A method for direct determination of α- and γ-glutamyl peptides in skeletal muscle using LC-MS and isotope dilution assay

Abstract

The present invention discloses a method for directly determining α and γ-glutamyl peptides in skeletal muscle using LC-MS and isotope dilution assay. The method of the present invention comprises the following steps: 1) using dithiothreitol aqueous solution to prepare standard stock solutions and internal standard storage solutions of α- and γ-glutamyl-cysteinyl-glycine, other α- and γ-glutamyl peptide standards are dissolved in deionized water to prepare standard stock solutions, and all standard stock solutions are mixed and diluted in deionized water containing internal standards to prepare mixed working solutions; 2) fresh skeletal muscle samples to be tested are processed, extracted by adding an extraction reagent containing an internal standard, and diluted to obtain an extraction solution; 3) the mixed working solution and the extraction solution are respectively determined by LC-MS method, and the samples to be tested are quantitatively and qualitatively detected according to the liquid chromatography and mass spectrometry data of the extraction solution and the data analysis of the mixed working solution. The present invention accurately analyzes α- and γ-glutamyl peptides in skeletal muscle using LC-MS method, with high selectivity and high sensitivity.

Description

Technical Field

[0001] The present invention relates to a method for directly determining alpha and gamma-glutamyl peptides in skeletal muscle using LC-MS and isotope dilution assay. Background Art

[0002] α- and γ-glutamyl peptides are distinguished by the formation of peptide bonds by the α- or γ-carboxyl groups of glutamic acid. They are widely distributed in organisms in nature and participate in metabolic regulation and cell signal transduction. They have various biological activities such as antioxidant and anti-inflammatory. However, due to structural differences, they exhibit different physiological properties. For example, γ-glutamyl-leucine can be used as a biomarker for the clinical diagnosis of various cardiovascular diseases. The corresponding α-glutamyl-leucine was found to have antioxidant activity. Currently, researchers have found that γ-glutamyl peptides can impart a full, complex and lasting delicious taste, known as "kokumi" (i.e. thick taste), by activating calcium-sensing receptors (CaSR). These peptides can enhance salty, sweet and umami tastes, thereby improving the palatability of food. In contrast, α-glutamyl peptides do not have a thick taste. Instead, they have been shown to have umami, sour or bitter tastes. Interestingly, as taste-active compounds, glutamyl peptides have inconsistent taste descriptions in different studies. Glutamyl-leucine tasted both bitter and salty in the study by R. Kuramitsu et al., but exhibited a kokumi taste in the study by Liu et al. These differences in taste can be largely attributed to the difficulty in distinguishing between α- and γ-glutamyl peptides by conventional analytical methods.

[0003] With the advancement of analytical technology, many methods for analyzing α- and γ-glutamyl peptides have emerged. Alexander Bollenbach, Dimitrios Tsikas and others developed analytical methods by derivatizing γ-glutamyl peptides with pentafluorophthalic anhydride and pentafluorophenyl bromide using gas chromatography tandem mass spectrometry (GC-MS). Akiyoshi Hirayama and others established a method for quantitative determination of γ-glutamyl peptides using capillary electrophoresis tandem mass spectrometry (CE-MS), which showed high accuracy. However, the widespread application of these analytical methods has been hindered by the complex sample pretreatment and long chromatographic separation time. Liquid chromatography tandem mass spectrometry (LC-MS) methods have the advantages of high efficiency and high sensitivity and are often used to detect and analyze small peptides in complex biological matrices. However, due to the similar structures and strong polarity of the isomers of α-glutamyl peptides and γ-glutamyl peptides, they are difficult to be retained and separated on traditional reversed-phase columns. Motonaka Kuroda and others used C 18The column was used for LC-MS analysis to detect and quantify γ-glutamyl-valyl-glycine, but the sample still needed to be derivatized with 6-aminoquinoyl-N-hydroxysuccinimide carbamate (AQC, a commercial kit).

