Method for detecting trimethylamine in excrement sample

Abstract

The invention relates to the technical field of metabolite detection, and particularly discloses a method for detecting trimethylamine in an excrement sample. By improving a sample treatment method and optimizing headspace-gas chromatography conditions, the invention provides the method for measuring trimethylamine in the excrement sample, which is simple and convenient to operate, high in accuracy, excellent in precision and strong in applicability. According to the detection method, an acidic medium-chain alcohol extract liquid compounded by n-hexanol and an inorganic strong acid dilute solution is selected to treat an excrement sample. The acidic medium-chain alcohol extract liquid not only can efficiently extract, enrich and fix trimethylamine in a sample, but also can block metabolic transformation of trimethylamine and a precursor thereof by quenching a microbial reaction, so that the anti-interference capability is improved. In cooperation with optimized headspace-gas chromatography detection conditions, the detection sensitivity and accuracy are further improved, and a stable and reliable technical support is provided for risk assessment of diseases related to intestinal flora metabolism.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of metabolite detection, and relates to a method for detecting trimethylamine in a fecal sample. BACKGROUND

[0002] Trimethylamine (TMA) in feces is a characteristic product of intestinal flora metabolism of substrates such as choline and L-carnitine, and its content level can directly reflect the structure and metabolic activity of intestinal flora. TMAO is generated after TMA is oxidized, and TMAO in blood is an independent risk factor for AS, thrombosis and myocardial infarction, and is a key biomarker for evaluating the risk of cardiovascular diseases, metabolic syndrome and other chronic diseases. It can also be used for early screening, etiological analysis and intervention effect monitoring of diseases related to intestinal microecological imbalance such as inflammatory bowel disease and non-alcoholic fatty liver, and has important clinical value for realizing the prevention and control of chronic diseases and health management.

[0003] However, the following problems are encountered when determining the TMA content in feces: (1) The fecal matrix is complex and contains a large amount of microorganisms, food residues, bile salts and pigments, which can seriously interfere with the extraction and detection of TMA, and the complex microbial environment can lead to variable TMA precursor metabolic pathways, further reducing the extraction efficiency. (2) Since TMA is an extremely volatile basic amine compound, it is easy to volatilize and lose during sample processing, storage and extraction, and may also be oxidized or react with matrix components, resulting in low recovery and inaccurate quantitative results; (3) The existing method often uses high-concentration sodium hydroxide to treat the sample, and the residual choline and L-carnitine in the feces are converted to TMA under the condition of alkali solution above 20 mol / L, resulting in high determination value and unable to accurately reflect the true content of TMA in feces. Due to the above problems in the determination of trimethylamine in feces, the accuracy and reliability of the detection results are greatly reduced, and it is difficult to meet the needs of clinical diagnosis and scientific research analysis for quantitative detection of fecal TMA. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a method for detecting trimethylamine in a fecal sample with good accuracy, precision and sensitivity by improving the sample processing method and optimizing the headspace-gas chromatography conditions.

[0005] To achieve the above-mentioned application purposes, the embodiments of the present application adopt the following technical solutions: A method for detecting trimethylamine in a fecal sample, the detection method comprising the following steps: S1. Mixing the fecal sample with an acidic medium-chain alcohol extractant, extracting and separating the layers to obtain an organic phase; S2. Adding a dilute solution of inorganic strong acid to the organic phase, and performing solid-liquid separation, and the supernatant is the test solution; S3. Determining the content of trimethylamine in the test solution by headspace-gas chromatography. The acid medium-chain alcohol extraction solution comprises n-hexanol and a dilute solution of a strong inorganic acid. The gas chromatography conditions comprise: The amine analysis modified siloxane chromatographic column is used. The injection port temperature is 195-205 DEG C, and the FID detector temperature is 245-255 DEG C. The column temperature rising program is as follows: 48-52 DEG C is kept for 5-7 min, then raised to 115-125 DEG C at a speed of 9-12 DEG C / min, then raised to 155-165 DEG C at a speed of 4-6 DEG C / min, then raised to 235-245 DEG C at a speed of 13-17 DEG C / min and kept for 3.5-4.5 min.

[0006] The fecal sample trimethylamine detection method provided by the application has the advantages of simple operation, high accuracy, high precision, strong applicability and the like, and can meet the detection requirements of a large number of samples. The acid medium-chain alcohol extraction solution is prepared by creatively compounding n-hexanol and a dilute solution of a strong inorganic acid. The extraction solution breaks through the limitation of traditional single extraction function, can efficiently extract and enrich trimethylamine, can avoid trimethylamine metabolic loss by quenching microbial reaction, can reduce the volatilization loss of trimethylamine by protonation, can further enrich trimethylamine in the organic phase, can efficiently separate water-soluble impurities and solid particles in the feces, and can greatly enhance the anti-interference performance of detection. In combination with headspace-gas chromatography detection, the detection sensitivity of the method is further improved, and stable and reliable technical support is provided for risk assessment of intestinal flora metabolism related diseases.

