CN111537648A - Kit for detecting anti-tuberculosis drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology - Google Patents
Kit for detecting anti-tuberculosis drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology Download PDFInfo
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Abstract
A kit for detecting anti-tuberculosis drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the anti-tuberculosis drugs comprise Cycloserine (CYS), Pyrazinamide (PZN), Isoniazid (INZ), P-aminosalicylic acid (P-ASA), Ethionamide (ETN), Ethambutol (ETB), Clofazimine (CFM), Bedaquiline (BDQ), Rifampicin (RFP), Rifabutin (RFB) and Rifapentine (RFT); the kit comprises the following reagents: eluent A, eluent B, mixed standard substance stock solution, mixed internal standard working solution, protein precipitator and quality control product; when the kit provided by the invention is used for detecting the anti-tuberculosis drugs in serum, the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and detection of the anti-tuberculosis drugs can be completed within 5min, the accuracy and the precision basically meet the requirements, and a reliable detection method is provided for the clinical monitoring of the treatment concentration of the anti-tuberculosis drugs.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a kit for detecting an anti-tuberculosis drug in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology.
Background
Tuberculosis is an infectious disease caused by infection of patients with mycobacterium tuberculosis, and tuberculosis is still the infectious disease causing the highest mortality rate of human except AIDS. Drugs against tuberculosis are mainly classified into four categories: first line antitubercular drugs, second line antitubercular drugs, novel antitubercular drugs and compound preparation drugs. In this patent, the first-line antitubercular drugs mainly include Rifampicin (RMP), Isoniazid (INH), pyrazinamide (Pvrazinamide, PZA) and Ethambutol (Ethambutol, EMB); first-line antituberculosis drugs remain the first choice drugs for the current treatment of tuberculosis. Isoniazid has been the first choice drug for treating tuberculosis since the world, and ethionamide is used as a derivative of isoniazid, and the action mechanism is similar to that of isoniazid. Ethionamide, rifapentine, rifabutin, cycloserine, para-aminosalicylic acid, clofazimine are used as second-line antitubercular drugs. Second-line antitubercular drugs are mainly used to treat tuberculosis patients with first-line antitubercular drug resistance. Bedaquiline is a new anti-tuberculosis drug approved to be marketed in the us in 2012, and is the first drug approved by the FDA for the treatment of multi-drug resistant tuberculosis. In recent years, drug-resistant tuberculosis cases have increased, and drug combinations have become widely used. In addition, tuberculosis patients often have other diseases or infectious diseases, and multiple drugs are used simultaneously, so that the concentration of the antituberculosis drugs entering the body is greatly different due to the difference of individual patients. The concentration of the anti-tuberculosis drug in the body is very easy to be too low, so that the concentration of the anti-tuberculosis drug in the body can not reach the therapeutic drug concentration, or the toxic reaction and other phenomena are caused by the too high concentration of the anti-tuberculosis drug in the body, so that the monitoring of the concentration of the anti-tuberculosis drug in the body of a patient is very important for ensuring the therapeutic effect and reducing the drug resistance.
At present, the method for detecting the in vivo concentration of the antituberculosis drugs mainly adopts an ultra-high performance liquid chromatography-tandem mass spectrometry method, the literature reports mostly detect 1-3 drugs at one time, and the patent reports also detect 5 antituberculosis drugs; chinese patent application (publication number: CN 110146620A) discloses a method for simultaneously detecting five antitubercular drugs in blood plasma by using UPLC-MS/MS method, wherein the dosage of the blood plasma is 90 mu L, the pretreatment steps are more, the filtration is needed, and the cost is high; chinese patent application (publication No. CN 109682915A) discloses a method for determining the concentration of pyrazinamide in blood plasma by liquid chromatography-mass spectrometry, wherein the dosage of the blood plasma is 100 mu L, only one drug is detected at one time, but the types of antituberculosis drugs are various, and the detection process is complicated.
