Quantitative detection method for short-chain fatty acid marked by Girard reagent P and application of quantitative detection method

Abstract

The invention discloses a short-chain fatty acid quantitative detection method based on a Girard reagent P label and application, and the method comprises the following steps: mixing a biological sample containing short-chain fatty acid (SCFAs) with GP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and pyridine, and incubating at room temperature to make the SCFAs form a GP-labeled derivative with permanent positive charges; gP-labeled deuterated SCFAs (AA-d4, PA-d5 and the like) is used as an internal standard, a liquid chromatography-tandem mass spectrometry (LC-MS / MS) positive ion mode is adopted for detection, and an internal standard method is adopted for quantification. The method is suitable for high-sensitivity synchronous detection of trace SCFAs (acetic acid, propionic acid, butyric acid, valeric acid and hexanoic acid) in complex matrixes such as pig manure, the detection limit reaches 0.1 pmol / g, the recovery rate is 80-104%, operation is easy and convenient, and stability is high.

Description

Technical Field

[0001] This invention relates to a method and application for the quantitative detection of short-chain fatty acids based on Girard's reagent P labeling. Background Technology

[0002] Short-chain fatty acids (SCFAs) are a class of carboxylic acid compounds with fewer than 6 carbon atoms (Postler et al., 2017), and are the main metabolites of dietary fiber fermentation by gut microbiota (Smith et al., 2023). Therefore, the concentration of SCFAs directly reflects the activity and diversity of the gut microbiota and can be used to assess gut health and microbiota function (Oh et al., 2021). In addition, by monitoring SCFAs, feed composition can be optimized to improve nutrient utilization efficiency, reduce energy loss, and optimize environmental management (Ratanpaul et al.; Zhao et al., 2020; Fuertes et al., 2025; Maher et al., 2025). Acetic acid, propionic acid, and butyric acid account for 90% of the total SCFAs produced in the intestine (Vinolo et al., 2011). After SCFAs are produced in the intestine, some are excreted through feces. Therefore, pig feces are often used as a typical specimen for detecting SCFAs. Acetic acid accounts for more than 50% of the total SCFAs (Metzler-Zebeli et al., 2021; Yang Yunnian et al., 2021; Mei Huadi et al., 2022).

[0003] The earliest description of chromatographic analysis of short-chain fatty acids was the direct determination by gas chromatography (GC) (James and Martin, 1952), but its limits of quantitation and resolution were low. For volatile SCFAs such as acetic acid, due to their hydrophilicity and low sensitivity, it has been proposed to convert fatty acids into more volatile derivatives for detection by GC and gas chromatography-mass spectrometry (GC-MS) (Gu et al., 2021; Park et al., 2021; Mulat et al., 2015; Zhang et al., 2015; Amer et al., 2015; García-Villalba et al., 2012), although these methods are time-consuming but more efficient (Gutnikov, 1995; Brondz, 2002). Various chemical derivatization reagents have been used, such as pentafluorobenzyl bromide (PFBBr), trimethylsilyl (TMS), chloroformate, and 1-(tert-butyldimethylsilyl)imidazole (TBDMS) (He et al., 2008; Li et al., 2020; Park et al., 2017; Pouteau et al., 2001). However, pentafluorobenzyl bromide derivatization may result in the loss of volatile derivatives through volatilization. Furthermore, trimethylsilyl derivatization must be performed under anhydrous conditions, which is unfavorable for the processing of biological samples (Li et al., 2020). These derivatization conditions somewhat limit the application of GC-MS in the analysis of SCFAs.

[0004] In recent years, liquid chromatography-tandem mass spectrometry (LC-MS) has also been frequently used for the determination of SCFAs. Reports indicate that SCFAs can be directly detected using LC-MS selected ion monitoring (SIM) mode, but this requires a demanding mobile phase (1.5 mM HCl) and post-column neutralization (van et al., 2009). Furthermore, considering that SCFAs contain carboxyl groups, they can be directly detected in negative ion LC-MS multiple reaction monitoring (MRM) mode (LC-MS / MS). However, SCFAs have low isoionization efficiency and are prone to water loss during the ionization of low molecular weight organic acids, making it difficult to quantitatively analyze the production of SCFAs using mass spectrometry in experiments (Song WS et al., 2020). To improve the sensitivity of SCFA detection, several chemical derivatization methods based on LC-MS / MS analysis have been reported. Han et al. used 3-nitrophenylhydrazine (3NPH) to derivatize SCFAs, converting them into their 3-nitrophenylhydrazone derivatives. To achieve accurate quantification, they utilized... 13 C6-3NPH was synthesized as a stable isotope label of the derivative and used as an internal standard to compensate for the matrix effect in electrospray ionization. Finally, it was quantified by negative ion LC-MS / MS (Han et al., 2015).

