Method for detecting NAD < + > in multiple samples

Abstract

The invention provides a method for detecting NAD < + > in various samples. The method can be used for qualitative and quantitative detection of NAD < + > in plasma, whole blood samples and serum samples. The method is characterized in that sample pretreatment is easy to operate, and sample pretreatment conditions are as follows: preferably, no acid solution is contained; detecting by adopting liquid chromatography-tandem mass spectrometry; wherein the detection conditions for completing mass spectrum detection and liquid chromatography within 7 minutes are as follows: the mobile phase does not contain an acidic solution; the lower quantification limit is 10ng / mL; the anti-interference capability is high; the cost is low. The scheme has the advantages of high detection efficiency, high precision and high sensitivity, is beneficial to large-scale popularization, and provides technical support for evaluating the concentration of NAD + in various body fluid samples.

Description

Technical Field

[0001] The present invention relates to the field of sample analysis, and in particular to a method for detecting NAD in multiple samples. + method. 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] NMN is a substance inherent in the human body and is also rich in some fruits and vegetables. In the human body, NMN is NAD + The precursor of NAD + NAD + Also known as coenzyme I, full name nicotinamide adenine dinucleotide, NAD + It plays a key role in cell metabolism. It is present in all living cells and plays an important role in a variety of biological processes such as energy production, DNA repair and cell signaling. Three Nobel Prize winners have been born from the study of NAD. In 1904, British biochemist Arthur Harden first discovered and named NAD + , and thus won the 1929 Nobel Prize in Chemistry. In 1920, Nobel Prize winner in Chemistry Euler Scherpin first isolated and purified NAD + In 1930, Nobel Prize winner in Physiology or Medicine Otto Heinrich Warburg first discovered NAD + It plays a key role as a coenzyme in material and energy metabolism.

[0004] NAD +Dysregulation of NAD levels has been linked to metabolic diseases as well as age-related disorders, including neurodegenerative diseases, immune response defects, and cancer. + By interacting with NADH as a cofactor, it plays an important role in enzymatic reactions of energy metabolism such as glycolysis, oxidative phosphorylation, fatty acid oxidation, and TCA cycle. + It also plays a role in the deacetylation of Sirtuins and ADP-ribosylation of PARP proteins during DNA damage / repair. + NAD cannot be directly absorbed from food. + Under physiological conditions, extracellular NAD in mammalian serum + The concentration is maintained at a low level, and the oxidized form of NAD + The concentration in the cell is maintained at a high level, but its concentration may fluctuate.

[0005] In order to better understand the different roles played by these energy metabolism small molecules 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. Quantitative analysis methods based on mass spectrometry play an important role in medical laboratories. With the continuous development of liquid chromatography-mass spectrometry (LC-MS), these emerging detection and quantification metabolite technologies have played an important role in clinical testing, mainly for small molecule analytes, and the sample matrix includes plasma, serum, whole blood and urine. This method uses stable isotope dilution liquid chromatography tandem mass spectrometry, which is one of the current mainstream international traceability reference methods.

[0006] There are two main types of NAD + The detection technology is to use the receptor protein of the recombinant protein probe to bind to the NAD after blood sampling. + Combine and emit corresponding fluorescence, and analyze NAD in the human body based on different fluorescence + concentration; the second is the WST-8 method, which uses the WST-8 colorimetric reagent to + / NADH catalyzes the reduction of formazan, and NAD is calculated based on the absorbance of formazan + / NADH concentration. Both methods are indirect detection in terms of mechanism. The WST-8 colorimetric reagent may also be reduced by metabolites such as NADPH, formic acid DH2, sodium bisulfite, cysteine, and glutathione, resulting in NAD + The test results are high. + The mass spectrometry quantitative method is also based on the mass spectrometry platform, because NAD + The content is extremely low, and NAD + It is an oxidized form that accepts electrons and carries a positive charge.+ ) obtains electrons together, it will become the electron donor NADH, affecting the test results. Summary of the invention

[0007] The present invention aims to overcome the above-mentioned defects and develops a method for the efficient and accurate quantitative detection of NAD from various sample sources by using ultra-high performance liquid chromatography-tandem mass spectrometry and F5 special chromatographic column. + 's detection scheme.

[0008] The present invention provides a method for detecting NAD in various samples. + The method can be used to detect NAD in plasma, whole blood samples, and serum samples. + , characterized in that: liquid chromatography tandem mass spectrometry is used for detection;

[0009] wherein the protein precipitation reagent for sample extraction is selected from a solution that does not contain acid;

[0010] The detection conditions of liquid chromatography are: the mobile phase must not contain acidic solutions.