Disclosure of Invention
The invention aims to solve the technical problem of providing a kit for detecting anti-tuberculosis drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a kit for detecting anti-tuberculosis drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology is disclosed, wherein the anti-tuberculosis drugs are respectively as follows: cycloserine (CYS), Pyrazinamide (PZN), Isoniazid (INZ), para-Aminosalicylic acid (4-Aminosalicylic acid, P-ASA), Ethionamide (Ethionamide, ETN), Ethambutol (Ethambutol, ETB), Clofazimine (CFM), Bedaquiline (BDQ), Rifampicin (RFP), Rifabutin (Rifabutin, RFB), Rifapentine (RFT).
The isotope internal standard substances corresponding to the antituberculosis drugs are respectively as follows: isoniazid-d 4(INZ-d4), pyrazinamide-d 3(PZN-d3), ethionamide-d 3(ETN-d3), ethambutol-d 4(ETB-d4), clofazimine-d 7(CFM-d7), bedaquiline-d 6(BDQ-d6), cycloserine- [15N,2H3] (CYS- [15N,2H3]), rifabutin-d 7(RFB-d7), P-aminosalicylic acid-13C 6(P-ASA-13C6), rifampin-d 8(RFP-d8), rifapentine-d 8(RFT-d 8).
The kit comprises the following reagents:
eluent: eluent A: formic acid, ammonium formate-water solution; eluent B: acetonitrile;
mixing standard stock solution: an aqueous methanol solution containing CYS, PZN, INZ, P-ASA, ETN, ETB, CFM, BDQ, RFP, RFB and RFT;
mixing internal standard solutions: aqueous methanol solutions containing CYS- [15N,2H3], PZN-d3, INZ-d4, P-ASA-13C6, ETB-d4, ETN-d3, CFM-d7, BDQ-d6, RFB-d7, RFP-d8 and RFT-d 8;
protein precipitant: a mixed solution of methanol and acetonitrile;
quality control product: quality control product: blank serum matrix solution containing anti-tuberculosis drugs is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H);
QC (L) QC (M) 10-fold dilution with blank serum matrix;
QC (M) is the above-mentioned mixed standard substance stock solution and is diluted to 200 times with blank serum matrix;
qc (h) was a 50-fold dilution of the above mixed standard stock with blank serum base.
Wherein the serum is human or animal serum.
Wherein the blank serum matrix is blank serum without anti-tuberculosis drugs.
Wherein the eluent A is water containing 0.01-0.2% of formic acid and 1-10 mM of ammonium formate.
Wherein the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-3.
Wherein the mixed standard stock solution is a methanol aqueous solution containing INZ 400000ng/mL, P-ASA10000ng/mL, ETN200000ng/mL, ETB 200000ng/mL, CFM 80000ng/mL, BDQ200000ng/mL, RFP800000ng/mL, RFB20000ng/mL, PZN 2000000ng/mL, RFT 1000000ng/mL and CYS 4000000ng/mL standard substances.
Wherein the mixed internal standard solution contains methanol aqueous solution of CYS- [15N,2H3]250000ng/mL, PZN-d350000ng/mL, INZ-d410000ng/mL, P-ASA-13C6800ng/mL, ETB-d45000ng/mL, ETN-d35000ng/mL, CFM-d72000ng/mL, BDQ-d65000ng/mL, RFB-d71000ng/mL, RFP-d810000ng/mL and RFT-d810000 ng/mL.
