CN115480008A

CN115480008A - Method for simultaneously detecting components of multiple antituberculosis drugs by high performance liquid chromatography-tandem mass spectrometry

Info

Publication number: CN115480008A
Application number: CN202211111314.XA
Authority: CN
Inventors: 郑佳; 应洪波; 栗琳; 周继福; 张新星; 周立; 丁沛瑜; 苏营雪; 丁亮
Original assignee: Hunan Haosi Biotechnology Co ltd; Jiangsu Haosi Muke Biotechnology Co ltd; Beijing Haosi Biotechnology Co ltd
Current assignee: Hunan Haosi Biotechnology Co ltd; Jiangsu Haosi Muke Biotechnology Co ltd; Beijing Haosi Biotechnology Co ltd
Priority date: 2022-09-13
Filing date: 2022-09-13
Publication date: 2022-12-16

Abstract

The invention relates to the technical field of pharmacy, in particular to a method for simultaneously detecting various antituberculosis drug components by high performance liquid chromatography-tandem mass spectrometry. The method for detecting four anti-tuberculosis drugs in human serum in high throughput by the high performance liquid chromatography-tandem mass spectrometry provided by the invention can simultaneously obtain four first-line anti-tuberculosis drugs of pyrazinamide, isoniazid, ethambutol and rifampicin by one-time detection, and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity and the like. The Coefficient of Variation (CV) of repeatability of the obtained low-value quality control product is less than or equal to 10 percent, the Coefficient of Variation (CV) of repeatability of the medium-value quality control product is less than or equal to 10 percent, and the Coefficient of Variation (CV) of repeatability of the high-value quality control product is less than or equal to 10 percent; the relative deviation (B) of the accuracy is less than or equal to +/-15 percent, and the standard recovery rate is 98-113 percent.

Description

Method for simultaneously detecting components of multiple antituberculosis drugs by high performance liquid chromatography-tandem mass spectrometry

Technical Field

The invention relates to the technical field of pharmacy, in particular to a method for simultaneously detecting various anti-tuberculosis medicine components by high performance liquid chromatography-tandem mass spectrometry.

Background

Tuberculosis is an infectious disease caused by infection of a patient with mycobacterium tuberculosis, and antituberculosis drugs are mainly classified into four types: first line antituberculosis drugs, second line antituberculosis drugs, novel antituberculosis drugs and compound preparation drugs. The first-line antitubercular drugs mainly comprise Rifampicin (RMP), isoniazid (INH), pyrazinamide (PZA) and Ethambutol (EMB).

The anti-tuberculosis drugs are mainly tested by a liquid chromatography-ultraviolet method, a liquid chromatography-evaporative light scattering method, a liquid chromatography-tandem mass spectrometry method and the like. As the ethambutol in the four first-line antituberculosis drugs has no ultraviolet absorption, an evaporative light scattering detector is required for detection, so that the simultaneous accurate detection of the four components is difficult.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a high performance liquid tandem mass spectrometry method capable of simultaneously detecting four antituberculosis drug components so as to solve the technical problem that ethambutol cannot be used for ultraviolet detection.

In order to solve the technical problems and achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the present invention provides a method for separating an antituberculous pharmaceutical ingredient containing at least one of pyrazinamide, isoniazid, ethambutol or rifampicin;

the separation method comprises the steps of carrying out methanol precipitation and impurity removal on a sample to be detected containing the anti-tuberculosis drug components, and then carrying out high performance liquid chromatography on the sample to be detected by using an F5 chromatographic column or a C18 chromatographic column and adopting a mobile phase prepared from methanol and an ammonium formate aqueous solution to realize the separation of the anti-tuberculosis drug components.

In alternative embodiments, the mobile phase A of the high performance liquid chromatography is an aqueous ammonium formate solution containing 1/1000 (v/v) formic acid, the ammonium formate concentration being between 5 and 15mM; the mobile phase B is methanol.

In an alternative embodiment, the mobile phase conditions of the high performance liquid chromatography are that the volume ratio of the mobile phase A is 87% to 95% in 0-2 min, the volume ratio of the mobile phase A is 10% in 2.1-4.5 min, and the volume ratio of the mobile phase A is 95% in 4.51-5 min.

In an alternative embodiment, the liquid phase conditions of the high performance liquid chromatography are that the flow rate of the mobile phase is 0.3-0.6 mL/min, the column temperature is 30-45 ℃, the temperature of the sample injector is 5-20 ℃, and the volume ratio of the needle washing liquid is 1: (0.5-2): (0.5-2) a mixed solution of methanol, acetonitrile and isopropanol, wherein the sample volume is 1-20 mu L.

In alternative embodiments, the liquid phase conditions of the hplc are a mobile phase flow rate of 0.5mL/min, a column temperature of 40 ℃, a sample injector temperature of 10 ℃, and a needle wash in a volume ratio of 1:1:1, and a mixed solution of methanol, acetonitrile and isopropanol, wherein the sample volume is 1 mu L.

In an alternative embodiment, the internal standards corresponding to pyrazinamide, isoniazid, ethambutol and rifampicin are pyrazinamide- ¹⁵ N,D ₃ isoniazid-D ₄ ethambutol-D ₄ And rifampin-D ₄ 。

In an alternative embodiment, the test sample comprises human serum.

