Method for detecting soil microbial marker-amino sugar by direct ion chromatography

Abstract

The invention discloses a method for detecting a soil microbial marker-amino sugar by direct ion chromatography, and relates to the technical field of biomarker analysis, and the method comprises the following steps: adding a soil sample into a 6mol / L hydrochloric acid solution, and hydrolyzing at 105 DEG C for 8 hours; drying the hydrolysate with nitrogen, redissolving with ultrapure water, centrifuging, taking supernate, and filtering with a 0.22 mu m filter membrane to obtain a to-be-detected solution; the method comprises the following steps: injecting a to-be-detected solution into an ion chromatography system provided with an electrochemical detector, and carrying out gradient elution by adopting an anion exchange chromatographic column and taking a mixed solution of sodium hydroxide and sodium acetate as a mobile phase to realize separation and quantitative detection of glucosamine (GlcN), galactosamine (GalN), aminomannose (ManN) and muramic acid (Mur). A highly toxic derivatization step necessary for a traditional method is omitted, a sample can be directly injected through three-step purification after acid hydrolysis, and efficient separation of four amino sugars is realized by matching with a special ion chromatography system.

Description

Technical Field

[0001] This invention relates to the technical field of biomarker analysis, and more specifically, to a method for detecting the soil microbial biomarker amino sugar using direct ion chromatography. Background Technology

[0002] Currently, the detection of amino sugars in soil mainly relies on gas chromatography-mass spectrometry (GC-MS), but this technique has significant limitations: samples need to be hydrolyzed continuously at 105°C for 8 hours with strong acid (such as 6M HCl); subsequent purification steps include pH adjustment, centrifugation, rotary evaporation drying, and methanol extraction; the derivatization step is particularly critical, requiring chemical derivatization reactions with highly toxic reagents (pyridine, acetic anhydride) at 78-80°C, followed by dichloromethane extraction. This process takes more than 3 days and involves over 20 steps, demanding stringent professional skills from laboratory personnel and posing significant safety hazards due to the large-scale use of Group I carcinogens (such as pyridine), while also generating highly toxic organic waste with high subsequent treatment costs. Furthermore, the complex manual operation process is prone to human error, leading to unstable method reproducibility.

[0003] Based on this, the present invention provides a method for detecting soil microbial markers—amino sugars—using direct ion chromatography. Summary of the Invention

[0004] To address the problems mentioned in the background section, this invention provides a method for detecting amino sugars, a soil microbial biomarker, using direct ion chromatography.

[0005] The method for detecting soil microbial markers—amino sugars—provided by this invention adopts the following technical solution:

[0006] A method for detecting soil microbial markers—amino sugars—using direct ion chromatography includes the following steps:

[0007] S1. Add the soil sample to a 6 mol / L hydrochloric acid solution and hydrolyze it at 105℃ for 8 hours;

[0008] S2. After drying the hydrolysate with nitrogen, redissolve it with ultrapure water, centrifuge to collect the supernatant and filter it through a 0.22μm filter membrane to obtain the test solution;

[0009] S3. Inject the test solution into an ion chromatography system equipped with an electrochemical detector. Use an anion exchange column and a mixed solution of sodium hydroxide and sodium acetate as the mobile phase for gradient elution to achieve the separation and quantitative detection of glucosamine (GlcN), galactosamine (GalN), mannose (ManN), and muramic acid (Mur).

[0010] Preferably, in S1, the amount of soil sample used is 0.2-0.5g, and the amount of hydrochloric acid solution added is 5mL.

[0011] Preferably, in S3, the chromatographic column is a Dionex. TM CarboPac TM PA20 anion exchange column (150mm × 3.0mm).

[0012] Preferably, the gradient elution process includes:

[0013] Mobile phase A is a 0.1 mol / L NaOH solution;

[0014] Mobile phase B is a 0.1 mol / L NaOH + 0.2 mol / L NaAc solution;

[0015] The elution gradient is:

[0016] At 0 min, A:B = 95:5 → At 15 min, A:B = 80:20 → At 15.1 min, A:B = 60:40 → At 25 min, A:B = 60:40 → At 25.1 min, A:B = 95:5 → At 40 min, A:B = 95:5.