The preparation method of the kit for detecting the anti-tuberculosis drugs in the serum by the ultra-high performance liquid chromatography tandem mass spectrometry technology;
(1) eluent A: taking 0.5mL of formic acid and 250 mu L of 10M ammonium formate, adding 500mL of ultrapure water, and uniformly mixing to obtain an eluent A;
eluent B: acetonitrile;
(2) mixing standard stock solution: respectively preparing mother liquor of standard products of antituberculotic drugs, wherein the concentrations are respectively as follows: INZ5mg/mL, P-ASA 1mg/mL, ETN 10mg/mL, ETB 10mg/mL, CFM 2.5mg/mL, BDQ 2.5mg/mL, RFP20mg/mL, RFB 5mg/mL, PZN10 mg/mL, RFT 4mg/mL, CYS20 mg/mL, then respectively removing INZ 80 mu L, P-ASA10 mu L, ETN20 mu L, ETB 20 mu L, CFM 32 mu L, BDQ 80 mu L, BDQ mu L, RFP 40 mu L, RFB 4 mu L, PZN 200 mu L, PZN mu L, RFT250 mu L, CYS200 mu L, adding 64 mu L methanol aqueous solution, and uniformly vortexing to obtain 1mL mixed standard stock solution;
(3) mixing internal standard solutions: CYS- [15N,2H3]1mg/mL, PZN-d31mg/mL, INZ-d41mg/mL, P-ASA-13C60.1mg/mL, ETB-d41mg/mL, ETN-d31mg/mL, CFM-d70.5mg/mL, BDQ-d60.5mg/mL, RFB-d70.1mg/mL, RFP-d80.5mg/mL, RFT-d80.5mg/mL mother liquor, then respectively transferring CYS- [15N,2H3]250 μ L, PZN-d350 μ L, INZ-d410 μ L, P-ASA-13C68 μ L, ETB-d 2 μ L, ETN-d35 μ L, CFM-d74 μ 356-d 610 μ L, RFB-d710 μ L, RFP-d 36820 μ L, RFT-d820 μ L into mixed water containing CYS- [15 mL, 2H3] 31 μ L and 15 μ L internal standard, 2H3]250000ng/mL, PZN-d350000ng/mL, INZ-d410000ng/mL, P-ASA-13C6800ng/mL, ETB-d45000ng/mL, ETN-d35000ng/mL, CFM-d72000ng/mL, BDQ-d65000ng/mL, RFB-d71000ng/mL, RFP-d810000ng/mL, RFT-d810000 ng/mL.
(4) Protein precipitant: mixing methanol and acetonitrile according to the volume ratio of 1: 1-3, and uniformly swirling;
(5) quality control product: preparing the mixed standard substance stock solution into QC (L), QC (M) and QC (H) with three different concentrations by using a blank serum substrate, wherein the corresponding concentrations of the anti-tuberculosis drug quality control substances of the QC (L), the QC (M) and the QC (H) are shown in a table 1 (the concentration is ng/mL);
TABLE 1 corresponding concentration of quality control product of antituberculosis drugs
The application of the kit in detecting the anti-tuberculosis drugs in the serum by using the ultra-performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting anti-tuberculosis drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology is as follows: cycloserine (CYS), Pyrazinamide (PZN), Isoniazid (INZ), para-aminosalicylic acid (P-ASA), Ethionamide (ETN), Ethambutol (ETB), Clofazimine (CFM), Bedaquiline (BDQ), Rifampicin (RFP), Rifabutin (RFB), Rifapentine (RFT).
Detecting the anti-tuberculosis drugs in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly separating a target substance to be detected from interfering components in a serum matrix by utilizing the ultra-high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the anti-tuberculosis drugs in the serum, wherein the specific chromatographic conditions are as follows:
(1) chromatographic conditions are as follows:
mobile phase A: water containing 0.01 to 0.2 percent of formic acid and 1 to 10mM of ammonium formate;
mobile phase B: acetonitrile;
a chromatographic column: phenomenex Kintex C18 (2.1X 100mm, 2.6 μm);
the gradient elution mode is adopted, and is shown in the table 2;
the flow rate is 0.3mL/min, the collection time of each sample is 5min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;
TABLE 2 mobile phase gradient elution parameters
(2) Mass spectrum conditions: an electrospray ionization positive ion mode and a mass spectrum scanning mode of multi-reaction monitoring; spraying voltage: 3.0 kV; source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard product and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 3;
TABLE 3 Mass spectrometric parameters
Wherein the serum is human or animal serum.
Wherein the pre-treated serum is prepared according to the following method: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing an internal standard into the centrifuge tube, and oscillating at high speed for 5 min; 14000r/min, centrifuging at 4 ℃ for 5min, taking 60 mu L of supernatant to a plastic lining tube, and injecting samples.