[0004] Nowadays, with the improvement of people's health awareness and the continuous pursuit of delicious food, α- and γ-glutamyl peptides in skeletal muscle have attracted more and more attention due to their biological activity and taste activity. Therefore, a method for detecting α- and γ-glutamyl peptides in skeletal muscle is urgently needed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for directly determining α- and γ-glutamyl peptides in skeletal muscle by using LC-MS and isotope dilution assay; the present invention is a highly selective and highly sensitive method for accurately analyzing α- and γ-glutamyl peptides in skeletal muscle by using LC-MS method.

[0006] To achieve the above-mentioned purpose, the present invention provides a method for directly determining α and γ-glutamyl peptides in skeletal muscle using LC-MS and isotope dilution assay, comprising the following steps:

[0007] 1) Prepare standard stock solutions of α- and γ-glutamyl-cysteinyl-glycine and internal standard γ-glutamyl-cysteinyl-glycine (glutathione) using dithiothreitol aqueous solution 13 C2 15 N1 stock solution, other α- and γ-glutamyl peptide standards were dissolved in deionized water to prepare standard stock solutions, and all α- and γ-glutamyl peptide standard stock solutions were mixed and diluted in deionized water containing internal standards to prepare a mixed working solution for use;

[0008] 2) The fresh skeletal muscle samples to be tested were trimmed to remove the inedible parts, minced and stored at -80°C, and the internal standard γ-glutamyl-cysteinyl-glycine (glutathione) was added. 13 C2 15 The extraction reagent of N1 is homogenized, centrifuged to remove the precipitate, the supernatant is collected, and diluted with the extraction reagent containing the internal standard to obtain an extraction solution;

[0009] 3) The mixed working solution in step 1) and the extraction solution in step 2) are respectively measured by LC-MS method, and the sample to be tested is quantitatively and qualitatively detected according to the liquid chromatography and mass spectrometry data of the extraction solution and the data analysis of the mixed working solution.

[0010] In the above method, the other α- and γ-glutamyl peptides include α- / γ-glutamyl-alanine, α- / γ-glutamyl-leucine, α- / γ-glutamyl-valine, α- / γ-glutamyl-phenylalanine, α- / γ-glutamyl-valyl-glycine;

[0011] The dithiothreitol aqueous solution of α- and γ-glutamyl-cysteinyl-glycine is prepared by 0.1% DTT aqueous solution to prepare 1.0 mg·mL -1 of α- and γ-glutamyl-cysteinyl-glycine;

[0012] The concentration of the internal standard stock solution is 7 μg mL -1 ;

[0013] The concentration of the mixed working solution is 0.5 to 1000 ng·mL -1 .

[0014] In the above method, in step 2), the sample extraction step is as follows:

[0015] Weigh the sample into a centrifuge tube and add 100 ng mL -1 The sample was mixed with an internal standard extractant containing 100 ng·mL -1 The mass volume ratio of the internal standard extraction reagent is 500 mg: 2-5 mL;

[0016] The mixture is homogenized at 60 Hz for 2 to 5 minutes and then placed at -80 to -20°C for 6 to 24 hours; it is then centrifuged at 10,000 to 14,000 rpm for 10 to 15 minutes at 4 to 10°C, the precipitate is removed, and the supernatant is collected, filtered and transferred to a sample bottle for analysis. Glutathione is detected by diluting it with an extraction reagent.

[0017] In the above method, the sample to be tested includes at least one of pork, chicken, beef and mutton;

[0018] The extraction reagent is selected from at least one of deionized water, ethanol, methanol, acetonitrile and methanol-water solution.

[0019] In the above method, in step 3), the liquid chromatography conditions are as follows:

[0020] Pentafluorophenyl (PFP) column, 2.1 × 100 mm, 1.7 μm;

[0021] The temperature of the column was maintained at 40°C;

[0022] Mobile phase A was deionized water, and mobile phase B was acetonitrile, both containing 0.2‰ formic acid by volume; the final gradient elution program was set as follows: 0-1min, 2% B; 1.5min, 10% B; 3min, 20% B; 7-8min, 98% B; 8.1-10min, 2% B;

[0023] The total running time can be 10 min and the flow rate can be 0.3 mL min -1 , the injection volume can be 5μL.

