Blank blood matrix, dried blood smears and their preparation and application in the detection of targets

Abstract

This invention relates to the field of detection, specifically to a blank blood matrix, which is a mixed solution containing hemoglobin, serum protein, pH adjuster, stabilizer, surfactant, and phosphate buffer; wherein the concentration of hemoglobin is 20%-60% (m / v), the concentration of serum protein is ≤20% (m / v), the concentration of stabilizer is ≤10% (m / v), the concentration of surfactant is ≤0.5% (m / v), and the concentration of pH adjuster is ≤0.5% (m / v). This invention provides a fully compounded blank blood matrix, which, based on the combined control of components and proportions, achieves synergy and can well simulate whole blood. Furthermore, it is essentially free of endogenous target substances such as amino acids, carnitine, organic acids, hormones, fatty acids, vitamins, neurotransmitters, or drugs, thus combining excellent whole blood simulation with low matrix effects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a blank blood matrix. Background Technology

[0002] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) boasts significant advantages, including high specificity, high sensitivity, wide linear range, and the ability to detect multiple compounds in a single injection. As an important detection technique, it is widely used in clinical testing and drug development. Dried blood spot (DBS) is a method for collecting blood samples that is simple to collect, convenient to transport, and can be stored for a long time. For infants and patients who cannot have large or frequent blood draws, LC-MS / MS offers significant advantages for the clinical detection of dried blood spots. However, research on dried blood spots using LC-MS / MS is not as extensive as that on matrices such as serum, plasma, and urine. This is due not only to the need for further updates to high-sensitivity instruments, but also to the significant challenge of the lack of human-derived blank blood matrices.

[0003] Human blood shares the same biological characteristics as the test organism, making it an ideal matrix for calibrators and quality controls in human disease diagnostic kits and methods. However, human blank blood has certain limitations. Firstly, human blood collection is strictly controlled worldwide, and transportation and storage costs are high. Furthermore, Chinese law strictly prohibits the sale of human blood (referring to whole blood, blood components, and raw plasma used in clinical applications and blood product manufacturing). Secondly, while normal human samples can be used as blank matrices for detecting exogenous substances, it is difficult to obtain human blank matrices for preparing standard curves and quality controls for detecting endogenous substances. Current clinical testing methods and in vitro diagnostic kits often use synthetic blood by processing human and animal whole blood to replace the matrix. While this method effectively removes endogenous substances from plasma, it cannot remove endogenous substances present in red blood cells. The high baseline values ​​used for calibrators and quality controls prevent accurate results within the clinically required detection concentration range. The difficulty in obtaining blank blood matrix has, to some extent, limited the development of clinical testing methods for dried blood smears and the production of in vitro diagnostic kits.

[0004] Therefore, it is of great significance to find a blank blood matrix that can replace human whole blood with similar biological characteristics and extremely low endogenous matrix. Summary of the Invention

[0005] In view of the problems of existing blank blood matrix materials containing large matrix effects and non-eliminable endogenous substances, as well as unsatisfactory target testing accuracy, the primary objective of this invention is to provide a fully compounded blank blood matrix material that can effectively simulate human whole blood and has ultra-low matrix effects.

[0006] A second objective of the present invention is to provide a dried blood sheet comprising the blank blood matrix.

[0007] A third objective of this invention is to provide the application of the aforementioned blank blood matrix or dried blood smear in the preparation of in vitro diagnostic kits for detecting targets.

[0008] To better simulate the characteristics of whole blood, the industry commonly uses adsorption, centrifugation, and other methods to separate whole blood to obtain the matrix. However, existing methods still result in matrices with significant matrix effects and unremovable endogenous substances, leading to unsatisfactory accuracy and recovery rates in target testing. To address this issue, this invention departs from industry norms and, for the first time, provides a method for obtaining a blank matrix through complete preparation. However, research has revealed that achieving this novel approach requires overcoming technical challenges such as effectively simulating the characteristics of whole blood and achieving effective measurement. To address these challenges, this invention, through in-depth research, provides the following solutions:

[0009] A blank blood matrix is ​​a mixed solution containing hemoglobin, serum proteins, pH adjuster, stabilizer, surfactant, and phosphate buffer.

[0010] The concentrations of hemoglobin, serum protein, stabilizer, surfactant, and pH adjuster are ≤20% (m / v), ≤10% (m / v), ≤0.5% (m / v), and ≤0.5% (m / v), respectively.

[0011] This invention provides a fully compounded blank blood matrix that, based on the combined control of components and proportions, can achieve synergy and can well simulate whole blood. Moreover, it is basically free of endogenous target substances such as amino acids, carnitine, organic acids, hormones, fatty acids, vitamins, neurotransmitters or drugs, thus combining excellent whole blood simulation with low matrix effect.

