Detection method for formic acid, methanol and ethanol based on automatic headspace gas chromatography-mass spectrometry technology

Abstract

The invention discloses a method for detecting formic acid, methanol and ethanol based on an automatic headspace gas chromatography-mass spectrometry technology. The detection method comprises the following steps: S1, preparing a mixed standard working solution of formic acid, methanol and ethanol; s2, adding an esterification reagent into the mixed standard working solution or the to-be-detected sample, and sealing the mixed standard working solution or the to-be-detected sample in a headspace bottle; s3, the headspace bottle is placed in an automatic headspace sampler, heating and heat preservation are conducted, formic acid and isopropanol are subjected to an esterification reaction to generate isopropyl formate, and meanwhile the isopropyl formate, methyl alcohol and ethyl alcohol are volatilized to the top space of the headspace bottle; s4, the headspace sample injector automatically extracts gas in the headspace and injects the gas into the gas chromatography system; and S5, carrying out separation through a chromatographic column, carrying out heating, carrying out detection by using a mass spectrometry detector, and carrying out qualitative and quantitative analysis on formic acid, methanol and ethanol in the to-be-detected sample. The detection method provided by the invention realizes rapid, automatic, high-sensitivity and high-specificity synchronous detection of formic acid, methanol and ethanol in a blood sample.

Description

Technical Field

[0001] This invention relates to the field of detection technology for poisons and their metabolites in biological samples, and in particular to a method for detecting formic acid, methanol and ethanol based on automated headspace gas chromatography-mass spectrometry. Background Technology

[0002] Methanol is a widely used industrial solvent, commonly found in chemical production, transportation, and the pharmaceutical and cosmetic industries that use methanol as a raw material. In recent years, occupational and everyday acute methanol poisoning incidents have occurred frequently. Foodborne methanol poisoning is mostly caused by accidental ingestion of industrial alcohol or counterfeit alcohol containing methanol, resulting in high mortality and disability rates. Formic acid is the main toxic metabolite of methanol in the human body; optic nerve damage and metabolic acidosis caused by methanol poisoning are mainly related to formic acid accumulation. Blood methanol and formic acid concentrations are not only key indicators for diagnosing methanol poisoning but also important bases for determining whether hemodialysis and other clinical treatments should be initiated.

[0003] Clinically, ethanol is often used to treat methanol poisoning because it has a much higher affinity for alcohol dehydrogenases than methanol, competitively inhibiting the metabolism of methanol into formic acid. Therefore, close monitoring of blood ethanol concentration is necessary during treatment. In actual poisoning cases, ethanol, methanol, and their metabolite formic acid are often present in the patient's blood simultaneously. Rapid and accurate detection of these three substances is crucial for the rapid differential diagnosis of the cause of poisoning and for establishing and adjusting treatment plans.

[0004] Currently, although there are reports of headspace gas chromatography being used to detect such substances, the following problems exist: (1) the use of a flame ionization detector (FID) relies solely on retention time for qualitative analysis, resulting in poor specificity and susceptibility to interference from complex impurities in biological samples, which may lead to false positives; (2) the method has a high detection limit, which cannot meet the testing needs of patients with low-concentration exposure or delayed medical treatment; (3) the pretreatment steps are cumbersome, requiring the sequential addition of multiple reagents, which affects detection efficiency and reproducibility. Therefore, there is an urgent need for a highly automated, simple pretreatment, highly sensitive, and interference-resistant method that can simultaneously perform accurate qualitative and quantitative detection of formic acid, methanol, and ethanol in blood. Summary of the Invention

[0005] This invention solves the problems of cumbersome pretreatment, poor qualitative ability, low sensitivity, and inability to effectively eliminate matrix interference in existing technologies. It provides a method for the detection of formic acid, methanol, and ethanol based on automated headspace gas chromatography-mass spectrometry, which enables rapid, automated, highly sensitive, and highly specific simultaneous detection of formic acid, methanol, and ethanol in blood samples.

