Abstract:

Objective: To establish a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of the plasma concentrations of bedaquiline (Bdq), pretomanid (Pa), and linezolid (Lzd), providing a method for monitoring clinical therapeutic drugs. Methods: Plasma samples were precipitated using acetonitrile, meanwhile, propranolol (PR) was added as an internal standard, followed by centrifugation to obtain the supernatant. Using a gradient elution method for HPLC-MS/MS analysis of Bdq, Pa, and Lzd concentrations in the supernatant with mobile phases A (0.1% formic acid and 5 mM ammonium formate in water) and B (acetonitrile). Chromatographic separation conditions were determined based on peak shape and resolution with a column temperature of 35 ℃, flow rate of 0.4 ml/min, and analysis time of 4 min. Mass spectrometry detection was performed using an electrospray ionization source (ESI) in positive ion mode with multiple reaction monitoring (MRM). The established HPLC-MS/MS method was validated through specificity, standard curve establishment and limit of quantification determination, precision and accuracy, recovery and matrix effect, and stability studies. Finally, the method was applied and validated using experimental mice. Results: MRM mode scanning of plasma samples showed that the declustering voltages for Bdq, Pa, Lzd, and PR were 80, 91, 80, and 100 V, respectively, and the collision energies were 80, 32, 28, and 30 V, respectively, with parent ion/product ion pairs of 555.2/58.2, 360.2/175.2, 338.1/296.3, and 260.2/116.2, respectively. Methodological validation results showed that the retention times for Bdq, Pa, and Lzd were 1.93, 1.79, and 1.53 min, respectively, with good peak shapes and no interfering peaks signals at the elution positions; the linear correlation coefficients R² were 0.993, 0.999, and 0.999, respectively, with good linear relationships in the concentration ranges of 0.05-12.5, 0.1-25, and 0.2-50 μg/ml, respectively; the intra-day and inter-day accuracies for the three drugs were above 90% and the precision fluctuations were less than 15%; the extraction recoveries of three drugs and the internal standard PR at different concentrations ranged from 78.45% to 108.78%. The method has the following characteristics: little affected by plasma processing methods. Matrix effects above 90%, meeting the ±15% requirement and stability errors under different storage conditions were within ±15%; the method successfully detected plasma concentrations of Bdq, Pa, and Lzd in 36 mouse plasma samples, with ranges of 0.03-1.82, 0.11-14.21, and 0.02-16.54 μg/ml, respectively. Conclusion: The established HPLC-MS/MS method can stably and rapidly detect the drug concentrations of Bdq, Pa, and Lzd in plasma samples simultaneously. Methodological validation showed that the method is stable, accurate, and sensitive. This method can serve as a reference for clinical application.

Key words: Tuberculosis, Chromatography, liquid, Mass spectrometry, Dose-response relationship, drug, Bedaquiline, Pretomanid, Linezolid