[0017] Preferably, the chromatographic conditions include a column temperature of 30°C, a flow rate of 0.5 mL / min, and an injection volume of 5 μL.

[0018] Preferably, the quantitative detection employs the external standard method, establishing a concentration-peak area standard curve by mixing standard solutions, and the amino sugar content is determined according to the formula. calculate;

[0019] Where C is the concentration measured by the instrument (μg / mL);

[0020] V is the final volume of the sample extract (mL);

[0021] F is the dilution factor;

[0022] M represents the soil sample mass (mg).

[0023] Preferably, in S2, the purification process only includes nitrogen drying, ultrapure water reconstitution, centrifugation and filtration, omitting pH adjustment, organic solvent extraction and derivatization steps.

[0024] Preferably, the method is applicable to the detection of amino sugars in solid soil, sediments, or liquid environmental samples that have undergone concentration and drying.

[0025] In summary, the present invention has the following beneficial technical effects:

[0026] This invention eliminates the highly toxic derivatization step required by traditional methods, allowing direct sample injection after three purification steps following acid hydrolysis. Combined with a dedicated ion chromatography system, it achieves efficient separation of four amino sugars. In terms of experimental efficiency, the operation process is streamlined from over ten steps to four, reducing analysis time to 1 / 8 of traditional methods (9 hours vs. 72 hours), while maintaining excellent linear correlation coefficients and detection sensitivity (1 μg / mL). Regarding environmental safety, it completely eliminates the use of carcinogenic reagents such as pyridine, reducing the organic solvent content in waste liquid by over 90%. Operationally, it significantly lowers the technical threshold, saving 110 yuan per sample, and provides universal applicability to both solid and liquid samples. It offers a safe, efficient, and economical standardized detection solution for soil microbial residue research, making it highly valuable for widespread application.

[0027] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0028] Figure 1 This is a flowchart of the method for detecting soil microbial markers—amino sugars—using direct ion chromatography in an embodiment of the present invention;

[0029] Figure 2 This is the standard ion chromatogram from Embodiment 1 of the present invention;

[0030] Figure 3 This is the total ion current chromatogram obtained by gas chromatography-mass spectrometry in Comparative Example 1 of this invention;

[0031] Figure 4 This is a data comparison and function transformation relationship diagram between Embodiment 1 and Comparative Example 1 of the present invention. Detailed Implementation

[0032] The following is in conjunction with the appendix Figures 1 to 4 The present invention will be described in further detail below.

[0033] This invention discloses a method for detecting amino sugars, a soil microbial biomarker, using direct ion chromatography. (Refer to...) Figure 1 A method for detecting soil microbial markers—amino sugars—using direct ion chromatography includes the following steps:

[0034] S1. Add the soil sample to a 6 mol / L hydrochloric acid solution and hydrolyze it at 105℃ for 8 hours;

[0035] S2. After drying the hydrolysate with nitrogen, redissolve it with ultrapure water, centrifuge to collect the supernatant and filter it through a 0.22μm filter membrane to obtain the test solution;

[0036] S3. Inject the test solution into an ion chromatography system equipped with an electrochemical detector. Use an anion exchange column and a mixed solution of sodium hydroxide and sodium acetate as the mobile phase for gradient elution to achieve the separation and quantitative detection of glucosamine (GlcN), galactosamine (GalN), mannose (ManN), and muramic acid (Mur).

[0037] Specifically, in S1, the amount of soil sample used is 0.2-0.5g, and the amount of hydrochloric acid solution added is 5mL.

[0038] Specifically, in S3, the chromatographic column is a Dionex. TM CarboPac TM PA20 anion exchange column (150mm × 3.0mm).