[0011] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0012] The mobile phase A is a solution without acid (such as pure water solution, etc.), and the mobile phase B is methanol.

[0013] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0014] A gradient elution method was used.

[0015] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0016] The above gradient elution conditions are as follows:

[0017] The mobile phase composition at 0 min was 98% A + 2% B;

[0018] 2.5min mobile phase composition is 98% A + 2% B;

[0019] 3.2min mobile phase composition is 10% A + 90% B;

[0020] 5.0min mobile phase composition is 10% A + 90% B;

[0021] 5.2min mobile phase composition is 98% A + 2% B;

[0022] 7.0min mobile phase composition is 98% A + 2% B.

[0023] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0024] The internal standard method was used, with NAD + -d4 is the internal standard.

[0025] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0026] The chromatographic column is selected with specifications of 2.6-3μm, 100×2.1-3mm;

[0027] The flow rate was 0.18 mL / min; the column temperature was 40°C.

[0028] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0029] The mass spectrometry conditions were:

[0030] Select electrospray ion source, positive and negative ion mode;

[0031] The capillary voltage was 5.5 kV and the desolvation temperature was 550 °C.

[0032] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0033] When the sample is plasma or serum, add NAD + -d4 stable isotope internal standard, then add acetonitrile for protein precipitation, shake and mix for 30-60 seconds, centrifuge at 12000-14000rpm, 0-4℃ for 5-10 minutes, aspirate the supernatant to obtain the sample to be tested.

[0034] Furthermore, the present invention provides a method for detecting NAD in multiple samples. + The method is characterized in that:

[0035] When the sample is a whole blood sample, the frozen and thawed whole blood is mixed with ddH2O and NAD is added. + -d4 stable isotope internal standard, then add acetonitrile, shake and mix for 30-60 seconds, centrifuge at 12000-14000rpm, 0-4℃ for 5-10 minutes, aspirate the supernatant and mix with ddH20 to obtain the sample to be tested.

[0036] In addition, the present invention also provides the above-mentioned detection of NAD in various samples+ The method was used to analyze the NAD in plasma, whole blood and serum samples. + For qualitative and quantitative applications.

[0037] Function and effect of the present invention:

[0038] Based on the existing NAD + The method uses acid-free extraction and chromatographic conditions for analysis, combined with mass spectrometry reaction monitoring scanning mode to achieve NAD + The quantitative requirements of NAD + The minimum value of the calibration curve is 10ng / mL, which breaks through the critical point of traditional technology. For example, the minimum detection value of patents such as 202410066739.6 is 390ng / ml.

[0039] At the same time, this method has strong sample adaptability, including blood, urine, tissue extracts, etc. In addition, the present invention also solves the problem that the existing method uses deionized water to prepare a concentration calibration curve and uses a matrix-free solution to calculate the experimental parameters of biological samples with complex matrices, which may not be suitable for clinical testing.

[0040] In summary, the present invention is based on a triple quadrupole liquid chromatography-tandem mass spectrometry platform to achieve the detection of NAD in a variety of samples. + It can detect qualitative and quantitative factors with low invasiveness, low quantitative limit, simple operation and controllable cost, providing a technical theoretical basis for the prevention, treatment and scientific research of various aging-related diseases in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 NAD + Chromatogram;

[0042] Figure 2 NAD + -d4 chromatogram;

[0043] Figure 3 Graphical representation of HPLC parameters;

[0044] Figure 4 Chromatograms after sample pretreatment with formic acid or trifluoroacetic acid. From top to bottom, they are S8-S1 chromatograms, with NAD listed on the left. + Chromatogram, NAD on the right + d4 chromatogram, NAD + The d4 signal intensity decreased by about 1 / 30, and NAD + The calibration curve S6-S5 shows a signal. DETAILED DESCRIPTION

[0045] 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.

[0046] 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:

[0047] S1. Methods and Materials

[0048] 1.1 Reagents

[0049] The standards and corresponding stable isotope internal standards are as follows:

[0050]

[0051] 1.2 Instrument: ABSciex 4500MD platform (National Medical Device Registration No. 20172401554) and supporting software

[0052] 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;

[0053]

[0054] 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. After collection, whole blood samples should be stored at -20°C, and cells should be lysed by repeated freezing and thawing, i.e., whole blood samples should be frozen below -20°C and thawed at room temperature for 3-5 times. The formation of ice particles in cells and the increase in salt concentration of the remaining cell fluid will cause swelling, which will break the cell structure and avoid the use of various lysis reagents.

