Detection method of anti-tuberculosis drugs and their metabolites in kits and samples

Abstract

The present invention specifically provides a kit and a method for detecting anti-tuberculosis drugs and their metabolites in a sample. The kit is used to detect anti-tuberculosis drugs and their metabolites in a sample, including calibrators, quality control products, instrument quality control products and isotope internal standards; the calibrators and quality control products contain rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and deacetyl rifampicin; the instrument quality control product includes a methanol solution containing rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and deacetyl rifampicin; the isotope internal standard contains an internal standard corresponding to the substance contained in the calibrator. The kit of the present invention can detect the concentration of anti-tuberculosis drugs and their metabolites in various sample types.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tuberculosis drug detection, and in particular relates to a kit and a method for detecting anti-tuberculosis drugs and metabolites thereof in a sample. Background Art

[0002] Tuberculosis has the characteristics of multidrug resistance (MDR-TB) and extreme drug resistance (XDR-TB), so how to suppress the drug resistance of tuberculosis is very important. Therapeutic drug concentration monitoring (TDM) is helpful for clinicians to provide reference for early personalized treatment and optimized treatment of tuberculosis patients. The existing anti-tuberculosis drug concentration monitoring method is generally detected by a kit, but the current kit can only perform single drug analysis or evaluate first-line anti-tuberculosis drugs at the same time, and can only detect the concentration of anti-tuberculosis drugs in plasma or serum. The number of detectable samples is small, and according to the test results of the current kit, the drug metabolism status of the patient cannot be obtained, nor can it be effectively analyzed whether there is any interaction between drugs when the patient uses multiple anti-tuberculosis drugs at the same time. Summary of the invention

[0003] The first purpose of the embodiment of the present invention is to provide a kit to solve the problem that the existing anti-tuberculosis drug detection can only monitor the concentration of the anti-tuberculosis drug but cannot monitor its corresponding metabolic status.

[0004] The technical solution adopted in the embodiment of the present invention is as follows:

[0005] A kit for detecting anti-tuberculosis drugs and their metabolites in a sample;

[0006] The kit comprises:

[0007] A calibrator, wherein the calibrator contains rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline, and desacetyl rifampicin;

[0008] Quality control products, wherein the quality control products contain rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and desacetyl rifampicin;

[0009] Instrument quality control products, including methanol solutions containing rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and desacetyl rifampicin;

[0010] And, isotope internal standards, the isotope internal standards include rifampicin-internal standard, isoniazid-internal standard, rifapentine-internal standard, pyrazinamide-internal standard, ethambutol-internal standard, clofazimine-internal standard, cycloserine-internal standard, moxifloxacin-internal standard, levofloxacin-internal standard, linezolid-internal standard, acetylisoniazid-internal standard, bedaquiline-internal standard and desacetylrifampicin-internal standard.

[0011] Preferably, the kit further comprises a C18-PFP mixed chromatographic column and a calibration diluent.

[0012] Preferably, the kit further comprises a mobile phase A and a mobile phase B; the mobile phase A comprises deionized water and an additive dissolved in the deionized water; the mobile phase B comprises a mixture of methanol and acetonitrile and an additive dissolved in the mixture.

[0013] Preferably, the additive includes any one of formic acid, acetic acid, triethylamine and trifluoroacetic acid.

[0014] Preferably, the kit further comprises a microplate, a pre-treatment plate sealing film and an upper plate sealing film.

[0015] Preferably, the calibrator further contains at least one of primanid, rifabutin, amikacin, terizidone, ethionamide, propylthioacetamide, para-aminosalicylic acid, pasniazid and meropenem;

[0016] The quality control product also contains at least one of primanib, rifabutin, amikacin, terizidone, ethionamide, propylthioacetamide, para-aminosalicylic acid, pasniazid and meropenem;

[0017] The isotope internal standard also contains at least one of premanid-internal standard, rifabutin-internal standard, amikacin-internal standard, terizidone-internal standard, ethionamide-internal standard, propylthioacetamide-internal standard, para-aminosalicylic acid-internal standard, pasniazid-internal standard and meropenem-internal standard.

[0018] Compared with the prior art, the kit provided in the embodiment of the present invention can not only monitor the concentration of anti-tuberculosis drugs in patients, but also monitor the metabolites of anti-tuberculosis drugs in patients and the mutual influence relationship between multiple anti-tuberculosis drugs in patients. At the same time, it can also monitor anti-tuberculosis drugs through plasma, serum, cerebrospinal fluid and urine, which can effectively improve the throughput of anti-tuberculosis drug monitoring and obtain more drug administration information and drug metabolism information of patients.

[0019] The second purpose of the embodiment of the present invention is to provide a method for detecting anti-tuberculosis drugs and their metabolites in a sample, and the specific technical scheme adopted is as follows:

[0020] The detection is performed using the above-mentioned kit, comprising the following steps:

[0021] Dissolving the isotope internal standard with a solvent to obtain a releasing agent;

[0022] dissolving the sample in the releasing agent and subjecting it to purification treatment to obtain a supernatant;

[0023] After the supernatant is diluted, it is tested in a liquid chromatography tandem mass spectrometer to obtain the anti-tuberculosis drug in the sample and the metabolite status of the anti-tuberculosis drug.

[0024] Preferably, the sample includes any one of plasma, serum, cerebrospinal fluid and urine.

[0025] Preferably, the detection index of the on-machine detection includes the concentration of at least one of isoniazid, rifampicin, rifapentine, cycloserine, ethambutol, levofloxacin, moxifloxacin, pyrazinamide, clofazimine, bedaquiline, and linezolid;

[0026] and detecting the concentration of at least one of acetylisoniazid, isonicotinic acid, 25-O-desacetylrifampicin, 25-O-desacetylrifapentine, pyrazinoic acid, 5-hydroxypyrazinoic acid, and bedaquiline M2.

[0027] Preferably, the method further comprises detecting the concentration of at least one of primanib, rifabutin, amikacin, terizidone, ethionamide, propylthioacetamide, para-aminosalicylic acid, pasniazid, and meropenem; and detecting the concentration of 25-O-desacetylrifabutin;

[0028] And / or, the purification process comprises subjecting the sample dissolved in the releasing agent to precipitation, freezing and centrifugation.

