CN112326824A

CN112326824A - Method for simultaneously determining blood concentration of 6 first-line antituberculosis drugs and antifungal drug voriconazole in plasma

Info

Publication number: CN112326824A
Application number: CN202011178860.6A
Authority: CN
Inventors: 赵冠人; 陈明; 车玲; 彭明丽; 孟瑾; 吴漫
Original assignee: 8th Medical Center of PLA General Hospital
Current assignee: 8th Medical Center of PLA General Hospital
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2021-02-05

Abstract

The invention discloses a method for simultaneously measuring blood concentrations of 6 first-line antituberculosis drugs and antifungal drugs voriconazole in plasma, and relates to the technical field of clinical blood concentration monitoring and control. The method adopts an HPLC-MS/MS method, wherein acetaminophen is taken as an internal standard, plasma is subjected to protein precipitation by methanol, is subjected to elution separation by HPLC under a specific gradient elution condition according to a specific mobile phase, and then enters a mass spectrum system, and the accurate quantification of the blood concentration of 7 antibiotics is realized by adopting multi-reaction monitoring analysis. The method has the advantages of strong specificity, high sensitivity, good precision and accuracy, good stability, high extraction recovery rate, no obvious matrix effect, simple operation, convenience and rapidness. The method can realize high-throughput measurement of clinical samples of tuberculosis patients and guide clinical individualized medication.

Description

Method for simultaneously determining blood concentration of 6 first-line antituberculosis drugs and antifungal drug voriconazole in plasma

Technical Field

The invention relates to the technical field of monitoring and controlling clinical blood concentration, in particular to a method for simultaneously measuring blood concentration of 6 first-line antituberculosis drugs and antifungal drug voriconazole in blood plasma.

Background

Tuberculosis is a chronic respiratory infectious disease caused by mycobacterium tuberculosis, the prevention and treatment of the tuberculosis mainly depend on long-term combined chemotherapy for the mycobacterium tuberculosis, and first-line antitubercular drugs such as isoniazid, rifampin, rifapentine, ethambutol, pyrazinamide and the like have the dominant position in the treatment of the tuberculosis. In addition, the antituberculosis activity of the fluoroquinolone antibiotics is increasingly paid attention, and the levofloxacin serving as a representative drug has a good effect in the treatment practice of tuberculosis. These antituberculosis drugs belong to concentration-dependent antibiotics, and their blood concentration is too low to achieve bactericidal effect, even resulting in drug resistance, and too high blood concentration can cause severe adverse reactions. Therefore, monitoring the blood concentration of these drugs in the clinic is of great importance for the treatment of tuberculosis.

In addition, tuberculosis patients have a tendency to develop secondary infections due to reduced autoimmunity, with fungal infections being one of the more common types. Voriconazole (Voriconazole) is frequently used clinically to effectively control fungal infections due to its low resistance to various fungi. However, the first-line antituberculosis drug rifamycin, as a strong CYP enzyme inducer, can significantly reduce the steady-state blood concentration of voriconazole, and the AUC of the rifamycin is even reduced by 95.5%. For tuberculosis patients infected by fungi, voriconazole is required to be used in combination with the conventional antituberculosis treatment, and the voriconazole can play a role in sterilization only by maintaining a certain blood concentration in vivo. Therefore, it is very important to control the blood concentration in vivo.

At present, the concentration of the medicines is mostly determined by an HPLC method, but the method is easily interfered by endogenous substances, the lower limit of detection is higher, and only one medicine can be determined at a time. However, the treatment scheme of tuberculosis is a long-term combined application of multiple drugs, and an HPLC method cannot meet the requirement. The development of a method capable of detecting the concentrations of various medicaments simultaneously has important significance for improving the detection efficiency and realizing high-throughput detection of the blood sample of a patient.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for simultaneously measuring blood concentrations of 6 first-line antituberculosis drugs and antifungal drugs voriconazole in plasma.

