Sample stable storage method and kit for dried blood spot anti-tuberculosis drug detection

Abstract

The application belongs to the technical field of biological sample analysis, and particularly relates to a sample stable storage method and kit for dry blood spot anti-tuberculosis drug detection, which comprises a sample collection card, a desiccant bag, a deoxidizer bag and an aluminum foil composite film sealing bag; the sample collection card comprises cellulose filter paper and vitamin C, citric acid and butylated hydroxyanisole attached to the cellulose filter paper; the amount of the desiccant bag and the deoxidizer bag is matched with the volume of the aluminum foil composite film sealing bag, so that the relative humidity in the sealed package is maintained below 20%, and the oxygen concentration is maintained below 0.1%. The chemical antioxidant of the collection card and the physical oxygen exclusion and dehumidification of the aluminum foil bag are coupled in depth, so that the six drugs, namely isoniazid, rifampicin, pyrazinamide, ethambutol, linezolid and levofloxacin, are simultaneously stabilized in the DBS at normal temperature.

Description

Technical Field

[0001] This invention belongs to the field of biological sample analysis technology, specifically relating to a method and kit for stable preservation of samples for detecting anti-tuberculosis drugs in dried blood spots. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Standard chemotherapy regimens for drug-resistant tuberculosis typically involve the combined use of multiple drugs. Isoniazid, rifampin, pyrazinamide, and ethambutol, as first-line core drugs, differ significantly in physicochemical properties from second-line core drugs linezolid and levofloxacin. They differ in pH, polarity, photosensitivity, and oxidative sensitivity, resulting in vastly different degradation kinetics for the six target substances on the same collection card (dried blood spot). Rifampin is sensitive to oxidation, isoniazid is easily oxidized and degraded, and levofloxacin is photostable; their degradation pathways and rates differ. This multi-drug coexistence system places extremely high demands on the chemical inertness of the sample collection medium and the microclimate control of the storage environment, rendering conventional filter paper carriers and simple packaging solutions inadequate. This has become a core bottleneck restricting its widespread adoption in the field of anti-tuberculosis TDM.

[0004] Linezolid and levofloxacin have extremely narrow therapeutic windows. Linezolid's blood concentration is closely related to its serious adverse reactions such as bone marrow suppression and peripheral neuropathy, while excessively high concentrations of levofloxacin may induce cardiotoxicity such as QT interval prolongation. Therefore, the accuracy of detection for these two substances is crucial. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method and kit for stable sample preservation for the detection of anti-tuberculosis drugs in dried blood spots.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots, comprising a sample collection card, a desiccant pack, an oxygen absorber pack, and an aluminum foil composite film sealing bag; The sample collection card includes cellulose filter paper and vitamin C, citric acid and butylated hydroxyanisole (BHA) attached to the cellulose filter paper; The amount of desiccant and oxygen absorber packets is matched with the volume of the aluminum foil composite film sealing bag to maintain the relative humidity inside the sealed packaging below 20% and the oxygen concentration below 0.1%.

[0007] Secondly, the present invention provides a method for stable preservation of samples for the detection of anti-tuberculosis drugs in dried blood spots, comprising the following steps: Place the fingertip blood or heel blood to be tested onto the sample collection card, and air dry it in a clean environment with a temperature of 20-30℃ and a humidity of 30%-50% in the dark to obtain a dried blood spot collection card. The dried blood spot collection card was placed in an aluminum foil composite film sealed bag, and a desiccant packet and an oxygen absorber packet were added. The bag was then heat-sealed under a vacuum of not less than -0.08 MPa to obtain the heat-sealed packaging. The desiccant pack is used to maintain the relative humidity inside the heat-sealed packaging below 20%; the oxygen absorber pack is used to reduce the oxygen concentration inside the heat-sealed packaging to below 0.1%. Store the heat-sealed packaging at 15-30℃ away from light.

[0008] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: This invention is the first to modify a DBS data acquisition card by combining vitamin C (free radical scavenging), citric acid (metal ion chelation), and BHA (lipid-soluble photo-oxidative protection), endowing the card with active protection capabilities. These three components complement each other in both the aqueous / lipid phase and free radical scavenging / metal ion chelation dimensions, forming a complete active protection network that cannot be achieved by a single antioxidant or a single type of protectant.