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

In a second aspect, the present invention provides the use of the isolation method of any one of the preceding embodiments in the development of a medicament comprising at least one of pyrazinamide, isoniazid, ethambutol or rifampin, in drug screening or in scientific research.

In a third aspect, the invention provides a method for detecting the concentration of an antituberculous drug component, wherein the antituberculous drug component contains at least one of pyrazinamide, isoniazid, ethambutol or rifampicin;

the detection method comprises the steps of separating the components of the anti-tuberculosis drugs contained in a sample to be detected by adopting the separation method of any one of the previous embodiments, and then detecting the concentration of each component by mass spectrometry.

In an alternative embodiment, the ion source parameters for mass spectrometry detection are: the ion source is ESI +, the air curtain air pressure is 30-40 psi, the collision air pressure is medium, the spraying voltage is 5500V, the temperature is 450-550 ℃, the pressure of the spraying gas GS1 is 40-55 psi, and the pressure of the spraying gas GS2 is 40-60 psi.

Preferably, the ion source parameters are: the ion source is ESI +, the air curtain pressure is 40psi, the collision pressure is 7psi, the spray voltage is 500V, the temperature is 500 ℃, the pressure of the spray GS1 is 55psi, and the pressure of the spray GS2 is 60psi.

The method for detecting four anti-tuberculosis drugs in human serum in high throughput by the high performance liquid chromatography-tandem mass spectrometry provided by the invention can simultaneously obtain four first-line anti-tuberculosis drugs of pyrazinamide, isoniazid, ethambutol and rifampicin by one-time detection, and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity and the like. The linear range of the pyrazinamide obtained by detection is 15.000-105.000 mug/mL, the isoniazide is 1.000-19.000 mug/mL, the ethambutol is 1.000-14.500 mug/mL, the rifampicin is 0.400-13.000 mug/mL, and the correlation coefficient r is more than or equal to 0.990; the Coefficient of Variation (CV) of repeatability of the low-value quality control product is less than or equal to 10 percent, the Coefficient of Variation (CV) of repeatability of the medium-value quality control product is less than or equal to 10 percent, and the Coefficient of Variation (CV) of repeatability of the high-value quality control product is less than or equal to 10 percent; the relative deviation (B) of the accuracy is less than or equal to +/-15 percent, and the labeling recovery rate is 98-113 percent.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram showing the results of direct sample injection after centrifugation of a methanol protein precipitated sample;

FIG. 2 is a diagram showing the results of acetonitrile extraction and direct sample injection after centrifugation;

fig. 3 is a graph of the results of methanol/acetonitrile =1/1 extraction, dilution with water by 4 times, centrifugation, and direct injection;

fig. 4 is a graph of the results of methanol/acetonitrile =2/1 extraction, dilution 4 times with water, centrifugation, and direct injection;

fig. 5 is a graph of the results of methanol/acetonitrile =3/1 extraction, dilution 4 times with water, centrifugation, and direct injection;

fig. 6 is a graph of the results of methanol/acetonitrile =4/1 extraction, dilution with water by 4 times, centrifugation, and direct injection;

FIG. 7 is a diagram showing the results of methanol extraction, dilution with water by 4 times, centrifugation, and direct sample injection;

FIG. 8 is a graph showing the results of the injection of mobile phase A without ammonium formate.

FIG. 9 is a graph showing the results of C18 column injection using acidic mobile phase.

Detailed Description

In order to make the objects, 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 with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention.

Thus, the following detailed description of the embodiments of the present invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments and features of the embodiments described below can be combined with each other without conflict.

The invention discloses a high performance liquid mass spectrometry method for detecting four anti-tuberculosis drugs in human serum in a high-throughput manner. Anti-tuberculosis drugs in human serum are subjected to protein precipitation by an organic solvent, centrifugation and dilution, then a sample loading test is carried out, all pretreatment can be carried out in a 96-well plate, and a discharge gun is used for transferring and taking, so that batch pretreatment is realized. After pretreatment, the sample enters a liquid chromatography-mass spectrometer for detection, and the test of four drugs can be completed within five minutes, so that the test is convenient and quick. As the human sample is difficult to obtain, the BSA is adopted as the substitute matrix of the human serum to prepare each concentration point and quality control of the standard curve.

1. The invention discloses a high-throughput detection method for four anti-tuberculosis drugs in human serum by a high performance liquid chromatography-tandem mass spectrometry method, which mainly comprises the following reagents:

TABLE 1 standards and reagents

2. The invention discloses a high-throughput detection method for four types of anti-tuberculosis drugs in human serum by a high performance liquid chromatography-tandem mass spectrometry method, which comprises the following equipment:

TABLE 2 instrumentation

Serial number	Device name	Model/specification
			1	Liquid chromatography tandem mass spectrometer	AB SCIEX Triple Quad TM4500MD
2	96-orifice plate oscillator	MB100-4A
			3	96-well plate centrifuge	Hunan instrument H2050R
4	10 mu L liquid shifter	0.5-10μL
			5	200 mu L liquid transfer device	20-200μL
6	1000 mu L liquid transfer device	100-1000μL
			7	300 mu L12-hole liquid transferring device	30-300μL
8	96-well plate	2mL
			9	96-well plate	1mL
10	Electronic balance	One in ten thousand

