A method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry

Abstract

This invention belongs to the field of analytical chemistry and biomedical detection, and discloses a method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry (LC-MS). The method includes: pretreatment of the biological sample by homogenization with a weakly basic ammonium acetate buffer, protein precipitation with organic solvent, vacuum concentration, and reconstitution; separation using an alkali-resistant C18 column and a weakly basic mobile phase with gradient elution; and detection by multiple reaction monitoring (MRM) in negative ion mode using an electrospray ionization source. This invention can simultaneously detect more than 40 target substances, covering bases, nucleosides, deoxynucleosides, and their phosphorylated forms and modified metabolites, with a limit of quantitation at the ng / pg / mL level. It exhibits strong resistance to matrix interference and excellent chromatographic separation. This method is rapid, highly sensitive, and stable, suitable for high-throughput and precise detection of nucleotides in biological samples, providing a powerful tool for disease biomarker screening and related basic research.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and biomedical detection technology, specifically to a method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry. Background Technology

[0002] Nucleotides, as core molecules of life activities, are widely involved in key biological processes such as DNA / RNA synthesis, energy metabolism, and cell signal transduction. Their concentration levels are closely related to various pathological states such as tumors, metabolic diseases, and immune diseases. They are highly promising biomarkers in the biomedical field, and their accurate detection is of great significance in early disease diagnosis, pathological mechanism research, and efficacy evaluation.

[0003] Currently, the main methods for detecting nucleotides in biological samples include liquid chromatography-ultraviolet (LC-UV) and liquid chromatography-mass spectrometry (LC-MS). However, existing technologies face several significant bottlenecks in practical applications, making it difficult to meet the demands for high-throughput, high-sensitivity, and high-accuracy detection.

[0004] The pretreatment process is lengthy: Traditional detection methods often require complex solid-phase extraction steps for sample purification, which is cumbersome, time-consuming and labor-intensive. This not only increases experimental costs but also severely limits sample processing throughput, making it unsuitable for the actual needs of large-scale biological sample screening.

[0005] Matrix interference is a significant problem: residual phospholipids in biological samples are the main cause of ion suppression during electrospray ionization. Existing pretreatment methods are insufficient to effectively remove such matrix impurities, leading to reduced response signals and poor quantitative repeatability of target analytes, severely impacting the accuracy and reliability of detection results.

[0006] Insufficient detection coverage and sensitivity: Nucleotides encompass various forms, including bases, nucleosides, deoxynucleosides, and their monophosphate, diphosphate, and triphosphate forms. These substances exhibit different physicochemical properties, making it difficult for existing methods to simultaneously quantify all types of compounds within the same detection system, particularly limiting their detection capabilities for nucleosides and deoxynucleotides. Furthermore, existing methods generally have low sensitivity, failing to meet the detection requirements for low-abundance nucleotides in biological samples.

[0007] Poor chromatographic separation: Conventional detection methods often use acidic mobile phase systems, but nucleotides are highly polar and have weak retention capacity under acidic conditions, making them prone to co-elution and severe peak tailing, which directly affects the accuracy of quantitative analysis.

[0008] With the advancement of epigenetic research, nucleoside-modified metabolites (such as N6-methyladenosine-m) have become increasingly important. 6 A, 5-methylcytidine-m 5C, 5-hydroxymethylcytidine-hm 5 The crucial role of modified metabolites (C, etc.) in gene expression regulation is being gradually revealed, and their potential as novel disease biomarkers is increasingly evident. However, the detection of such modified metabolites faces more severe challenges: on the one hand, modified metabolites are diverse and coexist with classical nucleotides in biological samples, with a wide range of polarity and acidity / basicity, making effective separation under the same chromatographic conditions extremely difficult; on the other hand, the abundance of modified metabolites is usually much lower than that of their parent nucleotides, placing extremely high demands on the sensitivity of detection methods; in addition, current research mostly focuses on the specific analysis of single or a few modified metabolites, lacking high-throughput methods that can perform panoramic, simultaneous analysis of classical nucleotide pools and modified metabolites.