[0005] Considering that in electrospray ionization (ESI)-mass spectrometry, the ionization efficiency of positive ion mode is generally higher than that of negative ion mode, resulting in higher sensitivity (Zhu et al., 2015), Song (Song WS et al., 2020) established a method for the chemical derivatization of short-chain fatty acids using Girard's reagent T (GT) with an inherently permanent positive charge, followed by quantitative analysis using positive ion LC-MS / MS. Summary of the Invention

[0006] The present invention aims to solve the problems of low detection sensitivity, large matrix interference, and inability to simultaneously quantify SCFAs under mild conditions in the prior art.

[0007] This invention is the first to propose that SCFAs can be efficiently derivatized at room temperature using GP and EDC.HCl in a pyridine buffer system to generate acylhydrazone derivatives with permanent positive charges, which significantly improves the ionization efficiency of ESI+ mode. Combined with HILIC separation and deuterated isotope internal standard, five SCFAs can be detected simultaneously with high sensitivity (LOD 0.1–0.5 pmol / g).

[0008] The key innovations of this invention are: ① GP replaces GT in SCFAs; ② EDC.HCl + pyridine achieves mild aqueous phase derivatization; ③ m / z 120 is a universal quantitative ion for all GP-SCFA derivatives.

[0009] The technical solution of the present invention is as follows: A method for quantitative detection of short-chain fatty acids based on Girard's reagent P labeling includes the following steps: (1) The biological sample containing short-chain fatty acids was mixed with the deuterated isotope internal standard, extracted with acetonitrile, and then centrifuged to obtain the supernatant; (2) Take the supernatant, add Girard's reagent P (GP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and pyridine aqueous solution, and incubate at room temperature for 1–3 hours to allow the short-chain fatty acid to form a permanently positively charged acylhydrazone derivative with GP; (3) The derivatized product was diluted with 50% methanol and filtered. It was detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in positive ion electrospray mode. The internal standard method was used to quantify the product by the ratio of the quantitative ion peak area of ​​the GP-labeled short-chain fatty acid to the corresponding deuterated internal standard derivative. The short-chain fatty acid is one or more of acetic acid, propionic acid, butyric acid, valeric acid, or hexanoic acid; The deuterated isotope internal standard is one or more of acetic acid-d4, propionic acid-d5, butyric acid-d7, valeric acid-d9, or hexanoic acid-d11; The mass spectrometry detection was performed in multiple reaction monitoring (MRM) mode, with all GP-labeled short-chain fatty acid derivatives using m / z 120 as the quantitative component ion and m / z 80 as the qualitative component ion.

[0010] The concentration of GP is 0.01–0.05 M, the concentration of EDC.HCl is 0.02–0.08 M, and the concentration of pyridine aqueous solution is 0.01–0.1 M.

[0011] The derivatization reaction takes 60–180 minutes and is carried out at a temperature of 20–37°C.

[0012] The LC-MS / MS used a HILIC column. Mobile phase A was an aqueous solution containing 2–5 mM ammonium formate and 0.05–0.1% formic acid, and mobile phase B was an aqueous solution of acetonitrile containing the same components. Gradient elution: phase B was 100% at 0 min, decreased to 50% and maintained within 1–3 min, and increased back to 100% at 3.1 min. The total run time was ≤5 min.

[0013] The biological sample was pig feces, with a sample volume of 50–500 mg and an internal standard concentration of 1–20 μM.

