Neary Megan, Arshad Usman, Tatham Lee, Pertinez Henry, Box Helen, Rajoli Rajith Kr, Valentijn Anthony, Sharp Joanne, Rannard Steve P, Biagini Giancarlo A, Curley Paul, Owen Andrew
Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, L7 3NY, UK.
Centre of Excellence in Long-acting Therapeutics (CELT), University of Liverpool, Liverpool, L7 3NY, UK.
bioRxiv. 2021 May 28:2021.05.27.445500. doi: 10.1101/2021.05.27.445500.
Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Unlike many other candidates being investigated, tizoxanide (the active metabolite of nitazoxanide) plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbeccos Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Therefore, a robustly validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6ng/mL to 1000ng/mL. Accuracy and precision ranged between 102.2% and 113.5%, 100.1% and 105.4%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.
目前，硝唑尼特正在作为治疗新型冠状病毒（SARS-CoV-2）的候选药物进行评估。与许多其他正在研究的候选药物不同，硝唑尼特的活性代谢产物替唑尼特在给予批准剂量后，血浆浓度可达到抗病毒水平，尽管在大多数患者中，预计需要更高的剂量才能在给药间隔内维持这些浓度。本文描述了一种已根据美国食品药品监督管理局（FDA）指南进行验证的液相色谱-串联质谱（LC-MS/MS）测定法。已评估了基本参数，包括准确度、精密度和灵敏度。该测定法已在含有不同浓度胎牛血清（FBS）的人血浆、小鼠血浆和杜氏改良伊格尔培养基（DMEM）中得到验证。基质效应是色谱分析中一个有充分记录的问题来源，有可能影响分析过程的各个阶段，包括抑制或增强电离。因此，本文提出了一种经过充分验证的LC-MS/MS分析方法，该方法能够在多种基质中定量替唑尼特，且基质效应的影响最小。本文介绍的经过验证的测定法在15.6ng/mL至1000ng/mL范围内呈线性。准确度和精密度分别在102.2%至113.5%、100.1%至105.4%之间。本文介绍的测定法在临床前和临床研究中均有应用，可用于促进对硝唑尼特治疗新型冠状病毒应用的进一步研究。
