Method for detecting concentrations of eight second-line antituberculous drugs based on HPLC-MS / MS

Abstract

The invention discloses a method for detecting the concentration of eight second-line antituberculous drugs based on HPLC-MS / MS. The eight second-line antituberculous drugs are divided into two groups for detection, one group takes levofloxacin as an internal standard substance, and an internal standard stock solution is prepared; the method comprises the following steps: detecting by taking standard substances of levofloxacin, ciprofloxacin and gatifloxacin as target substances; and in the other group, albendamide, deramanib, pritomanib, clarithromycin and azithromycin are used as target objects for detection. According to the method, the detection time is short, only 5 min is needed, and the detection time is saved; meanwhile, according to the method disclosed by the invention, the consumption of an organic solvent is reduced, so that the pollution to the environment is reduced, and the method has the characteristics of high accuracy and good precision, and provides the fastest and effective basis for clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS. Background Technology

[0002] Tuberculosis is a chronic, wasting infectious disease caused by Mycobacterium tuberculosis, also known as scabies or "white plague," and is an ancient zoonotic disease. The drugs used to treat this disease are called anti-tuberculosis drugs. 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. Second-line anti-tuberculosis drugs mainly include levofloxacin (LEV), ciprofloxacin (CIP), gatifloxacin (GAT), protonycinamide (PTO), delamanid (DLM), pretomanid (PA-824), clarithromycin (CAM), and azithromycin (ZAM).

[0003] Second-line anti-tuberculosis drugs are mainly used when first-line drugs such as isoniazid, rifampin, ethambutol, and pyrazinamide are ineffective or have resistance. Currently, drug concentration detection is mainly achieved through high-performance liquid chromatography (HPLC) and immunoassay. For example, Chinese patent CN121114275A discloses an HPLC method for detecting six second-line anti-tuberculosis drugs, including prothionamide, levofloxacin, linezolid, moxifloxacin, delamani, and bedaquiline. The gradient elution time is 25 minutes; however, this method has a long detection cycle and cannot meet the application requirements of faster detection in clinical emergencies.

[0004] Therefore, this application aims to provide a detection scheme for second-line anti-tuberculosis drugs with high sensitivity and short detection time. Summary of the Invention

[0005] To address at least one of the aforementioned problems, this invention provides a method for detecting the concentrations of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS.

[0006] To achieve the above objectives, the present invention employs the following technical means: This invention provides a method for detecting the concentrations of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS. The eight drugs include levofloxacin, ciprofloxacin, gatifloxacin, prothionamide, delamani, puttomani, clarithromycin, and azithromycin. The method involves dividing the drugs into two groups: a levofloxacin, ciprofloxacin, and gatifloxacin detection group, and a prothionamide, delamani, puttomani, clarithromycin, and azithromycin detection group, each for separate detection. The detection methods for levofloxacin, ciprofloxacin, and gatifloxacin are as follows: Levofloxacin was used as an internal standard to prepare an internal standard stock solution. A mixed standard stock solution was prepared using levofloxacin, ciprofloxacin, and gatifloxacin standards as target compounds. These solutions were then gradually diluted with a blank matrix to prepare the working solution for the standard curve. The sample to be tested was added to the internal standard solution, followed by the addition of methanol solution. After shaking and centrifugation, the supernatant was collected and analyzed by HPLC-MS / MS. For the levofloxacin, ciprofloxacin, and gatifloxacin detection groups, mobile phase A consisted of 0.1% formic acid and 5 mM ammonium acetate in water; mobile phase B consisted of 0.1% formic acid and 5 mM ammonium acetate in methanol. The detection methods for prothionamide, delamani, putomani, clarithromycin, and azithromycin are as follows: Using prothionamide, delamani, putomomani, clarithromycin, and azithromycin as target compounds, a mixed standard stock solution was prepared. This solution was then gradually diluted with a blank matrix to prepare the standard curve working solution. The sample to be tested was added to methanol solution, vortexed, centrifuged, and the supernatant was collected and diluted 10-fold with methanol solution. HPLC-MS / MS was used for detection. For the prothionamide, delamani, putomomani, clarithromycin, and azithromycin detection groups, mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was 0.1% formic acid methanol solution.

[0007] In some embodiments of the present invention, the chromatographic conditions for the detection of levofloxacin, ciprofloxacin, gatifloxacin, and prothionamide, delamani, putomani, clarithromycin, and azithromycin are as follows: Chromatographic column: ACE Excel-2 C18-PFP column, 100×2.1 mm, 2.6 μm; Wash solution: 50% isopropanol-water solution; Flow rate: 0.3 mL / min, column temperature: 40 ℃, injection volume: 2 μL; A gradient elution method was used, as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; the gradient elution time was 5.0 min before stopping. The volume fraction of mobile phase A was 99% at the beginning (0.3 min). Over 0.3–1.0 min, the volume fraction of mobile phase A decreased from 99% to 30%. The volume fraction of mobile phase A decreased from 30% to 2% over 1.0-2 minutes. The volume fraction of mobile phase A was maintained at 2% for 2.0-4.0 min. The volume fraction of mobile phase A increased from 2% to 99% over 4.0-4.01 min. The volume fraction of mobile phase A was maintained at 99% for 4.01-5 min.