The invention is realized by the following steps:

in a first aspect, embodiments of the present invention provide a method for simultaneously determining blood concentrations of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma, comprising:

the 6 first-line antituberculosis drugs are isoniazid, rifampicin, rifapentine, ethambutol, pyrazinamide and levofloxacin, and the antifungal drug is voriconazole;

respectively adding methanol to the 6 first-line antituberculosis drugs and the antifungal drug reference substances to dissolve the 6 first-line antituberculosis drugs and the antifungal drug reference substances to prepare 7 reference substance solutions with different gradient concentrations, then respectively mixing the 7 reference substance solutions and an internal standard acetaminophen stock solution, adding a protein precipitator to precipitate, taking supernate, carrying out sample injection analysis on high performance liquid chromatography-mass spectrometry combined equipment, taking the drug concentration as a horizontal coordinate, taking the ratio of the drug peak area to the internal standard peak area as a vertical coordinate, and calculating a regression equation to serve as a standard curve;

adding an internal standard acetaminophen stock solution into a clinical plasma sample, adding a protein precipitator for precipitation, taking supernatant, carrying out sample injection analysis on high performance liquid chromatography-mass spectrometry combined equipment, and calculating the concentration of each drug in the clinical plasma sample according to a standard curve;

the liquid chromatography parameters were:

mobile phase: the mobile phase A is acetic acid water solution with volume concentration of 0.1-0.3%, the mobile phase B is mixed solution of methanol and acetonitrile, gradient elution is adopted, and the elution procedure is as follows:

at 0-2.5min, the proportion of the mobile phase A and the mobile phase B is 92-98% → 80-86%: 2-8% → 14-20%; at 2.5-5min, the proportion of the mobile phase A and the mobile phase B is 71-77% → 42-48%: 23-29% → 52-58%; and when the time is 5-7.3min, the proportion of the mobile phase A to the mobile phase B is 42-48%: 52 to 58 percent; at 7.3-10min, the proportion of the mobile phase A and the mobile phase B is 27-33%: 67-73%; at 10-13min, the proportion of the mobile phase A and the mobile phase B is 92-98%: 2 to 8 percent.

In an alternative embodiment, the elution procedure is:

at 0-2.5min, the proportion of the mobile phase A and the mobile phase B is 92-95% → 80-83%: 5-8% → 17-20%; at 2.5-5min, the proportion of the mobile phase A and the mobile phase B is 71-74% → 42-45%: 26-29% → 55-58%; and when the time is 5-7.3min, the proportion of the mobile phase A to the mobile phase B is 42-45%: 55 to 58 percent; at 7.3-10min, the proportion of the mobile phase A and the mobile phase B is 27-30%: 70-73%; the proportion of the mobile phase A and the mobile phase B is 92-95% at 10-13 min: 5 to 8 percent.

In an alternative embodiment, the elution procedure is:

at 0-2.5min, the ratio of mobile phase a and mobile phase B is 95% → 83%: 5% → 17%; at 2.5-5min, the ratio of the mobile phase A and the mobile phase B is 74% → 45%: 26% → 55%; at 5-7.3min, the proportion of the mobile phase A and the mobile phase B is 45%: 55 percent; at 7.3-10min, the proportion of the mobile phase A and the mobile phase B is 30%: 70 percent; at 10-13min, the proportion of the mobile phase A and the mobile phase B is 95%: 5 percent.

In an alternative embodiment, the volume ratio of methanol to acetonitrile in the mobile phase B is 1:0.5 to 1.5.

In an alternative embodiment, the chromatography column is an Agilent Poroshell 120SB C₁₈A column;

preferably, the flow rate is 0.3-0.7 mL/min;

preferably, the sample size is 5-10 uL.

In an alternative embodiment, the mass spectrometry conditions are: the electrospray ion source, the positive ion mode and the multi-reaction monitoring mode are adopted for analysis, and the mass spectrum detection working parameters are as follows: the detection ion pair of parent ion/characteristic ion of isoniazid is 138.0/121.0, the detection ion pair of parent ion/characteristic ion of rifampicin is 823.2/791.2, the detection ion pair of parent ion/characteristic ion of rifapentine is 877.5/845.5, the detection ion pair of parent ion/characteristic ion of ethambutol is 205.2/116.1, the detection ion pair of parent ion/characteristic ion of pyrazinamide is 124.1/79.0, the detection ion pair of parent ion/characteristic ion of levofloxacin is 362.2/261.1, the detection ion pair of parent ion/characteristic ion of voriconazole is 350.1/281.3, and the detection ion pair of parent ion/characteristic ion of internal standard acetaminophen is 152.0/110.0;

the de-clustering voltage of isoniazid is 44V, and the cleavage energy is 22 eV;

the clustering removal voltage of the rifampicin is 62V, and the cleavage energy is 29 eV;

the cluster removing voltage of rifapentine is 67V, and the cleavage energy is 36 eV;

the declustering voltage of the ethambutol is 39V, and the cleavage energy is 21 eV;

the declustering voltage of the pyrazinamide is 37V, and the cleavage energy is 23 eV;

the cluster removing voltage of the levofloxacin is 63V, and the cleavage energy is 28 eV;

the declustering voltage of voriconazole is 61V, and the cracking energy is 17 eV;

the declustering voltage of the internal standard acetaminophen is 53V, and the cleavage energy is 24 eV;

the ion jet voltage is 5000V; the ion source temperature is 450 ℃; the atomizing gas was nitrogen, the source gas 1 was 30psi, the source gas 2 was 45psi, and the gas curtain pressure was 30 psi.

