Method for simultaneous detection of multiple anti-tuberculosis drug components by high performance liquid chromatography-tandem mass spectrometry

Abstract

The present invention relates to the field of pharmaceutical technology, and in particular to a method for simultaneously detecting multiple anti-tuberculosis drug components by high performance liquid chromatography tandem mass spectrometry. The method provided by the present invention is a method for high throughput detection of four first-class anti-tuberculosis drugs in human serum by high performance liquid chromatography tandem mass spectrometry, which can simultaneously obtain four first-line anti-tuberculosis drugs, namely pyrazinamide, isoniazid, ethambutol and rifampicin, in one detection, and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity, etc. The coefficient of variation (CV) of the repeatability of the low-value quality control product obtained by the detection is ≤10%, the coefficient of variation (CV) of the repeatability of the median quality control product is ≤10%, and the coefficient of variation (CV) of the repeatability of the high-value quality control product is ≤10%; the relative deviation (B) of the accuracy is ≤±15%, and the spike recovery rate is 98% to 113%.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical technology, in particular to a method for simultaneously detecting multiple anti-tuberculosis drug components using high performance liquid phase tandem mass spectrometry. Background Art

[0002] Tuberculosis is an infectious disease caused by infection with Mycobacterium tuberculosis. Anti-tuberculosis drugs are mainly divided into four categories: first-line anti-tuberculosis drugs, second-line anti-tuberculosis drugs, new anti-tuberculosis drugs and combination drugs. The first-line anti-tuberculosis drugs involved mainly include rifampicin (Rifamoicin, RMP), Isoniazid (Isoniazid, INH), Pyrazinamide (Pyrazinamide, PZA) and Ethambutol (Ethambutol, EMB).

[0003] The main methods for testing anti-tuberculosis drugs include liquid chromatography-ultraviolet method, liquid chromatography-evaporative light scattering method, liquid chromatography-tandem mass spectrometry, etc. Since ethambutol among the above four first-line anti-tuberculosis drugs has no ultraviolet absorption, it must be tested with an evaporative light scattering detector, which makes it difficult to accurately detect the four components at the same time.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a high performance liquid phase tandem mass spectrometry method capable of simultaneously detecting four anti-tuberculosis drug components, so as to solve the technical problem that ethambutol cannot be used for ultraviolet detection.

[0006] In order to solve the above technical problems and achieve the above objectives, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for separating anti-tuberculosis drug components, wherein the anti-tuberculosis drug components contain at least one of pyrazinamide, isoniazid, ethambutol or rifampicin;

[0008] The separation method comprises the following steps: a sample to be tested containing anti-tuberculosis drug components is subjected to methanol precipitation to remove impurities, and then an F5 chromatographic column or a C18 chromatographic column is used to prepare a mobile phase of methanol and an aqueous solution of ammonium formate to perform high performance liquid chromatography to separate the anti-tuberculosis drug components.

[0009] In an optional embodiment, the mobile phase A of the HPLC is an aqueous solution of ammonium formate containing 1 / 1000 (v / v) formic acid, and the concentration of the ammonium formate is 5-15 mM; and the mobile phase B is methanol.

[0010] In an optional embodiment, the mobile phase conditions of the HPLC are: the volume ratio of mobile phase A is 87% to 95% from 0 to 2 min, 10% from 2.1 to 4.5 min, and 95% from 4.51 to 5 min.

[0011] In an optional embodiment, the liquid phase conditions of the high performance liquid chromatography are: a mobile phase flow rate of 0.3 to 0.6 mL / min, a column temperature of 30 to 45°C, an injector temperature of 5 to 20°C, a needle wash solution of a mixed solution of methanol, acetonitrile and isopropanol in a volume ratio of 1:(0.5 to 2):(0.5 to 2), and an injection volume of 1 to 20 μL.

[0012] In an optional embodiment, the liquid phase conditions of the high performance liquid chromatography are: a mobile phase flow rate of 0.5 mL / min, a column temperature of 40°C, an injector temperature of 10°C, a needle wash solution of a mixed solution of methanol, acetonitrile and isopropanol in a volume ratio of 1:1:1, and an injection volume of 1 μL.

[0013] In an optional embodiment, the internal standards corresponding to pyrazinamide, isoniazid, ethambutol and rifampicin are pyrazinamide- 15 N,D3, isoniazid-D4, ethambutol-D4, and rifampicin-D4.

[0014] In an optional embodiment, the sample to be tested includes human serum.

[0015] Preferably, the hemoglobin concentration in the human serum is less than 0.04 g / L.

[0016] In a second aspect, the present invention provides an application of the separation method described in any one of the aforementioned embodiments in drug development, drug screening or scientific research, wherein the drug contains at least one of pyrazinamide, isoniazid, ethambutol or rifampicin.

[0017] In a third aspect, the present invention provides a method for detecting the concentration of an anti-tuberculosis drug component, wherein the anti-tuberculosis drug component contains at least one of pyrazinamide, isoniazid, ethambutol or rifampicin;

[0018] The detection method comprises separating the anti-tuberculosis drug components contained in the sample to be tested by using the separation method described in any of the aforementioned embodiments, and then obtaining the concentration of each component by mass spectrometry detection.

