建立并验证了一种液质联用色谱法，用于定量检测人血浆中的直接作用抗病毒药物 glecaprevir 和 pibrentasvir。
Development and validation of a liquid chromatographic-tandem mass spectrometric assay for the quantification of the direct acting antivirals glecaprevir and pibrentasvir in plasma.
机构信息
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
出版信息
J Pharm Biomed Anal. 2023 Oct 25;235:115629. doi: 10.1016/j.jpba.2023.115629. Epub 2023 Aug 19.
BACKGROUND
Direct acting antiviral (DAA) therapies are effective in the treatment and management of chronic HCV infections. Glecaprevir (GLE) and pibrentasvir (PIB) are pangenotypic DAAs that are delivered alone or as a fixed-dose oral formulation to treat chronic HCV infections with or without cirrhosis. Sensitive and dynamic bioanalytical assays are needed to understand the pharmacology of GLE and PIB.
METHODS
Drug free KEDTA plasma was spiked with GLE, PIB, and their internal standards. Drugs were extracted from plasma via protein precipitation, and subsequently quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated according to regulatory recommendations, and evaluated in remnant plasma samples from individuals prescribed GLE and PIB.
RESULTS
The analytical measuring ranges for GLE and PIB were 0.25-2000 ng/mL and 0.25-1000 ng/mL, respectively. The method showed acceptable accuracy and precision for both DAAs. GLE and PIB in plasma were stable following six freeze thaw cycles and at room temperature for up to 67 h. All analytes were stable in whole blood incubated at room temperature for 24 h, and at 40 °C and 100% humidity for 2 h. Negligible percent matrix effects were observed for PIB and PIB-IS across the measuring range of the assay. Significant ion suppression was observed for GLE, with an average matrix effects of 27.9%. However, relative matrix effects were < 6.3% between drug and internal standard, and deemed acceptable. Assay validation assessments in alternative matrices also met acceptance criteria. Both DAAs were successfully measured in remnant plasma samples from individuals administered GLE and PIB.
CONCLUSIONS
An LC-MS/MS method for GLE and PIB quantification in plasma has been developed and validated. The assay met acceptable performance criteria and may be used in downstream applications to characterize DAA pharmacology for HCV treatment.
背景
直接作用抗病毒（DAA）疗法在治疗和管理慢性 HCV 感染方面非常有效。格卡瑞韦（GLE）和哌仑他韦（PIB）是泛基因型 DAA，可单独使用或作为固定剂量口服制剂，用于治疗伴有或不伴有肝硬化的慢性 HCV 感染。需要灵敏和动态的生物分析检测法来了解 GLE 和 PIB 的药理学。
方法
无药物的 KEDTA 血浆中加入 GLE、PIB 及其内标物。药物通过蛋白沉淀从血浆中提取，然后使用液相色谱-串联质谱法（LC-MS/MS）定量。该方法根据监管建议进行了验证，并在接受 GLE 和 PIB 治疗的个体的剩余血浆样本中进行了评估。
结果
GLE 和 PIB 的分析测量范围分别为 0.25-2000ng/mL 和 0.25-1000ng/mL。该方法对两种 DAA 的准确性和精密度均表现出可接受的结果。GLE 和 PIB 在经过 6 次冻融循环以及在室温下放置长达 67 小时后，在血浆中保持稳定。在室温下孵育 24 小时以及在 40°C 和 100%湿度下孵育 2 小时的全血中，所有分析物均保持稳定。在整个分析测量范围内，PIB 和 PIB-IS 对基质的影响可忽略不计。GLE 观察到明显的离子抑制作用，平均基质效应为 27.9%。然而，药物与内标物之间的相对基质效应<6.3%，被认为是可接受的。替代基质中的检测验证评估也符合接受标准。GLE 和 PIB 在接受 GLE 和 PIB 治疗的个体的剩余血浆样本中均成功检测到。
结论
已开发并验证了用于 GLE 和 PIB 定量的 LC-MS/MS 方法。该方法符合可接受的性能标准，可用于下游应用，以描述 HCV 治疗中 DAA 的药理学。
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