[0007] Preferably, the dilute solution of a strong inorganic acid comprises dilute sulfuric acid.

[0008] Further preferably, the concentration of the dilute sulfuric acid is 0.04-0.07 mol / L.

[0009] Preferably, the volume ratio of the n-hexanol and the dilute solution of a strong inorganic acid is 95:5-99:1.

[0010] Illustratively, the acid medium-chain alcohol extraction solution is prepared according to a volume ratio of n-hexanol to dilute sulfuric acid of 98:2.

[0011] Preferably, the amine analysis modified siloxane chromatographic column is a CP-Volamine chromatographic column with a size of 30 m*0.32 mm*5 mu m.

[0012] Preferably, in step S1, the mass-to-volume ratio of the fecal sample to the acid medium-chain alcohol extraction solution is 1 g:3 mL-1 g:5 mL.

[0013] Preferably, in step S2, the volume ratio of the organic phase to the dilute inorganic strong acid solution is 1:3-1:5.

[0014] Preferably, in the headspace-gas chromatography method, the headspace injection conditions include: furnace temperature 78℃-82℃, quantitative loop temperature 85℃-95℃, sample equilibration time 35min-45min, and injection time 0.6s-1.2s.

[0015] Preferably, in the headspace-gas chromatography method, the injection volume is 0.8 mL-1.2 mL; the hydrogen flow rate is 25 mL / min-35 mL / min; the nitrogen flow rate is 25 mL / min-35 mL / min; the air flow rate is 250 mL / min-350 mL / min; the column flow rate is 1.4 mL / min-1.6 mL / min; and the split ratio is 8:1-12:1.

[0016] Preferably, the carrier gas is nitrogen. Exemplarily, it is high-purity nitrogen.

[0017] Preferably, step S3 includes: adding 4 mL-6 mL of the test solution and 4 mL-7 mL of sodium hydroxide solution with a concentration of 3.5 mol / L-6.5 mol / L to the headspace vial, tightening the cap, and determining the trimethylamine content using an HS-GC instrument.

[0018] This invention utilizes an acidic medium-chain alcohol extract composed of n-hexanol and a dilute solution of a strong inorganic acid for pretreatment of fecal samples. This acidic medium-chain alcohol extract possesses multiple advantages: it not only efficiently extracts and enriches trimethylamine from the sample, but also inhibits the metabolic transformation of trimethylamine and its precursors by quenching microbial reactions, and firmly immobilizes trimethylamine molecules through protonation, significantly reducing its volatilization loss. After treatment, trimethylamine is specifically enriched in the organic phase, effectively separating from water-soluble impurities and solid particles in the fecal matrix, significantly improving the detection's anti-interference capability. The trimethylamine detection method for fecal samples provided by this invention features high accuracy, excellent precision, and strong sensitivity, while also offering advantages such as ease of operation and wide applicability. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a gas chromatogram of the trimethylamine content in a fecal sample determined by the detection method provided in Example 1 of this invention; Figure 2This is the gas chromatogram of the reference standard concentration of 3.48 mg / L in Example 1 of the present invention; Figure 3 This is the trimethylamine standard curve provided in Example 1 of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are preferred examples of this invention and are only used to explain the invention and do not limit the invention.

[0022] To better illustrate the embodiments provided by the present invention, further examples are given below.

[0023] Example 1 This embodiment provides a method for detecting trimethylamine in fecal samples, the method comprising the following steps: S1. Slowly add 200 μL of 0.06 mol / L sulfuric acid-water solution (denoted as 0.06 mol / L sulfuric acid solution) to 9.8 mL of n-hexanol, and treat under ultrasonic conditions of 40 kHz and 300 W for 5 min until a transparent reverse microemulsion is formed, yielding an acidic medium-chain alcohol extract. Note that this extract should be prepared immediately before use to prevent component separation or inactivation. Take 1g of homogenized fecal sample and add it to 4mL of the above acidic medium-chain alcohol extract. Vortex for 5min to ensure sufficient contact between the two phases. Then centrifuge at 1000× speed for 2min at room temperature to promote phase separation and obtain the organic phase. S2. Mix the organic phase with dilute sulfuric acid at a volume ratio of 1:4, shake thoroughly to mix, centrifuge at 13000×g for 10min, take the supernatant, filter through a membrane, and obtain the test solution; Accurately weigh 0.09 g of trimethylamine hydrochloride, dissolve it in 0.06 mol / L sulfuric acid solution and dilute to 80 mL to prepare a 696 mg / L TMA standard stock solution. Dilute the TMA standard stock solution with 0.06 mol / L sulfuric acid solution to prepare a 69.6 mg / L TMA standard working solution. Dilute the TMA standard working solution with blank matrix solution to obtain five TMA standard solutions with concentrations of 6.96 mg / L, 3.48 mg / L, 1.74 mg / L, 0.87 mg / L, and 0.174 mg / L, respectively. The blank matrix solution was the supernatant obtained by repeatedly extracting the fecal sample with 0.06 mol / L sulfuric acid solution four times.