[0006] The purpose of this invention is to provide a method for detecting formic acid, methanol, and ethanol based on automated headspace gas chromatography-mass spectrometry, comprising the following steps:

[0007] S1: Prepare a mixed standard working solution of formic acid, methanol and ethanol;

[0008] S2: Add sulfuric acid-isopropanol esterification reagent to the mixed standard working solution or the sample to be tested, and seal it in a headspace vial;

[0009] S3: Place the headspace vial in the automatic headspace sampler, heat and keep it warm so that formic acid and isopropanol undergo an esterification reaction to produce volatile isopropyl formate, which evaporates into the top space of the headspace vial along with methanol and ethanol.

[0010] S4: The headspace sampler automatically extracts gas from the headspace and injects it into the gas chromatography system;

[0011] S5: Separation is performed using a medium-polarity capillary column, with programmed temperature rise and detection using a mass spectrometer detector to perform qualitative and quantitative analysis of formic acid, methanol, and ethanol in the sample.

[0012] The detection method proposed in this invention uses the following detection system (such as...) Figure 1 The detection system (as shown) includes a sample pretreatment device, a gas chromatography separation device, a mass spectrometry data acquisition device, and a qualitative and quantitative analysis module.

[0013] The sample pretreatment device (automatic headspace autosampler) includes a sample loading tray, a heating and insulation furnace, an automatic sampling needle, and an automatic sample transfer line; it has a built-in sample vial heating unit with a temperature control range of 40-90℃; a built-in gas pressure control unit; and a built-in automatic quantitative sampling and injection unit, equipped with a heating and insulation transfer line.

[0014] The gas chromatography separation device includes a sample vaporization chamber and a separation column system with a gas pressure controller and a column oven; it is connected to a headspace sampler through an injection port equipped with a gas pressure control unit; the chromatographic column is a medium polarity capillary column (such as Rxi-624Sil); and the column oven is equipped with a programmed temperature control system.

[0015] The mass spectrometry data acquisition device includes a vacuum pump, an automatic tuning fluid injection unit, an electron impact ion source, a quadrupole mass analyzer, and an electron multiplier; it is connected to the gas chromatography separation device via a heated and insulated transmission line, and is equipped with an external vacuum pump, an internal automatic quantitative tuning fluid injection unit, an internal electron impact ion source, an internal quadrupole mass analyzer, and an internal electron multiplier to detect ion signals;

[0016] The qualitative and quantitative analysis modules include a qualitative analysis module that integrates a mass spectrometry database (such as NIST) spectral library search program, and a quantitative analysis module that integrates standard curve / working curve generation algorithms such as internal standard method, external standard method, and standard addition method.

[0017] Preferably, the mixed standard working solution described in step S1 is prepared by the following steps:

[0018] S11: Pipette 350 μL, 350 μL, and 100 μL of chromatographic grade standard solutions of methanol, ethanol, and formic acid into 10.0 mL volumetric flasks, respectively, and dilute to the mark with water to obtain a mixed standard stock solution with methanol, ethanol, and formic acid concentrations of 27699 μg / mL, 27626 μg / mL, and 12200 μg / mL, respectively.

[0019] S12: Take 0.2 mL of the mixed standard stock solution prepared in step S11 into a 10 mL volumetric flask, and dilute to the mark with blank serum to obtain a mixed standard working solution with methanol, ethanol and formic acid concentrations of 553.98 μg / mL, 552.51 μg / mL and 244.0 μg / mL, respectively.

[0020] S13: Take seven 20 mL headspace vials and add 0.0 µL, 50 µL, 150 µL, 250 µL, 500 µL, 750 µL, and 1000 µL of the methanol, ethanol, and formic acid mixed standard working solution prepared in step S12, respectively. Dilute to 1.0 mL with blank serum and shake well to prepare mixed standard working solutions of formic acid, methanol, and ethanol. The concentrations of methanol are 0.00, 27.70, 83.10, 138.50, 276.99, 415.49, and 553.98 μg / mL, respectively; the concentrations of ethanol are 0.00, 27.63, 82.88, 138.13, 276.26, 414.38, and 552.51 μg / mL, respectively. μg / mL; the concentrations of formic acid were 0.00, 12.20, 36.60, 61.00, 122.00, 183.00, and 244.00 μg / mL, respectively.