[0019] In an optional embodiment, the ion source parameters of the mass spectrometry detection are: the ion source is ESI+, the curtain gas pressure is 30-40psi, the collision gas pressure is medium, the spray voltage is 5500V, the temperature is 450-550°C, the spray gas GS1 pressure is 40-55psi, and the spray gas GS2 pressure is 40-60psi.

[0020] Preferably, the ion source parameters are: the ion source is ESI+, the curtain gas pressure is 40psi, the collision gas pressure is 7psi, the spray voltage is 500V, the temperature is 500°C, the spray gas GS1 pressure is 55psi, and the spray gas GS2 pressure is 60psi.

[0021] The method provided by the present invention for high-throughput detection of four first-class anti-tuberculosis drugs in human serum by high-performance liquid chromatography tandem mass spectrometry can simultaneously obtain four first-line anti-tuberculosis drugs of pyrazinamide, isoniazid, ethambutol and rifampicin in one detection, and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity, etc. The linear range of pyrazinamide detected is 15.000-105.000 μg / mL, isoniazid is 1.000-19.000 μg / mL, ethambutol is 1.000-14.500 μg / mL, and rifampicin is 0.400-13.000 μg / mL, and the correlation coefficients are all r≥0.990; the coefficient of variation (CV) of the repeatability of the low-value quality control product is ≤10%, the coefficient of variation (CV) of the repeatability of the mid-value quality control product is ≤10%, and the coefficient of variation (CV) of the repeatability of the high-value quality control product is ≤10%; the relative deviation (B) of the accuracy is ≤±15%, and the spike recovery rate is 98%-113%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is the result of direct injection after centrifugation of the methanol protein precipitation sample;

[0024] Figure 2 This is the result of acetonitrile extraction and direct injection after centrifugation;

[0025] Figure 3 The result of extraction with methanol / acetonitrile = 1 / 1, dilution 4 times with water, centrifugation and direct injection;

[0026] Figure 4 The result of extraction with methanol / acetonitrile = 2 / 1, dilution 4 times with water, centrifugation and direct injection;

[0027] Figure 5 The result of extraction with methanol / acetonitrile = 3 / 1, dilution 4 times with water, centrifugation and direct injection;

[0028] Figure 6The result of direct injection after methanol / acetonitrile extraction = 4 / 1 and dilution 4 times with water and centrifugation;

[0029] Figure 7 The result of methanol extraction, dilution 4 times with water, centrifugation and direct injection;

[0030] Figure 8 This is the injection result when mobile phase A does not contain ammonium formate.

[0031] Fig. 9 Figure 1. Injection results of C18 column using acidic mobile phase. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0033] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Some embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0035] The present invention discloses a high-throughput detection method of four types of anti-tuberculosis drugs in human serum by high-performance liquid mass spectrometry. The anti-tuberculosis drugs in human serum are subjected to protein precipitation by organic solvent, centrifuged and diluted for sample loading test, and all pre-treatments can be carried out in a 96-well plate, and a row gun is used for transfer to achieve batch pre-treatment. After the pre-treatment, the sample enters a liquid chromatography-mass spectrometer for detection, and the test of four drugs can be completed within five minutes, which is convenient and fast. Since human samples are difficult to obtain, the present invention uses BSA as a substitute matrix for human serum to prepare various concentration points and quality control of the standard curve.

[0036] 1. The present invention discloses a high-throughput method for detecting four types of anti-tuberculosis drugs in human serum by high-performance liquid chromatography tandem mass spectrometry, which mainly includes the following reagents:

[0037] Table 1 Standards and reagents

[0038]

[0039]

[0040] 2. The present invention discloses a high-throughput method for detecting four types of anti-tuberculosis drugs in human serum by high-performance liquid chromatography tandem mass spectrometry, comprising the following equipment:

[0041] Table 2 Instruments and equipment

[0042] Serial number Device Name Model / Specification 1 Liquid chromatography tandem mass spectrometry AB SCIEX Triple Quad TM4500MD 2 96-well plate shaker MB100-4A 3 96-well plate centrifuge Xiangyi H2050R 4 10μL pipette 0.5-10μL 5 200μL pipette 20-200μL 6 1000μL pipette 100-1000μL 7 300μL 12-well pipette 30-300μL 8 96-well plate 2mL 9 96-well plate 1mL 10 Electronic balance One in 100,000

[0043] 3. The present invention discloses a high-throughput method for detecting four types of anti-tuberculosis drugs in human serum by high-performance liquid chromatography tandem mass spectrometry, and the preparation of standard products and quality control products:

[0044] 3.1. Preparation of anti-tuberculosis drug stock solution

[0045] Preparation of pyrazinamide stock solution: Use a 1 / 100,000 electronic balance to accurately weigh 100 mg of pyrazinamide, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a 10.00 mg / mL stock solution, transfer it to a 15 mL HDPE plastic bottle, label it, and store it at -80°C.

[0046] Preparation of isoniazid stock solution: Use a 1 / 100,000 electronic balance to accurately weigh 100 mg of isoniazid, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a stock solution with a concentration of 10.00 mg / mL, transfer it to a 15 mL HDPE plastic bottle, label it, and store it at -80°C.

[0047] Preparation of ethambutol stock solution: Use a 1 / 100,000 electronic balance to accurately weigh 100 mg of ethambutol, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a 10.00 mg / mL stock solution, transfer it to a 15 mL HDPE plastic bottle, label it, and store it at -80°C.