[0012] In this invention, the hemoglobin is at least one of bovine hemoglobin, porcine hemoglobin, or human hemoglobin.

[0013] In this invention, the pH adjuster includes acidic and / or alkaline substances;

[0014] The acidic substances mentioned include at least one of formic acid, acetic acid, ammonium fluoride, and hydrochloric acid;

[0015] The alkaline substance mentioned includes at least one of ammonia and sodium hydroxide.

[0016] In this invention, the stabilizer is at least one of trehalose and mannitol.

[0017] In the method, the stabilizer is a component of the matrix that has a protective effect;

[0018] In this invention, the surfactant is at least one of Tween-20 and Tween-80.

[0019] In the method described above, the surfactant has a solubilizing and diffusion-enhancing effect;

[0020] In this invention, the phosphate buffer is a PBS buffer, which can further be a PBS buffer containing 100mM sodium phosphate, 9.0% NaCl, and pH 6.8±0.1 (25℃).

[0021] In this invention, the concentration of each component can be further adjusted using ultrapure water as needed.

[0022] The blank blood matrix described in this invention is a blank blood matrix that simulates whole blood.

[0023] Preferably, the hemoglobin is at least one of bovine hemoglobin or porcine hemoglobin, with bovine or porcine hemoglobin being more readily available.

[0024] Preferably, the concentration of hemoglobin in the blank blood matrix is ​​30%-50% (m / v);

[0025] The concentration of the serum protein is 2%-10% (m / v);

[0026] The concentration of the pH adjuster is 0.1–0.5% (m / v);

[0027] The concentration of the stabilizer is 1%-10% (m / v);

[0028] The concentration of the surfactant is 0.1%-0.5% (v / v);

[0029] The solvent is a mixture of PBS buffer solution and water;

[0030] More preferably, the concentration of hemoglobin in the blank blood sample is 40%-45% (m / v);

[0031] The concentration of the serum protein is 3%-5% (m / v);

[0032] The concentration of the pH adjuster is 0.3–0.5% (m / v);

[0033] The concentration of the stabilizer is 4%-6% (m / v);

[0034] The concentration of the surfactant is 0.3%-0.5% (v / v);

[0035] The solvent is a mixture of PBS buffer solution and water in a volume ratio of 5–15:85–95.

[0036] The present invention also provides a method for preparing the blank blood matrix, wherein the components are mixed evenly to obtain the matrix.

[0037] For example, a typical preparation process of the present invention is as follows: obtain hemoglobin, serum protein, phosphate buffer solution and acid / alkaline substances, take hemoglobin and serum protein, add acid / alkaline substances, phosphate buffer solution and ultrapure water, add trehalose and Tween-20, and mix thoroughly with a mixer to obtain blank blood matrix for use as blank matrix.

[0038] The present invention also provides a blank blood dried blood sheet, which includes a carrier and the dry matter of the blank blood matrix described in the present invention loaded thereon.

[0039] In this invention, the carrier is a paper-based material, more specifically filter paper, and even more specifically, 903# filter paper sheet.

[0040] In this invention, the method for preparing the blank blood dried blood smear is, for example, to add a blank blood matrix to a carrier and then dry it. For example, 60 μL of blank blood from the previous step is pipetted onto an international 903# filter paper and dried in the dark to obtain the blank matrix for the dried blood smear.

[0041] The present invention also provides the application of the blank blood matrix or the blank dried blood smear in the preparation of a test kit for testing targets.

[0042] In this invention, the blank blood matrix or the blank dried blood smear can be used for the determination of targets, particularly targets in whole blood samples. For example, the blank blood matrix or the blank dried blood smear can be used to prepare standard samples or quality control samples for testing targets; and then used for the determination of target substances.

[0043] In this invention, the target may be, for example, an amino acid, carnitine, an organic acid, a hormone, a fatty acid, a vitamin, a neurotransmitter, or a drug.

