An analytical method for polymeric lignin structural units in watermelon rind

Abstract

This invention discloses an analytical method for polymeric lignin structural units in watermelon rind, belonging to the technical field of biomaterial composition determination methods. The method includes the following steps: S1, protective pretreatment: mixing the watermelon rind sample with a protective solution containing triphenylmethanethiol; S2, thioacid hydrolysis: adding a reaction solution containing boron trifluoride and ethanethiol, followed by heating to depolymerize; S3, in-situ neutralization and derivatization: adding a buffer solution, extracting and drying to obtain the organic phase, then adding a silanizing reagent for derivatization; S4, qualitative and quantitative analysis. This invention, through a multi-level design of structure protection, efficient derivatization, and system integration, establishes an analytical method that exhibits significant advantages in inhibiting condensation reactions, improving operational safety, shortening the analysis cycle, and achieving quantification. It effectively overcomes the technical bottlenecks of existing technologies, such as low recovery rate, high toxicity, long processing time, and incomplete detection, providing an excellent technical solution for the analysis of the composition of plant polymeric lignin.

Description

Technical Field

[0001] This invention belongs to the technical field of methods for determining the composition of biological substances, and relates to an analytical method for polymeric lignin structural units in watermelon peel. Background Technology

[0002] Lignin is a high-molecular-weight aromatic compound containing hydroxyl and methoxy groups, and is a derivative of phenylpropane. As a core chemical component of plants, it, together with cellulose and hemicellulose, forms the skeleton of the plant. It not only significantly improves the mechanical strength of cell walls, but also facilitates water transport in vascular tissues, and provides plants with key physiological functions such as support, environmental adaptation, resistance to diseases and pests, and lodging resistance.

[0003] Natural lignin is a high-molecular-weight polymer formed by the oxidative coupling of three aromatic alcohol precursors—p-coumarol, coniferyl alcohol, and sinapyl alcohol. These precursors correspond to the three basic structural units in polymeric lignin: the p-hydroxyphenyl structural unit (H-type), the guaiacol structural unit (G-type), and the syringyl structural unit (S-type). Accurately determining the content and ratio of these three structural units is crucial for in-depth research on plant growth and development, stress resistance mechanisms, and postharvest quality control of agricultural products.

[0004] The research value of the physiological functions and structural unit composition of polymeric lignin is particularly prominent in crops such as watermelon, where rind quality is the core commercial attribute. Specifically, the hardness, toughness, and disease resistance of watermelon rind directly determine its commercial value, storage and transportation losses, and post-harvest shelf life. These mechanical and preservation properties are closely related to the composition and structure of its cell wall, especially the deposition and distribution patterns of lignin. Therefore, in-depth research on the composition and ratio of polymeric lignin in watermelon rind is key to understanding the mechanisms of fruit development, ripening and softening, post-harvest browning, and disease. Current research indicates that the content of lignin and the ratio of its three structural units have a significant impact on the mechanical properties and chemical stability of the rind. For example, the accumulation of specific structural units (such as the H-type) has been reported to be related to young fruit development and epidermal toughness; while the S-type / G-type ratio has been reported to be related to the ease of degradation of lignin polymers, affecting the softening rate and disease resistance of the fruit under ripening or stress conditions.

[0005] Existing technologies include standard methods for determining total lignin content, such as acid hydrolysis and acetyl bromide methods. However, these methods cannot separate and qualitatively and quantitatively analyze the structural units of polymeric lignin. There are significant technical deficiencies in the detection of polymeric lignin structural units. For example, Chinese patent CN104535678A, entitled "A Method for Determining the Content of Lignin Monomers in Pear Peel," provides a method for determining the content of G-type and S-type lignin monomers in pear peel using thioacid hydrolysis coupled with gas chromatography-mass spectrometry. While this method achieves the determination of two specific structural units in pear peel, it fails to quantitatively detect H-type structural units. However, H-type structural units are widely found in many plants (especially herbaceous plants such as those in the Cucurbitaceae family) and are an important component of their lignin. Ignoring the detection of H-type structural units will fail to comprehensively reflect the complete lignin composition information of cucurbit crops such as watermelons.

