugal time is 10 min.

[0014] During the three ultrasonic extractions in step (2), the ratio of the sludge sample to ethyl acetate in the first ultrasonic extraction is 0.4 g:15 mL, and the volumes of ethyl acetate used in the three extractions are the same.

[0015] The internal standards added in the step (2) are C7-HSL, C9-HSL and C10-HSL. The volume ratio of C7-HSL, C9-HSL, C11-HSL and ethyl acetate in the first ultrasonic extraction is 1:1:1:300.

[0016] In the step (3), the volume ratio of methanol to ethyl acetate in the step (2) is 1:15. The volume ratio of ultrapure water to methanol is 0.4-0.5:1.

[0017] In the step (4), the volume ratio of dichloromethane to ultrasonic extraction concentrate is 2:1; the shaking and centrifugation treatment is performed at 280 rpm for 30 min and at 5000 rpm for 10 min.

[0018] The ratio of methanol used in the re-dissolution in step (4) and the re-dissolution in step (3) is 2:1.

[0019] The centrifugation after reconstitution in step (4) is performed at a speed of 12000 rpm for 5 minutes.

[0020] The liquid phase conditions of the liquid chromatography tandem mass spectrometry in the step (5) are: flow rate 0.3mL / min, column temperature from 40°C, injection volume 3μL; gradient elution is calculated by volume ratio with 15% mobile phase B and 85% mobile phase A as the starting mobile phase, gradually increasing mobile phase B to 70% within 5min, reducing mobile phase A to 30%, continuing to gradually increase mobile phase B, reducing mobile phase A, to 9min mobile phase B is adjusted to 95%, mobile phase A is adjusted to 5%, and the mobile phase ratio is maintained for 4min, gradually reducing mobile phase B, increasing mobile phase A, to 13.1min mobile phase B is adjusted to 15%, mobile phase A is adjusted to 85%, and the mobile phase ratio is maintained for 4min. The mobile phase A is formic acid water with a volume concentration of 0.1% (the volume ratio of formic acid to water is 1:999), and the mobile phase B is formic acid methanol with a volume concentration of 0.1% (the volume ratio of formic acid to methanol is 1:999).

[0021] The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry in step (5) are as follows: detection is performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and positive ion mode scanning; the drying gas temperature is 200° C., the sheath gas temperature is 250° C., the nebulizer pressure is 20 psi, the capillary voltage is 3000 V, and the nozzle voltage is 1500 V; the quantitative ion pair, collision voltage (CE), and retention time (RT) of each substance are:

[0022]

[0023]

[0024] In the step (5), different internal standards are used to calculate the contents of the 10 AHLs in the sample to be tested. The internal standard of C4-HSL, C6-HSL, 3-oxo-C6-HSL and 3-oxo-C8-HSL is C7-HSL, the internal standard of C8-HSL is C9-HSL, and the internal standard of C10-HSL, C12-HSL, 3-oxo-C12-HSL, C14-HSL and 3-oxo-C14-HSL is C11-HSL.

[0025] The present invention adds an internal standard to the sludge sample, continuously performs ultrasonic extraction and liquid-liquid extraction, and then uses high performance liquid chromatography tandem mass spectrometry for detection, and uses different internal standards to determine the concentrations of 10 AHLs in the sludge sample. At the same time, a method for simultaneously detecting 13 AHLs signal molecules in the mud phase of the sewage treatment system is established by using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method has a good linear relationship in the range of 0.5 to 500 ppb (R 2 >0.999).

[0026] The present invention has the advantages of being rapid, accurate and highly sensitive, and is suitable for simultaneous precise and efficient quantitative analysis of a variety of AHLs signal molecule compounds, effectively overcoming the shortcomings of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the chromatogram of 13 AHLs on the T3 column. DETAILED DESCRIPTION

[0028] The instruments and reagents used in the present invention are specifically:

[0029] Instrument: Triple quadrupole LC / MS system: Agilent 6495D; Chromatographic column: Waters CORTECS T3 Column, 1.6 μm, 2.1 mm*100 mm.

[0030] Reagents: methanol (HPLC grade), ethyl acetate (HPLC grade), dichloromethane (HPLC grade).

[0031] The process of the quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction of the present invention is described in detail below.