The preparation method comprises the following steps of preparing standard solutions by a gradient dilution method, and preparing the mixed standard stock solution into seven calibrator solutions with different concentration points by using a blank serum matrix, wherein the preparation process comprises the following steps:
adding 10 μ L of the mixed standard stock solution into 190 μ L of blank serum matrix as a first high-value concentration point (S7);
taking the first high-value concentration point (S7) and diluting the first high-value concentration point with an equal volume of blank serum matrix to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S7) with 3 times volume of blank serum substrate to obtain a third high-value concentration point (S5); diluting the second high-value concentration (S6) point with 9 times volume of blank serum substrate to obtain a fourth high-value concentration point (S4); diluting the third high concentration point (S5) with 9 times volume of blank serum matrix to obtain a fifth high concentration point (S3); diluting the fourth high concentration point (S4) with blank serum matrix of 4 times volume to obtain a sixth high concentration point (S2); diluting the fifth high concentration point (S3) with blank serum matrix 4 times the volume to obtain a seventh high concentration point (S1), which is described in Table 4 below (unit: ng/mL);
TABLE 4 Standard Curve formulation and concentrations
Has the advantages that: when the kit is used for detecting the anti-tuberculosis drugs in the serum, 11 common anti-tuberculosis drugs can be detected at one time; the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and the detection of the anti-tuberculosis drugs are completed within 5min, the accuracy and the precision basically meet the requirements, and the method can be used for the quantitative analysis of the anti-tuberculosis drugs in clinic and provides a reliable detection method for the monitoring of the treatment concentration of the anti-tuberculosis drugs in clinic.
Drawings
FIG. 1 is an ion flow chart of the extraction of anti-tuberculosis drug standard;
FIG. 2 is an ion flow chart of the extraction of anti-tuberculosis drugs in serum.
Detailed Description
For the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
Example 1
1. Material
(1) The samples for the research experiments in the reagent kit science are obtained from serum samples collected from the 8 month clinic of 2019 in the Nanjing drummer hospital.
(2) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like.
(3) Reagent consumables: MS grade methanol (Fisher, usa); HPLC grade methanol (Honeywell, usa); MS grade acetonitrile (Fisher, usa); HPLC grade acetonitrile (Honeywell, usa); MS grade formic acid (Fisher, usa); MS grade ammonium formate (Sigma, usa) column: phenomenex Kintex C18 (2.1X 100mm, 2.6 μm) (Phenomenex, USA).
(4) And (3) standard substance: the standards and their corresponding internal standards are shown in table 5 below.
TABLE 5 Standard and internal standards
(5) Quality control product: the blank serum matrix containing anti-tuberculosis drugs is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H), which are shown in table 1.
The upper and lower peripheries of the kit are coated, the shockproof and heat preservation are carried out, mobile phases A and B are placed at the upper left part, 11 ampoule bottles are respectively placed at the lower left part, and the standard solution, the quality control product and the mixed internal standard solution are respectively contained; to the right, 25mL of protein precipitant was placed.
2. Method of producing a composite material
(1) Chromatographic conditions
Mobile phase A: water containing 0.1% formic acid, 5mM ammonium formate;
mobile phase B: acetonitrile;
a chromatographic column: phenomenex Kintex C18 (2.1X 100mm, 2.6 μm);
the gradient elution mode is adopted, and is shown in the table 2;
the flow rate is 0.3mL/min, the collection time of each sample is 5min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;
(2) mass spectrum conditions: an electrospray ionization positive ion mode and a mass spectrum scanning mode of multi-reaction monitoring; spraying voltage: 3.0 kV; source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard product and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 3;
(3) preparing a mixed standard stock solution: accurately transferring a certain volume of mother liquor of the anti-tuberculosis drug standard substance, adding 64 mu L of 80% methanol aqueous solution, and fully and uniformly mixing to obtain 1mL of mixed standard substance stock solution, wherein the concentration is shown in Table 6.
TABLE 6 stock solution formulation for mixed standards
The mixed standard stock solution is prepared into calibration solution of seven different concentration points by using a blank serum matrix solution, and the concentration of each calibration point is shown in table 4.
(4) Preparing a mixed internal standard solution: accurately transferring a certain volume of isotope internal standard mother liquor of the anti-tuberculosis drugs respectively, adding 608 mu L of 80% methanol aqueous solution, and uniformly mixing to obtain 1mL of mixed internal standard solution with the concentration shown in the following table 7.