[0048] Preparation of rifampicin stock solution: Use a 1 / 100,000 electronic balance to accurately weigh 100 mg of pyrazinamide, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a stock solution with a concentration of 10.00 mg / mL, transfer it to a 15 mL HDPE plastic bottle, label it, and store it at -80°C.

[0049] 3.2 Preparation of secondary stock solution of anti-tuberculosis drugs

[0050] Preparation of pyrazinamide secondary stock solution: Use a pipette to accurately transfer 1 mL of pyrazinamide stock solution to a 10 mL volumetric flask, dilute to the mark with methanol to prepare a 1.00 mg / mL secondary stock solution, transfer to a 15 mL HDPE plastic bottle, label, and store at -80°C.

[0051] Preparation of isoniazid secondary stock solution: Use a pipette to accurately transfer 100 μl of isoniazid stock solution to a 10 mL volumetric flask, dilute to the mark with methanol to prepare a 0.10 mg / mL secondary stock solution, transfer to a 15 mL HDPE plastic bottle, label, and store at -80°C.

[0052] Preparation of ethambutol secondary stock solution: Use a pipette to accurately transfer 100 μl of ethambutol stock solution to a 10 mL volumetric flask, dilute to the mark with methanol to prepare a 0.10 mg / mL secondary stock solution, transfer to a 15 mL HDPE plastic bottle, label, and store at -80°C.

[0053] Preparation of rifampicin secondary stock solution: Use a pipette to accurately transfer 100 μl of rifampicin stock solution to a 10 mL volumetric flask, dilute to the mark with methanol to prepare a 0.10 mg / mL secondary stock solution, transfer to a 15 mL HDPE plastic bottle, label, and store at -80°C.

[0054] 3.3 Preparation of Calibrators S1 to S6

[0055] 3.3.1. Preparation of diluent

[0056] Preparation of PBS diluent: Measure 10mL of 10*PBS into a 100mL volumetric flask, then add pure water to the mark, mix well to obtain 1*PBS, and transfer to a glass container for later use.

[0057] Preparation of 0.1% BSA solution: Weigh 0.1 g BSA to an accuracy of 0.0001 g and put it into a glass container, add 100 mL PBS diluent, cover the bottle cap, and shake up and down until the BSA is completely dissolved.

[0058] The standard products were prepared according to the high-low preparation mode, that is, after preparing S1 and S6, they were mixed in proportion to prepare S2 to S5 and LQC, MQC and HQC.

[0059] Preparation of calibrator S6: Use a pipette of appropriate specifications to take 2.625 mL of pyrazinamide stock solution, 0.475 mL of isoniazid stock solution, 0.3625 mL of ethambutol stock solution, and 0.325 mL of rifampicin stock solution, respectively, and place them in a suitable HDPE tube, add 21.2125 mL of 0.1% BSA solution, and prepare 105.000 μg / mL of pyrazinamide, 19.000 μg / mL of isoniazid, 14.500 μg / mL of ethambutol, and 13.000 μg / mL of rifampicin standard solution. After thorough mixing, label and store at -20°C.

[0060] Preparation of calibrator S1: Use a pipette of appropriate specifications to take 0.75 mL of pyrazinamide secondary stock solution, 0.5 mL of isoniazid secondary stock solution, 0.5 mL of ethambutol secondary stock solution, and 0.2 mL of rifampicin secondary stock solution, place them in a suitable HDPE tube, add 48.05 mL of 0.1% BSA solution, and prepare 15.000 μg / mL pyrazinamide, 1.000 μg / mL isoniazid, 1.000 μg / mL ethambutol, and 10.400 μg / mL standard solutions. After thorough mixing, label and store at -20°C.

[0061] Preparation of calibrator S2: Accurately pipette 19 mL of S1 standard and 3.5 mL of S6 standard, mix thoroughly, and prepare standard solutions of 29.000 μg / mL pyrazinamide, 3.800 μg / mL isoniazid, 3.100 μg / mL ethambutol, and 2.360 μg / mL rifampicin. After thorough mixing, label and store at -20°C.

[0062] Preparation of calibrator S3: Accurately pipette 16 mL of S1 standard and 63.5 mL of S6 standard, mix thoroughly, and prepare standard solutions of 41.000 μg / mL pyrazinamide, 6.200 μg / mL isoniazid, 4.900 μg / mL ethambutol, and 4.040 μg / mL rifampicin. After thorough mixing, label and store at -20°C.

[0063] Preparation of calibrator S4: Accurately pipette 12 mL of S1 standard and 10.5 mL of S6 standard, mix thoroughly, and prepare standard solutions of pyrazinamide 57.000 μg / mL, isoniazid 9.400 μg / mL, ethambutol 7.300 μg / mL, and rifampicin 6.280 μg / mL. After thorough mixing, label and store at -20°C.

[0064] Preparation of calibrator S5: Accurately pipette 7 mL of S1 standard and 15.5 mL of S6 standard, mix thoroughly, and prepare standard solutions of pyrazinamide 77.000 μg / mL, isoniazid 13.400 μg / mL, ethambutol 10.300 μg / mL, and rifampicin 9.080 μg / mL. After thorough mixing, label and store at -20°C.

