A method for rapid detection of indoleacetic acid in livestock meat using gas chromatography-mass spectrometry and its application

Abstract

The present invention belongs to the field of analytical chemistry, and specifically relates to a method for rapidly detecting indoleacetic acid in livestock meat by gas chromatography-mass spectrometry and its application. The method comprises the following steps: (1) sample preparation and extraction; (2) purification and concentration by using a SinChERS purification column; (3) rapid derivatization and extraction of derivatization products; (4) gas chromatography-mass spectrometry analysis; (5) formulation of a standard working curve and quantitative analysis. The present invention is simple, rapid and accurate, greatly shortens the sample purification and derivatization time, reduces the experimental cost, improves the sample pretreatment efficiency and can achieve accurate detection, and plays an important role in the detection of indoleacetic acid content in livestock meat.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and specifically relates to a method for rapidly detecting indoleacetic acid in livestock meat by utilizing gas chromatography-mass spectrometry and an application thereof. Background Art

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Indoleacetic acid is a metabolite of tryptophan in organisms. Both mammalian tissues and intestinal microorganisms can decompose tryptophan into indoleacetic acid. In animals, the main functions of indoleacetic acid are: anti-oxidative stress, anti-inflammatory and apoptosis-mediating effects. At present, the analysis and research of indoleacetic acid mainly focuses on its effects and metabolism in animals, and there are no reports on the detection method in livestock meat. Therefore, it is urgent to develop accurate detection technology for indoleacetic acid in livestock meat.

[0004] The detection methods of indoleacetic acid include gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography and high performance liquid chromatography-mass spectrometry. Liquid chromatography analysis is mostly carried out under strong acid conditions, but strong acid conditions will affect the performance of the chromatographic column. Indoleacetic acid can be converted into a derivative with less polarity for gas chromatography-mass spectrometry analysis and detection through a derivatization reaction. However, common derivatization reagents have some defects: although diazomethane is fast, it is highly toxic and complicated to make; 10% boron trifluoride methanol and 10% boron trifluoride ether are expensive and have high experimental costs. In addition, the meat matrix is ​​complex and usually contains more impurities such as oil and fat. Common pretreatment techniques include solid phase extraction, gel permeation chromatography and freeze-degreasing. The first two methods have problems such as complex pretreatment process and large consumption of organic solvents. Although the freeze-degreasing method is simple, it requires a long freezing time and low temperature conditions. Summary of the invention

[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a method for rapidly detecting indoleacetic acid in livestock meat by gas chromatography-mass spectrometry, which has the advantages of high extraction efficiency, fast purification speed, short derivatization time, etc.

[0006] The second object of the present invention is to provide an application of the above method, wherein the method can be used to detect the content of indoleacetic acid in livestock meat.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for rapidly detecting indoleacetic acid in livestock meat by gas chromatography-mass spectrometry, the method comprising the following steps:

[0009] (1) Sample preparation and extraction: Weigh 5 g of ground meat sample, add 10 mL of water, adjust the pH value, add 20 mL of extracting solution, add 2-4 g of sodium chloride, vortex for 5 min, centrifuge at 8000 r / min for 3 min, and the supernatant is the extract;

[0010] (2) Purify and concentrate using a SinChERS purification column: insert the SinChERS purification column vertically into the extract from step (1) and slowly press down on the top of the purification column. Accurately transfer 4 mL of the purified solution from the SinChERS purification column to a 15 mL centrifuge tube and blow dry with nitrogen at 40°C to obtain a concentrated solution.

