A method for high-resolution mass spectrometric characterization of chromatographically co-eluting dimethylphenol isomers and applications thereof

Abstract

The application discloses a chromatographic co-elution dimethyl phenol isomer high-resolution mass spectrometry characterization method and application, and the method comprises the following steps: according to the petroleum content range in a sample, selecting a corresponding sample pretreatment process to prepare a sample; under a methanol-5.0-6.5 mmol / L ammonia water mobile phase system, using LC-HRMS to detect a standard sequence sample, a conversion factor calibration sample and the sample, collecting primary mass spectrometry (MS 1 ) and selected ion secondary mass spectrometry (MS 2 ) data; according to MS 1 data, calculating the first normalized concentration of co-eluted 3,5-dimethyl phenol and 2,6-dimethyl phenol, and the first conversion factor of 2,6-dimethyl phenol to 3,5-dimethyl phenol; and the concentration of other 11 kinds of phenols and alkyl-substituted phenols; according to MS 2 data, calculating the second normalized concentration and the second conversion factor of the co-eluted components, and then calculating the concentration of 3,5-dimethyl phenol and 2,6-dimethyl phenol. The application realizes quantitative characterization of 13 kinds of C0-C2-alkyl-substituted phenol single components.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental detection, and particularly relates to a chromatographic co-elution dimethyl phenol isomer high-resolution mass spectrometry characterization method and application. BACKGROUND

[0002] The stripping water discharged by the sulfur-containing and salt-containing sewage stripping device belongs to oil-containing wastewater, which is the most important process wastewater of a petroleum refinery, and the unit discharge amount reaches 0.2 m 3 / t crude oil, and the volatile phenol content is as high as 0.2-0.5 kg / m 3 (Yan Song, Li Lingbo, Han Congbi, Zhou Yanhong. Study on Organic Components of High-sulfur and High-acid Crude Oil Wastewater [J]. Contemporary Chemical Industry, 2015, 44(09): 2081-2083). In modern coal chemical projects, coal gasification is an important process, and the Lurgi pressurized gasification is a relatively mature gasification process. The scrubbing gas wastewater generated by gasification contains high-concentration and refractory organic pollutants such as phenol and ammonia (Li Xueyuan, Wei Danqing. Detection of Phenolic Compounds in Coal Gasification Wastewater by GC-MS [J]. Modern Chemical Industry, 2015, 35(09): 187-188). The high-content volatile phenols existing in the stripping water and the coal gasification wastewater mainly include phenol, methyl phenol (three isomers) and dimethyl / ethyl phenol (nine isomers), and have recycling and reuse value. Clarifying the composition of the volatile phenols is the basis for developing the recycling and comprehensive utilization of the volatile phenols.

[0003] The volatile phenol component contains numerous isomers. When the chromatography-mass spectrometry technology is used for quantification, the chromatographic separation of the isomers needs to be realized. The elution order and the chromatographic co-elution of the dimethyl phenol isomers in the same reversed-phase C18 liquid chromatography column change under two different mobile phase systems. A calculation method for the single-component content of the chromatographic co-elution isomers is established, and the quantitative characterization of the above-mentioned 13 kinds of alkyl-substituted phenol single components in the oil-containing wastewater is realized (Liu Yulong, Wang Zixian, Zhang Xiaofei, Shao Zhiguo, Tong Kun. A Characterization Method of C0-C2-alkyl Substituted Phenol and Application. Chinese Patent: 202211482228.X; Liu Yulong, Shao Zhiguo, Tong Kun, Zhang Xiaofei, Wang Zixian. A Detection Method of C1-C2-alkyl Substituted Phenol Isomers and Application. Chinese Patent: 202211482206.3). In the prior art, whether the gas chromatography separation or the liquid chromatography separation technology is used, there is rarely a method for solving the accurate quantification problem of the chromatographic co-elution of the isomer single components in only one mobile phase system. SUMMARY

[0004] The application aims to provide a chromatographic co-flow dimethyl phenol isomer high-resolution mass spectrometry characterization method and application, which adopts liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined technology, collects primary mass spectrometry full scan (MS 1 ) and selected ion secondary mass spectrometry (MS 2 ) data under a methanol-ammonia water mobile phase system, and realizes quantitative characterization of 13 kinds of C0~C2-alkyl substituted phenol single components in oily wastewater. 1 2 In addition, the application is also suitable for quantitative characterization of 13 kinds of C0~C2-alkyl substituted phenol single components in drinking water, surface water and underground water, coal gasification wastewater, and oily solid waste.

[0005] To achieve the above-mentioned purpose, the application provides the following technical scheme.

