A drug-containing biological matrix lyophilized reagent and a preparation method thereof

Abstract

The present application relates to the field of biological detection, and provides a preparation method of a drug-containing biological matrix freeze-dried reagent, steps comprising: S1, performing first freeze-drying treatment on the biological matrix; S2, performing resolubilization treatment on the biological matrix after the first freeze-drying treatment, and performing second freeze-drying treatment; S3, after the step S2 is cycled for several times, slowly adding a fully dissolved drug into the biological matrix after the second freeze-drying treatment and performing final freeze-drying treatment, thereby obtaining the drug-containing biological matrix freeze-dried reagent; the present application adopts step-by-step freeze-drying to perform low-temperature dehydration on the biological matrix, thereby maximally maintaining the biological activity of the biological matrix and the stability of the heat-labile compound therein; the present application adds the drug after the second freeze-drying treatment, thereby solving the problem of drug loss caused by temperature loss and vacuum during the freeze-drying treatment; the drug-containing biological matrix freeze-dried reagent prepared by the present application has the characteristics of anhydrous and oxygen-free, so that the biological matrix reagent can be stably and long-term stored without preservatives.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a drug-containing biological matrix freeze-dried reagent and its preparation method. Background Technology

[0002] Lyophilized reagents containing drug-containing biological matrices are important reference reagents applicable to fields such as forensic medicine and clinical drug monitoring. They are of great value for accurately determining the content of corresponding drugs in test biological matrix samples. In current testing work, laboratories often mix drugs with known content in proportion (mass fraction or mass concentration) with biological matrices in a certain configuration method to prepare their own reference reagents. This operation and the prepared reagents have the following defects: (1) there is a large operational error in a single preparation; (2) the biological matrix of each self-made reagent often comes from different sources and is not standardized. These two defects will lead to inconsistent analytical results.

[0003] In recent years, some developed countries and regions have gradually begun to pay attention to and value this issue. For example, the human serum abuse substance standard (NIST SRM 1959) demonstrates a typical process for preparing drug-containing blood matrix reagents by additive method: collecting drug-free serum from healthy adult volunteers and mixing it, filtering it using a 0.2 μm filter, adding a target compound at a concentration of about 1 μg / mL, mixing, aliquoting, and storing. However, this biomatrix has the following defects: (1) the biomatrix used is different from the real analytical matrix, resulting in inconsistent matrix effect parameters during practical analysis; (2) the prepared product is a water-containing biomatrix reagent, which is easily perishable and cannot be stored and transported for a long time; at the same time, the drug-containing biomatrix reagent used as a reference reagent is not allowed to have preservatives added. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a drug-containing biological matrix freeze-dried reagent and its preparation method.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of this invention is to provide a method for preparing a drug-containing biological matrix lyophilized reagent, comprising the steps of:

[0007] S1. Perform initial freeze-drying on the biological substrate;

[0008] S2. The biological matrix after the first freeze-drying treatment is reconstituted and then freeze-dried again;

[0009] S3. After repeating step S2 several times, the fully dissolved drug is slowly added to the biological matrix after the second freeze-drying treatment and the final freeze-drying treatment is performed to obtain the drug-containing biological matrix freeze-dried reagent.

[0010] The biological matrix includes one of blood, urine, or tissue homogenate; the drug includes at least one of organophosphorus pesticides, benzodiazepines, or tetrodotoxin.

[0011] Preferably, the steps further include:

[0012] S0. Provide a preparation environment, which is located indoors, with a temperature of 18℃-26℃, a relative humidity of 45%-65%, an air exchange rate of more than 15 times / hour, a positive pressure of more than 10Pa, and a unidirectional flow of more than 2% of the total air volume; the materials used in the preparation environment include: a first material; the weight of the first material is more than 99.98% of the total material weight; the first material is composed of metal, polyester, terrazzo, epoxy resin, anti-mildew coating, glass, and rubber.

[0013] Preferably, the blood matrix is ​​fresh whole blood containing an anticoagulant, the blood is stored at a temperature of 4°C, a humidity of 45%-65%, and a storage time of no more than one year.

[0014] Preferably, the urine matrix is ​​fresh urine, the urine is stored at a temperature of 4°C, a humidity of 45%-65%, and a storage time of no more than 72 hours.