[0009] In summary, existing detection methods suffer from problems such as complex pretreatment, weak resistance to matrix interference, narrow detection coverage, insufficient sensitivity, and poor chromatographic separation, failing to meet the demand for efficient and accurate detection of nucleotides (especially modified metabolites) in biological samples. Therefore, there is an urgent need in this field to develop a nucleotide detection method that features simple and rapid pretreatment, strong resistance to interference, broad coverage, and high sensitivity to fill the gaps in existing technologies and provide strong support for related biomedical research and clinical applications. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of existing technologies by providing a simple, rapid, highly sensitive, and matrix-resistant LC-MS / MS method for detecting nucleotides.

[0011] To solve the above-mentioned technical problems, the following technical solution is adopted: A method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry is provided, comprising the following steps:

[0012] Biological samples were homogenized in a weakly alkaline buffer solution, and organic solvents were added to precipitate proteins. The supernatant was then separated. After drying the supernatant, it was reconstituted with a weakly alkaline buffer solution to obtain the test solution.

[0013] The test solution was injected into the liquid chromatography system, separated using an alkali-resistant reversed-phase column, and a gradient elution program was used, wherein mobile phase A was a weakly alkaline buffer and mobile phase B was an organic solvent.

[0014] Tandem mass spectrometry was used to detect the separated nucleotides and obtain the sample detection results; the mass spectrometry detection was performed using an electrospray ionization source in negative ion mode with multiple reaction monitoring scanning.

[0015] The liquid chromatography conditions included: an ACQUITY UPLC BEH C18 column (1.8 μm, 2.1 × 150 mm); a column temperature of 40–45 °C; mobile phase A of 10 mM ammonium acetate aqueous solution (pH 8.0); mobile phase B of acetonitrile (mass spectrometry grade) containing 10 mM ammonium acetate; a flow rate of 0.2 mL / min; and an injection volume of 5 μL.

[0016] The gradient elution program was as follows: 0 min A / B ratio of 99:1 (V / V), 2 min A / B ratio of 99:1 (V / V), 10 min A / B ratio of 92:8 (V / V), 12 min A / B ratio of 10:90 (V / V), 13 min A / B ratio of 10:90 (V / V), 13.1 min A / B ratio of 99:1 (V / V), 16 min A / B ratio of 99:1 (V / V), and 0 min A / B ratio of 99:1 (V / V).

[0017] The mass spectrometry detection conditions include: an electrospray ion source, a negative ion scanning mode, a multiple reaction monitoring mode, an ion source temperature of 300~350℃, a spray voltage of -4500 V, a curtain gas of 35 psi, a collision gas of 9 psi, a nebulizing gas of 40 psi, and an auxiliary heating gas of 35 psi.

[0018] Furthermore, the pH value of the weakly alkaline buffer solution is 8.0~9.0.

[0019] Furthermore, the weakly alkaline buffer solution is a 5mM~25mM aqueous solution of ammonium acetate.

[0020] Furthermore, the organic solvent includes methanol or acetonitrile.

[0021] Furthermore, the alkali-resistant reversed-phase chromatographic column is a C18 column with a pH tolerance range of 1 to 12.

[0022] Furthermore, in the liquid chromatography separation step, mobile phase A is a 10 mM ammonium acetate aqueous solution (pH 8.0), and mobile phase B is acetonitrile or acetonitrile containing 10 mM ammonium acetate.

[0023] Furthermore, in the liquid chromatography separation step, the column temperature is 40℃~45℃ and the flow rate is 0.2mL / min.

[0024] Furthermore, the nucleotide substances include nucleosides, deoxynucleosides, nucleotides, deoxynucleotides, and their monophosphate, diphosphate, and triphosphate forms.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) This invention abandons the time-consuming and laborious solid phase extraction step in the prior art and adopts a simplified process of “weak alkaline buffer homogenization + organic solvent protein precipitation + vacuum concentration + reconstitution”. The entire process from biological sample homogenization to preparation of sample-ready test solution can be completed within 1.5 hours. The operation is simple and the steps are streamlined. It effectively solves the problem of the long pretreatment of traditional methods and the difficulty in meeting the needs of high-throughput screening. It significantly improves the sample processing efficiency and detection throughput and is suitable for batch analysis of large-scale biological samples.