[0006] In one aspect, the application provides a chromatographic co-flow dimethyl phenol isomer high-resolution mass spectrometry characterization method, which comprises the following steps:

[0007] According to the content range of petroleum in the sample, a corresponding sample pretreatment process is selected to prepare the sample;

[0008] Under a methanol-5.0~6.5mmol / L ammonia water mobile phase system, LC-HRMS is used to detect standard sequence samples, conversion factor calibration samples and the sample, collect primary mass spectrometry full scan (MS 1 ) and selected ion secondary mass spectrometry scan (MS 2 ) data;

[0009] According to MS 1 data, an internal standard calibration method is used to calculate the first normalized concentration of co-flow 3,5-dimethyl phenol and 2,6-dimethyl phenol, and the first conversion factor of 2,6-dimethyl phenol to 3,5-dimethyl phenol; and the concentration of phenol, 2-methyl phenol, 3-methyl phenol, 4-methyl phenol, 2,3-dimethyl phenol, 2,4-dimethyl phenol, 2,5-dimethyl phenol, 3,4-dimethyl phenol, 2-ethyl phenol, 3-ethyl phenol and 4-ethyl phenol;

[0010] According to MS 2 data, an internal standard calibration method is used to calculate the second normalized concentration of co-flow 3,5-dimethyl phenol and 2,6-dimethyl phenol, and the second conversion factor of 2,6-dimethyl phenol to 3,5-dimethyl phenol;

[0011] According to the first normalized concentration and the second normalized concentration of 3,5-dimethyl phenol and 2,6-dimethyl phenol, and the first conversion factor and the second conversion factor of 2,6-dimethyl phenol to 3,5-dimethyl phenol, the concentration of 3,5-dimethyl phenol and 2,6-dimethyl phenol is calculated.

[0012] As a preferred embodiment, the sample preparation procedure is selected according to the petroleum content range in the sample, which specifically includes:

[0013] When the petroleum content in the sample is not more than 50 mg / L, the sample is injected for detection after being filtered through a 0.22 μm filter;

[0014] When the petroleum content in the sample is more than 50 mg / L, the sample is alkalinized by NaOH and extracted by dichloromethane and n-hexane, and the organic phase is separated by centrifugation, and the water phase is filtered through a 0.22 μm filter, and then injected for detection after being neutralized by formic acid.

[0015] As a preferred embodiment, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia mobile phase system include:

[0016] The flow rate of the chromatographic column is 0.20 mL / min;

[0017] The gradient elution is as follows: maintaining zero gradient elution for 1 min, and then increasing the volume ratio of methanol to 40% within 30 min, and maintaining the volume ratio of methanol at 40% until all components are eluted;

[0018] The column temperature of the chromatographic column is 35-40℃.

[0019] As a preferred embodiment, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia mobile phase system include:

[0020] The flow rate of the chromatographic column is 0.20 mL / min;

[0021] The gradient elution is as follows: 0-1 min, 20% methanol; 1-31 min, 20%-40% methanol; 31-32 min, 40% methanol; 32-32.1 min, 40%-90% methanol; 32.1-37 min, 90% methanol; 37-37.1 min, 90%-20% methanol; 37.1-42 min, 20% methanol;

[0022] The column temperature of the chromatographic column is 40℃.

[0023] As a preferred embodiment, in the selected ion secondary mass spectrometry (MS 2 ) scan, the parent ion of 3,5-dimethylphenol-2,4,6-D3 is 124.08472 m / z, and the quantification daughter ion is 96.05342 m / z; the parent ion of 3,5-dimethylphenol and 2,6-dimethylphenol is 121.06589 m / z, and the quantification daughter ion is 93.03459 m / z.

[0024] As a preferred embodiment, the calculation formula of the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0025]

[0026] In the formula, k1 is the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，1 is the quantitative parent ion peak area of the co-eluted components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point of the calibration sample series, A 3,5-DMP，1 is the quantitative parent ion peak area of 3,5-dimethylphenol in the corresponding conversion factor calibration sample, A IS(3,5 / 2,6-DMP)，1 and A IS(3,5-DMP)，1 are the peak areas of the corresponding internal standard quantitative parent ions in the calibration point of the calibration sample series and the conversion factor calibration sample.

[0027] As a preferred embodiment, the calculation formula of the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0028]

[0029] In the formula, k2 is the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，2 is the quantitative daughter ion peak area of the co-eluted components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point of the calibration sample series, A 3,5-DMP，2 is the quantitative daughter ion peak area of 3,5-dimethylphenol in the corresponding conversion factor calibration sample, A IS(3,5 / 2,6-DMP)，2 and A IS(3,5-DMP)，2 are the peak areas of the corresponding internal standard quantitative daughter ions in the calibration point of the calibration sample series and the conversion factor calibration sample.