[0006] Therefore, there is an urgent need in this field to develop an analytical method that can be applied to a wider range of plant species and accurately quantify the three lignin structural units, including the H-type. Summary of the Invention

[0007] The purpose of this invention is to provide an analytical method that can be widely applied to various plants and accurately quantify the content of three types of lignin structural units: H-type, G-type, and S-type, in order to solve the problems existing in the prior art.

[0008] The technical solution adopted in this invention is an analytical method for polymeric lignin structural units in watermelon rind. The key aspect is that this analytical method achieves qualitative and quantitative analysis of hydroxyphenyl, guaiacyl, and syringyl structural units by depolymerizing and derivatizing the polymeric lignin in watermelon rind. The analytical method includes the following steps:

[0009] S1. Protective pretreatment: The watermelon rind sample is mixed with a protective solution containing triphenylmethanethiol for protective pretreatment.

[0010] S2, Thioacidolysis: Add a reaction solution containing boron trifluoride and ethanethiol directly to the reaction system of step S1, and heat to carry out the depolymerization reaction;

[0011] S3. In-situ neutralization and derivatization: After the depolymerization reaction is completed, a weakly alkaline buffer solution is added to the system to neutralize the excess acid. After extraction and drying to obtain the organic phase, a silanizing agent is added to carry out the derivatization reaction. The silanizing agent is composed of N-methyl-N-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane.

[0012] S4. Qualitative and quantitative analysis: Gas chromatography-mass spectrometry was used to analyze the derivatized products to complete the qualitative and quantitative analysis of the p-hydroxyphenyl structural unit, guaiacyl structural unit, and syringyl structural unit.

[0013] Furthermore, in step S1 above, the protective solution is a dioxane solution containing triphenylmethanethiol at a mass concentration of 4% to 6%; the volume of the protective solution used for each 50 mg watermelon rind sample is 1 mL.

[0014] Specifically, in step S1 above, the temperature for the protective pretreatment is 75℃~85℃, and the treatment time is 50min~70min.

[0015] Furthermore, in step S2 above, the reaction solution containing boron trifluoride and ethanethiol is a dioxane solution with a boron trifluoride mass concentration of 4% to 6% and an ethanethiol mass concentration of 18% to 22%; the volume of the reaction solution containing boron trifluoride and ethanethiol used to treat 50 mg of watermelon peel sample is 1 mL.

[0016] Specifically, in step S2 above, the reaction temperature of the depolymerization reaction is 98℃~102℃, and the reaction time is 3.5h~4.5h.

[0017] Furthermore, in step S3 above, the weakly alkaline buffer solution is an ammonium acetate buffer solution with a concentration of 0.8 mol / L to 1.2 mol / L, and the pH value of the neutralized system is 4.8 to 5.2.

[0018] Furthermore, in step S3 above, during the extraction step, a dichloromethane solution of the internal standard is added, and the pH of the system is adjusted to 3.0–4.0 with an acetic acid solution; the internal standard is tetracosane.

[0019] Furthermore, in step S3 above, the extraction involves adding ultrapure water and dichloromethane to the system, vortexing and mixing, allowing it to stand and separate into layers, then removing the lower organic phase and drying it with anhydrous Na2SO4 or anhydrous MgSO4.

[0020] Specifically, in step S3 above, the volume ratio of N-methyl-N-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in the silanizing reagent is (40-60):1; the volume of silanizing reagent used for each 50mg watermelon peel sample is 1mL.

[0021] More specifically, in step S3 above, the temperature of the derivatization reaction is 38℃~42℃, and the reaction time is 25min~35min.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention provides an analytical method for polymeric lignin structural units in watermelon rind. Through the synergistic effect of sample pretreatment with triphenylmethanethiol protectant and a green silanization derivatization system, it solves the technical problems of existing analytical methods for polymeric lignin structural units, such as severe condensation reactions, difficulty in accurately quantifying low-content lignin structural units like the H-type, cumbersome analytical procedures, and the use of highly toxic reagents.

