Composition for protein precipitation, protein precipitation agent containing same, protein precipitation combination product, detection kit, and preparation method and detection method of blood analysis sample

Abstract

The invention relates to the technical field of biological detection, in particular to a composition for protein precipitation, a protein precipitation agent containing the composition, a protein precipitation combined product, a detection kit and a preparation method and a detection method of a blood analysis sample. The composition for protein precipitation comprises zinc sulfate and ammonium acetate in a dosage ratio of (5-17) mg: (0.5-1) mmol. Compared with the prior art, the composition has the following beneficial effects that the zinc sulfate and the ammonium acetate are matched according to a specific proportion, so that the specific composition is formed, and hemoglobin in a blood sample can be effectively removed by taking the composition as a protein precipitation component.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a protein precipitation composition, a protein precipitant containing the same, a protein precipitation combination product, a test kit, and a method for preparing and detecting blood analysis samples. Background Technology

[0002] Immunosuppressants are drugs that suppress the body's immune response. They inhibit the proliferation and function of cells related to the immune response (such as T cells and macrophages like B cells), and reduce antibody immune responses. They are mainly used to treat and prevent organ transplant rejection, as well as to treat nephrotic syndrome, aplastic anemia, systemic lupus erythematosus, and other autoimmune diseases.

[0003] Immunosuppressants such as tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid are widely distributed in various tissues after administration and mainly bind to red blood cells in the blood. Therefore, whole blood samples are used clinically for drug concentration monitoring. However, due to the narrow therapeutic window of immunosuppressants and the fact that their pharmacokinetic characteristics are affected by various factors, such as gender, age, race, and drug-food interactions, it is necessary to carefully determine the dosage of immunosuppressants for each patient to reduce the side effects of drug treatment, such as nephrotoxicity and cardiotoxicity at high blood concentration levels. Therefore, therapeutic drug monitoring (TDM) is crucial in the clinical application of immunosuppressants such as tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid, helping to determine the dose adjustments required to achieve target blood drug concentrations, improve treatment efficacy, and reduce the occurrence of side effects.

[0004] Currently, total molecular weight (TDM) of immunosuppressants is mainly achieved through immunoassay, high-performance liquid chromatography-ultraviolet detection (HPLC-UV), or liquid chromatography-tandem mass spectrometry (LC-MS / MS). Immunoassay is simple to operate; however, it lacks specificity and sensitivity, and different immunoassays show significant differences in results, limiting its application. HPLC-UV requires long run times and instrument equilibration times to achieve chromatographic separation, resulting in insufficient throughput. LC-MS / MS, on the other hand, offers high specificity, high sensitivity, and high throughput, enabling the simultaneous detection of multiple compounds in low-volume samples. Its simple pretreatment steps and short run time make it widely accepted as the preferred technique for TDM.

[0005] Traditional immunosuppressant detection methods (LC-MS / MS) primarily employ blood sample pretreatment methods such as protein precipitation and liquid-liquid extraction. Liquid-liquid extraction is complex and invasive, therefore it is not the preferred method. Protein precipitation is simpler, as described in CN109187839A and CN113533555A, which use zinc sulfate and methanol as protein precipitants to pretreat whole blood samples before detecting immunosuppressants. However, current protein precipitation methods cannot effectively remove hemoglobin from whole blood. Therefore, effectively removing hemoglobin from blood samples is a critical technical problem that urgently needs to be solved in the detection of immunosuppressants in blood samples using LC-MS / MS. This application is therefore submitted. Summary of the Invention

[0006] Based on this, one or more embodiments of this application provide a protein precipitation composition, a protein precipitant containing the same, a protein precipitation combination product, a detection kit, and a method for preparing and detecting blood analysis samples. The technical solution includes:

[0007] One or more embodiments of this application provide a protein precipitation composition comprising zinc sulfate heptahydrate and ammonium acetate in a ratio of (5-17) mg:(0.5-1) mmol.

[0008] In some specific embodiments of this application, the protein precipitation composition comprises zinc sulfate heptahydrate and ammonium acetate in a ratio of (10-15) mg:(0.5-0.8) mmol / mL.

[0009] One or more embodiments of this application provide a protein precipitant comprising zinc sulfate heptahydrate at a solvent concentration of 5 mg / mL to 17 mg / mL and ammonium acetate at a concentration of 0.5 mmol / mL to 1 mmol / mL.

[0010] In some specific embodiments of this application, the protein precipitant includes the solvent, zinc sulfate heptahydrate at 10 mg / mL to 15 mg / mL, and ammonium acetate at 0.5 mmol / mL to 0.8 mmol / mL.

[0011] In some specific embodiments of this application, the solvent includes water.

