Method for detecting trans-4,5-epoxy-2(e)-decanal based on lc-ms / ms

Abstract

This invention provides a method for detecting trans-4,5-epoxy-2(E)-decaldehyde based on LC-MS / MS. First, trans-4,5-epoxy-2(E)-decaldehyde standards are prepared into standard solutions of different concentrations. Then, the standard solutions are derivatized by solid-phase extraction with 2,4-dinitrophenylhydrazine (DNPH) to obtain analytical samples containing standard solutions of different concentrations. Next, LC-MS / MS analysis is performed on the analytical samples of different concentrations of standard solutions to obtain a standard curve with standard concentration as the abscissa and peak area as the ordinate. The sample to be tested is detected using LC-MS / MS, and the content of trans-4,5-epoxy-2(E)-decaldehyde in the sample is determined using the standard curve. This invention achieves accurate qualitative and quantitative analysis of this compound and establishes a standardized platform for the role of this compound in systemic health and disease.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to a method for the detection of trans-4,5-epoxy-2(E)-decanoal based on LC-MS / MS. Background Technology

[0002] Volatile organic compounds (VOCs) are a class of carbon-based compounds that readily evaporate at room temperature, typically with boiling points ranging from 50°C to 250°C. This category includes extremely volatile organic compounds (VVOCs), volatile organic compounds (VOCs), and semi-volatile organic compounds (SVOCs), which are widely found in industrial emissions, environmental media, human respiration, and food. Although VOC concentrations are low, they can significantly impact ecosystems and human health, leading to respiratory diseases and carcinogenic effects. Conversely, they play a crucial indicative role in disease diagnosis (e.g., detecting respiratory biomarkers for lung cancer) and food flavor analysis. VOC detection is a key link connecting human health, environmental safety, and industrial production. Its significance lies not only in identifying pollution or disease markers but also in driving preventative interventions and sustainable development strategies through real-time and accurate monitoring data.

[0003] Significant progress has been made in the study of volatile organic compounds (VOCs) in blood for biomedical detection. Among them, trans-4,5-epoxy-2-decenal, a VOC found in mammalian blood, possesses unique chemical activity and olfactory sensitivity due to its molecular structure. Previous literature indicates that this substance has a distinctly characteristic "metallic, bloody" odor. This unique odor makes it play a crucial role in the overall composition of blood odor. This compound exhibits remarkable systemic stability, being detected not only in blood but also in various processed foods such as duck, French fries, tea, and wine. Its widespread presence suggests a common biochemical origin, with lipid peroxidation considered the primary formation pathway. Further research indicates that its formation is closely related to lipid peroxidation. This formation mechanism reasonably explains its coexistence in mammalian blood and thermally processed foods. The production of trans-4,5-epoxy-2(E)-decenal mainly originates from the oxidation of polyunsaturated fatty acids such as linoleic acid and linolenic acid. Taking linoleic acid as an example, its oxidation first forms various isomers. These isomers decompose to produce substances such as aldehydes, including (E,E)-2,4-decadienal. As a key intermediate, (E,E)-2,4-decadienal is further oxidized under heating conditions to produce trans-4,5-epoxy-(E)-2-decadienal. Additionally, heating phospholipids oxidizes their fatty acid components, which can also produce trans-4,5-epoxy-2(E)-decal.

[0004] Oxidative stress (OS), characterized by an imbalance between oxidation and antioxidation that favors oxidation, triggers neutrophil infiltration, increases protease secretion, and leads to excessive formation of oxidative byproducts. As a detrimental consequence of free radical accumulation, OS is considered a key factor in the pathogenesis of aging and diseases. Notably, OS imbalance and exacerbated lipid peroxidation are associated with various diseases, such as cardiovascular diseases, neurodegenerative diseases, and diabetes, including Alzheimer's and Parkinson's diseases. These pathological states may alter blood concentrations of aldehyde metabolites, including trans-4,5-epoxy-2(E)-decaldehyde. Simultaneously, during inflammation, metabolic shifts and redox imbalances further promote lipid peroxidation, potentially altering the production levels of this compound. Accurate blood concentration measurements can be achieved by establishing an LC-MS / MS method for detecting trans-4,5-epoxy-2(E)-decaldehyde. This analytical capability supports physicians in the early diagnosis, disease assessment, and monitoring of treatment efficacy for the aforementioned diseases and symptoms, enabling timely intervention and improved patient prognosis.