[0029] Compared with the prior art, the method for detecting anti-tuberculosis drugs and their metabolites in samples provided by the embodiment of the present invention is simple, fast and does not require step-by-step operation. It can not only detect plasma samples, but also detect serum samples, cerebrospinal fluid samples and urine samples, thereby effectively expanding the types of anti-tuberculosis drug sample detection. At the same time, the detection method uses isotope internal standards for normalization correction, which effectively improves the reliability of the detection results; more importantly, the detection method of the present invention can not only obtain the concentration of anti-tuberculosis drugs, but also obtain relevant information such as the concentration of metabolites of anti-tuberculosis drugs, thereby providing a reliable plan for patient medication, and can also obtain information such as the interaction relationship between multiple anti-tuberculosis drugs and whether the metabolism is abnormal. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 It is the extracted ion current chromatogram of 11 anti-tuberculosis drugs and metabolites of 2 anti-tuberculosis drugs in the calibration curve in Example 1 of the present invention;

[0032] Figure 2 is an extracted ion chromatogram of the anti-tuberculosis drug and its metabolites in a serum sample of a tuberculosis patient after taking the anti-tuberculosis drug according to Example 2 of the present invention;

[0033] Figure 3 is an extracted ion chromatogram of the anti-tuberculosis drug and its metabolites in a cerebrospinal fluid sample of a tuberculosis patient after taking the anti-tuberculosis drug in Example 3 of the present invention;

[0034] Figure 4 is an extracted ion chromatogram of the anti-tuberculosis drug and its metabolites in a plasma sample of a tuberculosis patient after taking the anti-tuberculosis drug in Example 4 of the present invention;

[0035] Figure 5 It is the extracted ion current chromatogram of the anti-tuberculosis drug and its metabolites in the urine sample of the tuberculosis patient after taking the anti-tuberculosis drug in Example 5 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] The present application involves at least two invention schemes, wherein the first invention scheme is to provide a kit for detecting anti-tuberculosis drugs and their metabolites in a sample.

[0038] Specifically, the kit includes calibrators, quality control products, instrument quality control products and isotope internal standards. Among them, the calibrators contain rifampicin (RFP), isoniazid (INZ), rifapentine (RFT), pyrazinamide (PZN), ethambutol (ETB), clofazimine (CFM), cycloserine (CYS), moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline (BDQ) and deacetyl rifampicin; the quality control products contain rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, bedaquiline (BDQ) and deacetyl rifampicin. The instrument quality control products include methanol solutions containing rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and desacetyl rifampicin; the isotope internal standards include rifampicin-internal standard, isoniazid-internal standard, rifapentine-internal standard, pyrazinamide-internal standard, ethambutol-internal standard, clofazimine-internal standard, cycloserine-internal standard, moxifloxacin-internal standard, levofloxacin-internal standard, linezolid-internal standard, acetylisoniazid-internal standard, bedaquiline-internal standard and desacetyl rifampicin-internal standard.

[0039] In some embodiments, the calibrator is blank plasma or blank serum or blank cerebrospinal fluid or urine containing rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and desacetyl rifampicin. In some embodiments, in the calibrator, the content of rifampicin is 40-60 mg / L, the content of isoniazid is 10-30 mg / L, the content of rifapentine is 40-60 mg / L, the content of pyrazinamide is 100-150 mg / L, the content of ethambutol is 15-35 mg / L, the content of clofazimine is 2-10 mg / L, the content of cycloserine is 40-80 mg / L, the content of moxifloxacin is 10-25 mg / L, the content of levofloxacin is 30-70 mg / L, the content of linezolid is 20-70 mg / L, the content of acetylisoniazid is 10-30 mg / L, the content of bedaquiline is 3-15 mg / L, and the content of deacetyl rifampicin is 10-30 mg / L. In some embodiments, the calibrator further contains at least one of primanib, rifabutin (RFB), amikacin, terizidone, ethionamide (ETN), propylthioacetamide, 4-aminosalicylic acid (P-ASA), pasniazid and meropenem. When the calibrator contains rifabutin-internal standard, it also contains 25-O-deacetyl rifabutin.

[0040] In some embodiments, the quality control material is blank plasma or blank serum or blank cerebrospinal fluid or urine containing rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and desacetyl rifampicin. In some embodiments, in the quality control product, the content of rifampicin is 5-20 mg / L, the content of isoniazid is 1-15 mg / L, the content of rifapentine is 8-25 mg / L, the content of pyrazinamide is 15-45 mg / L, the content of ethambutol is 1-10 mg / L, the content of clofazimine is 0.5-5 mg / L, the content of cycloserine is 10-30 mg / L, the content of moxifloxacin is 1-8 mg / L, the content of levofloxacin is 1-10 mg / L, the content of linezolid is 5-20 mg / L, the content of acetylisoniazid is 1-15 mg / L, the content of bedaquiline is 0.5-5 mg / L, and the content of deacetyl rifampicin is 0.5-10 mg / L. In some embodiments, the quality control product further contains at least one of primanib, rifabutin, amikacin, terizidone, ethionamide, propylthioacetamide, para-aminosalicylic acid, pasniazid and meropenem. When the quality control product contains rifabutin-internal standard, the quality control product also contains 25-O-deacetyl rifabutin.

[0041] In some embodiments, in the instrument quality control product, the methanol solution is an aqueous solution of methanol, and the volume content of methanol is 30% to 80%. In some embodiments, in the methanol solution, the content of rifampicin is 0.5-10 mg / L, the content of isoniazid is 0.1-5 mg / L, the content of rifapentine is 1-12 mg / L, the content of pyrazinamide is 5-15 mg / L, the content of ethambutol is 0.1-5 mg / L, the content of clofazimine is 0.1-1 mg / L, the content of cycloserine is 1-10 mg / L, the content of moxifloxacin is 0.1-2.5 mg / L, the content of levofloxacin is 0.5-5.5 mg / L, the content of linezolid is 1-10 mg / L, the content of acetylisoniazid is 0.5-8 mg / L, the content of bedaquiline is 0.1-2.5 mg / L, and the content of deacetyl rifampicin is 0.1-6 mg / L.

