Method for determining cyclic dipeptide epimer

Abstract

The invention relates to the field of analytical chemistry and biochemistry, and discloses a cyclic dipeptide epimer determination method, which comprises: S1, sample preparation: extracting a sample containing a cyclic dipeptide epimer, treating the sample with a solvent, filtering through a filtration membrane, and adding an internal standard substance with a known concentration for quantification; s2, adding an internal standard: adding an internal standard solution into the sample; s3, high performance liquid chromatography separation: injecting the sample into a high performance liquid chromatography system, and carrying out gradient elution by adopting a specific chromatographic column; s4, mass spectrometric detection: adopting an electrospray ionization source and a positive ion mode, and selecting specific parent ion and daughter ion pairs; and S5, data processing: comparing the peak area of the cyclic dipeptide epimer in the mass spectrum with the peak area of the internal standard. By combining HPLC and MS / MS technologies, efficient separation and high-sensitivity detection of the cyclic dipeptide epimer can be realized, and especially, the method has excellent detection performance on a low-concentration sample (the lowest detection limit can reach 50ng / mL).

Description

Technical Field

[0001] The invention relates to the fields of analytical chemistry and biochemistry, and in particular to a method for determining a cyclic dipeptide diastereomer. Background Art

[0002] Cyclic dipeptides are an important class of bioactive molecules that are widely present in nature and have a variety of pharmacological activities, such as antibacterial, anti-tumor, anti-inflammatory, and antioxidant effects. Therefore, cyclodipeptide compounds have important research and application value in the fields of medicine, food, and agriculture. Among the numerous cyclodipeptide compounds, the diastereomers of cyclodipeptides (such as L-silk-L-tyrosine cyclodipeptide and D-silk-L-tyrosine cyclodipeptide) often exhibit different biological properties and pharmacological effects due to their structural chiral differences. Therefore, accurately distinguishing and determining the content of cyclodipeptide diastereomers is of great significance for further research and development of their potential functions.

[0003] However, the structural similarity of cyclodipeptide isomers makes it difficult to effectively distinguish them using traditional detection methods (such as high performance liquid chromatography, HPLC). Especially when the physicochemical properties of the two isomers are very similar, it is difficult to obtain sufficient separation effect by relying solely on ordinary chromatography technology. In addition, existing chromatographic detection methods usually require long separation steps and high-cost chiral chromatographic columns, and have low sensitivity, making it difficult to detect low-concentration target compounds. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for determining diastereomers of cyclic dipeptides, which solves the problem that the existing chromatographic detection method has low sensitivity and is difficult to detect low-concentration target compounds.

[0005] To achieve the above object, the present invention is implemented by the following technical scheme: a method for determining the diastereomer of a cyclic dipeptide, comprising the following steps: S1: Sample preparation: extract samples containing cyclic dipeptide isomers, treat the samples with solvents, filter them through a membrane, and add internal standard substances of known concentrations for quantification; S2: Internal standard addition: add internal standard solution to the sample, the internal standard is used as a quantitative standard; S3: HPLC separation: the sample is injected into the HPLC system, a specific chromatographic column is used, and the mobile phase is a mixture of aqueous formic acid and aqueous formic acid-acetonitrile solution, and gradient elution is performed; S4: Mass spectrometry detection: using electrospray ionization source, positive ion mode, selecting specific parent ion and daughter ion pairs, and using multiple reaction monitoring mode for detection; S5: Data processing: By comparing the peak area of ​​the cyclic dipeptide diastereomer in the mass spectrum with the peak area of ​​the internal standard, the concentration of the cyclic dipeptide diastereomer is calculated according to the standard curve.

[0006] Preferably, the internal standard in step S2 is L-glycine-L-tyrosine cyclodipeptide, and the internal standard concentration is between 250-500 ng / ml.

[0007] Preferably, the chromatographic column in step S3 is a Kinetex F5 chromatographic column with a specification of 150 mm×3 mm and a particle size of 2 to 3 μm.

[0008] Preferably, in step S3, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% aqueous formic acid acetonitrile solution, the flow rate is between 0.2 mL / min and 0.4 mL / min, and the column temperature is between 20°C and 30°C.

[0009] Preferably, the gradient elution procedure in step S3 is: 0-3 minutes, mobile phase A is 98:2; 3-8 minutes, linear gradient transition to A is 80:20; 8-12 minutes, keep A is 80:20; 12-15 minutes, recovery A It is 98:2.