[0005] It must be emphasized that current methods for detecting trans-4,5-epoxy-2(E)-decanal remain significantly limited. Existing research has primarily focused on its olfactory properties and behavioral effects in animal models, providing an important foundation for understanding this compound, but achieving accurate quantification in complex biological matrices such as blood remains a major challenge. Currently, the main analytical techniques for trans-4,5-epoxy-2(E)-decanal in samples are gas chromatography-olfactometry-mass spectrometry (GC-O-MS) and isotope dilution assays (IDA), each with operational limitations. While GC-O-MS integrates the high-resolution separation of gas chromatography with structural description through mass spectrometry and human olfactory assessment, making it widely applicable for flavor analysis in the food and pharmaceutical industries, its clinical applicability is hampered by methodological subjectivity, inconsistent reproducibility, stringent technical requirements, and limited high-throughput capabilities. Similarly, isotope dilution assays face challenges of cost-effectiveness and standardization. Most importantly, current testing efforts primarily focus on simple samples such as duck meat, wine, and tea, while a reliable method for detecting trans-4,5-epoxy-2(E)-decaldehyde in human blood is lacking—a significant methodological shortcoming. This substance is both a volatile organic compound and a potential pathophysiological indicator, giving it dual importance.

[0006] Therefore, developing an efficient, sensitive, and accurate method for detecting blood-derived trans-4,5-epoxy-2(E)-decaldehyde has profound scientific and practical significance for clinical medicine and disease diagnosis. This volatile biomarker acts as a molecular bridge between physiological and pathological states, requiring precise quantitative methods to realize its promises in clinical diagnosis, treatment monitoring, and mechanistic studies of disease pathogenesis. Current methodological limitations particularly hinder its analysis in human blood—the most clinically relevant matrix. Summary of the Invention

[0007] The purpose of this invention is to provide a detection method for trans-4,5-epoxy-2(E)-decaldehyde based on LC-MS / MS (liquid chromatography-tandem mass spectrometry) to solve the technical problems of conventional detection methods such as gas chromatography-olfactometry-mass spectrometry (GC-O-MS), which are limited by inherent subjectivity, poor reproducibility, strict operating requirements and low throughput, as well as clinical reliability.

[0008] To achieve the above objectives, this invention provides a method for detecting trans-4,5-epoxy-2(E)-decanoal based on LC-MS / MS, the specific steps of which are as follows: (1) First, prepare standard solutions of different concentrations from trans-4,5-epoxy-2(E)-decaldehyde standard, and derivatize the standard solutions with 2,4-dinitrophenylhydrazine (DNPH) to obtain analytical samples containing standard solutions of different concentrations; (2) Then, LC-MS / MS analysis was performed on the standard solution samples with different concentrations to obtain a standard curve with the standard concentration as the abscissa and the peak area as the ordinate. (3) Solid-phase extraction and 2,4-dinitrophenylhydrazine (DNPH) derivatization were performed on the test sample, and the sample was detected by LC-MS / MS. The content of trans-4,5-epoxy-2(E)-decaldehyde in the test sample was determined by the standard curve obtained in step (2). The LC-MS conditions are as follows: Mobile phase: a mixture of (A) water and (B) acetonitrile; Flow rate: 0.3 mL / min; Injection volume: 1 μL; Column temperature: 40℃; The gradient elution conditions are shown in Table 1; Table 1 ; MS conditions are as follows: Detection method: Multiple reaction monitoring (MRM) Scanning method: Electrospray ionization negative ion mode Capillary voltage: 2.50 kV; Drying gas: nitrogen, flow rate 650 L / h; Collision gas: Helium; Tapered hole voltage: 10V; Mother-daughter ion pairs: 347 / 163, CE: 17 eV; 347 / 204, CE: 15 eV.

[0009] Preferably, in step (1), the standard solution is prepared by first dissolving trans-4,5-epoxy-2(E)-decanoal standard in acetonitrile to obtain stock solutions with concentrations of 20 μg / mL, 1 μg / mL, and 100 μg / mL, respectively, and then diluting the stock solutions with acetonitrile to obtain standard solutions of different concentrations. Specifically, the standard solution concentrations include: 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL.

