Method for detecting eight n-heptane metabolites in urine by gas chromatography / mass spectrometry

Abstract

The invention discloses a method for detecting eight n-heptane metabolites in urine by gas chromatography / mass spectrometry, which comprises the following steps: carrying out sample pretreatment on a urine sample to obtain a urine sample to be detected; preparing a standard curve sample and a linear regression equation of a standard curve; detecting eight n-heptane metabolites in the sample to be detected by using a gas chromatography / mass spectrometry method, and accurately recording peak areas of each metabolite and an internal standard substance; calculating the concentrations of eight n-heptane metabolites in the urine by combining a linear regression equation of the standard curve according to the peak areas of the metabolites and the internal standard substance; the accuracy and precision of the detection method are evaluated through a urine matrix labeling experiment; the eight n-heptane metabolites in the urine obtained by the invention are in respective linear ranges, the linear relationship is good, the correlation coefficients are all above 0.998, the detection limit is 5.7-17.3 [mu] g / L, and the lower quantification limit is 19.1-57.7 [mu] g / L; and the accuracy of the low-concentration sample, the accuracy of the medium-concentration sample and the accuracy of the high-concentration sample are 89.6%-107.5%, 91.8%-107.0% and 90.7%-108.3% respectively.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical detection, and more specifically, relates to a method for detecting 8 metabolites of n-heptane in urine by gas chromatography / mass spectrometry. Background Art

[0002] n-Heptane, with the chemical formula C 7 H 16 , is a hydrocarbon organic compound with a CAS number of 142-82-5 and is a colorless and volatile liquid at room temperature. n-Heptane can be separated by distillation during the petroleum refining process and prepared through alkane hydrogenation reaction. It has good lipophilicity and is widely used as an extraction solvent for animal fats and plants, a cleaning agent in offset printing and printing technology, a diluent in paint and varnish manufacturing, and a standard for measuring octane number. According to incomplete statistics, the total global production capacity of n-heptane in 2023 was approximately 1 million tons, with Asia-Pacific being the main production market (such as China and South Korea), accounting for more than 45% of the market share, followed by Europe and North America, etc. However, a large number of studies have shown that occupational exposure to n-heptane can affect the body's central nervous system, respiratory system, digestive system, and endocrine system, etc. In 2012, a domestic report on acute poisoning caused by occupational exposure to n-heptane showed that most poisoned patients had varying degrees of dizziness, headache, and fatigue and other symptoms of central nervous system damage, and a small number of patients also had abnormal triglycerides and respiratory system damage symptoms. In addition, relevant studies have shown that occupational exposure to n-heptane may also have a certain impact on hearing and eyes. Since there are mainly 8 metabolites of n-heptane in urine, it is necessary to develop a time-saving, labor-saving, highly specific, and applicable method for detecting a large number of samples, which can simultaneously detect multiple n-heptane metabolites in urine to protect the physical health of people exposed to n-heptane occupationally and carry out relevant epidemiological research and investigations.

[0003] Currently, there is no domestic research report on the detection method of n-heptane metabolites in urine, and the foreign detection methods for n-heptane metabolites in urine mainly include liquid-liquid extraction / gas chromatography / mass spectrometry, selective reagent ionization time-of-flight mass spectrometry combined with gas chromatography, and headspace solid-phase dynamic extraction / gas chromatography / mass spectrometry, etc. Among the above detection methods, selective reagent ionization time-of-flight mass spectrometry combined with gas chromatography can reduce the influence of urine matrix interference, has an extremely fast response speed, high resolution and sensitivity; but this method has expensive instrument equipment and high detection costs, and is not suitable for promotion in district and county-level units. Headspace solid-phase dynamic extraction / gas chromatography / mass spectrometry can determine 8 metabolites of n-heptane in urine, and has high sensitivity and accuracy; but the equipment cost of the headspace solid-phase dynamic extractor is high, and the sample analysis time is long, which is not suitable for widespread promotion and detection of a large number of samples. Summary of the Invention

[0004] In view of the above defects or improvement requirements of the prior art, the present invention provides a method for detecting 8 metabolites of n-heptane in urine by gas chromatography / mass spectrometry, which has high sensitivity, high accuracy, good precision and stability. At the same time, it is simple to operate and low in cost, can quickly and accurately detect the n-heptane metabolites in urine, helps to evaluate the health risks of occupationally exposed populations, and provides a scientific basis for formulating occupational health standards and protective measures; it is applicable to the detection of large-scale samples, can provide technical support for epidemiological investigations, and promote the research progress in related fields; it is suitable for popularization in testing institutions at all levels, and has significant scientific value and social benefits.

