A kind of NAD + High-throughput detection method for a molecule or its precursor

Abstract

The present invention belongs to the field of biological detection, and specifically relates to a NAD + A high-throughput detection method for a biological sample or a precursor molecule thereof, wherein the method uses high-performance liquid chromatography tandem mass spectrometry (LC‑MS / MS) to detect biological samples in a biological sample collection card without quantifying the sampled blood, has the characteristics of simple and rapid operation, high accuracy, and can simultaneously extract and process ten to dozens of dried blood spot samples, and is suitable for high-throughput sample preprocessing.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a high-throughput detection method for NAD or its precursor molecule. The method uses high-performance liquid chromatography tandem mass spectrometry (LC-MS / MS method) to detect biological samples in a biological sample collection card, which is convenient and efficient. Background Art

[0002] In 1962, Robert Guthrie published a method for screening neonatal phenylketonuria, which introduced the dried blood spot sampling technology (DBS) for the first time. Relying on the 903 and FTA patented blood collection filter papers, it has achieved great success in the fields of medical diagnosis and forensic identification. Dried blood spots using this type of blood collection filter paper can be used for analysis methods such as time-resolved immunofluorescence (TRFIA), tandem mass spectrometry (LC-MS / MS), enzyme chemistry, and fluorescence chemistry, and are an important means of screening and researching neonatal inherited metabolic diseases.

[0003] Nicotinamide adenine dinucleotide (NAD + ) is an important energy metabolite in the human body. It is an important electron transport medium for cellular glycolysis, tricarboxylic acid cycle and fatty acid oxidation. It is involved in almost all energy metabolism and transport processes in the human body. At the same time, a large number of studies have shown that with the continuous increase of age, NAD in body cells + The level will drop significantly, and the cell energy metabolism rate will decrease, causing the body to gradually show an aging state, and even eventually develop a series of aging-related diseases. At the same time, work pressure, excessive drinking, lack of sleep and many other unhealthy lifestyles will also cause the body's NAD + The level has dropped significantly. + The problem of low levels can be supplemented by supplementing with fresh vegetables, animal liver, eggs and milk.

[0004] Tandem mass spectrometry (LC-MS / MS) can be used to detect NAD + The concentration of precursor supplements such as (vitamin B3, NR, NMN) can accurately reflect the absorption of supplements, and NAD can also be detected + Changes in blood concentration levels reflect the energy metabolism level of the subject, and such tests are of great reference significance for the overall assessment of the subject's health status. However, the various blood collection filter papers currently available on the market cannot meet the requirements of NAD + Detection needs, mainly due to the high probability of sample NAD + There are problems of degradation, low elution rate and large detection error. In addition, the samples used for testing require the use of professional equipment such as pipettes, which has high requirements for users and poor convenience. Summary of the invention

[0005] In order to solve the above problems, the present invention now provides a NAD + The high-throughput detection method of the precursor molecule thereof does not require quantification of the sampled blood and has the characteristics of simple and rapid operation, high accuracy, ability to extract and process ten to dozens of dried blood spot samples at the same time, and suitability for high-throughput sample preprocessing.

[0006] The specific technical solutions are as follows:

[0007] A kind of NAD + A high-throughput detection method for a molecule or a precursor thereof, wherein the specific steps are as follows:

[0008] 1) Soaking the test paper in an inorganic salt solution and then drying it to prepare a biological sample collection card;

[0009] 2) Take samples containing different concentrations of NAD + or a standard substance of its precursor molecule and a sample to be tested are immersed in the biological sample collection card respectively to obtain a sampled card;

[0010] 3) taking the same area of ​​the sampled card in step 2) for elution, and detecting the eluate using high performance liquid chromatography tandem mass spectrometry (LC-MS / MS);

[0011] 4) Establish the relationship between ion chromatographic peak area and NAD using standard samples + or a standard curve of the concentration of a precursor molecule thereof;

[0012] 5) Enter the ion chromatogram peak area of ​​the sample to be tested and obtain the NAD in the sample to be tested + or the concentration of its precursor molecules.

[0013] In some embodiments, the inorganic salt in step 1) is selected from any one of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate, preferably any one of ammonium sulfate, magnesium chloride or ammonium chloride.

[0014] In some embodiments, the test paper in step 1) is a water-absorbent test paper, selected from any one of PET test paper, quantitative filter paper, FTA test paper or 903 test paper.