[0010] Preferably, in step (1), the specific method of solid phase extraction is as follows: the standard solution and the extract are mixed at a volume ratio of 1:4, sealed and shaken for 20 minutes; the extract is transferred to a centrifuge tube using a syringe, and the supernatant is collected by centrifugation; the supernatant is added to the solid phase extraction column and the effluent is collected; wherein, the extract is an acetonitrile solution containing 0.5% formic acid by mass.

[0011] Further preferred centrifugation conditions are: 10,000 rpm for 10 minutes.

[0012] In a further preferred embodiment, the solid-phase extraction column is activated using equal volumes of methanol and water.

[0013] Preferably, in step (1), 2,4-dinitrophenylhydrazine is prepared into a DNPH solution with a concentration of 2 mmol / L using acetonitrile, and then mixed with the solid phase extraction effluent; the volume ratio of the solid phase extraction effluent to the DNPH solution is 5:1.

[0014] Preferably, in step (1), the solid phase extract effluent is filtered using a 0.22 μm syringe filter and then mixed with DNPH solution.

[0015] Preferably, in step (1), the derivatization reaction conditions are: derivatization reaction at 40°C for 1 hour.

[0016] Preferably, in step (3), solid-phase extraction is performed using a solid-phase extraction column. The solid-phase extraction column is obtained by filling a syringe-type polypropylene hollow column with packing material, freeze-drying it, placing it on an upper sieve plate, and pressing the packing material. The packing material is prepared by the following method: (3-1) First, graphene oxide silica gel composite material was prepared using graphite powder and amino silica gel as raw materials; (3-2) The graphene oxide silica gel composite material was further modified using octadecyltrimethoxysilane to obtain the modified composite material; (3-3) Finally, the modified composite material is surface-modified using ionic liquid to obtain the filler.

[0017] More preferably, the length of the polypropylene hollow tube column is 7-8 cm, and the height of the compressed filler is 4-5 cm.

[0018] A further preferred method for step (3-1) is as follows: First, add sodium nitrate and concentrated sulfuric acid to graphite powder, stir for 30-40 minutes at 0-5℃ and 200-300 r / min, slowly add potassium permanganate, dilute with water, stir and mix well, add warm water and hydrogen peroxide, stir and mix well, perform the first post-treatment to obtain graphene oxide; then, ultrasonically disperse the graphene oxide in N,N-dimethylformamide, add amino silica gel and dicyclohexylcarbodiimide, heat under reflux, and perform the second post-treatment to obtain the final product.

[0019] More preferably, the ratio of graphite powder, sodium nitrate, concentrated sulfuric acid, potassium permanganate, dilution water, warm water, and hydrogen peroxide is 10g: 5-6g: 220-230mL: 28-32g: 480-500mL: 1-1.1L: 100-120mL, wherein the concentrated sulfuric acid has a mass concentration of 98%, the warm water temperature is 50-60℃, and the hydrogen peroxide has a mass concentration of 20-30%.

[0020] More preferably, the first post-treatment includes: centrifugation to collect the precipitate, washing with a 10% hydrochloric acid solution, washing with deionized water, and drying.

[0021] More preferably, the ratio of graphene oxide, N,N-dimethylformamide, aminosilicone, and dicyclohexylcarbodiimide is 1 mg: 2.3-2.5 mL: 20-22 mg: 0.85-0.95 mg.

[0022] More preferably, the heating reflux time is 25 to 30 hours.

[0023] In a further preferred embodiment, the second post-treatment includes: centrifugation to collect the precipitate, washing with deionized water, washing with methanol, and drying.

[0024] A further preferred method is as follows: the graphene oxide silica gel composite material is ultrasonically dispersed in anhydrous toluene containing triethylamine, then octadecyltrimethoxysilane is added, and the mixture is heated to reflux under nitrogen protection and kept at the reflux temperature for 24-26 hours. After post-treatment, the modified composite material is obtained.

[0025] More preferably, the ratio of graphene oxide silica gel composite material, anhydrous toluene containing triethylamine, and octadecyltrimethoxysilane is 1g:25-30mL:0.8-0.9mL; and in the anhydrous toluene containing triethylamine, the volume ratio of triethylamine to anhydrous toluene is 0.1:260-280.

[0026] In a further preferred embodiment, the post-processing includes: naturally cooling to room temperature, centrifuging to collect the precipitate, washing it sequentially with toluene, isopropanol, methanol, deionized water, and acetone, and extracting it with acetone for 10–12 hours to obtain the final product.