[0024] In the above method, in step 3), the mass spectrometry conditions are as follows:

[0025] The electrospray ion source was set to positive ion mode;

[0026] The ESI source parameters are as follows: the temperature of the drying gas N2 can be set to 250°C, and the flow rate can be set to 5 L min -1 ; Sheath gas temperature can be set to 150°C, flow rate can be set to 9L·min -1 ; Atomizer pressure can be set to 20psi; Capillary voltage can be set to 3500V; Nozzle voltage can be set to 0V.

[0027] In the above method, in step 3), when performing quantitative analysis, the mass spectrometry conditions are performed in a multiple reaction monitoring mode, and the dwell time of each multiple reaction monitoring (MRM) can be 50 ms.

[0028] In the above method, in step 3), the mass spectrometry data of the sample to be tested is compared with the data of the standard in the mixed working solution, and the transition from the precursor ion to the product ion with the highest response is used for the quantification of the target compound, while the remaining MS / MS transitions are used for the qualitative analysis of the target compound.

[0029] In the above method, in step 3), the quantitative analysis adopts an internal standard quantitative analysis method.

[0030] In the present invention, in step 3) quantitative analysis, the results are generally determined in accordance with EU Directive 2002 / 657 / EC, (EU) 2021 / 808, (1) the linear relationship of the standard curve (R 2 ) is greater than 0.99; (2) the recoveries (%) of high, medium and low levels are between 80% and 120%; (3) the relative standard deviation (RSD,%) of repeated test results is less than 15%.

[0031] The present invention has the following beneficial effects:

[0032] 1. The present invention uses a PFP chromatographic column to establish an HPLC-MS / MS method for separating and detecting α- and γ-glutamyl peptide isomers;

[0033] 2. The present invention separated 6 pairs of α- and γ-glutamyl peptide isomers within 10 minutes by optimizing the sample preparation method, chromatography and mass spectrometry conditions, and carried out quantitative analysis using the internal standard method, with the LOD lower than 1 ng·mL -1 , which shows that the method has extremely high sensitivity and detection efficiency, and the linearity, accuracy and precision all meet the detection requirements;

[0034] 3. The method of the present invention is successfully used to detect α- and γ-glutamyl peptide isomers in skeletal muscle samples. It is an effective method for accurately determining α- and γ-glutamyl peptide isomers in skeletal muscle. This application will greatly promote the research of healthy and delicious foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the chromatographic separation ability of α- and γ-glutamyl peptides on different chromatographic columns; Figure 1 A is the inverted C 18 Chromatographic column, B is a normal phase amide chromatographic column, and C is a PFP chromatographic column.

[0036] Figure 2 for the storage stability of all compounds and the extraction efficiency of different reagents; Figure 2 A is the extraction efficiency of endogenous glutathione using different extraction solvents, and B is the stability of all compounds; the data are expressed as mean ± standard deviation.

[0037] Figure 3 is the content of α- / γ-glutamyl peptide in the sample; Figure 3 A is black pork tenderloin, B is pork tenderloin, C is chicken breast, D is beef tenderloin and E is lamb tenderloin.

[0038] Figure 4 The effect of acetonitrile / water mobile phase system with different formic acid contents on the performance of the chromatographic column is shown in Figure 2. Figure 4 A represents the effect of different formic acid contents on the retention and separation of α- / γ-glutamyl-alanine, B represents the effect of different formic acid contents on the retention and separation of α- / γ-glutamyl-leucine, C represents the effect of different formic acid contents on the retention and separation of α- / γ-glutamyl-valine, D represents the effect of different formic acid contents on the retention and separation of α- / γ-glutamyl-phenylalanine, E represents the effect of different formic acid contents on the retention and separation of α- / γ-glutamyl-valyl-glycine, F represents the effect of different formic acid contents on the retention and separation of α- / γ-glutamyl-cysteinyl-glycine, and G represents the effect of mobile phase systems with different formic acid contents on the mass spectrometric response of α- / γ-glutamyl peptides.

[0039] Figure 5 is the structural formula of α- / γ-glutamyl peptide standard and internal standard.

