Storage protective agent, collection card, and storage and detection method for biological sample

Abstract

A storage protective agent, collection card, and storage and detection method for a biological sample, relating to the fields of biological preservation and biological detection. The storage protective agent at least contains an inorganic salt solution, which can maintain the stability of various metabolic small molecules in blood and / or cells, and is used for preserving a blood sample and / or a cell sample collected after administration of an anti-aging component such as NAD+ or a precursor molecule thereof. On the basis of the storage protective agent, also provided is a biological sample collection card, which can maintain the stability of NAD+ content in the blood sample while keeping a high elution rate, thereby enabling the measurement of NAD+ concentration via a dried blood spot method. Further provided is a high-throughput detection method for NAD+ or a precursor molecule thereof, which improves the detection efficiency of the NAD+ or the precursor molecule thereof.

Description

Storage protectant, collection card, storage and detection method of biological samples Technical Field

[0001] The invention belongs to the field of biological preservation and biological detection, and relates to a storage protectant, a collection card, and a storage and detection method for biological samples. Background Art

[0002] The immune system changes with age. After the age of 40, the body's immune cells begin to age significantly, reducing the ability to fight viruses and bacteria, and increasing the risk of infections, chronic diseases, and cancer. + Nicotinamide adenine dinucleotide (coenzyme I) is a coenzyme of many dehydrogenases in the body, connecting the tricarboxylic acid cycle and the respiratory chain. Its function is to transfer hydrogen removed during metabolism to flavoproteins, NAD + It can provide cell energy and promote cell regeneration. + Metabolism in cells is shown in Figure 1.

[0003] Taking NAD + Or its precursor molecules help delay aging, in order to feedback people taking NAD + or its precursor molecules and NAD + / NADH conversion rate, which is convenient for evaluating the aging status, requires the conversion of NMN and NAD in the body + / NADH content is monitored in real time. The current detection method mainly collects whole blood samples from subjects, extracts PBMCs, and then detects them using an ELISA test kit. This method uses a large amount of blood and requires the extraction of PBMCs. The operation is cumbersome and the workload is large. In addition, the blood and PBMCs must be kept fresh, cannot be frozen, and cannot be transported over long distances. After obtaining the sample, it must be measured as soon as possible, otherwise NAD + How to prolong the storage time of the sample to be tested and optimize the detection method at the same time is still a technical problem that needs to be solved in this field.

[0004] Based on this, the inventors developed a biological sample storage protectant that stabilizes metabolites in the sample and enables low-temperature transportation. Based on this, they then developed a new generation of biological sample collection cards to enable room-temperature storage of biological samples.

[0005] The biological sample collection card is a specially designed plant fiber or synthetic fiber paper that can be used for the collection, transportation, and storage of various biological fluid samples (including but not limited to blood, semen, saliva, cell culture fluid, microorganisms, plant tissue fluid, and virus culture fluid) at room temperature. The collection card uses a special treatment method to rupture the cell membranes and denature proteins in the biological sample, and can adsorb and fix the marker to be tested. The dried blood spots on this type of blood collection filter paper can be used for analytical methods such as time-resolved immunofluorescence (TRFIA), tandem mass spectrometry (LC-MS / MS), enzymatic chemistry, and fluorescence chemistry.

[0006] Finally, the inventors further improved the tandem mass spectrometry (LC-MS / MS) method to reduce the strict requirements on the amount of blood collected from biological samples and achieve the NAD + or high-throughput detection of its precursor molecules. Summary of the Invention

[0007] The purpose of the present invention is to provide a storage protectant for biological samples, which can stabilize the metabolites in the samples and realize low-temperature transportation. On this basis, the inventors further provide a biological sample collection card that can be stored at room temperature, and improve the detection method to realize high-throughput detection.

[0008] The specific technical solutions are as follows:

[0009] One of the objectives of the present invention is to provide a storage protectant for biological samples, wherein the storage protectant contains at least an inorganic salt solution.

[0010] In some embodiments, the inorganic salt solution is a saturated inorganic salt solution.

[0011] In some specific embodiments, the inorganic salt is selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate.

[0012] In some embodiments, the biological sample contains a metabolic small molecule, preferably NAD + or its precursor molecule, 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).

[0013] In some embodiments, the biological sample is a blood sample and / or a cell sample. Preferably, the blood sample is a whole blood sample.

[0014] In some specific embodiments, the volume ratio of the storage protectant to the biological sample is ≥3:1.

[0015] A second object of the present invention is to provide a method for storing biological samples, wherein the storage method uses the storage protective agent described in the first object.

[0016] In some embodiments, the storage method is low-temperature storage, and the low temperature is ≤0°C, preferably ≤-20°C, more preferably ≤-78°C, and even more preferably ≤-80°C.

[0017] The third purpose of the present invention is to provide a biological sample collection card that uses dry blood spot sampling technology to achieve the detection of NAD in blood and other various biological samples. + The biological sample collection card is a test paper pretreated with the storage protective agent described in the first purpose.

[0018] In some embodiments, the pretreatment refers to soaking the test paper in a storage protectant and then drying it.

[0019] In some specific embodiments, the storage protectant comprises an inorganic salt solution.

[0020] In some embodiments, the inorganic salt is selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride, or sodium dihydrogen phosphate.

[0021] In some specific embodiments, the test paper is a water-absorbent test paper, preferably any one selected from PET test paper, quantitative filter paper, FTA test paper or 903 test paper.

[0022] In some embodiments, the biological sample contains a metabolic small molecule, preferably NAD + or its precursor molecule, 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).

[0023] In some embodiments, the biological sample is a blood sample and / or a cell sample. Preferably, the blood sample is a whole blood sample.

[0024] A fourth object of the present invention is to provide an application of the above-mentioned biological sample collection card in the collection, transportation, analysis, identification and / or storage of biological samples.

[0025] A fifth object of the present invention is to provide a kit for biological sample detection, wherein the kit contains the biological sample collection card described in the fourth object.

[0026] A sixth object of the present invention is to provide a method for detecting a biological sample, having any one or more of the following characteristics:

[0027] 1) The biological sample is stored using the storage method described in Purpose 2;

[0028] 2) The biological sample collection card described in Purpose 3 of the test;

[0029] 3) The detection uses the kit described in purpose five.

[0030] In some embodiments, the detection method has any one or more of the following characteristics:

[0031] 1) The detection method is high performance liquid chromatography tandem mass spectrometry (LC-MS / MS);

[0032] 2) The LC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard of the molecule to be measured, preferably β-NMN-d4 and β-NAD- 13 C5.

