Preparation method of blank blood matrix and standard substance, and detection method of vitamin

Abstract

The present invention provides a method for preparing a blank blood matrix and a standard product, and a method for detecting vitamins. The method for preparing the blank blood matrix comprises: obtaining anticoagulated whole blood from a healthy person, and separating red blood cells; diluting the red blood cells, freezing, and crushing the red blood cells; removing compounds bound to the crushed red blood cells in the solution; and adding serum albumin to prepare the blank blood matrix. The present invention has the advantages of low background value, high preparation efficiency, and the like.

Description

Technical Field

[0001] The invention relates to vitamin detection, and in particular to a preparation method of a blank blood matrix and a standard product, and a vitamin detection method. Background Art

[0002] Vitamin A and vitamin E are two important fat-soluble vitamins (FSVs) and are basic nutrients required by the human body. They are widely present in various foods and are essential substances for maintaining normal metabolism and function of the body. They are closely related to animal growth, development, reproduction and immune function.

[0003] Dried blood spot sampling technology (DBS) is to collect peripheral whole blood on filter paper. Compared with serum, it has many advantages such as convenient storage and transportation, low professional and technical requirements for operators, and minimal invasiveness. It is very suitable for blood sample collection for vitamin nutritional status assessment of a large population, especially for remote areas where there is no local vitamin quantitative detection equipment and specimens need to be transported.

[0004] For the detection of vitamin A and vitamin E, some technical solutions are currently provided in the technology, such as:

[0005] 1. Patent CN 108195984 A provides a method and system for detecting multiple vitamins in dried blood spots. It also uses dried blood spot technology and claims to solve the time-consuming and labor-intensive technical defects of current methods for detecting multiple vitamins through a two-step pretreatment process (including extraction). However, the solution does not involve any research on stability.

[0006] 2. Patent CN 110174476 A provides a method for detecting multiple fat-soluble vitamins in dried blood smears by liquid chromatography-tandem mass spectrometry; this solution only provides a detection method based on dried blood smears and does not involve stability research.

[0007] 3. Patent CN 111983244 A proposes a detection method and detection kit for four vitamins in dried blood spots, which can provide calibrators, reagents involved in sample pretreatment and quality control products; this solution also only provides a detection method based on dried blood spots and does not involve stability research.

[0008] In summary, the prior art has the following deficiencies:

[0009] 1. The existing routine detection methods for vitamins A and E use serum samples, and some samples used for testing are peripheral blood samples. The transportation of these serum and whole blood samples requires cold chain transportation and the sample storage time is short, which makes it impossible to meet the sample testing needs of special populations such as infants and young children and remote areas.

[0010] 2. The amount of serum samples used is mostly 100-500 microliters. In addition to the need for retesting, the required amount of venous blood samples makes it difficult to collect blood from infants and other groups.

[0011] 3. The serum sample is subjected to organic solvent protein precipitation, followed by liquid-liquid extraction with weakly polar organic solvents such as n-hexane / tert-butyl methyl ether / ethyl acetate, and then concentrated and redissolved with nitrogen blown before being tested on a machine. This detection method is very time-consuming and labor-intensive.

[0012] 4. Vitamins A and E have poor stability, and their stability in dried blood spot samples is even worse than in solution states such as serum.

[0013] 5. Because sample matrices such as serum and red blood cells contain background values ​​of vitamins A and E, a perfect blank matrix cannot be obtained during mass spectrometry detection; in routine serum sample testing, the matrices of calibrators and quality control materials are surrogate matrices such as bovine serum albumin or human serum albumin phosphate buffer solution; there are large differences between the surrogate matrix and the sample matrix, which may lead to deviations in the test results.

[0014] Patent CN 112014186 A provides a method for more completely removing the substance to be tested from the blood sample to obtain a cleaner blank blood matrix sample. However, this solution is time-consuming, labor-intensive and expensive, and it cannot solve the problem of background values ​​of vitamins A and E. Summary of the invention

[0015] In order to solve the deficiencies in the above-mentioned prior art solutions, the present invention provides a method for preparing a blank blood matrix.