[0039] Specifically, the gradient elution procedure includes:

[0040] Mobile phase A is a 0.1 mol / L NaOH solution;

[0041] Mobile phase B is a 0.1 mol / L NaOH + 0.2 mol / L NaAc solution;

[0042] The elution gradient is:

[0043] At 0 min, A:B = 95:5 → At 15 min, A:B = 80:20 → At 15.1 min, A:B = 60:40 → At 25 min, A:B = 60:40 → At 25.1 min, A:B = 95:5 → At 40 min, A:B = 95:5.

[0044] Specifically, the chromatographic conditions included a column temperature of 30°C, a flow rate of 0.5 mL / min, and an injection volume of 5 μL.

[0045] Specifically, the quantitative detection employs the external standard method, establishing a concentration-peak area standard curve by mixing standard solutions. The amino sugar content is determined according to the formula... calculate;

[0046] Where C is the concentration measured by the instrument (μg / mL);

[0047] V is the final volume of the sample extract (mL);

[0048] F is the dilution factor;

[0049] M represents the soil sample mass (mg).

[0050] Specifically, in S2, the purification process only includes nitrogen drying, ultrapure water reconstitution, centrifugation and filtration, omitting pH adjustment, organic solvent extraction and derivatization steps.

[0051] Specifically, the method is applicable to the detection of amino sugars in solid soil, sediments, or liquid environmental samples that have undergone concentration and drying.

[0052] Example 1

[0053] Typical soil sample testing:

[0054] Take 0.30 g (accurate to 0.1 mg) of air-dried and ground farmland soil samples, totaling 54 portions. Add 5 mL of 6 mol / L HCl solution and place in a polytetrafluoroethylene hydrolysis tube. Hydrolyze at 105 °C for 8 hours. After cooling, take 500 μL of the hydrolysate and purge with nitrogen until completely dry (about 20 min). Redissolve the residue in 1 mL of ultrapure water, vortex for 1 min, centrifuge at 12000 rpm for 5 min, and filter the supernatant through a 0.22 μm nylon filter membrane to obtain the test solution.

[0055] Chromatographic analysis conditions

[0056]

[0057] Quantitative analysis: A mixed standard solution of GalN, ManN, GlcN, and Mur was prepared (concentration gradient: 1, 5, 10, 20, 30 μg / mL); the linear equation was GlcN: y = 0.218x - 0.015 (R²). 2 =0.999), Mur:y=0.192x+0.008(R 2 =0.998); the soil sample results showed a GlcN content of 12.3 μg / mg and an RSD of 1.8% (n=3).

[0058] Quantitative results:

[0059]

[0060] Example 2

[0061] Sediment sample analysis:

[0062] Take 0.25 g (accurate to 0.1 mg) of air-dried and ground river and lake sediment sample, add 5 mL of 6 mol / L HCl solution, place in a polytetrafluoroethylene hydrolysis tube, hydrolyze at 105 °C for 8 hours, cool, take 500 μL of hydrolysate, purge with nitrogen until completely dry (about 25 min), add 0.5 mL of ultrapure water to redissolve the residue (due to the low organic matter content, reduce the volume to increase the concentration), vortex for 1 min, centrifuge at 12000 rpm for 5 min, take the supernatant and filter through a 0.22 μm nylon filter membrane to obtain the test solution.

[0063] Chromatographic analysis conditions

[0064]

[0065]

[0066] Quantitative analysis: Prepare mixed standard solutions of GalN, ManN, GlcN, and Mur (concentration gradient: 1, 5, 10, 20, 30 μg / mL); Limit of detection (LOD): Mur is 0.8 μg / mL (signal-to-noise ratio S / N = 3); Spike recovery: With the addition of 10 μg / mL standard, the recovery rate of GalN is 98.2%, and the recovery rate of ManN is 94.7%.

[0067] Its standard ion chromatogram, such as Figure 2 As shown.