[0024] Specific technical effects include:

[0025] First, this invention establishes a lignin structure stabilization mechanism based on acid-instable protecting groups such as triphenylmethanethiol, effectively suppressing condensation side reactions during depolymerization. Addressing the critical issue of existing thioacid hydrolysis methods easily inducing the condensation of active intermediates during the degradation of polymeric lignin, leading to low structural unit recovery rates, this invention innovatively employs triphenylmethanethiol as a protecting reagent. Its unique triphenylmethyl structure can preferentially occupy the active sites of polymeric lignin through reversible chemical bonding during the pretreatment stage, and can be rapidly removed in the subsequent acidic depolymerization environment. This protects the native structure of lignin from damage without interfering with the normal depolymerization reaction pathway, laying a solid technical foundation for the complete and accurate analysis of polymeric lignin structural units.

[0026] Secondly, this invention employs a novel silanization reagent system, successfully replacing the traditional, highly toxic, and inefficient pyrimidine / BSA derivatization scheme. The pyrimidine used in the traditional method not only has a strong odor and toxicity, posing a hazard to laboratory personnel and the environment, but also requires a derivatization time of up to 4 hours when combined with BSA, resulting in low efficiency. In contrast, the silanization reagent system used in this invention is not only less toxic and has a milder odor, but also shortens the derivatization reaction time from 4 hours to 30 minutes. This significantly improves experimental safety while simultaneously enhancing analytical efficiency, demonstrating the green, environmentally friendly, and highly efficient advantages of this invention.

[0027] Third, this invention establishes a complete and reliable analytical method for polymeric lignin structural units through the systematic integration of protective pretreatment and green derivatization. This method not only achieves stable detection of G-type and S-type lignin structural units in watermelon rind but also overcomes the technical bottleneck of effectively quantifying H-type structural units. The synergistic design among the steps of this invention ensures optimization throughout the entire process, from sample pretreatment to final instrumental analysis, demonstrating significant advantages in method precision, accuracy, and operational safety, and providing strong technical support for in-depth research on the structural composition of polymeric lignin.

[0028] In summary, through a three-tiered innovative design of "structural protection - efficient derivatization - system integration," the analytical method established by this invention demonstrates significant advantages in suppressing condensation reactions, improving operational safety, shortening the analysis cycle, and achieving accurate quantitative analysis of complete polymeric lignin structural units. This invention effectively overcomes the technical bottlenecks of low recovery rate, high toxicity, long processing time, and incomplete detection results in existing technologies, providing an excellent technical solution for the qualitative and quantitative analysis of plant lignin composition. Attached Figure Description

[0029] Figure 1 This is a partial test spectrum of Embodiment 1 of the present invention.

[0030] Figure 2 This is a partial test spectrum of Embodiment 2 of the present invention.

[0031] Figure 3 This is a partial test spectrum of Embodiment 3 of the present invention. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] To ensure the consistency of experimental materials, all examples and comparative experiments used rind samples from the same mature and intact Meijia Red Honey watermelon, a variety bred by the Institute of Economic Crops, Hebei Academy of Agricultural and Forestry Sciences. Example 1

[0038] This embodiment provides a method for analyzing polymeric lignin structural units in watermelon rind, the specific steps of which include:

[0039] S1. Protective pretreatment:

[0040] Weigh 50 mg of the ground watermelon rind sample, add 1 mL of protective solution containing triphenylmethanethiol and mix, and perform protective pretreatment at 80 °C for 60 min.

[0041] The protective solution is a dioxane solution containing 5% triphenylmethanethiol by mass.

[0042] S2, Thioacidolysis:

[0043] Add 1 mL of a reaction solution containing boron trifluoride and ethanethiol directly to the reaction system in step S1, heat to 100°C, and carry out the depolymerization reaction for 4.0 h.