[0008] In some embodiments of the present invention, the mass spectrometry conditions used for the two sets of detections are as follows: Ion source: Electrospray ion source, positive ion mode; Spray capillary voltage: 2.7 kV; Desolvation gas temperature: 450℃; Desolvation gas flow rate: 900 L / Hr; Ion source temperature: 150℃; Desolvation gas pressure: 8 bar; Collision gas pressure: 0.7 bar; Scan mode: MRM.

[0009] In some embodiments of the present invention, the MRM parameters of the levofloxacin, ciprofloxacin, and gatifloxacin detection group are as follows:

[0010] In some embodiments of the present invention, the MRM parameters of the prothionamide, delamani, putomani, clarithromycin, and azithromycin detection groups are as follows:

[0011] In some embodiments of the present invention, the concentration ranges of levofloxacin, ciprofloxacin, and gatifloxacin in the standard curve working solutions of the levofloxacin, ciprofloxacin, and gatifloxacin detection groups are 0.5-25 μg / mL, 0.203-10.14 μg / mL, and 0.194-9.7 μg / mL, respectively.

[0012] In some embodiments of the present invention, the levofloxacin internal standard stock solution is diluted with a methanol-acetonitrile solution at a volume ratio of 1:2, and the concentration of the internal standard stock solution is 5 μg / mL.

[0013] In some embodiments of the present invention, the concentration ranges of prothionamide, delamanide, puttamani, clarithromycin, and azithromycin in the standard curve working solutions of the detection group are 0.18-9 μg / mL, 0.2-10 μg / mL, 0.2-10 μg / mL, 0.2-9.78 μg / mL, and 0.11-5.43 μg / mL, respectively.

[0014] In some embodiments of the present invention, the standard curve working solution includes nine concentrations, W1-W9, which are prepared by using a blank matrix and the corresponding mixed standard stock solution (M0); the mixed standard stock solution (M0) is prepared by using a methanol solution.

[0015] In the mixed standard stock solution (M0) of the levofloxacin, ciprofloxacin, and gatifloxacin detection group, the concentrations of levofloxacin, ciprofloxacin, and gatifloxacin were 500 μg / mL, 202.8 μg / mL, and 194 μg / mL, respectively.

[0016] In the mixed standard stock solution (M0) of the detection group of prothionamide, delamanide, putomanide, clarithromycin and azithromycin, the concentrations of prothionamide, delamanide, putomanide, clarithromycin and azithromycin were 180 μg / mL, 200 μg / mL, 201 μg / mL, 196 μg / mL and 109 μg / mL, respectively.

[0017] In some embodiments of the present invention, W1 of the standard curve working solution is obtained by diluting M0 with a blank matrix, W2-W7 are obtained by stepwise diluting W1 with a blank matrix, and W8-W9 are obtained by stepwise diluting W7 with a blank matrix.

[0018] In some embodiments of the present invention, a quality control step is further included. The quality control samples in this step are prepared by diluting and mixing standard solutions with a blank matrix. These include high-concentration, medium-concentration, and low-concentration quality control samples, with concentrations of: levofloxacin 20 μg / mL, 7.5 μg / mL, and 2.5 μg / mL; ciprofloxacin 8.112 μg / mL, 3.042 μg / mL, and 1.014 μg / mL; gatifloxacin 7.76 μg / mL, 2.91 μg / mL, and 1.014 μg / mL, respectively. μg / mL, 0.97μg / mL; Prothionamide 7.2μg / mL, 2.7μg / mL, 0.9μg / mL; Delamanide 8μg / mL, 3μg / mL, 1μg / mL; Putomomanide 8μg / mL, 3μg / mL, 1μg / mL; Clarithromycin 7.828μg / mL, 2.93μg / mL, 0.98μg / mL; Azithromycin 4.35μg / mL, 1.63μg / mL, 0.54μg / mL.

[0019] In some embodiments of the present invention, the blank matrix is ​​filtered human serum.