[0027] (2) Residual phospholipids in biological samples are key factors leading to inhibition of electrospray ionization and reduced response signals of target analytes, which are difficult to remove effectively using traditional pretreatment methods. This invention, through the synergistic effect of optimizing the pretreatment system and weakly alkaline chromatographic separation conditions, can efficiently reduce the residue of matrix impurities such as phospholipids, significantly reduce the ion inhibition effect, make the response signal of the target analyte more stable, greatly improve quantitative repeatability, and significantly improve detection accuracy, thus solving the pain points of severe matrix interference and poor reliability of quantitative results in traditional methods.

[0028] (3) Overcoming the limitations of existing methods in simultaneously detecting multiple types of nucleotides, this invention can simultaneously detect more than 40 target analytes, covering bases, nucleosides, deoxynucleosides, nucleotides, deoxynucleotides and their monophosphate, diphosphate, and triphosphate forms, while also being compatible with low-abundance nucleoside modified metabolites such as RNA-modified nucleosides and DNA-modified bases. For the first time, it has achieved simultaneous and accurate quantification of classical nucleotide metabolic pathways and deoxynucleotide pools, as well as panoramic analysis of classical nucleotides and modified metabolites, solving the problems of narrow detection range and inability to meet the needs of multi-dimensional research in existing technologies, and providing a comprehensive detection tool for nucleotide metabolic network research.

[0029] (4) In view of the problems of weak retention, easy co-elution and peak tailing of polar nucleotides in conventional acidic mobile phase systems, this invention innovatively adopts a combination of weak alkaline mobile phase and alkali-resistant hybrid particle C18 chromatographic column, combined with optimized gradient elution program, which effectively improves the chromatographic retention behavior of polar nucleotides (especially triphosphate form), realizes baseline separation of each target, and produces symmetrical and sharp peaks. It completely solves the quantitative error caused by co-elution of target and peak distortion in traditional methods, and greatly improves the accuracy and reliability of detection.

[0030] (5) A reverse-phase chromatographic column with a wide pH tolerance range is selected to adapt to the weakly alkaline detection system of the present invention, which avoids damage to the chromatographic column under alkaline conditions and significantly extends the service life of the chromatographic column. At the same time, through systematic optimization of the parameters of each link of sample pretreatment, chromatographic separation and mass spectrometry detection, the method maintains excellent reproducibility under different batches of samples and different detection cycles, and the detection results are stable and reliable, reducing the uncertainty of experimental operation and making it suitable for long-term and large-scale detection applications. Attached Figure Description

[0031] Figure 1 This is a general flowchart of the detection process provided in an embodiment of the present invention;

[0032] Figure 2 The extracted ion chromatogram of some nucleotides in the mixed standard;

[0033] Figure 3 The image shows the extracted ion chromatogram of some nucleotides detected in rat liver samples using the method of this invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0035] Example:

[0036] I. Materials and Reagents:

[0037] Table 1 Compound Information Table

[0038]

[0039]

[0040]

[0041] (ii) Reagents

[0042] Methanol (mass spectrometry grade), acetonitrile (mass spectrometry grade), ammonium acetate (mass spectrometry grade), ultrapure water (mass spectrometry grade).

[0043] II. Instruments and Equipment

[0044] Liquid chromatograph: Shimadzu LC-30A system;

[0045] Mass spectrometer: AB SCIEX 5500 + triple quadrupole mass spectrometer;

[0046] Fully automated rapid sample grinder;

[0047] High-speed refrigerated centrifuge;

[0048] Vacuum concentrator;

[0049] Electronic analytical balance (accuracy 0.0001 g);

[0050] Vortex oscillator;

[0051] Thermostatic water bath;

[0052] Pipettes (10 μL, 100 μL, 1000 μL).

[0053] III. Experimental Procedure

[0054] (a) Preparation of standard solutions

[0055] like Figure 1 As shown, accurately weigh each nucleotide standard and prepare a mixed standard stock solution with a concentration of 50 μg / mL using 10 mM ammonium acetate aqueous solution (pH 8.0). Seal and store at -20°C protected from light. Before use, serially dilute the mixed standard stock solution with 10 mM ammonium acetate aqueous solution (pH 8.0) to prepare a series of standard working solutions with concentrations ranging from 3.2 ng / mL to 50 μg / mL. Prepare and use immediately.