[0044] In the reaction solution containing boron trifluoride and ethanethiol, the mass concentration of boron trifluoride is 5%, the mass concentration of ethanethiol is 20%, and the solvent is a dioxane solution.

[0045] S3, in-situ neutralization and derivatization:

[0046] After the depolymerization reaction was completed, ammonium acetate with a concentration of 1 mol / L was added to neutralize the excess acid, and the pH value of the neutralized system was 4.9.

[0047] Add 0.2 mL of dichloromethane solution containing 0.1 mg / mL tetradecane internal standard, and adjust the pH to 3.6 with 0.4 mol / L acetic acid solution;

[0048] Add 3 mL of ultrapure water and 1 mL of dichloromethane, vortex mix for 1.5 min, let stand for 30 min to separate the layers, remove the lower organic phase, add anhydrous Na2SO4 to the organic phase and dry to obtain the organic phase extract;

[0049] Add 1 mL of silanizing reagent and carry out the derivatization reaction at 40 °C for 30 min. Then, place the system at -20 °C for 5 min to terminate the reaction.

[0050] The silanizing agent consists of N-methyl-N-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in a volume ratio of 50:1, without any other solvent.

[0051] S4. Qualitative and Quantitative Analysis:

[0052] Take 1 mL of the reaction solution after derivatization in step S3 and analyze it directly using gas chromatography-mass spectrometry.

[0053] Gas chromatography detection conditions:

[0054] Column: TG-5MS (30m × 0.25mm × 0.25μm);

[0055] Temperature program: Initial temperature 50℃, increase to 220℃ at a rate of 35℃ / min, continue to increase to 230℃ at a rate of 0.5℃ / min, and finally increase to 280℃ at a rate of 50℃ / min, and hold for 7 min;

[0056] Inlet temperature: 250℃;

[0057] Carrier gas flow rate: 1.2 mL / min;

[0058] Flow split ratio: 20:1;

[0059] Injection volume: 2 μL.

[0060] Mass spectrometry conditions:

[0061] Ion source temperature: 280℃, transfer line temperature: 280℃, solvent delay time: 5.00 min, scan range: 40-650 amu, ion source: EI source 70 eV.

[0062] After the on-machine inspection is completed, the content of each type of lignin structural unit is calculated;

[0063] The same watermelon rind sample was selected, and the operation of this embodiment was repeated for a total of 6 tests. The results are shown in Table 1, and some test chromatograms are shown in [reference needed]. Figure 1 . Example 2

[0064] This embodiment provides a method for analyzing polymeric lignin structural units in watermelon rind, the specific steps of which include:

[0065] S1. Protective pretreatment:

[0066] Weigh 50 mg of the ground watermelon rind sample, add 1 mL of protective solution containing triphenylmethanethiol and mix, and perform protective pretreatment at 75 °C for 70 min.

[0067] The protective solution is a dioxane solution containing 6% triphenylmethanethiol by mass.

[0068] S2, Thioacidolysis:

[0069] Add 1 mL of a reaction solution containing boron trifluoride and ethanethiol directly to the reaction system in step S1, heat to 102°C, and carry out the depolymerization reaction for 3.5 h.

[0070] In the reaction solution containing boron trifluoride and ethanethiol, the mass concentration of boron trifluoride is 6%, the mass concentration of ethanethiol is 18%, and the solvent is a dioxane solution.

[0071] S3, in-situ neutralization and derivatization:

[0072] After the depolymerization reaction was completed, ammonium acetate at a concentration of 0.8 mol / L was added to neutralize the excess acid, and the pH of the neutralized system was 5.2.

[0073] Add 0.2 mL of dichloromethane solution containing 0.1 mg / mL tetracosane internal standard, and adjust the pH to 4.0 with 0.5 mol / L acetic acid solution;

[0074] Add 3 mL of ultrapure water and 1 mL of dichloromethane, vortex mix for 2 min, let stand for 40 min to separate the layers, remove the lower organic phase, add anhydrous MgSO4 to the organic phase and dry to obtain the organic phase extract;

[0075] Add 1 mL of silanizing reagent and carry out the derivatization reaction at 42 °C for 25 min. Then place the system at -20 °C for 5 min to terminate the reaction.

