Liquid chromatography tandem mass spectrometry detection method for simultaneously detecting spermine and spermidine in multiple samples

Abstract

The invention provides a liquid chromatography-tandem mass spectrometry detection method for simultaneously detecting spermine and spermidine in various samples, which can be used for simultaneously detecting spermine and spermidine in plasma, whole blood samples and serum samples, and is characterized in that liquid chromatography-tandem mass spectrometry is adopted for detection; wherein a protein precipitation reagent for sample extraction is selected from an acid-containing solution; the detection condition of the liquid chromatography is as follows: a mobile phase is selected from an acid-containing solution. The scheme is high in detection efficiency, high in precision, high in sensitivity and beneficial to large-scale popularization.

Description

Technical Field

[0001] The present invention relates to the field of sample analysis, and in particular to a liquid chromatography tandem mass spectrometry detection method for simultaneously detecting spermine and spermidine in multiple samples. Background Art

[0002] The number and proportion of elderly people in countries around the world are on the rise. WHO data show that by 2030, one in six people in the world will be over 60 years old. From 2020 to 2030, the population over 60 will increase from 1 billion to 1.4 billion. By 2050, the world's population over 60 will double to 2.1 billion. Between 2020 and 2050, the number of people over 80 is expected to triple to 426 million. With the extension of human life expectancy and the intensification of global aging problems, studying the mechanisms of aging and its potential interventions has become the focus of the scientific community. The identification and evaluation of human longevity interventions has become a key goal in this field. Biomarkers of aging are crucial tools to achieve these goals within a realistic time frame. On October 20, 2021, the National Institutes of Health (NIH) launched a Cellular Senescence Network (SenNet) program for a rare non-dividing cell called "senescent" in the human body, which will provide $125 million in funding to 16 institutions over 5 years. It should be noted that currently no aging biomarker has been approved by US regulatory agencies for clinical use, and the same is true in China.

[0003] It was not until 250 years after the discovery of spermine and spermidine that the correct chemical composition of these two crystalline substances was determined and named, and the early research on polyamines began. In 2009, an article entitled "Spermidine induces autophagy to prolong life" published in Nature cell biology caused a sensation in the anti-aging of spermidine. In 2018, Science published a review "The role of spermidine in health and disease". Spermidine is not only present in semen, but also widely distributed in other tissues and cells of the human body. The concentration of spermidine in cells mainly depends on four factors: arginine → putrescine → spermidine ← spermine. Arginine is the main raw material for the synthesis of spermidine in cells. It generates ornithine and urea under the catalysis of arginase. Ornithine then generates putrescine under the action of ornithine decarboxylase 1 (ODC1) (this is the rate-limiting step), and putrescine generates spermidine under the action of spermidine synthase (SPDS). Spermidine can also be produced by the degradation of spermine. The concentration of spermidine changes with age. Some researchers have measured the concentration of polyamines in various tissues and organs of mice aged 3, 10 and 26 weeks, and found that it remains basically unchanged in the pancreas, brain and uterus, and slightly decreases with age in the intestine. In order to better understand the different roles played by these small molecules such as energy metabolism, biogenic amines, and amino acids in the aging process in the human body, it is necessary to accurately quantify these molecules and establish the metabolic spectrum of aging-related small molecule biomarkers in the human body, so as to provide a technical theoretical basis for the prevention, treatment and scientific research of various clinical aging-related diseases.

[0004] Spermine and spermidine are biogenic amine compounds. These substances lack chromophores and have neither ultraviolet absorption nor fluorescence and electrochemical activity, making conventional analysis and determination difficult. Currently, both the liquid chromatography method and the spectrophotometer method of GB 5009.208-2016 must be completed by derivatization. However, derivatization is often cumbersome and complicated, the derivatization conditions are strict, the derivatization products are unstable, and the method reproducibility is poor. The analysis and detection time of the GB 5009.208-2016 method is long, requiring 35 minutes, and the commercialization time cost is high. Summary of the invention

[0005] The present invention aims to overcome the above-mentioned defects and provide a method for efficiently and accurately detecting spermine and spermidine in multiple samples at the same time.

[0006] The present invention provides a liquid chromatography tandem mass spectrometry detection method for simultaneously detecting spermine and spermidine in multiple samples, which can be used to simultaneously detect spermine and spermidine in plasma, whole blood samples, and serum samples, and is characterized in that: liquid chromatography tandem mass spectrometry is used for detection;

[0007] The protein precipitation reagent for sample extraction is selected from a solution containing 0.1-0.2% formic acid; the detection conditions of liquid chromatography are: the mobile phase is selected from a solution containing 0.1-0.2% formic acid.