[0014] A GP-labeled short-chain fatty acid derivative has the following structure: R–C(O)–NH–N=CH–(CH2)3–N+(CH3)2 Wherein, R is acetyl (–CH3), propionyl (–C2H5), butyryl (–C3H7), valeryl (–C4H9), or hexanoyl (–C5H). 11 ); The derivatives are isotopic labels containing 4 to 11 deuterium atoms on R, namely: GP-AA-d4, GP-PA-d5, GP-BA-d7, GP-VA-d9, or GP-HA-d 11 .

[0015] A kit for the method, comprising: (i) GP acetonitrile solution 0.01–0.05 M; (ii) EDC.HCl acetonitrile solution 0.02–0.08 M; (iii) Aqueous solution of pyridine, 0.01–0.1 M; (iv) GP-labeled internal standard mixtures of deuterated SCFAs: GP-AA-d4, GP-PA-d5, GP-BA-d7, GP-VA-d9, GP-HA-d 11 .

[0016] The beneficial effects of this invention are: This invention is the first to use GP, which has an inherent permanent positive charge, to chemically derivatize SCFAs, and then combine it with positive ion LC-MS / MS for analysis, so as to achieve simultaneous quantification of five short-chain fatty acids in pig feces.

[0017] The results showed that within the range of 0.05–2 μmol / g -1 Within a specified concentration range, the average recoveries of short-chain fatty acids (SCFAs) in five pig fecal samples ranged from 80.1% to 103.8%, with relative standard deviations (RSDs) less than 10%. The limits of detection (LODs) for the five SCFAs ranged from 0.5 to 0.1 pmol, and the limits of quantitation (LOQs) ranged from 1.0 to 5.0 pmol. This method is suitable for the simultaneous determination of five short-chain fatty acids (acetic acid, propionic acid, butyric acid, valeric acid, and hexanoic acid) in pig feces. The method is highly sensitive, accurate, and easy to operate. Attached Figure Description

[0018] Figure 1 The chemical structures of GP-AA, GP-PA, GP-BA, GP-VA, GP-HA, GP-AA-d4, GP-PA-d5, GP-BA-d7, GP-VA-d9, and GP-HA-d11 are shown (drawn using ChemDraw Ultra).

[0019] Figure 2 These are chromatograms of GP-AA, GP-PA, GP-BA, GP-VA, and GP-HA (mobile phase: ammonium formate-formic acid-water-acetonitrile solution).

[0020] Figure 3 The chromatograms are for GP-AA-d4, GP-PA-d5, GP-BA-d7, GP-VA-d9, and GP-HA-d11 (mobile phase: acetonitrile-water).

[0021] Figure 4 These are chromatograms of GP-AA in different mobile phases.

[0022] Figures 5a-5j are mass spectra of GP-SCFAs and their isotopic derivatives. a: GP-AA, b: GP-AA-d4, c: GP-PA, d: GP-PA-d5, e: GP-BA, f: GP-BA-d7, g: GP-VA, h: GP-VA-d9, i: GP-HA, j: GP-HA-d11.

[0023] Figure 6 This is a possible cleavage pathway for SCFAs and their isotope derivatives.

[0024] Figure 7The effect of different concentrations of GP combined with EDC.HCl on the peak area of ​​SCFAs derivatives.

[0025] Figure 8a Figure 8C shows the effect of different solvents on the peak area of ​​SCFAs and their isotope derivatives.

[0026] Figure 9 a- Figure 9b The effect of different concentrations of pyridine on the peak area of ​​SCFAs and their isotope derivatives is shown.

[0027] Figure 10 This describes the effect of different incubation temperatures on the peak area of ​​SCFAs and their isotope derivatives. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0029] SCFAs and their isotopes were chemically derivatized using GP, which has an inherent permanent positive charge, to bind positive ions to the SCFAs. Separation was then performed using a HILIC column, followed by positive ion MS / MS analysis, enabling simultaneous quantification of five SCFAs in pig feces. The key to this invention lies in the condensation reaction between the hydrazide group (–NH–NH2) in GP and the carboxyl group of SCFAs under EDC.HCl activation, forming a stable acylhydrazone bond (–NH–N=CH–C(O)–), while retaining the quaternary ammonium cation (N) in the GP molecule. + This structure imparts a permanent positive charge to the derivatives. The ionization efficiency of this structure in ESI+ mode is significantly higher than that of free SCFAs, and it is less prone to dehydration or fragmentation. More importantly, all GP-SCFA derivatives co-fracture in collision-induced dissociation (CID) to generate an N-vinylformylpyridine cation with an m / z of 120. This fragment is independent of the fatty acid chain length and is a characteristic ion of the GP backbone. Therefore, this invention achieves, for the first time, the simultaneous detection of five SCFAs using only a single universal quantitative ion (m / z 120), eliminating the need for separate MRM channels for each acid and significantly improving detection throughput.