[0005] To achieve the above object, one aspect of the present invention provides a method for detecting 8 metabolites of n-heptane in urine by gas chromatography / mass spectrometry, comprising the following steps:

[0006] S1: Solution preparation: Prepare 1 moL / L sodium acetate buffer solution, 15% methanol dichloromethane solution, standard application solution including 8 n-heptane metabolites, and internal standard application solution respectively;

[0007] S2: Perform sample pretreatment on the urine sample to obtain the urine sample to be tested;

[0008] S3: Prepare standard curve samples, and establish a linear regression equation of the standard curve according to the concentrations of the standard curve samples and the peak areas of each metabolite and internal standard in the standard curve samples;

[0009] S4: Use gas chromatography / mass spectrometry (GC-MS) to detect 8 metabolites of n-heptane in the sample to be tested, and accurately record the peak areas of each metabolite and internal standard;

[0010] S5: Calculate the concentrations of 8 metabolites of n-heptane in urine based on the peak areas of each metabolite and internal standard and the linear regression equation of the standard curve established in step S3;

[0011] S6: Evaluate the accuracy and precision of the detection method for 8 metabolites of n-heptane through urine matrix spike experiments;

[0012] The 8 metabolites of n-heptane include 4-heptanone, 3-heptanone, 2-heptanone, 4-heptanol, 3-heptanol, 2-heptanol, 1-heptanol and 2,5-heptanedione.

[0013] Further, the preparation of the standard application solution in step S1 includes: taking 2 μL from each of the standard solutions of 8 n-heptane metabolites, adding them to 1 mL of methanol solution to prepare a standard stock solution; then taking 25 μL from the stock solution and mixing it with 1 mL of methanol solution to prepare a standard application solution, and storing it at -20 °C for later use.

[0014] Further, in step S1, isotope-labeled 1-heptanol-d 15 is used as a quantitative internal standard;

[0015] The preparation of the internal standard application solution includes: taking 2 μL from the 1-heptanol-d 15 internal standard solution and adding it to 1 mL of methanol solution to prepare the 1-heptanol-d 15 internal standard stock solution. Then, take 10 μL from the 1-heptanol-d 15 internal standard stock solution and mix it with 1 mL of methanol solution to prepare the 1-heptanol-d 15 internal standard application solution, and store it at -20 °C for later use.

[0016] Further, in step S2, the urine sample is pre-treated to obtain a sample to be tested; it includes:

[0017] S21: Thaw the urine sample;

[0018] S22: Acid hydrolysis. Take 2 mL of the thawed urine sample in a 15 mL centrifuge tube, add 100 μL of concentrated hydrochloric acid solution, and then place the centrifuge tube in a 100 °C water bath for 1.5 hours for acid hydrolysis;

[0019] S23: After the acid hydrolysis is completed, sequentially add 25 μL of the internal standard application solution, 1 mL of sodium acetate buffer solution, and 3 mL of 15% methanol dichloromethane solution to the centrifuge tube in S32;

[0020] S24: After the solution is fully mixed, place it in a three-dimensional multi-point vortex oscillator and shake it at 2500 rpm for 10 min;

[0021] S25: Centrifuge it at 10000 rpm for 5 min with a medical centrifuge, remove the upper aqueous phase solution, and take 2.5 mL of the lower organic phase solution;

[0022] S26: Use a dry bath nitrogen blower to nitrogen-blow the organic phase solution to about 0.5 mL to obtain the sample to be tested.

[0023] Further, in step S3, the standard curve samples are prepared, and a linear regression equation of the standard curve is established according to the concentration of the standard curve samples, the peak areas of each metabolite and internal standard in the standard curve samples; it includes:

[0024] S31: Respectively take 0, 1, 3, 10, 25 μL from the prepared standard application solution;

[0025] S32: Respectively add 25 μL of the internal standard application solution to each standard application solution;

[0026] S33: Perform sample pretreatment in the same manner as the urine sample in step S2 to obtain standard curve samples with standard concentrations of 0, 19, 58, 195, 485 μg / L respectively and an internal standard concentration of 200 μg / L;

[0027] S34: Detect the standard curve samples using gas chromatography / mass spectrometry (GC-MS), and record the peak areas of each metabolite and internal standard in the standard curve samples;

[0028] S35: Establish a linear regression equation of the standard curve based on the concentration of the standard curve samples, the peak areas of each metabolite and internal standard in the standard curve samples.

[0029] Further, the standard curve in step S3 is fitted with the relative concentration of each target substance to the internal standard as the abscissa and the relative peak area of each target substance to the internal standard as the ordinate. The linear regression equation of the standard curve is represented by formula (1):

[0030] Y = bX + a (1)

[0031] Wherein, Y is the concentration of each target substance; b is the curve slope; X is the ratio of the peak area of each target substance to the peak area of the internal standard; a is the curve intercept.