[0044] The specific amino acids mentioned are lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine ​​(Cys), ethanolamine phosphate (PEtN), gamma-aminobutyric acid (GABA), and taurine (Tau). ), β-aminoisobutyric acid (bAib), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3MHis), hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine ​​(Hcy), α-aminoadipic acid (Aad), α-aminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), β-alanine (β-Ala), kynurenine (Kyn), arginine succinate (Asa), cystathionine (Cth). The carnitine specifically comprises free carnitine, acetylcarnitine, propionylcarnitine, malonylcarnitine / 3-hydroxy-butyrylcarnitine, butyrylcarnitine, methylmalonylcarnitine / 3-hydroxy-isovalerylcarnitine, isovalerylcarnitine, isopentenylcarnitine, glutarylcarnitine / 3-hydroxy-hexanoylcarnitine, hexanoylcarnitine, adipicylcarnitine, capryloylcarnitine, and octenylcarnitine. The organic acid is 2-hydroxyisovaleric acid, 2-ketoisohexanoic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 2-ketoisovaleric acid, methylmalonic acid, and methylcitric acid. The vitamin specifically comprises vitamin A and vitamin E. The hormone specifically comprises dehydroepiandrosterone sulfate, cortisol, and androstenedione. The fatty acid specifically comprises γ-linolenic acid, linoleic acid, oleic acid, eicostrienoic acid, and docosahexaenoic acid.

[0045] The beneficial effects of the invention are:

[0046] This invention departs from the technical instruction of whole blood matrix purification and proposes for the first time the technical concept of fully prepared blank matrix. It also innovatively solves the problems faced by fully prepared matrix in terms of difficulty in effectively simulating human blood samples and unsatisfactory test results by jointly controlling the components and proportions.

[0047] The blank matrix described in this invention has at least the following characteristics: First, the materials are readily available, stable in performance, and inexpensive, and can be commercially purchased; it has similar biological characteristics to human blood samples; third, compared to human or animal blood, the synthesized blank blood matrix is ​​easier to store and transport. Blank blood matrix can be stored at -20°C for more than 2 months, and the prepared blank dried blood spots can be stored at -20°C for more than 6 months; fourth, the synthesized endogenous compounds and drugs have extremely low basis values. For some endogenous compounds, centrifugation and adsorption of human or animal blood can only reduce the basis value to a certain extent, but cannot completely remove them. The basis value of the blank blood matrix synthesized in this method is much lower than that of the matrix after processing human or animal blood, which is the commonly used method for obtaining blank matrix in clinical blood testing.

[0048] Blood is composed of plasma and blood cells, with blood cells accounting for approximately 45% of the blood volume. In the synthesis of common blank blood matrices, human or animal blood is often purified and partially replaced with other matrices. Plasma is typically treated with activated carbon or montmorillonite adsorption to remove baseline values, while blood cells are typically treated with washing, adsorption, or freeze-rupture to remove baseline values. As mentioned above, for endogenous compounds, the recombinant treatment of human or animal blood only reduces the baseline value to a certain extent. The residual baseline values ​​remaining in the plasma and blood cells can affect the accuracy of clinical blood test calibrators when used as clinical measurement ranges. When used as quality control materials, a high reference target value will not accurately monitor the accuracy of the method. Therefore, this invention uses a substitute plasma matrix prepared with PBS, BSA, acid / alkali, etc., and a certain proportion of commercially available hemoglobin to prepare a blank blood matrix with similar biological characteristics and extremely low baseline values ​​to human blood samples.

[0049] The blank blood matrix preparation method of the present invention can be used for the preparation, production and application of blank samples in existing detection systems, platforms, detection devices or kits for amino acids, carnitine, organic acids, hormones, fatty acids, vitamins, neurotransmitters or drugs. Attached Figure Description

[0050] Figure 1 Blank matrix dried blood smears prepared by dropping blank blood matrix with different formulations

[0051] Figure 2 This is a standard curve of amino acids prepared from blank dried blood slices using the blank blood matrix preparation method of this application in an embodiment of the present invention.

[0052] Figure 3 This is a comparison chart of the organic acid base value of blank dried blood smears prepared using the blank blood matrix preparation method of this application and the base value of negative samples from normal individuals in an embodiment of the present invention.

[0053] Figure 4 and Figure 5 These are standard curves of organic acids methylmalonic acid (MMA) and methylcitric acid (MCA) prepared from blank dried blood slices obtained using the blank blood matrix preparation method of this application, as shown in the embodiments of the present invention. Figure 4 The standard curve is Y = 0.587229x + 0.181801, r^2 = 0.999221; Figure 5 The standard curve is: Y = 0.803267x + 0.177482, r^2 = 0.997493.

[0054] Figure 6 This is a comparison chart of the baseline values ​​of vitamin A and vitamin E in blank dried blood slices prepared using the blank blood matrix preparation method of this application in an embodiment of the present invention, and the baseline values ​​in negative samples from normal individuals.

[0055] Figure 7 This is a comparison chart of hormone baseline values ​​in blank dried blood smears prepared using the blank blood matrix preparation method of this application and baseline values ​​in negative samples from normal individuals, as shown in this embodiment of the invention.