[0021] Preferably, the volume ratio of the mixed standard working solution or the sample to be tested to the sulfuric acid-isopropanol esterification reagent in step S1 is 1:1.

[0022] Preferably, the sample to be tested in step S2 is an in vitro biological sample, and the sulfuric acid-isopropanol esterification reagent is a mixed aqueous solution of concentrated sulfuric acid and isopropanol, wherein the volume fraction of concentrated sulfuric acid in the mixed aqueous solution is 5%-20% and the volume fraction of isopropanol is 10%-40%.

[0023] The esterification reagent is prepared by the following steps: Isopropanol (chromatographic grade) is placed in a volumetric flask and diluted to the mark with pure water to obtain an isopropanol aqueous solution with a volume fraction of 10%-40%; concentrated sulfuric acid is slowly added to the isopropanol aqueous solution while the mixture is slowly shaken to dissipate heat. After standing and cooling, the esterification reagent is obtained.

[0024] Further preferred, in step S2, the volume fraction of concentrated sulfuric acid in the mixed aqueous solution is 10%, and the volume fraction of isopropanol is 20%.

[0025] The detection target of this invention is an ex vivo biological sample, such as blood, urine, or tissue fluid. Taking blood samples as an example, the pretreatment steps for blood samples are as follows: collect approximately 5 mL of whole blood using a clean, dry tube and store it in a refrigerator at -4°C or below for 7 days for analysis. After removing the blood sample from the refrigerator and allowing it to return to room temperature, centrifuge it at 5000 r / min for 5 min, and then aspirate the supernatant serum to obtain the blood sample used for detection.

[0026] Preferably, the heating and heat preservation conditions in step S3 are: heating and heat preservation at 60℃-90℃ for 5-20 minutes.

[0027] Further preferred, the heating and heat preservation conditions in step S3 are: heating and heat preservation at 80°C for 15 minutes.

[0028] Preferably, the operating parameters of the automatic headspace sampler in step S3 are: furnace temperature 80 ℃, holding time 15 min, sampling needle temperature 105 ℃, transfer line temperature 120 ℃, pressurization 1.0 min, injection 0.2 mL, needle withdrawal 0.2 min, and column pressure 15.0 psi.

[0029] Preferably, the gas chromatography conditions described in step S4 or S5 are as follows: a RESTEK Rxi-624Sil medium polarity column (60 m × 250 μm × 1.4 μm) with an injection port temperature of 180°C; an initial column temperature of 45°C for 7 minutes, followed by a rise to 140°C at a rate of 20°C / min and a holding time of 1 minute; high-purity helium as the carrier gas with a flow rate of 0.8 mL / min; and a split ratio of 10:1.

[0030] Preferably, the mass spectrometry conditions described in step S5 are: an Agilent 5977C MSD detector, equipped with an electron impact ion source, with an electron energy of 69.9 eV, an ion source temperature of 230 °C, a quadrupole temperature of 150 °C, an auxiliary interface temperature of 280 °C, a mass scan range of 10–350 amu (m / z), simultaneous acquisition in Scan and SIM modes, and a solvent delay of 6.0 min.

[0031] The detection was performed using a mass spectrometer (MSD); simultaneous acquisition was conducted in full scan mode and selected ion monitoring mode (SIM) to improve specificity and sensitivity; and formic acid (calculated as isopropyl formate), methanol and ethanol were quantified using an external standard working curve method.

[0032] The SIM mode group monitoring time periods were set as follows: Group 1: methanol: 6.0-7.5 min, fragment ion m / z: 31.00, residence time 100 ms, low resolution; Group 2: ethanol: 7.5-9.0 min, fragment ion m / z: 31.00, residence time 100 ms, low resolution; Group 3: formic acid: 9.0-12.75 min, fragment ion m / z: 73.00, residence time 100 ms, low resolution.