[0008] Furthermore, the method provided by the present invention is also characterized in that:

[0009] The mobile phase A is a solution containing 0.1-0.2% formic acid, and the mobile phase B is 100% methanol. Furthermore, the method provided by the present invention is also characterized in that:

[0010] A gradient elution method was used.

[0011] Furthermore, the method provided by the present invention is also characterized in that:

[0012] The above gradient elution conditions are as follows:

[0013] The mobile phase composition at 0 min was 98% A + 2% B;

[0014] 2.5min mobile phase composition is 98% A + 2% B;

[0015] 3.2min mobile phase composition is 10% A + 90% B;

[0016] 5.0min mobile phase composition is 10% A + 90% B;

[0017] 5.2min mobile phase composition is 98% A + 2% B;

[0018] 7.0min mobile phase composition is 98% A + 2% B.

[0019] Furthermore, the method provided by the present invention is also characterized in that:

[0020] The internal standard method was adopted, with spermine-d20 and spermidine-d8 as internal standards.

[0021] Furthermore, the method provided by the present invention is also characterized in that:

[0022] The chromatographic column is selected with specifications of 2.6-3μm, 100×2.1-3mm;

[0023] The flow rate was 0.18 mL / min; the column temperature was 40°C.

[0024] Furthermore, the method provided by the present invention is also characterized in that:

[0025] The mass spectrometry conditions were:

[0026] Select electrospray ion source, positive and negative ion mode;

[0027] The capillary voltage was 5.5 kV and the desolvation temperature was 550 °C.

[0028] Furthermore, the method provided by the present invention is also characterized in that:

[0029] When the sample is plasma or serum, add a mixed internal standard of spermine-d20 and spermidine-d8 to the sample, then add acetonitrile to precipitate the protein, shake and mix for 30-60 seconds, centrifuge at 12000-14000 rpm, 0-4°C for 5-10 minutes, aspirate the supernatant, and obtain the sample to be tested.

[0030] Furthermore, the method provided by the present invention is also characterized in that:

[0031] When the sample is a whole blood sample, pipette the frozen-thawed whole blood and ddH 2 O, add spermine-d20 and spermidine-d8 mixed internal standards, then add acetonitrile, shake and mix for 30-60 seconds, centrifuge at 12000-14000rpm, 0-4℃ for 5-10 minutes, and aspirate the supernatant and ddHO. 2 0 after mixing to obtain the sample to be tested.

[0032] In addition, the present invention also provides the application of the above method in simultaneously performing qualitative and quantitative analysis on spermine and spermidine in plasma, whole blood samples and serum samples.

[0033] Functions and effects of the present invention:

[0034] The method does not require derivatization, saves the cost of pre-treatment, has good reproducibility, and greatly reduces the experimental cost and experimental error of pre-treatment; the analysis time is short, the anti-matrix interference ability is strong, and one detection is completed in 7 minutes; the concentration ranges of spermine and spermidine are 1-500 ng / mL and 10-5000 ng / mL, with good linear correlation, r is greater than 0.99, and the detection limit of this experimental method can far meet the national standard; at low, medium and high concentrations, the method CV (n=6) is less than 15%; a fast, simple, highly sensitive and reproducible biogenic amine detection method is provided, and the method has important reference significance in the field of biological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 SPM and SPM-d20 chromatograms;

[0036] Figure 2 SPD and SPD-d8 chromatograms. DETAILED DESCRIPTION

[0037] The present invention is capable of various modifications and various embodiments, and therefore specific embodiments are illustrated and described in the accompanying drawings. However, this is not intended to limit the present invention to specific implementations, but should be understood to include all modifications, equivalents, and even substitutes that fall within the concept and technical scope of the present invention.

[0038] The small molecule standards used in the methodology are all certified reference materials (CRMs), which ensure the accuracy and repeatability of the data, and are of great significance to the understanding of aging and the specific degree and monitoring of aging in the body. The specific methods are as follows:

[0039] S1. Methods and Materials

[0040] 1.1 Reagents

[0041] The standards and corresponding stable isotope internal standards are as follows:

[0042]

[0043]

[0044] 1.2 Instrument: ABSciex 4500MD platform (National Medical Device Registration No. 20172401554) and supporting software

[0045] 1.3 Establishing the calibration curve: NAD prepared with 5% BSA + Calibration curve working solution and establish calibration curve, NAD + The calibration curve working solution and calibration curve working solution were stored at -20°C;

[0046]

[0047] S2. Sample collection and preparation: Use EDTA vacuum blood collection tubes to collect plasma and whole blood samples, and use ordinary serum blood collection tubes to collect serum samples. Centrifuge plasma and serum samples at 4°C, 3000-3500rpm for 5 minutes, collect supernatant, and store at -20°C. Repeated freezing and thawing times should be less than 3 times. Whole blood samples are stored at -20°C, and cells are lysed by repeated freezing and thawing, i.e., whole blood samples are frozen below -20°C, thawed at room temperature, and repeated 3-5 times. The formation of ice particles in the cells and the increase in the salt concentration of the remaining cell fluid cause swelling, which breaks the cell structure and avoids the use of various lysis reagents.