[0009] In this invention, the chemical anti-oxidation of the acquisition card is deeply coupled with the physical oxygen isolation and dehumidification of the aluminum foil bag. The low-oxygen microenvironment not only directly inhibits the oxidation of the target drug, but also delays the consumption of antioxidants so that they can continue to play a protective role, forming a positive feedback protection cycle. This achieves the synchronous stability (>90% for 3 months) of six drugs, including isoniazid, rifampin, pyrazinamide, ethambutol, linezolid, and levofloxacin, in room temperature DBS.

[0010] By controlling the microenvironment within the packaging (RH≤20%, O2≤0.1%), samples can be stably stored for more than 8 weeks under simulated tropical climate (37℃ / 75%RH), completely eliminating reliance on the cold chain.

[0011] In this invention, components such as pretreatment acquisition card, desiccant, deoxidizer, and aluminum foil bag are integrated into a standardized reagent kit. The proportions of each group are precisely matched with the packaging volume to ensure the reliability of microenvironment parameters, reduce operational variability, and make it suitable for promotion in grassroots areas and resource-limited regions. Detailed Implementation

[0012] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0013] In a first aspect, the present invention provides a sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots, comprising a sample collection card, a desiccant pack, an oxygen absorber pack, and an aluminum foil composite film sealing bag; The sample collection card includes cellulose filter paper and vitamin C, citric acid and butylated hydroxyanisole (BHA) attached to the cellulose filter paper; The amount of desiccant and oxygen absorber packets is matched with the volume of the aluminum foil composite film sealing bag to maintain the relative humidity inside the sealed packaging below 20% and the oxygen concentration below 0.1%.

[0014] As a water-soluble free radical antioxidant, vitamin C can effectively diffuse in the aqueous phase of blood spots, directly capturing and eliminating reactive oxygen species such as hydroxyl radicals and superoxide anions generated during oxidation. It can also specifically inhibit the oxidative degradation of anti-tuberculosis drugs such as isoniazid and pyrazinamide, blocking the chain transmission pathway of oxidation reactions.

[0015] Fe naturally present in blood 3+ Cu 2+ Transition metal ions act as catalysts in the Fenton reaction, continuously catalyzing the generation of reactive oxygen species and accelerating drug oxidation. Citric acid can form stable chelates with these metal ions, interrupting the catalytic generation pathway of free radicals at its source, preventing the chain reaction of drug oxidation initiated by metal ions, and reducing the ineffective consumption of vitamin C.

[0016] Butyl hydroxyanisole (BHA), as a lipid-soluble free radical scavenger, can accumulate in the lipid region of cell membranes in dried blood spots. On the one hand, it protects lipid-soluble anti-tuberculosis drugs such as rifampin from the damage of lipid peroxidation chain reactions. On the other hand, it can block the photo-oxidative degradation pathway of the photosensitizing drug levofloxacin and make up for the blind spot of vitamin C in the lipid phase environment.

[0017] Vitamin C directly scavenge existing free radicals, citric acid interrupts the catalytic pathway of free radical generation, and BHA blocks the chain reaction of lipid peroxidation and photo-oxidation. These three components synergistically inhibit oxidative degradation, avoiding the blind spots of single protective mechanisms. The synergistic effect of these three components covers the entire degradation pathway of six anti-tuberculosis drugs (isoniazid, rifampin, pyrazinamide, ethambutol, linezolid, and levofloxacin), ultimately achieving a recovery rate of >90% for all drugs after 3 months of storage at room temperature, thus avoiding the limitation of single protective agents being effective only for specific drugs.