[0011] (3) Rapid derivatization and extraction of derivatization products: Add 200 μL of concentrated sulfuric acid-methanol mixed solution to the concentrated solution obtained in step (2), and vortex for 1 min; add 1.0 mL of n-hexane, and vortex for 1 min; then add 0.5 mL of saturated sodium chloride solution, vortex for 0.5 min, and allow to stand for stratification; filter the supernatant through a 0.22 μm organic phase filter to obtain an analysis solution;

[0012] (4) Gas chromatography-mass spectrometry analysis: the analysis liquid in step (3) is analyzed by gas chromatography-mass spectrometry;

[0013] (5) Preparation of standard working curve and quantitative analysis: Take a blank sample and process it according to steps (1) to (2). Add different volumes of standard solution to the concentrate of the blank sample. After drying with nitrogen, process it according to step (3) to prepare matrix standard solutions of different concentrations. Then perform gas chromatography-mass spectrometry analysis. Perform linear analysis on the peak area of ​​the target substance and the corresponding concentration. Calculate the standard working curve and regression equation of indoleacetic acid. Substitute the peak area of ​​indoleacetic acid obtained after the sample extract is tested into the corresponding regression equation to obtain the concentration of indoleacetic acid in the sample extract. Calculate the content of indoleacetic acid in the sample.

[0014] Furthermore, in step (1), the sample is pork, beef or mutton, and the pH value is adjusted to 1-6.

[0015] Furthermore, in step (1), the extracting solution is ether, ethanol, acetone or acetonitrile.

[0016] Furthermore, in step (2), the SinChERS purification column is a SP1101, SP1201, SP1501 or SP1601 purification column.

[0017] Furthermore, in step (3), the concentrated sulfuric acid-methanol mixed solution is composed of concentrated sulfuric acid and methanol in a volume ratio of 1:4.

[0018] Furthermore, in step (3), the vortexing time is 1 min.

[0019] Furthermore, in step (4), the gas chromatography conditions are: HP-5MS capillary column, 30 m×250 μm×0.25 μm; injection port temperature: 250°C; injection mode: splitless injection; injection volume: 1 μL; carrier gas: high-purity helium, purity 99.999%, flow rate: 1 mL / min; programmed temperature conditions: initial temperature 120°C, hold for 1 min; increase to 150°C at 10°C / min; increase to 280°C at 5°C / min, hold for 2.5 min.

[0020] Furthermore, the conditions of mass spectrometry were: ion source temperature: 300°C; transfer line temperature: 280°C; quadrupole temperature: 180°C; ionization energy: 70 eV; solvent delay: 5 min; monitoring mode: selected ion monitoring mode.

[0021] The application of the above method can be used to quickly detect the content of indoleacetic acid in meat products.

[0022] The present invention adjusts the pH value of the sample solution to 1-6, adds an extracting solution to extract the sample, purifies it using a SinChERS purification column, adds 200 μL of concentrated sulfuric acid methanol solution to derivatize it under vortex-assisted conditions for 1 min after drying with nitrogen, extracts the derivatized product with n-hexane, adds a saturated sodium chloride solution for purification, and determines it by gas chromatography-mass spectrometry EI source. The advantages of this method are simple operation, high extraction efficiency, fast purification speed, efficient and fast derivatization, low solvent consumption, and it is a green and environmentally friendly sample pretreatment method.

[0023] The present invention establishes a method for rapid detection of indoleacetic acid content in meat products by gas chromatography-mass spectrometry. The present invention investigates whether water is added to the sample or not, pH value, type of extraction solution, SinChERS purification column, volume of concentrated sulfuric acid and methanol, and derivatization time. The results show that indoleacetic acid has a good linear relationship in the range of 0.01-5.0 ug / mL, and the correlation coefficient is greater than 0.999. The detection limit of the method adopted by the present invention is 0.003 mg / kg, and the quantitative limit is 0.01 mg / kg. The average recovery rate of blank samples at three addition levels (0.01, 0.05, 0.1 mg / kg) is 92.1%-102.1%, and the relative standard deviation (n=6) is 2.6%-5.7%. The method provided by the present invention has simple derivatization steps, high sensitivity, and short pretreatment time, and can be used for the rapid determination of indoleacetic acid in livestock meat.

[0024] Beneficial effects:

[0025] (1) Filling the gap

[0026] The research on indoleacetic acid mainly focuses on plant-derived foods, and its detection method in livestock meat has not been reported. This method has a detection limit of 0.003 mg / kg and a quantification limit of 0.01 mg / kg, which fills the gap in the quality standard of indoleacetic acid in livestock meat and can assist in the study of the role and metabolism of indoleacetic acid in animals.