[0056] Figure 8 This is a comparison chart of the fatty acid base values ​​in blank dried blood smears prepared using the blank blood matrix preparation method of this application and the base values ​​in negative samples from normal individuals in an embodiment of the present invention. Detailed Implementation

[0057] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] Example 1: Preparation of blank blood matrix

[0060] Serum proteins, phosphate buffer, ammonium fluoride, trehalose, and Tween-20 were purchased from Sigma, and hemoglobin was purchased from Aladdin or Yuan Ye, with the hemoglobin being a dark red crystalline powder.

[0061] Hemoglobin, serum protein, ammonium fluoride, trehalose and other raw materials were selectively dissolved in water in advance to obtain their respective solutions. Then, according to the formula in Table 1, the components were mixed and the solutions were thoroughly mixed and completely dissolved using a vortex mixer or an ultrasonic mixer. The solutions were then centrifuged at 3000 rpm for 15 minutes and vortexed to mix, thus obtaining a blank blood sample.

[0062] As shown in Table 1 below:

[0063]

[0064]

[0065] m / v refers to the mass-to-volume ratio, and in this case, the unit is g / mL.

[0066] See the blood spots for each formula. Figure 1 ,pass Figure 1 It can be seen that the physical forms of formulas three, four, five, seven, eight and nine are all quite close to human blood, with formula four being the closest to human blood.

[0067] Example 2: Preparation of blank dried blood smear matrix

[0068] Prepare 903# filter paper under the conditions of ambient temperature of 18-25℃ and humidity of ≤55%;

[0069] Use a 100μL or 200μL pipette to draw 60-100μL of blank blood sample solution (Formula 4 of Example 1) and drop it onto the corresponding position on the filter paper;

[0070] Air dry naturally overnight. The same filter paper can be dripped multiple times to ensure that the blood spots do not come into contact with each other, thus obtaining a dry blood spot blank matrix.

[0071] Store in sealed aluminum foil bags at -20±5℃, with silicone deoxygenating and dehydrating agents added inside for vacuum packaging.

[0072] Example 3: Detection of the amino acid matrix of the above-mentioned dried blood smear blank matrix using tandem mass spectrometry.

[0073] PerkinElmer Non-Derivatized Multiple Amino Acids, Carnitine, and Succinoacetone Assay Kit (Tandem Mass Spectrometry) NeoBase TM The amino acid content of dried blood slices in Example 2 (Formula 4) was detected using a non-derivatized MSMS Kit (National Medical Device Registration Certificate No. 20173400071). This included blank matrix dried blood slices and quality control dried blood slices prepared from blank matrix dried blood slices (preparation method same as in Example 11). The concentrations of normal human samples were also compared. The results are shown in Table 2 below.

[0074]

[0075]

[0076] The results are shown in Table 2. It is evident that the amino acid content of the blank dried blood smear prepared from the blank blood matrix is ​​significantly lower than that of normal dried blood smear samples from newborns. Commonly used quality control products for amino acid screening on the market often exceed the clinical reference range in concentration, failing to effectively monitor the accuracy of the method within the specified concentration range. In contrast, the amino acid dried blood smear quality control product prepared using the aforementioned blank blood matrix covers both the negative and positive ranges, ensuring accurate measurement even within the negative range. The application of this quality control product in the detection method and kit can effectively monitor the accuracy and robustness of the method.

[0077] Example 4: Detection of carnitine matrix in the blank matrix of the above-mentioned dried blood smear using tandem mass spectrometry.

[0078] PerkinElmer Non-Derivatized Multiple Amino Acids, Carnitine, and Succinoacetone Assay Kit (Tandem Mass Spectrometry) NeoBase TM The results of detecting carnitine content in dried blood slices in Example 2 (Formula 4) using the Non-derivatized MSMS Kit (National Medical Device Registration Certificate No. 20173400071) and comparing the concentration in normal human samples with the carnitine matrix in the blank matrix of the above dried blood slices are shown in Table 3 below:

[0079]

[0080]

[0081] The results are shown in Table 3. It can be seen that the carnitine content of the blank dried blood smear is much lower than that of the normal dried blood smear sample of newborns. The carnitine content of the blank dried blood smear prepared with the blank blood matrix is ​​0 except for C0, which is 0.3 μmol / L.

[0082] Example 5: Comparison with normal human sample concentrations, the results of organic acid in the blank matrix of dried blood smears in Example 2 (Formula 4) are as follows: Figure 3 As shown.