3. The invention discloses a high-throughput detection method of four anti-tuberculosis drugs in human serum by a high performance liquid chromatography-tandem mass spectrometry method, which comprises the following steps of:

3.1 preparation of antituberculosis drug stock solution

Preparing a pyrazinamide stock solution: accurately weighing 100mg of pyrazinamide by using a one-ten-thousandth electronic balance, placing the pyrazinamide in a 10mL volumetric flask, dissolving the pyrazinamide in methanol, fixing the volume to a scale mark, preparing stock solution with the concentration of 10.00mg/mL, transferring the stock solution into a 15mLHDPE plastic bottle, labeling, and storing at-80 ℃.

Preparing isoniazid stock solution: accurately weighing 100mg of isoniazid by using a one-ten-thousandth electronic balance, placing the isoniazid in a 10mL volumetric flask, dissolving the isoniazid by using methanol, fixing the volume to a scale mark, preparing a stock solution with the concentration of 10.00mg/mL, transferring the stock solution into a 15mLHDPE plastic bottle, labeling, and storing at-80 ℃.

Preparing an ethambutol stock solution: accurately weighing 100mg of ethambutol by using a ten-thousandth electronic balance, placing the ethambutol into a 10mL volumetric flask, dissolving the ethambutol by using methanol, metering the volume to a scale mark, preparing a stock solution with the concentration of 10.00mg/mL, transferring the stock solution into a 15mLHDPE plastic bottle, labeling, and storing at-80 ℃.

Preparing a rifampicin stock solution: accurately weighing 100mg of pyrazinamide by using a one-ten-thousandth electronic balance, placing the pyrazinamide in a 10mL volumetric flask, dissolving the pyrazinamide in methanol, fixing the volume to a scale mark, preparing stock solution with the concentration of 10.00mg/mL, transferring the stock solution into a 15mLHDPE plastic bottle, labeling, and storing at-80 ℃.

3.2 preparation of Secondary stock solution of antituberculotic drug

Preparing a pyrazinamide secondary stock solution: accurately transferring 1mL of pyrazinamide stock solution by using a pipette, transferring to a 10mL volumetric flask, diluting to a constant volume with methanol to a scale mark, preparing into 1.00mg/mL secondary stock solution, transferring to a 15mLHDPE plastic bottle, labeling, and storing at-80 ℃.

Preparing an isoniazid secondary stock solution: accurately transferring 100 mu l of isoniazid stock solution by using a liquid transfer gun, transferring to a 10mL volumetric flask, metering to a scale mark by using methanol to prepare 0.10mg/mL secondary stock solution, transferring to a 15mLHDPE plastic bottle, labeling and storing at-80 ℃.

Preparing secondary ethambutol stock solution: accurately transferring 100 μ l of ethambutol stock solution by using a pipette, transferring to a 10mL volumetric flask, diluting to a scale mark with methanol to obtain a 0.10mg/mL secondary stock solution, transferring to a 15mLHDPE plastic bottle, labeling, and storing at-80 deg.C.

Preparing a rifampicin secondary stock solution: accurately transferring 100 μ l rifampicin stock solution by using a pipette, transferring to a 10mL volumetric flask, adding methanol to constant volume to scale mark, preparing into 0.10mg/mL secondary stock solution, transferring to a 15mLHDPE plastic bottle, labeling, and storing at-80 deg.C.

3.3 preparation of calibrators S1 to S6

3.3.1 preparation of Diluent

Preparing PBS diluent: measuring 10mL 10 × PBS in a 100mL volumetric flask, adding pure water to the scale mark, uniformly mixing to obtain 1 × PBS, and transferring to a glass container bottle for later use.

0.1% BSA solution preparation: weighing 0.1g BSA, accurately placing into a glass container bottle until the weight is 0.0001g BSA, adding 100ml PBS diluent, covering the bottle cap, and shaking up and down until the BSA is completely dissolved.

The preparation of the standard products is carried out according to a high-low preparation mode, namely S1 and S6 are prepared and then are mixed in proportion to prepare S2 to S5, LQC, MQC and HQC.

Preparing a calibrator S6: selecting a pipetting gun with a proper specification, respectively taking 2.625mL of pyrazinamide stock solution, 0.475mL of isoniazid stock solution, 0.3625mL of ethambutol stock solution and 0.325mL of rifampicin stock solution, placing the materials into a proper HDPE tube, adding 0.1 percent of BSA solution 21.2125mL, and preparing 105.000 mu g/mL of pyrazinamide, 19.000 mu g/mL of isoniazid, 14.500 mu g/mL of ethambutol and 13.000 mu g/mL of rifampicin standard solution. Mixing, labeling, and storing at-20 deg.C.