[0027] A further preferred method for step (3-3) is as follows: first, 2-bromoethylamine hydrobromide and 1-methylimidazole are added to acetonitrile and reacted to obtain an ionic liquid; then, the modified composite material and the ionic liquid are added to methanol, stirred and mixed, N,N-diisopropylethylamine is added, the mixture is heated and reacted, and then post-processed to obtain the final product.

[0028] More preferably, the molar ratio of 2-bromoethylamine hydrobromide to 1-methylimidazole is 1:1, and the amount of acetonitrile is 6 to 8 times the mass of 2-bromoethylamine hydrobromide; the ratio of the amount of modified composite material, ionic liquid, methanol, and N,N-diisopropylethylamine is 1 mg: 0.4 to 0.5 mg: 18 to 20 mL: 0.5 to 0.6 mL.

[0029] More preferably, the heating reaction conditions are: heating at 65-75°C for 20-24 hours.

[0030] More preferably, the post-treatment includes: natural cooling to room temperature, washing with deionized water, and drying.

[0031] The present invention has the following beneficial effects: This invention provides a method for detecting trans-4,5-epoxy-2(E)-decaldehyde based on LC-MS / MS, which enables accurate qualitative and quantitative analysis of this compound and establishes a standardized platform for the role of this compound in systemic health and disease.

[0032] Given that aldehydes are almost impossible to detect directly by LC-MS / MS, this invention employs 2,4-dinitrophenylhydrazine (DNPH) derivatization to enhance the chromatographic behavior and retention of the target compound. Furthermore, this invention utilizes solid-phase extraction (SPE) for purification to improve the sensitivity of LC-MS / MS for the detection of trans-4,5-epoxy-2(E)-decanal. Specifically, this invention uses a SPE column for solid-phase extraction. The SPE column is obtained by filling a syringe-type polypropylene hollow column with packing material, freeze-drying, placing it on an upper sieve plate, and pressing the packing material. The packing material is prepared by: first, using graphite powder and amino silica gel as raw materials to prepare a graphene oxide silica gel composite material; then, modifying the graphene oxide silica gel composite material with octadecyltrimethoxysilane to obtain a modified composite material; finally, surface-modifying the modified composite material with an ionic liquid. Using the SPE column filled with the packing material of this invention for solid-phase extraction achieves efficient extraction of the target analyte, removes interfering substances, and improves detection results.

[0033] Following derivatization, trans-4,5-epoxy-2(E)-decaldehyde exhibited strong retention in chromatographic separation. Compared to the combination of acetonitrile and ammonium acetate buffer, the mobile phase of this invention, composed of acetonitrile and ultrapure water, demonstrated significantly superior performance. Under optimized conditions, the LC-MS / MS method exhibited excellent linearity for trans-4,5-epoxy-2(E)-decaldehyde in the range of 1-300 ng / ml, with a correlation coefficient (r0.05). 2 The limit of detection (LOD) was 0.9979, the limit of detection (LOD) was 0.3 ng / ml, and the relative standard deviation (RSD) was 0.1253.

[0034] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0035] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The derivation principle of this invention; Figure 2 Chromatograms of trans-4,5-epoxy-2(E)-decaldehyde analyzed by LC-MS / MS using different mobile phases (percentages are by volume): (a) 70% acetonitrile and 30% ammonium acetate buffer; (b) 85% acetonitrile and 15% ammonium acetate buffer; (c) 50% acetonitrile and 50% ammonium acetate buffer; the ammonium acetate buffer had a pH of 5.2 and a concentration of 2 mol / L. Figure 3Chromatograms of trans-4,5-epoxy-(E)-2-decene at gradient concentrations; Figure 4 Chromatograms of fragment ions at gradient concentrations; Figure 5 Discussion on the dosage of derivatizing reagents; Figure 6 Standard curve of trans-4,5-epoxy-2(E)-decanoal; Figure 7 Flowchart of the present invention. Detailed Implementation

[0036] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, the present invention can be implemented in many different ways as defined and covered by the claims.