[0020] Beneficial effects of the present invention This patent utilizes the following drugs to treat serum levofloxacin (LEV), ciprofloxacin (CIP), gatifloxacin (GAT), protonamide (PTO), delamanid (DLM), pretomanid (PA-824), clarithromycin (CAM), and azithromycin. The detection method for eight second-line anti-tuberculosis drugs (ZAM) is based on the high sensitivity and specificity of LC-MS / MS: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) uses triple quadrupole mass spectrometry. The secondary mass spectrometer detects fragmented ions according to their mass-to-charge ratio (m / z), achieving two detections with higher sensitivity and specificity. After sample pretreatment, the samples are directly separated by liquid chromatography and detected by tandem mass spectrometry in multiple reaction detection (MRM) mode, achieving rapid and efficient detection of the eight anti-tuberculosis drugs. This method has a short detection time, requiring only 5 minutes, saving detection time. Simultaneously, this method reduces the amount of organic solvent used, thus reducing environmental pollution. The method also features high accuracy and precision, providing the fastest and most effective evidence for clinical treatment. Attached Figure Description

[0021] Figure 1 The method of the present invention is shown in the MRM extraction ion chromatograms of three anti-tuberculosis drugs. Figure 2 The standard curve of levofloxacin is shown; Figure 3 The standard curve of ciprofloxacin is shown; Figure 4 The standard curve of gatifloxacin is shown; Figure 5 The method of the present invention is shown in the MRM extraction ion chromatograms of five anti-tuberculosis drugs. Figure 6 The standard curve of propylthionamide is shown; Figure 7 The standard curve of Delamani is shown; Figure 8 The standard curve of Putmani is shown; Figure 9 The standard curve of clarithromycin is shown; Figure 10 The standard curve of azithromycin is shown.

[0022] Figure 11 The standard curve of levofloxacin obtained from the detection of 8 mixed groups in Example 3 is shown; Figure 12 The standard curve of ciprofloxacin obtained from the detection of 8 mixed groups in Example 3 is shown; Figure 13 The standard curve of gatifloxacin obtained from the detection of 8 mixed groups in Example 3 is shown; Figure 14 The standard curve of prothionamide obtained from the detection of the 8 mixed groups in Example 3 is shown; Figure 15 The standard curve of deltamani obtained from the detection of the 8 mixed groups in Example 3 is shown; Figure 16 The standard curve of putomovir obtained from the detection of 8 mixed groups in Example 3 is shown; Figure 17 The standard curve of clarithromycin obtained from the detection of 8 mixed groups in Example 3 is shown; Figure 18 The standard curve of azithromycin obtained from the detection of 8 mixed groups in Example 3 is shown; Figure 19 The extracted ion chromatograms of five anti-tuberculosis drugs, MRM, are shown under the comparative mobile phase. Detailed Implementation

[0023] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0025] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0026] Instruments, equipment, reagents and consumables (1) Instruments and equipment Waters Xevo TQD triple quadrupole mass spectrometer (USA, Waters), including the Accuracy UPLC system; KQ-500E Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China); 1-14KS / 3-18KS Tabletop High-Speed ​​Refrigerated Centrifuge (Germany, SIGMA); EVortex-Genie2 vortex mixer (Scientific Industries Company, USA). Cascada I Ultrapure Water Preparation System (Canada, Pallford Bioanalytical Instruments (Shanghai) Co., Ltd.); QUINTIX125D-1CN electronic balance (Germany, Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0027] (2) Reagents and consumables The basic information of the standard products is shown in Table 1 below.

[0028] Table 1 Basic Information of Standard Products

[0029] reagents Methanol: Purity ≥98%, HPLC grade (Catalog No. 1.06007.4008, Merk, Germany); Ammonium acetate: 99.0% purity, HPLC grade (Catalog No. A-801000, Sigma-Aldrich (FLuka), Germany); Formic acid: purity ≥98.0%, chromatographic grade (catalog number F112034-100mL, Aladdin, China) Isopropanol: Purity ≥98%, HPLC grade (Catalog No. 1.01040.4008, Merk, Germany) Acetonitrile: Acetonitrile: Purity ≥99.9%, HPLC grade (Catalog No. 1.00030.4000, Merk, Germany) 250 μL 96-well plates and sealing gaskets were purchased from Bona Axygen; 1.5 mL centrifuge tubes were purchased from Axygen.

[0030] Sample Requirements Sampling requirements Serum samples were collected using vacuum negative pressure yellow cap blood collection tubes (separation gel); After collecting the blood sample using an EDTAK2 anticoagulant tube, invert the tube to ensure the blood and anticoagulant are thoroughly mixed. Plasma must be separated and dispensed within 2 hours using centrifugation (4000 rpm, 10 min); Sample usage Fasting venous blood collection is required, with a blood volume of 1.0 mL. Sample storage and transportation Plasma or serum must be separated and aliquoted within 2 hours using centrifugation (4000 rpm, 10 min); It can be stored for 72 days under refrigeration.

[0031] Example 1: Detection of levofloxacin, ciprofloxacin, and gatifloxacin groups I. Testing Conditions 1. Chromatographic column: ACE Excel-2 C18-PFP column (100 × 2.1 mm, 2.6 μm); Mobile phase A: 5 mM ammonium acetate solution (containing 0.1% formic acid); Mobile phase B: 5 mM ammonium acetate methanol (containing 0.1% formic acid); Wash solution: 50% isopropanol-water solution Flow rate: 0.3 mL / min, column temperature: 40 ℃, injection volume: 2 μL; The gradient elution procedure is shown in Table 2.