[0015] In some embodiments, the standard in step 2) is a human serum albumin (HSA) solution containing NAD+ or its precursor molecules.

[0016] In some embodiments, the LC-MS / MS method in step 3) contains an internal standard, which is an isotope-labeled internal standard of the molecule to be detected, preferably β-NAD- 13 C5.

[0017] In some embodiments, the mobile phase A of the HPLC is an ammonium formate solution, and the mobile phase B is acetonitrile. Preferably, the mobile phase A is an ammonium formate solution containing 0.01% formic acid.

[0018] In some embodiments, the mobile phase flow rate of the HPLC is 0.1-0.5 mL / min, preferably 0.3 mL / min.

[0019] In some embodiments, the ion source used in the mass spectrometry is electrospray ionization (ESI).

[0020] In some embodiments, NAD + The precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof, and more preferably nicotinamide mononucleotide (NMN).

[0021] In some embodiments, the biological sample is a blood sample, preferably a whole blood sample.

[0022] The beneficial effects of the present invention are:

[0023] 1. The biological sample collection card of the present invention can maintain NAD in blood samples. + The content is stable while maintaining a high elution rate, thus achieving NAD + The existing dry blood spot method for sampling, such as the 903 test paper and FTA test paper, cannot achieve this function, and there is a certain error in the test results of NAD+ concentration.

[0024] 2. The preparation requirements of biological sample collection cards are low and the operation is simple. The detection results of NAD or its precursor molecules are not affected by the pH of the inorganic salt solution. The concentration only needs to ensure that no crystallization occurs after the test paper is soaked and dried. At the same time, it does not need to be frozen, which is more suitable for the collection, transportation, detection and storage of large quantities of samples.

[0025] 3. The LC-MS / MS method for detecting NAD+ in dried blood spots does not require quantitative analysis of the sampled blood. It is simple and fast to operate, has high accuracy, can simultaneously extract and process ten to dozens of dried blood spot samples, and is suitable for high-throughput sample pretreatment. The method uses HSA solution to simulate the blank whole blood matrix and establish a gradient standard. It is easy to configure and the measurement results are accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows NAD in dried blood spots made with different test strips + Content comparison;

[0027] Figure 2 It shows that the biological sample collection card pretreated with inorganic salt solution increases NAD + The preservation rate;

[0028] Figure 3 Shows the NAD of dried blood spots made from 903 test paper pretreated with different inorganic salt solutions + Content comparison;

[0029] Figure 4 The ion scanning group diagram of the blank matrix dry blood spot sample detection in the present invention is shown;

[0030] Figure 5 The ion scan group diagram of the detection of the dried blood spot sample with the limit of quantification (500 ng / mL) in the present invention is shown;

[0031] Figure 6 The standard curve diagram obtained by using the quantitative ion chromatography peak area-concentration as coordinates of the present invention is shown;

[0032] Figure 7 The ion scanning group diagram of the detection of the dried blood spot sample with the detection limit (25 ng / mL) in the present invention is shown;

[0033] Figure 8 The dried blood spot NAD prepared by the blood sample collection card of the present invention is shown. + 14 days stable;

[0034] Fig. 9 The results of the high-throughput detection method and the quantitative blood sampling method on 31 volunteers are shown;

[0035] Fig.10 A comprehensive comparison of the high-throughput detection method and the quantitative blood sampling method is shown. DETAILED DESCRIPTION

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] One of the objects of the present invention is to provide a NAD + The method is described below as an example of a high throughput detection method for a molecule or a precursor thereof.

[0038] Preparation of biological sample collection card: soak the test paper in an inorganic salt solution and then dry it to obtain the biological sample collection card.

[0039] In some embodiments, the inorganic salt is selected from sulfate, hydrochloride, phosphate, for example, selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate. In some specific embodiments, the inorganic salt is preferably any one of ammonium sulfate, magnesium chloride or ammonium chloride. The inorganic salt solution can be used in any concentration and any pH.

[0040] The test paper can be a test paper with water absorption function commonly used in the art, including various materials such as synthetic fiber materials, plant fiber materials, etc. In some embodiments, the test paper includes but is not limited to any one of PET test paper, quantitative filter paper, FTA test paper or 903 test paper.