[0016] The objective of the present invention is achieved through the following technical solutions:

[0017] The preparation method of blank blood matrix is ​​as follows:

[0018] Anticoagulated whole blood from healthy individuals was obtained and red blood cells were separated;

[0019] diluting the red blood cells, freezing, and disrupting the red blood cells;

[0020] Removes compounds bound to broken red blood cells from the solution;

[0021] Serum albumin was added to prepare blank blood matrix.

[0022] Another object of the present invention is to provide a method for preparing a standard product using the above-mentioned blank blood matrix preparation method. This object of the invention is achieved through the following technical solutions:

[0023] The preparation method of the standard product is as follows:

[0024] Add a certain amount of blank blood matrix as described in the present patent application, and different concentrations of vitamin A and vitamin E to prepare different standard solutions;

[0025] The standard solution was added to the filter paper and dried in the dark to obtain the standard.

[0026] Another object of the present invention is to provide a standard product, and this object of the invention is achieved by the following technical solutions:

[0027] The standard product is obtained by the method for preparing the standard product of this patent application.

[0028] Another object of the present invention is to provide a method for detecting vitamins, which is achieved by the following technical solutions:

[0029] A method for detecting vitamin content, the method comprising a sampling stage and a detection stage;

[0030] In the sampling stage, whole blood of the person to be tested is collected and added to the sample position of the filter paper, and dried in the dark;

[0031] In the detection stage, the sample position is cut to obtain a sample;

[0032] The content of vitamin A or vitamin E in the blood of the person to be tested is obtained by using the sample and the standard product of the present patent application.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. Low background value;

[0035] The blank blood matrix prepared by this patent has a vitamin A content of less than 50ng / mL and a vitamin E content of less than 100ng / mL, which significantly reduces the background value when detecting vitamin A and vitamin E.

[0036] 2. Good stability;

[0037] The preparation of the standard is kept dry and protected from light, and the handling of the sample during the sampling phase improves stability and facilitates transportation;

[0038] 3. High detection efficiency and good accuracy;

[0039] In vitamin testing, sample processing during the sampling phase and the use of low-background standard substances during the testing phase improve testing efficiency and accuracy;

[0040] 4. Low blood collection volume;

[0041] For vitamin measurement, only 20 μL of blood is required, which significantly reduces the amount of blood collected. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The disclosure of the present invention will become easier to understand with reference to the accompanying drawings. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. In the drawings:

[0043] Figure 1 is a schematic flow chart of a method for preparing a blank blood matrix according to an embodiment of the present invention;

[0044] Figure 2 is a schematic flow chart of a method for preparing a standard product according to an embodiment of the present invention;

[0045] Figure 3 is a schematic flow chart of a method for detecting vitamins according to an embodiment of the present invention;

[0046] Figure 4 Schematic diagram of the structure of a cutting device according to an embodiment of the present invention. DETAILED DESCRIPTION

[0047] Figure 1-4 The following description describes the optional embodiments of the present invention to teach those skilled in the art how to implement and reproduce the present invention. In order to teach the technical solution of the present invention, some conventional aspects have been simplified or omitted. It should be understood by those skilled in the art that variations or substitutions derived from these embodiments will be within the scope of the present invention. It should be understood by those skilled in the art that the following features can be combined in various ways to form multiple variations of the present invention. Thus, the present invention is not limited to the following optional embodiments, but is only limited by the claims and their equivalents.

[0048] Embodiment 1:

[0049] Figure 1 A schematic flow chart of a method for preparing a blank blood matrix according to an embodiment of the present invention is provided. Figure 1 As shown, the preparation method of the blank blood matrix is:

[0050] Anticoagulated whole blood from healthy individuals was obtained and red blood cells were separated;

[0051] diluting the red blood cells, freezing, and disrupting the red blood cells;

[0052] Removes compounds bound to broken red blood cells from the solution;

[0053] A blank blood matrix is ​​prepared by adding serum albumin, such as human serum albumin or bovine serum albumin.

[0054] To separate the red blood cells, further, the red blood cells are separated by:

[0055] The anticoagulated whole blood is centrifuged to remove plasma and obtain red blood cells;

[0056] The red blood cells are washed with saline and the saline is removed to separate the red blood cells.