[0032] Further, the gas chromatography (GC) detection conditions in step S4 are as follows: chromatographic column selection, VF-WAX ms 60m×25mm×5μm chromatographic column; injection port temperature: 250 °C; injection mode: split injection at a ratio of 10:1, split flow rate is 10 mL / min; carrier gas (helium) flow rate: 15 mL / min, helium (purity ≥ 99.999%); injection volume: 1 μL;

[0033] Temperature programming, initial temperature 60 °C, hold for 2 min, increase the temperature to 130 °C at a rate of 20 °C / min, increase the temperature to 165 °C at a rate of 2.5 °C / min, increase the temperature to 220 °C at a rate of 25 °C / min, and finally maintain for 2 min.

[0034] Further, the mass spectrometry (MS) detection conditions in step S4 are as follows: ion source, EI ion source; ion source temperature, 230 °C; MS quadrupole temperature, 150 °C; scan mode: selective ion mode (SIM) scan; solvent delay time: 8.20 min.

[0035] Further, in step S4, the full scan (SCAN) mode in the gas chromatography / mass spectrometry instrument is adopted to collect ion data for each mass-to-charge ratio. After determining the retention time of each metabolite, the single ion detection scan (SIM) mode is used to determine the optimal quantitative and qualitative ion pairs.

[0036] Further, step S4 further includes preparing 10 urine matrix spike samples repeatedly and calculating the standard deviation of the measurement results of the peak areas of each metabolite and internal standard in step S4; wherein, the method detection limit is 3 times the standard deviation, and the method quantification lower limit is 10 times the standard deviation.

[0037] Generally speaking, compared with the prior art through the above technical solutions conceived by the present invention, the following beneficial effects can be achieved:

[0038] (1) The method for detecting 8 metabolites of n-heptane in urine by gas chromatography / mass spectrometry of the present invention can effectively detect 8 metabolites of n-heptane in urine through optimized gas chromatography and mass spectrometry detection conditions, combined with internal standard quantitative analysis. It has a good linear relationship (correlation coefficient r≥0.998), a low detection limit (5.7 - 17.3 μg / L), and a low quantification lower limit (19.1 - 57.7 μg / L), and can meet the detection requirements of low-concentration metabolites. Using the internal standard 1-heptanol-d 15 corrects experimental errors and significantly improves the accuracy and reliability of the detection results. The RSD value range of within-batch precision is 1.7% - 14.6%, and the RSD value range of between-batch precision is 4.1% - 11.5%, both within the acceptable range (usually ≤15%), indicating that the method performs excellently in terms of repeatability and stability and is suitable for large-scale sample detection.

[0039] (2) The method for detecting 8 metabolites of n-heptane in urine by gas chromatography / mass spectrometry of the present invention can effectively extract the metabolites in urine by steps such as acid hydrolysis, vortex oscillation, centrifugation, and nitrogen blowing concentration, while removing impurities and interfering components, improving the enrichment degree of target metabolites, and ensuring the accuracy of detection results. The sample pretreatment process is standardized and consistent with the standard curve sample treatment, further improving the reliability of quantitative analysis.

[0040] (3) The method for detecting 8 metabolites of n-heptane in urine by gas chromatography / mass spectrometry of the present invention is applicable to the detection of samples at different concentration levels. Through urine matrix spike experiments, the accuracies of low-, medium-, and high-concentration samples are verified to be 89.6% - 107.5%, 91.8% - 107.0%, and 90.7% - 108.3% respectively, indicating that the method has wide applicability in practical applications. The accuracy and precision of the detection method remain stable at different time periods and are suitable for epidemiological investigations and health monitoring of occupationally exposed populations.

[0041] (4) The method for detecting 8 metabolites of n - heptane in urine by gas chromatography / mass spectrometry of the present invention uses a conventional gas chromatography / mass spectrometry instrument and simple organic solvents, with low equipment cost and simple operation. The method of the present invention fills the domestic blank in the detection of n - heptane metabolites in urine and provides reliable technical support for related research and practical applications. The method of the present invention has high sensitivity, high accuracy, good precision and stability, and at the same time is simple to operate and low in cost, suitable for popularization in detection institutions at all levels, and has significant scientific value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 FIG. is a schematic flow chart of the method for detecting 8 metabolites of n - heptane in urine by gas chromatography / mass spectrometry according to an embodiment of the present invention;

[0043] Figure 2 FIG. is a schematic diagram of the retention times of 8 metabolites of n - heptane and internal standard in urine on a VF - WAX ms chromatographic column in the method for detecting 8 metabolites of n - heptane in urine by gas chromatography / mass spectrometry according to an embodiment of the present invention.