[0010] Preferably, the parent ion and daughter ion pairs detected by mass spectrometry in step S4 are respectively: L-Silk-L-tyrosine cyclodipeptide: parent ion m / z 251.0, daughter ion m / z 136.0; D-silk-L-tyrosine cyclodipeptide: parent ion m / z 251.1, daughter ion m / z 136.3; Internal standard L-Gly-L-tyrosine cyclic dipeptide: parent ion m / z is 221.1, and daughter ion m / z is 107.3.

[0011] Preferably, in step S1, the solvent is a mixed solvent of methanol and water in a volume ratio of 50:50, and the pore size of the filter membrane is 0.22 μm.

[0012] Preferably, in step S3, the sample injection volume is 10 μL to 50 μL.

[0013] Preferably, in the step S4, the curtain gas pressure used in mass spectrometry detection is 20 to 25 psi, and the spray gas pressure is 14 to 18 psi.

[0014] The present invention provides a method for determining diastereomers of cyclic dipeptides. The method has the following beneficial effects: 1. By combining HPLC and MS / MS technology, the present invention can achieve efficient separation and high-sensitivity detection of cyclic dipeptide diastereomers, especially for low-concentration samples (the minimum detection limit can reach 50ng / mL). Compared with traditional high-performance liquid chromatography, HPLC-MS / MS has higher sensitivity and can effectively detect extremely low-concentration compounds, which is suitable for detecting cyclic dipeptide diastereomers in complex biological samples.

[0015] 2. The present invention avoids the high cost of chiral chromatographic columns by using non-chiral chromatographic columns (Kinetex F5 chromatographic columns) while maintaining efficient isomer separation. Traditional methods require expensive chiral chromatographic columns or the addition of chiral reagents to achieve separation of diastereomers, while the present invention uses optimized chromatographic conditions to achieve efficient separation without chiral columns, greatly reducing experimental costs.

[0016] 3. Compared with some traditional detection methods that rely on chiral chromatographic columns, the present method significantly shortens the analysis time through optimized gradient elution program and flow rate control. In a relatively short time, the complete separation of L-silk-L-tyrosine cyclodipeptide and D-silk-L-tyrosine cyclodipeptide can be achieved, and accurate quantitative analysis can be provided, thereby improving the work efficiency of the laboratory.

[0017] 4. The present invention optimizes the mass spectrometry detection parameters to ensure efficient detection of the parent ion and daughter ion pairs of the cyclic dipeptide isomers in the positive ion mode. By selecting appropriate collision voltage and curtain gas pressure, the stable cleavage of the target compound and high-quality signal output are guaranteed, ensuring the stability and accuracy of the detection results.

[0018] 5. The present invention is not only applicable to the detection of L-silk-L-tyrosine cyclodipeptide and D-silk-L-tyrosine cyclodipeptide, but can also be applied to the detection of other cyclodipeptide compounds with similar structures by corresponding adjustments. Therefore, the present invention has strong versatility and can be widely used in the fields of drug development, biomarker detection, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the mass spectrum of L-silk-L-tyrosine cyclic dipeptide of the present invention; Figure 2 is the mass spectrum of D-silk-L-tyrosine cyclic dipeptide of the present invention; Figure 3 is the mass spectrum of L-Gly-L-tyrosine cyclic dipeptide of the present invention; Figure 4 This is the extracted ion chromatogram of the present invention. DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Please see attached Figure 1 - Attachment Figure 4 The embodiment of the present invention provides a method for determining the diastereomers of a cyclic dipeptide, comprising the following steps.

[0022] S1: Sample preparation Samples containing cyclodipeptide diastereomers (e.g., biological samples or synthetic samples) are treated with a solvent. The solvent is a mixture of methanol and water in a volume ratio of 50:50 to ensure that the cyclodipeptide in the sample can be effectively dissolved. The treated sample is filtered through a filter membrane with a pore size of 0.22 μm to remove particles or other impurities. In order to improve the quantitative accuracy, a known concentration of the internal standard substance L-gly-L-tyrosine cyclodipeptide is added, and its concentration range is 250-500 ng / ml.