[0057] In order to remove the test compound bound to the broken red blood cells, further, the method of removing the compound bound to the broken red blood cells in the solution is:

[0058] An adsorbent, such as activated carbon, is added to the solution and mixed;

[0059] Centrifuge to obtain red blood cell solution.

[0060] Figure 2 A schematic diagram of the preparation method of the standard sample of the present invention is given, as shown in FIG. Figure 2 As shown, the preparation method of the standard is:

[0061] A certain amount of blank blood matrix of this embodiment and different concentrations of vitamin A and vitamin E are added to prepare different standard solutions;

[0062] Add the standard solution to the filter paper and dry it in the dark to obtain a standard, such as a calibrator or quality control.

[0063] In order to improve the stability of the standard, the filter paper is further processed, and the processing method is as follows:

[0064] Add stabilizer to the blood spot on the blank filter paper and dry it away from light.

[0065] In order to improve stability, the standard product is further packed and sealed.

[0066] The standard product of the embodiment of the present invention is the standard product obtained by the preparation method of the standard product of the embodiment of the present invention.

[0067] Figure 3 A schematic diagram of the structure of the method for detecting the vitamin content of an embodiment of the present invention is given, as shown in FIG. Figure 3 As shown, the method for detecting the vitamin content includes:

[0068] During the sampling phase, the whole blood of the person to be tested is collected and added to the sample position of the filter paper, and then dried away from light;

[0069] In the detection stage, the sample position is cut to obtain a sample;

[0070] The content of vitamin A or vitamin E in the blood of the test subject is obtained by using the sample and the standard product of this embodiment.

[0071] In order to improve cutting efficiency and accuracy, further, as Figure 4As shown, the cutting device comprises:

[0072] A container 11, such as an EP tube, for containing a sample cut from a sample carrier 31;

[0073] A sampling unit, comprising:

[0074] A stopper 20, which is disposed on the upper side of the container 11 and has a channel 21 for allowing the sample carrier 31 to pass through; a through hole 22 is disposed on the lower wall of the channel 21;

[0075] A cutting member 44 and a rotating arm 41 , wherein the cutting member 44 is disposed on the rotating arm 41 , and when the rotating arm 41 rotates, the cutting member 44 enters the through hole 22 .

[0076] In order to improve the sampling efficiency, the sampling unit further includes:

[0077] The elastic member 42 is disposed between the rotating arm 41 and the limiting member 20 , and is used to hinder the rotation of the rotating arm 41 .

[0078] In order to improve the sampling efficiency, further, the through hole 22 and the cutting member 44 are both multiple, and when the rotating arm 41 rotates, the multiple cutting members 44 enter the through hole 22 respectively.

[0079] In order to improve the sampling efficiency, further, a plurality of through holes 22 are arranged in a matrix on the limiting member 20 , and a plurality of cutting members 44 are arranged in a matrix on the rotating arm 41 .

[0080] In order to improve the sampling efficiency, further, the end of the cutting piece 44 is annular and has a conical groove 45 .

[0081] For the convenience of maintenance, further, the cutting member 44 is fixed in the threaded hole 43 of the rotating arm 41 by means of threads.

[0082] In order to improve sampling accuracy, further, in a direction perpendicular to the moving direction of the sample carrier 31 in the channel 21 , the length of the channel 21 is the same as the length of the sample carrier 31 .

[0083] In order to improve the sampling accuracy, further, in a direction perpendicular to the moving direction of the sample carrier 31 in the channel 21 , the positions of the through holes 22 correspond one-to-one to the sample positions 32 on the sample carrier 31 .

[0084] Embodiment 2:

[0085] This is an example of application of the blank blood matrix and standard preparation method and vitamin detection method according to Example 1 of the present invention in whole blood detection.