In an alternative embodiment, the protein precipitating agent is methanol.

In alternative embodiments, the ranges for each drug concentration in the standard curve plasma sample are as follows: 0.20-9.81 mu g/mL of isoniazid; 0.60-30.12 mu g/mL of rifampicin; 0.60-30.12 mu g/mL of rifapentine; 0.10-4.89 mu g/mL of ethambutol; 1.01-50.60 mu g/mL of pyrazinamide; 0.10-5.00 mu g/mL of levofloxacin; voriconazole 0.05-10.20 mug/mL, standard curve plasma sample internal standard acetaminophen concentration is 1.5-1.7 mug/mL.

In an alternative embodiment, the concentrations of isoniazid are: 0.20. mu.g/mL, 0.49. mu.g/mL, 0.98. mu.g/mL, 1.96. mu.g/mL, 4.90. mu.g/mL, 7.85. mu.g/mL, 9.81. mu.g/mL;

the concentrations of rifampicin are respectively as follows: 0.60. mu.g/mL, 1.51. mu.g/mL, 3.01. mu.g/mL, 6.02. mu.g/mL, 15.06. mu.g/mL, 24.10. mu.g/mL, 30.12. mu.g/mL;

the concentrations of rifapentine are respectively as follows: 0.60. mu.g/mL, 1.51. mu.g/mL, 3.01. mu.g/mL, 6.02. mu.g/mL, 15.06. mu.g/mL, 24.10. mu.g/mL, 30.12. mu.g/mL;

the concentration of the ethambutol is respectively as follows: 0.10. mu.g/mL, 0.24. mu.g/mL, 0.49. mu.g/mL, 0.98. mu.g/mL, 2.44. mu.g/mL, 3.91. mu.g/mL, 4.89. mu.g/mL;

the concentrations of the pyrazinamide are respectively as follows: 1.01. mu.g/mL, 2.53. mu.g/mL, 5.06. mu.g/mL, 10.12. mu.g/mL, 25.30. mu.g/mL, 40.48. mu.g/mL, 50.60. mu.g/mL;

the concentration of the levofloxacin is respectively as follows: 0.10. mu.g/mL, 0.25. mu.g/mL, 0.50. mu.g/mL, 1.00. mu.g/mL, 2.50. mu.g/mL, 4.00. mu.g/mL, 5.00. mu.g/mL;

the concentration of voriconazole is as follows: 0.05. mu.g/mL, 0.51. mu.g/mL, 1.02. mu.g/mL, 2.55. mu.g/mL, 5.10. mu.g/mL, 8.16. mu.g/mL, 10.20. mu.g/mL;

the concentration of acetaminophen as the internal standard was 1.62. mu.g/mL.

In alternative embodiments, precipitating the control solution or the plasma sample with a protein precipitating agent comprises: adding a protein precipitator into the control solution for precipitation, then carrying out vortex for 10-30s, then adding the protein precipitator for precipitating the protein, carrying out vortex for 0.5-1.5min, then centrifuging for 4-6min at 13000-15000rad/min at 3-5 ℃, taking the supernatant into a sample tube, and carrying out sample introduction analysis.

The invention has the following beneficial effects:

the inventor designs an HPLC-MS/MS method for simultaneously measuring blood concentrations of 6 first-line antituberculosis drugs and antifungal drugs voriconazole in plasma, which comprises the following steps: the method is characterized in that acetaminophen is used as an internal standard, plasma is subjected to protein precipitation by methanol, then is separated by HPLC, and then enters a mass spectrum system, and Multiple Reaction Monitoring (MRM) analysis is adopted to realize accurate quantification of blood concentrations of 6 first-line antitubercular drugs (isoniazid, rifampicin, rifapentine, ethambutol, pyrazinamide and levofloxacin) and antifungal drugs voriconazole. The method has the advantages of strong specificity, high sensitivity, good precision and accuracy, good stability, high extraction recovery rate, no obvious matrix effect, simple operation, convenience and rapidness, and can realize high-throughput measurement of clinical samples of tuberculosis patients and guide clinical individualized medication.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a chromatogram of blank plasma obtained by the method provided in the examples of the present application;