[0042] In some embodiments, rifampicin-internal standard, isoniazid-internal standard, rifapentine-internal standard, pyrazinamide-internal standard, ethambutol-internal standard, clofazimine-internal standard, cycloserine-internal standard, moxifloxacin-internal standard, levofloxacin-internal standard, linezolid-internal standard, acetylisoniazid-internal standard, bedaquiline-internal standard, and desacetyl rifampicin-internal standard are respectively rifampicin-d 8 , isoniazid-d 4 , Rifapentine-d 8 , pyrazinamide- 13 C 2 , 15 N 2 , ethambutol-d 4 , clofazimine-d 7 , cycloserine-d 3 , 15 N, Moxifloxacin-d 4 , levofloxacin-d 3 , linezolid-d 3 , acetyl isoniazid-d 4 , bedaquiline-d 6 , desacetyl rifampicin-d 8 The components of the isotope internal standard are divided into the following groups according to their mass ratio: rifampicin-d 8 :Isoniazid-d 4 : Rifapentine-d 8 :Pyrazinamide- 13 C 2 , 15 N 2 :Ethambutol-d 4 :Clofazimine-d 7 : Cycloserine-d 3 , 15N: Moxifloxacin-d 4 : Levofloxacin-d 3 :Linezolid-d 3 :Acetylisoniazid-d 4 :Bedaquiline-d 6 : Desacetyl rifampicin-d 8 =2.5~7.5:2~4:2.5~7.5:5~8:0.7~1.8:0.7~1.8:5~7.5:0.5~3:0.5~3:0.1~1.5:2~9:0.5~3:2.5~8. For example, in some embodiments, the isotope internal standard contains 10 μg of rifampicin-d 8 , 6.25 μg of isoniazid-d 4 , 10 μg of rifapentine-d 8 , 12.5 μg of pyrazinamide- 13 C 2 , 15 N 2 , 2.5 μg of ethambutol-d 4 , 2.5 μg of clofazimine-d 7 , 12.5 μg of cycloserine-d 3 , 15 N, 4 μg of moxifloxacin-d 4 , 4 μg of levofloxacin-d 3 , 2 μg of linezolid-d 3 , 7.5 μg of acetyl isoniazid-d 4 , 2 μg bedaquiline-d 6 and 10 μg of desacetyl rifampicin-d 8 In some embodiments, the isotope internal standard further contains at least one of primanib-internal standard, rifabutin-internal standard, amikacin-internal standard, terizidone-internal standard, ethionamide-internal standard, propylthioacetamide-internal standard, para-aminosalicylic acid-internal standard, pasniazid-internal standard, and meropenem-internal standard. When the isotope internal standard contains rifabutin-internal standard, the isotope internal standard also contains 25-O-deacetylrifabutin-internal standard.

[0043] In some embodiments, the kit further comprises a C18-PFP mixed chromatographic column and a calibration diluent. When detecting anti-tuberculosis drugs and metabolites of anti-tuberculosis drugs in a sample, it is necessary to combine the liquid chromatography tandem mass spectrometry for testing. Since the kit comprises a C18-PFP mixed chromatographic column, and the C18-PFP mixed chromatographic column has a chromatographic retention mechanism different from that of the C18 chromatographic column and the PFP chromatographic column, and the analysis effect is complementary, it can effectively solve the problem that the C18 chromatographic column and the PFP chromatographic column are not compatible with the simultaneous analysis of multiple compounds.

[0044] Based on the above C18-PFP mixed chromatographic column, the kit further comprises mobile phase A and mobile phase B; the mobile phase A comprises deionized water and an additive dissolved in the deionized water; the mobile phase B comprises a mixture of methanol and acetonitrile and an additive dissolved in the mixture. In some embodiments, the additive comprises any one of formic acid, acetic acid, triethylamine, and trifluoroacetic acid.

[0045] In some embodiments, the kit further comprises a microplate, a pre-treatment plate sealing film and an upper plate sealing film. Preferably, the microplate is a 96-well plate. Using a 96-well plate as a component of the kit can effectively increase the detection throughput and facilitate automated detection.

[0046] Based on the above-mentioned kit, the invention scheme of the second aspect of the present application is to provide a method for detecting anti-tuberculosis drugs and their metabolites in a sample.

[0047] The method for detecting anti-tuberculosis drugs and their metabolites in a sample is to use the kit described in the first aspect for detection.

[0048] Specifically, the detection method comprises the following steps:

[0049] (1) dissolving the isotope internal standard with a solvent to obtain a releasing agent;

[0050] In step (1), specifically, methanol, acetonitrile, isopropanol, deionized water or other solvents are added to the isotope internal standard so that the isotope internal standard is dissolved, thereby obtaining the releasing agent.

[0051] (2) dissolving the sample in the releasing agent and purifying the sample to obtain a supernatant;

[0052] In step (2), the sample can be any of the plasma, serum, cerebrospinal fluid or urine obtained by sampling. When the sample is purified, the sample, the quality control product and the calibrator are first added to the 96-well plate respectively, and then the release agent obtained in step (1) is added; then the pre-treatment plate is sealed with a film; then the 96-well plate after the film sealing is transferred to an oscillator for vortex oscillation treatment; then the sample obtained after the vortex oscillation treatment is transferred to an environment of -30℃ to -15℃℃ and allowed to stand for 10min to 360min; finally, the sample after the standing treatment is centrifuged at 2℃ to 12℃, the centrifugation time is 10min to 30min, the centrifugation speed is 2000rpm to 4000rpm, and the supernatant is taken after centrifugation. After freezing treatment and then centrifugation, lipids, proteins and other substances in the sample can be effectively removed, reducing the interference of lipids, proteins and other substances on the detection, so that the matrix effect between different sample types is reduced, and the isotope internal standard shows a better correction effect on the differences between different matrices. In addition, the sample recovery rate can meet the requirements without the need to add isotope internal standards.

[0053] (3) After diluting the supernatant, the supernatant is tested in a liquid chromatography tandem mass spectrometer to obtain the anti-tuberculosis drug in the sample and the metabolite status of the anti-tuberculosis drug.

[0054] In step (3), ultrapure water is added to the supernatant obtained in step (2), and the supernatant and ultrapure water are mixed at a mass ratio of 1:1 to 1:10, thereby achieving dilution of the supernatant, and then the film is sealed using an on-machine plate. During the on-machine test, the chromatographic column of the liquid chromatography tandem mass spectrometer is a C18-PFP mixed chromatographic column. Since the mobile phase B contains both methanol and acetonitrile, more substances can be separated and analyzed.