[0040] Figure 6 is the mass spectrum of the standard; Figure 6 AL are the mass spectra of α-Glu-Ala, γ-Glu-Ala, α-Glu-Leu, γ-Glu-Leu, α-Glu-Val, γ-Glu-Val, α-Glu-Phe, γ-Glu-Phe, α-Glu-Val-Gly, γ-Glu-Val-Gly, α-Glu-Cys-Gly and γ-Glu-Cys-Gly respectively. DETAILED DESCRIPTION

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Example

[0044] 1. Materials and Methods

[0045] 1.1 Chemicals and Materials

[0046] Methanol, acetonitrile, formic acid and dithiothreitol (DTT) were all HPLC grade and purchased from Thermo Fisher Scientific (Shanghai, China). Standards α- / γ-glutamyl-alanine (α- / γ-Glu-Ala), α- / γ-glutamyl-leucine (α- / γ-Glu-Leu), α- / γ-glutamyl-valine (α- / γ-Glu-Val), α- / γ-glutamyl-phenylalanine (α- / γ-Glu-Phe), α- / γ-glutamyl-valyl-glycine (α- / γ-Glu-Val-Gly) and α-glutamyl-cysteinyl-glycine (α-Glu-Cys-Gly) were purchased from Shanghai Shengong Biotechnology Co., Ltd., and γ-glutamyl-cysteinyl-glycine (γ-Glu-Cys-Gly) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The structural formulas are shown in Table 1. Figure 5 As shown. Internal standard γ-glutamyl-cysteinyl-glycine (glutathione)- 13 C2 15 N1 (English abbreviation γ-Glu-Cys-Gly- 13 C2 15 N1) was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. The deionized water used was purified by Milli-Q Advantage A10 water system (Merck).

[0047] 1.2 Storage solution

[0048] To prevent sulfhydryl oxidation, 1.0 mg mL -1 α- and γ-glutamyl-cysteinyl-glycine and 7 μg mL -1 The internal standard stock solutions of α- and γ-glutamyl peptides were prepared in deionized water. 0.5, 1, 5, 10, 25, 50, 100, 250, 500, and 1000 ng mL -1 All solutions were stored in a 4°C refrigerator away from light.

[0049] 1.3 Sample preparation

[0050] The samples used in this application were purchased from Beijing Meilimei Chain Commercial Co., Ltd. (Beijing, China). The samples included pork tenderloin, black pork tenderloin, chicken breast, beef tenderloin and lamb tenderloin. The purchased fresh samples were transferred to the laboratory on ice, then trimmed to remove inedible parts, minced and stored at -80°C.

[0051] The sample extraction steps are as follows: weigh 500 mg of sample into a centrifuge tube, then add 2 mL of 100 ng mL -1 The extract of the internal standard. The mixture was homogenized at 60 Hz for 3 min and then placed at -20 ° C for 12 h. It was then centrifuged at 14,000 rpm for 15 min at 4 ° C to remove the precipitate. The supernatant was collected, filtered and transferred to a sample bottle for analysis, and glutathione was detected by diluting 100 times with the extract. In order to improve the extraction efficiency, five extraction solutions including deionized water, ethanol, methanol, acetonitrile and methanol / deionized water (4:1, υ:υ) were compared.

[0052] 1.4 Instruments

[0053] The qualitative and quantitative analysis of α- and γ-glutamyl peptides was performed using an Agilent 1290 Infinity II ultra-high performance liquid chromatograph and an Ultivo triple quadrupole system (Cernusco sul Naviglio, Italy).

[0054] 1.4.1 Liquid chromatography conditions

[0055] In order to better separate α- and γ-glutamyl peptides, the mobile phase and chromatographic column were optimized. The optimal conditions are as follows: a pentafluorophenyl (PFP) chromatographic column (2.1×100mm, 1.7μm) was selected. The temperature of the chromatographic column was maintained at 40°C. Mobile phase A was deionized water and mobile phase B was acetonitrile. Both contained 0.2‰ formic acid (υ:υ). The final gradient elution program was set as follows: 0-1min, 2% B; 1.5min, 10% B; 3min, 20% B; 7-8min, 98% B; 8.1-10min, 2% B. The total running time was 10min and the flow rate was 0.3mL·min -1 , injection volume is 5μL. 100μg·L -1 The chromatogram of the standard solution is shown in Figure 1 Middle C.