[0018] The chemical protection of the collection card and the subsequent physical protection of the aluminum foil bag—low oxygen, low humidity, and light protection—form a positive feedback loop: the low-oxygen microenvironment of the aluminum foil bag not only directly inhibits drug oxidation but also significantly delays the oxidative consumption of antioxidants such as vitamin C and BHA, allowing the three components to continue to exert their protective function during the 3-month shelf life, avoiding drug degradation caused by rapid antioxidant failure. By inhibiting the differential degradation of different drugs, the concentration ratio of each drug in the dried blood spot is ensured to be consistent with the actual in vivo concentration at the time of blood collection, avoiding concentration detection deviations caused by the degradation of some drugs. In particular, it ensures the detection accuracy of linezolid and levofloxacin, which have narrow therapeutic windows, providing a reliable sample basis for individualized clinical dosing and drug resistance assessment.

[0019] In some embodiments, the mass ratio of vitamin C, citric acid and butylated hydroxyanisole on the cellulose filter paper is 5-20:1-10:1-5.

[0020] Preferably, the mass ratio of vitamin C, citric acid and butylated hydroxyanisole on the cellulose filter paper is 5-20:1-5:1-3.

[0021] More preferably, the mass ratio of vitamin C, citric acid and butylated hydroxyanisole on the cellulose filter paper is 8-15:2-5:1-3.

[0022] In some embodiments, the desiccant package is a silica gel desiccant package.

[0023] In some embodiments, the deoxidizer package is an iron-based deoxidizer package.

[0024] In some embodiments, the sample stabilization kit for detecting anti-tuberculosis drugs in dried blood spots also includes instructions for use.

[0025] Secondly, the present invention provides a method for stable preservation of samples for the detection of anti-tuberculosis drugs in dried blood spots, comprising the following steps: Place the fingertip blood or heel blood to be tested onto the sample collection card, and air dry it in a clean environment with a temperature of 20-30℃ and a humidity of 30%-50% in the dark to obtain a dried blood spot collection card. The dried blood spot collection card was placed in an aluminum foil composite film sealed bag, and a desiccant packet and an oxygen absorber packet were added. The bag was then heat-sealed under a vacuum of not less than -0.08 MPa to obtain the heat-sealed packaging. The desiccant pack is used to maintain the relative humidity inside the heat-sealed packaging below 20%; the oxygen absorber pack is used to reduce the oxygen concentration inside the heat-sealed packaging to below 0.1%. Store the heat-sealed packaging at 15-30℃ away from light.

[0026] In some embodiments, the fingertip blood or heel blood to be tested refers to fingertip blood or heel blood from a patient with drug-resistant tuberculosis during drug treatment; the drugs taken include isoniazid, rifampin, pyrazinamide, ethambutol, linezolid, and levofloxacin.

[0027] In some embodiments, the sample collection card is prepared by immersing cellulose filter paper in a mixed solution containing 0.5%-2% vitamin C (ascorbic acid), 0.1%-1% citric acid and 0.1%-0.5% butylated hydroxyanisole (BHA) for 1-3 hours and then drying it at 40-50°C.

[0028] If the soaking time is too short, the pores of the cellulose filter paper cannot be completely wetted by the pretreatment solution, and the three components cannot be uniformly loaded into the three-dimensional fiber network of the filter paper. This will result in a large difference in the antioxidant content in different areas of the sampling card, leading to significant differences in drug stability at different sampling points. Vitamin C is prone to oxidative degradation in aqueous solution. If the soaking time is too long, it will lead to excessive loss of effective active ingredients in the pretreatment solution, reducing the protective effect of the final sampling card, while increasing the preparation cost and production cycle.

[0029] The drying temperature is 40-50℃, which ensures drying efficiency while reducing the loss of the three modifiers.

[0030] Preferably, the concentration of citric acid is 0.1%-0.5%, where % is a mass percentage.

[0031] Preferably, the concentration of butylated hydroxyanisole is 0.1%-0.3%, where % is a mass percentage.

[0032] The invention will be further described through the following embodiments. The detailed descriptions below are illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] Example 1 The sample collection card was prepared as follows: Whatman 903 cellulose filter paper was soaked in a pretreatment solution (containing 1% vitamin C, 0.3% citric acid and 0.2% BHA, % being mass percentages) for 2 hours at room temperature, then removed and dried at 40℃. It was then cut to the standard DBS collection card size (4 sampling rings, 12mm in diameter).