[0055] Through on-machine detection, the concentration of at least one of isoniazid, rifampicin, rifapentine, cycloserine, ethambutol, levofloxacin, moxifloxacin, pyrazinamide, clofazimine, bedaquiline and linezolid can be obtained; at the same time, when the sample contains isoniazid, the concentration of isoniazid metabolites acetylisoniazid and isonicotinic acid can also be detected; when the sample contains rifampicin, the concentration of rifampicin metabolite 25-O-desacetylrifampicin can also be detected; when the sample contains rifapentine, the concentration of rifapentine metabolite 25-O deacetylrifapentine can also be detected; when the sample contains pyrazinamide, the concentration of pyrazinoic acid and 5-hydroxypyrazinoic acid of pyrazinamide metabolites can also be detected; when the sample contains bedaquiline, the concentration of bedaquiline metabolite bedaquiline M2 can also be detected. Therefore, the detection method provided in the embodiment of the present invention can not only detect anti-tuberculosis drugs and metabolites of anti-tuberculosis drugs in plasma, serum, cerebrospinal fluid and urine, but also judge the pharmacokinetics (PK), the therapeutic effects and adverse reactions (PD) of the drugs on the body according to the detection results, and accurately reflect the action process of anti-tuberculosis drugs on patients according to the PK / PD parameters, and formulate targeted dosing plans accordingly, so as to improve the treatment effect of tuberculosis and prevent the occurrence of drug resistance; more importantly, according to the test results, information on the ratio of metabolites to prototype drugs can be obtained, as well as information on whether there is interaction when patients take multiple anti-tuberculosis drugs at the same time and whether there is metabolic abnormality.

[0056] In order to better illustrate the technical solution of the present application, further explanation is given below through multiple embodiments.

[0057] Embodiment 1

[0058] A method for detecting anti-tuberculosis drugs and anti-tuberculosis drugs in a plasma sample comprises the following steps:

[0059] S10. Test kit status

[0060] Kit components: Calibrator, 1 tube, 200μL / tube; Quality control, 1 tube, 200μL / tube; Instrument quality control, 1 tube, 200μL / tube; Isotope internal standard, 1 tube, dry powder / tube; Mobile phase additive, 1 tube, 2mL / tube; Calibrator diluent, 1 tube, 1mL / tube; 96-well microplate, 2 pieces; Pre-treatment plate sealing film, 1 sheet; Machine plate sealing film, 1 sheet; Operating Instructions, 1 copy;

[0061] The calibrator is blank plasma without anti-tuberculosis drugs, and contains 51.2 mg / L rifampicin, 19.2 mg / L isoniazid, 51.2 mg / L rifapentine, 128 mg / L pyrazinamide, 25.6 mg / L ethambutol, 6.4 mg / L clofazimine, 64 mg / L cycloserine, 16.6 mg / L moxifloxacin, 41.7 mg / L levofloxacin, 50 mg / L linezolid, 19.2 mg / L acetylisoniazid, 8.3 mg / L bedaquiline and 20.4 mg / L desacetyl rifampicin.

[0062] The quality control product is blank plasma without anti-tuberculosis drugs, and the quality control product contains 11 mg / L rifampicin, 6.88 mg / L isoniazid, 13.75 mg / L rifapentine, 34.83 mg / L pyrazinamide, 5.5 mg / L ethambutol, 1.15 mg / L clofazimine, 19.25 mg / L cycloserine, 2.5 mg / L moxifloxacin, 6 mg / L levofloxacin, 12.32 mg / L linezolid, 6.88 mg / L acetylisoniazid, 1.47 mg / L bedaquiline and 4.4 mg / L desacetyl rifampicin;

[0063] The instrument quality control product is a 50% methanol aqueous solution, and the methanol aqueous solution contains 2.75 mg / L rifampicin, 1.72 mg / L isoniazid, 3.44 mg / L rifapentine, 8.71 mg / L pyrazinamide, 1.38 mg / L ethambutol, 0.29 mg / L clofazimine, 4.81 mg / L cycloserine, 0.63 mg / L moxifloxacin, 1.5 mg / L levofloxacin, 3.08 mg / L linezolid, 1.72 mg / L acetylisoniazid, 0.37 mg / L bedaquiline and 1.10 mg / L desacetyl rifampicin;

[0064] The isotope internal standard contains 10 μg of rifampicin-d 8 , 6.25 μg of isoniazid-d 4 , 10 μg of rifapentine-d 8 , 12.5 μg of pyrazinamide- 13 C 2 , 15 N 2 , 2.5 μg of ethambutol-d 4 , 2.5 μg of clofazimine-d 7 , 12.5 μg of cycloserine-d 3 , 15 N, 4 μg of moxifloxacin-d 4 , 4 μg of levofloxacin-d 3, 2 μg of linezolid-d 3 , 7.5 μg of acetyl isoniazid-d 4 , 2 μg bedaquiline-d 6 and 10 μg of desacetyl rifampicin-d 8 ;

[0065] The mobile phase additive was formic acid.

[0066] S11. Release agent configuration

[0067] 0.5 mL of methanol was added to the isotope internal standard for reconstitution, and then the isotope internal standard and methanol (V / V) were mixed to prepare a release agent.

[0068] S12. Sample extraction

[0069] Take 50 μL of each plasma sample, quality control product, and calibrator and add them to the corresponding 96-well microplate. Then add 150 μL of the release agent in step S11. Seal the plate with the pre-treatment film, then place it on the shaker and vortex for 5 minutes; place it in a -20°C environment for 20 minutes; then centrifuge it at 8°C for 20 minutes to obtain the supernatant.

[0070] S13, dilution and loading

[0071] Take out 50 μL of the supernatant in step S12, add 50 μL of ultrapure water, and place it on a sealing plate for film sealing.

[0072] S14, on-machine testing

[0073] Prepare mobile phase A and mobile phase B, wherein mobile phase A consists of 1000 mL of deionized water and 1 mL of mobile phase additive; mobile phase B consists of 500 mL of methanol, 500 mL of acetonitrile, and 1 mL of mobile phase additive;

[0074] Using C18-PFP hybrid chromatographic column as the chromatographic column, take 80 μL from the instrument quality control bottle and directly put it into the instrument to test the system's adaptability; according to the instrument setting program, the concentration of each analyte is automatically obtained, and the results are as follows Figure 1 Repeat the test to obtain the %CV of the anti-tuberculosis drugs in the quality control products, and the results are shown in Table 1.