[0032] 1. Pretreatment of sludge samples

[0033] (1) Prepare the sample to be tested

[0034] The muddy and water mixed liquid samples in the sewage treatment system were collected, placed in a centrifuge tube and centrifuged at 10000 rpm for 10 min, and the upper liquid was removed to obtain sludge.

[0035] (2) Freeze-drying the sludge to obtain a sludge sample. Before testing and analysis, the sludge sample was stored at -20°C and tested and analyzed within 1 week.

[0036] 2. Ultrasonic extraction (UE)

[0037] (1) Adding internal standard

[0038] Weigh 0.4 g of sludge sample into a brown glass bottle, add 15 mL of ethyl acetate, and add three internal standards, C7-HSL, C9-HSL and C11-HSL. The internal standard concentration is 500 μg / L, and the volume ratio of internal standards C7-HSL, C9-HSL, C11-HSL to ethyl acetate is 1:1:1:300, which are 0.05 mL, 0.05 mL, 0.05 mL and 15 mL respectively. A pipette can be used to take and transfer 0.05 mL of trace solution.

[0039] (2) Continuous ultrasonic extraction

[0040] Wrap the mouth of the glass bottle with tin foil and place it in a water bath ultrasonic pot for 10 minutes. After the extraction is completed, use a glass pipette to transfer the ethyl acetate extract to a centrifuge tube, and add 15 mL of ethyl acetate to the sludge sample again, repeat the ultrasonic extraction, and repeat the ultrasonic extraction process 3 times in a row, and combine the ethyl acetate extracts obtained 3 times.

[0041] (3) Nitrogen blowing concentration

[0042] The obtained ethyl acetate extract was continuously purged with nitrogen until it was evaporated to dryness, and 1 mL of methanol was used for vortex reconstitution. 0.4 mL of ultrapure water was added to the reconstituted methanol solution to change its physical and chemical properties, and the volume was fixed to 1.5 mL with methanol to obtain an ultrasonic extraction concentrate.

[0043] 3. Liquid-Liquid Extraction

[0044] (1) Add 3 mL of dichloromethane to the ultrasonic extraction concentrate, shake at 280 rpm for 30 min, and then centrifuge at 5000 rpm for 10 min;

[0045] (2) After the liquid-liquid extraction is completed, the upper solution is removed, and the lower solution is transferred to a 2 mL centrifuge tube and evaporated to dryness under nitrogen. The solution is re-dissolved in 0.5 mL of methanol by vortexing and centrifuging at 12,000 rpm for 5 min to obtain the test solution.

[0046] 4. Quantitative analysis of AHLs in the test solution by liquid chromatography-tandem mass spectrometry

[0047] Liquid chromatography-tandem mass spectrometry was used to detect the standard solution and the test solution, respectively, and the chromatograms of 13 AHLs standards were obtained. The retention time of the standard solution and the test solution was compared for qualitative analysis, and the peak position and peak area of ​​AHLs in the test solution were determined. The standard curve method combined with the internal standard recovery rate was then used for quantitative analysis to determine the content of AHLs in the test solution.

[0048] The liquid phase conditions are: the flow rate is 0.3mL / min, the column temperature is 40°C, and the injection volume is 3μL; the gradient elution is selected by volume ratio, with 15% mobile phase B and 85% mobile phase A as the starting mobile phase, gradually increasing mobile phase B to 70% within 5 minutes, reducing mobile phase A to 30%, continuing to gradually increase mobile phase B, reducing mobile phase A, to 9min mobile phase B is adjusted to 95%, mobile phase A is adjusted to 5%, and the mobile phase ratio is maintained for 4min, gradually reducing mobile phase B, increasing mobile phase A, to 13.1min mobile phase B is adjusted to 15%, mobile phase A is adjusted to 85%, and the mobile phase ratio is maintained for 4min. The mobile phase A is formic acid water with a volume concentration of 0.1% (the volume ratio of formic acid to water is 1:999), and the mobile phase B is formic acid methanol with a volume concentration of 0.1% (the volume ratio of formic acid to methanol is 1:999).