[0027] (2) Easy and quick operation

[0028] This method uses concentrated sulfuric acid methanol solution to carry out the derivatization reaction under vortex-assisted conditions, and the reaction can be completed in just 1 min. This method uses a push-pin SinChERS cleanup column, and it takes 5 s to complete the purification of one sample. The pretreatment of one sample only takes about 15 min, which greatly shortens the sample inspection time and improves work efficiency.

[0029] (3) Green and environmentally friendly

[0030] The derivatization reagent of this method only needs 200 μL, and the purification using SinChERS purification column does not require activation and equilibration, which reduces the use of organic reagents. The derivatization reagent is concentrated sulfuric acid and methanol solution, and saturated sodium chloride solution is selected for purification of the derivatized product. The reagent is easy to obtain and low in cost. The present invention greatly reduces the amount of organic reagents used, and is more environmentally friendly.

[0031] The invention has the advantages of being simple, rapid, green, and low-cost, and is suitable for rapid detection of indoleacetic acid in livestock meat, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 is the total ion current of indoleacetic acid derivatives;

[0034] Figure 2 The mass spectrum and chemical structure diagram of indoleacetic acid derivatives;

[0035] Figure 3 This is a graph showing the effect of adding water and acid to the sample on the extraction efficiency;

[0036] Figure 4 This is a graph showing the effect of pH value of sample solution on extraction efficiency;

[0037] Figure 5 This is a diagram for investigating the effect of the type of extraction solution on the extraction efficiency;

[0038] Figure 6 This is a comparison chart of the purification effects of four types of SinChERS purification columns;

[0039] Figure 7 This is a graph showing the effect of methanol volume on derivatization efficiency;

[0040] Figure 8 This is a graph showing the effect of concentrated sulfuric acid volume on derivatization efficiency;

[0041] Fig. 9 This is a graph showing the effect of derivatization time on derivatization efficiency. DETAILED DESCRIPTION

[0042] The following description further describes the specific details of the present invention for a full understanding of the present invention. The terms used in the description of the present invention are only used to illustrate the advantages and features of the present invention and are not intended to limit the present invention.

[0043] Unless otherwise defined, all professional and scientific terms used in the present invention have the same meanings as those understood by technicians in the technical field of the present invention. Unless otherwise specified, the drugs or reagents used in the present invention are used in accordance with the product instructions or the conventional methods of use in the field. The technical solution of the present invention is further described according to the drawings and specific embodiments of the specification.

[0044] The instruments used in the following examples are: Agilent 7890-5977B gas chromatograph-mass spectrometer (Agilent, USA), equipped with an electron impact ion source (EI); electronic balance (precision 0.01 g and 0.0001 g, Beijing Sartorius Scientific Instrument Co., Ltd.); MS 3 vortex mixer (IKA, Germany); 3-18K centrifuge (SIGMA, Germany); Milli-Q ultrapure water machine (Millipore, Germany).

[0045] Reagents and samples used in the following examples: indoleacetic acid standard: (1000 mg / L, Beijing Manhag Biotechnology Co., Ltd.); n-hexane, acetone, ether, ethanol, acetonitrile, methanol (chromatographic grade, Merck, Germany); concentrated sulfuric acid, formic acid, sodium chloride (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); SinChERS purification column (Tianjin Shi'an Technology Development Co., Ltd.); organic microporous filter membrane (0.22 μm, Shanghai Anpu Scientific Instrument Co., Ltd.); mutton, pork, beef and other samples were purchased from local supermarkets.

[0046] Preparation of the standard solution in the following examples: accurately pipette 100 μL of the standard with a concentration of 1000 mg / L, dissolve it in methanol and make up to 10 mL to prepare a standard solution with a mass concentration of 10 mg / L, and store it below 4°C;

[0047] The following example is the preparation of the matrix-matched standard working solution: After the blank sample is treated according to steps (1) to (2) in Example 1, 1, 10, 20, 50, 100, and 500 uL of the standard solution are added to a 15 mL centrifuge tube, dried with nitrogen gas, and derivatized, extracted, and impurity-removed according to step (3) in Example 1 to obtain matrix standard working solutions with concentrations of 0.01, 0.1, 0.2, 0.5, 1.0, and 5.0 μg / mL.