[0044] It should be noted that:

[0045] Gas chromatography / mass spectrometry (GC - MS) is an analytical method that combines two techniques of gas chromatography (GC) and mass spectrometry (MS); it uses the high - efficiency separation ability of gas chromatography and the high - sensitivity detection ability of mass spectrometry to qualitatively and quantitatively analyze the components in complex mixtures.

[0046] The RSD value (Relative Standard Deviation) is a statistical index used to measure the precision and repeatability of data. It represents the ratio of the standard deviation (SD) to the mean value (Mean) of a set of data, usually expressed as a percentage. The smaller the RSD value, the higher the repeatability and precision of the data. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0048] As Figure 1 shown, one aspect of the present invention provides a method for detecting 8 metabolites of n - heptane in urine by gas chromatography / mass spectrometry, including the following steps:

[0049] S1: Solution Preparation: Prepare 1 moL / L sodium acetate buffer solution, 15% methanol dichloromethane solution, standard application solution including 8 metabolites of n-heptane, and internal standard application solution respectively;

[0050] S2: Perform sample pretreatment on urine samples to obtain urine samples to be tested;

[0051] S3: Prepare standard curve samples, and establish a linear regression equation of the standard curve based on the concentrations of the standard curve samples, the peak areas of each metabolite and internal standard in the standard curve samples;

[0052] S4: Use gas chromatography / mass spectrometry (GC-MS) to detect 8 metabolites of n-heptane in the sample to be tested, and accurately record the peak areas of each metabolite and internal standard;

[0053] S5: Calculate the concentrations of 8 metabolites of n-heptane in urine based on the peak areas of each metabolite and internal standard, combined with the linear regression equation of the standard curve established in step S3;

[0054] S6: Through urine matrix spike experiments, evaluate the accuracy and precision of the detection method for 8 metabolites of n-heptane;

[0055] The 8 metabolites of n-heptane include 4-heptanone, 3-heptanone, 2-heptanone, 4-heptanol, 3-heptanol, 2-heptanol, 1-heptanol and 2,5-heptanedione.

[0056] Further, step S1 includes:

[0057] Instrument and Reagent Preparation:

[0058] Instruments include: Gas Chromatograph (7890B) - Tandem Mass Spectrometer (5977B); VF-WAX ms 60m×25mm×5μm chromatographic column; Dry Bath Nitrogen Blower; BY-G20 Medical Centrifuge; Multi-Reax Three-Dimensional Multi-Point Vortex Oscillator; β-Glucuronidase / Arylsulfatase; Concentrated Hydrochloric Acid; Sodium Acetate; Methanol, Dichloromethane; Laboratory Water: Ultra-Pure Water;

[0059] The preparation of 1 moL / L sodium acetate buffer solution includes: Take 82 g of solid sodium acetate, add it to 1 L of ultra-pure water, and prepare a sodium acetate buffer solution with a concentration of 1 moL / L (pH value of 5.5);

[0060] The preparation of 15% methanol dichloromethane solution includes: Use a 100 mL graduated cylinder to take 15 mL of methanol solution and 85 mL of dichloromethane solution respectively, and mix them to prepare a 15% methanol dichloromethane solution;

[0061] The preparation of the standard application solution includes: taking 2 μL from the standard solutions of 8 metabolites of n-heptane, adding them to 1 mL of methanol solution to prepare a standard stock solution (which can be stored in a -20°C refrigerator for 30 days), and then taking 25 μL from the standard stock solution and mixing it with 1 mL of methanol solution to prepare a standard application solution, which is stored at -20°C for later use; among them, the 8 metabolites of n-heptane include 4-heptanone, 3-heptanone, 2-heptanone, 4-heptanol, 3-heptanol, 2-heptanol, 1-heptanol, and 2,5-heptanedione; as shown in Table 1:

[0062]

[0063] Taking 1-heptanol-d of the isotope-labeled substance 15 as a quantitative internal standard; the preparation of the internal standard application solution includes: taking 2 μL from the 1-heptanol-d 15 internal standard solution, adding it to 1 mL of methanol solution to prepare a 1-heptanol-d 15 internal standard stock solution (which can be stored in a -20°C refrigerator for 30 days), and then taking 10 μL from the 1-heptanol-d 15 internal standard stock solution and mixing it with 1 mL of methanol solution to prepare a 1-heptanol-d 15 internal standard application solution, which is stored at -20°C for later use.