[0033] Preferably, the specific steps of the quantitative analysis in step S5 are as follows: Mass spectra of formic acid, methanol, and ethanol at gradient concentrations are obtained by analyzing the mixed standard working solution using gas chromatography-mass spectrometry (GC-MS). The characteristic fragment ion signal intensities at each gradient concentration are obtained from the mass spectra. Standard curves for formic acid, methanol, and ethanol are plotted with the gradient concentrations of the standard working solutions as the abscissa and the peak areas of the chromatographic peaks extracted by specific selected ions as the ordinate, and the standard curve equations are fitted. The sample to be tested is detected by GC-MS to obtain a sample mass spectrum. The characteristic fragment ion signal intensities of formic acid, methanol, and ethanol in the sample mass spectrum are obtained. The concentrations of formic acid, methanol, and ethanol are obtained through the standard curve fitting equations for formic acid, methanol, and ethanol, thereby achieving qualitative and quantitative analysis of formic acid, methanol, and ethanol in the sample to be tested.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. Simultaneous detection and high specificity: By optimizing headspace and chromatographic conditions and employing mass spectrometry detectors (Scan and SIM modes), this invention can simultaneously and accurately characterize and quantify formic acid, methanol, and ethanol in blood within a single analysis cycle (approximately 30 minutes), effectively avoiding interference from common coexisting substances in complex biological matrices (such as acetone, acetic acid, acetaldehyde, diethyl ether, ethyl formate, ethyl acetate, propanol, etc.).

[0036] 2. High sensitivity and wide linear range: The method has low detection limits (methanol: 5.83 μg / mL, ethanol: 3.14 μg / mL, formic acid: 3.90 μg / mL) and a wide linear range (methanol: 0-550 μg / mL, ethanol: 0-550 μg / mL, formic acid: 0-244 μg / mL), which can meet the needs of screening for low-concentration poisoning and monitoring for high-concentration poisoning.

[0037] 3. Good precision and accuracy: High accuracy (methanol spiked recovery: 100.95%~108.17%; ethanol spiked recovery: 97.50%~108.06%; formic acid spiked recovery: 92.29%~96.61%), high intra-batch precision (methanol: 2.03%~3.05%; ethanol: 1.87%~3.71%; formic acid: 4.82%~5.60%), and high inter-batch precision (methanol: 2.035%~5.19%; ethanol: 2.63%~4.72%; formic acid: 4.82%~9.14%).

[0038] 4. Automated and simplified sample preparation: Pre-preparing the esterification reagent simplifies the sample preparation steps, automates sample processing, and improves analytical efficiency and reproducibility. The entire method is rapid and can meet the urgent detection speed requirements of acute poisoning events.

[0039] 5. Significant application value: This method can provide timely and reliable laboratory evidence for the rapid diagnosis of methanol poisoning, the monitoring of the effect of ethanol treatment, and the judgment of hemodialysis indications, and has important clinical application value. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the detection system structure of the present invention.

[0041] Figure 2 These are interference test chromatograms (Scan and SIM modes) for methanol, ethanol, and formic acid (in the form of isopropyl formate); where: Figure 2 A is the total ion chromatogram in Scan mode of the interference test. 1 is acetaldehyde, 2 is methanol, 3 is ethanol, 4 is diethyl ether, 5 is acetone, 6 isopropanol, 7 is ethyl formate, 8 isopropyl formate, 9 isopropanol, and 10 is ethyl acetate. Figure 2 B is the SIM ion chromatogram from the interference experiment, 1 is methanol, 2 is ethanol, 3 is diethyl ether, 4 isopropyl formate, and 5 is ethyl acetate.