[0048] S3, plasma / serum sample pretreatment: protein precipitation method is used. Take the plasma / serum collected in S2, draw 100μL of plasma / serum, add 20μL of spermine-d20 and spermidine-d8 mixed internal standard, and then add 200μL of acetonitrile containing 0.2% formic acid (the acid content is 0.1-0.2%, lower and higher than this content will lead to poor detection results or increased detection limit), shake and mix for 30 seconds, centrifuge at 12000-14000rpm4℃ for 10 minutes, draw 100μL of supernatant, and obtain the sample to be tested;

[0049] S4. Whole blood sample pretreatment: protein precipitation method was used. Take the whole blood collected in S2, absorb 20 μL of frozen and thawed whole blood and 80 μL of ddH 2 O, add 20 μL of a mixed internal standard of spermine-d20 and spermidine-d8, and then add 200 μL of acetonitrile containing 0.2% formic acid (the acid content is 0.1-0.2%, and a content lower than or higher than this will lead to poor detection results or increased detection limits), shake and mix for 30 seconds, centrifuge at 12000-14000 rpm and 4°C for 10 minutes, and aspirate 100 μL of the supernatant to obtain the sample to be tested;

[0050] S5. Sample loading test: Add the sample to be tested into a sample injection vial or a 96-well plate for liquid chromatography tandem mass spectrometry detection. The mass analyzer used is a triple quadrupole. The internal standard method is adopted, and spermidine-d8hydrochloride and spermine-d20 are used as internal standards to calculate the concentration of spermine and spermidine in the sample to be tested;

[0051] The conditions of liquid chromatography are:

[0052] The column model is UPLC F5 (2.6μm, 100×2.1mm), the chromatographic column can be replaced with other types, the best choice is F5; mobile phase A is a pure water solution containing 0.1-0.2% formic acid (the acid content is 0.1-0.2%, lower and higher content will lead to poor detection results or increased detection limit), mobile phase B is 100% methanol (no acid, otherwise it will lead to poor detection results); flow rate is 0.18mL / min; column temperature is 40℃; gradient elution program is:

[0053] The mobile phase composition at 0 min was 98% A + 2% B, at 2.5 min it was 98% A + 2% B, at 3.2 min it was 10% A + 90% B, at 5.0 min it was 10% A + 90% B, at 5.2 min it was 98% A + 2% B, at 7.0 min it was 98% A + 2% B;

[0054] Without gradient elution, accurate chromatographic results cannot be obtained.

[0055] Table 1 Liquid chromatography elution conditions

[0056] Time (min) Mobile phase A% Mobile phase B% 0 98 2 2.5 98 2 3.2 10 90 5.0 10 90 5.2 98 2 7.0 98 2

[0057] The detection parameters of the mass spectrometer are: select the electrospray ion source, positive ion mode; the capillary voltage is 5.5KV, the desolvation temperature is 550℃, and the mass spectrometer ion information is shown in the following table:

[0058]

[0059] Specific steps:

[0060] ① Accurately weigh 1 mg SPM and SPD and dissolve them in 1 mL pure water to prepare 1000 μg / mL SPM and SPD stock solutions; accurately weigh 100 μg SPM-d20 and 20 μg SPD-d8HCl and dissolve them in 100 μL pure water to prepare 1000 μg / mL SPM-d20 and 200 μg / mL SPD-d8HCl stock solutions, and add 10 μL SPM-d20 and 30 μL SPD-d8HCl stock solutions to 9.96 mL methanol solution as internal standard working solutions. Each stock solution was divided into 2 mL centrifuge tubes and stored at -80°C for future use.

[0061] Three concentrations of QC 5% BSA samples were prepared: low, medium, and high, with SPM of 5, 80, and 400 ng / mL, and SPD of 50, 800, and 4000 ng / mL. All QC samples were packaged after preparation and stored at -80°C for future use.