[0064] Among them, 1 mol / L sodium acetate buffer solution is used to adjust the acidity and alkalinity of the solution subsequently, providing a suitable environment for the reaction; 15% methanol dichloromethane solution, as an organic solvent, helps to extract the target metabolites; the standard application solution, which contains 8 metabolites of n-heptane with known concentrations, is used to establish a standard curve subsequently; the internal standard application solution, the addition of the internal standard can be used as a reference to correct the errors in the experimental process and improve the accuracy of the detection results.

[0065] Furthermore, in step S2, the urine sample is subjected to sample pretreatment to obtain a sample to be tested; it includes:

[0066] S21: Thaw the urine sample. Specifically, take out the urine sample stored in a -80°C refrigerator for thawing treatment. Low-temperature storage can effectively prevent the degradation and deterioration of metabolites in the sample, ensuring the stability and reliability of the sample in subsequent detections. The thawing process needs to be gentle and uniform to avoid adverse effects on the sample components caused by sudden temperature changes;

[0067] S22: Acid hydrolysis. Take 2 mL of the thawed urine sample into a 15 mL centrifuge tube, add 100 μL of concentrated hydrochloric acid solution, and then place the centrifuge tube in a 100°C water bath for 1.5 hours for acid hydrolysis; the acid hydrolysis step aims to use the strong acidity of concentrated hydrochloric acid to decompose the conjugated metabolites in the urine into free forms, enabling them to be better extracted by subsequent organic solvents and improving the sensitivity and accuracy of the detection;

[0068] S23: After the acid hydrolysis is completed, 25 μL of internal standard application solution, 1 mol / L sodium acetate buffer solution, and 3 mL of 15% methanol dichloromethane solution are successively added to the centrifuge tube in S32. Among them, the addition of the internal standard application solution provides a reference for subsequent quantitative analysis; the sodium acetate buffer solution is used to adjust the pH value of the solution to reach the pH range suitable for subsequent extraction operations; the methanol dichloromethane solution serves as an organic solvent to provide a medium for the extraction of metabolites.

[0069] S24: After the solution is fully mixed, it is placed in a three-dimensional multi-point vortex oscillator and shaken at 2500 rpm for 10 min. Among them, the three-dimensional multi-point vortex oscillator can provide an all-round, uniform and efficient oscillation effect, enabling the components in the solution to fully contact, accelerating the dissolution and dispersion of metabolites in the organic phase, improving the extraction efficiency, and ensuring the stability of subsequent separation effects.

[0070] S25: Centrifuge at 10000 rpm for 5 min using a medical centrifuge to remove the upper aqueous phase solution, and take 2.5 mL of the lower organic phase solution. Through centrifugal separation, impurities and interfering components can be effectively removed, improving the enrichment degree of target metabolites in the organic phase and providing a purer sample for subsequent detection.

[0071] S26: Use a dry bath nitrogen blower to blow the organic phase solution to about 0.5 mL to obtain the sample to be tested. Specifically, place the separated organic phase solution in a dry bath nitrogen blower and blow the solution to about 0.5 mL by nitrogen purging to obtain the sample to be tested. The nitrogen blowing and concentration process can remove most of the organic solvents, significantly increase the concentration of metabolites, and at the same time avoid the interference of solvent residues on subsequent detection, ensuring that the sample to be tested meets the injection requirements of gas chromatography / mass spectrometry.

[0072] Furthermore, the establishment of the standard curve in step S3 is a key link in quantitative analysis. By measuring the response values of standard solutions with different concentrations, the relationship curve between concentration and response value is plotted, providing a basis for calculating the metabolite concentration in subsequent urine samples. In step S3, the standard application solution and the internal standard application solution are subjected to the same pretreatment process as the urine sample to ensure the consistency between the standard curve sample and the actual urine sample during the processing, thereby improving the accuracy and reliability of quantitative analysis.

[0073] By detecting the peak areas of the standard curve samples, the linear relationship between concentration and peak area can be obtained, thereby providing a basis for the quantitative analysis of metabolites in urine samples.

[0074] Furthermore, step S3 includes:

[0075] S31: Take 0, 1, 3, 10, and 25 μL respectively from the prepared standard application solution.

[0076] S32: Add 25 μL of internal standard application solution to each standard application solution.

[0077] S33: Perform sample pretreatment on the standard curve samples in the same way as the urine samples in step S2 to obtain standard curve samples with standard concentrations of 0, 19, 58, 195, 485 μg / L and an internal standard concentration of 200 μg / L.

[0078] S34: Detect the standard curve samples using gas chromatography / mass spectrometry (GC-MS), and record the peak areas of each metabolite and internal standard in the standard curve samples.

[0079] S35: Establish a linear regression equation for the standard curve based on the concentrations of the standard curve samples and the peak areas of each metabolite and internal standard in the standard curve samples.