[0030] As a preferred embodiment, the calculation formula of the concentration of 3,5-dimethylphenol and 2,6-dimethylphenol is:

[0031]

[0032]

[0033] In the formula, c 2,6-DMP represents the concentration of 2,6-dimethylphenol, c 3,5-DMP represents the concentration of 3,5-dimethylphenol, c STD1 represents the first normalized concentration of the co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, c STD2represents the second normalized concentration of the co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, k1 represents the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, and k2 represents the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

[0034] In another aspect, the application provides an application of a chromatographic co-eluted dimethylphenol isomer high-resolution mass spectrometry characterization method to the quantitative characterization of 13 C0-C2-alkyl-substituted phenol single components in oily wastewater, coal gasification wastewater, oily solid waste, drinking water, surface water and groundwater.

[0035] The technical effects and advantages of the application are as follows:

[0036] (1) One-time injection is adopted, and full-scan MS 1 and selected ion MS 2 data are collected simultaneously, so that accurate quantification of chromatographic co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol isomer single components is realized.

[0037] (2) Based on the parent ion 121.06589 m / z and the characteristic daughter ion 93.03459 m / z of 3,5-dimethylphenol and 2,6-dimethylphenol, a chromatographic co-eluted isomer single component quantitative method is established by solving equations.

[0038] (3) The sample is not enriched or derivatized, and the method detection limit of 13 C0-C2-alkyl-substituted phenol single components is lower than 1.0 μg / L; compared with the standard HJ744-2015 (derivative-GC-MS method) for testing phenolic compounds, the sensitivity of the method is 1-2 orders of magnitude higher.

[0039] Other features and advantages of the application will be set forth in the following description of the application, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the application. The objects and other advantages of the application can be realized and obtained by the structure indicated in the specification and the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 A chromatographic co-eluted dimethylphenol isomer high-resolution mass spectrometry characterization method flowchart of the application;

[0041] Figure 2 Extraction ion map of 9 C2-alkyl-substituted phenol [M-H] - in the methanol-6.0 mmol / L ammonia mobile phase system in the embodiment of the application;

[0042] Figure 3Secondary mass spectrum of 3,5-dimethylphenol parent ion 121.06589 m / z under normalized collision energy (NCE = 60, 80, 100).

[0043] Figure 4 Secondary mass spectrum of 2,6-dimethylphenol parent ion 121.06589 m / z under normalized collision energy (NCE = 60, 80, 100). DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be apparently and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0045] To solve the problems in the prior art, the present application discloses a high-resolution mass spectrometry characterization method for chromatography co-eluted dimethylphenol isomers, Figure 1 A high-resolution mass spectrometry characterization method flow chart for chromatography co-eluted dimethylphenol isomers according to the present application is shown in Figure 1 The method comprises the following steps:

[0046] Step S1, according to the petroleum content range in the sample, a corresponding sample pretreatment process is selected to prepare a test sample;

[0047] Step S2, under a methanol-5.0-6.5 mmol / L ammonia mobile phase system, the standard sequence sample, the conversion factor calibration sample and the test sample are detected by using LC-HRMS, and the first mass spectrum full scan (MS 1 ) and the selected ion secondary mass spectrum scan (MS 2 ) data are collected;

[0048] Step S3, according to the MS 1 data, the internal standard calibration method is used to calculate the first normalized concentration of the co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, and the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol; and the concentrations of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, 2-ethylphenol, 3-ethylphenol and 4-ethylphenol;

[0049] Step S4, according to the MS 2Data, the second normalized concentration of 3,5-dimethylphenol and 2,6-dimethylphenol co-flowing is calculated by using internal standard calibration method, and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol;

[0050] Step S5, according to the first normalized concentration and the second normalized concentration of 3,5-dimethylphenol and 2,6-dimethylphenol; and the first conversion factor and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, the concentration of 3,5-dimethylphenol and 2,6-dimethylphenol is calculated.

[0051] In step S1 of the present application, the corresponding sample preparation process is selected according to the content range of petroleum in the sample, specifically including:

[0052] When the content of petroleum in the sample is not more than 50 mg / L, the sample is detected after passing through a 0.22 μm filter;

[0053] When the content of petroleum in the sample is more than 50 mg / L, the sample is alkalinized by NaOH and extracted by dichloromethane and n-hexane, and the organic phase is separated by centrifugation, and then the water phase is filtered through a 0.22 μm filter, and then the sample is detected after being neutralized by formic acid.

[0054] In step S2 of the present application, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia mobile phase system include:

[0055] The flow rate of the chromatographic column is 0.20 mL / min;

[0056] The gradient elution is: maintaining zero gradient elution for 1 min, and then increasing the volume ratio of methanol to 40% within 30 min, and maintaining the volume ratio of methanol to 40% until all components flow out;

[0057] The column temperature of the chromatographic column is 35-40℃.