[0015] Preferably, the tissue homogenate is prepared from fresh tissue through homogenization treatment, and the tissue homogenate is stored at a temperature of 4°C, a humidity of 45%-65%, and a storage time of no more than 24 hours; the fresh tissue is stored for no more than 72 hours.

[0016] Preferably, the freeze-drying process for the initial freeze-drying treatment includes:

[0017]

[0018] Preferably, the reconstitution treatment includes: adding deionized water to the biological matrix after the first freeze-drying treatment, while simultaneously performing an ultrasonic ice-water bath; the volume ratio of the deionized water to the biological matrix is ​​(2-100):100; the ultrasonic frequency of the ultrasonic bath is 20kHz-40kHz, and the ultrasonic power is not less than 1000W.

[0019] Preferably, the freeze-drying process for the second freeze-drying treatment includes:

[0020]

[0021] Preferably, the final freeze-drying process includes: placing the biological matrix after adding the fully dissolved drug into a nitrogen-filled freeze-drying device and drying it for 2-12 hours.

[0022] Preferably, the method for preparing the fully dissolved drug includes: dissolving a drug with a purity greater than 95% in a neutral reagent, an acidic reagent, or an alkaline reagent; wherein the volume ratio of the neutral reagent, the acidic reagent, or the alkaline reagent to the biological matrix does not exceed 0.02.

[0023] Preferably, after the final freeze-drying process, the product is further packaged.

[0024] More preferably, the encapsulation process includes: sealing the biological matrix after the final freeze-drying treatment in an environment with a humidity of less than 30%.

[0025] A second aspect of the present invention is to provide a drug-containing biological matrix lyophilized reagent prepared by the above-described preparation method.

[0026] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0027] This invention employs stepwise freeze-drying to dehydrate biological matrices at low temperatures, maximizing the preservation of the bioactivity of the matrices and the stability of thermally unstable compounds. Stepwise freeze-drying achieves progressive dehydration of the biological matrix and also enables concentration, allowing for rapid freeze-drying of large-volume samples and improving freeze-drying efficiency. Furthermore, the addition of drugs after the second freeze-drying process solves the problem of drug loss due to temperature loss and vacuum during freeze-drying, preventing changes in drug traceability. The drug-containing biological matrix freeze-dried reagent prepared by this invention is anhydrous and oxygen-free, enabling stable long-term storage without preservatives. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0030] This specific embodiment provides a method for preparing a drug-containing biological matrix lyophilized reagent, the steps of which include:

[0031] S0. Provide a preparation environment, which is located indoors, with a temperature of 18℃-26℃, a relative humidity of 45%-65%, an air exchange rate of more than 15 times / hour, a positive pressure of more than 10Pa, and a unidirectional flow of more than 2% of the total air volume; the materials used in the preparation environment include: a first material; the weight of the first material is more than 99.98% of the total material weight; the first material is composed of metal, polyester, terrazzo, epoxy resin, anti-mildew coating, glass, and rubber.

[0032] S1. Perform a first freeze-drying treatment on at least 1 L of the biological substrate; the freeze-drying procedure for the first freeze-drying treatment includes:

[0033]

[0034] S2. The biological matrix after the first freeze-drying treatment is reconstituted and then freeze-dried again;

[0035] The reconstitution process includes: adding deionized water to the biological matrix after the initial freeze-drying treatment, while simultaneously performing an ultrasonic ice-water bath; the volume ratio of the deionized water to the biological matrix is ​​(2-100):100; the ultrasonic frequency of the ultrasonic bath is 20kHz-40kHz, and the ultrasonic power is not less than 1000W.

[0036] The freeze-drying process for the second freeze-drying treatment includes:

[0037]

[0038] S3. After repeating step S2 several times, the fully dissolved drug is slowly added to the biological matrix after the second freeze-drying treatment, and then the final freeze-drying treatment and encapsulation treatment are performed to obtain the drug-containing biological matrix freeze-dried reagent.

[0039] The final freeze-drying process includes: placing the biological matrix, after adding the fully dissolved drug, into a nitrogen-filled freeze-drying device and drying it for 2-12 hours;

[0040] The method for preparing the fully dissolved drug includes: dissolving a drug with a purity greater than 95% in a neutral reagent, an acidic reagent, or an alkaline reagent; the volume ratio of the neutral reagent, the acidic reagent, or the alkaline reagent to the biological matrix does not exceed 0.02;

[0041] The encapsulation process includes: sealing the biological matrix after the final freeze-drying treatment in an environment with a humidity of less than 30%.