[0033] In some specific embodiments, the biological sample is stored using the storage method described in Objective 2, and the biological sample is frozen at least once before testing. Failure to freeze-thaw can result in incomplete cell lysis and low NAD+ concentration.

[0034] In other specific embodiments, the detection uses the biological sample collection card described in purpose three, or the kit described in purpose five, and the detection steps include:

[0035] 1) Immersing the biological sample in the biological sample collection card and then drying it to obtain a sampled card;

[0036] 2) Elute the sampled card and test the eluate.

[0037] The seventh object of the present invention is to provide a NAD + Or a high-throughput detection method for its precursor molecules, which does not require quantification of the sampled blood, has the characteristics of simple and fast operation, high accuracy, can simultaneously extract and process ten to dozens of dried blood spot samples, and is suitable for high-throughput sample preprocessing.

[0038] The specific steps are as follows:

[0039] 1) soaking the test paper in an inorganic salt solution and then drying it to prepare a biological sample collection card;

[0040] 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 of the third object respectively to obtain a sampled card;

[0041] 3) taking the same area of ​​the sampled card from step 2) for elution, and detecting the eluate using high performance liquid chromatography tandem mass spectrometry (LC-MS / MS);

[0042] 4) Establish the relationship between ion chromatography peak area and NAD using standard + or a standard curve of the concentration of a precursor molecule thereof;

[0043] 5) Input 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.

[0044] In some embodiments, the inorganic salt in step 1) is selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate.

[0045] 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.

[0046] In some embodiments, the standard in step 2) is a product containing NAD + or a human serum albumin (HSA) solution thereof.

[0047] 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 B-NAD- 13 C5.

[0048] 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.

[0049] In some embodiments, the mobile phase flow rate of the HPLC is 0.1-0.5 mL / min, preferably 0.3 mL / min.

[0050] In some embodiments, the ion source used in the mass spectrometry is electrospray ionization (ESI).

[0051] 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, more preferably nicotinamide mononucleotide (NMN).

[0052] In some embodiments, the biological sample is a blood sample, preferably a whole blood sample.

[0053] The beneficial effects of the present invention are:

[0054] 1. By adding a storage protectant to biological samples, metabolites are stabilized and transported at low temperatures, extending the storage time to more than 7 days, up to approximately 3 months. After thawing the frozen biological sample, the storage protectant can protect the metabolites in the sample from decomposition, allowing the sample to be stored for more than 24 hours, which is beneficial for actual sample testing.

[0055] 2. Further optimize the storage mode of biological samples and provide a biological sample collection card that can maintain NAD in blood samples. + While maintaining content stability, it also maintains a high elution rate, thereby achieving NAD + The existing dry blood spot method for sampling, such as 903 test paper and FTA test paper, cannot achieve this function. + There are certain errors in the concentration detection results.

[0056] 3. In addition, the preparation requirements of biological sample collection cards are low, the operation is simple, and the NAD + The detection results of the biomarker or its precursor molecules are not affected by the pH of the inorganic salt solution; the concentration only needs to ensure that no crystals form after the test paper is soaked and dried. Biological sample collection cards use a lower blood sample volume, simplify the collection method, and do not require freezing, making them more suitable for the collection, transportation, testing, and storage of large quantities of samples.

[0057] 4. The inventors further optimized the LC-MS / MS detection method and provided a high-throughput detection method for detecting NAD in dried blood spots. + The method eliminates the need for quantification of sampled blood. It offers simple, rapid operation, high accuracy, and the ability to simultaneously extract and process tens to dozens of dried blood spot samples, making it suitable for high-throughput sample pretreatment. The method uses HSA solution to simulate a blank whole blood matrix to establish a gradient standard, which is easy to configure and provides accurate measurement results. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows the NAD + Schematic diagram of metabolism in cells;

[0059] Figure 2 shows NAD in whole blood, plasma, and blood cells + Concentration detection comparison;

[0060] Figure 3 shows the NAD of the sample without storage protectant after freezing and thawing for 30 minutes. + Complete decomposition;

[0061] Figure 4 shows that the addition of storage protectants does not affect NAD + Determination of content;

[0062] Figure 5 shows that storage at 4°C has a significant impact on the detection of metabolic small molecules in the sample;

[0063] Figure 6 shows the short-term preservation effect of the samples after adding storage protectants;

[0064] Figure 7 shows the long-term preservation effect of the samples after adding storage protectants;

[0065] Figure 8 shows that the sample can withstand multiple freeze-thaw cycles after adding a storage protectant;

[0066] Figure 9 shows the stability test results of whole blood samples after pretreatment;

[0067] Figure 10 shows the NAD content in dried blood spots made with different test strips + Content comparison;

[0068] Figure 11 shows that the biological sample collection card pretreated with inorganic salt solution increased NAD + preservation rate;

[0069] Figure 12 shows the NAD of dried blood spots made with 903 test paper pretreated with different inorganic salt solutions. + Content comparison;

[0070] Figure 13 shows the dried blood spot NAD prepared using the biological sample collection card + Stable for up to 14 days;

[0071] Figure 14 shows the dried blood spot NAD prepared using the biological sample collection card + 7 days stable;

[0072] Figure 15 shows a comparison of biological sample collection cards pretreated with inorganic salt solutions of different concentrations;

[0073] Figure 16 shows a comparison of biological sample collection cards pretreated with salt solutions of different pH values;

[0074] FIG17 shows an ion scan group diagram of the blank matrix dry blood spot sample detection in the present invention;

[0075] FIG18 shows an ion scan group diagram of the detection of dried blood spot samples with a limit of quantification (500 ng / mL) in the present invention;

[0076] FIG19 shows a standard curve obtained by using quantitative ion chromatographic peak area-concentration as coordinates according to the present invention;

[0077] FIG20 shows an ion scan group diagram of the detection limit (25 ng / mL) of the dried blood spot sample in the present invention;

[0078] FIG21 shows the dried blood spot NAD prepared by the blood sample collection card of the present invention.+ 14 days stable;

[0079] Figure 22 shows the test results of 31 volunteers using the high-throughput detection method and the quantitative blood sampling method;

[0080] FIG23 shows the comprehensive comparison results of the high-throughput detection method and the quantitative blood sampling method. DETAILED DESCRIPTION

[0081] 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.

[0082] One of the objectives of the present invention is to provide a storage protectant for biological samples, wherein the storage protectant contains at least an inorganic salt solution.