Preparing a calibrator S1: selecting a pipetting gun with a proper specification, respectively taking 0.75mL of pyrazinamide secondary stock solution, 0.5mL of isoniazid secondary stock solution, 0.5mL of ethambutol secondary stock solution and 0.2mL of rifampicin secondary stock solution, placing the materials into a proper HDPE tube, adding 48.05mL of 0.1-percent BSA solution, and preparing standard solutions of pyrazinamide 15.000 mu g/mL, isoniazid 1.000 mu g/mL, ethambutol 1.000 mu g/mL and rifampicin 10.400 mu g/mL. Mixing, labeling, and storing at-20 deg.C.

Preparing a calibrator S2: accurately transferring 11 mL of S1 standard substance and 3.5mL of S6 standard substance, and fully mixing to prepare 29.000 mu g/mL of pyrazinamide, 3.800 mu g/mL of isoniazid, 3.100 mu g/mL of ethambutol and 2.360 mu g/mL of rifampicin standard solution. Mixing, labeling, and storing at-20 deg.C.

Preparing a calibrator S3: accurately transferring the S1 standard substance 16mL and the S6 standard substance 63.5mL, and fully mixing to prepare a standard solution of pyrazinamide 41.000 mu g/mL, isoniazid 6.200 mu g/mL, ethambutol 4.900 mu g/mL and rifampicin 4.040 mu g/mL. After mixing well, labeling and storing at-20 ℃.

Preparing a calibrator S4: accurately transferring 12mL of an S1 standard substance and 10.5mL of an S6 standard substance, and fully mixing to prepare a 57.000 mu g/mL pyrazinamide standard solution, 9.400 mu g/mL isoniazid, 7.300 mu g/mL ethambutol and 6.280 mu g/mL rifampicin standard solution. Mixing, labeling, and storing at-20 deg.C.

Preparing a calibrator S5: accurately transferring 7mL of S1 standard and 15.5mL of S6 standard, and fully mixing to prepare 77.000 mu g/mL of pyrazinamide, 13.400 mu g/mL of isoniazid, 10.300 mu g/mL of ethambutol and 9.080 mu g/mL of rifampicin standard solution. Mixing, labeling, and storing at-20 deg.C.

3.4 preparation of quality control product

Preparing a low-concentration quality control product LQC: accurately transferring 21.2mL of S1 standard substance and 1.3mL of S6 standard substance, fully mixing to prepare a standard solution with 20.200 mu g/mL of pyrazinamide, 2.040 mu g/mL of isoniazid, 1.780 mu g/mL of ethambutol and 1.128 mu g/mL of rifampicin. After mixing well, labeling and storing at-20 ℃.

Preparing a medium-concentration quality control product MQC: accurately transferring 1 standard substance 12mL and 10.5mL of S6 standard substance, and mixing completely to prepare 57.000 mu g/mL of pyrazinamide, 9.400 mu g/mL of isoniazid, 7.300 mu g/mL of ethambutol and 6.280 mu g/mL of rifampicin standard solution. After mixing well, labeling and storing at-20 ℃.

Preparing a high-concentration quality control product HQC: accurately transferring 6mL of S1 standard substance and 16.5mL of S6 standard substance, fully mixing to prepare a standard solution of 81.000 mu g/mL of pyrazinamide, 14.200 mu g/mL of isoniazid, 10.900 mu g/mL of ethambutol and 9.640 mu g/mL of rifampicin. After mixing well, labeling and storing at-20 ℃.

4. The invention discloses a high-throughput detection method for four anti-tuberculosis drugs in human serum by a high performance liquid chromatography-tandem mass spectrometry method, and preparation of a sample extract.

Preparing a sample extraction liquid stock solution: 10mg of pyrazinamide- ¹⁵ N,D ₃ 10mg of isoniazid-D ₄ 10mg of ethambutol-D ₄ 10mg of rifampin-D ₄ Accurate to 0.01mg, respectively placing in 10mL volumetric flasks, adding methanol to scale mark, preparing into 1.00mg/mL stock solution, labeling, and storing at-20 deg.C.

Preparing a sample extraction liquid: transferring 1000 μ L of pyrazinamide- ¹⁵ N,D ₃ 100 μ L of isoniazid-D ₄ 10 μ L of ethambutol-D ₄ 500 μ L of rifampin-D ₄ Putting the mixture into a 500mL volumetric flask, adding methanol to the scale mark, and fully and uniformly mixing the mixture to prepare the pyrazinamide- ¹⁵ N,D ₃ 0.20. Mu.g/mL isoniazid-D ₄ 0.02. Mu.g/mL ethambutol-D ₄ ，1.00μgrifampin-D/mL ₄ The sample extract of (1). Labeling, and storing at-20 ℃.

5. The invention discloses a high-throughput detection method of four anti-tuberculosis drugs in human serum by a high performance liquid chromatography-tandem mass spectrometry method, which comprises the following steps:

preparation of 5M ammonium formate solution: accurately weighing 15.765g of ammonium formate by using an electronic balance, adding 50mL of pure water to prepare 50mL of 5mol/L ammonium formate solution, and placing the solution in a refrigerator at 4 ℃ for later use.

Preparing a mobile phase A liquid: and accurately transferring 1000mL of water by using the measuring cylinder, adding 1000 mu L of formic acid solution and 1000 mu L of 5M ammonium formate solution, uniformly mixing, filtering and ultrasonically degassing for later use.