[0076] The silanizing agent consists of N-methyl-N-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in a volume ratio of 60:1, without any other solvent.

[0077] S4. Qualitative and Quantitative Analysis:

[0078] Take 1 mL of the reaction solution after derivatization in step S3 and analyze it directly using gas chromatography-mass spectrometry. The analysis method and conditions are the same as those in step S4 of Example 1.

[0079] The same watermelon rind sample was selected, and the operation of this embodiment was repeated for a total of 6 tests. The results are shown in Table 1, and some test chromatograms are shown in [reference needed]. Figure 2 . Example 3

[0080] This embodiment provides a method for analyzing polymeric lignin structural units in watermelon rind, the specific steps of which include:

[0081] S1. Protective pretreatment:

[0082] Weigh 50 mg of the ground watermelon rind sample, add 1 mL of protective solution containing triphenylmethanethiol and mix, and perform protective pretreatment at 85 °C for 50 min.

[0083] The protective solution is a dioxane solution containing 4% by mass of triphenylmethanethiol.

[0084] S2, Thioacidolysis:

[0085] Add 1 mL of a reaction solution containing boron trifluoride and ethanethiol directly to the reaction system in step S1, heat to 98°C, and carry out the depolymerization reaction for 4.5 h.

[0086] In the reaction solution containing boron trifluoride and ethanethiol, the mass concentration of boron trifluoride is 4%, the mass concentration of ethanethiol is 22%, and the solvent is a dioxane solution.

[0087] S3, in-situ neutralization and derivatization:

[0088] After the depolymerization reaction was completed, ammonium acetate at a concentration of 1.2 mol / L was added to neutralize the excess acid, and the pH of the neutralized system was 4.8.

[0089] Add 0.2 mL of dichloromethane solution containing 0.1 mg / mL tetracosane internal standard, and adjust the pH to 3.0 with 0.3 mol / L acetic acid solution;

[0090] Add 3 mL of ultrapure water and 1 mL of dichloromethane, vortex mix for 1 min, let stand for 20 min to separate the layers, remove the lower organic phase, add anhydrous Na2SO4 to the organic phase and dry to obtain the organic phase extract;

[0091] Add 1 mL of silanizing reagent and carry out the derivatization reaction at 38 °C for 35 min. Then place the system at -20 °C for 5 min to terminate the reaction.

[0092] The silanizing agent consists of N-methyl-N-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in a volume ratio of 40:1, with no other solvents.

[0093] S4. Qualitative and Quantitative Analysis:

[0094] Take 1 mL of the reaction solution after derivatization in step S3 and analyze it directly using gas chromatography-mass spectrometry. The analysis method and conditions are the same as those in step S4 of Example 1.

[0095] The same watermelon rind sample was selected, and the operation of this embodiment was repeated for a total of 6 tests. The results are shown in Table 1, and some test chromatograms are shown in [reference needed]. Figure 3 . Comparative Example 1

[0096] This comparative example provides an analytical method for polymeric lignin structural units in watermelon rind. The main difference from Example 1 is that step S1 is omitted. The specific steps include:

[0097] S1, Thioacidolysis:

[0098] Add 1 mL of a reaction solution containing boron trifluoride and ethanethiol to 50 mg of ground watermelon rind sample, heat to 100 °C, and carry out the depolymerization reaction for 4.0 h.

[0099] In the reaction solution containing boron trifluoride and ethanethiol, the mass concentration of boron trifluoride is 5%, the mass concentration of ethanethiol is 20%, and the solvent is a dioxane solution.

[0100] S2, In-situ neutralization and derivatization: Same as step S3 in Example 1.