[0075] Table 1 %CV of each anti-tuberculosis drug in quality control products

[0076] Analytes Batch 1 (n=6) Batch 2 (n=6) Batch 3 (n=6) Between batches (n=18) Isoniazid 8.53％ 3.79％ 8.61％ 8.23％ Rifampin 8.22％ 7.28％ 7.37％ 7.11％ Rifapentine 7.97％ 5.96％ 4.47％ 9.66％ Cycloserine 4.73％ 5.88％ 10.11％ 9.32％ Ethambutol 4.35％ 2.28％ 4.43％ 5.83％ Levofloxacin 8.00％ 2.81％ 3.91％ 7.19％ Moxifloxacin 13.77％ 6.24％ 4.53％ 12.63％ Pyrazinamide 2.57％ 4.93％ 7.78％ 6.15％ Clofazimine 4.33％ 3.47％ 2.37％ 4.94％ Bedaquiline 5.21％ 4.18％ 6.22％ 5.12％ Acetylisoniazid 2.57％ 3.93％ 7.77％ 5.18％ Rifampicin desacetyl 9.00％ 7.10％ 9.36％ 8.81％ Linezolid 7.65％ 6.39％ 5.52％ 7.88％

[0077] Note: n in Table 1 represents the number of repetitions.

[0078] As can be seen from Table 1, the %CV of each analyte is less than 15%, indicating that the method is stable and the data collection is reliable, indicating that the quality control product in this example can be used to evaluate the stability of the method.

[0079] from Figure 1 It can be seen from the calibration curve extracted ion current chromatogram that 11 anti-tuberculosis drugs and two anti-tuberculosis drug metabolites can be detected.

[0080] Embodiment 2

[0081] The method for detecting anti-tuberculosis drugs and anti-tuberculosis drugs in serum samples of tuberculosis patients comprises the following steps:

[0082] S20, test kit status

[0083] Kit components: Calibrator, 1 tube, 200μL / tube; Quality control, 1 tube, 200μL / tube; Instrument quality control, 1 tube, 200μL / tube; Isotope internal standard, 1 tube, dry powder / tube; Mobile phase additive, 1 tube, 2mL / tube; Calibrator diluent, 1 tube, 1mL / tube; 96-well microplate, 2 pieces; Pre-treatment plate sealing film, 1 sheet; Machine plate sealing film, 1 sheet; Operating Instructions, 1 copy;

[0084] The calibrator is a blank serum without anti-tuberculosis drugs, and contains 51.2 mg / L rifampicin, 19.2 mg / L isoniazid, 51.2 mg / L rifapentine, 128 mg / L pyrazinamide, 25.6 mg / L ethambutol, 6.4 mg / L clofazimine, 64 mg / L cycloserine, 16.6 mg / L moxifloxacin, 41.7 mg / L levofloxacin, 50 mg / L linezolid, 19.2 mg / L acetylisoniazid, 8.3 mg / L bedaquiline and 20.4 mg / L desacetyl rifampicin.

[0085] The quality control product is blank serum without anti-tuberculosis drugs, and contains 11 mg / L rifampicin, 6.88 mg / L isoniazid, 13.75 mg / L rifapentine, 34.83 mg / L pyrazinamide, 5.5 mg / L ethambutol, 1.15 mg / L clofazimine, 19.25 mg / L cycloserine, 2.5 mg / L moxifloxacin, 6 mg / L levofloxacin, 12.32 mg / L linezolid, 6.88 mg / L acetylisoniazid, 1.47 mg / L bedaquiline, and 4.4 mg / L desacetyl rifampicin;

[0086] The instrument quality control product is a 50% methanol aqueous solution, and the methanol aqueous solution contains 2.75 mg / L rifampicin, 1.72 mg / L isoniazid, 3.44 mg / L rifapentine, 8.71 mg / L pyrazinamide, 1.38 mg / L ethambutol, 0.29 mg / L clofazimine, 4.81 mg / L cycloserine, 0.63 mg / L moxifloxacin, 1.5 mg / L levofloxacin, 3.08 mg / L linezolid, 1.72 mg / L acetylisoniazid, 0.37 mg / L bedaquiline and 1.10 mg / L desacetyl rifampicin;

[0087] The isotope internal standard contains 10 μg of rifampicin-d 8 , 6.25 μg of isoniazid-d 4 , 10 μg of rifapentine-d 8 , 12.5 μg of pyrazinamide- 13 C 2 , 15 N 2 , 2.5 μg of ethambutol-d 4 , 2.5 μg of clofazimine-d 7 , 12.5 μg of cycloserine-d 3 , 15 N, 4 μg of moxifloxacin-d 4 , 4 μg of levofloxacin-d 3 , 2 μg of linezolid-d 3 , 7.5 μg of acetyl isoniazid-d 4 , 2 μg bedaquiline-d 6 and 10 μg of desacetyl rifampicin-d 8 ;

[0088] The mobile phase additive was formic acid.

[0089] S21. Release agent configuration

[0090] 0.5 mL of methanol was added to the isotope internal standard for reconstitution, and then the isotope internal standard and methanol (V / V) were mixed to prepare a release agent.

[0091] S22. Sample extraction

[0092] Take 50 μL of each serum sample, quality control product, and calibrator and add them to the corresponding 96-well microplate. Then add 150 μL of the release agent in step S21. Seal the plate with the pre-treatment film, then place it on the shaker and vortex for 5 minutes; place it in a -20°C environment for 20 minutes; then centrifuge it at 8°C for 20 minutes to obtain the supernatant.

[0093] S23, dilution and loading

[0094] Take out 50 μL of the supernatant in step S22, add 50 μL of ultrapure water, and place it on a sealing plate for film sealing treatment.

[0095] S24, on-machine testing

[0096] Prepare mobile phase A and mobile phase B, wherein mobile phase A consists of 1000 mL of deionized water and 1 mL of mobile phase additive; mobile phase B consists of 500 mL of methanol, 500 mL of acetonitrile, and 1 mL of mobile phase additive;

[0097] Using C18-PFP hybrid column as the chromatographic column, take 80 μL from the instrument quality control bottle and directly test the adaptability of the system on the instrument; the concentration of each analyte is automatically obtained according to the instrument setting program. The results are as follows Figure 2 shown.

[0098] from Figure 2 It can be seen that the status of anti-tuberculosis drugs and their metabolites after the patients take anti-tuberculosis drugs can be obtained by testing the serum of tuberculosis patients, so as to analyze the information of the ratio of metabolites to prototype drugs, as well as the interaction between the anti-tuberculosis drugs after the patients take multiple anti-tuberculosis drugs at the same time and whether there are metabolic abnormalities.

