A method for analyzing phospholipid fatty acids in soil

Abstract

The application discloses a method for analyzing phospholipid fatty acid in soil, comprising the following steps: S1, weighing soil sample, adding extraction solution, vortex oscillation extraction, adding layered solvent and inorganic salt layering, taking organic phase drying and concentrating to obtain soil lipid extract; S2, dissolving soil lipid extract, purifying solution through zirconium peroxide solid phase extraction column, sequentially eluting fatty acid, neutral glycerin, glycolipid, betaine lipid and phospholipid to obtain high-purity soil phospholipid extract, mixing and concentrating after adding internal standard; S3, performing thermal catalytic transesterification treatment on soil phospholipid, simultaneously performing saponification and methylation of phospholipid to obtain fatty acid methyl ester product; S4, transferring fatty acid methyl ester product into a pipette gun head, eluting fatty acid methyl ester with organic solvent and concentrating; and S5, performing gas chromatography-mass spectrometry analysis on fatty acid methyl ester. The application provides a more environmental, efficient, accurate and stable analysis method with strong separation and enrichment capacity for soil phospholipid analysis.

Description

Technical Field

[0001] This invention relates to a method for analyzing phospholipid fatty acids in soil using zirconium oxide solid-phase extraction combined with thermocatalytic transesterification, belonging to the field of methods for analyzing soil microbial communities and functions. Background Technology

[0002] Soil phospholipid fatty acids (PLFAs) originate from the hydrolysis of phospholipids. Phospholipids consist of a glycerol backbone, hydrophobic fatty acid chains at the sn-1 and sn-2 positions, and a phosphate group at the sn-3 position, linked to the glycerol hydroxyl group via ester or ether bonds. Phospholipids are major components of microbial cell membranes, participating in cell signaling, maintaining membrane integrity and stability, cell proliferation, and survival. Due to their strong chemotactic capacity and specificity, and their rapid degradation after cell death, phospholipid fatty acid analysis has been widely used as a method independent of microbial culture and gene analysis to analyze changes in microbial communities and biomass. PLFA profiling analysis can be used to monitor microbial community composition and its relationship with nutrient availability, and has been widely applied to the analysis of PLFAs in soil, aquatic environments, and waste under environmental stress conditions.

[0003] Existing analytical methods for PLFAs mainly include silica gel column separation and gas chromatography-mass spectrometry (GC-MS) analysis following homogeneous acid / base catalytic transesterification. Silica gel column separation, a classic method for phospholipid separation and enrichment, uses chloroform, acetone, and methanol to separate neutral lipids, glycolipids, and phospholipids, respectively. However, due to the non-specific adsorption of silica gel, the separation of the phospholipid fraction is incomplete. Homogeneous acid / base catalytic transesterification typically uses sulfuric acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, etc., which increases the environmental risk during the experiment. Furthermore, homogeneous acid / base catalytic transesterification is time-consuming, requires multiple liquid-liquid extractions for pretreatment, and has low tolerance to impurities such as water. The use of strong acid / base catalysts for certain fatty acids with specific structures may also lead to side reactions, such as rearrangement and addition, resulting in the formation of new organic acids or the loss of some organic acids, further complicating the interpretation of the results. Summary of the Invention

[0004] This invention provides a method for analyzing phospholipid fatty acids in soil using zirconia solid-phase extraction combined with thermocatalytic transesterification. The aim is to design a method that specifically separates soil phospholipids, is simple and environmentally friendly in thermocatalytic transesterification, and has extremely high transesterification efficiency. This method can comprehensively reflect the changes in soil microbial communities and functions, providing a more environmentally friendly, efficient, accurate, and stable analytical method for soil phospholipid fatty acids with strong separation and enrichment capabilities.

[0005] The applicant's research found that existing methods for analyzing phospholipid fatty acids, particularly PLFA spectra obtained from silica gel column separation, include not only fatty acid methyl esters generated from the hydrolysis of phospholipids but also fatty acid methyl esters generated from the hydrolysis of betaine and other lipids. Since the adsorption principle of silica gel columns is based on the different adsorption forces of substances on silica gel, substances with higher polarity interact strongly with silica gel and have longer retention times, while substances with lower polarity interact weakly with silica gel and have shorter retention times. Substances are separated through repeated adsorption and desorption processes between the stationary and mobile phases. When betaine lipids are separated on silica gel columns, due to the polar similarity between the COO- of betaine lipids and the PO- of phospholipids, the methanol eluted fraction from low-phosphorus soils contains a relatively large amount of betaine lipids, along with trace amounts of glycolipids. If subsequent analysis does not selectively distinguish between phospholipids and other lipids, incomplete separation will complicate the subsequent analysis of microbial communities and functions.