[0055] S3. Plasma / serum sample pretreatment: protein precipitation method was used. Take 100 μL of plasma / serum collected in S2, add 20 μL of NAD +-d4 stable isotope internal standard, then add 200 μL of acetonitrile (preferably without formic acid or other acidic solutions, can contain up to 0.2% formic acid, but it will affect the detection limit), shake and mix for 30 seconds, centrifuge at 12000-14000rpm at 4℃ for 10 minutes, and aspirate 100 μL of supernatant to obtain the sample to be tested;

[0056] 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 mix with 80 μL ddH2O, add 20 μL NAD + -d4 stable isotope internal standard, then add 200 μL of acetonitrile (preferably without formic acid or other acidic solutions, and can contain up to 0.2% formic acid, but this will affect the detection limit), shake and mix for 30 seconds, centrifuge at 12000-14000 rpm and 4°C for 10 minutes, and aspirate 100 μL of supernatant to obtain the sample to be tested;

[0057] S5. Sample loading test: The sample to be tested is added to the injection vial or 96-well plate for liquid chromatography tandem mass spectrometry detection. The mass analyzer used is a triple quadrupole, and the internal standard method is adopted with NAD + -d4 is the internal standard, which is used to calculate the NAD in the sample to be tested + concentration;

[0058] The conditions of liquid chromatography are:

[0059] The column model is UPLC F5 (2.6 μm, 100 × 2.1 mm), the chromatographic column can be replaced with other types, the best choice is F5; mobile phase A is pure aqueous solution (preferably without acidic solutions such as formic acid, and at most 0.1-0.2% formic acid is added), mobile phase B is 100% methanol; flow rate is 0.18 mL / min; column temperature is 40 ° C; gradient elution program is:

[0060] 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;

[0061] Without gradient elution, accurate chromatographic results cannot be obtained.

[0062] Table 1 Liquid chromatography elution conditions

[0063] 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

[0064] 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:

[0065]

[0066] Specific steps:

[0067] ① Accurately weigh 1 mg NAD + Dissolve in 1 mL of pure water to prepare 1000 μg / mL NAD + Stock solution; accurately weigh 100 μg NAD + -d4 was dissolved in 100 μL of pure water to prepare 1000 μg / mL NAD + -d4 stock solution, add 40 μL NAD + -d4 stock solution was used as the internal standard working solution. Each stock solution was dispensed into 1.5 mL centrifuge tubes and stored at -80°C for future use.

[0068] Preparation of NAD + The three concentrations of QC 5% BSA samples were low, medium and high, 100, 800 and 4000 ng / mL, respectively. All QC samples were prepared and packaged and stored at -80°C for future use.

[0069] ② Sample pretreatment

[0070] For plasma / serum samples, pipette 100 μL accurately and add 20 μL NAD + -d4 internal standard working solution, add 200μL acetonitrile protein precipitation, shake and mix, centrifuge at 4℃ 12000-14000rpm×10min, aspirate 100μL supernatant, transfer to injection vial or 96-well plate for injection analysis. Repeat the sample test 5 times and take the average value.

[0071] For whole blood samples, accurately draw 20 μL and mix with 80 μL pure water, add 20 μL NAD + -d4 mixed internal standard working solution, add 200μL acetonitrile protein precipitation, shake and mix, centrifuge at 4℃ 12000-14000rpm×10min, aspirate 100μL 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.

[0072] ③Method verification

[0073] 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.

[0074] √ Exclusiveness

[0075] 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 sample pretreatment method was used to obtain the corresponding chromatographic mass spectrum to examine the method specificity. NAD + and NAD + -d4 retention time is about 1.6 minutes.

[0076] √Standard curve and linear range

[0077] Figure 1 and Figure 2 NAD + and NAD + -d4 calibration curve calibration minimum point S8 point chromatogram, quantification by internal standard method, using NAD + -d4 is the internal standard, using the NAD + Theoretical concentration (x) and NAD + The calibration curve was drawn by the ratio of the peak area of ​​NAD in the sample to be tested to the peak area of ​​the internal standard. The ratio of the peak area of ​​NAD in the sample to be tested to the peak area of ​​the internal standard was substituted into the fitted calibration curve equation to calculate the NAD in the sample to be tested. + Concentration. The regression operation is performed using weighted least squares method, with the weight factor being 1 / x 2 Calculating NAD + The calibration range is 10-5000 ng / mL. The response of the instrument to the analyte is evaluated within this range. The back-calculated concentration deviation of the calibration standard at each concentration point of the calibration curve is within ±15%.