[0032] Table 2 Gradient elution program

[0033] 2. Mass spectrometry conditions Ion source: Electrospray ionization (ESI), positive ion mode; Capillary voltage of spray nozzle: 2.7 kV; Desolvation temperature: 450 ℃; Desolvation flow rate: 900 L / Hr; Ion source temperature: 150℃; Desolvation gas pressure (pressure reducer): 8 bar; Collision air pressure (pressure relief gauge): 0.7 bar; Scanning mode: MRM. Quantitative ion pair information for each compound is shown in Table 3.

[0034] Table 3 Quantitative Ion Pair Information

[0035] II. Preparation of Calibration Solution 1. Preparation of single-standard stock solution The preparation method is shown in Table 4. After use, the single standard stock solution should be sealed and stored in a -80°C refrigerator.

[0036] Table 4 Preparation of Single Standard Stock Solutions

[0037] 2. Preparation of single-standard stock solution (M0) The preparation method is shown in Table 5. After preparation, seal and store in a refrigerator at -20°C for later use.

[0038] Table 5 Preparation method of mixed standard stock solution (M0)

[0039] Note: A mixed standard stock solution can be prepared according to the actual usage ratio. 3. Preparation of internal standard stock solution The preparation method is shown in Table 6. After use, the single standard stock solution should be sealed and stored in a -20°C refrigerator.

[0040] Table 6 Preparation of Internal Standard Stock Solution

[0041] 4. Preparation of internal standard stock solution (LEV-D8-P0) The preparation method is shown in Table 7. After preparation, seal and store in a refrigerator at 4°C for later use.

[0042] Table 7 Preparation method of internal standard stock solution (LEV-D8-P0)

[0043] 5. Preparation of W1 Blank matrix: filtered human serum.

[0044] Preparation of W1: Take 100 µL of M0, mix it with 1900 µL of filtered human serum, and store at -20°C. 6. Preparation of working solution for serum standard curve W1 was serially diluted with blank matrix to obtain standard curve working solutions of various concentrations. The preparation method is shown in Table 8, and the concentrations of the standard curve working solutions are shown in Table 9. After preparation, each sample was aliquoted into 1.5 mL centrifuge tubes at a rate of 20 μL and stored in sealed containers at -20°C.

[0045] Table 8. Preparation of working solutions for standard curves

[0046] Table 9 Concentration of working solution for serum standard curve

[0047] III. Preparation of Quality Control Samples The blank matrix was serially diluted to obtain high-concentration quality control (HQC), medium-concentration quality control (MQC), and low-concentration quality control (LQC) samples, respectively. The preparation method is shown in Table 10, and the concentration of the quality control samples is shown in Table 11. After preparation, each sample was aliquoted into 1.5 mL centrifuge tubes at a rate of 20 μL and stored in sealed containers at -20°C.

[0048] Table 10 Preparation of Quality Control Samples

[0049] Table 11 Preparation of Quality Control Samples

[0050] IV. Sample Pretreatment Add 50 μL of internal standard solution to 20 μL of sample, then add 1000 μL of methanol solution, vortex for 5 min, centrifuge for 5 min, and take the supernatant for loading.

[0051] V. HPLC-MS / MS Detection The processed test samples were analyzed by HPLC-MS / MS, with a sample loading volume of 2 μL each time. The chromatographic and mass spectrometry parameters were set as described above.

[0052] Levofloxacin was quantified using the internal standard method, while ciprofloxacin and gatifloxacin were quantified using the external standard method. MRM ion chromatograms were extracted from the samples for the target analytes and isotope-labeled substances, as shown below. Figure 1 The peak areas are obtained by integrating the results. Based on the obtained working curves, the concentration of the target analyte can be calculated.

[0053] The linear regression equations and linear correlation coefficients for levofloxacin, ciprofloxacin, and gatifloxacin are shown in Table 12, and the working curves are shown in Table 12. Figure 2-4 .

[0054] The linear regression equations and linear correlation coefficients of the drugs in Table 123

[0055] Procedure for handling data outside the linear range: Analyze the data exceeding the limits, and retest if necessary; For samples with concentrations exceeding the highest point of the standard curve, dilution was performed using a blank matrix. Based on the test results, an appropriate dilution factor (2x, 5x, 10x) was selected. For samples with concentrations below the lowest point of the standard curve, the results are given according to the calculated values; For samples with detection limits below the minimum detection limit, the results should be given according to the minimum detection limit of the method.

[0056] The limits of detection and limits of quantitation for levofloxacin, ciprofloxacin, and gatifloxacin are shown in Table 13 below.