[0065] 3.4. Preparation of quality control products

[0066] Preparation of low-concentration quality control LQC: Accurately pipette 21.2 mL of S1 standard and 1.3 mL of S6 standard, mix thoroughly, and prepare standard solutions of pyrazinamide 20.200 μg / mL, isoniazid 2.040 μg / mL, ethambutol 1.780 μg / mL, and rifampicin 1.128 μg / mL. After thorough mixing, label and store at -20°C.

[0067] Preparation of medium concentration quality control MQC: Accurately pipette 12mL of S1 standard and 10.5mL of S6 standard, mix thoroughly, and prepare standard solutions of pyrazinamide 57.000μg / mL, isoniazid 9.400μg / mL, ethambutol 7.300μg / mL, and rifampicin 6.280μg / mL. After thorough mixing, label and store at -20℃.

[0068] Preparation of high-concentration quality control HQC: Accurately pipette 6 mL of S1 standard and 16.5 mL of S6 standard, mix thoroughly, and prepare standard solutions of pyrazinamide 81.000 μg / mL, isoniazid 14.200 μg / mL, ethambutol 10.900 μg / mL, and rifampicin 9.640 μg / mL. After thorough mixing, label and store at -20°C.

[0069] 4. The present invention discloses a high-performance liquid chromatography tandem mass spectrometry method for high-throughput detection of four class I anti-tuberculosis drugs in human serum and the preparation of sample extracts.

[0070] Preparation of sample extract stock solution: Use a 100,000th balance to accurately weigh 10 mg of pyrazinamide- 15 N, D3, 10 mg isoniazid-D4, 10 mg ethambutol-D4, 10 mg rifampicin-D4, accurate to 0.01 mg, are placed in 10 mL volumetric flasks respectively, and methanol is added to the scale line to prepare a 1.00 mg / mL stock solution, which is labeled and stored at -20°C.

[0071] Preparation of sample extract: Use a pipette of appropriate specifications to transfer 1000 μL of pyrazinamide- 15 N, D3, 100 μL isoniazid-D4, 10 μL ethambutol-D4, 500 μL rifampicin-D4, placed in a 500 mL volumetric flask, added methanol to the scale, and mixed thoroughly to prepare a pyrazinamide-D4 solution with a concentration of 2.00 μg / mL. 15 N, D3, 0.20μg / mL isoniazid-D4, 0.02μg / mL ethambutol-D4, 1.00μg / mL rifampicin-D4 sample extract. Label and store at -20℃.

[0072] 5. The present invention discloses a high-throughput detection method of four types of anti-tuberculosis drugs in human serum by high-performance liquid chromatography tandem mass spectrometry, wherein the mobile phase is prepared as follows:

[0073] Preparation of 5M ammonium formate solution: Use an electronic balance to accurately weigh 15.765 g of ammonium formate, add 50 ml of pure water to prepare 50 mL of 5 mol / L ammonium formate solution, and place in a 4°C refrigerator for use.

[0074] Preparation of mobile phase A: Use a measuring cylinder to accurately transfer 1000 mL of water, add 1000 μL of formic acid solution and 1000 μL of 5 M ammonium formate solution, mix well, filter and degas by ultrasonication, and set aside.

[0075] Preparation of fluidity liquid B: methanol solution, filter and degas by ultrasonication, and set aside.

[0076] 6. The present invention discloses a high-throughput detection method of four class I anti-tuberculosis drugs in human serum by high-performance liquid chromatography tandem mass spectrometry, comprising the following pretreatment process:

[0077] 1) Add calibrator and quality control solution: Accurately pipette 100 μL of calibrator and quality control solution and add them into 2 mL 96-well plates respectively;

[0078] 2) Add serum sample: Accurately pipette 100 μL of serum sample and add it into 2 mL 96-well plates respectively;

[0079] 3) Add sample extract: Accurately pipette 400 μL of sample extract and add it to the corresponding 96-well plates above;

[0080] 4) Oscillation: Cover the 96-well plate with a lid, place on a 96-well plate oscillator, and oscillate thoroughly at 1500 rpm for 15 min;

[0081] 5) Centrifugation: In a refrigerated centrifuge, set the temperature to 4°C and the speed to 4000 rpm for 10 min;

[0082] 6) Transfer and dilute: After centrifugation, use a pipette to transfer 100 μL of the supernatant into a 1 mL 96-well U-shaped plate, and add 300 μL of water respectively;

[0083] 7) Oscillation: Cover the 96-well plate with a lid, place on a 96-well plate oscillator, and oscillate thoroughly at 1500 rpm for 5 min.

[0084] 8) Centrifugation: In a refrigerated centrifuge, set the temperature to 4°C and the speed to 4000 rpm for 10 min. After centrifugation, the sample is ready for testing.