[0041] The "PET test paper" is made of polyester fiber material with low fiber density. "PET" is specifically polyethylene terephthalate, with an irregular fiber mesh structure on the surface and a textile density of 66 g / m 2 .

[0042] During the manufacturing process of "quantitative filter paper", the pulp is treated with hydrochloric acid and hydrofluoric acid, and then washed with distilled water to remove most of the impurities in the paper fibers. Therefore, there is very little residual ash after burning, which has almost no effect on the analysis results, making it suitable for precise quantitative analysis.

[0043] "FTA Test Paper" (Flinders Technology Associates) is a special filter paper. It is soaked with a patented formula of strong denaturants and chelating agents, and contains special chemicals on the fiber matrix. When cells are captured, they are automatically lysed and combined with nucleic acids to maintain the integrity of the DNA in the sample, protect nucleic acids from degradation, damage from nucleases, oxidants and ultraviolet rays, and prevent the growth of bacteria and other microorganisms. It is mostly used for criminal investigation DNA collection and detection.

[0044] "903 test paper" can also be called "903 protein preservation card", which means that whatman 903 sample collection paper is made of 100% pure cotton wool without any wet strength additives, and is used for the collection and transfer of human fluid specimens. 903 test paper can fully absorb blood samples and form uniformly dried blood spots, which is more preferred in the present invention.

[0045] Preparation of standard curve

[0046] Take different concentrations of NAD + The standard sample of the biological sample collection card is immersed in the standard sample of the biological sample collection card to obtain the standard sample card. For example, the standard sample is dripped on the biological sample collection card and then dried. The drying method is, for example, natural drying at room temperature.

[0047] In some embodiments, the standard is a human serum albumin (HSA) solution containing NAD+ or its precursor molecules.

[0048] The same area of ​​the standard sample card is taken for elution, and the elution is detected using high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). The high performance liquid chromatography-tandem mass spectrometry technology uses a liquid chromatography system to separate the substance to be tested and then connects it to a mass spectrometer. After the substance to be tested is ionized, it is converted into an electrical signal and processed by a computer. It is analyzed according to the mass spectrum peak, and it has the excellent separation ability of liquid chromatography and the high sensitivity and high selectivity of mass spectrometry.

[0049] For example, a puncher is used to remove a disc of the same area from the part containing the biological sample. When testing is required, the sampled card can be eluted and the eluate can be tested. For ease of testing, a solution containing an internal standard can be used as an eluent. The elution method can be oscillation, ultrasound, etc.

[0050] According to the test results of the standard, the relationship between ion chromatographic peak area and NAD + or a standard curve of the concentration of its precursor molecule.

[0051] Sample processing and testing

[0052] The biological sample to be tested is immersed in the biological sample collection card to obtain the sample card to be tested. For example, the sample to be tested is dropped on the biological sample collection card and then dried. The drying method is, for example, natural drying at room temperature.

[0053] In some embodiments, the biological sample is a blood sample, preferably a whole blood sample.

[0054] The term "whole blood" refers to the mixture formed by collecting blood from the human body into a blood collection bag, which includes all components of blood cells and plasma.

[0055] Take the same area of ​​the sample card to be tested as the standard sample card, perform elution, and use high performance liquid chromatography tandem mass spectrometry (LC-MS / MS) to detect the eluate. The detection method can be the same as the detection method of the standard sample card mentioned above.

[0056] Substitute the ion chromatogram peak area of ​​the sample to be tested into the standard curve to obtain the NAD + or the concentration of its precursor molecules.

[0057] In some embodiments, the LC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard of the molecule to be detected, preferably β-NAD- 13 C5.

[0058] In some embodiments, the mobile phase A of the HPLC is an ammonium formate solution, and the mobile phase B is acetonitrile. Preferably, the mobile phase A is an ammonium formate solution containing 0.01% formic acid.

[0059] In some embodiments, the mobile phase flow rate of the HPLC is 0.1-0.5 mL / min, preferably 0.3 mL / min.

[0060] In some embodiments, the LC conditions are: C18 column; column temperature: 25-40°C; injection volume: 1-10 μL; mobile phase A: 1-5 mM ammonium formate aqueous solution, formic acid concentration 0.01-0.1%; mobile phase B: acetonitrile; flow rate 0.21-0.8 mL / min; gradient elution.