[0056] 1.4.2 Mass spectrometry conditions

[0057] The electrospray ion source was set to positive ion mode. The ESI source parameters were as follows: the drying gas (N2) temperature was set to 250 °C, the flow rate was 5 L min -1 ; The sheath gas temperature was set to 150°C and the flow rate was 9 L·min -1 ; Nebulizer pressure was set to 20 psi; capillary voltage was set to 3500 V; nozzle voltage was set to 0 V. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode with a dwell time of 50 ms for each MRM. The final MRM parameters and retention times of all standard compounds are shown in Table 1.

[0058] Table 1 Optimized MRM parameters and retention times for α- and γ-glutamyl peptides

[0059]

[0060] 1.5 Method Validation

[0061] The analytical method was validated in terms of linearity, sensitivity, matrix effects (ME), recovery, precision, selectivity, and stability. Spiked samples were prepared by adding appropriate standard solutions directly to the matching matrix followed by homogenization.

[0062] 1.5.1 Linearity and Sensitivity

[0063] The internal standard stock solution was gradually diluted with the matrix sample extraction solution and then diluted with 100 ng mL -1 The internal standard deionized water was gradually diluted with the mixed standard solution for analysis. The solvent and matrix matching calibration curves were fitted by the least squares regression method and the correlation coefficient (R 2) Linearity was evaluated by the limit of detection (LOD, S / N=3) and the limit of quantification (LOQ, S / N=10).

[0064] 1.5.2 Recovery, precision and matrix effect

[0065] Precision was evaluated based on recovery and matrix effect (ME). Recovery was evaluated by the ratio between the internal standard response after spiked samples and the internal standard response after blank samples were spiked. All recovery tests were performed at three concentration levels: high, medium, and low, 25, 50, and 250 ng mL -1 Each level was measured five times. The calculation formula of ME is as follows: ME = (slope of matrix matching calibration curve of internal standard / slope of solvent calibration curve of internal standard) × 100%.

[0066] 1.5.3 Selectivity and stability

[0067] 1mg·L -1 The mixed standard solution was used as a quality control (QC) sample to monitor stability. The selectivity of the method was verified by analyzing the QC sample in MRM mode and comparing the retention time and mass spectral secondary fragmentation of each glutamyl peptide.

[0068] 1.6 Data processing and statistical analysis

[0069] Data processing was performed using MassHunter Quantitative Analysis 10.1 and MassHunter Qualitative Analysis 10.0 (Agilent, Cernusco sul Naviglio, Italy). GraphPadPrism 10.2 software was used for data visualization and statistical analysis.

[0070] 2. Results and Discussion

[0071] 2.1 Optimization of ESI-MS / MS method

[0072] α- and γ-glutamyl peptides were quantitatively and qualitatively detected by LC-MS / MS in MRM mode. The optimization and selection of mass spectrometric conditions are crucial to achieve high sensitivity and specificity of the method. In this study, product ions were obtained by fragmentation in positive ion mode at 30 V collision energy. The mass spectra of all analytes are shown in Figure 6, indicating that both α- and γ-glutamyl peptides exhibit the same fragmentation pattern, making it challenging to distinguish isomers based solely on characteristic ions. Another interesting finding is that all glutamyl peptides have the same fragmentation with a mass-to-charge ratio (m / z) of 84.0, inferring that the product ion is a glutamyl residue after amide bond cleavage and loss of HCOOH, which may be a characteristic signature of glutamyl peptides.

[0073] When selecting MRM parameters, two of the highest responses were selected from all ion fragments of each compound as product ions. Specifically, the transition from the precursor ion to the highest response product ion was used for the quantification of the target compound, while the remaining MS / MS transitions were used for the qualitative analysis of the target compound. Then, the ion source parameters and other MRM parameters were optimized using Agilent MassHunter SourceOptimizer and Optimizer software. The MRM parameters for each glutamyl peptide are shown in Table 1.