Preparing a fluid B solution: filtering the methanol solution, and ultrasonically degassing for later use.

6. The invention discloses a high-throughput detection method of four anti-tuberculosis drugs in human serum by a high performance liquid chromatography-tandem mass spectrometry method, which comprises the following pretreatment processes:

1) Adding a calibrator and a quality control solution: precisely transferring 100 mu L of calibrator and quality control solution, and respectively adding into a 2mL 96-well plate;

2) Adding a serum sample: accurately transferring 100 mu L of serum samples, and respectively adding the serum samples into 2mL 96-well plates;

3) Adding the extract: precisely transferring 400 mu L of sample extraction liquid, and respectively adding the sample extraction liquid into the corresponding 96-well plates;

4) Oscillating: covering a 96-hole plate cover, placing on a 96-hole plate oscillator, and fully oscillating for 15min at 1500 rpm;

5) Centrifuging: centrifuging at 4000rpm for 10min at 4 deg.C in a refrigerated centrifuge;

6) Transferring and diluting: after centrifugation, 100 mu L of supernatant is transferred by a pipette gun and placed in 1mL of U-shaped plates with 96 holes, and 300 mu L of water is respectively added;

7) Oscillating: cover 96-well plate cover, place on 96-well plate shaker, shake well for 5min at 1500 rpm.

8) Centrifuging: setting the temperature at 4 ℃ in a refrigerated centrifuge, centrifuging at the rotation speed of 4000rpm for 10min, and testing the centrifuged sample.

7. The invention discloses a high-throughput detection method for four anti-tuberculosis drugs in human serum by a high performance liquid chromatography-tandem mass spectrometry method, which comprises the following steps:

liquid phase test condition table

1) A chromatographic column: kinetex 2.6 μm F5

(3.0*100mm)

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

3) Elution gradient:

TABLE 3 gradient conditions

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

4) Flow rate: 0.5mL/min

5) Column temperature: 40 deg.C

6) Autosampler temperature: 10 deg.C

7) Needle washing liquid: methanol: acetonitrile: isopropanol (1

8) Sample introduction amount: 1 μ L

The mass spectrometry conditions were as follows:

ion source parameter table

TABLE 4 ion Source parameters

Ion information

TABLE 5 ion information

The method adopts high performance liquid chromatography tandem mass spectrometry to detect four anti-tuberculosis drugs in human serum in high flux, can simultaneously obtain four first-line anti-tuberculosis drugs of pyrazinamide, isoniazid, ethambutol and rifampicin by one-time detection, and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity and the like. The linear range of pyrazinamide in the method is 15.000-105.000 mug/mL, isoniazid is 1.000-19.000 mug/mL, ethambutol is 1.000-14.500 mug/mL, rifampicin is 0.400-13.000 mug/mL, and the correlation coefficient r is more than or equal to 0.990; the repeatability Coefficient of Variation (CV) of the low-value quality control product is less than or equal to 10 percent, the repeatability Coefficient of Variation (CV) of the medium-value quality control product is less than or equal to 10 percent, and the repeatability Coefficient of Variation (CV) of the high-value quality control product is less than or equal to 10 percent; the relative deviation (B) of the accuracy is less than or equal to +/-15 percent, and the standard recovery rate is 98-113 percent.

Experimental data

1. Selection of pretreatment method

1.1 selection of precipitating agent

And respectively carrying out protein precipitation treatment on the sample by using methanol, acetonitrile and a mixed solution of acetonitrile and methanol, centrifuging the sample after protein precipitation, directly loading the sample to be detected, and judging that the sample after protein precipitation has a bifurcation phenomenon on a pyrazinamide peak as shown in figure 1, wherein the phenomenon is caused by a chromatographic solvent effect, so that the sample extract liquid after centrifugation is further diluted by water to enable the proportion in the solution to be as close as possible to the initial proportion of a mobile phase. Acetonitrile extraction, centrifugation and direct sample injection result are shown in figure 2, and the order of peaks in the figure from left to right are respectively ethambutol, isoniazid, pyrazinamide and rifampicin. The results of direct injection after methanol/acetonitrile =1/1 extraction, dilution 4 times with water and centrifugation are shown in fig. 3, wherein the peak sequence from left to right is respectively ethambutol, isoniazid, pyrazinamide and rifampicin. The results of methanol/acetonitrile =2/1 extraction, dilution with water by 4 times, centrifugation and direct sample injection are shown in fig. 4, in which the peak sequence from left to right is respectively ethambutol, isoniazid, pyrazinamide and rifampicin. Methanol/acetonitrile =3/1 extraction, diluted 4 times with water, centrifuged and directly injected as shown in fig. 5, wherein the peak sequence from left to right is respectively ethambutol, isoniazid, pyrazinamide and rifampicin. Methanol/acetonitrile =4/1 extraction, diluted 4 times with water, centrifuged and directly injected as shown in fig. 6, wherein the peak sequence from left to right is respectively ethambutol, isoniazid, pyrazinamide and rifampicin. Extracting with methanol, diluting with water by 4 times, centrifuging, and directly injecting sample as shown in figure 7, wherein the peak sequence is respectively ethambutol, isoniazid, pyrazinamide and rifampicin from left to right. FIG. 8 is a graph showing the results of the injection without ammonium formate in mobile phase A, wherein the order of the peaks from left to right is ethambutol, isoniazid, pyrazinamide and rifampicin. FIG. 9 is a graph showing the results of the C18 column using acidic ammonium formate as the mobile phase injection, in which the order of the peaks from left to right is ethambutol, isoniazid, pyrazinamide and rifampicin, respectively.