TABLE 7 Mixed internal standard working solution formulation
(5) Preparing a quality control product: preparing the mixed standard substance stock solution into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without the anti-tuberculosis drugs, wherein the corresponding concentrations of the anti-tuberculosis drug quality control products of the QC (L), the QC (M) and the QC (H) are shown in a table 1;
(6) sample treatment:
1) pre-treating a calibration product: taking 50 mu L of each concentration point sample, putting the sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:2), and oscillating at high speed for 5 min; centrifuging at 14000r/min at 4 deg.C for 5min, transferring 60 μ L of supernatant into plastic lined tube, and sampling 1 μ L.
2) Pretreatment of a serum sample: putting 50 μ L of serum into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol to acetonitrile is 1:2), and shaking at high speed for 5 min; centrifuging at 14000r/min at 4 deg.C for 5min, collecting supernatant 60 μ L, and introducing into plastic liner tube with sample amount of 1 μ L.
3) Pretreatment of quality control products: the quality control solutions QC (L), QC (M), QC (H) are respectively taken and 50 μ L of each quality control solution QC (L), QC (M), QC (H) are respectively put into a 1.5mL centrifuge tube, and then the quality control solutions QC (L), QC (M), QC (H) are consistent with the pretreatment of the serum sample, and the details are not.
The components of the assay kit are shown in Table 8.
TABLE 8 preparation of kit Components for analysis of antituberculosis drugs (100 persons)
Remarking: the protein precipitant containing the internal standard is prepared by the following method, 200 mu L of the mixed internal standard solution is added into 19.8mL of protein precipitant to obtain the protein precipitant containing the internal standard.
3. Method verification
1) Extracting an ion current chromatogram: the peak shapes of the standard substance of the anti-tuberculosis drug and the serum sample are symmetrical, and no interference of a hybrid peak exists, which indicates that the good detection can be obtained under the condition.
2) Calibration curve: and (3) establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the serum by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of the anti-tuberculosis drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, which is shown in Table 9.
TABLE 9 Linear regression equation and Linear correlation coefficient for antituberculotic drugs
3) Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample is prepared, 3 concentrations of mixed standard stock solutions of low, medium and high are added respectively, the treatment is repeated for 5 times by the same steps, and the result shows that the adding standard recovery rate of the anti-tuberculosis drugs is between 86.94% and 110.93%, the RSD of 5 repeated tests is in the range of 0.75% to 8.01%, and the statistical result is shown in the table 10.
TABLE 10 results of recovery of antituberculotic drugs with standard addition
4) And (3) precision test: taking an interference-free blank serum sample, adding mixed standard substance stock solutions with different concentrations to obtain serum samples with low, medium and high concentrations, repeatedly processing 6 batches in one day for three days continuously, quantitatively determining the concentration of the anti-tuberculosis drug by an isotope internal standard method, wherein the batch precision is 2.15-12.59%, processing 3 batches in three days, and calculating the batch precision to be 2.51-11.26%, and the result is shown in Table 11. TABLE 11 results of the precision measurements within and between batches
4. Discussion of the related Art
The invention establishes a method for simultaneously determining the anti-tuberculosis drugs in human serum by ID-UPLC-MS/MS. The serum dosage is less (only 50 mu L), the pretreatment is simple, and the analysis of various substances by one injection only needs 5min, and the method is simple and quick.
The isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the result is not influenced by conditions such as a pretreatment process, instrument response fluctuation and the like, and accurate quantification can be achieved. The accuracy of the method is evaluated by a standard recovery test, and the result shows that the standard recovery of the anti-tuberculosis drug is 86.94-110.93%, the RSD of 5 repeated tests is 0.75-8.01%, and the accuracy is good.
The reproducibility result of the method shows that the intrabatch precision of the antituberculosis drug is 2.15-12.59%, the interbatch precision is 2.51-11.26%, and the reproducibility of the method is good.