[0030] Add 10 mL of acetonitrile extraction solution containing 10 μM SCFAs isotope internal standard to approximately 200 mg of wet fecal sample, and extract ultrasonically for 10 min at ambient temperature (24 ± 1°C). Then centrifuge the suspension at 8000 rpm for 5 min. Carefully remove the supernatant, and then take 400 μL aliquots, adding 200 μL of GP acetonitrile solution (0.025 M), 200 μL of EDTC·HCl (0.05 M) acetonitrile solution, and 200 μL of pyridine aqueous solution (0.05 M), and incubate at ambient temperature for 2 h. Dilute each aliquot 50-fold with 50% methanol. All samples were stored at 4°C until analysis by LC-MS / MS on the same day, with quantification using the internal standard method.

[0031] The instruments and equipment used were: an ultra-high performance liquid chromatography-mass spectrometry system (Acquity UPLC TQ MS, Waters Corporation, USA), an ultrasonic cleaner (KQ-500E, Kunshan Ultrasonic Instrument Co., Ltd.), an ultrapure water system (Milli-Q, EMDMillipore, Germany), an electronic analytical balance (ME204E, Mettler Toledo, Switzerland), and a centrifuge (ST 40R, Thermo Scientific, USA).

[0032] Acetic acid (purity ≥99.9%), acetic acid-d4 (purity ≥99.5%), propionic acid (purity ≥99.9%), propionic acid-d5 (purity ≥99.9%), butyric acid (purity ≥99.5%), butyric acid-d7 (purity ≥98.0%), valeric acid (purity ≥99.0%), valeric acid-d9 (purity ≥98.0%), hexanoic acid (purity ≥99.0%), hexanoic acid-d11 (purity ≥98.0%), Girard's reagent P (purity ≥98.0%), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (purity ≥98.0%), and pyridine (purity ≥99.9%) were purchased from Bailingwei Technology Co., Ltd. Chromatographic grade methanol, acetonitrile, ammonium formate, and formic acid were purchased from Sigma-Aldrich, USA. Analytical grade triethylamine and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd. Acuqity UPLC BEHC 18 The chromatographic columns (2.1 × 100 mm, 1.7 μm) and Cortecs UPLC HILIC (2.1 × 100 mm, 1.6 μm) were purchased from Waters Corporation, USA.

[0033] GP acetonitrile solution: Weigh 0.1876 g GP (purity ≥98%, molecular formula C10). 16 H 31 Dissolve N2O3·HCl in 2 mL of methanol, and dilute to 10 mL with acetonitrile to obtain a 0.1 M stock solution. Dilute with acetonitrile before use.

[0034] EDC.HCl acetonitrile solution: Weigh 0.1917 g of EDC.HCl (purity ≥98%) and dissolve it in 5 mL of acetonitrile. Make up to 10 mL with acetonitrile to obtain a 0.1 M stock solution. Dilute with acetonitrile before use.

[0035] Pyridine aqueous solution: Weigh 0.1978 g of pyridine (purity ≥99.9%, molecular weight 79.1) and dissolve it in 50 mL of ultrapure water to obtain a 0.05 M solution.

[0036] SCFAs isotope internal standard stock solutions: Prepare 10 mM acetonitrile stock solutions for AA-d4, PA-d5, etc., and dilute them to the working concentration.

[0037] The fecal samples were from Zhejiang Qinglian Food Co., Ltd.

[0038] The reaction solution of short-chain fatty acids labeled with Girard's reagent P was directly injected into the mass spectrometer at a flow rate of 20 μL / min using a syringe pump. The mass spectrometer was operated in positive ion mode, with the spray voltage set to 2.5 kV and the ion source temperature set to 550 °C. Parameters such as the temperature or flow rate of the desolvating gas, nebulizing gas, and drying gas were adjusted to obtain high-quality full-scan mass spectrometry and tandem mass spectrometry (MS / MS) spectra. Corresponding tuning files were created.