[0034] Sample collection and drying: Collect approximately 50 μL of blood from the subject's fingertip and drop it onto the prepared sample collection card. Dry the card in a clean bench at 25±2℃ and 40%±5% humidity for 3 hours in the dark to obtain the DBS card.

[0035] Vacuum sealing packaging of samples: Place the dried DBS card into an aluminum foil composite film sealed bag (15cm×20cm, volume about 300mL), add 1 packet of silica gel desiccant (3g) and 1 packet of iron-based deoxidizer (2g), and heat seal under vacuum of -0.09MPa.

[0036] Example 2 The sample collection card was prepared as follows: Whatman 903 cellulose filter paper was soaked in a pretreatment solution (containing 1.5% vitamin C, 0.5% citric acid, and 0.4% BHA, where % is by mass percentage) at room temperature for 1.5 hours, then removed and dried at 45°C. It was then cut to the standard DBS collection card size (4 sampling rings, 12mm in diameter).

[0037] Sample collection and drying: Collect approximately 50 μL of blood from the subject's fingertip and drop it onto the prepared sample collection card. Dry the card in a clean bench at 25±2℃ and 40%±5% humidity for 3 hours in the dark to obtain the DBS card.

[0038] Vacuum sealing packaging of samples: Place the dried DBS card into an aluminum foil composite film sealed bag (15cm×20cm, volume about 300mL), add 1 packet of silica gel desiccant (3g) and 1 packet of iron-based deoxidizer (2g), and heat seal under vacuum of -0.09MPa.

[0039] Example 3 The sample collection card was prepared as follows: Whatman 903 cellulose filter paper was soaked in a pretreatment solution (containing 2% vitamin C, 0.7% citric acid and 0.3% BHA, % being mass percentages) at room temperature for 2.5 hours, then removed and dried at 50℃. It was then cut to the standard DBS collection card size (4 sampling rings, 12mm in diameter).

[0040] Sample collection and drying: Collect approximately 50 μL of blood from the subject's fingertip and drop it onto the prepared sample collection card. Dry the card in a clean bench at 25±2℃ and 40%±5% humidity for 3 hours in the dark to obtain the DBS card.

[0041] Vacuum sealing packaging of samples: Place the dried DBS card into an aluminum foil composite film sealed bag (15cm×20cm, volume about 300mL), add 1 packet of silica gel desiccant (3g) and 1 packet of iron-based deoxidizer (2g), and heat seal under vacuum of -0.09MPa.

[0042] Comparative Example 1 Blank control, simulating the worst-case scenario of existing technology: Ordinary plastic self-sealing bags (Fair FG7055R (150×200mm)) were used without desiccant / oxygen absorber. Ordinary Whatman 903 cellulose filter paper was used as the collection card for blood spot collection.

[0043] Specifically: Cut the Whatman 903 cellulose filter paper to the standard DBS acquisition card size (4 sampling rings, 12mm in diameter) to obtain the sample acquisition card; Collect approximately 50 μL of blood from the subject's fingertip and drop it onto the sample collection card prepared above. Air dry in a clean bench at 25 ± 2℃ and 40% ± 5% humidity for 3 hours in the dark to obtain the DBS card. Place the dried DBS card into a regular plastic resealable bag and heat-seal it.

[0044] Comparative Example 2 The antioxidant pretreatment sample collection card prepared in Example 1 was used, but the packaging was only a regular plastic self-sealing bag (same as Comparative Example 1), without vacuum, desiccant / deoxidizer.

[0045] Specifically: Whatman 903 cellulose filter paper was soaked in a pretreatment solution (containing 1% vitamin C, 0.3% citric acid, and 0.2% BHA, % being mass percentages) for 2 hours at room temperature, then removed and dried at 40°C. The paper was then cut to the standard DBS data acquisition card size (4 sampling rings, 12mm in diameter) to obtain the sample acquisition card. Collect approximately 50 μL of blood from the subject's fingertip and drop it onto the sample collection card prepared above. Air dry in a clean bench at 25 ± 2℃ and 40% ± 5% humidity for 3 hours in the dark to obtain the DBS card. Place the dried DBS card into a regular plastic resealable bag and heat-seal it.