The protein precipitant is carried out on the mixed solution of methanol, acetonitrile and methanol acetonitrile, and the peak type result obtained by dilution is judged, so that the result obtained by using the mixed solution of methanol and methanol acetonitrile as the precipitant can meet the requirement, and the toxicity and price of acetonitrile are higher than those of methanol, so that the subsequent test selects methanol as the precipitant to carry out the test.

2. Selection of chromatographic conditions

2.1 chromatographic column selection

Kinetex F5 (100mm. About.3mm, 2.6 μm) and XBridgeBEH C18 (50mm. About.3mm, 2.5 μm) were used, respectively. The peak patterns were adjusted by adjusting the addition of ammonia or formic acid using methanol and an aqueous ammonium formate solution as mobile phases, and the results showed that the four compounds could be separated using two types of columns, and the F5 column was selected as the final test condition column in the present invention.

Under C18 conditions, using methanol as phase A and 10mM ammonium formate aqueous solution as phase B, the pH was adjusted to 8 by adding ammonia, and the gradient conditions are as shown in the following table, and the peak sequences are isoniazid, pyrazinamide, ethambutol and rifampicin from left to right.

TABLE 6 gradient program

Under the condition of F5, the phase A is methanol, the phase B is 15mM ammonium formate aqueous solution, the pH is adjusted by adding thousandth of formic acid, the peak shape is improved, the gradient condition is shown in the following table, and the peak appearance sequence is respectively ethambutol, isoniazid, pyrazinamide and rifampicin from left to right.

TABLE 7 gradient program

T(min)	％A	％B	Flow rate of flow
				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

2.2 selection of mobile phase

2.2.1 Mobile phase selection

Compounds were assayed using an F5 column by adjusting the pH and different concentrations of aqueous ammonium formate and methanol as mobile phases, finally 5mM ammonium formate containing one thousandth of an aqueous formic acid solution and methanol was selected as the final test condition mobile phase.

Using an F5 column, a phase A is 15mM ammonium formate aqueous solution containing 0.2% formic acid in water, a phase B is methanol, a gradient flow is shown in the following table, and the peak sequence is ethambutol, isoniazid, pyrazinamide and rifampin from left to right.

TABLE 8 gradient program

An F5 column was used, phase A was 10mM aqueous ammonium formate containing 0.1% formic acid in water, phase B was methanol, the gradient flow is shown in the following table, with the order of the peaks from left to right being ethambutol, isoniazid, pyrazinamide and rifampicin.

TABLE 9 gradient program

Using an F5 column, the phase A is 5mM ammonium formate aqueous solution containing 0.1% formic acid in water, the phase B is methanol, the gradient flow is shown in the following table, and the peak sequence is ethambutol, isoniazid, pyrazinamide and rifampicin from left to right.

TABLE 10 gradient program

2.2.2 comparison of the addition of ammonium formate to the mobile phase with the absence of ammonium formate

Using an F5 column, 5mM ammonium formate containing one thousandth of an aqueous formic acid solution was finally selected as the final test condition mobile phase by comparing the effect of adding ammonium formate to the mobile phase a on the test results.

The calibration material and the quality control material are pretreated according to the pretreatment operation method of the calibration material and the quality control material, and the treated liquid is respectively subjected to sample analysis by two mobile phases.

The mobile phase method one: the results of gradient conditions of 0.2% aqueous formic acid for phase A and methanol for phase B in Table 11 are shown in Table 13 and FIG. 8.

TABLE 11 method-gradient conditions

Mobile phase method two: the results are shown in Table 13 and FIG. 7 for phase A with 0.1% formic acid in 5mM aqueous ammonium formate and phase B with methanol under gradient conditions in Table 12.

TABLE 12 method two gradient conditions

TABLE 13 comparison of results of method one and method two

Comparing fig. 8 with fig. 7, the absence of ammonium formate had a greater effect on the first ethambutol peak pattern, with a cross-peak. A comparison of the results of method one and method two from table 13 shows that the addition of ammonium formate significantly improves the linearity of ethambutol and the associated linearity coefficient, for which reason an aqueous solution of ammonium formate and formic acid is finally selected as mobile phase a.

2.2.3 internal Standard selection

And evaluating the accuracy by detecting a rifampicin accuracy sample and calculating the recovery rate of the rifampicin accuracy sample, and evaluating whether the accuracy meets the requirement of performance evaluation. Adding a certain amount of working solution of the substance to be detected into the blank human-derived matrix to prepare 4 samples with the concentration to be recovered, wherein the samples with the concentration to be recovered have quantitative lower limit concentration, low concentration quality control, medium concentration quality control and high concentration quality control, and 3 samples are repeatedly measured for each concentration.