[0056] (II) Pretreatment of biological samples (taking rat liver tissue as an example)

[0057] Take approximately 50 mg of frozen liver tissue sample, place it in a centrifuge tube, add 200 μL of pre-cooled 10 mM ammonium acetate aqueous solution (pH 8.0) and an appropriate amount of grinding beads;

[0058] Place the sample into a fully automated rapid grinder and homogenize it at a frequency of 67 Hz for 300 seconds. Repeat this process 3 times to ensure that the sample is fully homogenized.

[0059] Add 800 μL of pre-cooled methanol (mass spectrometry grade) to the homogenate, vortex for 30 seconds, and let stand in an ice bath for 30 minutes to fully precipitate the protein.

[0060] Centrifuge at 4℃ and 15000 g for 10 minutes, carefully transfer 900 μL of supernatant to a new centrifuge tube, and centrifuge again under the same conditions to completely remove impurities;

[0061] Take 420 μL of the clarified supernatant, transfer it into a vacuum concentrator, and dry it under suitable conditions;

[0062] Just before using the instrument, add 150 μL of 10 mM ammonium acetate aqueous solution (pH 8.0) to reconstitute the solution, vortex for 600 seconds to ensure complete dissolution, let stand in an ice bath for 30 minutes, and then centrifuge at 4°C and 15000 g for 15 minutes.

[0063] Take 80 μL of supernatant and inject it into the sample vial for LC-MS / MS analysis.

[0064] (III) Liquid Chromatography Separation Conditions

[0065] Chromatographic column: ACQUITY UPLC BEH C18 (1.8 μm, 2.1 × 150 mm), with a pH tolerance range of 1–12;

[0066] Column temperature: 40~45 ℃;

[0067] Mobile phase A: 10 mM ammonium acetate aqueous solution (pH 8.0);

[0068] Mobile phase B: Acetonitrile (mass spectrometry grade) or acetonitrile containing 10 mM ammonium acetate;

[0069] Flow rate: 0.2 mL / min;

[0070] Injection volume: 5 μL;

[0071] Gradient elution procedure:

[0072] Table 2 Gradient elution program

[0073]

[0074] (iv) Mass spectrometry detection conditions

[0075] Ion source: Electrospray ionization (ESI);

[0076] Scanning mode: Negative ion mode;

[0077] Detection mode: Multiple Response Monitoring (MRM);

[0078] Ion source parameters: Ion source temperature 300~350℃; Spray voltage -4500 V; curtain gas 35 psi; collision gas 9 psi; atomizing gas 40 psi; auxiliary heating gas 35 psi;

[0079] Table 3. MRM parameters for some representative compounds:

[0080]

[0081]

[0082] IV. Methodological Validation

[0083] (a) Linearity and Range

[0084] The standard working solutions of a series of concentrations were analyzed under the chromatographic-mass spectrometric conditions described above. A standard curve was plotted with the peak area of ​​each compound as the ordinate (y) and the concentration as the abscissa (x), and linear regression analysis was performed. The results showed that all target analytes exhibited good linearity in the concentration range of 3.2 ng / mL to 50 μg / mL, with correlation coefficients (R²) greater than 0.99, meeting the requirements for quantitative analysis.

[0085] (ii) Sensitivity

[0086] The limits of quantitation (LOQ) of each compound were calculated using the signal-to-noise ratio (S / N=10). The results showed that the LOQ can reach the ng / mL level, and the LOQ of some compounds (such as modified nucleoside metabolites) can reach the pg / mL level. This indicates that the method has extremely high sensitivity and can meet the detection requirements of low-abundance nucleotides in biological samples.

[0087] (III) Precision and Accuracy

[0088] Three standard working solutions of low, medium, and high concentrations were used, and parallel determinations (n=6) were performed according to the above experimental steps to calculate intra-day precision. Inter-day precision was calculated after three consecutive days of determination. The results showed that the relative standard deviations (RSDs) of both intra-day and inter-day precision were less than 15%. Accuracy was verified using a spiked recovery experiment. Different concentrations of the standard were added to rat liver samples with known content, and the spiked recovery rate was calculated. The results showed that the recovery rate was between 85% and 115%, indicating that the method has good precision and accuracy.