[0042] The biological matrix includes one of blood, urine, or tissue homogenate; the drug includes at least one of organophosphorus pesticides, benzodiazepines, or tetrodotoxin.

[0043] After the preparation of the drug-containing biological matrix freeze-dried reagent is completed, its quality can be evaluated, including: uniformity test, short-term test, long-term test and accelerated test;

[0044] The homogeneity test includes: taking 10% of the total amount of the drug-containing biological matrix lyophilized reagent prepared at equal intervals (if less than 20 bottles, count as 20 bottles) for the homogeneity test; accurately reconstitute the drug-containing biological matrix lyophilized reagent with pure water according to the labeled amount; after the drug-containing biological matrix lyophilized reagent is fully reconstituted, perform a one-way ANOVA on the homogeneity of the product with reference to the "CNAS GL-003 Guidelines for Evaluation of Homogeneity and Stability of Proficiency Testing Samples", with the F value being less than the critical value Fα of the significance level α (α=0.05);

[0045] The short-term test includes: randomly selecting 20% ​​of the total prepared amount of drug-containing biological matrix lyophilized reagent (if less than 120 bottles, count as 120 bottles) for short-term testing; dividing the selected drug-containing biological matrix lyophilized reagent into 3 equal portions and storing them under three environmental conditions: -20℃±3℃ and humidity <65%, 4℃±3℃ and humidity <80%, and 20℃±3℃ and humidity <85% respectively; on days 0, 1, 5, 10, and 15 of the storage period, taking out 1 / 5 of the amount stored under each condition, and performing a parametric t-test on the product's storage stability according to the "CNAS GL-003 Guidelines for Evaluation of Homogeneity and Stability of Proficiency Testing Samples". The t-value should be less than the critical value tα of the significance level α (α=0.05), and the change in the quantitative value compared with the previous test should not exceed 2%.

[0046] The long-term test includes: randomly selecting 20% ​​of the total prepared amount of drug-containing biological matrix lyophilized reagent (if less than 192 bottles, count as 192 bottles) for the long-term test; dividing the selected drug-containing biological matrix lyophilized reagent into 3 equal portions and storing them under three environmental conditions: -20℃±3℃ and humidity <65%, 4℃±3℃ and humidity <80%, and 20℃±3℃ and humidity <85% respectively; on days 0, 15, 30, 60, 90, 120, 150, and 180 of the storage period, taking 1 / 8 of the amount stored under each condition, and performing a parametric t-test on the product's storage stability according to the "CNAS GL-003 Guidelines for Evaluation of Homogeneity and Stability of Proficiency Testing Samples". The t-value should be less than the critical value tα of the significance level α (α=0.05), and the quantitative value should not change by more than 2% compared with the previous value.

[0047] The accelerated testing includes: randomly selecting 20% ​​of the total prepared amount of drug-containing biological matrix lyophilized reagent (if less than 40 bottles, count as 40 bottles) for accelerated testing; storing the selected drug-containing biological matrix lyophilized reagent in an environment with a temperature of 65℃±3℃ and humidity >85%; on days 0, 1, 5, 10, and 15 of the storage period, taking out 1 / 5 of the amount stored under each condition; and performing a parametric t-test on the product's storage stability according to the "CNAS GL-003 Guidelines for Evaluation of Homogeneity and Stability of Proficiency Testing Samples". The t-value should be less than the critical value tα of the significance level α (α=0.05), and the quantitative value should not change by more than 2% compared with the previous value.

[0048] The present invention will be further described below with reference to specific embodiments, but these are not intended to limit the scope of the invention.

[0049] Example 1

[0050] This embodiment provides a method for preparing a lyophilized reagent containing dichlorvos-based human urine matrix, the steps of which include:

[0051] Ten healthy volunteers were recruited. For one week prior to providing urine samples, volunteers were required to maintain a healthy diet and regular lifestyle, abstain from smoking and alcohol, and refrain from using any medications or health supplements. Starting eight days after recruitment, 34 bottles of urine samples (approximately 5 liters in total) were collected from the volunteers within 24 hours. The urine samples were tested the following day under the aforementioned restrictive conditions. Drug-free testing showed that all urine samples were free of organophosphorus pesticides such as dichlorvos and their metabolites. Physiological indicators showed that the urine samples had normal pH (range 4.5-8), negative nitrite levels, negative white blood cell and red blood cell counts, negative bilirubin and urobilinogen levels, normal specific gravity (range 1.010-1.030), negative ketone bodies, and normal urine glucose levels.