[0042] Figure 3 These are chromatograms of different ratios of sulfuric acid and isopropanol, as well as esterification reaction temperatures and times. Figure (a) shows the chromatograms with esterifying agents of 10% sulfuric acid-10% isopropanol, 10% sulfuric acid-20% isopropanol, and 10% sulfuric acid-30% isopropanol; Figure (b) shows the chromatograms with esterifying agents of 5% sulfuric acid-20% isopropanol, 10% sulfuric acid-20% isopropanol, 15% sulfuric acid-20% isopropanol, and 20% sulfuric acid-20% isopropanol; Figure (c) shows the chromatograms in the headspace sampler at esterification temperatures of 50℃, 60℃, 70℃, 80℃, and 90℃; Figure (d) shows the chromatograms in the headspace sampler at esterification times of 5 min, 10 min, 15 min, 20 min, and 30 min. Detailed Implementation

[0043] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0044] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental materials and reagents used herein are commercially available products conventionally available in this technical field.

[0045] The esterification reagent in the following examples or comparative examples is prepared by the following steps: Isopropanol (chromatographic grade) is placed in a volumetric flask and diluted to the mark with pure water to obtain an isopropanol aqueous solution with a volume fraction of 10%-40%; concentrated sulfuric acid is slowly added to the isopropanol aqueous solution while the mixture is slowly shaken to dissipate heat. After standing and cooling, the esterification reagent is obtained.

[0046] The sample pretreatment device was a PerkinElmer Turbomatrix 40 automatic headspace sampler with a 40-position sample tray.

[0047] Example 1

[0048] like Figure 1 As shown, a method for detecting formic acid, methanol, and ethanol based on automated headspace gas chromatography-mass spectrometry includes the following steps:

[0049] S1: Prepare a mixed standard working solution of formic acid, methanol and ethanol;

[0050] S11: Pipette 350 μL, 350 μL, and 100 μL of chromatographic grade standard solutions of methanol, ethanol, and formic acid into 10.0 mL volumetric flasks, respectively, and dilute to the mark with water to obtain a mixed standard stock solution with methanol, ethanol, and formic acid concentrations of 27699 μg / mL, 27626 μg / mL, and 12200 μg / mL, respectively.

[0051] S12: Take 0.2 mL of the mixed standard stock solution prepared in step S11 into a 10 mL volumetric flask, and dilute to the mark with blank serum to obtain a mixed standard working solution with methanol, ethanol and formic acid concentrations of 553.98 μg / mL, 552.51 μg / mL and 244.0 μg / mL, respectively.

[0052] S13: Take seven 20 mL headspace vials and add 0.0 µL, 50 µL, 150 µL, 250 µL, 500 µL, 750 µL, and 1000 µL of the methanol, ethanol, and formic acid mixed standard working solution prepared in step S12, respectively. Dilute to 1.0 mL with blank serum and shake well to prepare mixed standard working solutions of formic acid, methanol, and ethanol. The concentrations of methanol are 0.00, 27.70, 83.10, 138.50, 276.99, 415.49, and 553.98 μg / mL, respectively; the concentrations of ethanol are 0.00, 27.63, 82.88, 138.13, 276.26, 414.38, and 552.51 μg / mL, respectively. μg / mL; formic acid concentrations were 0.00, 12.20, 36.60, 61.00, 122.00, 183.00, and 244.00 μg / mL, respectively.

[0053] S2: Pipette 1.0 mL of mixed standard working solution or test sample into a 20 mL headspace vial, add 1.0 mL of 10% sulfuric acid-20% isopropanol aqueous solution esterification reagent, and seal the vial; the test sample is obtained by the following steps: collect about 5 mL of whole blood using a clean, dry tube and store it in a refrigerator at -4℃ or below. Analyze the sample within 7 days. Remove the blood sample from the refrigerator and allow it to return to room temperature. After centrifuging at 5000 r / min for 5 min, aspirate the upper serum layer, which is the test sample.

[0054] S3: Place the headspace vial in the automated headspace sampler and heat it at 80°C for 15 minutes to allow for the esterification reaction of formic acid and isopropanol to produce volatile isopropyl formate. Simultaneously, the formic acid, methanol, and ethanol evaporate into the top space of the headspace vial. The operating parameters of the automated headspace sampler are: oven temperature 80°C, sampling needle 105°C, transfer line 120°C; timing settings: pressurization 1.0 min, needle withdrawal 0.2 min, holding temperature 15 min, GC analysis cycle 15 min; injection volume set to 0.2 mL; column pressure set to 15.0 psi.