[0062] ② Sample pretreatment

[0063] For plasma / serum samples, accurately draw 100 μL, add 20 μL SPM-d20 and SPD-d8HCL mixed internal standard working solution, add 200 μL acetonitrile protein precipitation, shake and mix, centrifuge at 4℃ 12000-14000rpm×10min, draw 100 μL supernatant, transfer to injection vial or 96-well plate for injection analysis. Repeat the sample test 5 times and take the average value.

[0064] For whole blood samples, accurately draw 50 μL and mix with 50 μL pure water, add 20 μL SPM-d20 and SPD-d8HCL mixed internal standard working solution, add 200 μL acetonitrile protein precipitation, shake and mix, centrifuge at 4℃ 14000rpm×10min, draw 100 μL supernatant, transfer to injection vial or 96-well plate for injection analysis. Repeat the sample test 5 times and take the average. For whole blood samples, the final concentration needs to be calculated by multiplying the dilution factor.

[0065] ③Method verification

[0066] According to the FDA and EMA bioanalytical method validation guidelines, the analytical method validation items for this experiment include specificity, standard curve and linear range, minimum detection limit and minimum quantification limit, precision and accuracy, sample recovery rate, matrix effect, and short-term stability.

[0067] √ Exclusiveness

[0068] Under the established chromatographic-mass spectrometric conditions, 100 μL of blank matrix was taken, and the sample was processed and analyzed according to the sample pretreatment method except that no internal standard was added, and the chromatogram of the blank matrix sample was obtained; the internal standard solution and mixed standard solution of the above concentration were added to the blank serum, and the corresponding chromatographic mass spectra were obtained according to the sample pretreatment method to examine the method specificity. The retention time of SPM and SPM-d20, SPD and SPD-d8HCL was about 1.1 minutes.

[0069] √Standard curve and linear range

[0070] Figure 1 and Figure 2 The chromatograms of the calibration lowest point S8 of the HCL calibration curves of SPM and SPM-d20, SPD and SPD-d8 are shown respectively. The internal standard method is used for quantification, and the stable isotope is used as the internal standard. The calibration curve is drawn using the theoretical concentration (x) of SPM and SPD of the calibrator and the ratio (y) of the peak area of ​​SPM and SPD to the corresponding internal standard peak area in the sample to be tested. The ratio of the peak area of ​​SPM and SPD to the corresponding internal standard peak area in the sample to be tested is substituted into the fitted calibration curve equation to calculate the concentration of SPM and SPD in the sample to be tested. The weighted least squares method is used for regression operation, and the weight factor is 1 / x 2 Calculation: The calibration range of SPM is 1-500 ng / mL and that of SPD is 10-5000 ng / mL. The response of the instrument to the analyte is evaluated within this range, and the calculated concentration of the calibration standard at each concentration point of the calibration curve is within ±15%.

[0071] √ Imprecision and accuracy

[0072] Precision is the most basic performance of an analytical method. The SPM and SPD tandem mass spectrometry detection methods were evaluated for imprecision and accuracy with reference to the relevant guidelines of WS / T492-2016 “Performance Verification of Precision and Correctness of Quantitative Measurement Items in Clinical Laboratories”, and the results met regulatory acceptance standards.

[0073] Three quality control samples with low, medium and high concentration levels were selected for intra-batch differences, and six copies of each quality control sample were prepared in parallel. The measured concentration of the sample was calculated based on the standard curve of the day, and the imprecision and accuracy of the quality control samples at the three concentration levels were within ±15%.

[0074] Table 2 Intra-batch variation

[0075]

[0076] The inter-batch differences were selected from three quality control samples with low, medium and high concentration levels. Six copies of each quality control sample were prepared in parallel and tested for 5 consecutive days. The measured concentration of the sample was calculated based on the standard curve of the day. The imprecision and accuracy of the quality control samples at the three concentration levels were within ±15%.

[0077] Table 3 Batch differences

[0078]

[0079]

[0080] √Matrix effect

[0081] This study used the isotope internal standard method to reduce the matrix effect. After pretreatment, the blank serum was added with low, medium and high concentration mixed working solutions containing internal standards to prepare matrix samples. The determination was repeated 5 times, and the peak areas of each analyte and internal standard were compared with the corresponding matrix-free samples prepared with solvents. The matrix effect CV corrected by the internal standard was calculated to be within ±15%.

[0082]

[0083] √ Sample recovery rate

[0084] In order to investigate the loss of SPM and SPD during sample analysis, SPM storage solution was added to blank 5% BSA to prepare mixed quality control products at 5, 80, and 400 ng / mL, and SPD storage solution was prepared into 50, 800, and 4000 ng / mL. The mixed quality control products were pre-treated according to the sample processing method; the blank 5% BSA was pre-treated according to the sample processing method, and then SPM and SPD storage solutions were added to prepare the above three levels of mixed quality control products. The concentrations of the samples prepared by the two methods were measured 5 times each, and the recovery was calculated by the ratio. The CV of the recovery rates of SPM and SPD at the three levels was <15%.