Pre-treating the calibrator and the quality control material according to the pre-treatment operation method of the calibrator and the quality control material, analyzing the sample of the treated liquid, and respectively adopting rifampicin D as the analysis result ₄ And pyrazinamide- ¹⁵ N,D ₃ Rifampicin was quantitatively detected and analyzed as an internal standard, and the analysis results are shown in table 14.

TABLE 14 Effect of different internal standards on rifampicin test results

As can be seen from the results in Table 14, the recovery rate of the human sample matrix is between 93.55% and 106.42% when rifampicin D4 is used as the internal standard, and pyrazinamide- ¹⁵ N,D ₃ As an internal standard, the result shows that the recovery rate of rifampicin added with the standard is 32.41% to 68.87%, for this reason rifampicin-D4 is finally selected as the internal standard in the scheme to test rifampicin.

3. Linear range test data

3.1, a verification method: processing the calibrator S1-S6 solution of the product to be tested according to a sample processing method described by high-throughput detection of four anti-tuberculosis drugs in human serum by high performance liquid chromatography-tandem mass spectrometry, and repeatedly testing each concentration for 3 times. The correlation coefficient R of the linear regression can be calculated by referring to a formula, and the correlation coefficient R2 is equal to or more than 0.990.

r: linear regression correlation coefficient

xi: concentration of S1 to S6

yi: peak area ratio mean value of calibrator and its internal standard in corresponding concentration solution

3.2, acceptance criteria: the correlation coefficients r of the linear regression of ethambutol, isoniazid, pyrazinamide and rifampicin are all more than or equal to 0.990.

3.3, results of the experiment

Linear range: the linear range of pyrazinamide is 15.000-105.00 mu g/mL, the linear range of isoniazid is 1.000-19.000 mu g/mL, the linear range of ethambutol is 1.000-10.300 mu g/mL, and the linear range of rifampicin is 0.400-13.000 mu g/mL.

TABLE 15 Linearity as data

3.4, conclusion: the correlation coefficients R2 of the linear regression of ethambutol, isoniazid, pyrazinamide and rifampicin are all more than or equal to 0.990, and meet the acceptance standard.

4. Repeatability test data

4.1, a verification method: and (3) treating the product to be detected by using the prepared quality control product according to a sample treatment method described by high-throughput detection of four anti-tuberculosis drugs in human serum by high performance liquid chromatography-tandem mass spectrometry, and repeatedly measuring each sample for 10 times. The Coefficient of Variation (CV) of repeatability can be calculated by referring to a formula, wherein the CV of the low-value quality control product is less than or equal to 20 percent, and the CV of the high-value quality control product is less than or equal to 15 percent.

CV: coefficient of variation of repeatability

Average of 10 measurements

S: standard deviation of 10 measurements

4.2, acceptance criteria: the coefficient of variation CV of the low-value quality control product is less than or equal to 20 percent, the coefficient of variation CV of the medium-value quality control product is less than or equal to 15 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent.

4.3 Experimental results

TABLE 16 Pyrazinamide repeatability data

TABLE 17 Isoniazid repeatability data

TABLE 18 ethambutol repeatability data

TABLE 19 Rifampicin repeatability data

4.4, conclusion: the coefficient of variation CV of the low-value quality control product is less than or equal to 15 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent, so that the acceptable standard is met.

5. Evaluation of matrix Effect

5.1 Experimental procedure

At least 6 batches of blank matrices from different donors were used, and for each batch of matrices, the matrix factor for each analyte and internal standard should be calculated by calculating the ratio of the peak area in the presence of matrix (measured by extraction of blank matrix followed by addition of analyte and internal standard) to the product of the corresponding peaks without matrix (pure solution of analyte and internal standard), and further calculating the matrix factor normalized to internal standard by dividing the matrix factor for the analyte by the matrix factor for the internal standard.

5.2, acceptance criteria

The variation coefficients of the internal standard normalized matrix factors calculated from 6 batches of matrix should all be less than 15%.

5.3 Experimental results

TABLE 20 pyrazinamide matrix effect data

TABLE 21 isoniazid substrate effect data

TABLE 22 ethambutol matrix effect data

TABLE 23 Rifampicin matrix Effect data

5.4 summary of the experiments

The variation coefficients of the internal standard normalized matrix factors of the batch matrix calculation are all less than 15 percent, and the requirements are met.

6. Blood sample interference evaluation

6.1 Experimental procedure

Screening for potential interfering substances by interference testing procedures, quantifying the effects of interference, confirming interference in patient samples, confirming susceptibility of analytical methods to interfering substances, assessing potential risks, and providing meaningful interference statements to customers.

According to the concentration requirement of interferents in experimental design, a certain amount of interferent storage solution is added to a basic sample to serve as a test sample, and the addition amount of the experimental scheme is unified to be 5% of that of the basic sample. The base sample was added with 5% of the solvent used for the stock solution as a control sample in the same volume as the test sample. The test and control samples are analyzed in an alternating sequence.

6.2 acceptance criteria

If Dev% = (Xtest-Xcontrol)/Xcontrol ≦ 15%, then the potential interfering substance has no effect on the analyte determination, otherwise the potential interference may be judged to be valid.