[0074] 2.2 Optimization of liquid chromatography separation method

[0075] Since α- and γ-glutamyl peptide isomers have no characteristic ions, chromatographic separation is essential for qualitative and quantitative analysis. However, the high polarity and structural similarity of these isomers pose challenges for their retention and separation on chromatographic columns. To address this issue, the chromatographic separation of α- and γ-glutamyl peptides on reversed-phase (RP) C 18 Separation efficiency on chromatographic columns, normal phase (NP) amide columns and PFP columns. Figure 1 As shown, α- and γ-glutamyl peptides in RP C 18 The retention capacity on the chromatographic column is weak and in RP C 18 Or it is difficult to separate on NP amide column.

[0076] Fortunately, all α- and γ-glutamyl peptides were baseline separated on the PFP column. The chromatographic results of α- and γ-glutamyl peptides analyzed using the PFP column and the optimized instrumental method are shown in Figure 6. Figure 1 As shown in C. Although the retention mechanism of α- and γ-glutamyl peptides on PFP columns is still unclear, some studies have shown that the retention of Lewis basic compounds (such as amino acids and peptides) on PFP columns may involve ion exchange mechanisms, which may mean that the pH value of the mobile phase may have a significant impact on the retention of α- and γ-glutamyl peptides. Therefore, the effect of acetonitrile / water mobile phase systems with different formic acid contents (0.1‰, 0.2‰, 0.3‰, 0.4‰, 0.5‰, υ:υ) on the performance of the chromatographic column was investigated. Figure 4 As shown, Figure 4AF in the figure shows the effect of different formic acid content mobile phase systems on the retention and separation ability of α- and γ-glutamyl peptides, and G shows the effect of different formic acid content mobile phase systems on the mass spectrometric response of α- and γ-glutamyl peptides. Figure 4 The results showed that when the formic acid content in the mobile phase was 0.2‰, the separation effect was good and the mass spectrometry response was the highest.

[0077] 2.3 Selection of extraction reagents

[0078] Appropriate extraction reagents can improve the extraction rate of peptides in biological samples and reduce protein interference during analysis. Deionized water, ethanol, methanol, acetonitrile and methanol / deionized water (4:1, υ:υ) were selected as extraction reagents to evaluate the extraction efficiency. In brief, 500 mg of pork tenderloin were treated with these pre-cooled extraction reagents according to the above steps. The extraction efficiency was evaluated by analyzing the response of endogenous glutathione in the extract. Figure 2 As shown in Figure A, the mass spectrometric response of glutathione in the methanol / deionized water (4:1,υ:υ) extract is better than that of other extraction agents. Therefore, methanol / deionized water (4:1,υ:υ) was selected as the best extraction reagent for extracting glutamyl peptides from skeletal muscle samples.

[0079] 2.4 Method Validation

[0080] Six pairs of glutamyl peptides and internal standard IS were used to verify the linearity, precision, recovery, stability and selectivity of the established method.

[0081] 2.4.1 Stability and selectivity

[0082] The identification of the six pairs of glutamyl peptides was based on their MRM and retention times. All α- and γ-glutamyl peptides were successfully detected at their assigned MRM and retention times, showing excellent selectivity.

[0083] Since α- and γ-glutamyl-cysteinyl-glycine and the internal standard contain sulfhydryl groups, they are easily oxidized to form disulfides, which affects their use. Therefore, DTT was used as an antioxidant to protect the sulfhydryl groups in this study. Figure 2 As shown in B, the results are satisfactory, and the peak areas of all glutamyl peptide analytes have no significant differences within 48 hours of storage at 4°C in the dark.