[0012] One or more embodiments of this application provide a protein precipitation combination product, the protein precipitation combination product comprising:

[0013] The protein precipitation composition or the protein precipitant described herein; and,

[0014] Other protein precipitants.

[0015] In some specific embodiments of this application, the other protein precipitant includes methanol.

[0016] One or more embodiments of this application provide a detection kit, the detection kit comprising:

[0017] The protein precipitation composition, the protein precipitation agent, or the protein precipitation combination product described herein.

[0018] In some specific embodiments of this application, the test kit further includes one or more of the following: standards, quality control products, internal standard solutions, mobile phase reagents, and glassware.

[0019] In some specific embodiments of this application, the standard and / or the quality control product includes an immunosuppressant; the internal standard solution includes an internal standard of the immunosuppressant.

[0020] In some specific embodiments of this application, the immunosuppressant includes one or more of tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid.

[0021] In some specific embodiments of this application, the internal standard solution comprises a mixture of the other protein precipitant and the internal standard.

[0022] In some specific embodiments of this application, the mobile phase reagent includes one or more of formic acid, ammonium acetate, and methanol.

[0023] In some specific embodiments of this application, the vessel includes a protein precipitation plate.

[0024] In some specific embodiments of this application, the standard and / or the quality control product includes blood substitute matrix and the immunosuppressant standard.

[0025] In some specific embodiments of this application, the blood replacement matrix comprises bovine serum albumin aqueous solution and bovine whole blood, with the volume ratio of the bovine serum albumin aqueous solution to the bovine whole blood being 1:(2.5 to 3.5), based on the concentration of bovine serum albumin in the bovine serum albumin aqueous solution being 1wt% to 5wt%.

[0026] In some specific embodiments of this application, the bovine serum albumin aqueous solution further includes one or more of preservatives and antioxidants;

[0027] Optionally, the preservative includes Proclin 300;

[0028] Optionally, the antioxidant includes one or more of ascorbic acid, sodium metabisulfite, 2,6-di-tert-butyl-p-cresol, and vitamin E.

[0029] In some specific embodiments of this application, the concentration of Proclin 300 in the bovine serum albumin aqueous solution is 0.05wt% to 0.1wt%; or / and, the concentrations of ascorbic acid and sodium metabisulfite in the bovine serum albumin aqueous solution are each independently 10 mg / mL to 20 mg / mL; or / and, the concentrations of 2,6-di-tert-butyl-p-cresol and vitamin E in the bovine serum albumin aqueous solution are each independently 0.5 mg / mL to 1 mg / mL.

[0030] In some specific embodiments of this application, the bovine whole blood contains an anticoagulant.

[0031] In some specific embodiments of this application, the anticoagulant includes one or more of sodium citrate, lithium heparin, and sodium heparin.

[0032] One or more embodiments of this application provide a method for preparing a blood analysis sample. The preparation method uses the protein precipitation composition, the protein precipitant, the protein precipitation combination product, or the detection kit to pretreat the blood to be tested and prepare the analysis sample.

[0033] In some specific embodiments of this application, the preparation method includes the following steps: mixing the internal standard solution, standard, quality control product and the blood sample to be tested, adding the precipitant to the resulting mixture, mixing well, collecting the supernatant, and preparing the analytical sample.

[0034] One or more embodiments of this application provide a method for detecting blood analysis samples, the method comprising the following steps:

[0035] The analytical sample was prepared using the aforementioned preparation method; and,

[0036] The analyzed sample was then tested.

[0037] In some specific embodiments of this application, detection is performed using liquid chromatography-tandem mass spectrometry.

[0038] In some specific embodiments of this application, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry method include:

[0039] The ionization mode was ESI+, the capillary high voltage was 5500V, the backflush gas was 30psi, the ion source temperature was 500℃, and the collision gas was 9psi. The heating gas was 60psi, and multiple reaction monitoring (MRM) mode was used.

[0040] In some specific embodiments of this application, the liquid chromatography-tandem mass spectrometry method includes the following liquid chromatography conditions:

[0041] Chromatographic columns include C18 columns;

[0042] Mobile phase A includes water, formic acid at a volume percentage of 0.08%–0.12%, and ammonium acetate at a volume percentage of 1.8 mmol / mL–2.2 mmol / mL;

[0043] Mobile phase B includes methanol;

[0044] The elution gradient includes:

[0045] From 0 min to 0.01 min, the volume percentage of the mobile phase A is 90%.

[0046] Within 0.01 min to 2 min, the volume percentage of mobile phase A decreased from 90% to 2%.

[0047] For 2 to 3 minutes, the volume percentage of mobile phase A is maintained at 2%.

[0048] Within 3 to 3.1 minutes, the volume percentage of the mobile phase A increased from 2% to 90%.