[0037] 1.1 Reagents and Materials 2,4-Dinitrophenylhydrazine (DNPH) (C6H6N4O4, 98%) was purchased from Macklin (Shanghai, China). Acetonitrile was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol (AR) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Ultrapure water was prepared using a direct-q pure water system (Millipore, Billerica, MA, USA). trans-4,5-epoxy-2(E)-decaldehyde ((C 10 H 16 O2 (98%) was supplied by Aladdin (Shanghai, China). Formic acid (≥99%) was supplied by Thermofishers Scientific (Waltham, MA, USA). Stock solutions of the standards were prepared by dissolving them in acetonitrile at concentrations of 20 μg / mL, 1 μg / mL, and 100 μg / mL and then stored frozen.

[0038] Prior to LC / MS-MS analysis, all samples were filtered using an RC13 mm 0.22 μm syringe from Jinlong Filtration™ Tianjin Keyilong Experimental Equipment Co., Ltd. (Tianjin, China).

[0039] 1.2 Instruments and Meters LC-MS / MS studies were performed using a Waters ACQUITY UPLC h-classplus and a Waters LC / ms-ms XEVO tq-s Micro. Target compounds were separated on a Waters ACQUITY UPLC® BEH C18 analytical column (1.7 µm, 2.1 × 100 mm).

[0040] The mobile phase was a mixture of (A) water and (B) acetonitrile. The flow rate was set at 0.3 mL / min, the injection volume was 1 μL, and the column temperature was 40 °C. The gradient elution program on the column is listed in Table 1.

[0041] Table 1 ; Mass spectrometry conditions: Electrospray ionization negative ion mode (ESI-); Multiple reaction monitoring (MRM) mode.

[0042] Capillary voltage: 2.50 kV; Drying gas: nitrogen, flow rate 650 L / h; Collision gas: Helium; Tapered hole voltage: 10V; Precursor / Production ion: 347 / 163 (CE: 17 eV), 347 / 204 (CE: 15 eV).

[0043] 1.3 Extraction and Derivatization Reactions To prepare a blood sample containing the desired concentration of the compound trans-4,5-epoxy-2(E)-decanal, a standard trans-formaldehyde solution of a specific concentration was added to blank blood. 500 μL of the above blood and 2 mL of extraction buffer (acetonitrile solution containing 0.5% formic acid) were added to a 5 mL vial. After sealing, the vial was sonicated for 40 minutes and agitated for 20 minutes. The acetonitrile extract containing 0.5% formic acid was transferred to a 2 mL centrifuge tube using a syringe and centrifuged at 10,000 rpm for 10 minutes. The supernatant was added to a 3 mL solid-phase extraction column (pre-activated with 3 mL methanol and 3 mL water), and the eluent was collected. Then, 1 mL of the eluent was aspirated and filtered through a 0.22 μm syringe filter into a 1.5 mL glass vial. Finally, 200 μL of derivatization reagent (DNPH dissolved in acetonitrile) was added, and the vial was incubated at 40 °C for 1 hour.

[0044] This invention uses a solid-phase extraction column for solid-phase extraction. The solid-phase extraction column is obtained by filling a syringe-type polypropylene hollow column with packing material, freeze-drying it, placing it on an upper sieve plate, and pressing the packing material. The packing material is prepared by the following method: (3-1) First, graphene oxide silica gel composite material was prepared using graphite powder and amino silica gel as raw materials; (3-2) The graphene oxide silica gel composite material was further modified using octadecyltrimethoxysilane to obtain the modified composite material; (3-3) Finally, the modified composite material is surface-modified using ionic liquid to obtain the filler.

[0045] The length of the polypropylene hollow tube column is 7cm, and the height of the pressed filler is 5cm.

[0046] The specific method of step (3-1) is as follows: First, add sodium nitrate and concentrated sulfuric acid to graphite powder, stir for 35 minutes at 4℃ and 300r / min, slowly add potassium permanganate, dilute with water, stir and mix well, add warm water and hydrogen peroxide, stir and mix well, perform the first post-treatment to obtain graphene oxide; then, ultrasonically disperse the graphene oxide in N,N-dimethylformamide, add amino silica gel and dicyclohexylcarbodiimide, heat under reflux, perform the second post-treatment to obtain the final product.

[0047] The ratio of graphite powder, sodium nitrate, concentrated sulfuric acid, potassium permanganate, dilution water, warm water, and hydrogen peroxide is 10g:5.5g:225mL:30g:490mL:1.05L:110mL, wherein the concentrated sulfuric acid has a mass concentration of 98%, the warm water temperature is 55℃, and the hydrogen peroxide has a mass concentration of 25%.