[0099] Embodiment 3

[0100] The method for detecting anti-tuberculosis drugs and anti-tuberculosis drugs in cerebrospinal fluid samples of tuberculosis patients comprises the following steps:

[0101] S30, Test kit status

[0102] Kit components: Calibrator, 1 tube, 200μL / tube; Quality control, 1 tube, 200μL / tube; Instrument quality control, 1 tube, 200μL / tube; Isotope internal standard, 1 tube, dry powder / tube; Mobile phase additive, 1 tube, 2mL / tube; Calibrator diluent, 1 tube, 1mL / tube; 96-well microplate, 2 pieces; Pre-treatment plate sealing film, 1 sheet; Machine plate sealing film, 1 sheet; Operating Instructions, 1 copy;

[0103] The calibrator is blank cerebrospinal fluid without anti-tuberculosis drugs, and contains 51.2 mg / L rifampicin, 19.2 mg / L isoniazid, 51.2 mg / L rifapentine, 128 mg / L pyrazinamide, 25.6 mg / L ethambutol, 6.4 mg / L clofazimine, 64 mg / L cycloserine, 16.6 mg / L moxifloxacin, 41.7 mg / L levofloxacin, 50 mg / L linezolid, 19.2 mg / L acetylisoniazid, 8.3 mg / L bedaquiline and 20.4 mg / L desacetyl rifampicin.

[0104] The quality control product is blank cerebrospinal fluid without anti-tuberculosis drugs, and the quality control product contains 11 mg / L rifampicin, 6.88 mg / L isoniazid, 13.75 mg / L rifapentine, 34.83 mg / L pyrazinamide, 5.5 mg / L ethambutol, 1.15 mg / L clofazimine, 19.25 mg / L cycloserine, 2.5 mg / L moxifloxacin, 6 mg / L levofloxacin, 12.32 mg / L linezolid, 6.88 mg / L acetylisoniazid, 1.47 mg / L bedaquiline and 4.4 mg / L desacetyl rifampicin;

[0105] The instrument quality control product is a 50% methanol aqueous solution, and the methanol aqueous solution contains 2.75 mg / L rifampicin, 1.72 mg / L isoniazid, 3.44 mg / L rifapentine, 8.71 mg / L pyrazinamide, 1.38 mg / L ethambutol, 0.29 mg / L clofazimine, 4.81 mg / L cycloserine, 0.63 mg / L moxifloxacin, 1.5 mg / L levofloxacin, 3.08 mg / L linezolid, 1.72 mg / L acetylisoniazid, 0.37 mg / L bedaquiline and 1.10 mg / L desacetyl rifampicin;

[0106] The isotope internal standard contains 10 μg of rifampicin-d 8 , 6.25 μg of isoniazid-d 4 , 10 μg of rifapentine-d 8 , 12.5 μg of pyrazinamide- 13 C 2 , 15 N 2 , 2.5 μg of ethambutol-d 4 , 2.5 μg of clofazimine-d 7 , 12.5 μg of cycloserine-d 3 , 15 N, 4 μg of moxifloxacin-d 4 , 4 μg of levofloxacin-d 3 , 2 μg of linezolid-d 3 , 7.5 μg of acetyl isoniazid-d 4 , 2 μg bedaquiline-d 6 and 10 μg of desacetyl rifampicin-d 8 ;

[0107] The mobile phase additive was formic acid.

[0108] S31, release agent configuration

[0109] 0.5 mL of methanol was added to the isotope internal standard for reconstitution, and then the isotope internal standard and methanol (V / V) were mixed to prepare a release agent.

[0110] S32. Sample extraction

[0111] Take 50 μL of each cerebrospinal fluid sample, quality control product, and calibrator and add them to the corresponding 96-well microplate. Then add 150 μL of the release agent in step S31. Seal the plate with the pre-treatment film, then place it on the shaker and vortex for 5 minutes; place it in a -20°C environment for 20 minutes; then centrifuge it at 8°C for 20 minutes to obtain the supernatant.

[0112] S33, dilution and loading

[0113] Take out 50 μL of the supernatant in step S32, add 50 μL of ultrapure water, and place it on a sealing plate for film sealing treatment.

[0114] S34, on-machine testing

[0115] Prepare mobile phase A and mobile phase B, wherein mobile phase A consists of 1000 mL of deionized water and 1 mL of mobile phase additive; mobile phase B consists of 500 mL of methanol, 500 mL of acetonitrile, and 1 mL of mobile phase additive;

[0116] Using C18-PFP hybrid chromatographic column as the chromatographic column, take 80 μL from the instrument quality control bottle and directly put it into the instrument to test the system's adaptability; according to the instrument setting program, the concentration of each analyte is automatically obtained, and the results are as follows Figure 3 shown.

[0117] from Figure 3 The results show that the cerebrospinal fluid of tuberculosis patients can be tested to obtain the status of anti-tuberculosis drugs and their metabolites after the patients take anti-tuberculosis drugs, so as to analyze the information on the ratio of metabolites to parent drugs, as well as the interaction between the anti-tuberculosis drugs after the patients take multiple anti-tuberculosis drugs at the same time and whether there are metabolic abnormalities.

[0118] Embodiment 4

[0119] The method for detecting anti-tuberculosis drugs and anti-tuberculosis drugs in plasma samples of tuberculosis patients comprises the following steps:

[0120] S40, test kit status

[0121] Kit components: Calibrator, 1 tube, 200μL / tube; Quality control, 1 tube, 200μL / tube; Instrument quality control, 1 tube, 200μL / tube; Isotope internal standard, 1 tube, dry powder / tube; Mobile phase additive, 1 tube, 2mL / tube; Calibrator diluent, 1 tube, 1mL / tube; 96-well microplate, 2 pieces; Pre-treatment plate sealing film, 1 sheet; Machine plate sealing film, 1 sheet; Operating Instructions, 1 copy;

[0122] The calibrator is blank plasma without anti-tuberculosis drugs, and contains 51.2 mg / L rifampicin, 19.2 mg / L isoniazid, 51.2 mg / L rifapentine, 128 mg / L pyrazinamide, 25.6 mg / L ethambutol, 6.4 mg / L clofazimine, 64 mg / L cycloserine, 16.6 mg / L moxifloxacin, 41.7 mg / L levofloxacin, 50 mg / L linezolid, 19.2 mg / L acetylisoniazid, 8.3 mg / L bedaquiline and 20.4 mg / L desacetyl rifampicin.