[0049] For 3.1 to 4 minutes, the volume percentage of the mobile phase A is maintained at 90%.

[0050] In some specific embodiments of this application, the liquid chromatography conditions further include: a flow rate of 0.4 mL / min to 0.6 mL / min; or / and a column temperature of 45°C to 60°C.

[0051] Compared with traditional technologies, this application has the following advantages:

[0052] This application combines zinc sulfate and ammonium acetate in a specific ratio to form a specific composition. Using this composition as a protein precipitant, it can effectively remove hemoglobin from blood samples, which is beneficial for the detection of immunosuppressants (such as tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid) in blood samples. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 Chromatograms of five immunosuppressants;

[0055] Figure 2 , Figure 3 , Figure 4 This is a comparative graph showing analytical samples prepared using different methods for different whole blood samples. Detailed Implementation

[0056] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0057] Unless otherwise defined, 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 application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0058] the term

[0059] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0060] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0061] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0062] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0063] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0064] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0065] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0066] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0067] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0068] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0069] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0070] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0071] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0072] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0073] A first aspect of this application provides a protein precipitation composition comprising zinc sulfate heptahydrate and ammonium acetate in a ratio of (5-17) mg:(0.5-1) mmol.

[0074] In this application, the amount of zinc sulfate heptahydrate in the protein precipitation composition is, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mg, and the corresponding amount of ammonium acetate is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mmol. It is understood that the zinc sulfate heptahydrate in this application can be replaced by zinc sulfate hexahydrate, zinc sulfate monohydrate, anhydrous zinc sulfate, etc., containing an equivalent amount of zinc sulfate.

[0075] In some examples of this application, the protein precipitation composition comprises zinc sulfate heptahydrate and ammonium acetate in a ratio of (10-15) mg:(0.5-0.8) mmol / mL.

[0076] A second aspect of this application provides a protein precipitant comprising zinc sulfate heptahydrate at a solvent concentration of 5 mg / mL to 17 mg / mL and ammonium acetate at a concentration of 0.5 mmol / mL to 1 mmol / mL.

[0077] In the protein precipitant described in this application, the concentration of zinc sulfate heptahydrate is, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mg / mL, and the amount of ammonium acetate is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mmol / mL. It is understood that the concentration of zinc sulfate heptahydrate in this application can be replaced by zinc sulfate hexahydrate, zinc sulfate monohydrate, anhydrous zinc sulfate, etc., containing an equivalent amount of zinc sulfate.

[0078] In some examples of this application, the protein precipitant includes the solvent, zinc sulfate heptahydrate at 10 mg / mL to 15 mg / mL, and ammonium acetate at 0.5 mmol / mL to 0.8 mmol / mL.

[0079] In some examples of this application, the solvent may include, but is not limited to, water.

[0080] A third aspect of this application provides a protein precipitation combination product, the protein precipitation combination product comprising:

[0081] The protein precipitation composition or the protein precipitant described herein; and,

[0082] Other protein precipitants.

[0083] This application does not specifically limit the types of other protein precipitants mentioned, including but not limited to methanol.

[0084] A fourth aspect of this application provides a detection kit, the detection kit comprising:

[0085] The protein precipitation composition, the protein precipitation agent, or the protein precipitation combination product described herein.

[0086] In some examples of this application, the test kit further includes one or more of the following: standards, quality control products, internal standard solutions, mobile phase reagents, and glassware.

[0087] In some examples of this application, the standard and / or the quality control products include immunosuppressants; the internal standard solution includes an internal standard of the immunosuppressant.

[0088] In some examples of this application, the immunosuppressants include one or more of tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid.

[0089] In some examples of this application, the internal standard solution comprises a mixture of the other protein precipitant and the internal standard.

[0090] In some examples of this application, the mobile phase reagent includes one or more of formic acid, ammonium acetate, and methanol.

[0091] In some examples of this application, the vessel includes a protein precipitation plate.

[0092] In some examples of this application, the standards and / or the quality control products include blood substitute matrix and the immunosuppressant standards.

[0093] In some examples of this application, the blood substitute matrix comprises bovine serum albumin aqueous solution and bovine whole blood. The volume ratio of the bovine serum albumin aqueous solution to the bovine whole blood is 1:(2.5-3.5), for example, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, or 1:3.5, based on the concentration of bovine serum albumin in the aqueous solution being 1 wt% to 5 wt% (e.g., 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%).

[0094] In some examples of this application, the bovine serum albumin aqueous solution further includes one or more of preservatives and antioxidants. This application does not specifically limit the type of preservative, but includes, but is not limited to, Proclin 300. This application does not specifically limit the type of antioxidant, but includes, but is not limited to, ascorbic acid, sodium metabisulfite, 2,6-di-tert-butyl-p-cresol, and vitamin E.