[0061] In some embodiments, MS conditions: tandem mass spectrometer: Waters I-Class ultra-high pressure liquid chromatograph; Sciex Triple Quad 4500 mass spectrometer; ion source: ESI (electrospray ion source); spray voltage: 5500 V; spray temperature: 450-600 °C; scan mode: multiple reaction ion monitoring scan mode (MRM mode), the parameters are as follows:

[0062] Compound Precursor ion Product ions Collision energy (V) Declustering voltage (V) β-NAD+ 664.2 524.1 27 81 <![CDATA[β-NAD+- 13 C5]]> 669.3 529.0 51 96

[0063] In some embodiments, the ion source used in the mass spectrometry is electrospray ionization (ESI), which is an ionization method. The electrospray ion source is a soft ionization source that can generate multi-charged ions from large-mass organic molecules.

[0064] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or alimentarily or pharmaceutically acceptable salts, derivatives or prodrugs thereof, and more preferably nicotinamide mononucleotide (NMN).

[0065] The term “NAD + Precursors are substances that can be used by the body for NAD + Any small molecule synthesized, including but not limited to corresponding homologues, isomers or isotopic derivatives thereof.

[0066] NAD + NAD is a cofactor that plays a central role in metabolism. + As levels decline with age, NAD + It can help repair DNA, regulate immune cell conduction, provide cell energy and reduce aging, etc. During the in vitro preparation of immune cells, NAD is added +Treatment can provide sufficient energy for immune cell proliferation, improve immune cell proliferation and reduce cell aging indicators. + The precursor is converted into NAD through chemical conversion + of compounds.

[0067] In some embodiments, the NAD + The precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof; preferably nicotinamide mononucleotide (NMN). NMN is used as NAD + The direct precursor of NAD + , easily absorbed by cells.

[0068] The detection method described in the present disclosure does not require volume quantification of the collected blood samples, and the blood sampling test strips can maintain the long-term stability of the NAD+ content in the blood sample, thereby realizing the dry blood spot method detection of NAD+ concentration. It has the characteristics of simple and fast operation, high accuracy, and can extract and process ten to dozens of dry blood spot samples at the same time, and is suitable for high-throughput sample preprocessing.

[0069] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.

[0070] I. Quantitative blood sampling method

[0071] 1. Preparation and pre-treatment of dried blood spots

[0072] 1.1 Preparation method of dried blood spots

[0073] Use a pipette to take a certain amount of the collected whole blood sample and drop it on the biological sample collection card. Place the dried blood spot card at room temperature to dry for 2-4 hours. After the dried blood spot is completely dry, transfer it to the sealing tape and store it at room temperature for later use.

[0074] 1.2 Pretreatment of dried blood spot samples

[0075] Use a sampler to remove the complete dried blood spot filter paper, put it into a 2 mL EP tube, and add 300 μL of internal standard solution (β-NAD- 13C5), shake evenly to make the internal standard solution immerse the dried blood spot filter paper; ultrasonicate in an ice water bath for 20 min; after ultrasonicate, add 1200 μL of saturated ammonium sulfate solution and shake evenly or ultrasonicate for 1 min; after ultrasonicate, put the EP tube into a centrifuge and centrifuge at 10000 rpm for 3 min; take the supernatant and filter it and put it into a sample bottle for later use.

[0076] 2. Pretreatment of whole blood samples

[0077] Add 1190 μL of saturated ammonium sulfate solution to a 1.5 mL centrifuge tube, add the whole blood sample from the same donor and shake evenly, and freeze the EP tube in liquid nitrogen for 2 min;

[0078] Take out the frozen EP tube and place it in an ice box to thaw. After thawing, add 300 μL of internal standard solution (β-NAD- 13 C5), shake evenly; put the EP tube into an ice water bath for ultrasonication for 10 min; immediately after ultrasonication, put the EP tube into a centrifuge and centrifuge at 10,000 rpm for 3 min; take the supernatant after centrifugation, filter it, and put it into a sample bottle for later use.

[0079] 3. LC-MS / MS method for calculating NAD+ concentration

[0080] When using the LC-MS / MS method, the internal standard method is used, and the internal standard is an isotope-labeled compound of the molecule to be tested. An isotope internal standard of known concentration is added to a certain amount of a standard reference substance and a sample to be tested, and the concentration of the molecule to be tested in the sample to be tested is calculated based on the relationship between the peak area, mass or concentration of the molecule to be tested, the reference substance and the internal standard in the sample to be tested:

[0081] F = (A s / m s ) / (A r / m r ),

[0082] m i = (F*A i ) / (A s / m s ),

[0083] Where F is the correction factor, A s , A r , A i is the peak area of ​​internal standard, reference substance and analyte, m s 、m r 、m i is the mass or concentration of the molecule to be tested in the internal standard, reference substance and sample to be tested. This calculation process can usually be completed automatically on the LC-MS / MS supporting software.