[0023] S2: Internal standard addition Add the internal standard substance L-Gly-L-Tyrosine Cyclodipeptide to the prepared sample, which acts as a quantitative standard to calibrate the quantification of cyclodipeptide diastereomers in the sample. The internal standard concentration in each sample is controlled between 250-500ng / ml to ensure the stability of the internal standard signal during detection and facilitate subsequent data analysis.

[0024] S3: HPLC separation The treated samples were injected into the HPLC system. The chromatographic column used was a Kinetex F5 column with a specification of 150 mm × 3 mm and a particle size of 2 to 3 μm to ensure adequate separation. The mobile phases for chromatographic separation included mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% aqueous formic acid acetonitrile solution, the flow rate was controlled between 0.2 mL / min and 0.4 mL / min, and the column temperature was controlled between 20°C and 30°C.

[0025] The specific gradient elution procedure is as follows: 0-3 minutes, mobile phase A is 98:2; 3-8 minutes, linear gradient transition to A is 80:20; 8-12 minutes, keep A is 80:20; 12-15 minutes, recovery A It is 98:2.

[0026] The sample injection volume is 10 μL to 50 μL to ensure that the signal intensity is sufficient for detection. The two isomers of L-silk-L-tyrosine cyclodipeptide and D-silk-L-tyrosine cyclodipeptide can be effectively separated by chromatographic separation.

[0027] S4: Mass spectrometry detection After chromatographic separation, the sample enters the mass spectrometry system for detection. Electrospray ionization (ESI) is used for detection in positive ion mode. Through the multiple reaction monitoring mode (MRM), specific parent ion and daughter ion pairs are selected for detection. The mass spectrometry parameters are as follows: The parent ion m / z of L-silk-L-tyrosine cyclodipeptide was 251.0, and the product ion m / z was 136.0; The parent ion m / z of D-silk-L-tyrosine cyclodipeptide was 251.1, and the product ion m / z was 136.3; The parent ion m / z of L-Gly-L-tyrosine cyclic dipeptide (internal standard) was 221.1, and the product ion m / z was 107.3.

[0028] The curtain gas pressure of mass spectrometry detection is 20 to 25 psi, and the spray gas pressure is 14 to 18 psi to ensure stable mass spectrometry signal output and accurate detection results.

[0029] S5: Data Processing The peak area detected by the mass spectrometer is standardized using the peak area of ​​the internal standard substance. The obtained data is compared with the standard curve to calculate the concentration of the cyclodipeptide diastereomer in the sample. This method can accurately quantify the content of L-silk-L-tyrosine cyclodipeptide and D-silk-L-tyrosine cyclodipeptide, and the minimum detection sensitivity can reach 50ng / mL, ensuring the detection accuracy of low-concentration isomers.

[0030] The materials used in this determination method include the following categories, and the purpose and function of each type of material are described below.

[0031] 1.Mixed solvent of methanol and water Application: Used for dissolution and preliminary processing of samples.

[0032] Function: Methanol as an organic solvent can effectively dissolve cyclodipeptide compounds, while water as a polar solvent can help dissolve water-soluble components. Mixed solvents can improve the solubility of samples and ensure that the cyclodipeptide isomers in the sample are completely dissolved, which is convenient for subsequent chromatographic separation and detection. A 50:50 volume ratio is usually selected, which not only ensures the solubility of organic matter, but also does not affect subsequent chromatography and mass spectrometry detection.

[0033] 2. Filter membrane (pore size 0.22μm) Application: Used for filtering samples.

[0034] Function: The filter membrane with a pore size of 0.22μm can effectively remove solid particles and insoluble impurities in the sample, preventing these impurities from causing blockage or interference in the HPLC system. This filtration step ensures the purity of the sample and ensures smooth flow of the mobile phase in the chromatographic column.

[0035] 3. L-Gly-L-Tyrosine Cyclic Dipeptide (Internal Standard) Uses: Added into samples as internal standard for quantitative analysis.

[0036] Function: The internal standard is a substance with known concentration and properties, which is added to the sample as a reference. L-Gly-L-Tyrosine Cyclic Dipeptide as an internal standard can generate a stable signal during mass spectrometry detection, and has similar physical and chemical properties to the target analyte, but will not be confused with the analyte. By comparing the internal standard signal with the target compound signal, accurate quantitative analysis can be performed.

[0037] 4. Kinetex F5 chromatographic column (specifications: 150 mm × 3 mm, particle size: 2 to 3 μm) Uses: Used for chromatographic separation.