[0083] In some embodiments, the inorganic salt solution is a saturated inorganic salt solution, which refers to a solution obtained by adding an inorganic salt to a certain amount of pure water and when the inorganic salt can no longer dissolve.

[0084] In some specific embodiments, the inorganic salt is selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate.

[0085] In some embodiments, the biological sample contains a metabolic small molecule, preferably NAD + or its precursor molecules.

[0086] The term "NAD + Precursor to NAD + Any small molecule added may be in reduced or non-reduced form.

[0087] NAD + NAD is a cofactor that plays a central role in metabolism. + 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.

[0088] 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 can be easily converted directly into NAD by NMNAT enzyme. + , and is easily absorbed by cells.

[0089] In some embodiments, the biological sample is a blood sample and / or a cell sample, wherein the blood sample is a whole blood sample, and the cell sample is a human cell, including nerve cells, white blood cells, red blood cells, platelets, phagocytes, epithelial cells, cardiomyocytes, stem cells, etc. For example, PBMC can be used.

[0090] 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.

[0091] The term "PBMC" stands for peripheral blood mononuclear cells (PBMCs), which are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes.

[0092] In some specific embodiments, the volume ratio of the storage protectant to the biological sample is ≥3:1, for example, the volume ratio of the storage protectant to the biological sample is 3:1, 4:1, 5:1, etc.

[0093] A second object of the present invention is to provide a method for storing biological samples, wherein the storage method uses the storage protective agent described in the first object.

[0094] In some embodiments, the storage method is low-temperature storage, and the low temperature is ≤0°C, preferably ≤-20°C, more preferably ≤-78°C, and even more preferably ≤-80°C. That is, the low temperature can be sufficient to freeze the biological sample, for example, ≤0°C, ≤-5°C, ≤-10°C, ≤-15°C, ≤-20°C, ≤-30°C, ≤-40°C, ≤-50°C, ≤-60°C, ≤-70°C, ≤-78°C (can be stored with dry ice), and ≤-80°C (can be stored at -80°C).

[0095] The term "dry ice storage" refers to storing samples in a foam box or insulated box filled with dry ice.

[0096] The term "-80°C storage" means that the sample was stored in a -80°C freezer.

[0097] The storage protectant can be added before the biological sample is frozen, or after the frozen biological sample is added to the storage protectant and thawed, or immediately after the frozen biological sample is thawed.

[0098] A third object of the present invention is to provide a biological sample collection card, wherein the biological sample collection card is a test paper pretreated with the storage protective agent described in the first object.

[0099] In some embodiments, the pretreatment involves soaking the test paper in a storage protectant and then drying it. The inorganic salt solution can be used at any concentration and any pH. In some embodiments, the concentration is 0.2-1.2 mol / L. In some embodiments, the pH is 2.5-10.5.

[0100] In some embodiments, the inorganic salt is selected from sulfates, hydrochlorides, and phosphates, for example, selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride, or sodium dihydrogen phosphate.

[0101] 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. Any test paper with water-absorbing function can be used, including various materials such as synthetic fiber materials and plant fiber materials.

[0102] 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 66g / m 2 .

[0103] 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 and is suitable for precise quantitative analysis.

[0104] FTA (Flinders Technology Associates) is a specialized filter paper. It's soaked in a patented formula of powerful denaturants and chelating agents. Its fiber matrix contains specialized chemicals that automatically lyse cells upon capture and bind to nucleic acids, maintaining the integrity of the DNA in the sample and protecting it from degradation, damage by nucleases, oxidants, and ultraviolet light. It also prevents the growth of bacteria and other microorganisms. It's often used for DNA collection and testing in criminal investigations.

[0105] "903 test paper" can also be called "903 protein preservation card", which means that the 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.

[0106] In some embodiments, the biological sample contains a metabolic small molecule, preferably NAD + or its precursor molecule, 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).

[0107] In some embodiments, the biological sample is a blood sample and / or a cell sample. Preferably, the blood sample is a whole blood sample.

[0108] A fourth object of the present invention is to provide an application of the above-mentioned biological sample collection card in the collection, transportation, analysis, identification and / or storage of biological samples.

[0109] A fifth object of the present invention is to provide a kit for biological sample detection, wherein the kit contains the biological sample collection card described in the fourth object.

[0110] A sixth object of the present invention is to provide a method for detecting a biological sample, having any one or more of the following characteristics:

[0111] 1) The biological sample is stored using the storage method described in Purpose 2;

[0112] 2) The biological sample collection card described in Purpose 3 of the test;

[0113] 3) The detection uses the kit described in purpose five.

[0114] In some embodiments, the detection method has any one or more of the following characteristics:

[0115] 1) The detection method is high performance liquid chromatography tandem mass spectrometry (LC-MS / MS);

[0116] 2) The LC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard of the molecule to be measured, preferably β-NMN-d4 and β-NAD- 13 C5.

[0117] The detection method is 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 data. The signal is then analyzed based on the mass spectrum peak. It has the excellent separation ability of liquid chromatography and the high sensitivity and high selectivity of mass spectrometry.

[0118] When using LC-MS / MS, the internal standard method is used, and the internal standard is an isotope-labeled compound of the molecule to be tested. An isotope-labeled internal standard of known concentration is added to a certain amount of a standard reference substance and the sample to be tested. The concentration of the molecule to be tested in the sample 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:

[0119] F=(A s / m s ) / (A r / m r )

[0120] m i =(F*A i ) / (A s / m s )

[0121] 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 being measured in the internal standard, reference substance, and sample. This calculation is typically automated by the LC-MS / MS software.

[0122] During sample processing, samples are usually diluted or concentrated. The concentration of the analyte detected by LC-MS / MS and the dilution factor can be used to calculate the concentration of the analyte in the biological sample:

[0123] D=V a / V i

[0124] m a =m i *D

[0125] 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.

[0126] In some specific embodiments, the molecule to be detected is NAD+, and the internal standard is β-NAD- 13 C5. The calculation method for the content of NAD+ in biological samples is:

[0127] Dried blood spot sample testing: m sample =m report *(Vs +V SAS ) / V sample

[0128] Whole blood sample testing: m sample =m report *(V s +V SAS +V sample ) / V sample

[0129] 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.

[0130] In some specific embodiments, the biological sample is stored using the storage method described in Objective 2, and the biological sample is frozen at least once before testing. Failure to freeze-thaw can result in incomplete cell lysis and low NAD+ concentration.