[0083] The results are as follows Figure 3As shown in the figure, comparing the negative samples of organic acids from normal individuals with the blank dried blood tablets of this invention, it can be seen that the contents of 2-hydroxyisovaleric acid, 2-ketoisocaproic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, and 2-ketoisovaleric acid in the blank dried blood tablets are all 1-2 orders of magnitude lower than those in the negative samples of normal individuals. Methylmalonic acid and methylcitric acid are almost absent in both the negative samples and the blank dried blood tablets, indicating that the blank dried blood tablets of this invention are endogenous compounds with low baseline values. Furthermore, the chromatographic retention times of each compound are basically consistent before and after sample processing.

[0084] Example 6: Comparison with normal human sample concentrations, the vitamin results in the blank matrix of dried blood smears from Example 2 (Formula 4) are as follows: Figure 6 As shown.

[0085] The results are shown in Figure 6. As can be seen from the figure, comparing the vitamin A and vitamin E content in the negative samples from normal individuals with that in the blank dried blood tablets of this invention, the content of vitamin A and vitamin E in the blank dried blood tablets is only 1 / 8 to 1 / 6 of that in the negative samples from normal individuals. This indicates that the blank dried blood tablets of this invention are endogenous compounds with low baseline values. Furthermore, the chromatographic retention times of each compound remained essentially the same before and after sample processing.

[0086] Example 7: Compared with the concentration in normal human samples, the hormone results in the blank matrix of the dried blood smear in Example 2 (Formula 4) are as follows: Figure 7 As shown.

[0087] The results are as follows Figure 7 As shown in the figure, comparing the negative organic acid samples from normal individuals with the blank dried blood sample of this invention, it can be seen that the contents of dehydroepiandrosterone sulfate, cortisol, and androstenedione in the blank dried blood sample are all very low and negligible, indicating that the blank dried blood sample of this invention contains endogenous compounds with low baseline values. Furthermore, the chromatographic retention times of each compound are basically consistent before and after sample processing.

[0088] Example 8: Comparison with normal human sample concentrations, the results of fatty acid content in the blank matrix of dried blood smears from Example 2 (Formula 4) are as follows: Figure 8 As shown.

[0089] The results are as follows Figure 8 As shown in the figure, comparing the negative organic acid samples from normal individuals with the blank dried blood tablets of this invention, the contents of γ-linolenic acid, linoleic acid, oleic acid, eicosatrienoic acid, and docosahexaenoic acid in the blank dried blood tablets are all 1-2 orders of magnitude lower than those in the negative organic acid samples from normal individuals. This indicates that the blank dried blood tablets of this invention are endogenous compounds with low baseline values. Furthermore, the chromatographic retention times of each compound are consistent before and after sample processing.

[0090] Example 9: Comparison of baseline values ​​between conventional whole blood matrix treatment methods and whole blood replacement matrices for different animals. Conventional treatment methods for whole blood replacement matrices include:

[0091] Method 1: Use commercially available anticoagulated animal whole blood instead of human whole blood.

[0092] Method 2: Purchase anticoagulated whole blood from animals and centrifuge at 3000 rpm for 10 minutes. After centrifugation, separate the plasma layer, leukocyte layer, and platelet layer. Add approximately the same volume of physiological saline as the red blood cell layer and mix thoroughly and gently using a mixer. Centrifuge at 3000 rpm for 10 minutes and separate the supernatant, leukocyte layer, and platelet layer. Repeat this step 3 times to obtain red blood cells. Adjust the hematocrit to 45%-55% using physiological saline to obtain substitute matrix blood. Use a pipette to add 60 μL of substitute matrix whole blood onto an international 903# filter paper disc and air dry overnight at room temperature to obtain a dried blood smear blank substitute matrix.

[0093] Method 3: Purchase anticoagulated whole blood from animals and centrifuge at 3000 rpm for 10 minutes. After centrifugation, separate the plasma layer, leukocyte layer, and platelet layer. Add an equal volume of physiological saline to the erythrocyte layer and mix thoroughly using a mixer. Centrifuge at 3000 rpm for 10 minutes and separate the supernatant, leukocyte layer, and platelet layer. Repeat this step 3 times to obtain erythrocytes. Freeze the erythrocytes at -20°C overnight, then thaw them. Adjust the hematocrit of the ruptured erythrocytes to 45%-55% using physiological saline to obtain substitute matrix blood. Use a pipette to add 60 μL of substitute matrix whole blood onto an international 903# filter paper disc and air dry at room temperature overnight to obtain a dried blood smear blank substitute matrix.

[0094]

[0095]

[0096] The results of detecting the substrate values ​​of different alternative matrices using the method described in patent CN114236025A are shown in Table 4 below.