[0085]

[0086] Comparative Experiment Example 1:

[0087] When the sample was pretreated without formic acid or trifluoroacetic acid, the signal intensity of SPM and SPD decreased by about 1 / 30.

[0088] Therefore, when this method is used to detect SPM and SPD, acidic solvents such as formic acid and trifluoroacetic acid are used starting from sample pretreatment, because under acidic conditions, the ionization efficiency of SPM and SPD is significantly enhanced, which reduces the detection limit and quantification limit.

[0089] Comparative Experiment Example 2:

[0090] Without gradient elution, the HPLC peak separation will be poor and the results will be poor. Gradient elution can achieve the best separation of all components in the sample in the shortest time.

[0091] Comparative Experiment Example 3:

[0092] Change the gradient elution conditions to the following:

[0093] Time (min) Mobile phase A% Mobile phase B% 0 98 2 2.5 98 2 3.2 30 70 5.0 30 70 5.2 98 2 7.0 98 2

[0094] or

[0095] Time (min) Mobile phase A% Mobile phase B% 0 98 2 2.5 98 2 3.2 50 50 5.0 50 50 5.2 98 2 7.0 98 2

[0096] or

[0097]

[0098]

[0099] The accuracy of its quantitative analysis is no higher than 70%.

[0100] Although the above description is centered on the embodiment, it is only an example and does not limit the present invention. It is clear to those skilled in the art that various modifications and applications not illustrated above can be made within the scope of the essential characteristics of the present embodiment. For example, each component specifically shown in the embodiment can be implemented after being modified. Moreover, various differences related to such modifications and applications should be interpreted as being included in the scope of the present invention as defined in the attached claims.

Claims

1. A liquid chromatography tandem mass spectrometry detection method for simultaneously detecting spermine and spermidine in multiple samples, which can be used to simultaneously detect spermine and spermidine in plasma, whole blood samples, and serum samples, characterized in that: Liquid chromatography-tandem mass spectrometry was used for detection; wherein the protein precipitation reagent for sample extraction is selected from a solution containing 0.1-0.2% formic acid; The detection conditions of liquid chromatography are as follows: the mobile phase is selected from a solution containing 0.1-0.2% formic acid.

2. The method according to claim 1, characterized in that: The mobile phase A is a solution containing 0.1-0.2% formic acid, and the mobile phase B is 100% methanol.

3. The method according to claim 2, characterized in that: A gradient elution method was used.

4. The method according to claim 3, characterized in that: The gradient elution conditions are as follows: The mobile phase composition at 0 min was 98% A + 2% B; 2.5min mobile phase composition is 98% A + 2% B; 3.2min mobile phase composition is 10% A + 90% B; 5.0min mobile phase composition is 10% A + 90% B; 5.2min mobile phase composition is 98% A + 2% B; 7.0min mobile phase composition is 98% A + 2% B.

5. The method according to claim 1, characterized in that: The internal standard method was adopted, with spermine-d20 and spermidine-d8 as internal standards.

6. The method according to claim 1, characterized in that: The chromatographic column is selected with specifications of 2.6-3μm, 100×2.1-3mm; The flow rate was 0.18 mL / min; the column temperature was 40°C.

7. The method according to claim 1, characterized in that: The mass spectrometry conditions were: Select electrospray ion source, positive ion mode; The capillary voltage was 5.5 KV and the desolvation temperature was 550 °C.

8. The method according to claim 1, characterized in that: When the sample is plasma or serum, add a mixed internal standard of spermine-d20 and spermidine-d8 to the sample, then add acetonitrile to precipitate the protein, shake and mix for 30-60 seconds, centrifuge at 12000-14000 rpm, 0-4°C for 5-10 minutes, aspirate the supernatant, and obtain the sample to be tested.

9. The method according to claim 1, characterized in that: When the sample is a whole blood sample, the frozen and thawed whole blood is aspirated and mixed with ddH2O, and a mixed internal standard of spermine-d20 and spermidine-d8 is added, and then acetonitrile is added. After shaking and mixing for 30-60 seconds, centrifuge at 12000-14000 rpm and 0-4°C for 5-10 minutes, the supernatant is aspirated and mixed with ddH20 to obtain the sample to be tested.

10. Use of the method according to any one of claims 1 to 9 for simultaneous qualitative and quantitative analysis of spermine and spermidine in plasma, whole blood samples and serum samples.

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