[0101] S3. Qualitative and quantitative analysis: The analytical conditions and operations are the same as step S4 in Example 1, but the samples are not repeated. The results are shown in Table 2. Comparative Example 2

[0102] This comparative example provides an analytical method for polymeric lignin structural units in watermelon rind. The main difference from Example 1 is that L-cysteine ​​solution is used instead of the protective solution in step S1. The specific steps include:

[0103] S1. Protective pretreatment:

[0104] Weigh 50 mg of the ground watermelon rind sample and add 1 mL of a mixed solution of dioxane and water containing 5% L-cysteine ​​(dioxane to water volume ratio of 9:1). Perform a protective pretreatment at 80 °C for 60 min.

[0105] S2, Thioacid hydrolysis: Same as step S2 in Example 1.

[0106] S3, In-situ neutralization and derivatization: Same as step S3 in Example 1.

[0107] S4. Qualitative and quantitative analysis: The analytical conditions and operations are the same as step S4 in Example 1, but the samples are not repeated. The results are shown in Table 2. Comparative Example 3

[0108] This comparative example provides an analytical method for polymeric lignin structural units in watermelon rind. The main difference from Example 1 is that pyrimidine and BSA reagents are used instead of the silanizing reagent used in step S3. The specific steps include:

[0109] S1. Protective pretreatment: Same as step S1 in Example 1.

[0110] S2, Thioacid hydrolysis: Same as step S2 in Example 1.

[0111] S3, in-situ neutralization and derivatization:

[0112] After the depolymerization reaction was completed, ammonium acetate with a concentration of 1 mol / L was added to neutralize the excess acid, and the pH value of the neutralized system was 4.9.

[0113] Add 0.2 mL of dichloromethane solution containing 0.1 mg / mL tetradecane internal standard, and adjust the pH to 3.6 with 0.4 mol / L acetic acid solution;

[0114] Add 3 mL of ultrapure water and 1 mL of dichloromethane, vortex mix for 1.5 min, let stand for 30 min to separate the layers, remove the lower organic phase, add anhydrous Na2SO4 to the organic phase and dry to obtain the organic phase extract;

[0115] The dried organic phase extract was gently evaporated under nitrogen blowing at 45°C. The evaporated product was then redissolved in 0.4 mL of dichloromethane, followed by the addition of 50 μL of pyrimidine and 100 μL of BSA. The mixture was allowed to stand at 25°C for 4 h to carry out the derivatization reaction. The resulting solution was the derivatization reaction solution.

[0116] S4. Qualitative and quantitative analysis: The analytical conditions and operations are the same as step S4 in Example 1, but the samples are not repeated. The results are shown in Table 2.

[0117] Table 1: Summary of Analysis Results of Examples 1 to 3

[0118]

[0119] Table 2: Summary of Analysis Results for Comparative Examples 1 to 3

[0120]

[0121] As shown in Tables 1 and 2, the analytical method provided by this invention can achieve stable and accurate analysis of the three types of lignin structural units (H, G, and S) in watermelon rind. The test results of Example 1, in particular, exhibit excellent reproducibility while maintaining a high recovery rate. The coefficients of variation of the detected values ​​of each lignin structural unit in all three examples are less than 4%, demonstrating the high precision and reliability of the analytical method of this invention.

[0122] The results of each comparative example demonstrate the technical advantages of the analytical method of this invention from different perspectives. In Comparative Example 1, due to the lack of a protective pretreatment process, the polymerized lignin underwent severe condensation during depolymerization, resulting in a significant decrease in the recovery rates of G-type and S-type structural units compared to the embodiments of this invention. The content of the H-type structural unit was extremely low, making effective quantification difficult in conventional detection. This fully demonstrates that the protective pretreatment of this invention plays a crucial role in achieving accurate quantification of the three lignin structural units. In Comparative Example 2, L-cysteine ​​was used as a protective agent. The stable CS bonds formed by this reagent are difficult to break under subsequent thioacidolysis conditions, severely hindering the formation pathway of lignin structural unit derivatives. This resulted in the loss of signals for the G, H, and S structural units, a significant decrease in the recovery rate of the G-type, and the inability to detect the H and S-types. This indicates that only the unique design of using triphenylmethanethiol as a protective agent in this invention can achieve the effect of effective protection without interfering with subsequent detection processes. Comparative Example 3 uses traditional pyrimidine / BSA derivatization reagents. Although it can detect the three structural units, its more cumbersome operation process (such as nitrogen blowing concentration which may introduce losses), long derivatization time of up to 4 hours, and use of highly toxic reagents that are unfriendly to personnel and the environment, all contribute to its low analytical efficiency and lower recovery rate than the method of this invention. This demonstrates the comprehensive advantages of this invention in terms of derivatization efficiency and analytical sensitivity.