[0048] The first post-processing includes: centrifugation to collect the precipitate, washing with 10% hydrochloric acid solution, washing with deionized water, and drying.

[0049] The ratio of graphene oxide, N,N-dimethylformamide, aminosilicone, and dicyclohexylcarbodiimide was 1 mg: 2.4 mL: 21 mg: 0.9 mg.

[0050] The heating and reflux time is 28 hours.

[0051] The second post-processing includes: centrifugation to collect the precipitate, washing with deionized water, washing with methanol, and drying.

[0052] The specific method of step (3-2) is as follows: the graphene oxide silica gel composite material is ultrasonically dispersed in anhydrous toluene containing triethylamine, then octadecyltrimethoxysilane is added, and under nitrogen protection, it is heated to reflux and kept at the reflux temperature for 25 hours. After post-treatment, the modified composite material is obtained.

[0053] The ratio of graphene oxide silica gel composite material, anhydrous toluene containing triethylamine, and octadecyltrimethoxysilane is 1g:28mL:0.85mL; in the anhydrous toluene containing triethylamine, the volume ratio of triethylamine to anhydrous toluene is 0.1:270.

[0054] Post-processing includes: natural cooling to room temperature, centrifugation to collect the precipitate, washing sequentially with toluene, isopropanol, methanol, deionized water, and acetone, and extracting with acetone for 11 hours to obtain the final product.

[0055] The specific method of step (3-3) is as follows: First, 2-bromoethylamine hydrobromide and 1-methylimidazole are added to acetonitrile and reacted to obtain an ionic liquid; then, the modified composite material and the ionic liquid are added to methanol, stirred and mixed, N,N-diisopropylethylamine is added, heated and reacted, and then post-processed to obtain the final product.

[0056] The molar ratio of 2-bromoethylamine hydrobromide to 1-methylimidazole is 1:1, and the amount of acetonitrile used is 7 times the mass of 2-bromoethylamine hydrobromide; the ratio of the amount of modified composite material, ionic liquid, methanol, and N,N-diisopropylethylamine is 1 mg: 0.45 mg: 19 mL: 0.55 mL.

[0057] The heating reaction conditions were: heating at 70℃ for 22 hours.

[0058] Post-treatment includes: natural cooling to room temperature, washing with deionized water, and drying.

[0059] 1.4 Laboratory Verification 1.4.1 Standard Curve The prepared standard stock solutions were diluted with acetonitrile to gradient concentrations: 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL. For each concentration, mL of solution was transferred to a 1.5 mL glass vial, followed by the addition of 200 μL of derivatization reagent. The derivatization reaction was carried out at 40 °C for 1 hour, and the samples were then analyzed by LC-MS / MS.

[0060] 1.4.2 Method Stability Blank blood was prepared into blood containing 100 ng / mL standard, and extraction and derivatization reactions were performed using the method described in 1.3, with 5 parallel samples.

[0061] 1.5 Data Processing Data acquisition was performed using MassLynx software (version 4.2). Data and charts were processed using Microsoft Office 2021 and Origin Pro 2024.

[0062] 2. Results and Discussion 2.1 Optimization of LC-MS / MS analysis method Initially, studies found that LC-MS / MS techniques could not be directly used to analyze trans-4,5-epoxy-2(E)-decanoal. However, after derivatization with DNPH, this method could be used for its analysis. Currently, many studies involving LC-MS / MS analysis of aldehydes also employ derivatization, the principle of which lies in... Figure 1 As shown in the image.

[0063] In negative ion mode, the applicant optimized the mobile phase. When using a mixture of acetonitrile and ammonium acetate buffer as the mobile phase ( Figure 2 The chromatogram showed disordered peaks, and no sharp, interference-free target peak was observed. Therefore, the applicant switched to a mixed mobile phase of acetonitrile and ultrapure water. After repeated verification and adjustments, a unique chromatographic peak of the target analyte appeared at 6.64–6.65 minutes, exhibiting high response and minimal interference. Figure 3 , Figure 4 The chromatograms of the target analyte and its fragment ion peaks (0–300 ng) after adjusting the mobile phase are shown. The instrument response and peak prominence both increase with increasing concentration.

[0064] 2.2 Optimization of Preprocessing Methods In the initial phase of the study, trans-4,5-epoxy-2(E)-decanoal was extracted directly from blood using acetonitrile. However, during derivatization, a large amount of flocculent precipitate was generated in the extraction solution. The amount of precipitate increased over time, affecting subsequent LC-MS / MS analysis. It is speculated that the precipitation may be due to the complex reaction between the blood matrix and the derivatization reagent.