[0048] First embodiment

[0049] 1 Experimental part

[0050] 1.1 Instruments and Materials

[0051] Agilent 7890-5977B gas chromatograph-mass spectrometer (Agilent, USA), equipped with electron impact ion source (EI); electronic balance (precision 0.01 g and 0.0001 g, Beijing Sartorius Scientific Instrument Co., Ltd.); MS 3 vortex mixer (IKA, Germany); 3-18K centrifuge (SIGMA, Germany); Milli-Q ultrapure water machine (Millipore, Germany). Indoleacetic acid standard: (1000 mg / L, Beijing Manhag Biotechnology Co., Ltd.); n-hexane, ether, acetone, ethanol, acetonitrile, methanol (chromatographic grade, Merck, Germany); concentrated sulfuric acid, formic acid, sodium chloride (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); SinChERS purification column (Tianjin Shi'an Technology Development Co., Ltd.); organic microporous filter membrane (0.22 μm, Shanghai Anpu Scientific Instrument Co., Ltd.); mutton, pork, beef and other samples were purchased from local supermarkets.

[0052] 1.2 Preparation of standard solution

[0053] Accurately pipette 100 μL of the standard with a concentration of 1000 mg / L, dissolve it in methanol and make up to 10 mL to prepare a standard solution with a mass concentration of 10 mg / L, and store it below 4°C.

[0054] 1.3 Instrumental analysis conditions

[0055] Gas chromatography conditions: HP-5MS capillary column, 30 m×250 μm×0.25 μm; injection port temperature: 250°C; injection mode: splitless injection; injection volume: 1 μL; carrier gas: high-purity helium, purity 99.999%, flow rate: 1 mL / min; programmed temperature conditions: initial temperature 120°C, hold for 1 min; increase to 150°C at 10°C / min; increase to 280°C at 5°C / min, hold for 2.5 min;

[0056] Mass spectrometry conditions: ion source temperature: 300 °C; transfer line temperature: 280 °C; quadrupole temperature: 180 °C; ionization energy: 70 eV; solvent delay: 5 min; monitoring mode: selected ion monitoring mode;

[0057] 1.4 Sample pretreatment

[0058] 1.4.1. Sample preparation and extraction: mince the pork sample, and then use a tissue crusher to crush and mix. Weigh 5.0 g (accurate to 0.01 g) of the crushed sample into a 50 mL centrifuge tube, add 10 mL of water, add formic acid to adjust the pH value to 3, add 20 mL of acetonitrile as the extraction solvent, vortex extract for 5 min, then add 4 g of sodium chloride, vortex oscillate for 1 min, and then centrifuge at 8000 r / min for 3 min. The supernatant is the extract.

[0059] 1.4.2. Purify and concentrate using a SinChERS purification column: insert the SinChERS purification column vertically into the extract from step 1.4.1, and slowly press down on the top of the purification column to obtain a purified solution from the SinChERS purification column. Accurately transfer 4 mL of the purified solution to a 15 mL centrifuge tube, blow dry with nitrogen at 40°C to obtain a concentrated solution;

[0060] 1.4.3, rapid derivatization and extraction of derivatization products: add 200 μL of a mixed solution of concentrated sulfuric acid and methanol to the concentrated solution obtained in step 1.4.2, the mixed solution of 200 μL of concentrated sulfuric acid and methanol contains 40 μL of concentrated sulfuric acid and 160 μL of methanol, vortex for 1 min to carry out the derivatization reaction; add 1 mL of n-hexane and vortex for 1 min, then add 0.5 mL of saturated sodium chloride solution and vortex for 0.5 min, let stand for stratification, take the supernatant and filter it through a 0.22 μm organic phase filter membrane, and measure it by GC-MS;

[0061] 1.5 Development and quantitative analysis of standard working curve

[0062] Take a blank sample, and after using the treatment conditions of 1.4.1 and 1.4.2 above, add 1, 10, 20, 50, 100, 500uL of standard solution respectively, blow dry with nitrogen, and then treat in 1.4.3 to prepare matrix standard working solutions with concentrations of 0.01, 0.1, 0.2, 0.5, 1.0 and 5.0 μg / mL, and enter the gas chromatograph mass spectrometer in turn, record the peak area corresponding to each standard solution, and draw a standard curve with the concentration of the standard solution as the horizontal axis and the peak area as the vertical axis. Substitute the indoleacetic acid peak area obtained after the sample extract is detected into the standard curve regression equation to obtain the concentration of indoleacetic acid in the sample extract, and calculate the residual amount of indoleacetic acid in the sample.