[0131] In other specific embodiments, the detection uses the biological sample collection card described in purpose three, or the kit described in purpose five. Before the detection, a certain volume of biological sample is immersed in the biological sample collection card, for example, the biological sample is dropped on the biological sample collection card.

[0132] The collection card (sampled card) containing the collected biological sample can then be dried for easy storage and transportation to a testing site. The drying method may be, for example, natural drying at room temperature.

[0133] When testing is required, the sampled card can be eluted and the eluate can be tested. To facilitate testing, a solution containing an internal standard can be used as the eluent. Elution methods can include vibration, ultrasound, etc.

[0134] In some embodiments, the biological sample is a blood sample and / or a cell sample, wherein the blood sample is a whole blood sample. According to the disclosed method, a very small amount of blood can be collected for testing, such as 10 μL of blood from a fingertip. Quantitative capillary tubes can be used to quantitatively collect blood from the fingertip.

[0135] The seventh object of the present invention is to provide a NAD + A high-throughput detection method for a molecule or its precursor is described below.

[0136] Preparation of biological sample collection card: soak the test paper in an inorganic salt solution and then dry it to prepare the biological sample collection card.

[0137] In some embodiments, the inorganic salt is selected from sulfates, hydrochlorides, and phosphates, for example, 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 of any concentration and any pH.

[0138] The test paper can be a water-absorbing test paper 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.

[0139] Preparation of standard curve

[0140] Take different concentrations of NAD + Standard samples of the biological sample collection card or its precursor molecules are immersed in the biological sample collection card to obtain a standard sample card. For example, the standard sample is dropped onto the biological sample collection card and then dried. The drying method is, for example, natural drying at room temperature.

[0141] In some embodiments, the standard is a + or a human serum albumin (HSA) solution thereof.

[0142] The same area of ​​the standard sample card was eluted, and the eluate was detected using high-performance liquid chromatography tandem mass spectrometry (LC-MS / MS). The HPLC-MS technique uses a liquid chromatography system to separate the test substance, which is then connected to a mass spectrometer. The test substance is ionized and converted into an electrical signal. After computer data processing, analysis is performed based on mass spectrometry peaks. It combines the excellent separation capabilities of liquid chromatography with the highly sensitive and selective detection capabilities of mass spectrometry.

[0143] For example, a punch is used to remove a disc of equal size from the area containing the biological sample. When testing is required, the sampled card can be eluted and the eluate tested. To facilitate testing, a solution containing an internal standard can be used as the eluent. Elution methods can include vibration, ultrasound, and other methods.

[0144] 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.

[0145] Processing and testing of samples to be tested

[0146] Immerse the biological sample to be tested in the biological sample collection card to obtain the biological sample collection card. For example, drop the biological sample to be tested on the biological sample collection card and then dry it. The drying method is, for example, natural drying at room temperature.

[0147] The same area of ​​the sample card to be tested as that of the standard sample card is taken for elution, and the eluate is detected using high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). The detection method can be the same as the detection method for the standard sample card mentioned above.

[0148] Substitute the ion chromatogram peak area of ​​the sample to be tested into the standard curve to obtain the NAD in the sample to be tested. + or the concentration of its precursor molecules.

[0149] 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.

[0150] 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.

[0151] In some embodiments, the mobile phase flow rate of the HPLC is 0.1-0.5 mL / min, preferably 0.3 mL / min.

[0152] 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.

[0153] In some embodiments, the MS conditions are as follows: tandem mass spectrometer: Waters I-Class ultrahigh pressure liquid chromatograph; Sciex Triple Quad 4500 mass spectrometer; ion source: ESI (electrospray ionization source); spray voltage: 5500 V; spray temperature: 450-600° C.; scan mode: multiple reaction ion monitoring scan mode (MRM mode), and the parameters are as follows:

[0154] In some embodiments, the ion source used in the mass spectrometry is electrospray ionization (ESI), which is an ionization method. The electrospray ionization source is a soft ionization source that can generate multiply charged ions from large-mass organic molecules.

[0155] The detection method described in the present disclosure does not require volume quantification of the collected blood sample, and the blood test strip can maintain the NAD in the blood sample. + Long-term stability of NAD +The dry blood spot method for concentration detection has the characteristics of simple and fast operation, high accuracy, the ability to extract and process ten to dozens of dry blood spot samples at the same time, and is suitable for high-throughput sample preprocessing.

[0156] The present invention will be described below by way 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, conventional materials and methods in the art.

[0157] Example 1 Comparison of NAD in whole blood, plasma, and whole blood cells + concentration

[0158] The collected whole blood sample is subjected to plasma separation to obtain plasma and blood cells.

[0159] Add 850 μL of saturated ammonium sulfate solution and 50 μL of fresh sample to be tested (divided into 3 groups: whole blood / plasma / blood cells) to a 1.5 mL centrifuge tube, shake well, and freeze the EP tube in liquid nitrogen for 2 minutes;

[0160] Take out the frozen EP tube and place it in an ice box to melt. After melting, add 50 μL of internal standard solution (containing β-NMN-d4, β-NAD- 13 C5), shake evenly; place the EP tube in an ice water bath and sonicate for 10 minutes; immediately after sonication, place the EP tube in a centrifuge and centrifuge at 10,000 rpm for 3 minutes; take the supernatant after centrifugation, filter it, and place it in a sample bottle, and perform quantitative detection using LC-MS / MS method.

[0161] After testing whole blood, plasma, and blood cells (Figure 2), it was found that plasma contains almost no NMN / NAD. + The results of whole blood and blood cell tests are basically the same. Therefore, the following experiments all use whole blood samples for NMN / NAD + Detection.