[0058] Preferably, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia mobile phase system include:

[0059] The flow rate of the chromatographic column is 0.20 mL / min;

[0060] The gradient elution is: 0-1 min, 20% methanol; 1-31 min, 20%-40% methanol; 31-32 min, 40% methanol; 32-32.1 min, 40%-90% methanol; 32.1-37 min, 90% methanol; 37-37.1 min, 90%-20% methanol; 37.1-42 min, 20% methanol;

[0061] The column temperature of the chromatographic column is 40℃.

[0062] In step S3 of the present application, the selected ion secondary mass spectrum scan (MS 2 ) is performed, the parent ion of 3,5-dimethylphenol-2,4,6-D3 is 124.08472 m / z, and the quantitation daughter ion is 96.05342 m / z; the parent ions of 3,5-dimethylphenol and 2,6-dimethylphenol are 121.06589 m / z, and the quantitation daughter ion is 93.03459 m / z.

[0063] In step S3 of the present application, the calculation formula of the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0064]

[0065] In the formula, k1 is the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，1 is the peak area of the quantitation parent ion of the co-eluted components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point of the calibration sample series, A 3,5-DMP，1 is the peak area of the quantitation parent ion of 3,5-dimethylphenol in the corresponding conversion factor calibration sample, A IS(3,5 / 2,6-DMP)，1 and A IS(3,5-DMP)，1 are the peak areas of the corresponding internal standard quantitation parent ions in the calibration point of the calibration sample series and the conversion factor calibration sample.

[0066] In step S4 of the present application, the calculation formula of the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0067]

[0068] In the formula, k2 is the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，2 is the peak area of the quantitation daughter ion of the co-eluted components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point of the calibration sample series, A 3,5-DMP，2 is the peak area of the quantitation daughter ion of 3,5-dimethylphenol in the corresponding conversion factor calibration sample, A IS(3,5 / 2,6-DMP)，2 and A IS(3,5-DMP)，2 are the peak areas of the corresponding internal standard quantitation daughter ions in the calibration point of the calibration sample series and the conversion factor calibration sample.

[0069] In step S5 of the present application, the calculation formula of the concentration c 3,5-DMP of 3,5-dimethylphenol and the concentration c 2,6-DMP of 2,6-dimethylphenol is:

[0070]

[0071]

[0072] wherein, c 2,6-DMP represents the concentration of 2,6-dimethylphenol, c 3,5-DMP represents the concentration of 3,5-dimethylphenol, c STD1 represents the first normalized concentration of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, c STD2 represents the second normalized concentration of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, k1 represents the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, and k2 represents the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

[0073] Example 1

[0074] This example gives two sample pretreatment methods for LC-HRMS sample testing.

[0075] 1. Stripping water or coal gasification wastewater with petroleum content ≤ 50 mg / L

[0076] The water sample is passed through a 0.22 μm filter. Depending on the volatile phenol content, 10-900 μL of the filtrate is used to prepare the sample.

[0077] This procedure is also applicable to sample preparation of relatively clean water samples such as drinking water, underground water and surface water; or sample preparation of oil-water mixtures such as oily wastewater.

[0078] 2. Stripping water or coal gasification wastewater with petroleum content > 50 mg / L

[0079] 10.00 mL of the water sample is taken in a 20 mL round-bottom screw-neck sample bottle (sample cover with polytetrafluoroethylene gasket), 100 μL of 2.5 mol / L NaOH solution is added to adjust the pH to ≥ 12, 1-3 mL of dichloromethane-n-hexane (2+1) mixed solvent is added, and the bottle cover is tightly screwed. Shake for 30 min and centrifuge. The water phase is neutralized with formic acid, the water phase is passed through a 0.22 μm filter, and depending on the volatile phenol content, 10-900 μL of the filtrate is used to prepare the sample.

[0080] Example 2

[0081] This example optimizes the chromatographic conditions of 9 kinds of C2-alkyl substituted phenols under a methanol-ammonia water mobile phase system, and investigates the influence of the ammonia water mobile phase concentration in the range of 4.0-8.0 mmol / L on the chromatographic separation of C2-alkyl substituted phenols.

[0082] In this example and the following examples, ACQUITY UPLC BEH C 18The C1-C2-alkyl substituted phenols were separated by a chromatographic column (1.7 μm, 2.1 mm x 150 mm, Waters, USA).