[0049] The mass spectrometry conditions were as follows: detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and positive ion mode scanning; the drying gas temperature was 200°C, the sheath gas temperature was 250°C, the nebulizer pressure was 20 psi, the capillary voltage was 3000 V, and the nozzle voltage was 1500 V; the quantitative ion pairs, collision voltage (CE), and retention time (RT) of each substance were:

[0050]

[0051]

[0052] AHLs in different samples were analyzed by LC MS / MS. The analysis conditions were as follows: detection using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and positive ion mode scanning; the collision gas was 4 psi, the curtain gas was 35 psi, the nebulizer 1 was 45 psi, the nebulizer 2 was 45 psi, the spray needle voltage was 5500 V, and the drying temperature was 550°C.

[0053] The peak area of ​​AHLs in the sample is analyzed and combined with the standard curve to calculate the specific content of each AHLs in the sample.

[0054] In addition, according to the above instrument conditions, another 200 μL of AHLs solution with a concentration of 50 μg / L was added to the sample bottle, and other conditions were the same. Figure 1 As shown. Figure 1 It can be seen that under this instrument condition, the peak shape of the AHLs spectrum is good.

[0055] A series of AHLs methanol standard solutions with a concentration gradient of 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, and 500 μg / L were prepared. dMRM was used for quantitative analysis. The standard curve was drawn with the AHLs content as the abscissa and the AHLs peak area (quantitative ion response value) as the ordinate. The average relative response factor was calculated. The results are shown in Table 1.

[0056] Table 1 Standard curve of AHLs

[0057] Target Linear equation <![CDATA[R 2 ]]> RSD of RRF, % C4-HSL y = 515.4x + 104.9 0.99994 1.47 3-oxo-C6-HSL y = 324.4x + 73.8 0.99985 1.52 C6-HSL y = 400.4x + 55.9 0.99985 7.08 3-oxo-C8-HSL y = 372.2x - 4.3 0.99999 4.97 C7-HSL y = 443.6x + 88.2 0.99973 5.02 C8-HSL y = 596.4x - 49.5 0.99997 4.29 C9-HSL y = 709.6x - 86.4 0.99998 6.72 C10-HSL y = 626.5x - 77.6 0.99897 8.65 3-oxo-C12-HSL y = 338.0x - 35.4 0.99999 1.80 C11-HSL y = 701.6x - 144.3 0.99999 5.15 C12-HSL y = 889.5x - 196.7 0.99999 5.78 3-oxo-C14-HSL y = 454.4x - 92.7 0.99999 7.33 C14-HSL y = 533.5x - 76.7 0.99999 6.04

[0058] As shown in Table 1, the linear correlation coefficient R 2 The relative standard deviation (RSD) greater than 0.999 is less than 20%, which fully meets the requirements for the determination of trace levels of AHLs.

[0059] According to this method, a spiked experiment was conducted. 0.4 g of freeze-dried sludge sample was taken to make the target content 62.5 ng / g. The sample was pretreated according to the aforementioned method and measured four times in parallel. The results are shown in Tables 2 and 3.

[0060] Table 2 Different sample spike experiments

[0061] Target Average recovery rate / % RSD / % C4-HSL 68.80 7.30 3-oxo-C6-HSL 63.65 0.57 C6-HSL 78.25 6.04 3-oxo-C8-HSL 68.92 4.67 C7-HSL 85.41 6.06 C8-HSL 86.69 7.03 C9-HSL 83.06 7.51 C10-HSL 58.57 5.65 3-oxo-C12-HSL 46.23 6.53 C11-HSL 65.86 7.42 C12-HSL 49.81 6.94 3-oxo-C14-HSL 48.47 4.83 C14-HSL 47.44 9.70

[0062] Table 3 Relative recoveries after correction with different internal standards

[0063]

[0064] The results showed that the average recovery rate of freeze-dried samples with a target content of 62.5 ng / g was 46.23% to 86.69%, the relative standard deviation was 0.57% to 9.70%, and the relative recovery rate was 66.58% to 108.43%, indicating that the method has good accuracy and precision and is suitable for samples of different properties.

[0065] The experimental results show that the method of the present invention has good linear relationship, good recovery rate, extremely low detection limit, reliable method and good reproducibility. Secondly, the method has simple experimental process, more accurate qualitative analysis, and extremely low consumables cost and instrument cost.