[0084] During sample processing, samples are usually diluted or concentrated. The concentration of the molecule to be tested in the biological sample can be calculated by the concentration of the molecule to be tested and the dilution factor detected by LC-MS / MS:

[0085] D = V a / V i，

[0086] m a = m i *D,

[0087] Where D is the dilution factor, V a , V i are the volume of biological sample and the volume of the test fluid after sample processing, m a 、m i It is the mass or concentration of the molecule to be tested in the biological sample and the test solution.

[0088] When the molecule to be tested is NAD+, the internal standard is β-NAD- 13 C5. The calculation method of NAD+ content in biological samples is:

[0089] Dried blood spot sample testing: m sample = m report *(V s +V SAS ) / V sample，

[0090] Whole blood sample testing: m sample = m report *(V s +V SAS +V sample ) / V sample，

[0091] Where D is the dilution factor, m sample 、m report is the NAD+ concentration in biological samples and the NAD+ concentration detected by LC-MS / MS, V s 、V SAS 、V sample are the volumes of internal standard solution, saturated ammonium sulfate solution and biological sample, respectively.

[0092] The following Examples 1 to 3 all use this method to calculate the concentration of NAD+.

[0093] Example

[0094] Example 1 Comparison of dried blood spots made with different test strips

[0095] PET test paper (Labconco Replacement Filter Paper 7544810), quantitative filter paper (Titan, rapid quantitative filter paper 7 cm), FTA test paper (Whatman FTA standard card) and 903 test paper (Whatman903 protein storage card) were used to prepare 10 μL whole blood samples into dried blood spots according to method 1.1, and the samples were pre-treated according to method 1.2, and then injected into LC-MS / MS to detect NAD in the samples. + According to method 2, 10 μL of whole blood sample from the same donor was processed and the obtained NAD + content as a reference.

[0096] After testing several dried blood spots, it was found that ( Figure 1 ), NAD in dried blood spots on quantitative filter paper, 903 test paper, and FTA test paper + All of them have been degraded, and NAD in PET test paper + Content and NAD in fresh blood samples + The possible reasons are that the PET filter paper has the smallest thickness, the blood diffuses quickly, the drying time is short, and the NAD before the dried blood spot is dried + The enzymatic hydrolysis time is shorter. Therefore, NAD is guaranteed before the blood spots are dried. + Stability is the key to detecting NAD in dried blood spots + Necessary conditions for content.

[0097] Example 2 Comparison of biological sample collection cards after inorganic salt pretreatment

[0098] Test paper preparation: prepare 0.6 mol / L ammonium sulfate solution, soak PET test paper, quantitative filter paper and 903 test paper respectively, then lay them flat to dry for later use.

[0099] 10 μL of whole blood sample was prepared into dried blood spots using the above test paper according to method 1.1. After sample pretreatment according to method 1.2, the sample was injected into LC-MS / MS to detect the content of NAD+ in the sample. 10 μL of whole blood sample from the same donor was processed according to method 2, and the obtained NAD + content as a reference.

[0100] After drying blood spots NAD + Detection found ( Figure 2 ), NAD in dried blood spots made with PET test paper pretreated with ammonium sulfate solution + The content of NAD in the dried blood spots made of treated 903 test strips and quantitative filter paper is close to that of the untreated PET test strips. + The content was significantly higher than that of the untreated test paper. The results showed that pretreatment with saline solution can effectively ensure the NAD+ The stability of NAD + Preservation rate.

[0101] Example 3 Comparison of biological sample collection cards pretreated with different inorganic salt solutions

[0102] Preparation of biological sample collection cards: prepare 0.6 mol / L ammonium sulfate solution, sodium sulfate solution, sodium chloride solution, magnesium sulfate solution, ammonium chloride solution, and sodium dihydrogen phosphate solution respectively, soak 903 test paper, lay it flat and dry it for later use.