[0063] 1.6 Data Processing

[0064] Matrix effect (ME) refers to the phenomenon that components other than the analyte in the sample significantly interfere with the analyte and method detection during the analysis process. This study uses the ratio of the slope of the matrix matching standard curve to the slope of the solvent standard curve to quantitatively evaluate the matrix effect (ME) of indoleacetic acid. The formula is:

[0065] ;

[0066] The larger the absolute value of ME, the stronger the matrix effect. |ME|≤20% indicates a weak matrix effect, 20%<|ME|≤50% indicates a moderate matrix effect, and |ME|>50% indicates a strong matrix effect.

[0067] 2 Results and discussion

[0068] 2.1 Optimization of sample conditions before extraction

[0069] During the test, the addition of water and acid to the sample was optimized. Figure 3 .

[0070] 2.1 Optimization of pH

[0071] During the experiment, the pH value was optimized. Figure 4 .

[0072] 2.2 Optimization of extraction solvent

[0073] During the experiment, the type of extraction solvent was optimized. Figure 5 .

[0074] 2.2 Optimization of purification column

[0075] During the experiment, the SinChERS cleanup column was optimized. Figure 6 .

[0076] 2.3 Optimization of methanol volume

[0077] During the experiment, the volume of methanol was optimized. Figure 7 .

[0078] 2.4 Optimization of concentrated sulfuric acid volume

[0079] During the experiment, the volume of concentrated sulfuric acid was optimized. Figure 8 .

[0080] 2.5 Optimization of derivatization time

[0081] During the experiment, the derivatization time was optimized. Fig. 9 .

[0082] 3 Methodological validation

[0083] 3.1 Linear range, sensitivity and matrix effect of the method

[0084] Matrix effect is an important factor affecting the accurate quantification of pesticide residues. The matrix components of meat and meat product samples are complex. If the samples are not purified, pork, beef and mutton will have strong matrix effects. This study adjusted the pH value of the extract, extracted with acetonitrile, purified with a SinCHERS purification column, and added saturated sodium chloride to remove impurities, thereby purifying the on-machine solution and reducing the interference of the matrix effect. As shown in Table 1, beef, pork and mutton showed moderate matrix effects, and compensation measures were required. Therefore, in order to accurately quantify, this study used the blank matrix matching method to prepare the standard working curve.

[0085] Table 1 Investigation of matrix effect

[0086]

[0087] Using the above section 1.5, a matrix standard working solution with a concentration of 0.01-5.0 μg / mL was prepared. Under the optimized conditions, the limit of detection (LOD) and the limit of quantification (LOQ) of the method were determined by a blank sample spike test. The results are shown in Table 1: Indoleacetic acid has a good linear relationship in the range of 0.01-5.0 ug / mL, and the correlation coefficient is greater than 0.999. The method LOD is 0.003 mg / kg, and the method LOQ is 0.01 mg / kg.

[0088] 3.2 Method recovery and precision

[0089] The matrix-matched standard solution-external standard method was used for quantification. Pork, mutton and beef samples were selected. The content of indoleacetic acid was determined by the optimized method at the addition levels of 0.01, 0.05 and 0.1 mg / kg. The relative standard deviation (RSD) was 2.6%~5.7%.

[0090] Table 2 Precision and spike recovery experiment (n=6)

[0091]

[0092] 3.3 Analysis of actual samples

[0093] Using the optimized method, 30 batches of livestock meat from local supermarkets were tested, including 10 batches of pork, mutton and beef. The results are shown in Table 3. It can be seen from Table 3 that the detection rate of 30 batches of livestock meat is 80%, and the indoleacetic acid content is 0.01 mg / kg~0.08 mg / kg.