[0095] In some examples of this application, the concentration of Proclin 300 in the bovine serum albumin aqueous solution is 0.05 wt% to 0.1 wt%, for example, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, and 0.1 wt%.

[0096] In some examples of this application, the concentrations of the ascorbic acid and the sodium metabisulfite in the bovine serum albumin aqueous solution are each independently 10 mg / mL to 20 mg / mL, for example 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, and 20 mg / mL.

[0097] In some examples of this application, the concentrations of the 2,6-di-tert-butyl-p-cresol and the vitamin E in the bovine serum albumin aqueous solution are each independently 0.5 mg / mL to 1 mg / mL, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg / mL.

[0098] It is understood that the bovine whole blood contains anticoagulants. These anticoagulants include, but are not limited to, sodium citrate, lithium heparin, and sodium heparin.

[0099] A fourth aspect of this application provides a method for preparing a blood analysis sample, wherein the method uses the protein precipitation composition, the protein precipitant, the protein precipitation combination product, or the detection kit to pretreat the blood to be tested to prepare the analysis sample.

[0100] In some examples of this application, the preparation method includes the following steps: mixing the internal standard solution, standard, quality control product and the blood sample to be tested, adding the precipitant to the resulting mixture, mixing well, collecting the supernatant, and preparing the analytical sample.

[0101] In some examples of this application, the amount of protein precipitant used for every 50 μL of the blood sample to be tested is 45 μL-55 μL, for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 μL.

[0102] This application does not impose any particular limitation on the method of collecting the supernatant, including but not limited to filtration and centrifugation. For example, filtration can be performed using a positive pressure device / negative pressure device, and the volume of the filtered filtrate can be made up by adding ultrapure water as needed. For example, centrifugation can be performed, such as centrifuging at 5000 rpm for 10 minutes.

[0103] A fifth aspect of this application provides a method for detecting blood analysis samples, the method comprising the following steps:

[0104] The analytical sample was prepared using the aforementioned preparation method; and,

[0105] The analyzed sample was then tested.

[0106] In some specific embodiments of this application, detection is performed using liquid chromatography-tandem mass spectrometry.

[0107] In some specific embodiments of this application, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry method include:

[0108] The ionization mode was ESI+, the capillary high voltage was 5500V, the backflush gas was 30psi, the ion source temperature was 500℃, and the collision gas was 9psi. The heating gas was 60psi, and multiple reaction monitoring (MRM) mode was used.

[0109] In some specific embodiments of this application, the liquid chromatography-tandem mass spectrometry method includes the following liquid chromatography conditions:

[0110] Chromatographic columns include C18 columns;

[0111] Mobile phase A comprises water and formic acid at a volume percentage of 0.08% to 0.12% (e.g., 0.08%, 0.09%, 0.1%, 0.11%, 0.12%) and ammonium acetate at a volume percentage of 1.8 mmol / mL to 2.2 mmol / mL (e.g., 1.8, 1.9, 2.0, 2.1, 2.2 mmol / mL);

[0112] Mobile phase B includes methanol;

[0113] The elution gradient includes:

[0114] From 0 min to 0.01 min, the volume percentage of the mobile phase A is 90%.

[0115] Within 0.01 min to 2 min, the volume percentage of mobile phase A decreased from 90% to 2%.

[0116] For 2 to 3 minutes, the volume percentage of mobile phase A is maintained at 2%.

[0117] Within 3 to 3.1 minutes, the volume percentage of the mobile phase A increased from 2% to 90%.

[0118] For 3.1 to 4 minutes, the volume percentage of the mobile phase A is maintained at 90%.

[0119] In some specific embodiments of this application, the liquid chromatography conditions further include a flow rate of 0.4 mL / min to 0.6 mL / min, for example, 0.4, 0.5, or 0.6 mL / min.

[0120] In some specific embodiments of this application, the column temperature is 45℃~60℃, for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60℃.

[0121] This application can better precipitate proteins in human whole blood, thereby obtaining a cleaner solution for instrumentation. This not only reduces the wear and tear on the chromatographic column and mass spectrometer, but also reduces the interference of impurities in human whole blood with the analytes.

[0122] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0123] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0124] This application relates to detection kits and methods for five immunosuppressants (tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid). Samples are pretreated in a 96-well protein precipitation plate by mixing zinc sulfate heptahydrate, ammonium acetate, pure water, and methanol dilution containing internal standards. After positive or negative pressure filtration, the samples can be directly analyzed using liquid chromatography-tandem mass spectrometry. Sirolimus and everolimus use sirolimus-D3 as the internal standard, mycophenolic acid uses mycophenolic acid-D3 as the internal standard, and the internal standards for tacrolimus and cyclosporine A use their corresponding structural analogs, ascomycin and cyclosporine D, instead of isotopically labeled internal standards, thus reducing detection costs while ensuring accurate results.