[0006] The technical solution of this invention is: a method for analyzing phospholipid fatty acids in soil, comprising the following steps:

[0007] Extraction of lipids from soil S1: Weigh the soil sample dried under low temperature and vacuum, add the extraction solution, vortex and shake to extract, add the layering solvent and inorganic salt to separate the layers, take the organic phase, dry and concentrate to obtain the soil lipid extract.

[0008] S2 Soil Phospholipid Purification: Soil lipid extract was dissolved and purified using a zirconium peroxide solid-phase extraction column. Fatty acids, neutral triglycerides, glycolipids, betaine lipids, and phospholipids were eluted sequentially to obtain a high-purity soil phospholipid extract. After adding an internal standard, the extract was mixed, concentrated, and subjected to thermal catalytic transesterification.

[0009] S3 Soil Phospholipid Thermocatalytic Transesterification: Soil phospholipids are transesterified using a thermocatalytic transesterification method, while simultaneously undergoing saponification and methyl esterification of the phospholipids to obtain fatty acid methyl ester products.

[0010] Elution and enrichment of S4 soil phospholipids: The fatty acid methyl ester product was transferred into the pipette tip, eluted with organic solvent, concentrated and then analyzed by gas chromatography-mass spectrometry.

[0011] Detection of phospholipids in S5 soil: Gas chromatography-mass spectrometry was used to separate, qualitatively identify, and relatively quantify fatty acid methyl esters. The type of fatty acid methyl ester was qualitatively identified by comparison with retention time, retention index, standards, and mass spectra, and quantification was performed using the internal standard method.

[0012] To address the shortcomings of existing methods such as silica gel column separation and homogeneous acid / base catalyst transesterification, this invention employs zirconia solid-phase extraction (SPIE) for separation and purification. Zirconia exhibits strong Lewis acid-base interactions with the phosphate groups (PO-) of phospholipids, resulting in highly selective adsorption of phospholipids while having a weaker effect on other lipids. This characteristic makes it far superior to silica gel columns in separating betaine lipids and phospholipids. Thermocatalytic transesterification is a thermochemical methyl esterification transesterification pathway induced by an external heat source. This method simplifies the experimental procedure, reduces solvent usage and reaction time, and boasts extremely high transesterification efficiency. Combining zirconia SPIE purification with thermocatalytic transesterification significantly improves the analytical capabilities for phospholipid fatty acids in soil, overcoming the shortcomings of traditional phospholipid fatty acid analysis in separation and transesterification. This new method can more accurately and stably evaluate the composition and function of soil microbial communities.

[0013] Optionally, in step S1, the soil lipid extraction solution is chloroform:methanol:phosphate buffer (1:2:0.8v:v:v), and the layering solvent and inorganic salt are chloroform, ultrapure water and sodium chloride.

[0014] Optionally, in step S2, the solution for eluting fatty acids, neutral triglycerides, and glycolipids is 0.1% formic acid in methanol, the solution for eluting betaine lipids is 5% formic acid in methanol, and the solution for eluting phospholipids is 20% ammonia in methanol.

[0015] Optionally, in step S3, the thermocatalytic transesterification method is as follows: soil phospholipid extract and solvent are added to a thermocatalytic reactor containing porous material, sealed, and heated in a muffle furnace for transesterification.

[0016] Optionally, the solvent for the thermocatalytic transesterification is methanol, the porous material is diatomaceous earth, the reaction temperature is 360-380℃, and the time is 6-10 min.

[0017] Optionally, in step S4, after the soil phospholipids are converted into fatty acid methyl esters, the fatty acid methyl esters are enriched by elution with the organic solvent n-hexane.