[0052] After the urine matrix is ​​first lyophilized, it is reconstituted and then lyophilized again. After one cycle of reconstitution and lyophilization, fully dissolved dichlorvos is slowly added to the lyophilized urine matrix and then lyophilized and packaged to obtain a human urine matrix lyophilized reagent containing dichlorvos.

[0053] After being stored for 9 months, the lyophilized reagent containing dichlorvos-containing human urine matrix prepared by the method described in this embodiment was simultaneously delivered to two laboratories within 8 hours, along with fresh urine samples from individuals poisoned by dichlorvos. The two laboratories analyzed the fresh urine samples from the individuals poisoned by dichlorvos using both the lyophilized reagent containing dichlorvos-containing human urine matrix prepared by the method described in this embodiment and a laboratory-made temporary reagent containing dichlorvos. The analysis results showed that, using the lyophilized reagent containing dichlorvos-containing human urine matrix prepared by the method described in this embodiment as a reference, the dichlorvos content in the fresh urine samples from the individuals poisoned by dichlorvos obtained by the two laboratories was relatively consistent (difference between the two samples was 2.7%). However, using the laboratory-made temporary reagent containing dichlorvos as a reference, the dichlorvos content in the fresh urine samples from the individuals poisoned by dichlorvos obtained by the two laboratories differed significantly (difference between the two samples was 198.7%). This indicates that the preparation method of this invention provides a standardized supply of biological matrix and has a positive effect on analytical practice.

[0054] Example 2

[0055] This embodiment provides a method for preparing a lyophilized reagent containing diazepam and human blood matrix, the steps of which include:

[0056] Two batches of 1-liter blood matrix from healthy individuals, obtained through legal procedures, were received and tested. The first batch of blood matrix showed no presence of diazepam or other benzodiazepines and their metabolites in a drug-free test. Physiological indicators showed a blood glucose level of 8.7 mmol / L, serum potassium of 4.1 mmol / L, alanine aminotransferase (ALT) of 8 IU / L, and total bilirubin of 12 μmol / L, all within normal limits. Physical indicators showed a blood matrix annual value of 4.2 (normal).

[0057] The second batch of blood matrix drug-free test results showed that the blood matrix did not contain diazepam or other benzodiazepines and their metabolites; physiological index test results showed that the blood matrix blood glucose was 8.9 mmol / L, serum potassium was 3.9 mmol / L, alanine aminotransferase was 8 IU / L, and total bilirubin was 14 μmol / L, all of which were normal; physical index test results showed that the blood matrix annual value was 4.3 (normal).

[0058] After the blood matrix is ​​first lyophilized, it is reconstituted and then lyophilized again. The fully dissolved diazepam is slowly added to the lyophilized blood matrix and then lyophilized and packaged to obtain the first diazepam-containing human blood matrix lyophilized reagent and the second diazepam-containing human blood matrix lyophilized reagent.

[0059] Using the first and second diazepam-containing human blood matrix lyophilization reagents as reference reagents, blood drug concentrations were monitored in five patients treated with diazepam, with three monitoring sessions per patient. The results showed no statistically significant difference between the data obtained using the first and second diazepam-containing human blood matrix lyophilization reagents as reference reagents, indicating that the preparation method of the present invention has stable batch-to-batch consistency and has a positive effect on analytical practice.

[0060] Example 3

[0061] This embodiment provides a method for preparing a freeze-drying reagent for a homogenized fish meat matrix containing tetrodotoxin, the steps of which include:

[0062] 1500 grams of common grass carp were purchased, deboned and dissected, and the muscle tissue was homogenized. The homogenized fish meat matrix was then tested. The drug-free test results showed that the homogenized fish meat matrix did not contain tetrodotoxin.

[0063] After the homogenized fish meat matrix is ​​first freeze-dried, it is reconstituted and then freeze-dried again. After three cycles of reconstitution and freeze-drying, fully dissolved tetrodotoxin is slowly added to the freeze-dried homogenized fish meat matrix and then freeze-dried and packaged to obtain the tetrodotoxin-containing homogenized fish meat matrix freeze-dried reagent.