In a word, the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the compound within 5min, meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical serum anti-tuberculosis drugs, and provides a reliable detection method for monitoring related drug concentrations.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A kit for detecting anti-tuberculosis drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology is characterized in that the anti-tuberculosis drugs comprise CYS, PZN, INZ, P-ASA, ETN, ETB, CFM, BDQ, RFP, RFB and RFT;
the kit comprises the following reagents:
eluent: eluent A: formic acid, ammonium formate-water solution; eluent B: acetonitrile;
mixing standard stock solution: an aqueous methanol solution containing CYS, PZN, INZ, P-ASA, ETN, ETB, CFM, BDQ, RFP, RFB and RFT;
mixing internal standard solutions: aqueous methanol solutions containing CYS- [15N,2H3], PZN-d3, INZ-d4, P-ASA-13C6, ETB-d4, ETN-d3, CFM-d7, BDQ-d6, RFB-d7, RFP-d8 and RFT-d 8;
protein precipitant: a mixed solution of methanol and acetonitrile;
quality control product: quality control product: blank serum matrix solution containing anti-tuberculosis drugs is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H);
QC (L) QC (M) 10-fold dilution with blank serum matrix;
QC (M) is the above-mentioned mixed standard substance stock solution and is diluted to 200 times with blank serum matrix;
qc (h) was a 50-fold dilution of the above mixed standard stock with blank serum base.
2. The kit for detecting the anti-tuberculosis drugs in the serum according to the ultra performance liquid chromatography-tandem mass spectrometry technology of claim 1, wherein the serum is human or animal serum.
3. The kit for detecting the anti-tuberculosis drugs in the serum according to the ultra performance liquid chromatography-tandem mass spectrometry technology of claim 1, wherein the blank serum matrix is blank serum without the anti-tuberculosis drugs.
4. The kit for detecting the anti-tuberculosis drugs in the serum by the ultra-high performance liquid chromatography-tandem mass spectrometry technology according to claim 1, wherein the eluent A is water containing 0.01-0.2% of formic acid and 1-10 mM of ammonium formate.
5. The kit for detecting the anti-tuberculosis drug in the serum according to the ultra-high performance liquid chromatography tandem mass spectrometry technology of claim 1, wherein the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 1-3.
6. The kit for detecting the anti-tuberculosis drug in the serum according to the ultra-high performance liquid chromatography tandem mass spectrometry technology of claim 1, wherein the mixed standard stock solution is a methanol aqueous solution containing INZ 400000ng/mL, P-ASA10000ng/mL, ETN200000ng/mL, ETB 200000ng/mL, CFM 80000ng/mL, BDQ200000ng/mL, RFP800000ng/mL, RFB20000ng/mL, PZN 2000000ng/mL, RFT 1000000ng/mL, CYS 4000000 ng/mL.
7. The kit for detecting the anti-tuberculosis drugs in the serum by the ultra-high performance liquid chromatography tandem mass spectrometry technology according to claim 1, wherein the mixed internal standard solution is a methanol aqueous solution containing CYS- [15N,2H3]250000ng/mL, PZN-d350000ng/mL, INZ-d410000ng/mL, P-ASA-13C6800ng/mL, ETB-d45000ng/mL, ETN-d35000ng/mL, CFM-d72000ng/mL, BDQ-d65000ng/mL, RFB-d71000ng/mL, RFP-d810000ng/mL and RFT-d810000 ng/mL.