[0080] Further, in step S33, performing sample pretreatment on the standard curve samples in the same way as the urine samples in step S2 to obtain standard curve samples with standard concentrations of 0, 19, 58, 195, 485 μg / L and an internal standard concentration of 200 μg / L includes:

[0081] Add 2 mL of blank matrix solution (such as pure water or blank urine) to a 15 mL centrifuge tube.

[0082] Add 100 μL of concentrated hydrochloric acid solution.

[0083] Place the centrifuge tube in a 100 °C water bath for 1.5 hours for acid hydrolysis treatment.

[0084] After acid hydrolysis, add the following solutions to the centrifuge tube in sequence: 25 μL of internal standard application solution (internal standard concentration of 200 μg / L), 1 mL of sodium acetate buffer solution, and 3 mL of 15% methanol dichloromethane solution.

[0085] After thoroughly mixing the solution, place it in a three-dimensional multi-point vortex shaker and shake it at a speed of 2500 rpm for 10 minutes.

[0086] After shaking, centrifuge it at a speed of 10000 rpm for 5 minutes using a medical centrifuge.

[0087] Remove the upper aqueous phase solution, and take 2.5 mL of the lower organic phase solution.

[0088] Place the separated organic phase solution in a dry bath nitrogen evaporator, and evaporate the solution to approximately 0.5 mL by nitrogen purging to obtain the standard curve samples.

[0089] In step S3, the standard curve is fitted with the relative concentration of each target substance to the internal standard (the concentration of each target substance divided by the concentration of the internal standard) as the abscissa and the relative peak area of each target substance to the internal standard (the peak area of each target substance divided by the peak area of the internal standard) as the ordinate. The linear regression equation of the standard curve is expressed by formula (1):

[0090] Y = bX + a (1)

[0091] Wherein, Y is the concentration of each target substance; b is the curve slope; X is the ratio of the peak area of each target substance to the peak area of the internal standard; a is the curve intercept.

[0092] Furthermore, the gas chromatography (GC) detection conditions and mass spectrometry (MS) detection conditions in steps S34 and S4 are the same; among them, the gas chromatography (GC) detection conditions are: for chromatographic column selection, a VF-WAX ms 60m×25mm×5μm chromatographic column; inlet temperature: 250°C; injection mode: split injection at 10:1, split flow rate is 10 mL / min; carrier gas (helium) flow rate: 15 mL / min, helium (purity ≥ 99.999%); injection volume: 1 μL:

[0093] Temperature programming, starting temperature 60°C, holding for 2 min, heating to 130°C at 20°C / min, heating to 165°C at 2.5°C / min, heating to 220°C at 25°C / min, and finally maintaining for 2 min;

[0094] The mass spectrometry (MS) detection conditions are: ion source, EI ion source; ion source temperature, 230°C; MS quadrupole temperature, 150°C; scan mode: selective ion mode (SIM) scan; solvent delay time: 8.20 min;

[0095] In step S4, the pretreated sample to be tested is injected into a gas chromatography / mass spectrometry instrument. Using the high separation ability and high sensitivity of this instrument, 8 metabolites of n-heptane in urine are detected, and the peak areas of each metabolite and the internal standard are accurately recorded; the gas chromatography part effectively separates the complex sample components through a suitable chromatographic column and temperature program; the mass spectrometry part realizes the qualitative and quantitative analysis of the target metabolites through the detection of characteristic ion fragments; the peak area, as the key data of the detection result, reflects the content level of the metabolites in the sample and provides a direct basis for subsequent concentration calculation; the retention times of 8 metabolites of n-heptane and the internal standard in urine on the VF-WAX ms chromatographic column are as Figure 2 shown;

[0096] In step S4, the full scan (SCAN) mode of a gas chromatography / mass spectrometry (GC / MS) instrument is adopted to collect ion data for each mass-to-charge ratio. After determining the retention time of each metabolite, the single ion detection scan (SIM) mode is used to determine the optimal quantitative and qualitative ion pairs. The results are shown in Table 2;

[0097]

[0098] Furthermore, step S4 also includes preparing 10 replicated urine matrix spike samples and calculating the standard deviation of the measurement results of the peak areas of each metabolite and internal standard in step S4; among them, the method detection limit is 3 times the standard deviation, and the method quantitation limit is 10 times the standard deviation; the results are shown in Table 3, which is the working curve linearity, detection limit, and quantitation limit table for 8 metabolites of n-heptane;

[0099]

[0100] As can be seen from Table 3, for the 8 metabolites of n-heptane in urine in step S4, within their respective linear ranges, the linear relationships are good, and the correlation coefficients (r) are all above 0.998. The detection limits are 5.7 - 17.3 μg / L, and the quantitation limits are 19.1 - 57.7 μg / L; these results indicate that this detection method has high sensitivity and accuracy and can be applied to the quantitative analysis of n-heptane metabolites in urine.

