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CN-122017062-A - HPLC-MS/MS-based detection method for concentration of 8 two-wire antitubercular drugs

CN122017062ACN 122017062 ACN122017062 ACN 122017062ACN-122017062-A

Abstract

The invention discloses a method for detecting the concentration of 8 secondary antitubercular drugs based on HPLC-MS/MS, which comprises the steps of detecting the concentration of the 8 secondary antitubercular drugs in two groups, wherein one group uses levofloxacin as an internal standard substance to prepare an internal standard stock solution, uses the standard substances of levofloxacin, ciprofloxacin and gatifloxacin as target substances, and the other group uses the propylthioisoniazid, delamanib, protomanib, clarithromycin and azithromycin as target substances. The method has the advantages that the detection time is short, only 5 minutes is needed, and the detection time is saved; meanwhile, the method reduces the dosage of the organic solvent so as to reduce the pollution to the environment, has the characteristics of high accuracy and good precision, and provides the fastest and effective basis for clinical treatment.

Inventors

  • GAO FEI
  • LIU LINLIN
  • WANG MINGMING

Assignees

  • 杭州度安医学检验实验室有限公司

Dates

Publication Date
20260512
Application Date
20260126

Claims (10)

  1. 1. The detection method for the concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS is characterized in that the 8 two-wire antitubercular drugs comprise levofloxacin, ciprofloxacin, gatifloxacin, prothioisonicotinamide, delamanib, protomanib, clarithromycin and azithromycin, and the two-wire antitubercular drugs are respectively detected by the levofloxacin, ciprofloxacin, gatifloxacin detection group and prothioisonicotinamide, delamanib, protomanib, clarithromycin and azithromycin detection group; The detection method of the levofloxacin, ciprofloxacin and gatifloxacin detection group is as follows: Preparing an internal standard stock solution by taking levofloxacin as an internal standard substance, preparing a mixed standard stock solution by taking standard substances of levofloxacin, ciprofloxacin and gatifloxacin as target substances, gradually diluting with a blank matrix to prepare a standard curve working solution, adding a sample to be detected into the internal standard solution, adding a methanol solution, oscillating and centrifuging, and taking a supernatant, and performing on-line detection by adopting an HPLC-MS/MS (high performance liquid chromatography/mass spectrometry), wherein a mobile phase A of a levofloxacin, ciprofloxacin and gatifloxacin detection group is ammonium acetate water containing 0.1% formic acid and 5mM, and a mobile phase B is ammonium acetate methanol containing 0.1% formic acid and 5 mM; The detection method of the detection group of the prothioisonicotinamide, the delamanib, the protomanib, the clarithromycin and the azithromycin is as follows: preparing a mixed standard stock solution by taking prochloraz, delamanib, protomanib, clarithromycin and azithromycin as target substances, gradually diluting with a blank matrix to prepare a standard curve working solution, adding a methanol solution into a sample to be detected, oscillating and centrifuging, taking a supernatant, diluting 10 times with the methanol solution, and detecting by using an HPLC-MS/MS machine, wherein the mobile phase A of a detecting group of prochloraz, delamanib, protomanib, clarithromycin and azithromycin is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid methanol solution.