6.3 Experimental results

TABLE 24 interferent names and their concentrations

TABLE 25 results of bilirubin interference experiments

TABLE 26 results of triglyceride interference experiments

TABLE 27 results of cholesterol interference experiments

TABLE 28 hemoglobin interference test results

TABLE 29 results of rifampin hemoglobin interference experiments

6.4 summary of the experiments

The concentration of the experimental interferent mainly refers to the recommended experimental concentration of a common endogenous interferent in WS/T-2013 interference experimental guideline, and because the hemoglobin has larger influence on the detection of the rifampicin under the recommended concentration, the concentration of the hemoglobin is reduced to obtain the concentration of the hemoglobin which has no influence on the detection of the rifampicin. The test shows that when the concentration of the hemoglobin in the serum is 0.04g/L, the rifampicin detection is not affected in the method.

7. Accuracy of

7.1 test procedure

The scheme evaluates the accuracy by detecting accuracy samples of pyrazinamide, isoniazid, ethambutol and rifampicin and calculating the recovery rate of the samples. And evaluating whether the accuracy meets the requirement of performance evaluation. A certain amount of working solution of the object to be detected is added into the blank human-derived matrix to prepare 2 samples L and H (namely quality control samples) with the concentration to be recovered. Concentration selection included low concentration levels, high concentration levels, taking three analytical batches, each concentration being assayed in duplicate for 3 samples.

7.2 acceptance criteria

And (3) recovery rate: 85 to 115 percent.

TABLE 30 pyrazinamide accuracy

TABLE 31 Isoniazid accuracy

TABLE 32 ethambutol accuracy

TABLE 33 Rifampicin accuracy

7.3 summary of the experiments

The accuracy is within +/-15% of the marked value, and the requirement is met.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The method for separating the antituberculous drug components is characterized in that the antituberculous drug components contain at least one of pyrazinamide, isoniazid, ethambutol or rifampicin;

the separation method comprises the steps of carrying out methanol precipitation and impurity removal on a sample to be detected containing the anti-tuberculosis drug components, and then carrying out high performance liquid chromatography on the sample by using an F5 chromatographic column or a C18 chromatographic column and adopting a mobile phase prepared from methanol and an ammonium formate aqueous solution to realize the separation of the anti-tuberculosis drug components.

2. The separation method according to claim 1, wherein the mobile phase A of the high performance liquid chromatography is an aqueous ammonium formate solution containing 1/1000 (v/v) formic acid, and the concentration of the ammonium formate is 5-15 mM; the mobile phase B is methanol.

3. The separation method according to claim 2, wherein the mobile phase conditions of the high performance liquid chromatography are 87 to 95% by volume of mobile phase A at 0 to 2min, 10% by volume of mobile phase A at 2.1 to 4.5min and 95% by volume of mobile phase A at 4.51 to 5min.

4. The separation method according to claim 3, wherein the detection conditions of the high performance liquid chromatography are that the flow rate of the mobile phase is 0.3 to 0.6mL/min, the column temperature is 30 to 45 ℃, the temperature of the sample injector is 5 to 20 ℃, and the volume ratio of the needle washing solution is 1: (0.5-2): (0.5-2) a mixed solution of methanol, acetonitrile and isopropanol, wherein the sample volume is 1-20 mu L.

5. The separation method according to claim 4, wherein the detection conditions of the high performance liquid chromatography are that the flow rate of the mobile phase is 0.5mL/min, the column temperature is 40 ℃, the sample injector temperature is 10 ℃, and the volume ratio of the needle washing solution is 1:1:1, and a mixed solution of methanol, acetonitrile and isopropanol, wherein the sample volume is 1 mu L.

6. The separation method as claimed in any one of claims 1 to 5, wherein the internal standards corresponding to pyrazinamide, isoniazid, ethambutol and rifampicin are pyrazinamide- ¹⁵ N,D ₃ isoniazid-D ₄ ethambutol-D ₄ And rifampin-D ₄ 。

7. The separation method according to claim 6, wherein the sample to be tested comprises human serum;

8. Use of the isolation method of any one of claims 1 to 7 in the development of a drug containing at least one of pyrazinamide, isoniazid, ethambutol or rifampicin, drug screening or scientific research.

9. The method for detecting the concentration of the antituberculous drug components is characterized in that the antituberculous drug components contain at least one of pyrazinamide, isoniazid, ethambutol or rifampicin;

the detection method comprises the steps of separating the components of the anti-tuberculosis drugs contained in a sample to be detected by adopting the separation method of any one of claims 1 to 7, and then detecting the concentration of each component by mass spectrometry.

10. The detection method according to claim 9, wherein the ion source parameters of the mass spectrometric detection are: the ion source is ESI +, the air curtain air pressure is 30-40 psi, the collision air pressure is medium, the spraying voltage is 5500V, the temperature is 450-550 ℃, the pressure of the spraying gas GS1 is 40-55 psi, and the pressure of the spraying gas GS2 is 40-60 psi;

preferably, the ion source parameters are: the ion source is ESI +, the pressure of the gas curtain is 40psi, the pressure of the collision gas is 7psi, the spraying voltage is 500V, the temperature is 500 ℃, the pressure of the spraying gas GS1 is 55psi, and the pressure of the spraying gas GS2 is 60psi.