[0162] Example 2 Screening of storage and pretreatment methods for whole blood samples

[0163] Pretreatment method 1: Freeze the collected samples in liquid nitrogen for 2 minutes, and immediately add 850 μL of saturated ammonium sulfate solution and 50 μL of internal standard solution (containing β-NMN-d4, β-NAD- 13 C5), after mixing, ultrasonication, centrifugation and filtration, the sample was injected for analysis of NMN / NAD + concentration;

[0164] Pretreatment method 2: The collected samples were frozen in liquid nitrogen for 2 minutes, and after thawing, they were divided into groups and placed in a refrigerator at room temperature and 4°C for 30 minutes respectively, and then 850 μL of saturated ammonium sulfate solution and 50 μL of internal standard solution (containing β-NMN-d4, β-NAD- 13 C5), after mixing, ultrasonication, centrifugation and filtration, the sample was injected for analysis of NMN / NAD + concentration;

[0165] Pretreatment method 3: Add 850 μL of saturated ammonium sulfate solution and 50 μL of internal standard solution (containing β-NMN-d4, β-NAD- 13 C5) Mix well, freeze in liquid nitrogen for 2 minutes, thaw and place at room temperature for 30 minutes, mix well, sonicate, centrifuge and filter, and then sample and analyze NMN / NAD + concentration;

[0166] Experiment 1 (Pretreatment Method 1 and Pretreatment Method 2): Comparison of the test results of samples without storage protectant added before freezing, immediately after freeze-thaw and after 30 minutes of storage, showed that NAD+ was completely decomposed after 30 minutes at room temperature and 4°C (Figure 3).

[0167] Experiment 2 (Pretreatment Method 1 and Pretreatment Method 3): Comparison of samples without storage protectant and samples with storage protectant before freezing. The results show that freezing does not affect NAD + The content determination (Figure 4) shows that saturated ammonium sulfate can maintain the stability of various metabolic small molecules in the blood.

[0168] Experiment 3: Comparison of NAD in whole blood samples at different storage times at 4°C + The results show (Figure 5) that the content of NAD+ changes irregularly over time regardless of whether a storage protective agent is added or not. + The content of NMN increases with time. The possible reason is that NMN will continue to transfer to NAD under 4℃. + Conversion, NAD in the blood after leaving the human body + Consumption pathways are reduced, resulting in NAD + The synthesis rate is greater than the consumption rate. Therefore, cryopreservation of biological samples is an important way to ensure the NAD + Necessary conditions for concentration detection.

[0169] Example 3 Verification of the storage effect of cryoprotectants

[0170] In the following experiments, storage protectants were added to biological samples before freezing:

[0171] 3.1 Short-term freezing: Add 4 times the volume of saturated ammonium sulfate solution to the collected samples, mix them and store them on dry ice and at -80℃ respectively. Melt and take samples at 0h, 24h and 48h, add internal standard solution (containing β-NMN-d4, β-NAD- 13 C5), after mixing, ultrasonication, centrifugation and filtration, the sample was injected for analysis of NMN / NAD + Concentration, detection of NMN / NAD + The concentration changes over time. The results are shown in Figure 6. The storage protectant can ensure the stability of whole blood samples stored on dry ice for 48 hours, enabling blood samples from other provinces and cities to be delivered for testing. The storage protectant can ensure the stability of whole blood samples stored at -80℃ for 48 hours, enabling local storage of samples.

[0172] 3.2 Long-term freezing: Add 4 times the volume of saturated ammonium sulfate solution to the collected samples, store at -80℃ and -20℃, thaw and take samples at 0 days, 1 day, 2 days, 3 days, and 7 days, add internal standard solution (containing β-NMN-d4, β-NAD- 13 C5), after mixing, ultrasonication, centrifugation and filtration, the sample was injected for analysis of NMN / NAD + Concentration, detection of NMN / NAD + The change of concentration over time. The results are shown in Figure 7. After using storage protectant and freezing, the NMN / NAD content in blood samples can be guaranteed. + The method showed excellent stability with a variation of less than 8% within 7 days.

[0173] 3.3 Freeze-thaw times: Add 4 times the volume of saturated ammonium sulfate solution to the collected samples, freeze at -80℃ and thaw at room temperature repeatedly within 24 hours, take samples after 0, 1, 3, and 5 freeze-thaw cycles, add internal standard solution (containing β-NMN-d4, β-NAD- 13 C5), after mixing, ultrasonication, centrifugation and filtration, the sample was injected for analysis of NMN / NAD + The results are shown in Figure 8. The blood samples treated with the storage protectant can withstand multiple freeze-thaw cycles without affecting the NMN / NAD content in the blood samples. + The test results must be frozen. Direct storage at 4°C (without freeze-thaw and direct testing) will result in incomplete cell lysis and NAD + The concentration is low.

[0174] 3.4 Stability test after whole blood sample pretreatment: add 4 times the volume of saturated ammonium sulfate solution to the sample to be tested, freeze at -80℃, thaw at room temperature, add internal standard solution (containing β-NMN-d4, β-NAD- 13 C5), after mixing, ultrasonication, centrifugation and filtration, the test solution was stored in a sample chamber at 10°C for 24 hours. The results are shown in Figure 9.+ The concentration remains unchanged and there is no deterioration, which can ensure that when a large number of samples are tested, the test results will not be affected by the long testing time.

[0175] In order to improve the convenience of detection, the inventors developed a dried blood spot method (quantitative blood collection method) based on the above method.

[0176] 1. Preparation and pretreatment of dried blood spots

[0177] 1.1 Preparation method of dried blood spots

[0178] Use a pipette to take 10 μL 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 a sealed bag and store it at room temperature for later use.

[0179] 1.2 Pretreatment of dried blood spot samples

[0180] Use a sampler to remove the complete dried blood spot filter paper, place it in a 2mL EP tube, and add 300μL of internal standard solution (B-NAD- 13 C5), shake evenly so that the internal standard solution immerses the dried blood spot filter paper; sonicate in an ice-water bath for 20 minutes; after sonication, add 1200 μL of saturated ammonium sulfate solution and shake evenly or sonicate for 1 minute; after sonication, place the EP tube in a centrifuge at 10,000 rpm for 3 minutes; take the supernatant, filter it, and place it in a sample bottle for later use.

[0181] 2. Pretreatment of whole blood samples

[0182] Add 1190 μL of saturated ammonium sulfate solution to a 1.5 mL centrifuge tube, add 10 μL of whole blood sample from the same donor, and shake evenly. Place the EP tube in liquid nitrogen and freeze for 2 minutes.

[0183] Take out the frozen EP tube and place it in an ice box to melt. After melting, add 300 μL of internal standard solution (β-NAD- 13 C5), shake evenly; put the EP tube into an ice water bath and sonicate for 10 minutes; immediately after sonication, put the EP tube into a centrifuge at 10000 rpm for 3 minutes, take the supernatant after centrifugation, filter it, and put it into a sample bottle for later use.

