关键词: 抗结核药, 色谱法,液相, 药物监测, 评价研究

Abstract:

Objective: To establish a high-performance liquid chromatography (HPLC) method for the simultaneous determination of isoniazid (INH), pyrazinamide (PZA), and either rifapentine (RFT) or rifampicin (RFP) in plasma from tuberculosis patients. Methods: Chromatographic separation was performed on a StarCore Amide column (4.6 mm×100 mm, 2.6 μm) maintained at 20 ℃ with a ternary gradient elution system (A: 20 mmol/L ammonium acetate, pH 4.7; B: acetonitrile; C: ultrapure water; 0-3 min 97% B/3% C; 3-4 min 30% A/70% B; 4-7 min 50% A/50% B; 7-9 min 50% B/50% C; 9-15 min 97% B/3% C) at a flow rate of 1.2 ml/min. UV detection was set at 260 nm, and the injection volume was 30 μl. RFT or RFP was used as the internal standard. After full method validation, 147 plasma samples collected consecutively from tuberculosis patients undergoing therapeutic drug monitoring (TDM) were analyzed in parallel by the proposed HPLC-UV method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to evaluate clinical applicability. Results: Under the specified conditions, INH, PZA, and RFT (or RFP) achieved baseline separation within 15 minutes, with no interfering peaks from blank serum. When RFP was used as the internal standard, the linear ranges were 0.6-15 mg/L (R²=0.999) for INH, 2-100 mg/L (R²=0.999) for PZA, and 0.8-40 mg/L (R²=0.999) for RFT. Both intra-day and inter-day precision (relative standard deviation) were ≤12.14%, extraction recoveries ranged from 95.85% to 107.22%, and stability deviations under room temperature or refrigeration conditions were within ±15% of the nominal concentrations. When RFT was employed as the internal standard, the linear ranges of all analytes were consistent with their retention times (RFP: 3.39 min), demonstrating good specificity. Validation with clinical patient plasma samples showed high agreement with LC-MS/MS results (Bland-Altman bias <5%), confirming the reliability of the method. Conclusion: We developed a simple, specific, and sensitive HPLC method capable of simultaneously quantifying three first-line anti-tuberculosis drugs in plasma. It can be applied to TDM of the core agents in both the standard 6-month regimen and the 4-month short-course chemotherapy, thereby facilitating individualized dosing in tuberculosis patients.

Key words: Antitubercular agents, Chromatography, liquid, Drug monitoring, Evaluation studies