FIG. 2 is a chromatogram of 6 first-line antitubercular drugs, voriconazole and internal standard obtained by the method provided in the examples of the present application;

FIG. 3 is a chromatogram of ethambutol obtained by the method provided in the examples of the present application;

FIG. 4 is a chromatogram of isoniazid obtained by the method provided in the examples of the present application;

FIG. 5 is a chromatogram of pyrazinamide obtained by the assay provided in the examples of the present application;

FIG. 6 is a chromatogram of acetaminophen obtained by the assay provided in the examples herein;

FIG. 7 is a chromatogram of levofloxacin obtained by the assay provided in the examples herein;

FIG. 8 is a chromatogram of voriconazole obtained by the method provided in the examples of the present application;

FIG. 9 is a chromatogram of rifampicin obtained by the method provided in the examples of this application;

FIG. 10 is a chromatogram of rifapentine obtained from the determination of the method provided in the examples of this application;

FIG. 11 chromatograms of the 6 first line antitubercular drugs, voriconazole and internal standard obtained from the assay provided in comparative example 4 of the present application;

FIG. 12 chromatograms of the 6 first line antitubercular drugs, voriconazole and internal standard obtained by the method provided in comparative example 5 of the present application.

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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

In the experiment, a standard curve is prepared by using 7 antibiotic drug (isoniazid, rifampicin, rifapentine, ethambutol, pyrazinamide, levofloxacin and voriconazole) standard substances and blank plasma, and the drug concentration in the plasma of a patient is calculated according to the standard curve.

Firstly, experimental instruments and reagents are as follows:

the instrument comprises the following steps: ABCIEX 3200QTRAP type triple quadrupole tandem mass spectrometer (Applied biosystem Inc, USA); prominence LC-20A ultra-fast high performance liquid chromatography system (Shimadzu Technologies Inc., japan); a vortex mixer of model VDRTEX-5 (linbel instruments ltd); model 5420 bench-top high speed centrifuge (Eppendorf, Germany).

Reagent: isoniazid (China pharmaceutical biologicals institute, batch number 100578-201501); rifampicin (China institute for testing biological products of drugs, lot No. 130496-201502); rifapentine (China institute for drug and biological products, lot No. 130541-200401); ethambutol hydrochloride (China pharmaceutical biologicals institute, lot number 100165-201504); pyrazinamide (China pharmaceutical biologicals institute, lot number 100178-201503); levofloxacin (China pharmaceutical biologicals institute, batch No. 130455-201505); voriconazole (China institute for testing biological products of drugs, lot number 100862-; acetaminophen (chinese institute for drug and biological products, lot No. 100018-201508); methanol, acetonitrile and acetic acid are all chromatographically pure (Fisher), water is Milli-Q to prepare ultrapure water, the resistivity is 18.2M omega cm, a patient plasma sample is provided by tuberculosis research institute of eighth medical center of general Hospital of the liberation force, and blank plasma is provided by volunteers.

Second, Experimental methods

Preparation of standard curve plasma sample and quality control plasma sample

1.1 preparation of stock solutions

Using 50% methanol water solution as solvent, respectively preparing stock solutions of 7 antibiotics (isoniazid, rifampin, rifapentine, ethambutol, pyrazinamide, levofloxacin and voriconazole) and internal standard acetaminophen, and storing all the stock solutions in a refrigerator at-20 ℃, wherein the rifampin and the rifapentine need to be stored in a dark place.

1.2 Standard Curve plasma sample preparation

Taking 90 mu L of blank plasma, adding a certain volume of the drug stock solution and the internal standard stock solution prepared in the item 1.1 to prepare a standard curve plasma sample of the drug with a certain standard concentration, and respectively preparing the standard curve plasma samples of different standard concentrations of various drugs according to the method. The final plasma drug concentration in the standard curve plasma samples is shown in Table 1, with a final internal standard concentration of 1.62. mu.g/mL.

TABLE 1 Final concentration of Standard Curve plasma samples for each drug

1.3 quality control plasma sample preparation

Taking 90 mu L of blank plasma, adding a certain volume of the drug stock solution and the internal standard stock solution prepared in the above 1.1 to prepare a quality control plasma sample with a certain quality control concentration of the drug, and respectively preparing the quality control plasma samples with different quality control concentrations of each drug according to the method. In quality control plasma samples, the final plasma drug concentration is shown in Table 2, and the final internal standard concentration is 1.62. mu.g/mL.