[0039] Mobile phase A was an aqueous solution containing 2.5 mM ammonium formate and 0.1% formic acid, and mobile phase B was an aqueous solution of acetonitrile (containing 10% water) containing 2.5 mM ammonium formate and 0.1% formic acid. Short-chain fatty acids labeled with Girard's reagent P were separated on the analytical column at a flow rate of 0.5 mL / min. The HPLC gradient was set as follows: at 0 min, mobile phase B comprised 100%; at 1 min, the proportion of mobile phase B decreased to 50%, and remained at 50% at 3 min; at 3.1 min, the proportion of mobile phase B increased back to 100%; at 5 min, mobile phase B remained at 100%. The column temperature was set to 40℃. The injection volume was 5 µL.

[0040] Optimal derivatization conditions were determined by evaluating the concentrations of GP and the coupling agent EDC.HCl, the reaction solvent, the reaction time, and the temperature. UPLC-MS / MS was used to detect derivatized SCFAs in pure solution and in a biomatrix under optimized conditions to assess derivatization efficiency.

[0041] (1) Optimization of GP and EDC-HCl concentrations 200 μL of acetonitrile solutions of GP at different concentrations (0.005, 0.01, 0.025, 0.05 M) and 200 μL of EDC.HCl acetonitrile solutions at different concentrations (0.005, 0.01, 0.01, 0.025, 0.05 M) (see Table 1) were added to 400 μL of a 0.1 mM SCFAs mixed standard solution (containing a 0.01 mM SCFAs isotope mixed standard solution) and 200 μL of a 0.05 M pyridine aqueous solution, and incubated at ambient temperature for 2 hours. The reaction solution was diluted 50-fold with 50% methanol. The peak areas of SCFAs derivatives in different solvents were compared by LC-MS / MS analysis.

[0042] Table 1. Combinations of different concentrations of GP with EDC.HCl

[0043] (2) Optimization of reaction solvent A mixed standard solution of SCFAs and a mixed standard solution of SCFAs isotopes were added to 200 μL of 0.025 M GP, 200 μL of 0.05 M EDC·HCl, and 200 μL of different solvents (5 mM triethylamine aqueous solution, pyridine aqueous solutions of different concentrations, and HCl-pyridine aqueous solution), and incubated at ambient temperature for 2 hours. The reaction solution was then diluted 50-fold with 50% methanol. The peak areas of SCFAs derivatives in different solvents were compared by LC-MS / MS analysis.

[0044] (3) Optimization of reaction time Add 400 μL of 0.1 mM SCFAs mixed standard solution (containing 0.01 mM SCFAs isotope mixed standard solution), 200 μL of 0.025 M GP, 200 μL of 0.05 M EDC·HCl, and 200 μL of 0.05 M pyridine aqueous solution to capped centrifuge tubes and incubate at ambient temperature. Take 100 μL samples at 30, 60, 90, 120, 150, and 180 minutes, dilute 50 times with 50% methanol, and store at 4°C until analysis.

[0045] (4) Optimization of reaction temperature Add 400 μL of 0.1 mM SCFAs mixed standard solution (containing 0.01 mM SCFAs isotope mixed standard solution), 200 μL of 0.025 M GP, 200 μL of 0.05 M EDC·HCl, and 200 μL of 0.05 M pyridine aqueous solution to a capped centrifuge tube. Incubate at 25, 37, 50, and 70 °C for 2 hours. Dilute 50 times with 50% methanol and store at 4 °C until analysis.

[0046] Weigh 200 mg of fecal sample and add 10 mL of acetonitrile extraction solution containing 10 μM SCFAs isotope internal standard. Extract by sonication for 10 min, then centrifuge at 8000 rpm for 5 min. Transfer 400 μL of the supernatant to a 2 mL centrifuge tube, and then add 200 μL of 0.025 M GP, 200 μL of 0.05 M EDC.HCl, and 200 μL of 0.05 M pyridine aqueous solution sequentially. Incubate at ambient temperature for 2 hours. Dilute the reaction solution 50 times with 50% methanol solution for later use.

