■Remdesivir(GS-5734)是一种核苷类似物前药,对几种单链RNA病毒具有抗病毒活性,包括新型严重呼吸窘迫综合征病毒2(SARS-CoV-2)。它是目前唯一获得FDA批准的抗病毒药物,用于治疗由SARS-CoV-2引起的COVID-19患者。然而,remdesivir药代动力学/药效学(PK/PD)和人体毒性数据极其有限.它是必要的精确的分析方法,用于定量的remdesivir及其活性代谢物,GS-441524是为进一步研究而开发的。我们报告,在这里,首次验证的抗病毒纸喷雾质谱(PS-MS/MS)测定用于定量人血浆中的remdesivir和GS-441524。我们寻求强调PS-MS/MS技术和自动化进步的实用性,以便其在临床研究和临床实验室环境中的潜在未来用途。
■使用七种不同浓度的基于血浆的校准物和两种设定浓度的同位素内标创建remdesivir和GS-441524的校准曲线。以与校准物类似的方式制备四个基于血浆的质量对照并用于验证。不需要样品制备。简而言之,将血浆样品点样在预先制造的塑料盒板内的纸质基材上,然后使用PS-MS/MS直接分析样品1.2分钟。所有实验均利用自动化技术在ThermoScientificAltis三重四极质谱仪上进行。
■remdesivir和GS-441524的校准范围分别为20-5000和100-25000ng/mL。两种抗病毒剂的校准曲线显示出优异的线性(平均R2=0.99-1.00)。对于两种分析物,在四个QC水平下的验证运行的日间和日间精度(%CV)小于11.2%,并且精度(%偏差)在±15%内。评估血浆校准物稳定性,并在第7天开始观察4°C和室温样品的降解。血浆校准物在-20℃下是稳定的。无干扰,矩阵效应,或者在验证过程中发现了结转。
■PS-MS/MS代表了一种用于快速定量remdesivir和GS-441524的有用方法,可能对临床PK/PD有用,治疗药物监测(TDM),和毒性评估,特别是在当前的COVID-19大流行和未来的病毒爆发期间。
UNASSIGNED: Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2). It is currently the only FDA-approved antiviral agent for the treatment of individuals with COVID-19 caused by SARS-CoV-2. However, remdesivir pharmacokinetics/pharmacodynamics (PK/PD) and toxicity data in humans are extremely limited. It is imperative that precise analytical methods for the quantification of remdesivir and its active metabolite, GS-441524, are developed for use in further studies. We report, herein, the first validated anti-viral paper spray-mass spectrometry (PS-MS/MS) assay for the quantification of remdesivir and GS-441524 in human plasma. We seek to highlight the utility of PS-MS/MS technology and automation advancements for its potential future use in clinical research and the clinical laboratory setting.
UNASSIGNED: Calibration curves for remdesivir and GS-441524 were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Briefly, plasma samples were spotted on a paper substrate contained within pre-manufactured plastic cassette plates, and the spots were dried for 1 h. The samples were then analyzed directly for 1.2 min utilizing PS-MS/MS. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer utilizing automated technology.
UNASSIGNED: The calibration ranges were 20 - 5000 and 100 - 25000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process.
UNASSIGNED: PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir and GS-441524, which may be useful for clinical PK/PD, therapeutic drug monitoring (TDM), and toxicity assessment, particularly during the current COVID-19 pandemic and future viral outbreaks.