TABLE 2 Final concentration of quality control plasma samples of each drug

(II) Experimental procedures

2.1 plasma sample pretreatment

100uL of plasma sample is placed in a sample tube, vortexed for 20s, then 300 uL of methanol is added to precipitate protein, vortexed for 1min and then centrifuged for 5min at 14000rad/min at 4 ℃, 200 uL of supernatant is taken and placed in a sample tube, 5uL of automatic sample introduction is carried out for HPLC-MS/MS analysis, and quantitative detection is carried out by a peak area internal standard method.

2.2 chromatographic conditions

A chromatographic column: agilent Poroshell 120SBC₁₈Column: 4.6mm × 50mm, 2.7 μm; the flow rate was 0.5mL/min at room temperature, and the amount of sample was 5 uL.

Mobile phase: the mobile phase A is 0.2% acetic acid aqueous solution, the mobile phase B is mixed solution of methanol and acetonitrile according to the volume ratio of 1:1, gradient elution is adopted, and the elution procedure is as follows:

time of day	Mobile phase A	Mobile phase B
			0-2.5min	95％→83％	5％→17％
2.5-5min	74％→45％	26％→55％
			5-7.3min	45％	55％
7.3-10min	30％	70％
			10-13min	95％	5％

2.3 Mass Spectrometry conditions

The electrospray ion source, the positive ion mode and the multi-reaction monitoring mode are adopted for analysis, and the mass spectrum detection working parameters are as follows: the detection ion pair of parent ion/characteristic ion of isoniazid is 138.0/121.0, the detection ion pair of parent ion/characteristic ion of rifampicin is 823.2/791.2, the detection ion pair of parent ion/characteristic ion of rifapentine is 877.5/845.5, the detection ion pair of parent ion/characteristic ion of ethambutol is 205.2/116.1, the detection ion pair of parent ion/characteristic ion of pyrazinamide is 124.1/79.0, the detection ion pair of parent ion/characteristic ion of levofloxacin is 362.2/261.1, the detection ion pair of parent ion/characteristic ion of voriconazole is 350.1/281.3, and the detection ion pair of parent ion/characteristic ion of internal standard acetaminophen is 152.0/110.0; isoniazid has a declustering voltage (DP) of 44V, a Cleavage Energy (CE) of 22eV, a declustering voltage (DP) of 62V, a Cleavage Energy (CE) of 29eV, a declustering voltage (DP) of 67V, and a Cleavage Energy (CE) of 29eV36eV, a declustering voltage (DP) of ethambutol of 39V, a Cleavage Energy (CE) of 21eV, a declustering voltage (DP) of pyrazinamide of 37V, a Cleavage Energy (CE) of 23eV, a declustering voltage (DP) of levofloxacin of 63V, a Cleavage Energy (CE) of 28eV, a declustering voltage (DP) of voriconazole of 61V, a Cleavage Energy (CE) of 17eV, a declustering voltage (DP) of internal standard acetaminophen of 53V, and a Cleavage Energy (CE) of 24 eV; the ion ejection voltage (IS) IS 5000V; the ion source Temperature (TEM) is 450 ℃; the atomizing gas is nitrogen, and the source gas 1 (GS)₁) 30psi, Source gas 2 (GS)₂) At 45psi, and a curtain Gas pressure (Cur Gas) of 30 psi.

The detection chromatogram of the blank plasma is shown in figure 1, the detection chromatograms of the 6 first-line antitubercular drugs, voriconazole and the internal standard are shown in figure 2, the drugs are respectively as follows from left to right: ethambutol, isoniazid, pyrazinamide, acetaminophen, levofloxacin, voriconazole, rifampin, rifapentine. The chromatograms for specific individual drugs can be seen in figures 3-10, respectively.

(III) evaluation of methodology

The methodology verification mainly comprises linearity, precision, accuracy, extraction recovery rate, matrix effect and stability.

3.1 Standard Curve and lower quantitative Limit

For each of the standard curve plasma samples formulated for each standard concentration of a drug listed in "table 1", the following operations were performed: after the operation of '2.1 plasma sample pretreatment', HPLC-MS/MS (using MRM mode) measurement is carried out according to the conditions of '2.2' and '2.3', and chromatograms are recorded; calculating the ratio of the peak area of the drug to the peak area of the internal standard; after all samples are subjected to the above operation, the drug concentration is taken as the abscissa, the ratio of the drug peak area to the internal standard peak area is taken as the ordinate, and the weight is used (1/x)²) The least square method is used for regression operation to obtain a linear regression equation of the medicine, namely the standard curve of the medicine. Following the same procedure, a standard curve for each drug was prepared. The results of the linear regression equation and the lower limit of quantitation for each drug are shown in table 3.