[0047] The method of this invention was validated with reference to European Commission resolution 2002 / 657 / EC (2002 / 657 / EC), where accuracy (recovery), precision (reproducibility), linearity, and selectivity were evaluated, with minor modifications: the limit of detection (LOD) and limit of quantitation (LOQ) were used instead of the limit of determination (CCα) and detection capacity (CCβ) commonly used in samples (Frenich et al., 2011; Ying et al., 2013; Kiebooms et al., 2015).

[0048] Recovery was assessed using the standard addition method. Two groups of samples were set up: Sample A was feces containing endogenous SCFAs and SCFA isotope internal standards; Sample B was added according to the addition scheme in Table 2, with 1 mL of mixed standard solution of SCFAs containing three different concentration levels (low (FL1), medium (FL2), and high (FL3)) and SCFA isotope internal standards added to the sample (3 replicates). After processing according to the extraction and derivatization steps, quantitative analysis was performed by LC-MS / MS.

[0049] Table 2. Suggestions for adding SCFAs to pig fecal samples

[0050] The average recovery of SCFAs was assessed using equation (1). For an effective pretreatment method, the recovery should be between 80% and 110% (Molognoni et al., 2018).

[0051] Recovery rate (%) = (detected concentration / actual added concentration) * 100 (1) The precision of the method was determined by repeatability and expressed as relative standard deviation (RSD). The method was tested on feed samples with three different addition levels on the same day, with three replicates for each level. A good standard of precision was an RSD of less than 20% (Molognoni et al., 2018).

[0052] SCFAs were dissolved in acetonitrile (concentration 100 mmol), serially diluted, and then derivatized using Girard's reagent (GP). 5 μL of the derivatized solution was injected into a liquid chromatography-tandem mass spectrometer (LC-MS / MS) for analysis (n=3). The number of Girard's reagent P-labeled short-chain fatty acid molecules injected into the LC-MS / MS were 5 fmol, 10 fmol, 25 fmol, 50 fmol, 100 fmol, 250 fmol, and 500 fmol, respectively. The GP derivatization reaction conditions were as follows: 400 μL of a prepared SCFAs standard mixture (with a mixed internal standard concentration of 10 mM), 200 μL of 0.025 M GP, 200 μL of 0.05 M EDC.HCl, and 200 μL of 0.05 M pyridine aqueous solution were added and incubated at ambient temperature for 2 hours. The reaction solution was diluted 50-fold with 50% methanol before LC-MS / MS analysis. Quantitative standard curves of GP-labeled SCFAs were plotted and sensitivity tests were performed. The correlation coefficient (R²) of the alignment curves was determined. 2 The value should be greater than 0.98 (Molognoni et al., 2018).

[0053] Selectivity of SCFAs in pig feces was determined by LC-MS / MS method, and the results were compared by spiked samples (0.5 μmol / g). -1 The results were analyzed to verify the findings. Characteristic ions of the compound and mean retention time ± relative standard deviation were determined within an appropriate retention time range.

[0054] Method detection limit (LOD): The LOD of the SCFA is determined by a signal-to-noise ratio (S / N) of 3, i.e., the concentration at which the detected signal intensity is 3 times the background noise intensity. Limit of quantitation (LOQ): The LOQ of the SCFA is determined by a signal-to-noise ratio (S / N) of 10, i.e., the concentration at which the detected signal intensity is 10 times the background noise intensity.

[0055] (1) Optimization and determination of mobile phase

[0056] The samples were compared in a methanol-water mixture containing 0.1% formic acid at C 18 chromatographic column ( Figure 4 ) and HILIC column ( Figure 2 The study investigated the chromatographic retention behavior of GP-labeled short-chain fatty acids in different systems, including HILIC columns, and the presence of different concentrations of ammonium formate, formic acid, acetonitrile, and water. The results showed that GP-labeled short-chain fatty acids exhibited good retention in C... 18Almost no retention was observed on the column, but the highest sensitivity and optimal peak shape were obtained using an acetonitrile-water mobile phase system containing 2.5 mM ammonium formate and 0.1% formic acid on a HILIC column. This method uses an aqueous solution containing 2.5 mM ammonium formate and 0.1% formic acid, and an aqueous solution of acetonitrile as the mobile phase. Under optimized chromatographic conditions, the retention times of GP-AC, GP-PA, GP-BA, GP-VA, and GP-CA were 2.35 min, 2.33 min, 2.28 min, 2.21 min, and 2.19 min, respectively. Therefore, the detection of 10 Radd reagent P-labeled short-chain fatty acids and their isotopes can be achieved within 5 min.