[0018] Optionally, in step S5, gas chromatography-mass spectrometry (GC-MS) analysis is performed under the following conditions: DB-5 column (30m × 0.25mm i.d. × 0.25μm film thickness); injection port temperature 250℃, non-separation mode; carrier gas is ultrapure helium at a constant flow rate of 2.0mL / min; column temperature is maintained at 50℃ for 2min, then increased to 280℃ at 8℃ / min and maintained for 15min; transfer line temperature and solvent delay time are set to 280℃ and 5.0min, respectively; ion source and quadrupole temperatures are maintained at 230℃ and 150℃, respectively; ionization energy: 70eV; full scan mass range: 45-600aum; acquisition mode: full scan (Scan); MS libraries: NIST14 and Willy08, for qualitative and relative quantification of fatty acid methyl esters.

[0019] The beneficial effects of this invention are as follows: It provides a pretreatment method for highly selective separation and efficient transesterification of phospholipids in soil, mainly comprising three steps: extraction of phospholipids from soil, zirconium oxide solid-phase extraction purification, and thermocatalytic transesterification analysis of phospholipids. Subsequently, gas chromatography-mass spectrometry (GC-MS) is used to qualitatively and quantitatively identify 42 fatty acids in the soil, including 10 saturated fatty acids, 10 monounsaturated fatty acids, 12 branched-chain fatty acids, 3 10-methyl fatty acids, 2 cyclopropane fatty acids, and 5 other hydroxylated or polyunsaturated fatty acids. This analytical method accurately and stably reflects the community structure, abundance, and activity of microorganisms in soil and can be widely applied to soil microbial diversity research. This invention provides a more environmentally friendly, efficient, accurate, and stable analytical method for soil microbial evaluation, offering strong separation and enrichment capabilities.

[0020] Compared with existing technologies, this invention also has the following advantages: 1) Zirconia solid-phase extraction purification utilizes the strong interaction between Lewis acid and base and phospholipids to completely separate phospholipids from other lipids (betaine lipids, moderately polar glycolipids, etc.); 2) While analyzing phospholipid fatty acids, it can also analyze and evaluate betaine fatty acids in the soil; 3) Thermocatalytic transesterification of phospholipids does not require the use of homogeneous acid / base catalysts, and inexpensive and reusable porous diatomaceous earth materials can be used for complete and efficient transesterification; 4) Phospholipid transesterification time is short (8 min), and the separation and purification of fatty acid methyl esters is simple (direct rinsing); 5) Using the thermocatalytic transesterification method, there are fewer side reactions such as rearrangement and addition of certain fatty acids with special structures in phospholipids; 6) The phospholipid fatty acid analysis method has high accuracy and stability, and the microbial community and functional evaluation analysis is simpler and less complex. Attached Figure Description

[0021] Figure 1 Flowchart of a method for analyzing phospholipid fatty acids in soil by zirconium oxide solid-phase extraction combined with thermocatalytic transesterification;

[0022] Figure 2 Comparison and flow chart of zirconia and traditional silica gel solid phase extraction column for purifying soil phospholipids;

[0023] Figure 3 Optimization of thermocatalytic transesterification conditions: (A) reaction temperature; (B) reaction time; (C) amount of methanol in acyl acceptor. The optimization was carried out using the content of each biomarker phospholipid fatty acid (PLFA) group as an indicator. Bac: bacteria, G+: Gram-positive bacteria, G-: Gram-negative bacteria, Actino: actinomycetes, Fungi: fungi, Anae: anaerobic bacteria, AM: arbuscular mycorrhizal fungi.

[0024] Figure 4 Principal component analysis of fatty acid methyl esters in soil samples from quality control of thermocatalytic transesterification (qc1-qc6) and conventional acid / alkali transesterification (qc7-qc12) was conducted, and the stability of the methods was compared based on the fluctuation of the deviation (SD).