[0089] (iv) Matrix effect

[0090] The matrix effect was assessed using post-column infusion and post-extraction addition methods. The results showed that the ion inhibition or enhancement effect of the biological sample matrix treated by this method on the target analyte was less than 15%, indicating that the pretreatment method can effectively remove matrix-interfering substances such as phospholipids and has strong resistance to matrix interference.

[0091] V. Results and Analysis

[0092] (I) Chromatographic separation effect

[0093] Under the optimized liquid chromatography conditions of this invention, such as Figure 2 As shown, all nucleotides (including bases, nucleosides, deoxynucleosides and their monophosphate, diphosphate, and triphosphate forms) in the mixed standard can be effectively separated. The chromatographic peaks are symmetrical, with no obvious tailing or co-elution phenomenon, indicating that the combination of alkaline mobile phase system and alkali-resistant C18 column effectively improves the retention and separation of polar nucleotides.

[0094] (II) Results of biological sample testing

[0095] The method of this invention was used to detect rat liver samples, such as... Figure 3 As shown, multiple target nucleotide substances were successfully detected, with stable chromatographic peak signals and few interfering peaks, proving that this method is suitable for the detection of nucleotide substances in complex biological matrices and can achieve simultaneous quantification of more than 40 target analytes.

[0096] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry, characterized in that, Includes the following steps: Biological samples were homogenized in a weakly alkaline buffer solution, and organic solvents were added to precipitate proteins. The supernatant was then separated. After drying the supernatant, it was reconstituted with a weakly alkaline buffer solution to obtain the test solution. The test solution was injected into the liquid chromatography system, separated using an alkali-resistant reversed-phase column, and a gradient elution program was used, wherein mobile phase A was a weakly alkaline buffer and mobile phase B was an organic solvent. Tandem mass spectrometry was used to detect the separated nucleotides and obtain the sample detection results; the mass spectrometry detection was performed using an electrospray ionization source in negative ion mode with multiple reaction monitoring scanning. The liquid chromatography conditions included: an ACQUITY UPLC BEH C18 column (1.8 μm, 2.1 × 150 mm); a column temperature of 40–45 °C; mobile phase A of 10 mM ammonium acetate aqueous solution (pH 8.0); mobile phase B of acetonitrile (mass spectrometry grade) containing 10 mM ammonium acetate; a flow rate of 0.2 mL / min; and an injection volume of 5 μL. The gradient elution program was as follows: 0 min A / B ratio of 99:1 (V / V), 2 min A / B ratio of 99:1 (V / V), 10 min A / B ratio of 92:8 (V / V), 12 min A / B ratio of 10:90 (V / V), 13 min A / B ratio of 10:90 (V / V), 13.1 min A / B ratio of 99:1 (V / V), 16 min A / B ratio of 99:1 (V / V), and 0 min A / B ratio of 99:1 (V / V). The mass spectrometry detection conditions include: an electrospray ion source, a negative ion scanning mode, a multiple reaction monitoring mode, an ion source temperature of 300~350℃, a spray voltage of -4500 V, a curtain gas of 35 psi, a collision gas of 9 psi, a nebulizing gas of 40 psi, and an auxiliary heating gas of 35 psi.

2. The method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, The pH value of the weakly alkaline buffer solution is 8.0~9.

0.

3. The method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry according to claim 2, characterized in that, The weakly alkaline buffer solution is a 5mM~25mM aqueous solution of ammonium acetate.

4. The method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, The organic solvent is methanol or methanol:acetonitrile = 1:

1.

5. The method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, The alkali-resistant reversed-phase chromatographic column is a C18 column with a pH tolerance range of 1 to 12.

6. The method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, In the liquid chromatography separation step, mobile phase A is a 10 mM ammonium acetate aqueous solution (pH 8.0), and mobile phase B is acetonitrile or acetonitrile containing 10 mM ammonium acetate.

7. The method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, In the liquid chromatography separation step, the column temperature is 40℃~45℃ and the flow rate is 0.2mL / min.

8. The method for detecting nucleotides in biological samples based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, The nucleotides include nucleosides, deoxynucleosides, nucleotides, deoxynucleotides, and their monophosphate, diphosphate, and triphosphate forms.

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