[0038] Function: KinetexF5 column is a core component of high performance liquid chromatography separation, with a size of 150mm×3mm, which can provide efficient compound separation. The particle size of 2 to 3μm ensures a large surface area for interaction with the sample, which is suitable for separating small molecule compounds such as cyclic dipeptides. KinetexF5 column has good selectivity and can effectively separate the two isomers of L-silk-L-tyrosine cyclic dipeptide and D-silk-L-tyrosine cyclic dipeptide.

[0039] 5. 0.1% formic acid aqueous solution (mobile phase A) Uses: As one of the mobile phases in high performance liquid chromatography.

[0040] Function: Formic acid is a weak acid that can adjust the pH value of the chromatographic system, promote the ionization of compounds, and increase the sensitivity of mass spectrometry detection. 0.1% formic acid aqueous solution is used as mobile phase A, mainly to maintain the aqueous phase component of the sample and ensure good resolution and retention time during chromatographic separation.

[0041] 6.0.1% aqueous formic acid acetonitrile solution (mobile phase B) Uses: As another mobile phase for high performance liquid chromatography.

[0042] Function: Acetonitrile is an organic solvent with low polarity, which can promote the separation of hydrophobic substances. Adding 0.1% formic acid can further enhance the ionization effect of compounds in mass spectrometry detection. By mixing with mobile phase A (formic acid aqueous solution) in proportion to form a gradient elution system, mobile phase B can control the separation of different polar components in the sample.

[0043] 7. Electrospray ionization source (ESI) Application: Used for mass spectrometry detection.

[0044] Function: The electrospray ionization source is an ionization device in a mass spectrometer that can spray liquid samples through high voltage to form an aerosol of charged ions, so that the analyte is ionized and enters the mass spectrometer detector. The positive ion mode is for the positively charged ions in the target cyclodipeptide molecule, which can achieve higher sensitivity detection.

[0045] 8. Curtain gas and spray gas Application: Auxiliary gas in mass spectrometry.

[0046] Function: Curtain gas (curtain pressure is 20 to 25 psi) is used to control aerosol formation and ion transmission in the electrospray ionization source to ensure a stable ion flow into the mass spectrometer. Spray gas (pressure is 14 to 18 psi) is used to assist in the atomization of liquid samples, increase ionization efficiency, and thus improve the sensitivity and stability of mass spectrometry detection.

[0047] By using these materials, the entire determination method can efficiently and accurately achieve the separation and quantitative detection of cyclic dipeptide diastereomers. Each material plays a key role in its respective link, ensuring the success rate of the experiment and the reliability of the data.

[0048] Combine the following information and tables: Research objects: L-silk-L-tyrosine cyclic dipeptide and D-silk-L-tyrosine cyclic dipeptide Internal standard substance: L-Gly-L-tyrosine cyclodipeptide Research Method: HPLC-MS / MS Chromatographic conditions: chromatographic column (kinetex F5, 150×3 mm, 2.6 um); flow rate 0.2 mL / min; column temperature 20°C; mobile phase: 0.1% FA-H2O:0.1% FA-(CH3CN:H2O=95:5)=98:2.

[0049] Mass spectrometry conditions: electrospray ionization source, 4500 V; ion source temperature 550°C; positive ion mode; MRM detection; collision gas: medium; curtain gas: 20 psi; spray gas: 14 psi; auxiliary heating gas: 14 psi.

[0050] Table 1: Mass spectrometry detection parameters for cyclic dipeptide isomers Analytes Precursor ion Product ions Collision voltage L-Silk-L-Tyrosine Cyclic Dipeptide 251.0 136.0 24.0 D-Silk-L-Tyrosine Cyclic Dipeptide 251.1 136.3 24.0 L-Gly-L-Tyrosine Cyclic Dipeptide 221.1 107.3 20.0 It can be seen that: Target analytes: The table lists three cyclic dipeptide compounds, namely L-serine-L-tyrosine cyclic dipeptide, D-serine-L-tyrosine cyclic dipeptide and L-glycine-L-tyrosine cyclic dipeptide (as internal standard), which indicates that the objects of the experiment are these specific compounds.

[0051] Mass spectrometry parameters: Precursor ion (m / z): The mass-to-charge ratio (m / z value) of the precursor ion detected by the mass spectrometer for each compound. These values ​​can be used to confirm that the mass spectrometer analyzed ions of a specific mass-to-charge ratio during detection.