[0086] In this application example, Figure 1 A schematic diagram of the structure of the method for preparing a blank blood matrix according to an embodiment of the present invention is given. Figure 1 As shown, the preparation method of the blank blood matrix is:

[0087] First, centrifuge human red blood cells (4000 rpm, 5 min) and discard the upper liquid; redisperse with an equal amount of physiological saline; shake upside down to mix; centrifuge again (4000 rpm, 5 min) and discard the upper liquid, and repeat the washing process three times;

[0088] After washing, add an equal amount of 0.1% formic acid saline to the red blood cells, mix well, and place in a -20°C refrigerator for 2 hours. After taking out the hemolyzed red blood cells and returning them to room temperature, add 50 mg / mL activated carbon and vortex mix at 2500 rpm for 10 minutes; then centrifuge at 14000 rpm for 5 minutes to remove the compounds bound to the broken red blood cells; transfer the upper red blood cell solution;

[0089] The treated adsorbed red blood cell solution was added with human serum albumin at a mass concentration ratio of 5%, and the mixture was dissolved and mixed evenly to obtain a blank whole blood matrix.

[0090] The removal optimization effect is shown in the following table:

[0091]

[0092] Figure 2 A schematic diagram of the preparation method of the standard sample of the present invention is given, as shown in FIG. Figure 2 As shown, the preparation method of the standard is:

[0093] Calibrator intermediate solution matrix (10 mg / mL BHT methanol solution), weigh 1 g BHT and dissolve it in 200 mL methanol, mix well, and store at 2-8°C;

[0094] Preparation of calibrator intermediate solution: Use a 100μL pipette to transfer 50μL of 2mg / mL VA standard solution and 10μL of 20mg / mL VE standard solution into 1.5mL centrifuge tubes respectively; use a 1mL pipette to transfer 940μL of calibrator intermediate solution matrix (10mg / mL BHT methanol solution) into the above 1.5mL centrifuge tubes; use a multi-well plate vortex mixer at 700rpm to vortex mix for 5min to obtain working solution intermediate solution J1; the remaining concentrations were diluted in sequence according to the method in the table below.

[0095]

[0096] Preparation of whole blood matrix calibrators

[0097] Take enough 2 mL centrifuge tubes, number them, pipette the calibrator intermediate solution according to the preparation ratio in the table below, and dilute it with freshly prepared whole blood matrix to obtain the matrix calibrator solution.

[0098] Concentration level Pipetting Whole blood matrix volume Vitamin A (μg / mL) Vitamin E (μg / mL) J1 Intermediate solution J1 10μL 190μL 5 10 J2 Intermediate solution J2 10μL 190μL 2.5 5 J3 Intermediate solution J3 10μL 190μL 1 2 J4 Intermediate solution J4 10μL 190μL 0.5 1 J5 Intermediate solution J5 10μL 190μL 0.25 0.5 J6 Intermediate solution J6 10μL 190μL 0.1 0.2 J7 Intermediate solution J7 10μL 190μL 0.05 0.1

[0099] Preparation of filter paper dried blood spot calibrator:

[0100] Take the filter paper treated with antioxidant; use a 100 μL pipette to drop the whole blood matrix calibrant solution onto the treated filter paper, dry naturally for 2 hours away from light; seal the aluminum foil bag and store at 2-8°C; add silica gel deoxidizer and dehumidifier to the bag and vacuum pack it.

[0101] The filter paper was treated as follows: 60 μl of antioxidant (0.05% ascorbyl palmitate + 0.5% 2,6-di-tert-butyl-p-cresol ethanol solution) was added to each filter paper identification circle using a micropipette and dried in the dark for two hours;

[0102] The preparation of quality control products refers to the preparation of the above-mentioned calibration products.

[0103] Figure 3 A schematic diagram of the structure of the vitamin detection method of the embodiment of the present invention is given, as shown in FIG. Figure 2 As shown, the detection method of the vitamin is:

[0104] Sampling phase:

[0105] Massage or apply hot compress to the heel / fingertips for 3-5 minutes to increase blood flow. Disinfect the heel / fingertips with alcohol. After air drying, puncture the heel / fingertips with a disposable blood collection needle; wipe off the first drop of blood.

[0106] Use a quantitative capillary tube to collect and drop 50 μL of blood (minimum 20 μL) in the marked circle on the filter paper.

[0107] After blood collection, the filter paper is dried naturally in the air away from light for at least two hours to prevent the blood from coming into contact with other surfaces and being lost when it penetrates the filter paper. The dried blood is placed in a clean aluminum foil bag and vacuumed for storage. Antioxidants and desiccant are added to the bag.