[0125] Example 1

[0126] 1. Precipitator

[0127] The precipitant is an aqueous solution of zinc sulfate and ammonium acetate, wherein the concentration of zinc sulfate heptahydrate is 15 mg / mL and the concentration of ammonium acetate is 0.5 mmol / mL.

[0128] 2. Test kit

[0129] 2.1 Precipitator

[0130] As described in item 1.

[0131] 2.2 Calibrators and Quality Control Products

[0132] Both the calibrators and quality control products contain blood substitute matrix and immunosuppressant standards (tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid). The blood substitute matrix in the calibrators and quality control products has the same composition. The only difference between the two reagents is the concentration of the immunosuppressant standards they contain.

[0133] The blood substitute matrix is ​​a mixture of bovine serum albumin aqueous solution and calf whole blood in a volume ratio of 1:3. The bovine serum albumin aqueous solution contains bovine serum albumin at a concentration of 3 wt%, preservative Proclin 300 at a concentration of 0.05 wt%, and ascorbic acid at a concentration of 15 mg / mL. The bovine whole blood contains sodium citrate as an anticoagulant.

[0134] The calibrators include calibrators S1 to S6, and the quality control products include quality control products 1 to 3. The concentrations of the immunosuppressants are as follows:

[0135] Table 1

[0136]

[0137] Both calibrators and quality control samples were prepared using the following method: a mixture of calf whole blood and bovine serum albumin solution was used as the blood substitute matrix, and a methanol solution of mixed standards of different concentrations (tacrolimus, sirolimus, everolimus, cyclosporine A and mycophenolic acid) was added and shaken to mix. The volume ratio of the blood substitute matrix to the methanol solution of mixed standards of different concentrations was 49:1.

[0138] 2.3 Internal Standard Solution

[0139] The internal standard solution was a methanol solution containing sirolimus-D3, mycophenolic acid-D3, ascomycin, and cyclosporine D, with concentrations of each immunosuppressant being 2 ng / mL, 100 ng / mL, 2 ng / mL, and 2 ng / mL, respectively.

[0140] 2.4 Mobile Phase

[0141] Phase A consists of 0.1% (v / v) formic acid and 2 mmol / mL ammonium acetate aqueous solution; Phase B consists of methanol.

[0142] 2.5 Protein Precipitation Plate

[0143] The 96-well protein precipitation plate uses polypropylene packing material. Equipping the plate reduces manual steps, increases sample throughput, and facilitates integration with automated pretreatment platforms.

[0144] 3. Testing

[0145] The test kit is used to preprocess calibrators, quality control samples, and whole blood samples from the subjects. After preprocessing, the processed samples are analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to achieve accurate quantification of five immunosuppressant drugs in whole blood. Specifically,

[0146] The preprocessing steps include:

[0147] (1) Take 100 μL of internal standard solution into the corresponding well of the 96-well protein precipitation plate to be used;

[0148] (2) Add 50 μL of calibrator, 50 μL of quality control material and 50 μL of whole blood sample from the test subject;

[0149] (3) Add 50 μL of precipitant and seal (cover with a gasket);

[0150] (4) Place it on a microplate shaker and shake to mix (1500 rpm, 10 min);

[0151] (5) Filter using a positive pressure device / negative pressure device (0.22μm), and collect the filtrate using a 1mL 96-well receiving plate;

[0152] (6) Add 150 μL of ultrapure water, place it on a microplate shaker and shake to mix (500 rpm, 5 min) to prepare the analytical sample.

[0153] The above-mentioned samples were analyzed by liquid chromatography-tandem mass spectrometry.

[0154] In the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method, the mass spectrometry conditions included: ionization mode ESI+, capillary high pressure of 5500 V, backflush gas of 30 psi, ion source temperature of 500 °C, collision gas of 9 psi, and heating gas of 60 psi. Multiple reaction monitoring (MRM) mode was used, and the MRM parameters for the compounds were as follows:

[0155] Table 2

[0156]

[0157] In the liquid chromatography-tandem mass spectrometry method, the liquid chromatography conditions include:

[0158] chromatographic column used 2.6um C18 LC Column 50×2.1mm;

[0159] The mobile phase A consists of 0.1% (v / v) formic acid and 2 mmol / mL ammonium acetate aqueous solution, and phase B consists of methanol;

[0160] The elution gradients used are shown in the table below:

[0161] Table 3

[0162] Time (min) Mobile phase A% Mobile phase B% Flow rate (mL / min) 0 min~0.01 min 90 10 0.5 0.01 min ~ 2 min 2 98 0.5 2 min ~ 3 min 2 98 0.5 3 min ~ 3.1 min 90 10 0.5 3.1 min~4 min 90 10 0.5

[0163] According to the table above, the elution gradient is:

[0164] From 0 min to 0.01 min, the volume percentage of the mobile phase A is 90%.