TABLE 3 Linear regression equation, correlation coefficient, Linear Range and lower quantitative Limit for each drug

3.2 precision and accuracy

For each of the quality control plasma samples of each drug formulated at the respective quality control concentrations listed in "table 2" (6 samples prepared for each concentration of each drug, one sample prepared daily for 3 consecutive days, and 3 samples prepared in total as samples for precision and accuracy) the following operations were performed: after the operation of '2.1 plasma sample pretreatment', HPLC-MS/MS (using MRM mode) measurement is carried out according to the conditions of '2.2' and '2.3', and chromatograms are recorded; calculating the ratio of the peak area of the drug to the peak area of the internal standard; and substituting the standard curve obtained on the day to obtain the test concentration, and finally calculating the precision (qualified when the absolute value is less than 15%) and accuracy (qualified when the absolute value of the relative deviation is less than 15%) between batches. The specific results are shown in Table 4.

TABLE 4 accuracy and precision test results for each drug

As can be seen from Table 4, the absolute values of the accuracy relative deviations of the tested concentrations of each drug in plasma were less than 15%, and the absolute values of the precision within and between the batches of each tested concentration of each drug in plasma were less than 15%. Therefore, the method provided by the invention has good precision and accuracy in detection of the 7 drugs in the plasma.

3.3 matrix Effect and extraction recovery

Taking blank plasma, processing according to a plasma pretreatment method to obtain a blank matrix, and preparing 3 parts of quality control solutions of the 7 medicines with low, medium and high concentrations by using the blank matrix for HPLC-MS/MS analysis. Peak area (A)₁) Peak area (A) corresponding to the corresponding concentration of the drug standard solution₀) The ratio is the matrix effect result. Taking low, medium and high plasma samples (n is 6), and performing the operation according to the item of '2.1 plasma sample pretreatment' to obtain the peak area (A) of the drug in the plasma₂) Peak area (A) of a blank substrate prepared at the same concentration₁) The ratio is the extraction recovery rate of the medicine in the blood plasma. The results of matrix effect and extraction recovery are shown in Table 5.

Table 5 recovery of each drug and matrix effect test (n ═ 6)

As can be seen from the results in Table 5, the 7 drugs all have higher extraction recovery rates, and the matrix has little influence on the measurement.

3.4 stability

The ratio of the peak area of the drug measured under different conditions of the plasma sample to the initial peak area immediately after plasma preparation (0 hour) was observed, and the stability of each drug was measured by the relative deviation RE value. The stability of the plasma samples after being treated, placed for 3h and 24h and repeatedly frozen and thawed for three times is examined, and the results are shown in table 6.

TABLE 6 stability survey

From the results in table 6, it can be seen that the RE values of the plasma concentrations of the drugs are less than 10% under the above-mentioned examination conditions, indicating that the method of the present invention has good stability in detecting the above-mentioned 7 drugs in the plasma under the above-mentioned examination conditions.

Example 2

This example is substantially the same as example 1 except that the elution procedure is different, and the elution procedure of this example is:

time of day	Mobile phase A	Mobile phase B
			0-2.5min	92％→80％	8％→20％
2.5-5min	71％→42％	29％→57％
			5-7.3min	42％	58％
7.3-10min	27％	73％
			10-13min	92％	8％

Example 3

time of day	Mobile phase A	Mobile phase B
			0-2.5min	97％→85％	3％→15％
2.5-5min	76％→47％	24％→53％
			5-7.3min	47％	53％
7.3-10min	32％	68％
			10-13min	97％	3％

Example 4

This example is substantially the same as example 1 except that in this example, the mobile phase B was a mixed solution of methanol and acetonitrile at a volume ratio of 1: 0.5.

Example 5

This example is substantially the same as example 1 except that in this example, the mobile phase B was a mixed solution of methanol and acetonitrile at a volume ratio of 1: 0.7.

Comparative example 1

This comparative example is essentially the same as example 1 except that the elution procedure is different, and the elution procedure of this comparative example is:

time of day	Mobile phase A	Mobile phase B
			0-2.5min	95％→83％	5％→17％
2.5-5min	83％→65％	17％→35％
			5-6min	65％→30％	35％→70％
6.01-9min	20％→10％	80％→90％
			9.01-12min	95％	5％

Comparative example 2

This comparative example is essentially the same as example 1, except that in this example, mobile phase B is methanol.