[0097] The results are shown in Table 4. It is evident that conventional methods 1 to 3 have limited effectiveness in removing endogenous matrix, rendering animal blood substitutes unsuitable for accurate disease detection. In particular, compounds such as Cit, Gly, Tau, Hyp, and 1MHis are present in high concentrations in erythrocytes; even with the destruction of erythrocytes using method 3, these endogenous compounds remain. In contrast, blank dried blood smears prepared using our synthesized blank blood matrix show significantly lower endogenous compound matrix levels compared to human blood, demonstrating a substantial advantage over animal blood / processed animal blood.

[0098] Matrix equivalence study of the blank dried blood patch matrix described in Examples 10 and 2 (Formula 4) with matrix from 6 different randomized human dried blood patch samples.

[0099] The consistency / equivalence between blank dried blood smear matrix and human dried blood smear samples was demonstrated by examining the matrix effect between matrices. Using the method described in patent CN114236025A, human dried blood smear samples from six different individuals were randomly selected. The signal of the analyte spiked onto the extracted sample was compared with the signal of the analyte spiked onto the matrix of the extracted standard curve. The ratio of the peak area response in the presence of the matrix (measured by adding analyte and internal standard after extraction from the blank matrix, i.e., B) to the corresponding peak area response without the matrix (pure solution of analyte and internal standard, i.e., A) was calculated. The matrix factor for each analyte (low, medium, and high quality control concentrations) and internal standard was calculated, and the matrix factor normalized to the internal standard was calculated.

[0100] Formula: Matrix factor (ME) = Peak area response of the analyte after extraction of blank matrix / Peak area response of the analyte in pure solution * 100%; Internal standard normalized ME = analyte ME / internal standard ME * 100%.

[0101] The matrix factor (ME) calculation results for blank dried blood smear matrix and six different random human dried blood smear samples are shown in Table 5 below.

[0102]

[0103]

[0104] The results are shown in Table 5. It can be seen that the coefficient of variation of matrix factors (ME) between the blank dried blood smear matrix and the six different randomized human dried blood smear samples is ≤15%, and the ME%RE of the internal standard-normalized matrix factor is within ±15%. No significant inhibition or enhancement was observed between the blank dried blood smear matrix and the six different randomized human dried blood smear samples; they were quantitatively consistent, fully demonstrating that the simulated blank dried blood smear matrix can accurately quantify human dried blood smears, and that the two matrices have similar biological characteristics.

[0105] Example 11: Using the blank blood described in Example 2 (Formula 4) prepared according to the present invention, a matrix-dried blood patch correction line was obtained.

[0106] The steps for preparing dried blood spot calibration lines from blank dried blood spot matrix are as follows:

[0107] 1) Prepare 5 mL of calibrator intermediate solution. Weigh the materials shown in Table 6 below into the corresponding containers and dissolve them in the corresponding solutions. Vortex and sonicate until completely dissolved. The solutions can be stored at -80±5℃ for 12 months. Then, transfer the single standard solution into the corresponding containers according to the transfer volumes in the table, add 1215 μL of ultrapure water, mix well, and label accordingly. The solutions can be stored at -80±5℃ for 12 months.

[0108]

[0109]

[0110] 2) Dilution of Calibration Intermediate Solution: Transfer 50 μL of the calibration intermediate solution to a 1.5 ml centrifuge tube, add 450 μL of ultrapure water, and mix well to obtain a 10-fold diluted calibration intermediate solution. Then, dilute both the calibration intermediate solution and the 10-fold diluted calibration intermediate solution according to the concentration settings of calibration lines S0-S6 to obtain calibration solutions at different concentration levels S1-S6.

[0111] 3) Mix the calibrator solution with the blank blood solution at a ratio of 1:3. Mix S0 with ultrapure water and the blank blood solution at a ratio of 1:3 to obtain whole blood calibrator solutions of different concentration levels for the calibration lines S0-S6.

[0112] 4) Under the conditions of ambient temperature of 18-25℃ and humidity of ≤55%, prepare international 903# filter paper. Use a 100μL or 200μL pipette to take 60μL of calibrator solution at different concentration levels S1-S6 and drop it onto the corresponding position of the filter paper, one blood spot for each concentration. Let it air dry overnight. Use a 100μL or 200μL pipette to take 60μL of calibrator solution at concentration S0 and drop it onto the corresponding position of the filter paper. The same filter paper can be used for multiple drops to ensure that the blood spots do not come into contact with each other. Prepare once per batch and let it air dry overnight with the calibrator filter paper to obtain calibrator dried blood spots.

[0113] 5) After drying, arrange and stack the dried blood smears of the calibrator, divide them into 2 smears / packs, seal them in aluminum foil bags, add an equal amount of molecular sieve (non-woven paper), add silica gel deoxygenating and dehydrating agent to the bags, vacuum pack them, and store them sealed at -20±5℃.