[0165] Within 0.01 min to 2 min, the volume percentage of mobile phase A decreased from 90% to 2%.

[0166] For 2 to 3 minutes, the volume percentage of mobile phase A is maintained at 2%.

[0167] Within 3 to 3.1 minutes, the volume percentage of the mobile phase A increased from 2% to 90%.

[0168] 3.1 min to 4 min, the volume percentage of the mobile phase A is maintained at 90%;

[0169] The flow rate was 0.5 mL / min;

[0170] The column temperature is 55℃;

[0171] The data processing method involves recording the chromatograms of each test sample, the peak area of ​​the analyte, and the peak area of ​​the corresponding internal standard, and calculating the ratio of the peak area to the internal standard peak area. A standard curve is plotted and fitted using the ratio of the calibrator's labeled concentration (x) to the peak area (y). The ratio of the peak area of ​​the test sample to the internal standard peak area is then substituted into the fitted standard curve equation to calculate the concentration of the analyte in the sample. Figure 1 The chromatograms are of five immunomodulators.

[0172] 4. Comparison of different methods

[0173] 4.1 Sample Preparation

[0174] This application compares different pretreatment methods for human whole blood samples:

[0175] The above-mentioned samples were pretreated using traditional pretreatment methods, and the protein precipitation effect of human whole blood samples was observed.

[0176] (1) Preprocessing method of this application:

[0177] 1) Take 100 μL of internal standard solution into the corresponding well of the 96-well protein precipitation plate to be used;

[0178] 2) Add 50 μL of human whole blood sample;

[0179] 3) Add another 50 μL of precipitant and seal the film (cover with a gasket);

[0180] 4) Place the mixture on a microplate shaker and shake to mix (1500 rpm, 5 min). Centrifuge (5000 rpm, 10 min) to obtain the supernatant, and observe it.

[0181] (2) Traditional protein precipitation methods:

[0182] Take 50 μL of human whole blood sample, add 100 μL of methanol, vortex mix, then add 50 μL of 0.1 M zinc sulfate solution, place on a microplate shaker and shake to mix (1500 rpm, 5 min), centrifuge (5000 rpm, 10 min), and collect the supernatant for observation.

[0183] For a single person's whole blood sample, the analytical sample prepared using the two methods described above yielded the following results: Figure 2 As shown. According to Figure 2 It is evident that the pretreatment method of this application yields a clearer and more transparent supernatant solution, which shows no significant change after standing at room temperature for 24 hours. In contrast, the traditional method, which uses zinc sulfate and methanol for protein precipitation, produces a pale red solution with obvious impurities precipitating out after standing for 24 hours.

[0184] For another person's whole blood sample, the analytical results of the sample prepared using the above two methods are as follows: Figure 3 As shown. Figure 3 In the diagram, A and B correspond to the supernatant prepared by the method of this application, and E, F, and G correspond to the supernatant prepared by conventional methods. Figure 3 As can be seen, the supernatant prepared by the method of this application is clear and transparent, while the supernatant prepared by the traditional method is light red. This result shows that the method of this application can remove proteins and other impurities more effectively.

[0185] For another whole blood sample, the analytical results of the sample prepared using the two methods described above are as follows: Figure 4 As shown. Figure 4 In the diagram, A, B, C, and D correspond to the supernatant prepared by the method of this application, while E, F, and G correspond to the supernatant prepared by conventional methods. According to... Figure 4 As can be seen, the supernatant prepared by the method of this application is clear and transparent, while the supernatant prepared by the traditional method is light red. This result shows that the method of this application can remove proteins and other impurities more effectively.

[0186] 4.2 Accuracy Verification

[0187] The accuracy of the above-mentioned application and conventional methods is verified through the following steps:

[0188] Basic samples containing five immunomodulators and samples with high, medium and low concentration recoveries were prepared using whole blood samples from normal healthy individuals. Supernatants were prepared using the method of this application and conventional methods, respectively, as analytical samples. Three samples of each type were processed in parallel and analyzed. The parameters of liquid chromatography-tandem mass spectrometry are as described in Section 3.

[0189] The recovery rates of human whole blood samples were examined, as shown in the table below: In the method of this application, the recovery rate of immunomodulators in human whole blood was between 85% and 115%, which met the requirements; while in the traditional method, the recovery rate was significantly higher or lower, and after the analytical samples prepared by the traditional method were injected, the chromatographic column showed a significant increase in pressure (i.e., blockage).