[0085] 7. The present invention discloses a high-throughput detection method of four class I anti-tuberculosis drugs in human serum by high-performance liquid chromatography tandem mass spectrometry, comprising the following method settings:

[0086] Liquid phase test conditions table

[0087] 1) Chromatographic column: Kinetex 2.6μm F5 (3.0*100mm)

[0088] 2) Mobile phase: Phase A: water (containing 0.1% formic acid + 5 mM ammonium formate); Phase B: methanol

[0089] 3) Elution gradient:

[0090] Table 3 Gradient conditions

[0091] T(min) ％A ％B 0.00 95 5 2.00 87 13 2.10 10 90 4.50 10 90 4.51 95 5 5.00 95 5

[0092] 4) Flow rate: 0.5 mL / min

[0093] 5) Column temperature: 40°C

[0094] 6) Automatic sampler temperature: 10°C

[0095] 7) Needle washing solution: methanol: acetonitrile: isopropanol (1:1:1, v / v / v)

[0096] 8) Injection volume: 1 μL

[0097] The mass spectrometry test conditions are as follows:

[0098] Ion source parameter table

[0099] Table 4 Ion source parameters

[0100]

[0101] Ion information

[0102] Table 5 Ion information

[0103]

[0104] This method uses high-performance liquid chromatography tandem mass spectrometry to detect four first-class anti-tuberculosis drugs in human serum in high throughput. One test can simultaneously obtain four first-line anti-tuberculosis drugs, pyrazinamide, isoniazid, ethambutol, and rifampicin, with the characteristics of high sensitivity, good repeatability, high accuracy, and good specificity. The linear range of this method is 15.000-105.000μg / mL for pyrazinamide, 1.000-19.000μg / mL for isoniazid, 1.000-14.500μg / mL for ethambutol, and 0.400-13.000μg / mL for rifampicin. The correlation coefficients are all r≥0.990; the coefficient of variation (CV) of the repeatability of the low-value quality control product is ≤10%, the coefficient of variation (CV) of the repeatability of the mid-value quality control product is ≤10%, and the coefficient of variation (CV) of the repeatability of the high-value quality control product is ≤10%; the relative deviation (B) of the accuracy is ≤±15%, and the spike recovery rate is 98%-113%.

[0105] Experimental data

[0106] 1. Selection of pre-treatment method

[0107] 1.1. Selection of precipitant

[0108] Methanol, acetonitrile, and a mixture of acetonitrile and methanol were used to precipitate proteins from the samples. After centrifugation, the samples were directly loaded for testing. The results are shown in Figure 1 As shown in the figure, there is a bifurcation phenomenon in the pyrazinamide peak. It is inferred that this phenomenon is caused by the chromatographic solvent effect. Therefore, the sample extract after centrifugation is further diluted with water to make the ratio in the solution as close as possible to the initial ratio of the mobile phase. The results of acetonitrile extraction and direct injection after centrifugation are shown in the figure. Figure 2 As shown in the figure, the order of the peaks from left to right is ethambutol, isoniazid, pyrazinamide and rifampicin. Methanol / acetonitrile = 1 / 1 extraction, dilution 4 times with water, centrifugation and direct injection results are as follows Figure 3 As shown in the figure, the order of the peaks from left to right is ethambutol, isoniazid, pyrazinamide and rifampicin. Methanol / acetonitrile = 2 / 1 extraction, dilution 4 times with water, centrifugation and direct injection results are as follows Figure 4 As shown in the figure, the order of the peaks from left to right is ethambutol, isoniazid, pyrazinamide and rifampicin. Methanol / acetonitrile = 3 / 1 extraction, dilute 4 times with water, centrifuge and inject directly as shown in Figure 5 As shown in the figure, the order of the peaks from left to right is ethambutol, isoniazid, pyrazinamide and rifampicin. Methanol / acetonitrile = 4 / 1 extraction, dilution 4 times with water, centrifugation and direct injection as shown in the figure. Figure 6 As shown in the figure, the order of the peaks from left to right is ethambutol, isoniazid, pyrazinamide and rifampicin. Methanol extraction, dilution 4 times with water, centrifugation and direct injection as shown in Figure 7 As shown, the order of the peaks from left to right in the figure are ethambutol, isoniazid, pyrazinamide and rifampicin. Figure 8 This is the injection result diagram of the mobile phase A without ammonium formate. The order of the peaks from left to right in the figure is ethambutol, isoniazid, pyrazinamide and rifampicin. Fig. 9 This is the result of injecting sample into a C18 column using acidic ammonium formate as the mobile phase. The order of the peaks from left to right are ethambutol, isoniazid, pyrazinamide and rifampicin.

[0109] By using methanol, acetonitrile and a mixed solution of methanol and acetonitrile as protein precipitants and diluting the measured peak results, it can be judged that the results of using methanol and a mixed solution of methanol and acetonitrile as precipitants can meet the requirements. Since the toxicity and price of acetonitrile are higher than methanol, methanol was chosen as the precipitant for the subsequent experiments.

[0110] 2. Selection of chromatographic conditions

[0111] 2.1. Chromatographic column selection

[0112] Kinetex F5 (100mm*3mm, 2.6μm) chromatographic column and XBridgeBEH C18 (50mm*3mm, 2.5μm) were selected respectively. Methanol and ammonium formate aqueous solution were used as mobile phases, and the peak shape was adjusted by adding ammonia or formic acid. The results showed that the four compounds could be separated by using both chromatographic columns. The present invention selected F5 column as the final test condition chromatographic column.

[0113] Under C18 conditions, phase A was methanol, phase B was 10 mM ammonium formate aqueous solution, and ammonia was added to adjust its pH to 8. The gradient conditions are shown in the following table. The order of peaks from left to right is isoniazid, pyrazinamide, ethambutol and rifampicin.