[0101] Furthermore, in step S5, based on the peak areas of each metabolite and internal standard, combined with the standard curve established in step S3, the concentrations of 8 metabolites of n-heptane in urine are calculated through a linear regression equation; the standard curve in step S3 provides a mathematical model for the quantitative relationship between concentration and peak area. By substituting the peak area of the sample to be measured into this model, the actual concentration of the metabolite in urine can be accurately obtained, thus realizing the precise quantitative analysis of n-heptane metabolites in urine and providing reliable data support for relevant research and practical applications.

[0102] Furthermore, in step S6, the accuracy and precision of the detection method for 8 metabolites of n-heptane are evaluated through a urine matrix spike experiment; including:

[0103] S61: Samples with 3 different mass concentrations, low, medium, and high, are prepared by the method of spiking the urine matrix, and each concentration level of the sample is replicated 6 times (n = 6); among them, the low concentration refers to a spiking concentration of 125 μg / L; the medium concentration refers to a spiking concentration of approximately 250 μg / L; the high concentration refers to a spiking concentration of approximately 500 μg / L;

[0104] S62: The above 3 samples with different concentrations are detected to evaluate the accuracy and within-batch precision of samples with different concentrations; specifically,

[0105] Accuracy (%) was calculated by comparing the actually measured concentration with the theoretical spiked concentration;

[0106] Within the same batch, samples at each concentration level were repeatedly detected 6 times, and the standard deviation (SD) and relative standard deviation (RSD) were calculated to obtain the within-batch precision;

[0107] S63: Over different time periods, the concentrations of 5 different spiked samples were detected in total (n = 6 for each detection), and the between-batch precision was evaluated: The between-batch precision was obtained by comparing the detection results of different batches and calculating the standard deviation (SD) and relative standard deviation (RSD);

[0108] The experimental results are shown in Table 4, which is a table of the accuracy and precision of 8 metabolites of n-heptane:

[0109]

[0110] Through the above urine matrix spiking experiment design and result analysis, it can be seen that the accuracies of low, medium, and high concentration samples of 8 metabolites of n-heptane are 89.6% - 107.5%, 91.8% - 107.0%, and 90.7% - 108.3% respectively; this means that the deviation between the actually measured concentration and the theoretical spiked concentration is between -10.4% and +8.3%, indicating that the method has high accuracy; the RSD value range of the within-batch precision is 1.7% - 14.6%; this shows that within the same batch, the results of repeated detections have high precision, but the precision of some metabolites may be slightly worse. Generally, an RSD value ≤ 15% is usually considered acceptable, so the method performs well in terms of within-batch precision; the RSD value range of the between-batch precision is 4.1% - 11.5%; this shows that over different time periods, the repeatability of the detection results is good, and the RSD values are all within the acceptable range (usually ≤ 15%), indicating that the method has good stability and reproducibility.

[0111] These results indicate that the detection method has high accuracy and good precision and is suitable for the quantitative analysis of n-heptane metabolites in urine.

[0112] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention and is not used to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry, characterized in that: The steps include: S1: Solution preparation: prepare 1 mol / L sodium acetate buffer, 15% methanol-dichloromethane solution, standard application solutions including 8 metabolites of n-heptane, and internal standard application solution respectively; S2: performing sample pretreatment on the urine sample to obtain a urine sample to be tested; S3: Prepare standard curve samples, and establish a linear regression equation of the standard curve according to the concentration of the standard curve samples and the peak areas of each metabolite and internal standard in the standard curve samples; S4: using gas chromatography / mass spectrometry (GC-MS) to detect 8 metabolites of n-heptane in the sample to be tested, and accurately recording the peak area of ​​each metabolite and internal standard; S5: Calculate the concentrations of the eight metabolites of n-heptane in urine based on the peak areas of the metabolites and the internal standard in combination with the linear regression equation of the standard curve established in step S3; S6: The accuracy and precision of the detection method for eight metabolites of n-heptane were evaluated by urine matrix spike experiments; The eight metabolites of n-heptane include 4-heptanone, 3-heptanone, 2-heptanone, 4-heptanol, 3-heptanol, 2-heptanol, 1-heptanol and 2,5-heptanedione.

2. The method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to claim 1, characterized in that: The preparation of the standard application solution in step S1 includes: taking 2 μL of each standard solution of 8 metabolites in n-heptane, adding it to 1 mL of methanol solution to prepare a standard stock solution; taking another 25 μL from the stock solution and mixing it with 1 mL of methanol solution to prepare a standard application solution, which is stored at -20°C for future use.