[0123] In summary, this invention, through the synergistic innovation of using triphenylmethanethiol as a protective agent for sample pretreatment and silanization reagent derivatization system, successfully overcomes the core technical bottlenecks commonly found in existing technologies, such as low recovery rates due to condensation reactions, difficulty in effectively detecting low-content structural units, and complex operating procedures. It provides a reliable solution for achieving accurate, efficient, and complete analysis of the composition of polymeric lignin.

[0124] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for analyzing polymeric lignin structural units in watermelon rind, characterized in that, The analytical method describes the qualitative and quantitative analysis of hydroxyphenyl, guaiacyl, and syringyl structural units by depolymerizing and derivatizing the polymerized lignin in watermelon rind. The analytical method includes the following steps: S1. Protective pretreatment: The watermelon rind sample is mixed with a protective solution containing triphenylmethanethiol for protective pretreatment; the protective solution is a dioxane solution containing 4% to 6% triphenylmethanethiol by mass; the treatment temperature for the protective pretreatment is 75℃ to 85℃, and the treatment time is 50 min to 70 min. S2, Thioacidolysis: A reaction solution containing boron trifluoride and ethanethiol is directly added to the reaction system of step S1, and the reaction is heated to carry out the depolymerization reaction; the reaction solution containing boron trifluoride and ethanethiol is a dioxane solution with a boron trifluoride mass concentration of 4% to 6% and an ethanethiol mass concentration of 18% to 22%; the reaction temperature of the depolymerization reaction is 98℃ to 102℃, and the reaction time is 3.5h to 4.5h; S3. In-situ Neutralization and Derivatization: After the depolymerization reaction, a weakly alkaline buffer solution is added to the system to neutralize excess acid. After extraction and drying to obtain the organic phase, a silanizing reagent is added for derivatization. The silanizing reagent consists of N-methyl-N-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane. The weakly alkaline buffer solution is an ammonium acetate buffer solution with a concentration of 0.8 mol / L to 1.2 mol / L, and the pH of the neutralized system is 4.8 to 5.

2. In the extraction step, a dichloromethane solution containing an internal standard is added, and the pH of the system is adjusted to 3.0 to 4.0 with an acetic acid solution. The internal standard is tetracosane. The extraction involves adding ultrapure water and dichloromethane to the system, vortexing, allowing it to stand and separate into layers, then aspirating the lower organic phase and drying it with anhydrous Na2SO4 or anhydrous MgSO4. S4. Qualitative and quantitative analysis: Gas chromatography-mass spectrometry was used to analyze the derivatized products to complete the qualitative and quantitative analysis of the p-hydroxyphenyl structural unit, guaiacyl structural unit, and syringyl structural unit.

2. The analytical method according to claim 1, characterized in that, In step S1, the volume of protective liquid used for each 50mg watermelon rind sample is 1mL.

3. The analytical method according to claim 1, characterized in that, In step S2, the volume of the reaction liquid containing boron trifluoride and ethanethiol used to process 50 mg of watermelon rind sample is 1 mL.

4. The analytical method according to claim 1, characterized in that, In step S3, the volume ratio of N-methyl-N-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in the silanizing reagent is (40-60):1; the volume of silanizing reagent used for each 50mg watermelon peel sample is 1mL.

5. The analytical method according to claim 1, characterized in that, In step S3, the temperature of the derivatization reaction is 38℃~42℃, and the reaction time is 25min~35min.

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