[0114] Table 6 Gradient program

[0115]

[0116]

[0117] Under F5 conditions, phase A was methanol and phase B was 15 mM ammonium formate aqueous solution. The pH was adjusted by adding 1 part per thousand formic acid to improve the peak shape. The gradient conditions are shown in the following table. The order of peaks from left to right was ethambutol, isoniazid, pyrazinamide and rifampicin.

[0118] Table 7 Gradient program

[0119] T(min) ％A ％B Flow rate 0.00 95 5 0.5mL / min 2.00 87 13 0.5mL / min 2.10 10 90 0.5mL / min 4.50 10 90 0.5mL / min 4.51 95 5 0.5mL / min 5.00 95 5 0.5mL / min

[0120] 2.2 Mobile phase selection

[0121] 2.2.1 Mobile phase selection

[0122] Using an F5 column, the compounds were analyzed and detected by adjusting the pH and different concentrations of ammonium formate aqueous solution and methanol as the mobile phase. Finally, 5 mM ammonium formate containing 1 / 1000 formic acid aqueous solution and methanol were selected as the final test condition mobile phase.

[0123] An F5 column was used, with phase A being 15 mM ammonium formate aqueous solution containing 0.2% formic acid in water, and phase B being methanol. The gradient flow was as shown in the table below. The order of peaks from left to right was ethambutol, isoniazid, pyrazinamide, and rifampicin.

[0124] Table 8 Gradient program

[0125] T(min) ％A ％B Flow rate 0.00 95 5 0.5mL / min 2.00 87 13 0.5mL / min 2.10 10 90 0.5mL / min 4.50 10 90 0.5mL / min 4.51 95 5 0.5mL / min 5.00 95 5 0.5mL / min

[0126] An F5 column was used, phase A was 10 mM ammonium formate aqueous solution containing 0.1% formic acid, phase B was methanol, and the gradient process was shown in the table below. The peak order from left to right was ethambutol, isoniazid, pyrazinamide and rifampicin.

[0127] Table 9 Gradient program

[0128]

[0129]

[0130] An F5 column was used, phase A was 5 mM ammonium formate aqueous solution containing 0.1% formic acid, phase B was methanol, and the gradient process was shown in the table below. The order of peaks from left to right was ethambutol, isoniazid, pyrazinamide and rifampicin.

[0131] Table 10 Gradient program

[0132] T(min) ％A ％B Flow rate 0.00 95 5 0.5mL / min 2.00 87 13 0.5mL / min 2.10 10 90 0.5mL / min 4.50 10 90 0.5mL / min 4.51 95 5 0.5mL / min 5.00 95 5 0.5mL / min

[0133] 2.2.2 Comparison of adding ammonium formate to the mobile phase and not adding ammonium formate

[0134] Using the F5 column, by comparing the effect of adding ammonium formate to mobile phase A on the test results, 5 mM ammonium formate containing 1 / 1000 formic acid aqueous solution and methanol were finally selected as the final test condition mobile phase.

[0135] The calibration and quality control materials were pre-treated according to the calibration and quality control material pre-treatment operation methods, and the treated liquids were used for sample analysis using two mobile phases respectively.

[0136] Mobile phase method 1: Phase A is 0.2% formic acid aqueous solution, phase B is methanol, gradient conditions are shown in Table 11, results are shown in Table 13, and results are shown in Figure 14. Figure 8 shown.

[0137] Table 11 Gradient conditions of method 1

[0138] T(min) ％A ％B Flow rate 0.00 95 5 0.5mL / min 2.00 87 13 0.5mL / min 2.10 10 90 0.5mL / min 4.50 10 90 0.5mL / min 4.51 95 5 0.5mL / min 5.00 95 5 0.5mL / min

[0139] Mobile phase method 2: Phase A is 0.1% formic acid, 5 mM ammonium formate aqueous solution, phase B is methanol, gradient conditions are shown in Table 12, results are shown in Table 13, and results are shown in Figure 14. Figure 7 shown.

[0140] Table 12 Gradient conditions of method 2

[0141]

[0142]

[0143] Table 13 Comparison of results between method 1 and method 2

[0144]

[0145] Compare Figure 8 and Figure 7 , without adding ammonium formate, the first ethambutol peak was greatly affected, and a forked peak appeared. From the results in Table 13, the method 1 was compared with the method 2, and the results showed that the addition of ammonium formate significantly improved the linearity of ethambutol, and the related linear coefficient was significantly improved. Therefore, ammonium formate and formic acid aqueous solution were finally selected as mobile phase A.

[0146] 2.2.3 Internal Standard Selection

[0147] The accuracy of rifampicin was evaluated by testing the accuracy sample and calculating its recovery rate to assess whether the accuracy met the requirements of performance evaluation. A certain amount of the working solution of the analyte was added to the blank human matrix to prepare 4 concentrations of the lower limit of quantification of the recovered sample, low concentration quality control, medium concentration quality control, and high concentration quality control. Three samples were measured repeatedly for each concentration.

[0148] The calibration and quality control products were pre-treated according to the calibration and quality control product pre-treatment operation method, and the treated liquid was sampled for analysis. The analysis results were respectively Rifampicin D4 and Pyrazinamide- 15 N,D3 was used as the internal standard to perform quantitative detection and analysis of rifampicin. The analysis results are shown in Table 14.