[0055] S4: The headspace sampler automatically extracts 0.2 mL of gas from the headspace and injects it into the gas chromatography system;

[0056] S5: Separation is performed using a medium-polarity capillary column, with programmed temperature rise and detection using a mass spectrometer detector to perform qualitative and quantitative analysis of formic acid, methanol, and ethanol in the sample.

[0057] The gas chromatography separation device was an Agilent 7890A gas chromatograph equipped with a RESTEK Rxi-624Sil medium polarity column (60 m × 250 μm × 1.4 μm), with the injection port temperature set at 180℃; the column oven temperature was set to an initial temperature of 45℃, held for 7 minutes, then increased to 140℃ at a rate of 20℃ / min, and held for 1 minute; the carrier gas was high-purity helium, with a flow rate set at 0.8 mL / min; and the split ratio was 10:1.

[0058] The mass spectrometry data acquisition device was an Agilent 5977C MSD detector with an electron impact ion source. The electron energy was set to 69.9 eV, the ion source temperature to 230 ℃, the quadrupole temperature to 150 ℃, and the auxiliary interface temperature to 280 ℃. The mass scan range was 10–350 amu (m / z). The acquisition type was simultaneous Scan and SIM modes. The solvent delay was 6.0 min. The SIM mode group monitoring time periods were set as follows: Group 1: methanol: 6.0–7.5 min, fragment ion m / z: 31.00, residence time 100 ms, low resolution; Group 2: ethanol: 7.5–9.0 min, fragment ion m / z: 31.00, residence time 100 ms, low resolution; Group 3: formic acid: 9.0–12.75 min, fragment ion m / z: 73.00, residence time 100 ms, low resolution.

[0059] Based on the external standard working curve method, standard curves for formic acid, methanol, and ethanol were plotted with the gradient concentrations of the standard working solutions as the x-axis and the peak areas of the chromatographic peaks extracted by specific selected ions as the y-axis. The standard curve equations were then fitted to generate quantitative standard curves.

[0060] Methanol: The linear range of the method was 0.00~553.98 μg / mL, the regression equation was y=514.71x+1906.91, and the correlation coefficient was r=0.9992.

[0061] Ethanol: The linear range of the method was 0.00~552.51 μg / mL, the regression equation was y=954.69x+8511.08, and the correlation coefficient was r=0.9993.

[0062] Formic acid: The linear range of the method was 0.00~244.0 μg / mL, the regression equation was y=769.81x+7969.08, and the correlation coefficient was r=0.9994.

[0063] Comparative Example 1

[0064] Same as Example 1, except that the detector is FID. The same standard curve series and spiked recovered samples were analyzed by this method and automated headspace gas chromatography (FID detector) (the pretreatment methods were the same), and the comparison results are shown in Table 1 below.

[0065] Table 1

[0066] As shown in Table 1, the detection limit and correlation of the method proposed in this invention are significantly better than those of gas chromatography, and the recovery rate of methanol is better than that of gas chromatography.

[0067] Example 2

[0068] The test samples were tested using the method of Example 1. Eighteen 20 mL headspace vials were divided into three groups of six. Low-background blood samples were added to each group, followed by the addition of a mixed standard stock solution of methanol, ethanol, and formic acid to prepare low, medium, and high-concentration blood samples. The samples were then tested on the same day according to the method of Example 1. The results are shown in Table 2.

[0069] Table 2

[0070] Table 2 shows the recoveries of the spiked samples as follows: methanol: 100.95%–108.17%; ethanol: 97.50%–108.06%; formic acid: 92.29%–96.61%. The intra-batch precisions were as follows: methanol: 2.03%–3.05%; ethanol: 1.87%–3.71%; formic acid: 4.82%–5.60%. Twelve parallel samples were prepared and tested over three non-consecutive days, and the inter-batch precisions were as follows: methanol: 2.035%–5.19%; ethanol: 2.63%–4.72%; formic acid: 4.82%–9.14%. The method proposed in this invention exhibits good precision and accuracy.