[0052] Product ion (m / z): The mass-to-charge ratio of the product ion formed after mass spectrometry fragmentation. These values ​​indicate the specific fragment information produced by the collision fragmentation of the analyte through the mass spectrometry, which is used for subsequent quantitative and qualitative analysis.

[0053] Collision voltage: The table lists the collision voltage corresponding to each compound. This is the energy condition in the mass spectrometry fragmentation process. Different voltages will affect the fragmentation behavior of the compound, thereby generating different product ion information. The choice of collision voltage helps to optimize the mass spectrometry signal intensity and specificity.

[0054] Key details of the experimental method: HPLC-MS / MS detection conditions: Parameters such as electrospray voltage (4500 V), temperature (550 °C), curtain gas, and spray gas provide the operating conditions for the experiment and ensure the repeatability and detection sensitivity of the experiment.

[0055] Mobile phase and chromatographic column: The text in the background of the table describes the chromatographic separation conditions in the experiment, such as 0.1% FA-H2O and 0.1% FA-(CH3CN:H2O=95:5)=98:2 as the mobile phase, and the information of the chromatographic column used (kinetexF5, 150×3mm, particle size 2.6μm). These conditions help to achieve effective separation of the sample.

[0056] With this information, the table summarizes the specific parameters of each cyclic dipeptide in mass spectrometry detection and shows the optimized conditions selected during the experiment, which helps to perform accurate quantitative and qualitative analysis in the experiment.

[0057] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining a cyclic dipeptide epimer, characterized in that: The following steps are involved: S1: Sample preparation: extract samples containing cyclic dipeptide isomers, treat the samples with solvents, filter them through a membrane, and add internal standard substances of known concentrations for quantification; S2: Internal standard addition: add internal standard solution to the sample, the internal standard is used as a quantitative standard; S3: HPLC separation: the sample is injected into the HPLC system, a specific chromatographic column is used, and the mobile phase is a mixture of formic acid aqueous solution and formic acid acetonitrile aqueous solution, and gradient elution is performed; S4: Mass spectrometry detection: using electrospray ionization source, positive ion mode, selecting specific parent ion and daughter ion pairs, and using multiple reaction monitoring mode for detection; S5: Data processing: By comparing the peak area of ​​the cyclic dipeptide diastereomer in the mass spectrum with the peak area of ​​the internal standard, the concentration of the cyclic dipeptide diastereomer is calculated according to the standard curve.

2. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: The internal standard in step S2 is L-glycine-L-tyrosine cyclic dipeptide, and the internal standard concentration is between 250-500 ng / ml.

3. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: The chromatographic column in step S3 is a Kinetex F5 chromatographic column with a specification of 150 mm×3 mm and a particle size of 2 to 3 μm.

4. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: In the step S3, the mobile phase A is a 0.1% formic acid aqueous solution, the mobile phase B is a 0.1% aqueous formic acid acetonitrile solution, the flow rate is between 0.2 mL / min and 0.4 mL / min, and the column temperature is between 20°C and 30°C.

5. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: The gradient elution procedure in step S3 is: 0-3 minutes, mobile phase A is 98:2; 3-8 minutes, linear gradient transition to A is 80:20; 8-12 minutes, keep A is 80:20; 12-15 minutes, recovery A It is 98:

2.

6. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: The parent ion and daughter ion pairs detected by mass spectrometry in step S4 are: L-Silk-L-tyrosine cyclodipeptide: parent ion m / z 251.0, daughter ion m / z 136.0; D-silk-L-tyrosine cyclic dipeptide: parent ion m / z 251.1, daughter ion m / z 136.3; Internal standard L-Gly-L-tyrosine cyclic dipeptide: parent ion m / z is 221.1, and daughter ion m / z is 107.

3.

7. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: In the step S1, the solvent is a mixed solvent of methanol and water in a volume ratio of 50:50, and the pore size of the filter membrane is 0.22 μm.

8. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: In the step S3, the sample injection volume is 10 μL to 50 μL.

9. The method for determining a cyclic dipeptide epimer according to claim 1, characterized in that: In the step S4, the curtain gas pressure used in mass spectrometry detection is 20 to 25 psi, and the spray gas pressure is 14 to 18 psi.

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