[0149] Table 14 Effects of different internal standards on rifampicin test results

[0150]

[0151]

[0152] From the results in Table 14, it can be seen that when rifampicin D4 was used as the internal standard, the recovery rate of the human sample matrix spiked was between 93.55% and 106.42%, and when pyrazinamide- 15 N,D3 was used as the internal standard, and the results showed that the recovery rate of rifampicin was 32.41% to 68.87%. Therefore, rifampicin-D4 was finally selected as the internal standard in this plan to test rifampicin.

[0153] 3. Linear range test data

[0154] 3.1. Verification method: Treat the calibrants S1 to S6 of the tested product according to the sample processing method described in the high-throughput detection of four class I anti-tuberculosis drugs in human serum by HPLC-MS / MS, and repeat the test three times for each concentration. The correlation coefficient r of linear regression can be calculated by reference to the formula, and the correlation coefficient R^2 should be ≥0.990.

[0155]

[0156] r: linear regression correlation coefficient

[0157] xi: Concentration of S1 to S6

[0158] yi: The mean peak area ratio of the calibrator to its internal standard in the corresponding concentration solution

[0159] 3.2. Acceptance criteria: The linear regression correlation coefficients of ethambutol, isoniazid, pyrazinamide, and rifampicin were all ≥ 0.990.

[0160] 3.3 Experimental Results

[0161] Linear range: pyrazinamide linear range 15.000~105.00μg / mL, isoniazid linear range 1.000~19.000μg / mL, ethambutol linear range 1.000~10.300μg / mL, rifampicin linear range 0.400~13.000μg / mL.

[0162] Table 15 Linear data

[0163]

[0164] 3.4. Conclusion: The correlation coefficients R^2 of linear regression of ethambutol, isoniazid, pyrazinamide, and rifampicin were all ≥0.990, which met the acceptance criteria.

[0165] 4. Repeatability test data

[0166] 4.1. Verification method: Use the prepared quality control products and process the products to be tested according to the sample processing method described in the high-throughput detection of four class I anti-tuberculosis drugs in human serum by HPLC-MS / MS. Repeat the test 10 times for each sample. The coefficient of variation (CV) of repeatability can be calculated by reference to the formula. The CV of low-value quality control products is ≤20%, and the CV of high-value quality control products is ≤15%.

[0167]

[0168] CV: coefficient of variation of repeatability

[0169] The average value of 10 measurements

[0170] S: standard deviation of 10 measurements

[0171] 4.2. Acceptance criteria: The coefficient of variation CV of low-value quality control products is ≤20%, the coefficient of variation CV of mid-value quality control products is ≤15%, and the coefficient of variation CV of high-value quality control products is ≤15%.

[0172] 4.3 Experimental Results

[0173] Table 16 Pyrazinamide repeatability data

[0174]

[0175] Table 17 Isoniazid repeatability data

[0176]

[0177]

[0178] Table 18 Ethambutol repeatability data

[0179]

[0180] Table 19 Rifampicin repeatability data

[0181]

[0182]

[0183] 4.4. Conclusion: The coefficient of variation CV of the low-value quality control product is ≤15%, and the coefficient of variation CV of the high-value quality control product is ≤15%, which meets the acceptance criteria.

[0184] 5. Evaluation of matrix effects

[0185] 5.1 Experimental Process

[0186] Use at least 6 batches of blank matrix from different donors. For each batch of matrix, the matrix factor of each analyte and internal standard should be calculated by calculating the ratio of the peak area in the presence of matrix (measured by blank matrix extraction followed by addition of analyte and internal standard) to the corresponding peak area in the absence of matrix (pure solutions of analyte and internal standard). Further, the matrix factor normalized for the internal standard should be calculated by dividing the matrix factor of the analyte by the matrix factor of the internal standard.

[0187] 5.2 Acceptance criteria

[0188] The coefficient of variation of the internal standard normalized matrix factor calculated from 6 batches of matrix should all be less than 15%.

[0189] 5.3 Experimental Results

[0190] Table 20 Pyrazinamide matrix effect data

[0191]

[0192] Table 21 Isoniazid matrix effect data

[0193]

[0194] Table 22 Ethambutol matrix effect data

[0195]

[0196] Table 23 Rifampicin matrix effect data

[0197]

[0198] 5.4 Experimental Summary

[0199] The coefficient of variation of the matrix factors normalized by the internal standard calculated by batch matrix was less than 15%, which met the requirements.

[0200] 6. Blood sample interference evaluation

[0201] 6.1 Experimental Process

[0202] Screen potential interfering substances through interference experimental procedures, quantify interfering effects, confirm interference in patient samples, confirm the sensitivity of analytical methods to interfering substances, evaluate potential risks, and provide meaningful interference statements to customers.

[0203] According to the interference concentration requirements of the experimental design, a certain amount of interference storage solution is added to the basic sample as the test sample. The addition amount of this experimental scheme is uniformly 5% of the basic sample. Add 5% of the solvent used to prepare the storage solution to the basic sample as the control sample, and its addition volume is the same as that of the test sample. Analyze the test sample and the control sample in an alternating order.