[0184] Example 4 Comparison of dried blood spots prepared with different test papers

[0185] PET test paper (Labconco Replacement Filter Paper 7544810), quantitative filter paper (Titan, rapid quantitative filter paper 7 cm), FTA test paper (Whatman Waterman FTA standard card) and 903 test paper (Whatman 903 protein storage card) were used to prepare dried blood spots from whole blood samples according to method 1.1. Sample pretreatment was performed according to method 1.2 and the samples were injected into LC-MS / MS to detect NAD in the samples. + The NAD+ content of whole blood samples from the same donor was processed according to method 2 and used as a reference.

[0186] After testing several dried blood spots (Figure 10), it was found that NAD + All of them have been degraded, and NAD in PET test paper + Content and NAD in fresh blood samples + The content is relatively close. The possible reason is that the thickness of PET filter paper is the smallest, the blood diffuses quickly, the drying time is short, and the NAD before the blood spot is dried is high. + The enzymatic hydrolysis time is shorter. Therefore, ensuring the stability of NAD+ before the dried blood spot is the key to the detection of NAD+ in the dried blood spot. + Necessary conditions for content.

[0187] Example 5 Comparison of biological sample collection cards after inorganic salt pretreatment

[0188] 5.1 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.

[0189] The blood samples were prepared into dried blood spots using the above-mentioned test paper according to method 1.1. After sample pretreatment according to method 1.2, the samples were injected into LC-MS / MS to detect NAD + The NAD+ content of whole blood samples from the same donor was processed according to method 2 and used as a reference.

[0190] After drying blood spots NAD + The results (Figure 11) showed that NAD + The content of NAD in dried blood spots made of treated 903 test paper and quantitative filter paper is close to that of fresh blood test results. + The content is significantly higher than that of untreated test paper. The results show that pretreatment with saline solution can effectively ensure the NAD + The stability of NAD + Preservation rate.

[0191] Example 6 Comparison of biological sample collection cards pretreated with different inorganic salt solutions

[0192] 6.1 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 in water, lay it flat and dry, and set aside.

[0193] Blood samples were prepared into dried blood spots using the above-mentioned 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 + The whole blood samples of the same donor were processed according to method 2, and the NAD + content as a reference.

[0194] Comparison of NAD in dried blood spots of biological sample collection cards treated with different salts + The results show that the biological sample collection cards pretreated with different salts can ensure the NAD content of dried blood spots to a certain extent. + Stability, among which ammonium sulfate, magnesium chloride and ammonium chloride have the best effects.

[0195] Example 7 Stability of Dried Blood Spots Prepared Using Biological Sample Collection Cards

[0196] PET test paper and 903 test paper were used to verify the stability of dried blood spots on biological sample collection cards.

[0197] 7.1 Preparation of biological sample collection card: Prepare 0.6 mol / L ammonium sulfate solution, soak the PET test paper, and lay it flat to dry for later use.

[0198] Blood samples were prepared into dried blood spots using the above-mentioned test strips according to method 1.1. Untreated PET 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 + The whole blood samples of the same donor were processed according to method 2, and the NAD + content as a reference.

[0199] After 14 days of testing on dried blood spots (Figure 13), the pre-treated biological sample collection card NAD + The content change rate in 14 days was less than 10%, and the NAD was detected by dry blood spot test on day 14. + The content is about 96.63% of the fresh blood test results, while the NAD+ content in the dried blood spots made from untreated PET test paper dropped to 84.62% after 7 days and to 73.79% after 14 days. The results show that the biological sample collection card pretreated with inorganic salt solution can effectively ensure the NAD+ content in the dried blood spots. +Stability, extending the storage time of dried blood spot samples at room temperature.

[0200] 7.2 Preparation of biological sample collection card: Prepare 0.6 mol / L ammonium sulfate solution, soak 903 test paper in it, and lay it flat to dry for later use.

[0201] Blood samples were prepared into dried blood spots using the above-mentioned 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 + The whole blood samples of the same donor were processed according to method 2, and the NAD + content as a reference.

[0202] After 7 days of testing on dried blood spots (Figure 14), it was found that the pre-treated biological sample collection card was stored at room temperature for 7 days. + No obvious degradation, which can ensure the NAD in dried blood spots + Stable for 7 days.

[0203] Example 8 Comparison of biological sample collection cards pretreated with salt solutions of different concentrations

[0204] 8.1 Preparation of biological sample collection cards: Prepare 0.2, 0.4, 0.6, and 1.2 mol / L ammonium sulfate solutions, respectively. Soak PET test paper in them, lay them flat and dry, and set aside.

[0205] Blood samples were prepared into dried blood spots using the above-mentioned test strips according to method 1.1. Untreated PET 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 + The whole blood samples of the same donor were processed according to method 2, and the NAD + content as a reference.

[0206] After testing the dried blood spots stored for 1 day and 7 days (Figure 15), it was found that the NAD of the dried blood spots prepared by the biological sample collection cards pretreated with different salt concentrations + The content is stable for 7 days, and pretreatment of the biological sample collection card with salt solutions of different concentrations can extend the storage time of the dried blood spot samples at room temperature.

[0207] Example 9 Comparison of biological sample collection cards pretreated with salt solutions of different pH values

[0208] 9.1 Preparation of biological sample collection cards: Prepare 0.6 mol / L ammonium sulfate solutions with pH values ​​of 2.5, 4.5, 6.5, 8.5, and 10.5, respectively. Soak PET test paper in these solutions, lay them flat and air dry for later use.

[0209] Blood samples were prepared into dried blood spots using the above-mentioned test strips according to method 1.1. Untreated PET 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 + The whole blood samples of the same donor were processed according to method 2, and the NAD + content as a reference.

[0210] After testing the dried blood spots stored for 1 day and 7 days (Figure 16), it was found that the NAD of the dried blood spots prepared by PET filter paper pretreated with salt solutions of different pH values ​​was + The content showed no obvious degradation for 7 days, which could extend the storage time of dried blood spot samples at room temperature.

[0211] In order to further improve the detection convenience of the LC-MS / MS method, the inventors further developed a high-throughput detection method based on the above method.

[0212] Example 10 LC-MS / MS High Throughput Detection

[0213] 10.1 Instrument detection settings:

[0214] Chromatographic column: Yuexu Xtimate C18 column, 2.1*50mm; particle size 1.8μm;

[0215] Mobile phase A: 1 mM ammonium formate solution containing 0.01% formic acid; mobile phase B: acetonitrile; column temperature: 25°C.

[0216] The 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.

