El-Gendy Manal, Hefnawy Mohamed, Alrubia Sarah, Alnasser Abdulaziz, Alsegiani Amsha, Jardan Yousef Bin, El-Azab Adel, Abdel-Aziz Alaa, Attwa Mohamed, Alsarhani Emad
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia.
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia.
Anal Biochem. 2025 Mar;698:115741. doi: 10.1016/j.ab.2024.115741. Epub 2024 Dec 3.
The combination of venetoclax plus azacitidine (VTX-AZA) is FDA-approved to treat patients with acute myeloid leukemia (AML) aged ≥75 years and has become the standard of care for AML patients. However, the literature has not reported an analytical method for determining VTX-AZA in plasma samples. Therefore, developing an accurate and sensitive bioanalytical assay to quantify VTX-AZA in plasma is important. For the first time, this study describes the development of a new liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the simultaneous determination of VTX and AZC in plasma samples with its application to pharmacokinetic study in rats. The assay employs repaglinide (RPG) as an internal standard. The chromatographic separations of VTX, AZC, and RPG are achieved within 2.5 min at 25 °C on an Eclipse plus C18 column (100 mm × 2.1 mm, 1.8 μm) and an isocratic mobile phase consisted of water with 0.1 % formic acid and acetonitrile (50:50, v/v, pH 3.2) at a flow rate of 0.30 mL/min. VTX and AZC have been extracted from rat plasma using the solid-phase extraction (SPE) procedure without interference from plasma endogenous. The FDA guidelines were followed in the validation of the developed assay, and linearity in rat plasma was observed for AZC and VTX, respectively, ranging from 5 to 3000 and 5-1000 ng/mL, with r ≥ 0.998. The lower limits of detection (LLOD) were 2 ng/mL for both drugs. In addition, the inter-day and intra-day accuracy were 0.8-6.6 % and 2.2-5.7 %; the inter-day and intra-day precision were 3-6.6 % and 1.5-7.1 %, respectively. The validated assay was effectively used in a pharmacokinetic investigation including the simultaneous oral administration of 40 mg/kg of AZA and 100 mg/kg of VTX to rats. The maximum plasma concentration (C) for AZC and VTX was 794 ± 99.6 ng/mL and 641 ± 96.9 ng/mL achieved at 0.5 ± 0.03 h and 6 ± 0.05 h, respectively. The AUC for AZC and VTX was 1253 ± 252.6 and 4881 ± 745.4 ng/mL.h; respectively.
维奈托克联合阿扎胞苷（VTX - AZA）已获美国食品药品监督管理局（FDA）批准用于治疗年龄≥75岁的急性髓系白血病（AML）患者，并且已成为AML患者的标准治疗方案。然而，文献中尚未报道测定血浆样本中VTX - AZA的分析方法。因此，开发一种准确且灵敏的生物分析方法来定量血浆中的VTX - AZA非常重要。本研究首次描述了一种新的液相色谱 - 串联质谱法（LC - MS/MS）的开发，用于同时测定血浆样本中的VTX和AZC，并将其应用于大鼠的药代动力学研究。该分析方法采用瑞格列奈（RPG）作为内标。在25℃下，于Eclipse plus C18柱（100 mm×2.1 mm，1.8μm）上，VTX、AZC和RPG在2.5分钟内实现色谱分离，等度流动相由含0.1%甲酸的水和乙腈（50:50，v/v，pH 3.2）组成，流速为0.30 mL/min。已采用固相萃取（SPE）程序从大鼠血浆中提取VTX和AZC，且不受血浆内源性物质的干扰。在开发该分析方法的验证过程中遵循了FDA指南，在大鼠血浆中分别观察到AZC和VTX的线性范围为5至3000 ng/mL和5至1000 ng/mL，r≥0.998。两种药物的检测下限（LLOD）均为2 ng/mL。此外，日间和日内准确度分别为0.8 - 6.6%和2.2 - 5.7%；日间和日内精密度分别为3 - 6.6%和1.5 - 7.1%。经过验证的分析方法有效地用于药代动力学研究，包括对大鼠同时口服40 mg/kg的AZA和100 mg/kg的VTX。AZC和VTX的最大血浆浓度（C）分别在0.5±0.03小时和6±0.05小时达到794±99.6 ng/mL和641±96.9 ng/mL。AZC和VTX的曲线下面积（AUC）分别为1253±252.6和4881±745.4 ng/mL·h。