[0057] Table 13 Limit of Detection and Limit of Quantification

[0058] Six Methodological Performance Verification (1) Accuracy verification Accuracy experiment: Intergroup accuracy was obtained by analyzing two samples with different concentrations over at least three days (6 parallel samples were made for each concentration point). The results are shown in Table 14.

[0059] The standard for qualification is that the average concentration of each measured level should be within 15% of the theoretical value, that is, the accuracy range should be between 85% and 115%.

[0060] Quality control: LQC (low concentration quality control); MQC (medium concentration quality control); HQC (high concentration quality control).

[0061] Table 14. Accuracy data of quality control sample testing over three days.

[0062] The results show that the accuracy of the method of the present invention meets the requirements for quantitative analysis of biological samples.

[0063] (2) Precision verification Precision: Three different concentrations of QC were measured at one time, with a minimum of 5 samples (n=6) for each concentration. The qualified standard is that the CV of each concentration QC sample is ≤15%, as shown in Table 15.

[0064] Table 15 Precision Verification Results

[0065] Note: LQC is low-concentration quality control; MQC is medium-concentration quality control; HQC is high-concentration quality control.

[0066] The results show that the precision of the method of the present invention meets the requirements for quantitative analysis of biological samples.

[0067] Example 2: Detection of prothionamide, delamani, putomani, clarithromycin, and azithromycin. Other operations are the same as in Example 1, except that: 1. Chromatographic conditions Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid in methanol solution; 2. Mass spectrometry conditions The quantitative ion pair information for each compound is shown in Table 16.

[0068] Table 16 Quantitative Ion Pair Information

[0069] 3. Preparation of calibration solution 6.2.1 Preparation of single-standard stock solution The preparation method is shown in Table 17. After use, the single standard stock solution should be sealed and stored in a -80°C refrigerator.

[0070] Table 17 Preparation of Single Standard Stock Solutions

[0071] 6.2.2 Preparation of Single Standard Stock Solution (M0) The preparation method is shown in Table 18. After preparation, seal and store in a refrigerator at -20°C for later use.

[0072] Table 18 Preparation method of mixed standard stock solution (M0)

[0073] Note: A mixed standard stock solution can be prepared according to the actual usage ratio. 6.2.3 Preparation of W1 Blank matrix: filtered human serum.

[0074] Preparation of W1: Take 100 µL of M0, mix it with 1900 µL of filtered human serum, and store at -20°C. 6.2.4 Preparation of working solution for plasma standard curve W1 was serially diluted with blank matrix to obtain standard curve working solutions of various concentrations. The preparation method is shown in Table 19, and the concentrations of the standard curve working solutions are shown in Table 20. After preparation, each sample was aliquoted into 1.5 mL centrifuge tubes at a rate of 20 μL and stored in sealed containers at -20°C.

[0075] Table 19 Preparation of working solutions for standard curves

[0076] Table 20 Concentration of working solution for standard curve

[0077] 6.3 Preparation of quality control samples The blank matrix was serially diluted to obtain high-concentration quality control (HQC), medium-concentration quality control (MQC), and low-concentration quality control (LQC) samples, respectively. The preparation method is shown in Table 21, and the concentration of the quality control samples is shown in Table 22. After preparation, each sample was aliquoted into 1.5 mL centrifuge tubes at a rate of 20 μL and stored in sealed containers at -20°C.

[0078] Table 21 Preparation of quality control samples

[0079] Table 22 Concentration of Quality Control Samples

[0080] 6.4 Sample Pretreatment Add 1000 μL of methanol solution to 20 μL of sample, vortex for 5 min, centrifuge for 5 min, take 20 μL of supernatant and mix with 180 μL of methanol solution, then load the sample.

[0081] V. HPLC-MS / MS Detection The processed test samples were analyzed by HPLC-MS / MS, with a sample loading volume of 2 μL each time. The chromatographic and mass spectrometry parameters were set as described above.

[0082] Drug quantification was performed using the external standard method. MRM ion chromatograms were extracted from the sample for both the target analyte and the isotope-labeled analyte, as shown below. Figure 5 The peak areas are obtained by integrating the results. Based on the obtained working curves, the concentration of the target analyte can be calculated.

[0083] The linear regression equations and linear correlation coefficients for prothionamide, delamani, putomamani, clarithromycin, and azithromycin are shown in Table 23, and the working curves are shown in... Figure 6-10 .

[0084] Table 235 shows the linear regression equations and linear correlation coefficients for various drugs.

[0085] Data processing program for data outside the linear range Analyze the data that exceeds the limits, and retest if necessary.

[0086] For samples exceeding the highest concentration of the standard curve, dilution is performed using a blank matrix. Based on the test results, an appropriate dilution factor (2x, 5x, 10x) is selected.

[0087] For samples with concentrations below the lowest point of the standard curve, the results are given according to the calculated values.

[0088] For samples with detection limits below the minimum detection limit, the results should be given according to the minimum detection limit of the method.