[0071] Example 3

[0072] Similar to Example 1, except for the ratio of sulfuric acid to isopropanol in the esterification reagent. Interactive experiments were conducted with different sulfuric acid concentrations (5%, 10%, 15%, 20%) and corresponding isopropanol concentrations (10%, 20%, 30%, 40%). Based on the peak shape and response of each chromatographic peak, the optimal esterification reagent concentration was selected. Figure 3As shown, sulfuric acid concentration has no effect on the peak shape or response of methanol and ethanol, but it does affect the response of formic acid. When the isopropanol concentration is 10%, the formic acid response tends to stabilize when the sulfuric acid concentration reaches 15%, and when the isopropanol concentration is 20%, the response tends to stabilize when the sulfuric acid concentration reaches 10%. Isopropanol concentration has a significant impact on methanol, ethanol, and formic acid. The higher the isopropanol concentration, the flatter the peak shape of methanol and ethanol. When the isopropanol concentration reaches 30%, the peak shape becomes flat and cracked, affecting quantification. When the isopropanol concentration reaches 40%, the acquired signal is abnormal. The higher the isopropanol concentration, the larger the response value of formic acid. Considering all factors, a 10% sulfuric acid-20% isopropanol solution was selected as the esterification agent.

[0073] Example 4

[0074] Similar to Example 1, the difference lies in the different esterification reaction temperatures and times. Interactive experiments were conducted with different headspace temperatures (60℃, 70℃, 80℃, 90℃) and corresponding holding times (5 min, 10 min, 15 min, 20 min). The optimal esterification reaction temperature and time were selected based on the peak shape and response of each chromatographic peak. The results showed that higher temperatures resulted in a greater response, but after 90℃, the pressure inside the sample vial exceeded the needle pressure, leading to pre-injection and causing double peaks. Holding time affected the response of all three substances; the response increased with increasing temperature, and the reaction tended to stabilize after 15 min. Considering all factors, 80℃ was chosen as the holding temperature and 15 min as the holding time.

[0075] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for detecting formic acid, methanol, and ethanol based on automated headspace gas chromatography-mass spectrometry, characterized in that, Includes the following steps: S1: Prepare a mixed standard working solution of formic acid, methanol and ethanol; S2: Add sulfuric acid-isopropanol esterification reagent to the mixed standard working solution or the sample to be tested, and seal it in a headspace vial; S3: Place the headspace vial in the automatic headspace sampler, heat and keep it warm so that formic acid and isopropanol undergo an esterification reaction to produce volatile isopropyl formate, which evaporates into the top space of the headspace vial along with methanol and ethanol. S4: The headspace sampler automatically extracts gas from the headspace and injects it into the gas chromatography system; S5: Separation is performed using a medium-polarity capillary column, with programmed temperature rise and detection using a mass spectrometer detector to perform qualitative and quantitative analysis of formic acid, methanol, and ethanol in the sample.

2. The detection method according to claim 1, characterized in that, The mixed standard working solution mentioned in step S1 is prepared by the following steps: S11: Pipette 350 μL, 350 μL, and 100 μL of chromatographic grade standard solutions of methanol, ethanol, and formic acid into 10.0 mL volumetric flasks, respectively, and dilute to the mark with water to obtain a mixed standard stock solution with methanol, ethanol, and formic acid concentrations of 27699 μg / mL, 27626 μg / mL, and 12200 μg / mL, respectively. S12: Take 0.2 mL of the mixed standard stock solution prepared in step S11 into a 10 mL volumetric flask, and dilute to the mark with blank serum to obtain a mixed standard working solution with methanol, ethanol and formic acid concentrations of 553.98 μg / mL, 552.51 μg / mL and 244.0 μg / mL, respectively. S13: Take seven 20 mL headspace vials and add 0.0 µL, 50 µL, 150 µL, 250 µL, 500 µL, 750 µL, and 1000 µL of the methanol, ethanol, and formic acid mixed standard working solution prepared in step S12, respectively. Dilute to 1.0 mL with blank serum and shake well to prepare mixed standard working solutions of formic acid, methanol, and ethanol. The concentrations of methanol are 0.00, 27.70, 83.10, 138.50, 276.99, 415.49, and 553.98 μg / mL, respectively; the concentrations of ethanol are 0.00, 27.63, 82.88, 138.13, 276.26, 414.38, and 552.51 μg / mL, respectively. μg / mL; the concentrations of formic acid were 0.00, 12.20, 36.60, 61.00, 122.00, 183.00, and 244.00 μg / mL, respectively.