  2. 2. The method for detecting the concentration of 8 kinds of two-wire antitubercular drugs based on HPLC-MS/MS according to claim 1, wherein the chromatographic conditions of the detection of levofloxacin, ciprofloxacin, gatifloxacin and prothioisoniazid, delamanib, protomanib, clarithromycin and azithromycin are as follows: Chromatographic column, ACE Excel-2C 18-PFP column, 100×2.1 mm,2.6 μm; wash solution, 50% isopropyl alcohol-water solution; the flow rate is 0.3 mL/min, the column temperature is 40 ℃, and the sample injection amount is 2 mu L; the gradient elution mode is adopted, wherein the gradient elution mode comprises the following steps of volume fraction of mobile phase A and volume fraction of mobile phase B = 100%, and the gradient elution time is 5.0min, wherein: Starting to obtain a volume fraction of mobile phase A of 99% at 0.3 min; The volume fraction of the mobile phase A is reduced from 99% to 30% within 0.3-1.0 min; The volume fraction of the mobile phase A is reduced from 30% to 2% within 1.0-2 min; The volume fraction of the mobile phase A is maintained at 2% within 2.0-4.0 min; 4.0-4.01min, the volume fraction of the mobile phase A is increased from 2% to 99%; The volume fraction of mobile phase A was maintained at 99% for 4.01-5 min.
  3. 3. The method for detecting the concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS according to claim 1, wherein the mass spectrometry conditions of the detection are: Ion source, electrospray ion source, positive ion mode, spray capillary voltage 2.7 KV, desolvation gas temperature 450 ℃, desolvation gas flow rate 900L/Hr, ion source temperature 150 ℃, desolvation gas pressure 8 bar, collision gas pressure 0.7 bar, and scanning mode MRM.
  4. 4. The method for detecting the concentration of 8 kinds of two-wire antitubercular drugs based on HPLC-MS/MS according to claim 3, wherein the MRM parameters of the detection group of levofloxacin, ciprofloxacin and gatifloxacin are as follows: 。
  5. 5. The method for detecting the concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS according to claim 3, wherein the MRM parameters of the detection group of prothioisoniazid, delamanib, protomanib, clarithromycin and azithromycin are as follows: 。
  6. 6. the method for detecting the concentration of 8 kinds of two-wire antitubercular drugs based on HPLC-MS/MS according to claim 1, wherein the concentration ranges of the levofloxacin, the ciprofloxacin and the gatifloxacin in the standard curve working solution of the detection group of the levofloxacin, the ciprofloxacin and the gatifloxacin are 0.5-25 mug/mL, 0.203-10.14 mug/mL and 0.194-9.7 mug/mL respectively.
  7. 7. The method for detecting the concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS according to claim 1, wherein the internal standard solution of levofloxacin adopts methanol acetonitrile solution with the volume ratio of 1:2 as a diluent, and the concentration of the internal standard solution is 5 mug/mL.
  8. 8. The method for detecting the concentration of 8 kinds of two-wire antitubercular drugs based on HPLC-MS/MS according to claim 1, wherein the concentration ranges of the prochloraz, the delamani, the pratimanib, the clarithromycin and the azithromycin in the standard curve working solution of the detecting group of the prochloraz, the delamanib, the pratimanib, the clarithromycin and the azithromycin are 0.18-9 mug/mL, 0.2-10 mug/mL, 0.2-9.78 mug/mL and 0.11-5.43 mug/mL respectively.
  9. 9. The method for detecting the concentration of the 8 two-wire antitubercular drugs based on the HPLC-MS/MS according to claim 1, further comprising a quality control step, wherein the quality control of the quality control step is prepared by adopting a blank matrix to dilute a mixed standard solution, and comprises a high-concentration quality control, a medium-concentration quality control and a low-concentration quality control, wherein the concentrations of the quality control are 20 mug/mL, 7.5 mug/mL, 2.5 mug/mL, 8.112 mug/mL, 3.042 mug/mL, 1.014 mug/mL, 7.76 mug/mL, 2.91 mug/mL, 0.97 mug/mL, 7.2 mug/mL, 2.7 mug/mL, 0.9 mug/mL, 8 mug/mL, 3 mug/mL, 1 mug/mL, 8 mug/mL, 3 mug/mL, 62 mug/mL, 53 mug/mL, 0.54 mug/mL, 3.4 mug/mL, and 93 mug/mL of ciprofloxacin.
  10. 10. The method for detecting the concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS according to any one of claims 1 to 9, wherein said blank matrix is filtered human serum.