Claims

1. A quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction, characterized in that: The following steps are involved: (1) collecting the sludge-water mixture in the sewage treatment system, centrifuging and freeze-drying the sludge solid to obtain a sludge sample; (2) performing ultrasonic extraction, adding ethyl acetate and an internal standard to the sludge sample, and extracting by water bath ultrasonication; the above extraction process is repeated three times, and the ethyl acetate obtained three times is combined to obtain an ultrasonic extract; (3) evaporating the ultrasonic extraction liquid obtained in step (2) with nitrogen, then re-dissolving it with methanol, and adding ultrapure water to change its physical and chemical properties, thereby obtaining an ultrasonic extraction concentrate; (4) liquid-liquid extraction (LLE) is performed on the ultrasonic extraction concentrate obtained in step (3) using dichloromethane, and after mixing, the mixture is shaken and centrifuged, and the lower layer solution is transferred to a centrifuge tube and evaporated to dryness by nitrogen blowing, and then re-dissolved by vortexing with methanol, and then centrifuged after re-dissolution, and the upper layer of the supernatant is taken as the test solution; (5) The AHLs in the test solution are quantitatively analyzed by liquid chromatography-tandem mass spectrometry, the peak area of ​​the spectrum of AHLs in the test solution is measured, and the content of AHLs in the test sample is calculated based on the standard curve.

2. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: The rotation speed of the centrifugal treatment in step (1) is 10000 rpm and the centrifugal time is 10 min.

3. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: During the three ultrasonic extractions in step (2), the ratio of the sludge sample to ethyl acetate in the first ultrasonic extraction is 0.4 g:15 mL, and the volumes of ethyl acetate used in the three extractions are the same.

4. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: The internal standards added in the step (2) are C7-HSL, C9-HSL and C10-HSL. The volume ratio of C7-HSL, C9-HSL, C11-HSL and ethyl acetate in the first ultrasonic extraction is 1:1:1:

300.

5. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: In the step (3), the volume ratio of methanol to ethyl acetate in the step (2) is 1:15, and the volume ratio of ultrapure water to methanol is 0.4-0.5:

1.

6. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: In the step (4), the volume ratio of dichloromethane to ultrasonic extraction concentrate is 2:1; the shaking and centrifugation treatment is performed at 280 rpm for 30 min and at 5000 rpm for 10 min.

7. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: The ratio of methanol used in the re-dissolution in step (4) and the re-dissolution in step (3) is 2:1, and the centrifugation after re-dissolution is performed at a speed of 12000 rpm for 5 minutes.

8. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: The liquid phase conditions of the liquid chromatography tandem mass spectrometry in the step (5) are: flow rate 0.3mL / min, column temperature from 40°C, injection volume 3μL; gradient elution is calculated by volume ratio with 15% mobile phase B and 85% mobile phase A as the starting mobile phase, gradually increase mobile phase B to 70% within 5min, reduce mobile phase A to 30%, continue to gradually increase mobile phase B, reduce mobile phase A, to 9min mobile phase B is adjusted to 95%, mobile phase A is adjusted to 5%, maintain this mobile phase ratio to 4min, gradually reduce mobile phase B, increase mobile phase A, to 13.1min mobile phase B is adjusted to 15%, mobile phase A is adjusted to 85%, maintain this mobile phase ratio to 4min. The mobile phase A is formic acid water with a volume concentration of 0.1%, and the mobile phase B is formic acid methanol with a volume concentration of 0.1%.

9. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 1, characterized in that: The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry in step (5) are: detection using a triple quadrupole mass spectrometer in multiple reaction monitoring mode, electrospray ionization, and positive ion mode scanning; the drying gas temperature is 200° C., the sheath gas temperature is 250° C., the nebulizer pressure is 20 psi, the capillary voltage is 3000 V, and the nozzle voltage is 1500 V; the quantitative ion pairs, collision voltage, and retention time of each substance are:

10. The quantitative method for determining quorum sensing signal molecules by ultrasonic extraction and liquid-liquid extraction according to claim 9, characterized in that: In the step (5), different internal standards are used to calculate the contents of the 10 AHLs in the sample to be tested. The internal standard of C4-HSL, C6-HSL, 3-oxo-C6-HSL and 3-oxo-C8-HSL is C7-HSL, the internal standard of C8-HSL is C9-HSL, and the internal standard of C10-HSL, C12-HSL, 3-oxo-C12-HSL, C14-HSL and 3-oxo-C14-HSL is C11-HSL.

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