3. The detection method according to claim 1 or 2, characterized in that, The volume ratio of the mixed standard working solution or the sample to be tested to the sulfuric acid-isopropanol esterification reagent mentioned in step S1 is 1:1, and the total volume of liquid in the headspace vial does not exceed 1 / 3 of the total capacity of the headspace vial.

4. The detection method according to claim 1 or 2, characterized in that, The sample to be tested in step S2 is an in vitro biological sample. The sulfuric acid-isopropanol esterification reagent is a mixed aqueous solution of concentrated sulfuric acid and isopropanol, in which the volume fraction of concentrated sulfuric acid is 5%-20% and the volume fraction of isopropanol is 10%-40%.

5. The detection method according to claim 4, characterized in that, In step S2, the volume fraction of concentrated sulfuric acid in the mixed aqueous solution is 10%, and the volume fraction of isopropanol is 20%.

6. The detection method according to claim 1 or 2, characterized in that, The heating and heat preservation conditions described in step S3 are: heating and heat preservation at 60℃-90℃ for 5-20 minutes.

7. The detection method according to claim 1 or 2, characterized in that, The operating parameters of the automatic headspace sampler described in step S3 are as follows: furnace temperature 80 ℃, holding time 15 min, sampling needle temperature 105 ℃, transfer line temperature 120 ℃, pressurization 1.0 min, injection 0.2 mL, needle withdrawal 0.2 min, and column pressure 15.0 psi.

8. The detection method according to claim 1 or 2, characterized in that, The gas chromatography conditions described in step S4 or S5 are as follows: RESTEK Rxi-624Sil medium polarity column, 60 m × 250 μm × 1.4 μm, injection port temperature set at 180℃; column oven temperature set at an initial temperature of 45℃, held for 7 minutes, then increased to 140℃ at a rate of 20℃ / min, held for 1 minute; high-purity helium as carrier gas, carrier gas flow rate set at 0.8 mL / min; The split ratio is 10:

1.

9. The detection method according to claim 1 or 2, characterized in that, The mass spectrometry conditions described in step S5 are as follows: Agilent 5977C MSD detector, equipped with an electron impact ion source, with an electron energy of 69.9 eV, an ion source temperature of 230 ℃, a quadrupole temperature of 150 ℃, an auxiliary interface temperature of 280 ℃, a mass scan range of 10~350 amu (m / z), simultaneous acquisition in Scan and SIM modes, and a solvent delay of 6.0 min.

10. The detection method according to claim 1 or 2, characterized in that, The specific steps of the quantitative analysis described in step S5 are as follows: Mass spectra of formic acid, methanol, and ethanol at gradient concentrations are obtained by analyzing the mixed standard working solution using gas chromatography-mass spectrometry (GC-MS). The characteristic fragment ion signal intensities at each gradient concentration are obtained from the mass spectra. Standard curves for formic acid, methanol, and ethanol are plotted with the gradient concentrations of the standard working solutions as the abscissa and the peak areas of the chromatographic peaks extracted by specific selected ions as the ordinate, and the standard curve equations are fitted. The sample to be tested is then detected using GC-MS to obtain a sample mass spectrum. The characteristic fragment ion signal intensities of formic acid, methanol, and ethanol in the sample mass spectrum are obtained. The concentrations of formic acid, methanol, and ethanol are obtained through the standard curve fitting equations for formic acid, methanol, and ethanol, thereby achieving qualitative and quantitative analysis of formic acid, methanol, and ethanol in the sample to be tested.

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