Comparative example 3

This comparative example is substantially the same as example 1 except that mobile phase B, which is methanol, and the elution procedure are different from those of comparative example 1.

Comparative example 4

time of day	Mobile phase A	Mobile phase B
			0-2.5min	95％→83％	5％→17％
2.5-5min	74％→30％	26％→70％
			5-9min	30％	70％
9-12min	95％	5％

(ii) a Please refer to fig. 11 for its detection chromatogram.

Comparative example 5

time of day	Mobile phase A	Mobile phase B
			0-2.5min	95％→83％	5％→17％
2.5-5min	74％→45％	26％→55％
			5-10min	45％	55％
10-13min	95％	5％

(ii) a Please refer to fig. 12 for its detection chromatogram.

The results of the tests carried out by the methods of examples 1 to 5 and comparative examples 1 to 5 are as follows:

	example 1	Example 2	Example 3	Example 4	Example 5
							Residence time	Residence time	Residence time	Residence time	Residence time
Isoniazid	1.67	1.59	1.85	1.65	1.68
						Rifampicin	9.29	8.67	9.50	8.10	9.60
Rifapentine	9.87	9.55	10.21	9.38	10.36
						Ethambutol	0.88	0.85	0.87	0.86	0.86
Pyrazinamides	3.01	2.65	3.40	2.91	3.13
						Levofloxacin	4.85	4.72	4.97	4.74	4.95
Voriconazole	8.91	8.37	9.31	8.13	9.41
							Comparative example 1	Comparative example 2	Comparative example 3	Comparative example 4	Comparative example 5
	Residence time	Residence time	Residence time	Residence time	Residence time
						Isoniazid	1.71	1.97	2.00	1.67	1.64
Rifampicin	8.10	10.1	8.27	6.12	8.65
						Rifapentine	8.27	10.79	8.39	6.48	10.86
Ethambutol	0.86	0.86	0.86	0.88	0.90
						Pyrazinamides	3.14	3.73	3.69	2.91	2.88
Levofloxacin	5.85	5.31	6.49	4.73	4.80
						Voriconazole	8.18	9.93	8.35	6.46	8.24

The 7 antibiotics of isoniazid, rifampicin, rifapentine, ethambutol, pyrazinamide, levofloxacin and voriconazole are mainly rifampicin, rifapentine and voriconazole which are difficult to separate during separation, and peak shape coincidence is easy to occur. From the residence times of examples 1 to 5 and comparative examples 1 to 5 described above, it can be seen that the separation effect of example 1 of the present application is the best and the peak shape is good, while the separation effect of the elution procedure provided by examples 2 and 3 is not good enough compared to example 1 but is significantly better than that of the comparative example, whereas the separation effect obtained by changing the ratio of the mobile phase in examples 4 and 5 is also significantly worse than that of example 1. Further, the above comparative examples 1 to 3 were significantly inferior in separation effect to example 1, comparative example 4, in which rifampicin and voriconazole were overlapped, had good separation, and comparative example 5, in which the degree of separation was good but the peak shape was not good. Therefore, the best separation effect can be obtained in the embodiment 1 of the application, and the phenomena of incapability of baseline separation, tailing or poor peak shape exist in other embodiments or comparative examples, and the baseline separation, tailing or poor peak shape of voriconazole which is 6 first-line antituberculosis drugs and antifungal drugs can be effectively separated by adopting a specific mobile phase to carry out elution according to a specific elution program, and the accurate quantification of blood concentrations of 7 antibiotics can be realized simultaneously. The method has the advantages of strong specificity, high sensitivity, good precision and accuracy, good stability, high extraction recovery rate, no obvious matrix effect, simple operation, convenience and rapidness. The method can realize high-throughput measurement of clinical samples of tuberculosis patients and guide clinical individualized medication.