[0123] The quality control product is blank plasma without anti-tuberculosis drugs, and the quality control product contains 11 mg / L rifampicin, 6.88 mg / L isoniazid, 13.75 mg / L rifapentine, 34.83 mg / L pyrazinamide, 5.5 mg / L ethambutol, 1.15 mg / L clofazimine, 19.25 mg / L cycloserine, 2.5 mg / L moxifloxacin, 6 mg / L levofloxacin, 12.32 mg / L linezolid, 6.88 mg / L acetylisoniazid, 1.47 mg / L bedaquiline and 4.4 mg / L desacetyl rifampicin;

[0124] The instrument quality control product is a 50% methanol aqueous solution, and the methanol aqueous solution contains 2.75 mg / L rifampicin, 1.72 mg / L isoniazid, 3.44 mg / L rifapentine, 8.71 mg / L pyrazinamide, 1.38 mg / L ethambutol, 0.29 mg / L clofazimine, 4.81 mg / L cycloserine, 0.63 mg / L moxifloxacin, 1.5 mg / L levofloxacin, 3.08 mg / L linezolid, 1.72 mg / L acetylisoniazid, 0.37 mg / L bedaquiline and 1.10 mg / L desacetyl rifampicin;

[0125] The isotope internal standard contains 10 μg of rifampicin-d 8 , 6.25 μg of isoniazid-d 4 , 10 μg of rifapentine-d 8 , 12.5 μg of pyrazinamide- 13 C 2 ,15 N 2 , 2.5 μg of ethambutol-d 4 , 2.5 μg of clofazimine-d 7 , 12.5 μg of cycloserine-d 3 , 15 N, 4 μg of moxifloxacin-d 4 , 4 μg of levofloxacin-d 3 , 2 μg of linezolid-d 3 , 7.5 μg of acetyl isoniazid-d 4 , 2 μg bedaquiline-d 6 and 10 μg of desacetyl rifampicin-d 8 ;

[0126] The mobile phase additive was formic acid.

[0127] S41, release agent configuration

[0128] 0.5 mL of methanol was added to the isotope internal standard for reconstitution, and then the isotope internal standard and methanol (V / V) were mixed to prepare a release agent.

[0129] S42. Sample extraction

[0130] Take 50 μL of each plasma sample, quality control product, and calibrator and add them to the corresponding 96-well microplate. Then add 150 μL of the release agent in step S31. Seal the plate with the pre-treatment film, then place it on the shaker and vortex for 5 minutes; place it in a -20°C environment for 20 minutes; then centrifuge it at 8°C for 20 minutes to obtain the supernatant.

[0131] S43, dilution and loading

[0132] Take out 50 μL of the supernatant in step S12, add 50 μL of ultrapure water, and place it on a sealing plate for film sealing.

[0133] S44, on-machine testing

[0134] Prepare mobile phase A and mobile phase B, wherein mobile phase A consists of 1000 mL of deionized water and 1 mL of mobile phase additive; mobile phase B consists of 500 mL of methanol, 500 mL of acetonitrile, and 1 mL of mobile phase additive;

[0135] Using C18-PFP hybrid chromatographic column as the chromatographic column, take 80 μL from the instrument quality control bottle and directly put it into the instrument to test the system's adaptability; according to the instrument setting program, the concentration of each analyte is automatically obtained, and the results are as follows Figure 4 shown.

[0136] from Figure 4The results presented indicate that the status of anti-tuberculosis drugs and their metabolites after patients have taken anti-tuberculosis drugs can be obtained by testing the plasma of tuberculosis patients, thereby analyzing information on the ratio of metabolites to parent drugs, as well as analyzing the interactions between anti-tuberculosis drugs after patients take multiple anti-tuberculosis drugs at the same time and whether there are metabolic abnormalities.

[0137] Embodiment 5

[0138] The method for detecting anti-tuberculosis drugs and anti-tuberculosis drugs in urine samples of tuberculosis patients comprises the following steps:

[0139] S50, test kit status

[0140] Kit components: Calibrator, 1 tube, 200μL / tube; Quality control, 1 tube, 200μL / tube; Instrument quality control, 1 tube, 200μL / tube; Isotope internal standard, 1 tube, dry powder / tube; Mobile phase additive, 1 tube, 2mL / tube; Calibrator diluent, 1 tube, 1mL / tube; 96-well microplate, 2 pieces; Pre-treatment plate sealing film, 1 sheet; Machine plate sealing film, 1 sheet; Operating Instructions, 1 copy;

[0141] The calibrator is blank urine without anti-tuberculosis drugs, and contains 51.2 mg / L rifampicin, 19.2 mg / L isoniazid, 51.2 mg / L rifapentine, 128 mg / L pyrazinamide, 25.6 mg / L ethambutol, 6.4 mg / L clofazimine, 64 mg / L cycloserine, 16.6 mg / L moxifloxacin, 41.7 mg / L levofloxacin, 50 mg / L linezolid, 19.2 mg / L acetylisoniazid, 8.3 mg / L bedaquiline and 20.4 mg / L desacetyl rifampicin.

[0142] The quality control material is blank urine without anti-tuberculosis drugs, and the quality control material contains 11 mg / L rifampicin, 6.88 mg / L isoniazid, 13.75 mg / L rifapentine, 34.83 mg / L pyrazinamide, 5.5 mg / L ethambutol, 1.15 mg / L clofazimine, 19.25 mg / L cycloserine, 2.5 mg / L moxifloxacin, 6 mg / L levofloxacin, 12.32 mg / L linezolid, 6.88 mg / L acetylisoniazid, 1.47 mg / L bedaquiline, and 4.4 mg / L desacetyl rifampicin;

[0143] The instrument quality control product is a 50% methanol aqueous solution, and the methanol aqueous solution contains 2.75 mg / L rifampicin, 1.72 mg / L isoniazid, 3.44 mg / L rifapentine, 8.71 mg / L pyrazinamide, 1.38 mg / L ethambutol, 0.29 mg / L clofazimine, 4.81 mg / L cycloserine, 0.63 mg / L moxifloxacin, 1.5 mg / L levofloxacin, 3.08 mg / L linezolid, 1.72 mg / L acetylisoniazid, 0.37 mg / L bedaquiline and 1.10 mg / L desacetyl rifampicin;

[0144] The isotope internal standard contains 10 μg of rifampicin-d 8 , 6.25 μg of isoniazid-d 4 , 10 μg of rifapentine-d 8 , 12.5 μg of pyrazinamide- 13 C 2 , 15 N 2 , 2.5 μg of ethambutol-d 4 , 2.5 μg of clofazimine-d 7 , 12.5 μg of cycloserine-d 3 , 15 N, 4 μg of moxifloxacin-d 4 , 4 μg of levofloxacin-d 3 , 2 μg of linezolid-d 3 , 7.5 μg of acetyl isoniazid-d 4 , 2 μg bedaquiline-d 6 and 10 μg of desacetyl rifampicin-d 8 ;

[0145] The mobile phase additive was formic acid.