[0103] 10 μL whole blood samples were prepared into dried blood spots using the above test strips according to method 1.1. Untreated 903 test strips were used as the control group. After sample pretreatment according to method 1.2, the samples were injected into LC-MS / MS to detect NAD in the samples. + According to method 2, 10 μL of whole blood sample from the same donor was processed, and the NAD + content as a reference.

[0104] Comparison of NAD in dried blood spots of biological sample collection cards treated with different salts + Content found ( Figure 3 ), using biological sample collection cards pretreated with different salts can ensure the NAD concentration of dried blood spots to a certain extent. + Stability, among which ammonium sulfate, magnesium chloride and ammonium chloride have the best effects.

[0105] In order to improve the detection convenience of the LC-MS / MS method, the inventors further developed a high-throughput detection method based on the above method.

[0106] II. High-throughput detection methods

[0107] Example 4 LC-MS / MS high-throughput detection

[0108] 4.1 Instrument detection settings:

[0109] Chromatographic column: Yuexu Xtimate C18 chromatographic column, 2.1*50 mm; particle size 1.8 μm;

[0110] Mobile phase A: 1 mM ammonium formate solution containing 0.01% formic acid; mobile phase B: acetonitrile; column temperature: 25 °C.

[0111] LC liquid phase pump gradient settings, mass spectrometry conditions, multiple reaction monitoring ion pairs (MRM) and corresponding voltage parameters are shown in Tables 1 to 3, respectively.

[0112] Table 1 LC liquid phase pump gradient

[0113] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (mL / min) 0.0 100 0 0.3 1.0 100 0 0.3 5.0 20 80 0.3 6.0 20 80 0.3 6.1 100 0 0.3 8.0 100 0 0.3

[0114] Table 2 Mass spectrometry conditions

[0115] Ion source ESI Spray voltage 5500 V Spray temperature (TEM) 550 ℃ Atomizing gas (GS1) 55 psi Auxiliary heating gas (GS2) 60 psi Curtain Air (CUR) 35 psi Impingement Air (CAD) 9 psi

[0116] Table 3 Multiple reaction monitoring ion pairs (MRM) and corresponding voltage parameters

[0117] Compound Precursor ion Product ions Collision energy (V) Declustering voltage (V) β-NAD+ 664.2 524.1 27 81 <![CDATA[β-NAD+- 13 C5]]> 669.3 529.0 51 96

[0118] 4.2 Preparation of Dried Blood Spot Standards

[0119] Biological sample collection card: prepare 1.2 mol / L ammonium sulfate solution, soak 903 test paper (Whatman 903 protein storage card) and lay it flat to dry for later use.

[0120] Because blood cells contain higher levels of β-NAD + In this method, the blank whole blood matrix of the dried blood spots is human serum albumin (HSA).

[0121] Preparation of β-NAD + of human serum albumin solution, wherein the concentration of human serum albumin is 200 mg / mL; β-NAD + The linear range is 0.5 to 100 μg / mL:

[0122] 1) Blank matrix HSA solution: Weigh a certain amount of human serum albumin and dissolve it in saline to a concentration of 200 mg / mL;

[0123] 2) Prepare blank matrix sample: take 1 drop (about 20 μL) of HSA solution and drop it on the biological sample collection card;

[0124] 3) Preparation of standard dried blood spot S0: Weigh a certain amount of β-NAD + The standard is completely dissolved with the HSA solution in 1) to adjust to a 10 μg / mL standard stock solution, recorded as S0 solution, and 1 drop (about 20 μL) is dropped on the biological sample collection card;

[0125] 4) Prepare standard dried blood spot S1: accurately pipette 200 μL of S0 solution, add 200 μL of HSA solution, mix well, record as S1 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0126] 5) Prepare standard dried blood spot S2: accurately pipette 200 μL of S1 solution, add 200 μL of HSA solution, mix well, record as S2 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0127] 6) Prepare standard dried blood spot S3: accurately pipette 200 μL of S2 solution, add 200 μL of HSA solution, mix well, record as S3 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0128] 7) Prepare standard dried blood spot S4: accurately pipette 200 μL of S3 solution, add 50 μL of HSA solution, mix well, record as S4 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0129] 8) Prepare standard dried blood spot S5: accurately pipette 200 μL of S4 solution, add 200 μL of HSA solution, mix well, record as S5 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0130] 9) Prepare standard dried blood spot S6: accurately pipette 200 μL of S5 solution, add 200 μL of HSA solution, mix well, record as S6 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0131] 10) Prepare the standard dried blood spot S7: accurately pipette 100 μL of S6 solution, add 150 μL of HSA solution, mix well, record as S7 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0132] 11) Prepare standard dried blood spot S8: accurately pipette 200 μL of S7 solution, add 200 μL of HSA solution, mix well, record as S8 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0133] 12) Prepare the standard dried blood spot S9: accurately pipette 200 μL of S8 solution, add 200 μL of HSA solution, mix well, record as S9 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0134] 13) Prepare the standard dried blood spot S10: accurately pipette 100 μL of S9 solution, add 900 μL of HSA solution, mix well, record as S10 solution, take 1 drop (about 20 μL) and drop it on the biological sample collection card;