[0065] Subsequently, the method was improved. Utilizing the volatility of trans-4,5-epoxy-2(E)-decene as a VOC, and referencing the headspace gas chromatography method used for ethanol detection, sealed glass vials containing blood were placed in an oven and heated at 70 °C for 20 minutes to completely coagulate the blood. After removal, they were immediately frozen. After 30 minutes, they were removed and extracted using a syringe with an extraction buffer, yielding an extract with minimal blood matrix. While this method is theoretically feasible, several problems arose in practice. For example, it was impossible to ensure complete sealing of the glass vials and prevent the escape of the target substance. LC-MS / MS analysis showed poor stability of the method. Even after optimization, satisfactory results could not be obtained (Table 2). The specific pretreatment method is as follows: Method 1: Add 1 ml of spiked blood sample to a sample vial, seal, and heat at 70°C for 20 minutes. Immediately after removal, freeze for 30 minutes. Add 1 ml of extraction solution using a syringe, vortex to mix, filter through a microporous membrane, and then perform derivatization.

[0066] Method 2: Add 0.5 mL of spiked blood and 1.5 mL of extraction buffer to the sample vial. Seal the vial and mix by shaking. Heat at 70°C for 20 minutes. Remove and immediately freeze for 30 minutes. Then add 1 mL of extraction buffer using a syringe, vortex again to mix, filter through a microporous membrane, and perform derivatization.

[0067] Method 3: Inject 0.5 mL of spiked blood sample and 1 mL of acetonitrile into a sample vial. Seal and mix by shaking, then heat at 70°C for 20 minutes. Remove and immediately freeze for 30 minutes. Then add 1 mL of extraction solution using a syringe, mix by shaking again, filter through a microporous membrane, and finally perform derivatization.

[0068] Table 2. Stability Study of Three Pretreatment Methods To eliminate the influence of the blood matrix and achieve better stability, blood samples were extracted with 0.5% formic acid acetonitrile solution, followed by solid-phase extraction (SPE). This column primarily adsorbs lipids from the blood, resulting in a clearer extract. After removing interfering impurities, the extract underwent a DNPH derivatization reaction, during which no precipitation occurred.

[0069] Spiked recovery experiments were conducted (using a commercially available HLB solid-phase extraction column as a control, catalog number CASHLB6150, 6 mL, Beijing Naphthalene Chemical Technology Co., Ltd.), and the recovery rate was calculated. Recovery rate P = (c1 - c0) / c2 × 100%, where c0 is the concentration of the analyte in the sample, c1 is the concentration of the analyte in the spiked sample, and c2 is the spike concentration. The results are shown in Table 3.

[0070] Table 3 As can be seen from Table 3, the recovery rate and precision of the solid phase extraction column of this invention are significantly better than those of commercially available products.

[0071] 2.3 Discussion on the dosage of derivatization reagents During derivatization, different dosages (50 μL, 80 μL, 100 μL, 150 μL, 200 μL) of derivatizing reagent were used to select the optimal amount. The results are shown in... Figure 5 In the derivatization reaction, the peak area was largest and the instrument response was highest when the amount of derivatization reagent was 200 μL. Each sample was measured twice. Therefore, 200 μL of reagent was used for the derivatization reaction.

[0072] 2.4 Standard Curve Using the concentration of the standard as the x-axis and the peak area as the y-axis, a linear regression was performed on the results to obtain the standard curve. Figure 6 The linear regression equation for this curve is y = 247.15x - 938.15, and the coefficient of determination r² of the regression model is 0.9979, indicating a good linear fit.

[0073] 2.5 Method Stability The results for five sets of parallel samples are shown in Table 4. The average peak areas for the five sets are: 2950.00, 2455.55, 1974.10, 2427.61, and 2396.91. The average absolute deviation is 0.0792, and the relative standard deviation (RSD) is 0.1187. The results indicate that the method has good stability.

[0074] Table 4 3. Conclusion There is a lack of standardized and reliable detection methods for trans-4,5-epoxy-2(E)-decenal. To address this analytical gap, this invention innovatively develops and validates an LC-MS / MS method for the detection of Trans-4,5-epoxy-(E)-2-decenal in blood.

