用于分析尿液中异烟肼及其代谢物乙酰异烟肼的有效液相色谱-串联质谱分析法

IF 3.1 4区医学 Q2 MEDICAL LABORATORY TECHNOLOGY Journal of Mass Spectrometry and Advances in the Clinical Lab Pub Date : 2024-02-06 DOI:10.1016/j.jmsacl.2024.02.001

Sydwell Poulo Maputla, Willem Van Dalen, Anton Joubert, Jennifer Norman, Sandra Castel, Marthinus van der Merwe, Lubbe Wiesner

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引用次数: 0

摘要

导言INH（INsoniazid）是最有效、最强效的一线抗结核药物之一。INH 也可作为一种有效的预防性单一疗法，并已被证明可显著降低结核病的发病率。INH 主要在肝脏中代谢为乙酰异烟肼（AcINH）。该研究旨在开发并全面验证一种生物分析方法，该方法采用液相色谱-串联质谱法定量检测人体尿液中的 INH 和 AcINH。提取物经 Atlantis T3 分析柱色谱分析，流动相为等度流动相。检测时，使用 AB Sciex™ API 5500 三重四极杆质谱仪，在正电喷雾电离后，在多反应监测模式下进行单位分辨率检测。结果该分析方法具有足够的灵敏度，INH 和 AcINH 定量下限的平均信噪比分别为 7.07 和 6.23。在 INH 和 AcINH 的分析范围 0.234-30.0 µg/mL 内的峰面积比显示，该方法的准确度、精密度和整体稳健性均达到了要求。所有必要的验证实验均经过评估，符合美国食品药品管理局和欧洲药品管理局的验收标准指南。在一项 III 期开放标签多中心试验中，该验证方法被用来测定尿液中 AcINH 的浓度，以评估服用异烟肼预防结核病感染的依从性。

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A validated liquid chromatography-tandem mass spectrometry assay for the analysis of isoniazid and its metabolite acetyl-isoniazid in urine

Introduction

Isoniazid (INH) is one of the most effective and potent first-line anti-tubercular drug. INH is also effectively administered as a preventative monotherapy and has been shown to significantly reduce TB incidence. INH is primarily metabolised to acetyl-isoniazid (AcINH) in the liver. AcINH is mainly excreted in urine presenting as a target for monitoring adherence to INH therapy.

Objective

The study aimed to develop and fully validate a bioanalytical method using liquid chromatography-tandem mass spectrometry for the quantification of INH and AcINH in human urine.

Methods

The samples were prepared using solid phase extraction, with the internal standards isoniazid-d4 and acetyl-isoniazid-d4 being used. The extracts were chromatographed on an Atlantis T3 analytical column with an isocratic mobile phase. For detection, a AB Sciex™ API 5500 triple quadrupole mass spectrometer was used at unit resolution in the multiple reaction monitoring mode, following positive electrospray ionization.

Results

The analytical method demonstrated sufficient sensitivity, as indicated by average signal-to-noise ratios of 7.07 and 6.23 at the lower limit of quantification for INH and AcINH, respectively. Validation was performed over three consecutive batches, demonstrating accuracy, precision, and overall robustness based on peak area ratios within the analytical range of 0.234–30.0 µg/mL for both INH and AcINH. All required validation experiments were assessed and met the acceptance criteria guidelines of the US Food and Drug Administration and European Medicines Agency. The validated method was utilized to measure concentrations of AcINH in urine as a means of assessing adherence to the intake of isoniazid in order to prevent TB infection during a phase III open-label multicenter trial.