[0135] Place the above-mentioned standard dried blood spots in a clean, dry and ventilated condition and air-dry for 2-4 hours, then put them into a sealed bag and store them at room temperature away from light.

[0136] Table 4 β-NAD corresponding to standard dried blood spots + concentration

[0137] serial number <![CDATA[NAD + (ng / mL)]]> S0 100000 S1 50000 S2 25000 S3 12500 S4 10000 S5 5000 S6 2500 S7 1000 S8 500 S9 250 S10 25

[0138] 4.3 Preparation of internal standard

[0139] Weigh an appropriate amount of β-NAD + - 13 C5 pure product was dissolved and made up to 500 ng / mL.

[0140] 4.4 Preparation of whole blood dried blood spot samples

[0141] Take 1 drop (about 20 μL) of whole blood (venous blood or fingertip blood) and drop it on the biological sample collection card. Place the collection card in a clean, dry and ventilated condition to air-dry for 2-4 hours, then put it into a sealed bag and store it at room temperature away from light.

[0142] 4.5 Sample preparation

[0143] ① Sampling: For dried blood spot samples of S0-S10, use a hole punch to take 2 dried blood pieces with a diameter of 3 mm and add them into a 1.5 mL centrifuge tube;

[0144] ② Elution: Add 100 μL 500 ng / mL β-NAD + - 13 C5 internal standard solution, ultrasonically treated in an ice-water bath for 15 min, added with 400 μL saturated ammonium sulfate solution and ultrasonicated for 5 min, centrifuged and collected the supernatant or filtered it into a sample bottle and placed it in a high performance liquid chromatography tandem mass spectrometry system for detection.

[0145] Example 5 Detection of β-NAD in Dried Blood Spot Samples + Exclusive

[0146] Blank matrix dried blood spots and S8 standard dried blood spots were taken respectively and tested according to the sample pretreatment described in 4.5 and the LC-MS / MS determination conditions described in 4.1. + and β-NAD + - 13 The retention time of C5 is 2.58 min (see Figure 4 and Figure 5 ), there was no obvious interfering peak at the retention time of β-NAD+ in the blank matrix sample.

[0147] Example 6 Detection of β-NAD in Dried Blood Spot Samples + Linear relationship and detection limit

[0148] Take the standard dried blood spot samples of different concentrations prepared in 4.3, and test them according to the sample pretreatment described in 4.5 and the LC-MS / MS determination conditions described in 4.1. Plot the quantitative ion chromatogram peak area-concentration to obtain the standard curve Y=0.00097x+(-0.00439), see Figure 6 .

[0149] The results showed that the linear range and quantification limit of β-NAD+ in dried blood spots are as follows:

[0150] 1) β-NAD + Limit of detection (LOD): 25 ng / mL (see Figure 7 )

[0151] 2) β-NAD + Limit of quantitation (LOQ): 500 ng / mL

[0152] 3) β-NAD + Detection range: β-NAD + In the range of 500 ng / mL to 100000 ng / mL, the linearity was good, and the correlation coefficient R 2 >0.9800.

[0153] Example 7 Detection of NAD in Dried Blood Spot Samples + The recovery and precision

[0154] Dried blood spots from the same whole blood source were tested according to the methods described in 4.4 and 4.5 and the LC-MS / MS assay conditions described in 4.1. The test was repeated for 6 batches, and the precision was 5.58%.