[0083] Under the conditions of a column flow rate of 0.20 mL / min and an ammonia mobile phase concentration of 6.0 mmol / L, the initial volume ratio of methanol mobile phase was set to be 10%, 20% and 25% respectively, and maintained for 1 min, and then the volume ratio of methanol was increased to 40% within 30 min, and maintained at 40% until all components were eluted. The results showed that when the initial volume ratio of methanol was 10%, 20% and 25% respectively, all the C2-alkyl substituted phenols were eluted within 32 min; when the initial volume ratio of methanol was 10%, the separation of the first eluted components was relatively poor, while the separation of the later eluted components was relatively good; when the initial volume ratio of methanol was 25%, the separation of the first eluted components was relatively good, while the separation of the later eluted components was relatively poor. Therefore, the initial volume ratio of methanol was selected to be 20%.

[0084] The effects of column temperature on the chromatographic separation of C2-alkyl substituted phenols were compared. The column temperature was 30°C, 35°C, 40°C and 45°C respectively. The chromatographic separation of C2-alkyl substituted phenols was poor at 30°C and 45°C, and good at 35°C and 40°C. When the column temperature was 40°C, the column pressure drop was reduced by about 40-50 bar, so the column temperature was preferably 40°C.

[0085] The optimized chromatographic conditions were as follows: column flow rate was 0.20 mL / min; gradient elution was as follows: 0-1 min, 20% methanol; 1-31 min, 20%-40% methanol; 31-32 min, 40% methanol; 32-32.1 min, 40%-90% methanol; 32.1-37 min, 90% methanol; 37-37.1 min, 90%-20% methanol; 37.1-42 min, 20% methanol; column temperature was 35-40°C; and the preferred temperature was 40°C.

[0086] Under the optimized chromatographic conditions, the effects of ammonia mobile phase concentration on the chromatographic separation of C2-alkyl substituted phenols were investigated. When the ammonia concentration was 4.0 mmol / L, 9 kinds of C2-alkyl substituted phenols were separated into 7 peaks; when the ammonia concentration was 6.0 mmol / L, 9 kinds of C2-alkyl substituted phenols were separated into 8 peaks with good peak shape; and when the ammonia concentration was 8.0 mmol / L, 9 kinds of C2-alkyl substituted phenols were separated into 7 peaks.

[0087] The retention time and elution order of C2-alkyl substituted phenols were confirmed by a single standard method when the ammonia concentration was 6.0 mmol / L, and the co-elution of components was determined. Figure 2The ion extraction chromatogram of 9 C2-alkyl substituted phenols [M-H]- in methanol-6.0 mmol / L ammonia mobile phase system is shown in the embodiment of the present application, wherein the corresponding chromatographic peaks are: 1, 3,4-dimethylphenol; 2, 3-ethylphenol; 3, 3,5-dimethylphenol; 4, 2,6-dimethylphenol; 5, 2,3-dimethylphenol; 6, 4-ethylphenol; 7, 2,5-dimethylphenol; 8, 2-ethylphenol; 9, 2,4-dimethylphenol. -

[0088] As shown in Figure 2 , when the ammonia concentration is 6.0 mmol / L, only 3,5-dimethylphenol and 2,6-dimethylphenol are co-eluted. Further experiments have determined that when the ammonia concentration is 5.0-6.5 mmol / L, 9 C2-alkyl substituted phenols are co-eluted into 8 peaks, the peak shapes are good, and only 3,5-dimethylphenol and 2,6-dimethylphenol are co-eluted. Accordingly, it is determined that the suitable ammonia mobile phase concentration range is 5.0-6.5 mmol / L, and the ammonia mobile phase concentration selected for sample testing is 6.0 mmol / L.

[0089] Embodiment 3

[0090] The embodiment provides a method for detecting 13 C0-C2-alkyl substituted phenol single components in stripping water by using ultra-high performance liquid chromatography-electric field orbitrap high resolution mass spectrometry (UPLC-Orbitrap HRMS).

[0091] 1. Instrument conditions

[0092] UPLC-Orbitrap HRMS: Vanquish C-H10 type ultra-high performance liquid chromatograph, Q-Exactive Plus type electric field orbitrap high resolution mass spectrometer (ThermoFisher Company, USA).

[0093] (1) Liquid chromatography conditions

[0094] Mobile phase system: methanol-6.0 mmol / L ammonia. Column temperature: 40℃; column flow rate: 0.20 mL / min; sample chamber temperature: 6℃; sample injection amount: 2μL. Gradient elution: 0-1min, 20% methanol; 1-31min, 20%-40% methanol; 31-32min, 40% methanol; 32-32.1min, 40%-90% methanol; 32.1-37min, 90% methanol; 37-37.1min, 90%-20% methanol; 37.1-42min, 20% methanol.

[0095] (2) Orbitrap HRMS conditions

[0096] ​Heated electrospray ion source: negative ion mode; sheath gas flow rate: 35 Arb; auxiliary gas flow rate: 10 Arb; spray voltage: 2.6kV; ion transmission tube temperature: 320℃; S-lens RF voltage: 50V; auxiliary gas heater temperature: 380℃.