[0075] This invention relates to a comprehensive optimization of the following three aspects: (1) DNPH derivatization for enhanced aldehyde detection; (2) Solid-phase extraction for matrix purification; (3) Instrument parameters and sample pretreatment scheme.

[0076] This invention demonstrates robust performance through linear quantification (r²>0.99 at gradient concentrations) and excellent stability (RSD<5%), establishing the first reliable LC-MS / MS method for blood-derived trans-4,5-epoxy-2(E)-decaldehyde analysis, enabling rapid, ultra-sensitive, and accurate analysis of the pathological biomarker trans-4,5-epoxy-2(E)-decaldehyde in clinical settings.

[0077] This invention utilizes the derivatization reaction principle of 2,4-dinitrophenylhydrazine (DNPH) with aldehydes, combining LC-MS / MS technology with a purification method using a solid-phase extraction column to achieve the detection of the target substance trans-4,5-epoxy-2(E)-decanal. Furthermore, this invention optimizes the instrumental analysis methods and pretreatment methods to ensure the stability of the method.

[0078] In summary, this invention provides a new method for the quantitative detection of trans-4,5-epoxy-2(E)-decaldehyde in blood. Figure 7 It has the potential for clinical application.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting trans-4,5-epoxy-2(E)-decanoal based on LC-MS / MS, characterized in that, The specific steps are as follows: (1) First, prepare standard solutions of different concentrations from trans-4,5-epoxy-2(E)-decaldehyde standard, and then derivatize the standard solutions with 2,4-dinitrophenylhydrazine (DNPH) to obtain analytical samples containing standard solutions of different concentrations. (2) Then, LC-MS / MS analysis was performed on the standard solution samples with different concentrations to obtain a standard curve with the standard concentration as the abscissa and the peak area as the ordinate. (3) Solid-phase extraction and 2,4-dinitrophenylhydrazine (DNPH) derivatization were performed on the test sample, and the sample was detected by LC-MS / MS. The content of trans-4,5-epoxy-2(E)-decaldehyde in the test sample was determined by the standard curve obtained in step (2). The LC-MS conditions are as follows: Mobile phase: a mixture of (A) water and (B) acetonitrile; Flow rate: 0.3 mL / min; Injection volume: 1 μL; Column temperature: 40℃; The gradient elution conditions are shown in Table 1; Table 1 ； MS conditions are as follows: Detection method: Multiple reaction monitoring (MRM) Scanning method: Electrospray ionization negative ion mode Capillary voltage: 2.50 kV; Drying gas: nitrogen, flow rate 650 L / h; Collision gas: Helium; Tapered hole voltage: 10V; Mother-daughter ion pairs: 347 / 163, CE: 17 eV; 347 / 204, CE: 15 eV.

2. The detection method according to claim 1, characterized in that, The standard solutions are prepared by first dissolving trans-4,5-epoxy-2(E)-decaldehyde standard in acetonitrile to obtain stock solutions with concentrations of 20 μg / mL, 1 μg / mL and 100 μg / mL, respectively, and then diluting the stock solutions with acetonitrile to obtain standard solutions of different concentrations.

3. The detection method according to claim 1, characterized in that, The specific method of solid-phase extraction is as follows: the standard solution and the extract are mixed at a volume ratio of 1:4, sealed and shaken for 20 minutes; the extract is transferred to a centrifuge tube using a syringe, and the supernatant is collected by centrifugation; the supernatant is added to the solid-phase extraction column and the effluent is collected; wherein the extract is an acetonitrile solution containing 0.5% formic acid by mass.

4. The detection method according to claim 3, characterized in that, Centrifugation conditions: 10,000 rpm for 10 minutes.

5. The detection method according to claim 3, characterized in that, The solid-phase extraction column is activated using equal volumes of methanol and water.

6. The detection method according to claim 3, characterized in that, 2,4-Dinitrophenylhydrazine was prepared into a 2 mmol / L DNPH solution using acetonitrile, and then mixed with the solid-phase extraction effluent; the volume ratio of the solid-phase extraction effluent to the DNPH solution was 5:

1.

7. The detection method according to claim 1, characterized in that, The solid-phase extract effluent was filtered using a 0.22 μm syringe filter and then mixed with DNPH solution.

8. The detection method according to claim 1, characterized in that, The derivatization reaction conditions were: derivatization reaction at 40℃ for 1 hour.

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