[0025] Figure 5 An optimized zirconium oxide solid-phase extraction combined with thermocatalytic transesterification method was used to analyze the chromatograms of phospholipid fatty acid methyl esters in typical soils. Detailed Implementation

[0026] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0027] Example 1

[0028] A method for analyzing phospholipid fatty acids in soil using zirconium oxide solid-phase extraction combined with thermocatalytic transesterification, the detailed operation of each step is mainly divided into the following 5 aspects:

[0029] Step S1, Soil phospholipid extraction

[0030] Weigh 2g of freeze-dried soil sample (QC sample of tobacco-grown soil), and extract soil phospholipids using 18mL of chloroform:methanol:phosphate buffer (1:2:0.8v:v:v) as the Bligh-Dyer extraction solvent. Vortex extract for 1h, centrifuge at 4000rpm for 5min, transfer the liquid to a centrifuge tube, add 1mL of chloroform, 2mL of ultrapure water and 0.8g of sodium chloride to separate the aqueous and organic phases. After centrifugation at 3000rpm, transfer the organic phase to a 10mL centrifuge tube, add 1.00g of anhydrous Na2SO4 to dry, vortex for 30s, let stand for 20min, filter through an organic filter membrane to another 10mL centrifuge tube, and blow dry with high-purity N2.

[0031] Step S2: Purification and enrichment of soil phospholipids using a zirconium oxide solid-phase extraction column.

[0032] The dried extract was dissolved in 600 μL of 0.1% formic acid-methanol solution, vortexed for 30 s, and centrifuged at 4000 rpm for 1 min. A 5 mL methanol-activated zirconia solid-phase extraction column (Supelco Hybrid SPE-Phospholipid) (30 mg) was prepared. The prepared sample was transferred onto the column. The centrifuge tube was washed with 200 μL of 0.1% formic acid-methanol and transferred to the column. The column was eluted with 2 × 700 μL of 0.1% formic acid-methanol to remove fatty acids, neutral phospholipids, and glycolipids; 5 × 1 mL of 5% formic acid-methanol was used to remove betaine lipids, and the eluent was collected in a 10 mL centrifuge tube. 3 × 1 mL of methanol was used to remove residual formic acid. 5 × 1 mL of 20% ammonia-methanol was used to elute the phospholipids enriched in the packing material, and the eluent was collected in a 10 mL centrifuge tube. 24 μL of 0.58 mg / mL palmitic acid-d was added. 31 (Deuterated) internal standard, concentrated to 0.8 mL with dry nitrogen, and 400 μL was used for thermocatalytic transesterification analysis.

[0033] Step S3, thermocatalytic transesterification analysis of phospholipid fatty acids

[0034] 400 μL of the extract solution mixed with an internal standard was subjected to thermocatalytic transesterification, simultaneously undergoing saponification and methyl esterification. A Swagelok straight-through fitting (SS-400-61, 1 / 4 inch) with two fittings (SS-400-P, 1 / 4 inch) was used as the thermocatalytic reactor. One side of the straight-through fitting was sealed with a fitting and filled with 0.25 g of diatomaceous earth. Then, 400 μL of the extract solution and 200 μL of methanol were added to the reactor, followed by the addition of 0.25 g of diatomaceous earth, and the other side was sealed. The reactor was reacted in a muffle furnace at 370 °C for 8 min, and then quenched with water for cooling.

[0035] Step S4, Elution and enrichment of soil phospholipids

[0036] Pour the diatomaceous earth from the reactor in step S3 into a 10 mL pipette tip, rinse with 2 mL of n-hexane, concentrate the eluent to 100 μL, and then analyze by GC-MS.

[0037] Step S5: Gas chromatography-mass spectrometry analysis of fatty acid methyl esters

[0038] Qualitative and quantitative analysis of PLFA methyl esters was performed using an Agilent 7890A GC-5975C MS (Agilent Technologies, Palo Alto, CA, USA) with a DB-5 column (30 m × 0.25 mm id × 0.25 μm film thickness). The injection port temperature was 250 °C, in non-separation mode, with ultrapure helium as the carrier gas at a constant flow rate of 2.0 mL / min. The column temperature was held at 50 °C for 2 min, then increased to 280 °C at 8 °C / min and held for 15 min. The transfer line temperature and solvent delay time were set to 280 °C and 5.0 min, respectively, while the ion source and quadrupole temperatures were maintained at 230 °C and 150 °C, respectively. Ionization energy: 70 eV; full scan mass range: 45-600 aum; acquisition mode: full scan (Scan). MS libraries: NIST14 and Willy08 were used for qualitative and relative quantification of fatty acid methyl esters (palmitic acid-d...). 31 A total of 42 fatty acids were identified, including 10 saturated fatty acids, 10 monounsaturated fatty acids, 12 branched-chain fatty acids, 3 10-methyl fatty acids, 2 cyclopropane fatty acids, and 5 other hydroxylated and polyunsaturated fatty acids. This indicates that the method can determine a wider range of fatty acid methyl esters for evaluating microbial diversity. The composition of phospholipid fatty acids in soil is shown in Table 1.