[0024] S3. Add 5 mL of 5 mol / L sodium hydroxide solution to the headspace vial, mix with 5 mL of TMA standard solution of different concentrations respectively, immediately tighten the cap, determine by headspace-gas chromatography, and plot the trimethylamine standard curve. Add 5 mL of the test solution and 5 mL of 5 mol / L sodium hydroxide solution to the headspace vial, immediately tighten the cap, and determine the trimethylamine content in the test solution using headspace-gas chromatography. Calculate the trimethylamine content in the test solution based on the trimethylamine standard curve described above. Gas chromatography conditions: An Agilent CP-Volamine column (30m × 0.32mm × 5μm) was used. The injection port temperature is 200℃; the split ratio is 10:1; the FID detector temperature is 250℃. Column temperature rise program: 50℃ for 6 min, rise to 120℃ at a rate of 10℃ / min, rise to 160℃ at a rate of 5℃ / min, then rise to 240℃ at a rate of 15℃ / min and hold for 4 min; The carrier gas was high-purity nitrogen, and the column flow rate was 1.5 mL / min; The flow rate of the exhaust gas (nitrogen) is 30 mL / min, the flow rate of hydrogen is 30 mL / min, and the flow rate of air is 300 mL / min.

[0025] The injection method is headspace injection, and the specific headspace sampler conditions are as follows: The furnace temperature was 80℃, the quantitative loop temperature was 90℃, the transfer line temperature was 110℃, the sample equilibration time was 40 min, and the injection volume was 1 mL.

[0026] The gas chromatogram for determining the trimethylamine content in fecal samples using the detection method provided in this embodiment is shown below. Figure 1 As shown.

[0027] Comparative Example 1 This invention provides a method for detecting trimethylamine in fecal samples, the specific method being as follows: S1. Take 1g of homogenized fecal sample, add 4mL of 0.06 mol / L sulfuric acid solution, vortex for 5min, centrifuge at 13000×g for 10min, and collect the supernatant; add 4mL of 0.06 mol / L sulfuric acid solution to the centrifuged fecal precipitate again, centrifuge at 13000×g for 10min, and collect the supernatant; add 4mL of 0.06 mol / L sulfuric acid solution to the centrifuged fecal precipitate again, vortex thoroughly to mix, centrifuge at 13000×g for 10min, and collect the supernatant; combine the supernatants obtained from the three centrifugations, mix well, filter through a membrane, and obtain the test solution; S3. Add 5 mL of the test solution and 5 mL of 5 mol / L sodium hydroxide solution to the headspace vial, immediately tighten the cap, and determine the trimethylamine content in the test solution using headspace-gas chromatography. The gas chromatography conditions and headspace sampler conditions are the same as in Example 1.

[0028] Comparative Example 2 This invention provides a method for detecting trimethylamine in fecal samples. The detection method is basically the same as that in Example 1, except that n-hexanol in step S1 is replaced with an equal amount of n-octanol. All other operating parameters are the same as in Example 1.

[0029] Example of effect This invention provides a methodological investigation of the methods for detecting trimethylamine in fecal samples provided in Example 1 and Comparative Examples 1-2. Specifically, it includes the following: (1) Linear range The gas chromatogram for determining the concentration of the reference standard at 3.48 mg / L in Example 1 of this invention is shown below. Figure 2 As shown; the trimethylamine standard curve provided in Example 1 of this invention is shown in the figure. Figure 3 As shown. The standard curve formula is: Y = 39.935x + 1.2834(R²). 2 =0.999).

[0030] The results showed that the linear range of trimethylamine in this experiment was 0.174-6.96 mg / L, and the linear relationship was good within the linear range.