[0084] 2.4.2 Matrix Effects

[0085] When using HPLC-MS / MS to analyze complex sample matrices, the mass spectrometric response intensity of the target compound is usually affected by matrix effects (ME). These matrix effects originate from the interference of co-eluting components on the ionization of the target compound and can be divided into matrix suppression effect and matrix enhancement effect. This effect has a significant impact on the precision and sensitivity of the analytical method. In this study, the matrix suppression effect was evaluated by comparing the slopes of the internal standard calibration curves in the sample extract and the extraction reagent. The ME in the four sample matrices ranged from 47.80% to 70.6%, showing a strong matrix suppression effect. Therefore, adopting an appropriate strategy to reduce ME is the key to achieving accurate analysis. Therefore, an internal standard-based quantitative analysis method was used to correct the matrix effect and ensure the accuracy of sample analysis. Table 2 lists the ME test results for each matrix.

[0086] Table 2 Matrix effects, method recoveries and repeatability of different matrices

[0087]

[0088] 2.4.3 Linearity, LOD and LOQ

[0089] Table 3 shows that all 12 standards were in the range of 0.5 to 1000 ng·mL -1 The internal standard calibration curve showed good linearity in the range of 2 The detection limits (LOD) of α- and γ-glutamyl peptides were between 0.5 and 1.0 ng·mL -1 Their limits of quantification (LOQ) were between 1.0 and 5.0 ng·mL -1 Akiyoshi Hirayama et al. developed a method for detecting γ-glutamyl peptides using CE-MS. The detection limits of the six γ-glutamyl peptides reported in the paper ranged from 0.8 to 37.06 ng·mL -1 Naohiro MIYAMURA used AQC derivatization and LC / MS / MS to determine the LOQ of γ-glutamyl-valyl-glycine to be 400 ng·mL -1 Therefore, the sensitivity of the method established in this paper is better than that of existing methods, and the detection efficiency has significant advantages.

[0090] Table 3 Linear range, calibration curves and correlation coefficients of 12 standards and internal standards

[0091]

[0092] 2.4.4 Recovery rate test

[0093] Precision was assessed based on recovery. Four independent matrices, pork, chicken, beef, and lamb, were used at 25, 50, and 250 ng mL -1 The recovery was evaluated by comparing the internal standard response at three different concentration levels. The results are shown in Table 2. The average recoveries at the three added levels ranged from 80.16% to 105.59%, with RSDs ranging from 1.9% to 5.08%, showing good precision.

[0094] 2.5 Qualitative and quantitative analysis of α- and γ-glutamyl peptides in skeletal muscle samples

[0095] like Figure 3 As shown in Figure 2, glutamyl peptides in the collected samples were analyzed using the developed method. Among these peptides, γ-glutamyl-cysteinyl-glycine, a well-known bioactive compound known for its potent antioxidant properties, was quantified using the established method in the concentration range of 28.0 to 35.5 mg kg -1 . Notably, α-glutamyl-cysteinyl-glycine, γ-glutamyl-valyl-glycine, α-glutamyl-valyl-glycine, and α-glutamyl-valine were not detected in any skeletal muscle samples. γ-Glutamyl peptides have been reported to contribute to the production of a rich kokumi flavor (thick taste) and to have significant differences in their distribution in different skeletal muscles. Therefore, this method can be further used to monitor changes in taste-active compounds in meat or meat products, providing technical support for food sensory research.

[0096] 3 Conclusion

[0097] The present invention uses a PFP chromatographic column to establish an HPLC-MS / MS method for separating and detecting α- and γ-glutamyl peptide isomers. By optimizing the sample preparation method, chromatographic and mass spectrometric conditions, 6 pairs of α- and γ-glutamyl peptide isomers were separated within 10 minutes, and quantitative analysis was performed using the internal standard method, with LOD less than 1 ng·mL -1 . This indicates that the method has extremely high sensitivity and detection efficiency. The linearity, accuracy, and precision all meet the detection requirements. The method was successfully used to detect α- and γ-glutamyl peptide isomers in skeletal muscle samples. In conclusion, this study proposed an effective method for the accurate determination of α- and γ-glutamyl peptide isomers in skeletal muscle, and this application will greatly promote the research of healthy and delicious foods.