[0046] Comparative Example 3 Ordinary Whatman 903 cellulose filter paper without pretreatment was used as the collection card, but it was placed in an aluminum foil bag and vacuum sealed with desiccant and deoxidizer.

[0047] Specifically: Cut the Whatman 903 cellulose filter paper to the standard DBS acquisition card size (4 sampling rings, 12mm in diameter) to obtain the sample acquisition card; Collect approximately 50 μL of blood from the subject's fingertip and drop it onto the prepared sample collection card. Air dry the card in a clean bench at 25 ± 2℃ and 40% ± 5% humidity for 3 hours in the dark to obtain the DBS card.

[0048] Place the dried DBS card into an aluminum foil composite film sealed bag (15cm×20cm, volume about 300mL), add 1 packet of silica gel desiccant (3g) and 1 packet of iron-based deoxidizer (2g), and heat seal under vacuum of -0.09MPa.

[0049] Comparative Example 4 The difference from Example 1 is that vitamin C was replaced with citric acid in the sample collection card preparation process. The pretreatment solution after replacement contained 1.3% citric acid and 0.2% BHA, where % is a mass percentage. All other aspects are the same as in Example 1.

[0050] Comparative Example 5 The difference from Example 1 is that vitamin C was replaced with BHA during the sample collection card preparation process. The pretreatment solution after replacement contained 0.3% citric acid and 1.2% BHA, where % is a mass percentage. All other aspects are the same as in Example 1.

[0051] Comparative Example 6 The difference from Example 1 is that citric acid was replaced with vitamin C during the sample collection card preparation process. The pretreatment solution after replacement contained 1.3% vitamin C and 0.2% BHA, where % is a mass percentage. All other aspects are the same as in Example 1.

[0052] Comparative Example 7 The difference from Example 1 is that BHA was replaced with vitamin C during the sample collection card preparation process. The pretreatment solution after replacement contained 1.2% vitamin C and 0.3% citric acid, where % is a mass percentage. All other aspects are the same as in Example 1.

[0053] Comparative Example 8 The difference from Example 1 is that the vacuum sealing packaging process of the sample does not include the addition of silica gel desiccant; all other aspects are the same as in Example 1.

[0054] Comparative Example 9 The difference from Example 1 is that the vacuum sealing packaging process of the sample does not involve the addition of an iron-based deoxidizer; all other aspects are the same as in Example 1.

[0055] Comparative Example 10 The difference from Example 1 is that vacuum sealing is not performed in the final sealing and packaging operation; all other aspects are the same as in Example 1.

[0056] Specifically, place the dried DBS card into an aluminum foil composite film sealed bag (15cm×20cm, volume approximately 300mL), add one packet of silica gel desiccant (3g) and one packet of iron-based deoxidizer (2g), and heat seal.

[0057] All packages obtained in Example 1 and Comparative Examples 1-10 were stored under light-protected conditions at a temperature of 25°C. Samples were taken after 0, 1, 2, and 3 months of storage to determine the recovery rate of DBS samples of six anti-tuberculosis drugs after 3 months of storage under different storage conditions (with 0 months as 100%). The results are shown in Table 1.

[0058] Table 1. Recovery rate (%) of DBS samples of six anti-tuberculosis drugs after 3 months of storage.

[0059] Results Analysis: As shown in Table 1, although Comparative Examples 2 and 3 improved the drug recovery rate to varying degrees, the 3-month recovery rate of rifampicin did not exceed 90%, indicating that the protective efficacy of a single measure was significantly insufficient. In particular, the 3-month recovery rate of rifampicin in Comparative Example 1 was only 55.8%, confirming its characteristic of being easily oxidized and degraded at room temperature.