[0039] Table 1. Validation and comparison of methods for the analysis of phospholipid fatty acids in soil by zirconium oxide solid-phase extraction combined with thermocatalytic transesterification.

[0040]

[0041]

[0042] a Relative standard deviation; b Mean ± 6 standard deviations; c NS: Unspecified, Bac: Bacteria, G-: Gram-negative bacteria, G+: Gram-positive bacteria, Actino: Actinomycetes, Fungi: Fungi, AM: Arbuscular mycorrhizal fungi, Anae: Anaerobic bacteria

[0043] Compare with Example 1

[0044] Except for replacing step S2 with the following content, all other contents are the same as in Example 1.

[0045] Step S2, comparison of the purification and enrichment of soil phospholipids using silica gel solid-phase extraction column.

[0046] Dissolve the dried extract in 300 μL of CHCl3, vortex for 30 s, and centrifuge at 4000 rpm for 1 min. Activate a silica gel solid-phase extraction column (Bond Elut Si) (500 mg) with 5 mL of methanol, and then activate the column with 5 mL of CHCl3 until clear. Transfer the prepared sample to the column, wash the centrifuge tube with 1 mL of CHCl3 and transfer it to the column, rinse with 3 × 1 mL of CHCl3, and drain. Rinse with 4 × 1 mL of acetone, and drain. Rinse the bottom of the silica gel solid-phase extraction column with a small amount of methanol, rinse the phospholipids with 5 × 1 mL of methanol, collect the eluent in a 10 mL glass centrifuge tube, and add 24 μL of 0.58 mg / mL d. 31 Palmitic acid (deuterated) internal standard. After drying with high-purity N2, thermocatalytic transesterification analysis was performed.

[0047] Table 2 shows the comparison results of some detection results (betaine lipids and phospholipid fatty acids) in Example 1 and Control Example 1. Except for a few data anomalies, the phospholipid + betaine lipid fraction of the zirconia column and the phospholipid fraction of the silica column showed good consistency. The results proved that the methanol elution fraction of the silica column contained a high content of betaine lipids, and also proved that the zirconia solid phase extraction column can simultaneously separate betaine lipids and phospholipids, which can more accurately and precisely evaluate the microbial community and function.

[0048] Table 2 Comparison of betaine lipids and phospholipid fatty acids in soil samples separated by silica gel and zirconia solid-phase extraction columns.

[0049]

[0050]

[0051] a Fatty acid methyl esters and internal standard methyl palmitate-d 31 ratio

[0052] Example 2: Optimization of Zirconia Solid-Phase Extraction Purification

[0053] Thermocatalytic transesterification analysis of pure 2-oleoyl-1-palmitoyl-sn-glycerol-3-phosphocholine (PC) was performed, with the full scan ion peaks of each fatty acid and palmitic acid-d... 31The ratios of the m / z 77 ion peaks were calculated, and these ratios were used to calculate and evaluate the recovery rate and the degree of interference between lipids. The palmitic acid / trans-oleic acid ratio in the PC standard was 1.18, the palmitic acid / internal standard ratio was 14.5, and the trans-oleic acid / internal standard ratio was 12.4. Four common lipid standards were selected: neutral lipids—trans-oleic triglyceride (TR) 3 mg / mL, glycolipids—1,2-diac-cis-oleic acid, palmitic acid-3-O-(α-D-glucopyranosyl)-sn-glycerol (MGLc-DAG) 5 mg / mL, betaine lipids—1,2-palmitoyl betaine (DGTS) 2.5 mg / mL, and phospholipids—PC. 20 μL of each standard was mixed and separated using a zirconia solid-phase extraction column for optimization. As shown in Table 3, through optimization of the eluent, 5 mL of 20% ammonia-methanol was selected to elute PC. Under optimal conditions, the ratio of PC trans oleic acid to internal standard was approximately 12.0, the recovery rate was approximately 94%, and the fatty acid ratio was approximately 1.20. The results were similar to those of the standard, indicating that the interference from other lipids (neutral lipids, glycolipids, and betaine lipids) was relatively small (1,2-palmitoyl betaine (DGTS) contains palmitic acid, which can lead to an increase in the fatty acid ratio due to interference).