[0146] S51, release agent configuration

[0147] 0.5 mL of methanol was added to the isotope internal standard for reconstitution, and then the isotope internal standard and methanol (V / V) were mixed to prepare a release agent.

[0148] S52. Sample extraction

[0149] Take 50 μL of each urine sample, quality control product, and calibrator and add them to the corresponding 96-well microplate. Then add 150 μL of the release agent in step S51. Seal the plate with the pre-treatment film, then place it on the shaker and vortex for 5 minutes; place it in a -20°C environment for 20 minutes; then centrifuge it at 8°C for 20 minutes to obtain the supernatant.

[0150] S53, dilution and loading

[0151] Take out 50 μL of the supernatant in step S52, add 50 μL of ultrapure water, and place it on a sealing plate for film sealing treatment.

[0152] S54, on-machine testing

[0153] Prepare mobile phase A and mobile phase B, wherein mobile phase A consists of 1000 mL of deionized water and 1 mL of mobile phase additive; mobile phase B consists of 500 mL of methanol, 500 mL of acetonitrile, and 1 mL of mobile phase additive;

[0154] Using C18-PFP hybrid chromatographic column as the chromatographic column, take 80 μL from the instrument quality control bottle and directly put it into the instrument to test the system's adaptability; according to the instrument setting program, the concentration of each analyte is automatically obtained, and the results are as follows Figure 5 shown.

[0155] from Figure 5 As can be seen from the displayed graph, the status of anti-tuberculosis drugs and their metabolites after the patients take anti-tuberculosis drugs can be obtained by testing the urine of tuberculosis patients, so as to analyze the information on the ratio of metabolites to parent drugs, as well as the interaction between the anti-tuberculosis drugs after the patients take multiple anti-tuberculosis drugs at the same time and whether there are metabolic abnormalities.

[0156] In summary, the detection method of the present application can effectively expand the sample types for sampling and testing of tuberculosis patients. At the same time, it can not only detect the types of anti-tuberculosis drugs in tuberculosis patient samples, but also detect the metabolism of anti-tuberculosis drugs in the body, obtain information on the ratio of metabolites to prototype drugs, whether there are interactions when patients take multiple anti-tuberculosis drugs at the same time, and whether there are metabolic abnormalities, as well as PK / PD data, so that targeted dosing plans can be formulated.

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit, characterized in that: The kit is used to detect anti-tuberculosis drugs and their metabolites in samples; The kit comprises: A calibrator, wherein the calibrator contains rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline, and desacetyl rifampicin; Quality control products, wherein the quality control products contain rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and desacetyl rifampicin; Instrument quality control products, including methanol solutions containing rifampicin, isoniazid, rifapentine, pyrazinamide, ethambutol, clofazimine, cycloserine, moxifloxacin, levofloxacin, linezolid, acetylisoniazid, bedaquiline and desacetyl rifampicin; And, isotope internal standards, wherein the isotope internal standards include rifampicin-internal standard, isoniazid-internal standard, rifapentine-internal standard, pyrazinamide-internal standard, ethambutol-internal standard, clofazimine-internal standard, cycloserine-internal standard, moxifloxacin-internal standard, levofloxacin-internal standard, linezolid-internal standard, acetylisoniazid-internal standard, bedaquiline-internal standard and desacetylrifampicin-internal standard; Wherein, the kit also includes a C18-PFP mixed chromatographic column and a calibration diluent; The kit further comprises a mobile phase A and a mobile phase B; the mobile phase A comprises deionized water and an additive dissolved in the deionized water; the mobile phase B comprises a mixture of methanol and acetonitrile and an additive dissolved in the mixture.

2. The kit according to claim 1, characterized in that The additive includes any one of formic acid, acetic acid, triethylamine and trifluoroacetic acid.

3. The kit according to any one of claims 1 to 2, characterized in that The kit also includes a microplate, a pre-treatment plate sealing film and an upper plate sealing film.

4. The kit according to any one of claims 1 to 2, characterized in that The calibrator also contains at least one of primanib, rifabutin, amikacin, terizidone, ethionamide, propylthioacetamide, para-aminosalicylic acid, pasniazid and meropenem; The quality control product also contains at least one of primanib, rifabutin, amikacin, terizidone, ethionamide, propylthioacetamide, para-aminosalicylic acid, pasniazid and meropenem; The isotope internal standard also contains at least one of premanid-internal standard, rifabutin-internal standard, amikacin-internal standard, terizidone-internal standard, ethionamide-internal standard, propylthioacetamide-internal standard, para-aminosalicylic acid-internal standard, pasniazid-internal standard and meropenem-internal standard.

5. A method for detecting anti-tuberculosis drugs and their metabolites in a sample, characterized in that: The detection is performed using the kit according to any one of claims 1 to 4, comprising the following steps: Dissolving the isotope internal standard with a solvent to obtain a releasing agent; dissolving the sample in the releasing agent and subjecting it to purification treatment to obtain a supernatant; After the supernatant is diluted, it is tested in a liquid chromatography tandem mass spectrometer to obtain the anti-tuberculosis drug in the sample and the metabolite status of the anti-tuberculosis drug.

6. The detection method according to claim 5, characterized in that The sample includes any one of plasma, serum, cerebrospinal fluid and urine.

7. The detection method according to any one of claims 5 to 6, characterized in that The detection index of the on-machine detection includes the concentration of at least one of isoniazid, rifampicin, rifapentine, cycloserine, ethambutol, levofloxacin, moxifloxacin, pyrazinamide, clofazimine, bedaquiline, and linezolid; and detecting the concentration of at least one of acetylisoniazid, isonicotinic acid, 25-O-desacetylrifampicin, 25-O-desacetylrifapentine, pyrazinoic acid, 5-hydroxypyrazinoic acid, and bedaquiline M2.

8. The detection method according to claim 7, characterized in that It also includes detecting the concentration of at least one of primanib, rifabutin, amikacin, terizidone, ethionamide, propylthioacetamide, para-aminosalicylic acid, pasniazid, and meropenem; and detecting the concentration of 25-O-desacetylrifabutin; And / or, the purification process comprises subjecting the sample dissolved in the releasing agent to precipitation, freezing and centrifugation.

Images