3. The method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to claim 1, characterized in that: In step S1, the isotope labeled substance 1-heptanol-d 15 As a quantitative internal standard; The preparation of the internal standard application solution comprises: 15 Take 2 μL of the internal standard solution and add it to 1 mL of methanol solution to prepare 1-heptanol-d 15 Internal standard stock solution, then from 1-heptanol-d 15 Take 10 μL of the internal standard stock solution and mix it with 1 mL of methanol solution to prepare 1-heptanol-d 15 The internal standard application solution should be stored at -20℃ for future use.

4. A method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to any one of claims 1 to 3, characterized in that: In step S2, the urine sample is pre-treated to obtain a sample to be tested; include: S21: thawing the urine sample; S22: Acid hydrolysis: take 2 mL of thawed urine sample into a 15 mL centrifuge tube, add 100 μL of concentrated hydrochloric acid solution, and then place the centrifuge tube in a 100°C water bath for 1.5 hours for acid hydrolysis; S23: After the acid hydrolysis is completed, 25 μL of internal standard application solution, 1 mL of sodium acetate buffer and 3 mL of 15% methanol dichloromethane solution are added to the centrifuge tube of S32 in sequence; S24: After the solution is fully mixed, place it in a three-dimensional multi-point vortex oscillator and shake at 2500 rpm / 10 min; S25: Centrifuge at 10000 rpm / 5 min in a medical centrifuge to remove the upper aqueous phase solution and take 2.5 mL of the lower organic phase solution; S26: Use a dry bath nitrogen blower to blow nitrogen into the organic phase solution to about 0.5 mL to obtain a sample to be tested.

5. A method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to any one of claims 1 to 3, characterized in that: The step S3 is to prepare a standard curve sample, and establish a linear regression equation of the standard curve according to the concentration of the standard curve sample, the peak area of ​​each metabolite and the internal standard in the standard curve sample; including: S31: Take 0, 1, 3, 10, and 25 μL of the prepared standard application solution respectively; S32: Add 25 μL of internal standard application solution to each standard application solution; S33: performing sample pretreatment in the same manner as the urine sample in step S2 to obtain standard curve samples with standard concentrations of 0, 19, 58, 195, 485 μg / L and an internal standard concentration of 200 μg / L; S34: Detecting the standard curve samples using gas chromatography / mass spectrometry (GC-MS), and recording the peak areas of each metabolite and internal standard in the standard curve samples; S35: Establish a linear regression equation of the standard curve according to the concentration of the standard curve samples and the peak areas of each metabolite and the internal standard in the standard curve samples.

6. The method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to claim 4, characterized in that: The standard curve in step S3 is fitted with the relative concentration of each target substance and the internal standard as the abscissa and the relative peak area of ​​each target substance and the internal standard as the ordinate. The linear regression equation of the standard curve is expressed by formula (1): Y=bX+a (1) Wherein, Y is the concentration of each target substance; b is the slope of the curve; X is the ratio of the peak area of ​​each target substance to the peak area of ​​the internal standard; a is the intercept of the curve.

7. The method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to claim 1, characterized in that: The gas phase (GC) detection conditions in step S4 are: chromatographic column selection, VF-WAX ms 60m×25mm×5μm chromatographic column; injection port temperature: 250°C; injection mode: 10:1 split injection, split flow rate of 10mL / min; carrier gas (helium) flow rate: 15mL / min, helium (purity ≥99.999%); injection volume: 1μL: The heating program was as follows: the starting temperature was 60°C, maintained for 2 min, increased to 130°C at 20°C / min, increased to 165°C at 2.5°C / min, increased to 220°C at 25°C / min, and finally maintained for 2 min.

8. The method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to claim 1, characterized in that: The mass spectrometry (MS) detection conditions in step S4 are: ion source, EI ion source; ion source temperature, 230° C.; MS quadrupole temperature, 150° C.; scanning mode: selective ion mode (SIM) scanning; solvent delay time: 8.20 min.

9. The method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to claim 1, characterized in that: In step S4, the full scan (SCAN) mode of the gas chromatograph / mass spectrometer is used to collect ion data of each mass-to-charge ratio, and after determining the retention time of each metabolite, the single ion detection scan (SIM) mode is used to determine the optimal quantitative and qualitative ion pairs.

10. The method for detecting eight metabolites of n-heptane in urine by gas chromatography / mass spectrometry according to claim 1, characterized in that: Step S4 also includes repeatedly preparing 10 urine matrix spiked samples and calculating the standard deviation of the peak area determination results of each metabolite and internal standard in step S4; wherein the method detection limit is 3 times the standard deviation and the method quantitative lower limit is 10 times the standard deviation.

Images