Description

HPLC-MS/MS-based detection method for concentration of 8 two-wire antitubercular drugs Technical Field The invention belongs to the technical field of biological detection, and particularly relates to a detection method of concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS. Background Tuberculosis is a chronic wasting infectious disease caused by tubercle bacillus, also called scabies and "white plague", which is an old zoonotic infectious disease. The medicine for treating the disease is antituberculosis medicine. Antituberculosis drugs are mainly divided into four types, namely first-line antituberculosis drugs, second-line antituberculosis drugs, novel antituberculosis drugs and compound preparation drugs. The related two-line antitubercular drugs mainly comprise levofloxacin (Levofloxacin, LEV), ciprofloxacin (Ciprofloxacin, CIP), gatifloxacin (Gatifloxacin, GAT), prothioisonicotinamide (Protionamide, PTO), delamanib (DELAMANID, DLM), protomanib (Pretomanid, PA-824), clarithromycin (Clarithromycin, CAM) and azithromycin (Azithromycin, ZAM). The second-line antituberculosis drug is mainly used for the first-line drug, such as isoniazid, rifampicin, ethambutol and pyrazinamide, which is ineffective or resistant to treatment. Currently, the detection of drug concentration mainly includes high performance liquid chromatography and immunoassay. For example, chinese patent CN121114275A discloses a method for detecting six two-wire antitubercular drugs by high performance liquid chromatography, wherein the two-wire drugs are 6 kinds of such drugs as prothioisoniazid, levofloxacin, linezolid, moxifloxacin and dela Ma Niji bedaquiline, the gradient elution time is 25min, the detection period is long, and the application requirement cannot be satisfied when the requirements of faster detection are met in clinical emergency. Therefore, the application aims to provide a detection scheme of the two-wire antitubercular drug with high sensitivity and short detection time. Disclosure of Invention In order to solve at least one of the problems, the invention provides a detection method of the concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS. In order to achieve the above purpose, the invention adopts the following technical means: The invention provides a method for detecting the concentration of 8 two-wire antitubercular drugs based on HPLC-MS/MS, wherein the 8 two-wire antitubercular drugs comprise levofloxacin, ciprofloxacin, gatifloxacin, prothioisonicotinamide, delamanib, protomanib, clarithromycin and azithromycin, and the two-wire antitubercular drugs are respectively detected by the levofloxacin, ciprofloxacin, gatifloxacin detection group and prothioisonicotinamide, delamanib, protomanib, clarithromycin and azithromycin detection group; The detection method of the levofloxacin, ciprofloxacin and gatifloxacin detection group is as follows: Preparing an internal standard stock solution by taking levofloxacin as an internal standard substance, preparing a mixed standard stock solution by taking standard substances of levofloxacin, ciprofloxacin and gatifloxacin as target substances, gradually diluting with a blank matrix to prepare a standard curve working solution, adding a sample to be detected into the internal standard solution, adding a methanol solution, oscillating and centrifuging, and taking a supernatant, and performing on-line detection by adopting an HPLC-MS/MS (high performance liquid chromatography/mass spectrometry), wherein a mobile phase A of a levofloxacin, ciprofloxacin and gatifloxacin detection group is ammonium acetate water containing 0.1% formic acid and 5mM, and a mobile phase B is ammonium acetate methanol containing 0.1% formic acid and 5 mM; The detection method of the detection group of the prothioisonicotinamide, the delamanib, the protomanib, the clarithromycin and the azithromycin is as follows: preparing a mixed standard stock solution by taking prochloraz, delamanib, protomanib, clarithromycin and azithromycin as target substances, gradually diluting with a blank matrix to prepare a standard curve working solution, adding a methanol solution into a sample to be detected, oscillating and centrifuging, taking a supernatant, diluting 10 times with the methanol solution, and detecting by using an HPLC-MS/MS machine, wherein the mobile phase A of a detecting group of prochloraz, delamanib, protomanib, clarithromycin and azithromycin is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid methanol solution. In some embodiments of the invention, the chromatographic conditions for the detection of levofloxacin, ciprofloxacin, gatifloxacin and prothioisonicotinamide, delamanib, protomanib, clarithromycin, azithromycin detection group are: Chromatographic column, ACE Excel-2C 18-PFP column, 100×2.1 mm,2.6 μm; wash solution, 50% isopropyl alcohol-water solution; the flow rate is 0.3 mL/min, the column temperature is 40 ℃, and the