[0114] See results Figure 1 As shown in the figure, the blood smear calibrators prepared using the blank blood matrix preparation method of this application were used as the standard curve for liquid chromatography-mass spectrometry detection. The standard curve was plotted with concentration as the abscissa and the ratio of the peak area of ​​the analyte to the corresponding internal standard (AreaRatio) as the ordinate. The linear correlation coefficients R were all ≥0.995.

[0115] Quantitative analysis of the calibration lines prepared from the blank dried blood smear matrix described in Examples 12 and 2 (Formula 4) for the quality control of human-derived dried blood smears.

[0116] Using the method described in patent CN114236025A, the dried blood sample quality control product prepared by quantifying human whole blood matrix spiked with the above-mentioned dried blood sample calibrator was calculated, and the spike recovery rate and precision results are shown in Table 7.

[0117]

[0118]

[0119] The results are shown in Table 7. The dried blood sample calibrator was used to quantify the human dried blood sample quality control, and the precision and accuracy were calculated by comparing the results with theoretical values. The precision deviation of each intrinsic index was ≤15%, the spiked recovery rate was between 80% and 120%, and the accuracy bias was ≤15%. The dried blood sample calibrator exhibits good accuracy and precision for the quantification of human dried blood samples.

[0120] We also compared the blank blood spot matrix properties of Formula 2 and Formula 6. Using the same preparation and dropping methods as in Examples 1, 2, and 11, we dropped the control group dried blood spot calibrators. Using the detection method described in patent CN114236025A, we quantitatively dropped the dried blood spot quality control products prepared with human whole blood matrix spiked with the above dried blood spot calibrators. The spiked recovery rate test results of the two batches of QC are shown in Tables 8 and 9.

[0121] Results of quantitative QC recovery of the dried blood smear calibrator prepared by Formula 2:

[0122]

[0123]

[0124] Results of quantitative QC recovery of the dried blood smear calibrator prepared by Formula 6:

[0125]

[0126]

[0127] As can be seen from the table above, the quantitative QC recovery results of the dried blood spot calibrators prepared by Formula 2 and Formula 6 are not ideal. The quantitative QC recovery results of Formula 2 calibrator show that only a very few indicators have a spiked recovery rate between 80% and 120%, while the spiked recovery rates of more than 90% of the indicators are beyond the acceptable range. There is no obvious pattern in the low spiked recovery rate, while the recovery rate in the middle and high points generally shows a high trend, which is presumably related to the physical properties of the blood spot and the influence of the matrix.

[0128] The quantitative QC recovery results of Formula 6 calibrator showed that only a very few indicators had a spiked recovery rate between 80% and 120%, while the spiked recovery rate of more than 85% of the indicators was beyond the acceptable range. There was no obvious pattern in the low spiked recovery rate, and the overall recovery rate of the medium and high points showed a low trend, which is presumably related to the physical properties of blood spots and the influence of the matrix.

[0129] Uniformity of calibrators / quality control samples prepared from the blank dried blood smear matrix described in Examples 13 and 2 (Formula 4)

[0130] Twenty dried blood spots of each amino acid quality control sample were randomly selected, processed according to routine testing procedures, and analyzed using LC-MS / MS. RSD was calculated to determine the homogeneity of the calibrators / controls. The experimental results are shown in Table 10 below:

[0131]

[0132]

[0133] Experimental data show that the RSD of amino acids in the quality control products is between 3% and 15%, indicating that the quality control matrix is ​​uniform and has good homogeneity.

[0134] Stability of calibrators / quality control samples prepared from blank dried blood smear matrix in Examples 14 and 2 (Formula 4)

[0135] The accuracy of the amino acid dried blood spot quality control was determined using LC-MS / MS. The experimental results are shown in Table 11 below:

[0136] Target value 01:Lys 02:His 03:Arg 04:Ala 05:Glu 06:Ile 07:Phe 08:Ser 09:Val L 122.4 37.9 19.9 228.24 83.33 19.2 94 67.5 96 M 388.9 125.9 118.3 528.24 308.33 185.3 366.3 202.8 387.6 H 815.4 266.6 275.7 1008.2 668.33 451.2 801.9 419.3 854.2 Measurement -6M 01:Lys 02:His 03:Arg 04:Ala 05:Glu 06:Ile 07:Phe 08:Ser 09:Val L 130.3 39.2 21.1 242.2 92.1 20 106.7 68.3 102 M 388 129.5 117 550.4 293.7 184.7 364.9 204.5 383.4 H 807.6 254.9 265.7 985.2 648.6 413.3 774.4 412.9 826.4 L - Accuracy 106％ 103％ 106％ 106％ 111％ 104％ 113％ 101％ 106％ M - Accuracy 100％ 103％ 99％ 104％ 95％ 100％ 100％ 101％ 99％ H-Accuracy 99％ 96％ 96％ 98％ 97％ 92％ 97％ 98％ 97％