[0031] (2) Limit of detection (LOD) and limit of quantitation (LOQ) The detection limit and quantitation limit were calculated using a signal-to-noise ratio (S / N) of 3 and 10, respectively, where the noise was the response signal distribution at zero analyte concentration.

[0032] (3) Accuracy Accuracy was verified using a spiked recovery test. Three concentrations of standard solution (low, medium, and high) were added to the fecal sample within the standard curve range. A blank matrix solution was also added to achieve final concentrations of 0.174 mg / L (low), 0.87 mg / L (medium), and 1.74 mg / L (high). The accuracy of the method was assessed by calculating the measured values ​​of the fecal sample with added TMA standard, the measured TMA content in the fecal sample, and the measured value of the blank matrix with added TMA standard.

[0033] (4) Precision Intra-day precision and inter-day precision are both characterized by the coefficient of variation (CV%). Intra-day precision was determined as follows: within the same experimental day, fecal samples were analyzed repeatedly every 4 hours, for a total of 6 parallel measurements (n=6). Inter-day precision was determined as follows: the experiment was conducted for 3 consecutive days, with fecal samples analyzed every 4 hours each day, for a total of 18 measurements (n=18) over 3 days.

[0034] The sensitivity, accuracy, and precision of the detection methods for trimethylamine in the three fecal samples provided in Examples 1 and Comparative Examples 1-2 are shown in Table 1 below.

[0035] Table 1

[0036] As shown in Table 1, compared with Comparative Examples 1 and 2, the method for detecting trimethylamine in fecal samples provided in Example 1 of this invention exhibits the best overall performance in terms of sensitivity, accuracy, and precision. The detection limit of the method for detecting trimethylamine in fecal samples provided in Example 1 of this invention is 0.0021 mg / L, the quantitation limit is 0.007 mg / L, the average recovery rate reaches 99.19%, and the coefficients of variation for intra-day and inter-day precision are both less than 3%, demonstrating good performance.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting trimethylamine in fecal samples, characterized in that: The detection method includes the following steps: S1. Mix the fecal sample with an acidic medium-chain alcohol extract, extract and separate the layers to obtain the organic phase; S2. Add a dilute solution of a strong inorganic acid to the organic phase, separate the solid and liquid phases, and take the supernatant to obtain the test solution; S3. The trimethylamine content in the test solution was determined by headspace-gas chromatography; The acidic medium-chain alcohol extract includes n-hexanol and a dilute solution of a strong inorganic acid. Gas chromatography conditions include: A modified siloxane column for amine analysis was used. The injection port temperature is 195℃-205℃; the FID detector temperature is 245℃-255℃. Column temperature rise program: 48℃-52℃, hold for 5min-7min, rise to 115℃-125℃ at a rate of 9℃ / min-12℃ / min, then rise to 155℃-165℃ at a rate of 4℃ / min-6℃ / min, then rise to 235℃-245℃ at a rate of 13℃ / min-17℃ / min and hold for 3.5min-4.5min.

2. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: The inorganic strong acid dilute solution includes dilute sulfuric acid.

3. The method for detecting trimethylamine in fecal samples as described in claim 2, characterized in that: The concentration of the dilute sulfuric acid is 0.04 mol / L to 0.07 mol / L.

4. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: The volume ratio of n-hexanol to the dilute inorganic strong acid solution is 95:5-99:

1.

5. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: The modified siloxane column used for amine analysis is a CP-Volamine column, 30m × 0.32mm × 5μm.

6. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: In step S1, the mass-to-volume ratio of the fecal sample to the acidic medium-chain alcohol extract is 1g:3mL-1g:5mL.

7. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: In step S2, the volume ratio of the organic phase to the dilute inorganic strong acid solution is 1:3-1:

5.

8. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: In the headspace-gas chromatography method, the headspace injection conditions include: furnace temperature 78℃-82℃, quantitative loop temperature 85℃-95℃, and sample equilibration time 35min-45min.

9. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: Injection volume: 0.8 mL - 1.2 mL; and / or The hydrogen flow rate is 25 mL / min-35 mL / min; and / or Nitrogen flow rate is 25 mL / min-35 mL / min; and / or Air flow rate of 250 mL / min-350 mL / min; and / or Column flow rate 1.4 mL / min - 1.6 mL / min; and / or The split ratio is 8:1-12:

1.

10. The method for detecting trimethylamine in fecal samples as described in claim 1, characterized in that: Step S3 includes: adding 4 mL-6 mL of the test solution and 4 mL-7 mL of sodium hydroxide solution with a concentration of 3.5 mol / L-6.5 mol / L to the headspace vial, tightening the cap, and determining the trimethylamine content using an HS-GC instrument.

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