[0217] Table 1 LC liquid phase pump gradient

[0218] Table 2 Mass spectrometry conditions

[0219] Table 3 Multiple reaction monitoring ion pairs (MRM) and corresponding voltage parameters

[0220] 10.2 Preparation of Dried Blood Spot Standards

[0221] Biological sample collection card: Prepare 1.2 mol / L ammonium sulfate solution, soak 903 test paper (Whatman 903 protein storage card) with it, then lay it flat and dry it for later use.

[0222] 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).

[0223] Preparation of β-NAD + 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:

[0224] 1) Blank matrix HSA solution: Weigh a certain amount of human serum albumin and dissolve it in normal saline to a concentration of 200 mg / mL;

[0225] 2) Prepare a blank matrix sample: Place 1 drop (approximately 20 μL) of HSA solution on the biological sample collection card;

[0226] 3) Preparation of standard dried blood spot S0: Weigh a certain amount of β-NAD + The standard was completely dissolved in the HSA solution in step 1) to adjust to a 10 μg / mL standard stock solution, designated as S0 solution. One drop (approximately 20 μL) was placed on the biological sample collection card.

[0227] 4) Prepare the dried blood spot standard S1: Accurately pipette 200 μL of S0 solution, add 200 μL of HSA solution, mix well, and record it as S1 solution. Place one drop (approximately 20 μL) on the biological sample collection card;

[0228] 5) Prepare the dried blood spot standard S2: Accurately pipette 200 μL of S1 solution, add 200 μL of HSA solution, mix well, record as S2 solution, and place one drop (approximately 20 μL) on the biological sample collection card;

[0229] 6) Prepare the dried blood spot standard S3: Accurately pipette 200 μL of S2 solution, add 200 μL of HSA solution, mix well, and record it as S3 solution. Place one drop (approximately 20 μL) on the biological sample collection card;

[0230] 7) Prepare the dried blood spot standard S4: Accurately pipette 200 μL of S3 solution, add 50 μL of HSA solution, mix well, record as S4 solution, and place one drop (approximately 20 μL) on the biological sample collection card;

[0231] 8) Prepare the dried blood spot standard S5: Accurately pipette 200 μL of S4 solution, add 200 μL of HSA solution, mix well, record as S5 solution, and place one drop (about 20 μL) on the biological sample collection card;

[0232] 9) Prepare the dried blood spot standard S6: Accurately pipette 200 μL of S5 solution, add 200 μL of HSA solution, mix well, record as S6 solution, and place one drop (about 20 μL) on the biological sample collection card;

[0233] 10) Prepare the dried blood spot standard S7: Accurately pipette 100 μL of S6 solution, add 150 μL of HSA solution, mix thoroughly, and record it as S7 solution. Place one drop (approximately 20 μL) of this solution on the biological sample collection card.

[0234] 11) Prepare the dried blood spot standard S8: Accurately pipette 200 μL of S7 solution, add 200 μL of HSA solution, mix well, record as S8 solution, and place one drop (about 20 μL) on the biological sample collection card;

[0235] 12) Prepare the dried blood spot standard S9: Accurately pipette 200 μL of S8 solution, add 200 μL of HSA solution, mix well, and record it as S9 solution. Place one drop (approximately 20 μL) on the biological sample collection card;

[0236] 13) Prepare the dried blood spot standard S10: Accurately pipette 100 μL of S9 solution, add 900 μL of HSA solution, mix well, record as S10 solution, and place one drop (about 20 μL) on the biological sample collection card;

[0237] Place the above-mentioned standard dried blood spots in a clean, dry and ventilated environment and air-dry for 2-4 hours, then put them into a sealed bag and store them at room temperature away from light.

[0238] Table 4 β-NAD corresponding to standard dried blood spots + concentration

[0239] 10.3 Preparation of internal standard

[0240] Weigh an appropriate amount of β-NAD + - 13 Dissolve pure C5 to a volume of 500 ng / mL.

[0241] 10.4 Preparation of Whole Blood Dried Spot Samples

[0242] 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 environment to air dry for 2-4 hours, then put it into a sealed bag and store it at room temperature away from light.

[0243] 10.5 Sample Pretreatment

[0244] ① Sampling: For dried blood spot samples from S0-S10, use a hole punch to take two 3mm diameter dried blood smears and add them into a 1.5mL centrifuge tube;

[0245] ② Elution: Add 100 μL 500 ng / mL β-NAD + - 13 C5 internal standard solution was ultrasonicated in an ice-water bath for 15 minutes, 400 μL of saturated ammonium sulfate solution was added and ultrasonicated for 5 minutes, and the supernatant was collected after centrifugation or filtered and placed in a sample bottle for detection in a high performance liquid chromatography tandem mass spectrometry system.

[0246] Example 11 Detection of B-NAD in Dried Blood Spot Samples + Exclusivity

[0247] Take blank matrix dried blood spots and S8 standard dried blood spots respectively and perform the assay according to the sample pretreatment described in 10.5 and the LC-MS / MS assay conditions described in 10.1. + and β-NAD + - 13 The retention time of C5 was 2.58 min (see Figures 17 and 18). + There were no obvious interfering peaks at the retention time.

[0248] Example 12 Detection of β-NAD in Dried Blood Spot Samples + Linear relationship and detection limit

[0249] Take dried blood spot samples of the standard prepared in 10.3 at different concentrations and perform the assay according to the sample pretreatment described in 10.5 and the LC-MS / MS assay conditions described in 10.1. Plot the quantitative ion chromatogram peak area versus concentration to obtain a standard curve (Y = 0.00097x + (-0.00439), see Figure 19).

[0250] The results showed that the linear range and limit of quantification of β-NAD+ in dried blood spots are as follows:

[0251] 1) β-NAD + Limit of detection (LOD): 25 ng / mL (see Figure 20)

[0252] 2) β-NAD + Limit of quantification (LOQ): 500 ng / mL

[0253] 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.

[0254] Example 13 Detection of NAD in Dried Blood Spot Samples + The recovery and precision

[0255] Dried blood spots from the same whole blood source were tested according to the methods described in 10.4 and 10.5 and the LC-MS / MS assay conditions described in 10.1. The test was repeated for 6 batches, and the precision was 5.58%.

[0256] Take dried blood spots from the same whole blood source and add three concentrations of high, medium and low to perform sample recovery and precision experiments. According to the methods described in 10.4 and 10.5 and the LC-MS / MS determination conditions described in 10.1, repeat the test for three batches. + The recovery rates are shown in the table below:

[0257] Table 4

[0258] Example 14 NAD in dried blood spot samples + Stability

[0259] NAD in dried blood spots + The stability of the dried blood spot sample is to put it into a sealed bag after drying, store it at room temperature under non-light conditions, and detect NAD in the dried blood spot within 14 days. + Repeatability of content.