[0190] Table 4. Recovery Validation of this Method

[0191]

[0192]

[0193] Table 5. Recovery rate validation of traditional methods

[0194]

[0195] Example 2

[0196] This embodiment provides a detection method for whole blood analysis samples. The only difference from Example 1 is that the concentration of ammonium acetate in the precipitant is 0.5 mmol / mL, and the concentration of zinc sulfate heptahydrate is shown in the table below.

[0197] The chromatographic peak response intensities of whole blood samples from the same individual are shown in the table below. When the concentration of zinc sulfate heptahydrate is 20 mg / mL, the response intensities of the five immunomodulators are significantly lower.

[0198] Table 6

[0199]

[0200]

[0201] Example 3

[0202] This embodiment provides a detection method for whole blood analysis samples. The only difference from Example 1 is that the zinc sulfate heptahydrate precipitant used is 10 mg / mL, and the concentration of ammonium acetate is shown in the table below.

[0203] The chromatographic peak response intensities of whole blood samples from the same individual are shown in the table below. When the concentration of ammonium acetate is 1.5 mmol / mL, the response of the five immunomodulators is significantly lower.

[0204] Table 7

[0205] name Mycophenolic acid Tacrolimus Cyclosporine A Sirolimus Everolimus Ammonium acetate 0.5 mmol / mL 8.79E+05 2.00E+05 2.17E+06 1.42E+05 1.02E+05 Ammonium acetate 0.5 mmol / mL - 2 8.72E+05 1.71E+05 1.89E+06 1.14E+05 1.08E+05 Ammonium acetate 0.5 mmol / mL - 3 8.41E+05 1.68E+05 1.50E+06 1.17E+05 1.00E+05 Ammonium acetate 0.8 mmol / mL 8.36E+05 1.86E+05 2.15E+06 1.10E+05 1.02E+05 Ammonium acetate 0.8 mmol / mL - 2 9.36E+05 1.82E+05 2.24E+06 1.24E+05 1.15E+05 Ammonium acetate 0.8 mmol / mL - 3 8.56E+05 2.09E+05 2.69E+06 1.28E+05 1.30E+05 Ammonium acetate 1 mmol / mL 7.42E+05 2.79E+05 2.97E+06 1.27E+05 1.19E+05 Ammonium acetate 1 mmol / mL - 2 7.17E+05 2.25E+05 2.08E+06 1.12E+05 1.11E+05 Ammonium acetate 1 mmol / mL - 3 7.29E+05 2.96E+05 6.03E+06 1.29E+05 1.26E+05 Ammonium acetate 1.5 mmol / mL 7.11E+05 1.23E+05 1.38E+06 1.56E+05 1.02E+05 Ammonium acetate 1.5 mmol / mL - 2 7.19E+05 1.23E+05 1.33E+06 1.57E+05 1.06E+05 Ammonium acetate 1.5 mmol / mL - 3 7.10E+05 1.12E+05 1.23E+06 1.13E+05 1.02E+05 Ammonium acetate 0.5 mmol / mL (mean) 8.64E+05 1.80E+05 1.85E+06 1.24E+05 1.03E+05 Ammonium acetate 0.8 mmol / mean 8.76E+05 1.92E+05 2.36E+06 1.21E+05 1.16E+05 Ammonium acetate 1 mmol / mean 7.29E+05 2.67E+05 3.69E+06 1.23E+05 1.19E+05 Ammonium acetate 1.5 mmol / mL (mean) 7.13E+05 1.20E+05 1.31E+06 1.42E+05 1.03E+05

[0206] Comparative Example 1

[0207] This comparative example provides an analytical method for whole blood samples. The only difference from the sample preparation method in Example 1 is that the precipitant used is an aqueous solution of ammonium acetate, with a concentration of 1 mmol / mL. During the sample preparation process, the supernatant in step (5) contains many impurities and is therefore not used for analysis.

[0208] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0209] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A composition for protein precipitation, characterized in that, The protein precipitation composition comprises zinc sulfate heptahydrate and ammonium acetate in a ratio of (5-17) mg:(0.5-1) mmol. Optionally, the protein precipitation composition comprises zinc sulfate heptahydrate and ammonium acetate in a ratio of (10-15) mg:(0.5-0.8) mmol / mL.

2. A protein precipitant, characterized in that, The protein precipitant comprises zinc sulfate heptahydrate at a solvent concentration of 5 mg / mL to 17 mg / mL and ammonium acetate at a concentration of 0.5 mmol / mL to 1 mmol / mL. Optionally, the protein precipitant includes the solvent, zinc sulfate heptahydrate at a concentration of 10 mg / mL to 15 mg / mL, and ammonium acetate at a concentration of 0.5 mmol / mL to 0.8 mmol / mL. Optionally, the solvent includes water.