In conclusion, according to the method, acetaminophen is used as an internal standard, methanol is used for precipitating proteins in plasma, high performance liquid chromatography is used for separating drug components in supernatant, and then a high-resolution mass spectrum multi-reaction monitoring mode is used for performing drug targeting detection and quantification, so that the concentration of 7 antibiotic drugs in the plasma can be analyzed and determined simultaneously. The kit has the advantages of strong specificity, high sensitivity, good precision and accuracy, good stability, high extraction recovery rate, no obvious matrix effect, high flux, convenience, rapidness and the like, can realize high-flux measurement on clinical samples of tuberculosis patients, and guides clinical individual medication.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining blood concentration of 6 first-line antituberculosis drugs and antifungal drugs voriconazole in plasma is characterized by comprising the following steps:

respectively adding methanol to the 6 first-line antituberculosis drugs and the antifungal drug reference substances to dissolve the 6 first-line antituberculosis drugs and the antifungal drug reference substances to prepare 7 reference substance solutions with different gradient concentrations, then respectively mixing the 7 reference substance solutions with an internal standard acetaminophen stock solution, adding a protein precipitator to precipitate, taking supernate, carrying out sample injection analysis on high performance liquid chromatography-mass spectrometry combined equipment, taking the drug concentration as a horizontal coordinate, taking the ratio of the drug peak area to the internal standard peak area as a vertical coordinate, and calculating a regression equation to serve as a standard curve;

the liquid chromatography parameters were:

2. The method for simultaneously determining the blood concentration of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma according to claim 1, wherein the elution procedure is as follows:

3. The method for simultaneously determining the blood concentration of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma according to claim 1, wherein the elution procedure is as follows:

4. The method for simultaneously determining the blood concentration of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma of 6 drugs according to any one of claims 1 to 3, wherein the volume ratio of methanol to acetonitrile in the mobile phase B is 1: 0.5-1.5.

5. According to claim1-3, the method for simultaneously determining the blood concentration of 6 first-line antituberculosis drugs and antifungal drugs voriconazole in blood plasma is characterized in that a chromatographic column is Agilent Poroshell 120SBC₁₈A column;

preferably, the flow rate is 0.3-0.7 mL/min;

preferably, the sample size is 5-10 uL.

6. The method for simultaneously determining the blood concentration of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma according to any one of claims 1 to 3, wherein the mass spectrum conditions are as follows: the electrospray ion source, the positive ion mode and the multi-reaction monitoring mode are adopted for analysis, and the mass spectrum detection working parameters are as follows: the detection ion pair of parent ion/characteristic ion of isoniazid is 138.0/121.0, the detection ion pair of parent ion/characteristic ion of rifampicin is 823.2/791.2, the detection ion pair of parent ion/characteristic ion of rifapentine is 877.5/845.5, the detection ion pair of parent ion/characteristic ion of ethambutol is 205.2/116.1, the detection ion pair of parent ion/characteristic ion of pyrazinamide is 124.1/79.0, the detection ion pair of parent ion/characteristic ion of levofloxacin is 362.2/261.1, the detection ion pair of parent ion/characteristic ion of voriconazole is 350.1/281.3, and the detection ion pair of parent ion/characteristic ion of internal standard acetaminophen is 152.0/110.0;

7. The method for simultaneously determining the blood concentration of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma according to any one of claims 1 to 3, wherein the protein precipitant is methanol.

8. The method for simultaneously determining blood concentration of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma of 6 drugs according to any one of claims 1 to 3, wherein the concentration ranges of the drugs in the standard curve plasma sample are as follows: 0.20-9.81 mu g/mL of isoniazid; 0.60-30.12 mu g/mL of rifampicin; 0.60-30.12 mu g/mL of rifapentine; 0.10-4.89 mu g/mL of ethambutol; 1.01-50.60 mu g/mL of pyrazinamide; 0.10-5.00 mu g/mL of levofloxacin; voriconazole 0.05-10.20 mug/mL, standard curve plasma sample internal standard acetaminophen concentration is 1.5-1.7 mug/mL.

9. The method for simultaneously determining the blood concentration of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma according to claim 8,

the concentrations of isoniazid are respectively as follows: 0.20. mu.g/mL, 0.49. mu.g/mL, 0.98. mu.g/mL, 1.96. mu.g/mL, 4.90. mu.g/mL, 7.85. mu.g/mL, 9.81. mu.g/mL;

the concentration of acetaminophen as the internal standard was 1.62. mu.g/mL.

10. The method for simultaneously determining blood levels of voriconazole, a first-line antituberculosis drug and an antifungal drug, in plasma of 6 species according to any one of claims 1 to 3, wherein precipitating the control solution or the plasma sample with a protein precipitating agent comprises: adding a protein precipitator into the control solution for precipitation, then carrying out vortex for 10-30s, then adding the protein precipitator for precipitating the protein, carrying out vortex for 0.5-1.5min, then centrifuging for 4-6min at 13000-15000rad/min at 3-5 ℃, taking the supernatant into a sample tube, and carrying out sample introduction analysis.