[0068] Comparative Example 1

[0069] Take 0.30 g (accurate to 0.1 mg) of farmland soil sample from the same source as in Example 1, totaling 54 portions. Add 5 mL of 6 mol / L HCl solution and place in a polytetrafluoroethylene hydrolysis tube. Hydrolyze at 105 °C for 8 hours. After cooling, transfer the hydrolysate to a centrifuge tube and add 10 mol / L HCl solution dropwise. Adjust the pH to 7.0 with NaOH solution (consuming approximately 1.5g of NaOH). During this process, use an ice bath to prevent overheating. Centrifuge the solution at 12000 rpm for 10 min, collect the supernatant, and dry the supernatant at 60℃ for 2 hours using a rotary evaporator. Add 2 mL of methanol to the residue, vortex for 2 min, centrifuge at 12000 rpm for 5 min, collect the supernatant, and repeat the methanol extraction twice. Combine the three extracts, concentrate the methanol extract to near dryness using a rotary evaporator at 40℃, and perform derivatization: Add 50 μL of pyridine and 100 μL of acetic anhydride to the residue, seal, and react in a water bath at 80℃ for 1 hour. Extract with 1 mL of dichloromethane, vortex for 1 min, allow to stand for layering, collect the organic phase, concentrate the organic phase to 50 μL by nitrogen purging, and transfer to a vial. The total time is >24 hours.

[0070] GC-MS analysis

[0071]

[0072]

[0073] Quantitative analysis: Prepare a mixed standard solution of GalN, ManN, GlcN, and Mur (concentration of 5 μg / μL), add standard, and recoveries: add 5 μg / μL standard, and the correction factors are calculated to be 0.56, 0.44, 0.50, and 0.19.

[0074] Quantitative results:

[0075]

[0076] Its total ion chromatogram obtained by gas chromatography-mass spectrometry is as follows: Figure 3 As shown.

[0077] The comparison is as follows

[0078]

[0079]

[0080] Data comparison between Example 1 and Comparative Example 1 - function transformation relationship as follows: Figure 4 As shown.

[0081] Example 3

[0082] Take 10 mL of soil aqueous extract (containing dissolved organic matter), place it in a glass test tube, and concentrate it to 0.5 mL by purging with nitrogen at 60 °C (about 2 hours). Add 5 mL of 6 mol / L HCl solution to the concentrate, place it in a polytetrafluoroethylene hydrolysis tube, and hydrolyze it at 105 °C for 8 hours. After cooling, take 500 μL of the hydrolysate, purge it with nitrogen until completely dry (about 20 min), redissolve the residue in 1 mL of ultrapure water, vortex for 1 min, centrifuge at 12000 rpm for 5 min, and filter the supernatant through a 0.22 μm nylon filter membrane to obtain the test solution.

[0083] Chromatographic analysis conditions

[0084]

[0085] Quantitative analysis: A mixed standard solution of GalN, ManN, GlcN, and Mur was prepared (concentration gradient: 1, 5, 10, 20, 30 μg / mL); the concentration of Mur was detected at 0.65 μg / mL, and the recovery rate (with 5 μg / mL Mur added) was 92.4%; the chromatogram showed that humic acid eluted before 10 min, and Mur eluted at 22.5 min, with no co-elution interference.

[0086] As can be seen from the above, this invention solves the long-standing triangular problem of "poor safety-low efficiency-high cost" in the field of soil amino sugar analysis by using the dual core technologies of "direct detection by ion chromatography without derivatization" and "gradient elution program synergistic aqueous phase purification". For the first time, it eliminates the use of highly toxic reagents while ensuring analytical accuracy. By using derivatization-free design and direct injection into the aqueous phase, it skips the complex steps required by existing technologies, such as pH adjustment, organic solvent extraction, and derivatization reaction.