[0204] 6.2 Acceptance criteria

[0205] If Dev% = (Xtest-Xcontrol) / Xcontrol≤15%, the potential interfering substance has no effect on the determination of the analyte, otherwise it can be judged that the potential interference exists.

[0206] 6.3 Experimental Results

[0207] Table 24 Interfering substances and their concentrations

[0208]

[0209]

[0210] Table 25 Results of bilirubin interference experiment

[0211]

[0212] Table 26 Triglyceride interference test results

[0213]

[0214]

[0215] Table 27 Cholesterol interference test results

[0216]

[0217] Table 28 Hemoglobin interference test results

[0218]

[0219]

[0220] Table 29 Results of rifampicin hemoglobin interference experiment

[0221]

[0222] 6.4 Experimental Summary

[0223] The concentration of interference in this experiment mainly refers to the recommended experimental concentration of common endogenous interference in the "WS / T-2013 Interference Experiment Guide". Since hemoglobin has a greater impact on rifampicin detection at this recommended concentration, the hemoglobin concentration is reduced to obtain a hemoglobin concentration that has no effect on rifampicin detection. The test showed that when the hemoglobin concentration in serum is 0.04g / L, it has no effect on rifampicin detection in this method.

[0224] 7. Accuracy

[0225] 7.1 Test process

[0226] This protocol evaluates accuracy by testing accuracy samples of pyrazinamide, isoniazid, ethambutol and rifampicin and calculating their recovery rates. Evaluate whether the accuracy meets the requirements of performance evaluation. Add a certain amount of the working solution of the analyte to the blank human matrix to prepare two concentrations of recovered samples L and H (i.e., quality control samples). The concentration selection includes low concentration level and high concentration level. Take three analysis batches and repeat the measurement of 3 samples at each concentration.

[0227] 7.2 Acceptance criteria

[0228] Recovery rate: 85%~115%.

[0229] Table 30 Pyrazinamide Accuracy

[0230]

[0231] Table 31 Isoniazid Accuracy

[0232]

[0233] Table 32 Ethambutol Accuracy

[0234]

[0235]

[0236] Table 33 Rifampicin Accuracy

[0237]

[0238] 7.3 Experimental Summary

[0239] The accuracy is within ±15% of the indicated value, meeting the requirements.

[0240] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the concentration of anti-tuberculosis drug components, characterized in that: The anti-tuberculosis drug contains pyrazinamide, isoniazid, ethambutol and rifampicin; The detection method comprises using a separation method to separate the anti-tuberculosis drug components contained in the sample to be tested, and then obtaining the concentration of each component through mass spectrometry detection; the separation method comprises: after the sample to be tested containing the anti-tuberculosis drug components is subjected to methanol precipitation to remove impurities, an F5 chromatographic column is used, and a mobile phase is prepared using methanol and ammonium formate aqueous solution to perform high performance liquid chromatography to separate the anti-tuberculosis drug components; Among them, the internal standards corresponding to pyrazinamide, isoniazid, ethambutol and rifampicin are pyrazinamide- 15 N,D3, isoniazid-D4, ethambutol-D4, and rifampin-D4; Wherein, the mobile phase A of the HPLC is an aqueous solution of ammonium formate containing 1 / 1000 v / v formic acid, and the concentration of the ammonium formate is 5-15 mM; the mobile phase B is methanol; The mobile phase conditions of the high performance liquid chromatography are as follows: the volume ratio of mobile phase A from 0 to 2 min is 95% to 87%, the volume ratio of mobile phase A from 2.1 to 4.5 min is 10%, and the volume ratio of mobile phase A from 4.51 to 5 min is 95%; Among them, the ion source parameters of the mass spectrometry detection are: the ion source is ESI+, the curtain gas pressure is 30~40 psi, the collision gas pressure is medium, the spray voltage is 5500 V, the temperature is 450~550 ℃, the spray gas GS1 pressure is 40~55 psi, and the spray gas GS2 pressure is 40~60 psi.

2. The method for detecting the concentration of anti-tuberculosis drug components according to claim 1, characterized in that: The detection conditions of the high performance liquid chromatography are as follows: a mobile phase flow rate of 0.3-0.6 mL / min, a column temperature of 30-45°C, an injector temperature of 5-20°C, a needle wash solution of a mixed solution of methanol, acetonitrile and isopropanol in a volume ratio of 1:(0.5-2):(0.5-2), and an injection volume of 1-20 μL.

3. The method for detecting the concentration of anti-tuberculosis drug components according to claim 1, characterized in that: The detection conditions of the high performance liquid chromatography are as follows: a mobile phase flow rate of 0.5 mL / min, a column temperature of 40°C, an injector temperature of 10°C, a needle wash solution of a mixed solution of methanol, acetonitrile and isopropanol in a volume ratio of 1:1:1, and an injection volume of 1 μL.

4. The separation method according to claim 1, characterized in that The sample to be tested includes human serum.

5. The separation method according to claim 4, characterized in that The hemoglobin concentration in the human serum is less than 0.04 g / L.

6. The detection method according to claim 1, characterized in that The ion source parameters were as follows: ESI+ ion source, 40 psi curtain gas pressure, 7 psi collision gas pressure, 500 V spray voltage, 500 °C temperature, 55 psi spray gas GS1 pressure, and 60 psi spray gas GS2 pressure.

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