[0060] To quantify the synergistic effect, this invention, based on the assumption that chemical pretreatment and physical barrier packaging act on mutually independent degradation pathways, introduces a probability product model Rtheoretical = 1 - (1 - R2) × (1 - R3) to calculate the theoretical expected value of the recovery rate of Example 1 (i.e., the expected value when the protective effects of Comparative Example 2 and Comparative Example 3 are simply superimposed), and compares it with the actual value of Example 1. The results are shown in Table 2, where the protective effect is characterized by the degradation rate D (D = 100% - measured recovery rate). The lower the D value, the stronger the protection against six anti-tuberculosis drugs when stored at 25°C in the dark for 3 months.

[0061] Table 2 Comparison of theoretical and actual recovery rates (%) after storage at 25℃ for 3 months

[0062] Table 2 shows that the actual recovery rates of the six drugs in Example 1 significantly exceeded the theoretical expected values ​​calculated based on the probability product model, with the actual-expected difference ranging from +14.6 to +31.5 percentage points. This non-additive gain effect cannot be explained by "the simple addition of the two measures," proving that there is a genuine synergistic effect between active chemical protection and passive physical protection in this invention.

[0063] Mechanism analysis: While the antioxidants (vitamin C / BHA) on the data acquisition card scavenge free radicals, the continuously maintained low-oxygen microenvironment (O2≤0.1%) within the aluminum foil bag effectively delays the oxidative consumption of the antioxidants themselves, allowing them to maintain their protective effect for up to 3 months. This secondary protection mechanism for the antioxidants cannot be achieved under single-measure conditions, demonstrating that this invention, through the deep coupling of active and passive measures, produces a technical effect significantly superior to simple superposition.

[0064] Validation of levofloxacin photodegradation protection: To verify the protective effect of this invention against photosensitizing drugs, the following comparative experiment was conducted: Comparative Example 11 The difference from Example 1 is that ordinary transparent plastic self-sealing bags are used for packaging (without aluminum foil bags), while the other conditions are the same as in Example 1.

[0065] The samples from Example 1 and Comparative Example 11 were stored in an indoor lighting environment with an illuminance of 500±50 Lux for 4 weeks at an indoor temperature of 25℃. The recovery rate of levofloxacin was determined, and the results are shown in Table 3.

[0066] Table 3. Levofloxacin recovery rates in Example 1 and Comparative Example 11.

[0067] The results showed that the light-shielding function of the aluminum foil bag provided crucial protection for the photosensitizing drug levofloxacin, and the recovery rate was still above 95% after 4 weeks, verifying the effectiveness of the light-shielding protection.

[0068] Verification of the coupling effect between antioxidant retention rate and low-oxygen environment: To further reveal the mechanism of the synergistic effect, the percentage of residual vitamin C in the collection card under different storage conditions (with the initial amount of adhering vitamin C as 100%) and the changes in oxygen concentration inside the packaging were detected. The results are shown in Table 4.

[0069] Table 4. Trends in residual vitamin C content and oxygen concentration inside the packaging under different storage conditions.

[0070] Note: "--" indicates not measured; Group B's O2 concentration had risen to approximately 18.5% (v / v) after 3 months because ordinary packaging could not maintain a low-oxygen environment.

[0071] Results Analysis: The above experimental results further reveal the intrinsic mechanism of synergistic protection. In Comparative Example 2, which lacked hypoxia protection, vitamin C was largely consumed within 3 months, with a remaining rate of only 12.5%, and the antioxidant protective effect subsequently diminished. However, in the hypoxia packaging environment (O2≤0.1%) of Example 1, the oxidative consumption of vitamin C was significantly inhibited, and the remaining rate was still as high as 61.7% after 3 months.

[0072] The above data indicate that the hypoxic microenvironment not only directly inhibits the oxidative degradation of the target drug, but more importantly, it delays the oxidative consumption of the antioxidant itself, allowing it to continue to exert its free radical scavenging function during long-term storage. This secondary protective effect of the antioxidant cannot be achieved under single-measure conditions, forming the scientific basis for the synergistic gain effect of this invention.

[0073] Stability verification in high temperature and high humidity environments: The recovery rates of each drug were investigated by placing the sample from Example 1 in a constant temperature and humidity chamber at 37°C and 75%RH. The results are shown in Table 5.