Claims

1. A method for directly determining α-glutamyl peptides and γ-glutamyl peptides in skeletal muscle using LC-MS and isotope dilution assay, comprising the following steps: 1) Prepare the standard stock solutions of α-glutamyl-cysteinyl-glycine and γ-glutamyl-cysteinyl-glycine and the internal standard γ-glutamyl-cysteinyl-glycine using dithiothreitol aqueous solution. 13 C2 15 The stock solution of N1 and other α-glutamyl peptide and γ-glutamyl peptide standards were dissolved in deionized water to prepare standard stock solutions. All α-glutamyl peptide and γ-glutamyl peptide standard stock solutions were mixed and diluted in deionized water containing internal standards to prepare a mixed working solution for use; The other α-glutamyl peptides and γ-glutamyl peptides are α-glutamyl-alanine, γ-glutamyl-alanine, α-glutamyl-leucine, γ-glutamyl-leucine, α-glutamyl-valine, γ-glutamyl-valine, α-glutamyl-phenylalanine, γ-glutamyl-phenylalanine, α-glutamyl-valyl-glycine and γ-glutamyl-valyl-glycine; 2) Trim the fresh skeletal muscle sample to remove the inedible part, mince it and store it at -80℃; the sample extraction steps are as follows: Weigh the sample into a centrifuge tube and add 100 ng mL -1 The sample was mixed with an internal standard extract containing 100 ng·mL -1 The mass volume ratio of the extraction reagent for the internal standard is 500 mg: 2~5 mL; the mixture is homogenized at 60 Hz for 2~5 min and then placed at -80~-20℃ for 6~24 h; it is then centrifuged at 10,000~14,000 rpm for 10~15 min at 4~10℃, the precipitate is removed, and the supernatant is collected, filtered and transferred to a sample bottle for analysis; The extraction reagent is methanol / deionized water with a volume ratio of 4:1; 3) Performing quantitative and qualitative detection on the sample by LC-MS method; The liquid chromatography conditions were as follows: Pentafluorophenyl column, 2.1 × 100 mm, 1.7 μm; The temperature of the column was maintained at 40°C; Mobile phase A was deionized water, and mobile phase B was acetonitrile, both containing 0.2‰ formic acid by volume; the final gradient elution program was set as follows: 0-1 min, 2% B; 1.5 min, 10% B; 3 min, 20% B; 7-8 min, 98% B; 8.1-10 min, 2% B; The total run time was 10 min and the flow rate was 0.3 mL min -1 , injection volume was 5 μL; The mass spectrometry conditions were as follows: The electrospray ion source was set in positive ion mode; the mass spectrometry conditions were performed in multiple reaction monitoring mode.

2. The method according to claim 1, characterized in that The standard stock solutions of α-glutamyl-cysteinyl-glycine and γ-glutamyl-cysteinyl-glycine were prepared using 0.1% DTT aqueous solution to a concentration of 1.0 mg·mL -1 of α-glutamyl-cysteinyl-glycine and γ-glutamyl-cysteinyl-glycine; The concentration of the internal standard stock solution was 7 μg mL -1 ; The concentration of the mixed working solution is 0.5-1000 ng·mL -1 .

3. The method according to claim 1 or 2, characterized in that: The sample to be tested includes at least one of pork, chicken, beef and mutton.

4. The method according to claim 1 or 2, characterized in that: In step 3), the mass spectrometry conditions are as follows: The ESI source parameters were as follows: the temperature of the drying gas N2 was set to 250 °C and the flow rate was 5 L min -1 ; The sheath gas temperature was set to 150°C and the flow rate was 9 L·min -1 ; Nebulizer pressure was set to 20 psi; Capillary voltage was set to 3500 V; Nozzle voltage was set to 0 V.

5. The method according to claim 1 or 2, characterized in that In step 3), when performing quantitative analysis, the dwell time for each MRM was 50 ms.

6. The method according to claim 1 or 2, characterized in that In step 3), the mass spectrum data of the sample to be tested is compared with the data of the standard in the mixed working solution, and the transition from the precursor ion to the product ion with the highest response is used for the quantification of the target compound, while the remaining MS / MS transitions are used for the qualitative identification of the target compound.

7. The method according to claim 1 or 2, characterized in that: In step 3), the quantitative analysis adopts an internal standard quantitative analysis method.

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