[0054] Table 3. Effects of different ammonia ratios and elution volumes on the elution recovery rate and interference level of phospholipids (PC).

[0055]

[0056] Example 3: Optimization of thermocatalytic transesterification of phospholipids in soil and purification of fatty acid methyl esters

[0057] The efficiency of thermocatalytic transesterification of triglycerides is affected by reaction temperature, time, the amount of methanol (acyl acceptor), and the diatomaceous earth / phospholipid ratio. When the mass ratio of porous material to triglycerides is >8, the transesterification efficiency is not significantly different. The low phospholipid content in soil lipid extracts leads to a higher mass ratio (typically >100), which has almost no effect on transesterification efficiency. Based on optimized conditions for triglycerides and the thermal decomposition characterization and preliminary study of phospholipids in thermogravimetric analysis, the reaction temperature, time, and amount of methanol (acyl acceptor) were optimized within the ranges of 260-420℃, 2-12 min, and 100-600 μL, respectively. The transesterification efficiencies under different conditions are shown below. Figure 3 As shown, the total amount of PLFA for each biomarker class was evaluated. Regarding reaction temperature, each biomarker class of PLFA showed a significant variation between 260 and 360 °C, exhibiting an S-shaped pattern, indicating that the conversion of PLFA to fatty acid methyl esters was completely saturated at ≥360 °C. Figure 3A). However, different fatty acid compositions exhibit different thermocatalytic methyl esterification properties; Bac, Anae, G-, and AM showed slight decreases at 420℃, while fungal decreases were significant at 420℃. Regarding reaction time, the PLFA of each biomarker increased significantly from 2 min to 4 min, then increased slowly, remaining essentially unchanged at 8 min. Figure 3 B). For the amount of methanol required as the acyl acceptor, only 0.2 mL of methanol is needed for the highest transesterification efficiency. Figure 3 C). When the methanol content was increased to 0.4 mL, the transesterification efficiency began to decrease to some extent. Based on this, 370-380℃ was selected as the reaction temperature, 6-10 min as the reaction time, and 0.2 mL of methanol as the methyl esterification solvent.

[0058] Complete extraction of PLFA methyl ester after thermocatalytic transesterification is crucial for achieving high recovery and sensitivity. The elution solvents of different polarities (n-hexane, n-hexane:methyl tert-butyl ether 1:1, methyl tert-butyl ether, dichloromethane, and chloroform) and volumes (1-5 mL) were optimized. The results showed that the polarity of the elution solvent had little effect on the recovery rate, indicating that PLFA methyl ester has a weak adsorption capacity on diatomaceous earth. Furthermore, only 2 mL of organic solvent was required to provide the maximum recovery rate. Considering the need for a less toxic organic solvent and a shorter sample preparation and drying time, 2 mL of n-hexane was chosen for eluting PLFA methyl ester.

[0059] Example 4: Validation of a method for analyzing phospholipid fatty acids in soil by combining zirconium oxide solid-phase extraction purification with thermocatalytic transesterification.

[0060] The PLFA analysis method developed using the thermocatalytic transesterification of porous diatomaceous earth was validated for linearity, repeatability, and reproducibility. As shown in Table 1, Pearson correlation analysis was performed on microbial-associated fatty acid methyl esters in QC samples of different weights (0.5, 1, 2, and 4 g) to assess linearity. Repeatability was determined by the relative standard deviation of the analysis of 5 QC samples on the same day, while reproducibility was determined by the relative standard deviation of the analysis of 5 QC samples daily for 4 consecutive days. The correlation coefficients for the 29 microbially specified PLFA components ranged from 0.926 to 0.999, with repeatability standard deviations (RSDs) all less than 12.1%, of which 28 components had RSDs less than 10%. The reproducibility standard deviations (RSDs) were less than 16.2%, of which 21 components had RSDs less than 10%. Comparison of the optimized thermocatalytic transesterification method with the acid / base transesterification method showed Fisher's minimum significance, indicating no significant difference in the concentration of specified PLFAs between the two methods. To further verify the reliability of the method in sample analysis, six QC samples from thermocatalytic transesterification and six QC samples from acid / base ester exchange were uniformly analyzed in the analytical sequence to monitor analytical performance. PCA was performed on the QC samples. Figure 4 The results show that the deviations of the two methods are controlled within 2 SD, but the acid-base method exhibits larger fluctuations. This indicates that both methods meet the quantitative requirements, but the new method demonstrates higher precision in sample analysis. The high reproducibility is likely due to the simplified procedure and more stable transesterification efficiency. Therefore, the thermocatalytic transesterification analysis method for PLFA in soil using porous diatomaceous earth exhibits excellent linearity, repeatability, and reproducibility, making it suitable for profiling soil PLFA. The optimized chromatogram of typical soil phospholipid fatty acid methyl esters is shown below. Figure 5 As shown.