[0137] Target value 10:Thr 11:Tyr 12:Asn 13:Trp 14:Gln 15:Met 16:Asp 17:Gly 18:Leu L 58.2 39.5 2.6 8.4 21.03 36.91 12.99 65.8 217.83 M 228.2 203.1 34.7 40.7 321.03 224.41 50.49 599.2 405.33 H 500.2 464.9 85.9 92.3 801.03 524.41 110.49 1452.8 705.33 Measurement -6M 10:Thr 11:Tyr 12:Asn 13:Trp 14:Gln 15:Met 16:Asp 17:Gly 18:Leu L 61.5 41.8 2.3 8.8 25.6 37.1 13.7 66 248.2 M 226.8 197.4 36.4 40.1 329.5 222.7 48.8 616.8 407.6 H 484.2 452.8 83.3 88.8 780.3 492.5 109.5 1342 692 L - Accuracy 106％ 106％ 87％ 105％ 122％ 101％ 106％ 100％ 114％ M - Accuracy 99％ 97％ 105％ 99％ 103％ 99％ 97％ 103％ 101％ H - Accuracy 97％ 97％ 97％ 96％ 97％ 94％ 99％ 92％ 98％

[0138]

[0139]

[0140]

[0141]

[0142] The long-term stability of the amino acid dried blood spot quality control at -20℃ was verified using LC-MS / MS. The accuracy of the amino acid was between 80% and 120%. The amino acid blood spot quality control was stable for 6 months under conditions of -20℃ and humidity below 30%RH.

[0143] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A blank blood matrix, characterized in that, It is a mixed solution containing hemoglobin, serum albumin, pH adjuster, stabilizer, surfactant, phosphate buffer, and water; Among them, the concentration of hemoglobin is 20%~60% (m / v), the concentration of serum protein is ≤20% (m / v), the concentration of stabilizer is ≤10% (m / v), the concentration of surfactant is ≤0.5% (m / v), and the concentration of pH adjuster is ≤0.5% (m / v). The pH adjuster is ammonium fluoride; The stabilizer is trehalose; The surfactant is at least one of Tween-20 and Tween-80.

2. The blank blood matrix as described in claim 1, characterized in that, The hemoglobin mentioned is at least one of bovine hemoglobin, porcine hemoglobin, or human hemoglobin.

3. The blank blood matrix as described in claim 1, characterized in that, In the blank blood matrix, the concentration of hemoglobin is 30%~50% (m / v). The concentration of the serum protein is 2%~10% (m / v). The concentration of the pH adjuster is 0.1%~0.5% (m / v). The concentration of the stabilizer is 1%~10% (m / v). The concentration of the surfactant is 0.1% to 0.5% (m / v).

4. The blank blood matrix as described in claim 3, characterized in that, In the blank blood matrix, the concentration of hemoglobin is 40%~45% (m / v). The concentration of the serum protein is 3%~5% (m / v); The concentration of the pH adjuster is 0.3%~0.5% (m / v). The concentration of the stabilizer is 4%~6% (m / v); The concentration of the surfactant is 0.3%~0.5% (m / v).

5. A method for preparing a blank blood matrix according to any one of claims 1 to 4, characterized in that, Mix all ingredients thoroughly to obtain the final product.

6. A blank dried blood tablet, characterized in that, The dry matter of the blank blood matrix as described in any one of claims 1 to 4, including the carrier and its load.

7. The blank dried blood smear as described in claim 6, characterized in that, The carrier is a paper-based material.

8. The blank dried blood smear as described in claim 7, characterized in that, The carrier is filter paper.

9. The use of a blank blood matrix according to any one of claims 1 to 4 or a blank dried blood smear according to any one of claims 6 to 8 in the preparation of a test kit for testing a target.

10. An application according to claim 9, characterized in that, The blank blood matrix or the blank dried blood smear is used to prepare a test kit for testing target substances in whole blood samples.

11. The application as described in claim 10, characterized in that, The blank blood matrix or the blank dried blood smear is used to prepare standard samples or quality control samples for the test target.

12. The application as described in claim 11, characterized in that, The targets are amino acids, carnitine, organic acids, hormones, fatty acids, vitamins, neurotransmitters, or drugs.

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