[0057] (2) Elucidation of the mass spectrometry fragmentation pathway of GP-SCFAs derivatives Due to the pre-charged groups in Girard's reagent P, the 10 derivatives produce molecular ion peaks ([M]) under electrospray ionization (ESI) mode. + The mass spectra of the parent ions were obtained by bombarding them with m / z 194.0, m / z 197.2, m / z 208.1, m / z 213.2, m / z 222.0, m / z 229.2, m / z 236.2, m / z 245.2, m / z 250.2, and m / z 261.3 (Figures 5a-5j). The study found that the only difference in mass among the 10 derivatives during fragmentation was formed by the loss of the N-ethylpyridine ring. After fragmentation, the 10 derivatives produced common fragment ions including a pyridine ring cation (m / z 80), an N-vinylformylpyridine cation (m / z 120), and an N-(carboxymethyl)pyridine cation (m / z 138). The fragment ions are shown in Table 3, and the possible fragmentation pathways are listed in [Table 3]. Figure 6 Therefore, in multiple reaction monitoring (MRM) mode, the daughter ion with a higher response m / z 120 was selected as the quantitative ion, while the daughter ion m / z 80, common to the 10 derivatives, was selected as the qualitative ion. Using the compound optimization function of Masslynx software (Waters, version 4.2) combined with manual verification, the MRM analytical parameters for Girard reagent P-labeled short-chain fatty acids, including residence time, cone voltage, and collision energy, were determined (Table 4).

[0058] Table 3 Fragment ions of SCFAs

[0059] Table 4. Reference values ​​for qualitative ion pairs, quantitative ion pairs, and collision energies of short-chain fatty acids.

[0060] Among different combinations of GP and EDC.HCl concentrations, the combination of 0.025 M GP and 0.05 M EDC.HCl resulted in a stronger signal from SCFA derivatives. Figure 7 The coupling reaction between carboxyl groups and GP is more efficient under alkaline conditions, and the efficiency increases with increasing pH. Among different solvents, SCFA derivatives show stronger signals when using pyridine aqueous solution. Figure 8a -Figure 8C), and comparing different concentrations of pyridine aqueous solution, it was found that the SCFAs derivatives showed a stronger signal when using 50 mM pyridine aqueous solution (Figure 9a-Figure 9b). Simultaneously, experiments showed that EDC.HCl could rapidly activate the carboxyl group at room temperature; therefore, increasing the reaction temperature did not accelerate the reaction rate (Figure 10). Regarding the reaction time, it was found that the reaction reached equilibrium within 120 minutes. Figure 8a (Figure 8C), therefore, 2 hours was chosen as the derivatization reaction time. The optimal derivatization conditions were finally determined to be: 0.025 M GP and 0.05 M EDC·HCl at 25 °C, with the addition of 50 mM pyridine aqueous solution, and reaction for 2 hours.

[0061] Analytical results of SCFAs in fecal samples Following the implementation steps, three parallel experiments were performed. 0.200 mg of fecal sample was weighed, and 1 mL of 0.1 mMSCFAs isotope internal standard was added. Acetonitrile was then added to a total volume of 10 mL. After ultrasonic extraction for 10 min, the sample was centrifuged at 8000 rpm for 5 min. 400 μL of the supernatant was transferred to a 2 mL centrifuge tube, followed by the addition of 200 μL of GP, 200 μL, and 200 μL of pyridine aqueous solution. The mixture was incubated at ambient temperature for 2 hours. The reaction solution was diluted 50-fold with 50% methanol solution and filtered through a 0.22 μm filter membrane. The LC-MS / MS analysis results of the pig fecal samples are shown in Table 5.