[0089] The limits of detection and limits of quantitation for prothionamide, delamani, putomali, clarithromycin, and azithromycin are shown in Table 24 below.

[0090] Table 24 Limit of Detection and Limit of Quantification

[0091] VI. Methodological Performance Requirements (1) Accuracy verification Accuracy experiment: Intergroup accuracy was obtained by analyzing two samples of different concentrations over at least three days (6 parallel samples for each concentration point), as shown in Table 25.

[0092] The qualified standard requires that the average concentration of each measured level be within 15% of the theoretical value, that is, the accuracy range is between 85% and 115%.

[0093] Quality control: LQC (Low Concentration Quality Control); MQC (Medium Concentration Quality Control); HQC (High Concentration Quality Control) Table 25 Accuracy data of quality control sample testing over three days

[0094] (2) Precision verification Precision: Three different concentrations of QC were measured at once, with a minimum of 5 samples per concentration (n=6). The acceptable standard is that the CV of the QC samples at each concentration level is ≤ 15%, as shown in Table 26. Table 26 Precision Verification Results

[0095] Note: LQC is low-concentration quality control; MQC is medium-concentration quality control; HQC is high-concentration quality control. The results show that the accuracy and precision of the method of the present invention meet the requirements for quantitative analysis of biological samples.

[0096] Example 3: Detection of a combination of 8 second-line anti-tuberculosis drugs I. Testing Conditions 1. Chromatographic column: ACE Excel-2 C18-PFP column (100 × 2.1 mm, 2.6 μm); Mobile phase A: 5 mM ammonium acetate solution (containing 0.1% formic acid); Mobile phase B: 5 mM ammonium acetate methanol (containing 0.1% formic acid); Wash solution: 50% isopropanol-water solution Flow rate: 0.3 mL / min, column temperature: 40 ℃, injection volume: 2 μL; The gradient elution procedure is shown in Table 27.

[0097] Table 27 Gradient elution program

[0098] 2. Mass spectrometry conditions Ion source: Electrospray ionization (ESI), positive ion mode; Capillary voltage of spray nozzle: 2.7 kV; Desolvation temperature: 450 ℃; Desolvation flow rate: 900 L / Hr; Ion source temperature: 150℃; Desolvation gas pressure (pressure reducer): 8 bar; Collision air pressure (pressure relief gauge): 0.7 bar; Scan mode: MRM. Quantitative ion pair information for each compound is shown in Table 28.

[0099] Table 28 Quantitative Ion Pair Information

[0100] II. Preparation of Calibration Solution 1. Preparation of single-standard stock solution The preparation method is shown in Table 29. After use, the single standard stock solution should be sealed and stored in a -80°C refrigerator.

[0101] Table 29 Preparation of Single Standard Stock Solutions

[0102] 2. Preparation of single-standard stock solution (M0) The preparation method is shown in Table 30. After preparation, seal and store in a refrigerator at -20°C for later use.

[0103] Table 30 Preparation method of mixed standard stock solution (M0)

[0104] Note: A mixed standard stock solution can be prepared according to the actual usage ratio. 3. Preparation of internal standard stock solution The preparation method is shown in Table 31. After use, the single standard stock solution should be sealed and stored in a -20°C refrigerator.

[0105] Table 31 Preparation of Internal Standard Stock Solution

[0106] 4. Preparation of internal standard stock solution (LEV-D8-P0) The preparation method is shown in Table 32. After preparation, seal and store in a refrigerator at 4°C for later use.

[0107] Table 32 Preparation method of internal standard stock solution (LEV-D8-P0)

[0108] 5. Preparation of W1 Blank matrix: filtered human serum.

[0109] Preparation of W1: Take 100 µL of M0, mix it with 1900 µL of filtered human serum, and store it at -20℃.

[0110] 6. Preparation of working solution for serum standard curve W1 was serially diluted with blank matrix to obtain standard curve working solutions of various concentrations. The preparation method is shown in Table 33, and the concentrations of the standard curve working solutions are shown in Table 34. After preparation, each sample was aliquoted into 1.5 mL centrifuge tubes at a rate of 20 μL and stored in sealed containers at -20°C.

[0111] Table 33 Preparation of Standard Curve Working Solution

[0112] Table 34 Concentration of working solution for serum standard curve (μg / mL)

[0113] III. Preparation of Quality Control Samples The blank matrix was serially diluted to obtain high-concentration quality control (HQC), medium-concentration quality control (MQC), and low-concentration quality control (LQC) samples, respectively. The preparation method is shown in Table 35, and the concentration of the quality control samples is shown in Table 36. After preparation, each sample was aliquoted into 1.5 mL centrifuge tubes at a rate of 20 μL and stored in sealed containers at -20°C.