[0064] After being stored for one month, a lyophilized fish meat homogenate containing tetrodotoxin was mailed from Shanghai to Urumqi, Xinjiang Uygur Autonomous Region; Gonghe County, Qinghai Province; and Qujing, Yunnan Province. The packages were not specially insulated. The recipients returned the packages to Shanghai the following day. Upon receiving the returned packages, the lyophilized fish meat was analyzed using both the lyophilized fish meat homogenate containing tetrodotoxin from the package and a lyophilized fish meat homogenate containing tetrodotoxin stored at low temperature (4°C) at the inventor's facility as reference reagents. The experimental results showed… The analytical test results obtained using four reagents as reference reagents showed high consistency (the maximum difference between two samples was 4.4%), indicating that the drug-containing biological matrix lyophilized reagent prepared by the method of the present invention has high stability. In this example, the longest express delivery time reached 12 days, and the weather temperature during express delivery was above 28°C. The consistency of the analytical results shows that high temperature and long-term logistics transportation do not affect the key traceability indicators of the drug-containing biological matrix lyophilized reagent, indicating that the preparation method of the present invention solves the problem that biological matrix reagents are prone to spoilage and deterioration and are not suitable for long-term storage and transportation.

[0065] In summary, this invention employs stepwise freeze-drying for low-temperature dehydration of the biological matrix, maximizing the preservation of its bioactivity and the stability of thermally unstable compounds. Stepwise freeze-drying achieves progressive dehydration of the biological matrix and also enables concentration, allowing for rapid freeze-drying of large-volume samples and improving freeze-drying efficiency. Furthermore, the addition of drugs after the second freeze-drying process solves the problem of drug loss due to temperature loss and vacuum during freeze-drying, preventing changes in drug traceability. The drug-containing biological matrix freeze-dried reagent prepared by this invention is anhydrous and oxygen-free, enabling stable long-term storage without preservatives.

[0066] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content of this specification should be included within the protection scope of the present invention.

Claims

1. A method for preparing a lyophilized reagent of a medicated biological matrix characterized by the steps of include: S1. Perform initial freeze-drying on the biological substrate; S2. The biological matrix after the first freeze-drying treatment is reconstituted and then freeze-dried again; S3. After repeating step S2 several times, the fully dissolved drug is slowly added to the biological matrix after the second freeze-drying treatment and the final freeze-drying treatment is performed to obtain the drug-containing biological matrix freeze-dried reagent. The biological matrix includes one of blood, urine, or tissue homogenate; the drug includes at least one of organophosphorus pesticides, benzodiazepines, or tetrodotoxin.

2. The preparation method according to claim 1, characterized in that, The steps also include: S0. Provide a preparation environment, which is located indoors, with a temperature of 18℃-26℃, a relative humidity of 45%-65%, an air exchange rate of more than 15 times / hour, a positive pressure value of more than 10Pa, and a unidirectional flow of more than 2% of the total air supply volume.

3. The preparation method according to claim 1, characterized in that, The initial freeze-drying process includes: 。 4. The preparation method according to claim 1, characterized in that, The reconstitution process includes: adding deionized water to the biological matrix after the initial freeze-drying treatment, while simultaneously performing an ultrasonic ice-water bath; the volume ratio of the deionized water to the biological matrix is ​​(2-100):100; the ultrasonic frequency of the ultrasonic ice-water bath is 20kHz-40kHz, and the ultrasonic power is not less than 1000W.

5. The preparation method according to claim 1, characterized in that, The final freeze-drying process includes: placing the biological matrix, after adding the fully dissolved drug, into a nitrogen-filled freeze-drying device and drying it for 2-12 hours.

6. The preparation method according to claim 1, characterized in that, The method for preparing the fully dissolved drug includes: dissolving a drug with a purity greater than 95% in a neutral reagent, an acidic reagent, or an alkaline reagent; the volume ratio of the neutral reagent, the acidic reagent, or the alkaline reagent to the biological matrix does not exceed 0.

02.

7. The preparation method according to claim 1, characterized in that, After the final freeze-drying process, the product is further packaged.

8. The preparation method according to claim 7, characterized in that, The encapsulation process includes sealing the biological matrix after final freeze-drying in an environment with a humidity of less than 30%.

9. A drug-containing biological matrix lyophilized reagent prepared by the preparation method according to any one of claims 1-8.

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