8. A method for producing the kit according to any one of claims 1 to 7,
(1) eluent A: taking 0.5mL of formic acid solution and 250 mu L of 10M ammonium formate, adding 500mL of ultrapure water, and uniformly mixing to obtain eluent A;
eluent B: acetonitrile;
(2) mixing standard stock solution: respectively preparing mother liquor of standard products of antituberculotic drugs, wherein the concentrations are respectively as follows: INZ5mg/mL, P-ASA 1mg/mL, ETN 10mg/mL, ETB 10mg/mL, CFM 2.5mg/mL, BDQ 2.5mg/mL, RFP20mg/mL, RFB 5mg/mL, PZN10 mg/mL, RFT 4mg/mL and CYS20 mg/mL, then respectively transferring INZ 80 mu L, P-ASA10 mu L, ETN20 mu L, ETN mu L, ETB 20 mu L, CFM 32 mu L, BDQ 80 mu L, RFP 40 mu L, RFB 4 mu L, PZN 200 mu L, RFT250 mu L, CYS200 mu L, CYS mu 200 mu L, adding 64 mu L methanol aqueous solution, and uniformly vortexing to obtain 1mL mixed standard stock solution;
(3) mixing internal standard solutions: CYS- [15N,2H3]1mg/mL, PZN-d31mg/mL, INZ-d41mg/mL, P-ASA-13C60.1mg/mL, ETB-d41mg/mL, ETN-d31mg/mL, CFM-d70.5mg/mL, BDQ-d60.5mg/mL, RFB-d70.1mg/mL, RFP-d80.5mg/mL, RFT-d80.5mg/mL mother liquor, then respectively transferring CYS- [15N,2H3]250 μ L, PZN-d350 μ L, INZ-d410 μ L, P-ASA-13C68 μ L, ETB-d 2 μ L, ETN-d35 μ L, CFM-d74 μ 356-d 610 μ L, RFB-d710 μ L, RFP-d 36820 μ L, RFT-d820 μ L into mixed water containing CYS- [15 mL, 2H3] 31 μ L and 15 μ L internal standard, 2H3]250000ng/mL, PZN-d350000ng/mL, INZ-d410000ng/mL, P-ASA-13C6800ng/mL, ETB-d45000ng/mL, ETN-d35000ng/mL, CFM-d72000ng/mL, BDQ-d65000ng/mL, RFB-d71000ng/mL, RFP-d810000ng/mL, RFT-d810000 ng/mL;
(4) protein precipitant: mixing methanol and acetonitrile according to the volume ratio of 1: 1-3, and uniformly swirling;
(5) quality control product: preparing the mixed standard substance stock solution into three different concentrations of QC (L), QC (M) and QC (H) by using a blank serum substrate, wherein,
QC (L) includes: INZ 200ng/mL, P-ASA 5ng/mL, ETN 100ng/mL, ETB 100ng/mL, CFM40 ng/mL, BDQ 100ng/mL, RFP 400ng/mL, RFB 10ng/mL, PZN1000 ng/mL, RFT 500ng/mL and CYS2000 ng/mL;
QC (M) comprises: INZ 2000ng/mL, P-ASA 50ng/mL, ETN 1000ng/mL, ETB 1000ng/mL, CFM400ng/mL, BDQ 1000ng/mL, RFP 4000ng/mL, RFB 100ng/mL, PZN10000 ng/mL, RFT 5000ng/mL and CYS 20000 ng/mL;
QC (H) includes: INZ 8000ng/mL, P-ASA200 ng/mL, ETN 4000ng/mL, ETB 4000ng/mL, CFM1600ng/mL, BDQ 4000ng/mL, RFP 16000ng/mL, RFB 400ng/mL, PZN 40000ng/mL, RFT20000ng/mL and CYS 80000 ng/mL.
9. Use of the kit of any one of claims 1 to 8 for detecting anti-tuberculosis drugs in serum by ultra high performance liquid chromatography tandem mass spectrometry.
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| CN112730657A (en) * | 2020-12-18 | 2021-04-30 | 卓和药业集团有限公司 | Analysis and detection method for rifapentine content |
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| CN114354804A (en) * | 2021-12-31 | 2022-04-15 | 深圳市第三人民医院(深圳市肝病研究所) | Kit and method for detecting anti-tuberculosis drugs and metabolites thereof in sample |
| CN115389677A (en) * | 2022-09-13 | 2022-11-25 | 北京豪思生物科技股份有限公司 | Reagent for quantitatively detecting concentration of antituberculosis drug, application and kit |
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2020
- 2020-05-29 CN CN202010475211.6A patent/CN111537648A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112730657A (en) * | 2020-12-18 | 2021-04-30 | 卓和药业集团有限公司 | Analysis and detection method for rifapentine content |
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| CN113109493A (en) * | 2021-04-14 | 2021-07-13 | 安徽万邦医药科技股份有限公司 | Method for measuring rifampicin in plasma by high performance liquid chromatography-mass spectrometry |
| CN114354804A (en) * | 2021-12-31 | 2022-04-15 | 深圳市第三人民医院(深圳市肝病研究所) | Kit and method for detecting anti-tuberculosis drugs and metabolites thereof in sample |
| CN115389677A (en) * | 2022-09-13 | 2022-11-25 | 北京豪思生物科技股份有限公司 | Reagent for quantitatively detecting concentration of antituberculosis drug, application and kit |
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