[0087] This invention eliminates the highly toxic derivatization step required by traditional methods, allowing direct injection after three-step purification via acid hydrolysis. Combined with a dedicated ion chromatography system, it achieves efficient separation of four amino sugars. In terms of experimental efficiency, the procedure is streamlined from over ten steps to four, reducing analysis time to 1 / 8 of traditional methods (9 hours vs. 72 hours), while maintaining excellent linear correlation coefficients and detection sensitivity (1 μg / mL). Furthermore, a linear function conversion relationship between this method and traditional methods has been established (e.g., ...). Figure 4 (As shown); In terms of environmental safety, the use of carcinogenic reagents such as pyridine is completely eliminated, and the organic solvent content in waste liquid is reduced by more than 90%; in terms of operation, the technical threshold is significantly reduced, the cost per sample is reduced by 110 yuan, and it is universally applicable to both solid and liquid samples, providing a safe, efficient, and economical standardized detection solution for soil microbial residue research, which is of great promotional value.

[0088] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0089] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0090] The accompanying drawings of the embodiments disclosed in this invention only involve structures relevant to the embodiments disclosed in this invention. Other structures can be referred to with common designs. Unless otherwise specified, the same embodiment and different embodiments of this invention can be combined with each other.

[0091] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting soil microbial markers—amino sugars—using direct ion chromatography, characterized in that, Includes the following steps: S1. Add the soil sample to a 6 mol / L hydrochloric acid solution and hydrolyze it at 105℃ for 8 hours; S2. After drying the hydrolysate with nitrogen, redissolve it with ultrapure water, centrifuge to collect the supernatant and filter it through a 0.22μm filter membrane to obtain the test solution; S3. Inject the test solution into an ion chromatography system equipped with an electrochemical detector. Use an anion exchange column and a mixed solution of sodium hydroxide and sodium acetate as the mobile phase for gradient elution to achieve the separation and quantitative detection of glucosamine (GlcN), galactosamine (GalN), mannose (ManN), and muramic acid (Mur).

2. The method for detecting soil microbial markers—amino sugars—by direct ion chromatography according to claim 1, characterized in that: In S1, the amount of soil sample used was 0.2-0.5g, and the amount of hydrochloric acid solution added was 5mL.

3. The method for detecting soil microbial markers—amino sugars—by direct ion chromatography according to claim 1, characterized in that: In S3, the chromatographic column is a Dionex. TM CarboPac TM PA20 anion exchange chromatography column.

4. The method for detecting soil microbial markers—amino sugars—by direct ion chromatography according to claim 1, characterized in that: The gradient elution procedure includes: Mobile phase A is a 0.1 mol / L NaOH solution; Mobile phase B is a 0.1 mol / L NaOH + 0.2 mol / L NaAc solution; The elution gradient is: At 0 min, A:B = 95:5 → At 15 min, A:B = 80:20 → At 15.1 min, A:B = 60:40 → At 25 min, A:B = 60:40 → At 25.1 min, A:B = 95:5 → At 40 min, A:B = 95:

5.

5. The method for detecting soil microbial markers—amino sugars—by direct ion chromatography according to claim 4, characterized in that: Chromatographic conditions included a column temperature of 30℃, a flow rate of 0.5 mL / min, and an injection volume of 5 μL.

6. The method for detecting soil microbial markers—amino sugars—by direct ion chromatography according to claim 1, characterized in that: The quantitative detection employs the external standard method, establishing a concentration-peak area standard curve using mixed standard solutions. Amino sugar content is determined according to the formula... calculate; Where C is the concentration measured by the instrument (μg / mL); V is the final volume of the sample extract (mL); F is the dilution factor; M represents the soil sample mass (mg).

7. The method for detecting soil microbial markers—amino sugars—by direct ion chromatography according to claim 1, characterized in that: In S2, the purification process only includes nitrogen drying, ultrapure water reconstitution, centrifugation, and filtration.

8. The method for detecting soil microbial markers—amino sugars—by direct ion chromatography according to any one of claims 1-7, characterized in that: The method is applicable to the detection of amino sugars in solid soil, sediments, or liquid environmental samples that have undergone concentration and drying.

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