[0097] Full scan of mass spectrometry (MS) 1 Acquisition parameters: Resolution: 70000; Maximum C-trap ion count (AGCTarget): 1×10⁻⁶ 6 C-trap maximum injection time (Maximum IT): 100ms; Quality range: m / z 90~155;

[0098] Selected ion secondary mass spectrometry (MS) scan 2 Acquisition parameters: Selected ion frequencies of 121.06589 m / z and 124.08472 m / z; Resolution: 17500; Maximum C-trap ion count (AGC Target): 1 × 10⁻⁶ 5 C-trap maximum injection time (Maximum IT): 100ms; Isolation window: 1m / z; Normalized collision energy (steppedNCE): 60, 80, and 100.

[0099] 2. Preparation of standard sequence samples and conversion factor calibration samples

[0100] Single-standard stock solutions were prepared using 13 phenols (phenol, methylphenol, and dimethyl / ethylphenol) with a purity greater than 98% and internal standards (phenol-D5 and 3,5-dimethylphenol-2,4,6-D3). The 13 phenols mixed working solution, 3,5-dimethylphenol working solution, and mixed internal standard working solution were prepared by stepwise dilution with methanol. The standard sequence samples and conversion factor calibration samples were then prepared using the working solutions.

[0101] Standard series samples: Add 900 μL of pure water to each of six 2 mL sample vials. Using a microsyringe, sequentially add the mixed working solution of 13 phenols and the mixed internal standard working solution. Add methanol to bring the total methanol content in the calibration standard solution to 100 μL. Shake well and store at 4–6 °C until analysis. The concentrations of each component of the 13 phenols in the standard series are 10, 100, 500, 1000, 2500, and 5000 μg / L, respectively, and the concentration of each component of the internal standard is 200 μg / L.

[0102] Calibration sample: In a 2 mL sample bottle, add 900 μL of pure water, and then add 3,5-dimethylphenol working solution and mixed internal standard working solution in sequence using a microsyringe. Add methanol to make the total amount of methanol in the calibration standard solution 100 μL. Shake well and store at 4-6°C until testing. The concentration of 3,5-dimethylphenol in the calibration sample is 1000 μg / L, and the concentration of each component of the internal standard is 200 μg / L.

[0103] 3. Sample collection and sample preparation

[0104] The stripping water or coal gasification wastewater sample is collected in a 40 mL VOA sample bottle with a polytetrafluoroethylene gasket. The water sample is required to fill the sample bottle without overflowing and no protective agent is added. The sample is stored at 4-6°C and the sample preparation is completed within 48 h.

[0105] The water sample stored at low temperature is taken out and the sample temperature is restored to room temperature. The sample is filtered through a 0.22 μm nylon filter membrane. 90 μL of methanol is pre-added to a 2 mL sample bottle. 10-900 μL of the filtrate is added to the sample bottle according to the volatile phenol content, and pure water is added to make the total volume of the sample 990 μL. 10 μL of the mixed internal standard working solution is added, and the sample is shaken well and stored at 4-6°C until testing.

[0106] 4. Data acquisition and result calculation

[0107] Xcalibur 4.4 software is used to acquire data, and TraceFinder 5.1 software is used for data analysis. The target compounds in the sample are identified according to the retention time matching, and the concentration of the target compounds is calculated according to the peak area of the parent ion [M-H] - or daughter ion. Table 1 shows the retention time, quantitative parent ion and quantitative daughter ion information of the mixed internal standard and 13 phenolic components.

[0108] The influence of different collision energies on the secondary mass spectrum fingerprint characteristics of 3,5-dimethylphenol and 2,6-dimethylphenol is investigated under the mobile phase system of methanol-6.0 mmol / L ammonia water. The results show that when the collision energy increases from NCE=30 to NCE=100, the parent ion 121.06589 m / z of 3,5-dimethylphenol and 2,6-dimethylphenol both produces a characteristic daughter ion 93.03459 m / z ([C6H5O] - ) after fragmentation, and higher collision energy can obtain higher daughter ion abundance; the relative abundance of other daughter ions is low. Figure 3 The secondary mass spectrum of 3,5-dimethylphenol parent ion 121.06589 m / z under normalized collision energy (NCE=60, 80, 100). Figure 4This is a secondary mass spectrum of the 2,6-dimethylphenol parent ion at a normalized collision energy (NCE = 60, 80, 100) of 121.06589 m / z. Figure 3 and Figure 4 As shown, 3,5-dimethylphenol and 2,6-dimethylphenol do not have their own specific characteristic daughter ions that can be used for quantitative analysis; the parent ion of 3,5-dimethylphenol and 2,6-dimethylphenol is 121.06589 m / z ([C8H9O)). - After fragmentation, a high abundance of daughter ions (93.03459 m / z) was produced ([C6H5O]). - It can be applied to quantitative analysis.