[0061] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for analyzing phospholipid fatty acids in soil, characterized in that, Includes the following steps: Extraction of lipids from soil S1: Weigh the soil sample dried under low temperature and vacuum, add the extraction solution, vortex and shake to extract, add the layering solvent and inorganic salt to separate the layers, take the organic phase, dry and concentrate to obtain the soil lipid extract. S2 Purification of soil phospholipids: Soil lipid extracts were dissolved and purified using a zirconium peroxide solid-phase extraction column. Fatty acids, neutral triglycerides, glycolipids, betaine lipids, and phospholipids were eluted sequentially to obtain high-purity soil phospholipid extracts. After adding an internal standard, the extracts were mixed, concentrated, and subjected to thermal catalytic transesterification. The solution used to elute fatty acids, neutral triglycerides, and glycolipids was 0.1% formic acid in methanol; the solution used to elute betaine lipids was 5% formic acid in methanol; and the solution used to elute phospholipids was 20% ammonia in methanol. S3 Thermal catalytic transesterification of soil phospholipids: Soil phospholipids are transesterified using a thermal catalytic transesterification method, simultaneously undergoing saponification and methyl esterification to obtain fatty acid methyl ester products; wherein, the thermal catalytic transesterification method is as follows: soil phospholipid extract and solvent are added to a thermal catalytic reactor containing porous material, sealed, and heated in a muffle furnace for transesterification; the solvent is methanol, the porous material is diatomaceous earth, the reaction temperature is 360-380℃, and the time is 6-10 min; S4 Elution and enrichment of soil phospholipids: The fatty acid methyl ester product was transferred into the pipette tip, eluted with an organic solvent, concentrated, and then analyzed by gas chromatography-mass spectrometry. S5 Detection of soil phospholipids: Gas chromatography-mass spectrometry was used to separate, qualitatively and relatively quantify fatty acid methyl esters.

2. The method for analyzing phospholipid fatty acids in soil according to claim 1, characterized in that, In step S1, the extraction solution for the soil lipids is chloroform:methanol:phosphate buffer, with a volume ratio of 1:2:0.

8. v:v:v The layered solvent and inorganic salt are chloroform, ultrapure water and sodium chloride.

3. The method for analyzing phospholipid fatty acids in soil according to claim 1, characterized in that, In step S4, after the soil phospholipids are converted into fatty acid methyl esters, the fatty acid methyl esters are enriched by elution with the organic solvent n-hexane.

4. The method for analyzing phospholipid fatty acids in soil according to claim 1, characterized in that, In step S5, gas chromatography-mass spectrometry (GC-MS) analysis was performed using a DB-5 column with dimensions of 30 m × 0.25 mm id × 0.25 μm film thickness; the injection port temperature was 250 ℃, in non-separation mode; ultrapure helium was used as the carrier gas at a constant flow rate of 2.0 mL / min; the column temperature was held at 50 ℃ for 2 min, then increased to 280 ℃ at 8 ℃ / min and held for 15 min; the transfer line temperature and solvent delay time were set to 280 ℃ and 5.0 min, respectively; the ion source and quadrupole temperatures were maintained at 230 ℃ and 150 ℃, respectively; the ionization energy was 70 eV; the full scan mass number range was 45-600 aum; and the acquisition mode was full scan. MS libraries: NIST14 and Willy08, for qualitative and relative quantification of fatty acid methyl esters.

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