Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 3 Rokietnicka Street, 60-806 Poznań, Poland.
Doctoral School, Poznan University of Medical Sciences, 70 Bukowska Street, 60-812 Poznań, Poland.
Molecules. 2024 Jan 9;29(2):337. doi: 10.3390/molecules29020337.
Tuberculosis (TB) remains one of the leading global causes of mortality. Several methods have been established to detect anti-TB agents in human plasma and serum. However, there is a notable absence of studies analyzing TB drugs in urine. Thus, our objective was to validate a method for quantifying first-line anti-TB agents: isoniazid (INH), pyrazinamide (PZA), ethambutol (ETH), and rifampicin (RIF), along with its metabolite 25-desacetylrifampicin, and degradation products: rifampicin quinone and 3-formyl-rifampicin in 10 µL of urine. Chromatographic separation was achieved using a Kinetex Polar C18 analytical column with gradient elution (5 mM ammonium acetate and acetonitrile with 0.1% formic acid). Mass spectrometry detection was carried out using a triple-quadrupole tandem mass spectrometer operating in positive ion mode. The lower limit of quantification (LLOQ) was 0.5 µg/mL for INH, PZA, ETH, and RIF, and 0.1 µg/mL for RIF's metabolites and degradation products. The method was validated following FDA guidance criteria and successfully applied to the analysis of the studied compounds in urine of TB patients. Additionally, we conducted a stability study of the anti-TB agents under various pH and temperature conditions to mimic the urine collection process in different settings (peripheral clinics or central laboratories).
结核病 (TB) 仍然是全球主要的死亡原因之一。已经建立了几种方法来检测人血浆和血清中的抗结核药物。然而，分析尿液中 TB 药物的研究却明显缺乏。因此，我们的目标是验证一种定量分析一线抗结核药物的方法：异烟肼 (INH)、吡嗪酰胺 (PZA)、乙胺丁醇 (ETH) 和利福平 (RIF)，以及其代谢物 25-脱乙酰利福平，以及降解产物：利福平醌和 3-甲酰基利福平，检测量为 10 μL 尿液。采用 Kinetex Polar C18 分析柱进行梯度洗脱（5 mM 乙酸铵和乙腈，含 0.1%甲酸）实现色谱分离。采用三重四极杆串联质谱仪在正离子模式下进行质谱检测。INH、PZA、ETH 和 RIF 的定量下限 (LLOQ) 为 0.5 μg/mL，RIF 代谢物和降解产物的 LLOQ 为 0.1 μg/mL。该方法遵循 FDA 指导原则进行了验证，并成功应用于 TB 患者尿液中研究化合物的分析。此外，我们还进行了抗结核药物在不同 pH 值和温度条件下的稳定性研究，以模拟不同环境（周边诊所或中心实验室）中尿液采集过程。