[0094] Table 3 Detection of indoleacetic acid in different batches of livestock meat

[0095]

[0096] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials used in the present invention, addition of auxiliary raw materials, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for rapidly detecting indoleacetic acid in livestock meat using gas chromatography-mass spectrometry, characterized in that: The method comprises the following steps: (1) Sample preparation and extraction: add water to the ground meat sample, then add formic acid to adjust the pH value to 1-6, add extraction solvent, add sodium chloride, vortex, centrifuge, and the supernatant is the extract; the sample is pork, beef or mutton; the mass volume ratio of the sample, sodium chloride, water and extraction solvent is 5 g: 2 g: 10 mL: 20 mL; the extraction solvent is ether, ethanol, acetone or acetonitrile; (2) Purification and concentration using a SinChERS purification column: vertically insert the SinChERS purification column into the extract of step (1), slowly press down the top of the purification column to obtain a purified solution in the SinChERS purification column, remove the purified solution in the SinChERS purification column, blow dry with nitrogen gas, and obtain a concentrated solution; (3) Rapid derivatization and extraction of derivatization products: Add concentrated sulfuric acid and methanol mixed solution to the concentrated solution in step (2), vortex and oscillate for derivatization, and the vortexing time is 1 min; the concentrated sulfuric acid and methanol mixed solution is composed of concentrated sulfuric acid and methanol in a volume ratio of 1:4; add n-hexane and vortex; then add saturated sodium chloride solution, vortex, and stand to separate layers; the upper clear liquid is filtered through a 0.22 μm organic phase filter to obtain an analysis solution; the volume ratio of the purified solution in step (2) to the concentrated sulfuric acid and methanol mixed solution in step (3) is 4 mL:200 μL; (4) Gas chromatography-mass spectrometry analysis: the analysis liquid in step (3) is analyzed by gas chromatography-mass spectrometry; (5) Preparation of standard working curve and quantitative analysis: Take a blank sample and process it according to steps (1) to (2). Add different volumes of standard solution to the concentrate of the blank sample. After drying with nitrogen, process it according to step (3) to prepare matrix standard solutions of different concentrations. Then perform gas chromatography-mass spectrometry analysis. Perform linear analysis on the peak area of ​​the target substance obtained and the corresponding concentration. Calculate the standard working curve and regression equation of indoleacetic acid. Substitute the peak area of ​​indoleacetic acid obtained after the sample analysis solution is tested into the corresponding regression equation to obtain the concentration of indoleacetic acid in the sample analysis solution. Calculate the content of indoleacetic acid in the sample.

2. The method according to claim 1, characterized in that In step (1), the centrifugal speed is 8000 r / min and the time is 3 min.

3. The method according to claim 1, characterized in that In step (2), the SinChERS cleanup column is a SP1101, SP1201, SP1501 or SP1601 cleanup column.

4. The method according to claim 1, characterized in that: In step (3), the volume ratio of the concentrated sulfuric acid-methanol mixed solution, n-hexane and saturated sodium chloride solution is 200 μL:1 mL:0.5 mL.

5. The method according to claim 1, characterized in that After adding n-hexane in step (3), vortex for 1 min, then add saturated sodium chloride solution and vortex for 0.5 min, and let stand to separate the layers.

6. The method according to claim 1, characterized in that In step (4), the gas chromatography conditions are: HP-5MS capillary column, 30 m×250 μm×0.25 μm; injection port temperature: 250°C; injection mode: splitless injection; injection volume: 1 μL; carrier gas: high-purity helium, purity 99.999%, flow rate: 1 mL / min; programmed temperature conditions: initial temperature 120°C, hold for 1 min; increase to 150°C at 10°C / min; increase to 280°C at 5°C / min, hold for 2.5 min; Mass spectrometry conditions: ion source temperature: 300°C; transfer line temperature: 280°C; quadrupole temperature: 180°C; ionization energy: 70 eV; solvent delay: 5 min; monitoring mode: selected ion monitoring mode.

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