[0155] Dried blood spots from the same whole blood source were added with three concentrations of high, medium and low for recovery and precision experiments. The test was performed according to the methods described in 4.4 and 4.5 and the LC-MS / MS determination conditions described in 4.1. The test was repeated for three batches. + The recovery rates are shown in the following table:

[0156] Table 5 Recovery rate of NAD+

[0157] Spike Amount (ng / mL) Recovery rate Precision 5000 104.20% 2.12% 10000 109.43% 0.98% 20000 106.88% 1.35%

[0158] Example 8 NAD in Dried Blood Spot Samples + Stability

[0159] NAD in Dried Blood Spots + The stability of the dried blood spot samples is to put them into a sealed bag after drying, store them at room temperature without light, and detect NAD in the dried blood spots within 14 days. + Repeatability of content.

[0160] Dried blood spots from the same whole blood source were collected at different storage times and tested according to the methods described in 4.4 and 4.5 and the LC-MS / MS assay conditions described in 4.1. Six batches were tested at each storage time. The NAD content of 14-day dried blood spots was + Change rate <10% (see Figure 8 ), which can ensure that the samples are stored at room temperature for 14 days before testing, without the need for low-temperature storage.

[0161] Example 9 Comparison of high-throughput detection method and quantitative blood sampling method

[0162] 31 volunteers were recruited to use quantitative blood sampling and high-throughput detection methods for sampling at the same time, and the test results were cross-referenced. Fig. 9 and Fig.10 As shown, all numerical differences are within the range of ±20% of the mean of the two methods ( Fig. 9 ), combining the data of quantitative blood sampling method and high-throughput detection method of all volunteers, the two methods detected NAD + The results of content were consistent ( Fig.10 ).

[0163] In summary, the present invention provides a high-throughput detection method for biological samples, which can accurately detect the NAD+ concentration in blood samples by quantitative ion chromatography peak area and concentration. The method is flexible and convenient, easy for users to operate, and the subject to be tested does not need to use professional tools such as pipettes, but only needs to provide a drop of blood to prepare the sample to be tested, which greatly improves the convenience of the blood test method.

Claims

1. A type of NAD + or a high-throughput detection method of a precursor molecule thereof, characterized in that The steps include: The test paper is soaked in an inorganic salt solution and then dried to obtain a biological sample collection card, wherein the inorganic salt is selected from any one of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate; Take different concentrations of NAD + or a standard substance of its precursor molecule and a sample to be tested are immersed in the biological sample collection card respectively to obtain a sampled card; Taking the same area of ​​the sampled card containing the biological sample in step 2) for elution, and detecting the eluate using high performance liquid chromatography tandem mass spectrometry; Establish the relationship between ion chromatographic peak area and NAD by using standard + or a standard curve of the concentration of a precursor molecule thereof; Substitute the ion chromatogram peak area of ​​the sample to be tested to obtain the NAD in the sample to be tested. + or the concentration of its precursor molecules.

2. The high-throughput detection method according to claim 1, characterized in that: Step 1) The inorganic salt is selected from any one of ammonium sulfate, magnesium chloride or ammonium chloride.

3. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: Step 1) The test paper is a water-absorbent test paper, selected from any one of PET test paper, quantitative filter paper, FTA test paper or 903 test paper.

4. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: Step 2) The standard contains NAD + or a human serum albumin solution thereof.

5. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: Step 3) The HPLC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard of the molecule to be measured.

6. The high-throughput detection method according to claim 5, characterized in that: The internal standard is β-NAD-13C5.

7. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: The mobile phase A of the high performance liquid chromatography is ammonium formate solution, and the mobile phase B is acetonitrile.

8. The high-throughput detection method according to claim 7, characterized in that: The mobile phase A of the HPLC is an ammonium formate solution containing 0.01% formic acid.

9. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: The mobile phase flow rate for HPLC was 0.1-0.5 mL / min.

10. The high-throughput detection method according to claim 9, characterized in that: The mobile phase flow rate of the HPLC was 0.3 mL / min.

11. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: The ion source used in the mass spectrometry is electrospray ionization.

12. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: The NAD + The precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, nicotinamide riboside, or alimentarily or pharmaceutically acceptable salts, derivatives or prodrugs thereof.

13. The high-throughput detection method according to claim 12, characterized in that: The NAD + The precursor is nicotinamide mononucleotide.

14. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: The biological sample is a blood sample.

15. The high-throughput detection method according to any one of claims 1 to 2, characterized in that: The biological sample is a whole blood sample.

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