[0260] Dried blood spots from the same whole blood source were collected at different storage times and tested according to the methods described in 10.4 and 10.5 and the LC-MS / MS assay conditions described in 10.1. Six batches were tested at each storage time. The NAD concentration in the 14-day dried blood spots was 0. + The change rate is less than 10% (see Figure 21), which ensures that the samples can be stored at room temperature for 14 days before testing without the need for low-temperature storage.

[0261] Example 15 Comparison of high-throughput detection method and quantitative blood sampling method

[0262] 31 volunteers were recruited and sampled using both the quantitative blood sampling method and the high-throughput detection method. The test results were cross-referenced. The results are shown in Figures 22 and 23. All numerical differences were within the range of ±20% of the mean value of the two methods (Figure 22). Combining the data of all volunteers using the quantitative blood sampling method and the high-throughput detection method, the two methods for detecting NAD + The results of the content were consistent (Figure 23).

[0263] In summary, the present invention first provides a storage protectant for biological samples. ① Biological samples containing the storage protectant can be stored for more than 7 days, meeting the needs of long-distance transportation of samples; ② Biological samples containing the storage protectant can be frozen and thawed multiple times without affecting the experimental results; ③ Biological samples containing the storage protectant can also be stored for more than 24 hours after freezing and thawing, meeting the actual needs of sample testing. On this basis, the invention continues to provide a biological sample collection card, which can store dried blood spot samples at room temperature for 14 days and maintain NAD +Stable, meeting the needs of long-distance sample transportation and actual sample detection needs. Finally, the present invention also provides a high-throughput detection method for biological samples, which can accurately detect NAD in blood samples by quantitative ion chromatography peak area and concentration. + The method is flexible and convenient, and is easy for users to operate. The subject to be tested does not need to use professional tools such as a pipette, and only needs to provide a drop of blood to prepare the test sample, which greatly improves the convenience of the blood test method.

Claims

1. A biological sample storage protectant, characterized in that: The storage protective agent at least contains an inorganic salt solution.

2. The storage protective agent according to claim 1, wherein The inorganic salt is selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate.

3. The storage protective agent according to claim 1 or 2, characterized in that: The biological sample contains metabolic small molecules, preferably NAD + or its precursor molecule, 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, more preferably nicotinamide mononucleotide (NMN).

4. The storage protective agent according to any one of claims 1 to 3, characterized in that: The biological sample is a blood sample and / or a cell sample. Preferably, the blood sample is a whole blood sample.

5. The storage protective agent according to any one of claims 1 to 4, characterized in that: The inorganic salt solution is a saturated inorganic salt solution.

6. The storage protective agent according to any one of claims 1 to 5, characterized in that: The volume ratio of the storage protectant to the biological sample is ≥3:

1.

7. A method for storing biological samples, characterized in that: The storage method uses the storage protective agent according to any one of claims 1 to 6.

8. The storage method according to claim 7, wherein: The storage method is low-temperature storage, and the low temperature is ≤0°C, preferably ≤-20°C, more preferably ≤-78°C, and even more preferably ≤-80°C.

9. A biological sample collection card, characterized in that: The biological sample collection card is a test paper pretreated with the storage protective agent according to any one of claims 1 to 4.

10. The biological sample collection card according to claim 9, wherein: The pretreatment refers to soaking the test paper in a storage protective agent and then drying it.

11. The biological sample collection card according to claim 9 or 10, wherein: The test paper is a water-absorbent test paper.

12. The biological sample collection card according to claim 11, wherein: The water-absorbent test paper is selected from any one of PET test paper, quantitative filter paper, FTA test paper or 903 test paper.

13. Use of the biological sample collection card according to any one of claims 9 to 12 in the collection, transportation, analysis, identification and / or storage of biological samples.

14. A kit for biological sample detection, characterized in that: The kit contains the biological sample collection card according to any one of claims 9 to 12.

15. A method for detecting a biological sample, characterized in that: Have any one or more of the following characteristics: 1) The biological sample is stored using the storage method according to claim 7 or 8; 2) Detection using the biological sample collection card according to any one of claims 9 to 12; 3) The test kit according to claim 14 is used for detection.

16. The detection method according to claim 15, wherein The detection method has any one or more of the following characteristics: 1) The detection method is high performance liquid chromatography tandem mass spectrometry (LC-MS / MS); 2) The LC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard of the molecule to be measured, preferably β-NMN-d4 and β-NAD- 13 C5.

17. The detection method according to claim 16, wherein The biological sample is stored using the storage method according to claim 7 or 8, and the biological sample is frozen at least once before detection.

18. The detection method according to claim 16, wherein The detection uses the biological sample collection card according to any one of claims 9 to 12, or the kit according to claim 14, and the detection steps include: 1) Immersing a certain volume of biological sample into the biological sample collection card and then drying it to obtain a sampled card; 2) Elute the sampled card and test the eluate.

19. A type of NAD + or a high-throughput detection method for a precursor molecule thereof, characterized in that The steps include: 1) Take samples containing different concentrations of NAD + or a standard sample of its precursor molecule and a sample to be tested are immersed in the biological sample collection card according to any one of claims 9 to 12 to obtain a sampled card; 2) taking the same area of the sampled card from step 2) for elution, and detecting the eluate using high performance liquid chromatography tandem mass spectrometry (LC-MS / MS); 3) Establish the relationship between ion chromatographic peak area and NAD using standard + or a standard curve of the concentration of a precursor molecule thereof; 4) 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.

20. The high-throughput detection method according to claim 19, wherein Step 2) The standard contains NAD + or a human serum albumin (HSA) solution thereof.

21. The high-throughput detection method according to claim 19 or 20, wherein: Step 3) The LC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard of the molecule to be measured, preferably β-NAD- 13 C5.

22. The high-throughput detection method according to any one of claims 19 to 21, wherein: The mobile phase A of the high performance liquid chromatography is ammonium formate solution, and the mobile phase B is acetonitrile. Preferably, the mobile phase A is ammonium formate solution containing 0.01% formic acid.

23. The high-throughput detection method according to any one of claims 19 to 22, wherein: The mobile phase flow rate of HPLC is 0.1-0.5 mL / min, preferably 0.3 mL / min. The high-throughput detection method according to any one of claims 19 to 23, wherein: The ion source used in the mass spectrometry was electrospray ionization (ESI).

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