[0078] √ Imprecision and accuracy

[0079] Precision is the most basic performance of an analytical method. + The tandem mass spectrometry detection method was 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 the regulatory acceptance standards.

[0080] 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%.

[0081] Table 2 Intra-batch variation

[0082]

[0083] 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%.

[0084] Table 3 Batch differences

[0085]

[0086] √Matrix effect

[0087] 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%.

[0088]

[0089] √ Sample recovery rate

[0090] To investigate NAD + The extent of loss during sample analysis, NAD was added to blank 5% BSA + The storage solution was prepared into three levels of mixed quality control products: 100, 800, and 4000 ng / mL, and pre-treated according to the sample processing method; blank 5% BSA was pre-treated according to the sample processing method, and then NAD was added + The storage solution was prepared into 3 levels of mixed quality control products: 100, 800, and 4000 ng / mL. The concentrations of the samples prepared by the two methods were measured 5 times each, and the recovery was calculated by the ratio. + The recovery rate is between 80% and 120%.

[0091]

[0092] Comparative Experiment Example 1:

[0093] When formic acid or trifluoroacetic acid was used for sample pretreatment, NAD+ -d4 signal intensity decreased by about 1 / 30, NAD + The calibration curve S5 shows a signal ( Figure 4 ).

[0094] Therefore, this method was used to detect NAD + When preparing samples, avoid using acidic solvents such as formic acid and trifluoroacetic acid. Under acidic conditions, NAD + The ionization efficiency is significantly reduced. The same calibration curve is in the condition of 0.2% FA mobile phase, NAD + The concentration at which an obvious chromatographic peak appears is approximately at the S5 level (100 ng / mL).

[0095] Comparative Experiment Example 2:

[0096] 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.

[0097] Comparative Experiment Example 3:

[0098] Change the gradient elution conditions to the following:

[0099] 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

[0100] or

[0101] 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

[0102] or

[0103] Time (min) Mobile phase A% Mobile phase B% 0 50 50 2.5 50 50 3.2 10 90 5.0 10 90 5.2 50 50 7.0 50 50

[0104] The accuracy of its quantitative analysis is no higher than 70%.

[0105] 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. One method to detect NAD in multiple samples + The method can be used to detect NAD 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 that does not contain acid; The detection conditions of liquid chromatography are as follows: the mobile phase is selected from a solution containing no acid.

2. A method for detecting NAD in multiple samples as claimed in claim 1 + The method is characterized in that: The mobile phase A is a solution without acid, and the mobile phase B is 100% methanol.

3. A method for detecting NAD in multiple samples as claimed in claim 2 + The method is characterized in that: A gradient elution method was used.

4. A method for detecting NAD in multiple samples as claimed in claim 3 + The method is 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. A method for detecting NAD in multiple samples as claimed in claim 1 + The method is characterized in that: The internal standard method was used, with NAD + -d4 is the internal standard.

6. A method for detecting NAD in multiple samples as claimed in claim 1 + The method is 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. A method for detecting NAD in multiple samples as claimed in claim 1. + The method is characterized in that: The mass spectrometry conditions were: Select electrospray ion source, positive and negative ion mode; The capillary voltage was 5.5 kV and the desolvation temperature was 550 °C.

8. A method for detecting NAD in multiple samples as claimed in claim 1 + The method is characterized in that: When the sample is plasma or serum, add NAD + -d4 stable isotope internal standard, then add acetonitrile for protein precipitation, shake and mix for 30-60 seconds, centrifuge at 12000-14000rpm, 0-4℃ for 5-10 minutes, aspirate the supernatant to obtain the sample to be tested.

9. A method for detecting NAD in multiple samples as claimed in claim 1 + The method is characterized in that: When the sample is a whole blood sample, the frozen and thawed whole blood is mixed with ddH2O and NAD is added. + -d4 stable isotope internal standard, then add acetonitrile, shake and mix for 30-60 seconds, centrifuge at 12000-14000rpm, 0-4℃ for 5-10 minutes, aspirate the supernatant and mix with ddH20 to obtain the sample to be tested.

10. The method for detecting NAD in multiple samples according to any one of claims 1 to 9. + The method was used to analyze the NAD in plasma, whole blood and serum samples. + For qualitative and quantitative detection applications.

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