[0109] Table 1. Quantitative parent ions and quantitative daughter ions of internal standards and 13 phenolic components.

[0110]

[0111]

[0112] For MS 1 The data were processed, and the concentrations of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, 2-ethylphenol, 3-ethylphenol, and 4-ethylphenol were calculated using the internal standard method based on the peak area of ​​the parent ion.

[0113] The concentrations of the co-eluent 3,5-dimethylphenol and 2,6-dimethylphenol were calculated as follows:

[0114] At each calibration point, when the prepared concentrations of 3,5-dimethylphenol (3,5-DMP) and 2,6-dimethylphenol (2,6-DMP) are equal, a calibration curve is plotted with the sum of the concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol as the independent variable, and the sum of the response values ​​(e.g., peak areas) of the parent ion of 3,5-dimethylphenol and 2,6-dimethylphenol (121.06589 m / z) divided by the response value (e.g., peak area) of the associated internal standard parent ion as the dependent variable. The concentration value of the co-eluting component calculated from the calibration curve is defined as the first standardized concentration of co-eluting 3,5-dimethylphenol and 2,6-dimethylphenol, denoted by c. STD1 express.

[0115] The first conversion factor for 2,6-dimethylphenol to 3,5-dimethylphenol is represented by k1 and is calculated according to formula (1):

[0116]

[0117] Among them, A 3,5 / 2,6-DMP，1A is the quantitative daughter ion peak area of the co-eluted components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point (e.g. 1000 μg / L) of the calibration sample series. 3,5-DMP，1 A is the quantitative daughter ion peak area of 3,5-dimethylphenol in the corresponding conversion factor calibration sample (e.g. 1000 μg / L). IS(3,5 / 2,6-DMP)，1 and A IS(3,5-DMP)，1 A is the peak area of the corresponding internal standard quantitative daughter ion in the calibration point and conversion factor calibration sample of the above calibration sample series.

[0118] The related calibration sample of the first conversion factor can be repeatedly measured, and the average value is taken as k1.

[0119] Similarly, a calibration curve is plotted with the sum of the concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol as the independent variable, and the sum of the response values (e.g. peak area) of the daughter ions 93.03459 m / z of 3,5-dimethylphenol and 2,6-dimethylphenol divided by the response value (e.g. peak area) of the daughter ion of the associated internal standard as the dependent variable. The concentration value of the co-eluted components calculated by the calibration curve is defined as the second normalized concentration of the co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, denoted by c STD2 .

[0120] The second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is denoted by k2, and calculated according to formula (2):

[0121]

[0122] wherein A 3,5 / 2,6-DMP，2 is the quantitative daughter ion peak area of the co-eluted components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point (e.g. 1000 μg / L) of the calibration sample series. 3,5-DMP，2 A is the quantitative daughter ion peak area of 3,5-dimethylphenol in the corresponding conversion factor calibration sample (e.g. 1000 μg / L). IS(3,5 / 2,6-DMP)，2 and A IS(3,5-DMP)，2 A is the peak area of the corresponding internal standard quantitative daughter ion in the calibration point and conversion factor calibration sample of the above calibration sample series.

[0123] The related calibration sample of the second conversion factor can be repeatedly measured, and the average value is taken as k2.

[0124] The concentration c 3,5-DMP of 3,5-dimethylphenol and the concentration c 2,6-DMP of 2,6-dimethylphenol in the sample are related to c STD1 , c STD2 , k1 and k2, which can be represented by formula (3) and formula (4):

[0125]

[0126]

[0127] Solving equations (3) and (4) simultaneously, we have:

[0128]

[0129]

[0130] 5. Calibration range and detection limit

[0131] The first regression correlation coefficient (R) of 13 phenolic components in the range of 10–5000 μg / L 2 All values ​​were greater than 0.997. The detection limits for all 13 phenolic components were ≤1.0 μg / L. Compared with the standard HJ744-2015 (derivative-GC-MS method) for testing phenolic compounds, this method has a sensitivity that is 1 to 2 orders of magnitude higher.

[0132] It should be noted that this embodiment is merely a preferred example. This invention does not limit the type and specifications of the chromatographic column or the type of LC-HRMS instrument. Other similar reverse-phase chromatographic columns can be used to implement this invention based on the technical solutions disclosed herein.