[0114] Table 35 Preparation of Quality Control Samples

[0115] Table 36 Concentration of quality control samples (μg / mL)

[0116] IV. Sample Pretreatment For 20 samples, add 50 μL of internal standard solution, then add 400 μL of (methanol:acetonitrile = 1:2) solution, vortex for 5 min, centrifuge for 5 min, take 10 μL of supernatant, add 490 μL of methanol and mix well, then take the supernatant and load it onto the sample.

[0117] V. HPLC-MS / MS Detection The processed test samples were analyzed by HPLC-MS / MS, with a sample loading volume of 2 μL each time. The chromatographic and mass spectrometry parameters were set as described above.

[0118] Levofloxacin was quantified using the internal standard method, while other drugs were quantified using the external standard method. MRM ion chromatograms were extracted from the samples for both the target analyte and the isotope-labeled analyte, as shown below. Figure 1The peak areas are obtained by integrating the results. Based on the obtained working curves, the concentration of the target analyte can be calculated.

[0119] The linear regression equations and linear correlation coefficients of levofloxacin, ciprofloxacin, gatifloxacin, prothionamide, delamani, putomani, clarithromycin, and azithromycin are shown in Table 37.

[0120] Table 378 shows the linear regression equations and linear correlation coefficients for each drug.

[0121] The linear curves of the eight drugs—levofloxacin, ciprofloxacin, gatifloxacin, prothionamide, delamani, putomomani, clarithromycin, and azithromycin—are shown below. Figures 11-18 As shown. The linear fit was poor; except for azithromycin and clarithromycin, the linear coefficients R0 for the other six drugs were... 2 All are less than 0.8.

[0122] Example 4 Sample Preprocessing Optimization The other operating steps are the same as those in Example 1 for the levofloxacin, ciprofloxacin, and gatifloxacin detection groups, the difference being the sample pretreatment method: Scheme (1): Add 50 μL of internal standard solution to 20 μL of sample, then add 1000 μL of methanol solution, vortex for 5 min, centrifuge for 5 min, take 10 μL of supernatant and add 490 μL of methanol to mix well, take the supernatant and load the sample.

[0123] Scheme (2): Add 50 μL of internal standard solution to 20 μL of sample, then add 1000 μL of methanol solution, vortex for 5 min, centrifuge for 5 min, take 5 μL of supernatant and add 195 μL of methanol to mix well, then take the supernatant for sample loading.

[0124] Scheme (3): Add 50 μL of internal standard solution to 20 μL of sample, then add 400 μL of (methanol:acetonitrile = 1:2) solution, vortex for 5 min, centrifuge for 5 min, take 10 μL of supernatant and add 490 μL of methanol to mix well, take the supernatant and load the sample.

[0125] Scheme (4): Add 1000 μL of methanol solution to 20 μL of sample, vortex for 5 min, centrifuge for 5 min, take 20 μL of supernatant and mix with 180 μL of methanol solution and load the sample.

[0126] Results: Linear coefficient R of schemes (1), (2), and (3) 2 All are less than 0.8, and the linear coefficient R of scheme (4) is less than 0.8. 2The value is greater than 0.99, indicating a good linear fit. However, scheme (4) was diluted 10 times, and the MS response of the three drugs was not particularly high, so it was not necessary to dilute 10 times.

[0127] Example 5: Optimization of Chromatographic Conditions The other operating procedures are the same as in Example 2: the detection of prothionamide, delamani, putomani, clarithromycin, and azithromycin, the difference being: Mobile phase A: 5 mM ammonium acetate solution (containing 0.1% formic acid); Mobile phase B: 5mM ammonium acetate methanol (containing 0.1% formic acid).

[0128] The results are as follows Figure 19 Except for propylthionamide, which has a relatively good peak shape, the peak shapes of delamani, putomomani, clarithromycin, and azithromycin are all very messy.

[0129] Example 6: Clinical Sample Testing To verify the method of this experiment, we took clinical samples and tested them using the method of this application. The results are shown in Table 38 below.

[0130] Table 38 Sample Detection Results

[0131] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for detecting the concentrations of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS, characterized in that, The eight second-line anti-tuberculosis drugs include levofloxacin, ciprofloxacin, gatifloxacin, prothionamide, delamani, putomani, clarithromycin, and azithromycin. These second-line anti-tuberculosis drugs were divided into two groups for testing: levofloxacin, ciprofloxacin, and gatifloxacin, and prothionamide, delamani, putomani, clarithromycin, and azithromycin. The detection methods for levofloxacin, ciprofloxacin, and gatifloxacin are as follows: Levofloxacin was used as an internal standard to prepare an internal standard stock solution. A mixed standard stock solution was prepared using levofloxacin, ciprofloxacin, and gatifloxacin standards as target compounds. These solutions were then gradually diluted with a blank matrix to prepare the working solution for the standard curve. The sample to be tested was added to the internal standard solution, followed by the addition of methanol solution. After shaking and centrifugation, the supernatant was collected and analyzed by HPLC-MS / MS. For the levofloxacin, ciprofloxacin, and gatifloxacin detection groups, mobile phase A consisted of 0.1% formic acid and 5 mM ammonium acetate in water; mobile phase B consisted of 0.1% formic acid and 5 mM ammonium acetate in methanol. The detection methods for prothionamide, delamani, putomani, clarithromycin, and azithromycin are as follows: Using prothionamide, delamani, putomomani, clarithromycin, and azithromycin as target compounds, a mixed standard stock solution was prepared. This solution was then gradually diluted with a blank matrix to prepare the standard curve working solution. The sample to be tested was added to methanol solution, vortexed, centrifuged, and the supernatant was collected and diluted 10-fold with methanol solution. HPLC-MS / MS was used for detection. For the prothionamide, delamani, putomomani, clarithromycin, and azithromycin detection groups, mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was 0.1% formic acid methanol solution.

2. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 1, characterized in that, The chromatographic conditions for the detection of levofloxacin, ciprofloxacin, gatifloxacin, and prothionamide, delamani, putomani, clarithromycin, and azithromycin were as follows: Chromatographic column: ACE Excel-2 C18-PFP column, 100×2.1 mm, 2.6 μm; Wash solution: 50% isopropanol-water solution; Flow rate: 0.3 mL / min, column temperature: 40 ℃, injection volume: 2 μL; A gradient elution method was used, as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; the gradient elution time was 5.0 min before stopping. The volume fraction of mobile phase A was 99% at the beginning (0.3 min). Over 0.3–1.0 min, the volume fraction of mobile phase A decreased from 99% to 30%. The volume fraction of mobile phase A decreased from 30% to 2% over 1.0-2 minutes. The volume fraction of mobile phase A was maintained at 2% for 2.0-4.0 min. The volume fraction of mobile phase A increased from 2% to 99% over 4.0-4.01 min. The volume fraction of mobile phase A was maintained at 99% for 4.01-5 min.

3. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 1, characterized in that, The mass spectrometry conditions for the detection are as follows: Ion source: Electrospray ion source, positive ion mode; Spray capillary voltage: 2.7 KV; Desolvation gas temperature: 450 ℃; Desolvation gas flow rate: 900 L / Hr; Ion source temperature: 150℃; Desolvation gas pressure: 8 bar; Collision gas pressure: 0.7 bar; Scan mode: MRM.

4. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 3, characterized in that, The MRM parameters for the levofloxacin, ciprofloxacin, and gatifloxacin detection groups are as follows: 。 5. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 3, characterized in that, The MRM parameters for the detection groups of prothionamide, delamani, putomani, clarithromycin, and azithromycin are as follows: 。 6. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 1, characterized in that, In the standard curve working solutions of the levofloxacin, ciprofloxacin, and gatifloxacin detection groups, the concentration ranges of levofloxacin, ciprofloxacin, and gatifloxacin are 0.5-25 μg / mL, 0.203-10.14 μg / mL, and 0.194-9.7 μg / mL, respectively.

7. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 1, characterized in that, The levofloxacin internal standard stock solution was diluted with a methanol-acetonitrile solution at a volume ratio of 1:2, and the concentration of the internal standard stock solution was 5 μg / mL.

8. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 1, characterized in that, In the standard curve working solutions of the prothionamide, delamanide, puttamani, clarithromycin, and azithromycin detection groups, the concentration ranges of prothionamide, delamanide, puttamani, clarithromycin, and azithromycin are 0.18-9 μg / mL, 0.2-10 μg / mL, 0.2-10 μg / mL, 0.2-9.78 μg / mL, and 0.11-5.43 μg / mL, respectively.

9. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to claim 1, characterized in that, The process also includes quality control steps. The quality control samples are prepared by diluting and mixing standard solutions with a blank matrix. These include high-concentration, medium-concentration, and low-concentration quality control samples, with the following concentrations: levofloxacin 20 μg / mL, 7.5 μg / mL, 2.5 μg / mL; ciprofloxacin 8.112 μg / mL, 3.042 μg / mL, 1.014 μg / mL; gatifloxacin 7.76 μg / mL, 2.91 μg / mL, 0 μg / mL... 0.97 μg / mL; Prothionamide 7.2 μg / mL, 2.7 μg / mL, 0.9 μg / mL; Delamanide 8 μg / mL, 3 μg / mL, 1 μg / mL; Putomomanide 8 μg / mL, 3 μg / mL, 1 μg / mL; Clarithromycin 7.828 μg / mL, 2.93 μg / mL, 0.98 μg / mL; Azithromycin 4.35 μg / mL, 1.63 μg / mL, 0.54 μg / mL.

10. The method for detecting the concentration of eight second-line anti-tuberculosis drugs based on HPLC-MS / MS according to any one of claims 1-9, characterized in that, The blank matrix is ​​filtered human serum.

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