[0108] Detection phase:

[0109] Preparation of sample extract

[0110] 600 ml of methanol, 150 ml of ultrapure water, 150 ml of acetonitrile and 100 ml of isopropanol were measured respectively, and 500 μl of formic acid was added to prepare a sample extraction solution containing 0.05% formic acid, 60% methanol, 15% acetonitrile, 15% water and 10% isopropanol.

[0111] Preparation of sample extract containing internal standard

[0112] Use a 100μL pipette to transfer 50μL of 1mg / mL vitamin A-d8 standard solution and 50μL of 3mg / mL vitamin E-d6 standard solution into a 10mL volumetric flask; use the above sample extract to make up the volume, shake well, and obtain the internal standard working solution.

[0113] Take the calibrator, quality control and sample dried blood spots; use the cutting device to make a 6mm blood spot into a 2mL centrifuge tube.

[0114] Use a 200 μL pipette to add 200 μL of sample extract containing isotope internal standard, and use a multi-tube vortex mixer to vortex mix at 2500 rpm for 20 min.

[0115] The above solution was centrifuged at 13000r / min for 5min, and 150μL of the supernatant was transferred to a 96-well plate (1mL). It was then detected using the liquid chromatography tandem mass spectrometry system of the present application to obtain the content of vitamin A and vitamin E. The specific detection method is the existing technology.

[0116] like Figure 4 As shown, in the cutting device, the sample carrier 31 is a dry blood smear, such as a sampled blood smear, a calibration product, and a quality control product, and has three parallel blood spots (i.e., sample positions 32); correspondingly, the lower wall of the channel 21 has three through holes 22 (the channel 21 of this part has no upper wall), and in a direction perpendicular to the moving direction of the sample carrier 31 in the channel 21, the positions of the through holes 22 correspond to the sample positions 32 on the sample carrier 31 one by one, and the length of the channel 21 is the same as the length of the sample carrier 31;

[0117] The rotating arm 41 has three threaded holes 43 , in which the cutting member 44 is fixed; the end of the cutting member 44 is a conical groove 45 , thereby forming an annular cutting portion; the elastic member 42 is a torsion spring, and is arranged between the limit member 20 and the rotating arm 41 .

[0118] The working mode of the sampling device of this embodiment is:

[0119] The dried blood smear is pushed to move in the channel 21, and the dried blood smear has no displacement in the direction perpendicular to the moving direction;

[0120] When the blood spots are observed to be on the upper side of the through holes 22, the movement is stopped, the rotating arm 41 is driven forward, the cutting members 44 enter the through holes 22 respectively, parts of the three blood spots are cut off and enter the container; the rotating arm 41 is released, and the rotating arm 41 automatically returns to its original position in the reverse direction under the action of the elastic member 42.

[0121] Embodiment 3:

[0122] The application example of the vitamin detection method according to Example 1 of the present invention is different from that of Example 2 in that:

[0123] In the cutting device, the dry blood film has six blood spots (i.e., sample positions) distributed in a 2*3 matrix; correspondingly, the lower wall and the upper wall of the channel respectively have three through holes (the upper wall is made of transparent material); in the cutting state, in the direction perpendicular to the moving direction of the sample carrier in the channel, the positions of each through hole respectively correspond to the sample positions on the sample carrier, so that when cutting, the cutting member passes through the through holes of the upper wall and the lower wall in turn, and the sampling of the three sample positions is completed in one cutting, and the sampling of the six blood spots on the dry blood film is completed in two cuttings.

[0124] Embodiment 4:

[0125] The application example of the vitamin detection method according to Example 1 of the present invention is different from that of Example 2 in that:

[0126] In the cutting device, the dry blood film has six blood spots (i.e., sample positions) distributed in a 2*3 matrix; correspondingly, the lower wall and the upper wall of the channel respectively have six through holes distributed in a 2*3 matrix (the upper wall is made of transparent material). In the direction perpendicular to the moving direction of the sample carrier in the channel, the position of each through hole corresponds one by one to the sample position on the sample carrier, so that when cutting, the cutting member passes through the through holes of the upper wall and the lower wall in turn, and completes the sampling of 6 blood spots at one time.