[0074] Table 5. Recovery rates (%) of each drug

[0075] The results showed that even under extreme conditions, the recovery rate of all drugs remained above 91% within 8 weeks. The proposed method completely frees DBS samples from dependence on cold chain logistics.

[0076] Example 4: Kit Composition and Usage The kit contains: 20 pretreatment acquisition cards, 20 packets of silica gel desiccant (3g), 20 packets of iron-based deoxidizer (2g), 20 aluminum foil bags, and 1 instruction manual.

[0077] Instructions for use: (a) Collect blood drop card; (b) Air dry in the dark for 2-4 hours; (c) Place in an aluminum foil bag; (d) Add desiccant and oxygen absorber; (e) Vacuum heat seal (≤-0.08MPa); (f) Store and transport at room temperature; (g) Unpack and punch for inspection.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots, characterized in that: Includes sample collection card, desiccant pack, oxygen absorber pack, and aluminum foil composite film sealed bag; The sample collection card includes cellulose filter paper and vitamin C, citric acid and butylated hydroxyanisole (BHA) attached to the cellulose filter paper; The amount of desiccant and oxygen absorber packets is matched with the volume of the aluminum foil composite film sealing bag to maintain the relative humidity inside the sealed packaging below 20% and the oxygen concentration below 0.1%.

2. The sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots according to claim 1, characterized in that: The mass ratio of vitamin C, citric acid and butylated hydroxyanisole on cellulose filter paper is 5-20:1-10:1-5.

3. The sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots according to claim 2, characterized in that: The mass ratio of vitamin C, citric acid and butylated hydroxyanisole on cellulose filter paper is 5-20:1-5:1-3.

4. The sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots according to claim 3, characterized in that: The mass ratio of vitamin C, citric acid and butylated hydroxyanisole on cellulose filter paper is 8-15:2-5:1-3.

5. The sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots according to claim 1, characterized in that: The desiccant package is a silica gel desiccant package.

6. The sample stability preservation kit for detecting anti-tuberculosis drugs in dried blood spots according to claim 1, characterized in that: The deoxidizer package is an iron-based deoxidizer package.

7. A method for stable preservation of samples for the detection of anti-tuberculosis drugs in dried blood spots, characterized in that: Includes the following steps: The fingertip blood or heel blood to be tested is dropped onto the sample collection card described in any one of claims 1-4, and dried in a clean environment at a temperature of 20-30°C and a humidity of 30%-50% in the dark to obtain a dried blood spot collection card. The dried blood spot collection card was placed in an aluminum foil composite film sealed bag, and a desiccant packet and an oxygen absorber packet were added. The bag was then heat-sealed under a vacuum of not less than -0.08 MPa to obtain the heat-sealed packaging. The desiccant pack is used to maintain the relative humidity inside the heat-sealed packaging below 20%; the oxygen absorber pack is used to reduce the oxygen concentration inside the heat-sealed packaging to below 0.1%. Store the heat-sealed packaging at 15-30℃ away from light.

8. The method for stable preservation of samples for detecting anti-tuberculosis drugs in dried blood spots according to claim 7, characterized in that: The sample collection card is prepared by immersing cellulose filter paper in a mixed solution containing 0.5%-2% vitamin C, 0.1%-1% citric acid and 0.1%-0.5% butylated hydroxyanisole for 1-3 hours, and then drying it at 40-50°C.

9. The method for stable preservation of samples for detecting anti-tuberculosis drugs in dried blood spots according to claim 8, characterized in that: The concentration of citric acid is 0.1%-0.5%, where % is a mass percentage; Alternatively, the concentration of butylated hydroxyanisole is 0.1%-0.3%, where % is a mass percentage.

10. The method for stable preservation of samples for detecting anti-tuberculosis drugs in dried blood spots according to claim 8, characterized in that: The fingertip or heel blood to be tested refers to fingertip or heel blood from patients with drug-resistant tuberculosis during drug treatment; the drugs taken include isoniazid, rifampin, pyrazinamide, ethambutol, linezolid, and levofloxacin.

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