3. A protein precipitation combination product, characterized in that, The protein precipitation combination product includes: The protein precipitation composition of claim 1 or the protein precipitant of claim 2; and, Other protein precipitants; Optionally, the other protein precipitant includes methanol.

4. A test kit, characterized in that, The detection kit includes: The protein precipitation composition of claim 1, the protein precipitant of claim 2, or the protein precipitation combination product of claim 3.

5. The detection kit according to claim 4, characterized in that, The test kit also includes one or more of the following: standards, quality control products, internal standard solutions, mobile phase reagents, and glassware; Optionally, the standard and / or the quality control product includes an immunosuppressant; the internal standard solution includes an internal standard of the immunosuppressant; further optionally, the immunosuppressant includes one or more of tacrolimus, sirolimus, everolimus, cyclosporine A, and mycophenolic acid; further optionally, the internal standard solution includes a mixture of the other protein precipitants and the internal standard. Optionally, the mobile phase reagent includes one or more of formic acid, ammonium acetate, and methanol; Optionally, the vessel includes a protein precipitation plate.

6. The detection kit according to claim 5, characterized in that, The standard and / or the quality control products include blood substitute matrix and the immunosuppressant standard; Optionally, the blood substitute matrix comprises bovine serum albumin aqueous solution and bovine whole blood, wherein the volume ratio of the bovine serum albumin aqueous solution to the bovine whole blood is 1:(2.5-3.5), based on the concentration of bovine serum albumin in the bovine serum albumin aqueous solution being 1wt% to 5wt%. Optionally, the bovine serum albumin aqueous solution further includes one or more of preservatives and antioxidants; Optionally, the preservative includes Proclin 300; Optionally, the antioxidant includes one or more of ascorbic acid, sodium metabisulfite, 2,6-di-tert-butyl-p-cresol, and vitamin E; Optionally, the concentration of Proclin 300 in the bovine serum albumin aqueous solution is 0.05 wt% to 0.1 wt%; or / and, the concentrations of ascorbic acid and sodium metabisulfite in the bovine serum albumin aqueous solution are each independently 10 mg / mL to 20 mg / mL; or / and, the concentrations of 2,6-di-tert-butyl-p-cresol and vitamin E in the bovine serum albumin aqueous solution are each independently 0.5 mg / mL to 1 mg / mL; Optionally, the bovine whole blood contains an anticoagulant; Optionally, the anticoagulant includes one or more of sodium citrate, lithium heparin, and sodium heparin.

7. A method for preparing a blood analysis sample, characterized in that, The preparation method uses the protein precipitation composition of claim 1, the protein precipitant of claim 2, the protein precipitation combination product of claim 3, or the detection kit of any one of claims 4 to 6 to pretreat the blood to be tested and prepare the analytical sample.

8. The method for preparing blood analysis samples according to claim 7, characterized in that, The preparation method includes the following steps: mixing the internal standard solution, standard, quality control product and the blood sample to be tested, adding the precipitant to the resulting mixture, mixing well, collecting the supernatant, and preparing the analytical sample.

9. A method for detecting blood analysis samples, characterized in that, The detection method includes the following steps: The analytical sample was prepared using the preparation method described in any one of claims 7 to 8; and, The analyzed sample was then tested.

10. The method for detecting blood analysis samples according to claim 9, characterized in that, The detection was performed using liquid chromatography-tandem mass spectrometry. Optionally, in the liquid chromatography-tandem mass spectrometry method, the mass spectrometry conditions include: The ionization mode is ESI+, the capillary high voltage is 5500V, the backflush gas is 30psi, the ion source temperature is 500℃, the collision gas is 9psi, the heating gas is 60psi, and the multiple reaction monitoring (MRM) mode is used. Optionally, in the liquid chromatography-tandem mass spectrometry method, the liquid chromatography conditions include: Chromatographic columns include C18 columns; Mobile phase A includes water, formic acid at a volume percentage of 0.08%–0.12%, and ammonium acetate at a volume percentage of 1.8 mmol / mL–2.2 mmol / mL; Mobile phase B includes methanol; The elution gradient includes: From 0 min to 0.01 min, the volume percentage of the mobile phase A is 90%. Within 0.01 min to 2 min, the volume percentage of mobile phase A decreased from 90% to 2%. For 2 to 3 minutes, the volume percentage of mobile phase A is maintained at 2%. Within 3 to 3.1 minutes, the volume percentage of the mobile phase A increased from 2% to 90%. 3.1 min to 4 min, the volume percentage of the mobile phase A is maintained at 90%; Optionally, the liquid chromatography conditions also include: The flow rate is 0.4 mL / min to 0.6 mL / min; or / and the column temperature is 45℃ to 60℃.

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