Claims

1. A method for detecting vitamin content, comprising a sampling stage and a detection stage; characterized in that: In the sampling stage, whole blood of the person to be tested is collected and added to the sample position of the filter paper, and dried in the dark to obtain a sample dried blood film, wherein the sample position is a marking circle; In the detection stage, a cutting device is used to cut the sample site, standard and quality control product of the sample dried blood smear into a centrifuge tube, and a 200 μL pipette is used to add 200 μL of the sample extract containing the isotope internal standard respectively, and a multi-tube vortex mixer is used to vortex and mix at 2500 rpm for 20 minutes, and the above solution is centrifuged at 13000 r / min for 5 minutes, and 150 μL of the supernatant is transferred to a 96-well plate, and then detected by a liquid chromatography tandem mass spectrometry system to obtain the content of vitamin A and vitamin E; The sample extract containing the internal standard is prepared as follows: 600 ml of methanol, 150 ml of ultrapure water, 150 ml of acetonitrile and 100 ml of isopropanol are measured respectively, and then 500 microliters of formic acid are added to prepare a sample extract containing 0.05% formic acid, 60% methanol, 15% acetonitrile, 15% water and 10% isopropanol; 50 μL of 1 mg / mL vitamin A-d8 standard solution and 50 μL of 3 mg / mL vitamin E-d6 standard solution are respectively transferred to a 10 mL volumetric flask using a 100 μL pipette; the sample extract is made up to volume with the above sample extract, and shaken well to obtain a sample extract containing the internal standard; The preparation method of the standard is as follows: adding a certain amount of blank blood matrix and different concentrations of vitamin A and vitamin E to prepare different standard solutions; Add the standard solution to the filter paper and dry it away from light to obtain the standard product; The preparation method of the blank blood matrix: Obtain anticoagulated whole blood from a healthy person, centrifuge the anticoagulated whole blood, remove the plasma, and separate the red blood cells; Take human red blood cells and centrifuge to discard the upper layer of liquid, the centrifugal speed is 4000rpm, and the centrifugal time is 5min; use an equal amount of physiological saline to disperse again; shake upside down to mix; centrifuge again to discard the upper layer of liquid, the centrifugal speed is 4000rpm, and the centrifugal time is 5min; repeat the washing process three times; After washing, add an equal amount of 0.1% formic acid saline to the red blood cells, mix well, and place in a -20℃ refrigerator for 2 hours. After taking out the hemolyzed red blood cells and returning to room temperature, add 50 mg / mL activated carbon and vortex mix at 2500 rpm for 10 minutes. Then centrifuge at 14000 rpm for 5 minutes to remove the compounds bound to the broken red blood cells. Transfer the upper red blood cell solution. The treated adsorbed red blood cell solution was added with human serum albumin at a mass concentration ratio of 5%, and the mixture was dissolved and mixed evenly to obtain a blank whole blood matrix; The cutting method is: the dried blood piece is pushed to move in the channel, and the dried blood piece has no displacement in the direction perpendicular to the moving direction; When the blood spots are observed to be located on the upper side of the through holes, the movement is stopped, the rotating arm is driven forward, the cutting pieces enter the through holes respectively, and parts of the blood spots are cut off and enter the container; the rotating arm is released, and the rotating arm automatically returns to the reverse position under the action of the elastic piece; The cutting device includes: a container for accommodating a sample cut from a sample carrier; A sampling unit, comprising: A stopper, the stopper is arranged on the upper side of the container and has a channel for allowing the sample carrier to pass through; a through hole is arranged on the lower wall of the channel; in a direction perpendicular to the moving direction of the sample carrier in the channel, the length of the channel is the same as the length of the sample carrier, and the positions of the through holes correspond to the sample positions on the sample carrier one by one; A cutting member and a rotating arm, wherein the cutting member is arranged on the rotating arm, and when the rotating arm rotates, the cutting member enters the through hole; An elastic member is arranged between the rotating arm and the limiting member, and is used for hindering the rotation of the rotating arm.

2. The detection method according to claim 1, characterized in that: The filter paper is processed by: Add stabilizer to the blood spot on the blank filter paper and dry it away from light.

3. The detection method according to claim 1, characterized in that: The standard substance is a calibrator or a quality control substance.

4. The detection method according to claim 3, characterized in that: The standard products are packed and sealed.

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