[0133] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of high resolution mass spectrometric characterization of chromatographic co-eluting dimethylphenol isomers, characterized by, The method comprises the following steps: According to the content range of petroleum in the sample, the corresponding sample pretreatment process is selected to prepare the sample; In methanol-5.0-6.5 mmol / L ammonia mobile phase system, the standard sequence sample, conversion factor calibration sample and the sample are detected by LC-HRMS, and the full scan MS of primary mass spectrum is collected 1 and the secondary mass spectrum scan MS of selected ions 2 data; According to MS 1 The first normalized concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol co-eluted and the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol were calculated using the internal standard calibration method; and the concentrations of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, 2-ethylphenol, 3-ethylphenol, and 4-ethylphenol; According to MS 2 Data, the second normalized concentration of 3,5-dimethylphenol and 2,6-dimethylphenol co-flowing was calculated by using internal standard calibration method, and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol; According to the first and second standard concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol and the first and second conversion factors of 2,6-dimethylphenol to 3,5-dimethylphenol, the concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol are calculated; The calculation formula of the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is: ； wherein is a first conversion factor for 2,6-dimethylphenol to 3,5-dimethylphenol, is the quantitative parent ion peak area of the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point of the calibration sample series, is the quantitative parent ion peak area of 3,5-dimethylphenol in the calibration sample corresponding to the conversion factor, and is the peak area of the corresponding internal standard quantitative parent ion in the calibration point of the calibration sample series and the conversion factor calibration sample. The calculation formula of the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is: ； wherein is a second conversion factor for 2,6-dimethylphenol to 3,5-dimethylphenol, is the quantitative sub-ion peak area of the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration point of the calibration sample series, is the quantitative sub-ion peak area of 3,5-dimethylphenol in the calibration sample corresponding to the conversion factor, and is the peak area of the corresponding internal standard quantitative sub-ion in the calibration sample and the conversion factor calibration sample. The calculation formula of the concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol is: wherein denotes the concentration of 2,6-dimethylphenol, denotes the concentration of 3,5-dimethylphenol, denotes the first normalized concentration of the co-elution of 3,5-dimethylphenol and 2,6-dimethylphenol, denotes the second normalized concentration of the co-elution of 3,5-dimethylphenol and 2,6-dimethylphenol, denotes the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, denotes the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

2. A method of high resolution mass spectrometric characterization of co-eluting dimethylphenol isomers by chromatography according to claim 1, characterized in that, According to the content range of petroleum in the sample, the corresponding sample pretreatment process is selected to prepare the sample, specifically comprising: When the content of petroleum in the sample is not more than 50 mg / L, the sample is filtered through a 0.22 μm filter membrane and then injected for detection; When the content of petroleum in the sample is more than 50 mg / L, the sample is alkalinized by NaOH and extracted by dichloromethane and n-hexane, and the organic phase is separated by centrifugation, and then the water phase is filtered through a 0.22 μm filter membrane, and then injected for detection after neutralization by formic acid.

3. A method of high resolution mass spectrometric characterization of co-eluting dimethylphenol isomers by chromatography according to claim 1 or 2, characterized in that, The elution conditions of the methanol-5.0-6.5 mmol / L ammonia mobile phase system include: The flow rate of the chromatographic column is 0.20 mL / min; The gradient elution is as follows: maintaining zero gradient elution for 1 min, then increasing the volume ratio of methanol to 40 % within 30 min at the same gradient, and maintaining the volume ratio of methanol at 40 % until all components flow out; The column temperature of the chromatographic column is 35-40 ℃.

4. A method of high resolution mass spectrometric characterization of co-eluting dimethylphenol isomers by chromatography according to claim 3, characterized in that, The elution conditions of the methanol-5.0-6.5 mmol / L ammonia mobile phase system include: The flow rate of the chromatographic column is 0.20 mL / min; The gradient elution is as follows: 0-1 min, 20 % methanol; 1-31 min, 20 %-40 % methanol; 31-32 min, 40 % methanol; 32-32.1 min, 40 %-90 % methanol; 32.1-37 min, 90 % methanol; 37-37.1 min, 90 %-20 % methanol; 37.1-42 min, 20 % methanol; The column temperature of the chromatographic column is 40 ℃.

5. A method of high resolution mass spectrometric characterization of co-eluting dimethylphenol isomers by chromatography according to claim 1, characterized in that, The selected ion secondary mass spectrum scan MS 2 In this case, the parent ion of 3,5-dimethylphenol-2,4,6-D3 is 124.08472 m / z and the quantifying daughter ion is 96.05342 m / z; the parent ions of 3,5-dimethylphenol and 2,6-dimethylphenol are 121.06589 m / z and the quantifying daughter ion is 93.03459 m / z.

6. Use of a method of high resolution mass spectrometric characterization of the chromatographic co-elution of dimethylphenol isomers according to any one of claims 1 to 5, characterized in that, The characterization method is applied to the quantitative characterization of 13 kinds of C0-C2-alkyl substituted phenol monomers in oily wastewater, coal gasification wastewater, oily solid waste, drinking water, surface water and groundwater.

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