[0062] Table 5. Analytical results of SCFAs in fecal samples

[0063] In the prepared mixed series of standard working solutions, the concentrations of acetic acid, propionic acid, and butyric acid were 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mM, respectively; the concentrations of valeric acid and hexanoic acid were 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, and 0.1 mM, respectively. The mixed internal standard concentration was 0.01 mM. The correlation coefficient (R0) of the obtained standard curve was calculated. 2The LOD range of the five SCFAs was 0.5-0.1 pmol, and the LOQ range was 1.0-5.0 pmol, determined by the internal standard method. The recovery rate of SCFAs in feces was good, ranging from 80.1% to 103.8%, with an RSD of 1.8% to 10.0%. The results are shown in Table 6.

[0064] Table 6 Methodological Evaluation Results

[0065] RSD = Relative Standard Deviation In summary, this invention is the first to utilize GP, which possesses an inherent permanent positive charge, for chemical derivatization of SCFAs, followed by analysis using positive ion LC-MS / MS, achieving simultaneous quantification of five short-chain fatty acids in pig feces. Satisfactory results were obtained in terms of recovery rate, RSD, and detection limit. This method is suitable for trace analysis of SCFAs in feces.

[0066] 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 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 the present invention should be determined by the appended claims.

Claims

1. A method for quantitative detection of short-chain fatty acids based on labeling with Girard's reagent P, characterized in that The method comprises the following steps: (1) mixing a biological sample containing short-chain fatty acids with a deuterium isotope internal standard, extracting with acetonitrile, and centrifuging to obtain supernatant; (2) taking the supernatant, adding Girard's reagent P (GP), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and pyridine aqueous solution, and incubating at room temperature for 1-3 hours to form a permanently positively charged acylhydrazone derivative of the short-chain fatty acid and GP; (3) diluting the derivatization product with 50% methanol, filtering, and detecting by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in positive ion electrospray mode, and quantifying by internal standard method using the quantitative ion peak area ratio of GP-labeled short-chain fatty acid and corresponding deuterium internal standard derivative; The short-chain fatty acid is one or more of acetic acid, propionic acid, butyric acid, valeric acid or caproic acid; The deuterium isotope internal standard is one or more of acetic acid-d4, propionic acid-d5, butyric acid-d7, valeric acid-d9 or caproic acid-d11; The mass spectrometry detection adopts a multiple reaction monitoring (MRM) mode, all GP-labeled short-chain fatty acid derivatives have m / z 120 as the quantitative sub-ion and m / z 80 as the qualitative sub-ion.

2. The method of claim 1, wherein: The concentration of GP is 0.01-0.05 M, the concentration of EDC.HCl is 0.02-0.08 M, and the concentration of pyridine aqueous solution is 0.01-0.1 M.

3. The method of claim 1, wherein: The derivatization reaction time is 60-180 minutes and the temperature is 20-37°C.

4. The method of claim 1, wherein: The LC-MS / MS uses a HILIC chromatographic column, mobile phase A is an aqueous solution containing 2-5 mM ammonium formate and 0.05-0.1% formic acid, and mobile phase B is an aqueous acetonitrile solution containing the same components; gradient elution: B phase is 100% at 0 min, decreases to 50% within 1-3 min and maintains, rises to 100% at 3.1 min, and the total running time is ≤5 min.

5. The method of claim 1, wherein: The biological sample is pig feces, the sample dosage is 50-500 mg, and the internal standard concentration is 1-20 μM.

6. A GP-tagged short chain fatty acid derivative, characterized in that has the following structure: R-C(O)-NH-N=CH-(CH2)3-N+(CH3)2 wherein R is acetyl (-CH3), propionyl (-C2H5), butyryl (-C3H7), valeryl (-C4H9) or caproyl (-C5H 11 ); The derivatives are isotopically labeled with 4 to 11 deuterium atoms on R, i.e. GP-AA-d4, GP-PA-d5, GP-BA-d7, GP-VA-d9 or GP-HA-d 11 .

7. A kit for use in the method of claim 1, characterized in that comprises: (i) GP acetonitrile solution 0.01-0.05 M; (ii) EDC.HCl acetonitrile solution 0.02-0.08 M; (iii) pyridine aqueous solution 0.01-0.1 M; (iv) GP-labeled deuterated SCFAs internal standard mixture GP-AA-d4, GP-PA-d5, GP-BA-d7, GP-VA-d9, GP-HA-d 11 .

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