AIT Bioscience, LLC, 7840 Innovation Blvd, Indianapolis, IN 46278, USA.
Nacuity Pharmaceuticals, Inc., 306 W 7th Street, Suite 310, Ft Worth, TX 76102, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Mar 1;1109:25-36. doi: 10.1016/j.jchromb.2019.01.013. Epub 2019 Jan 23.
Studies of N-acetylcysteine amide (NACA) in nonclinical models have demonstrated various antioxidant, anti-apoptotic, anti-inflammatory and neuroprotective effects, and it is currently being developed as a treatment for retinitis pigmentosa. Sensitive LC-MS/MS methods were developed and validated to quantitate reduced and total NACA and its major metabolite, N-acetylcysteine (NAC), in human plasma to support clinical studies involving NACA. To trap and stabilize reduced NACA and NAC at the time of collection, whole blood was immediately treated with 2-chloro-1-methylpyridinium iodide (CMPI) to convert free thiols to 1-methylpyridinyl thioether derivatives. Plasma was harvested and frozen until samples were assayed using protein precipitation and an LC-MS/MS separation based on hydrophilic-interaction chromatography (HILIC). To process NACA and NAC present as disulfides, an intermediate portion of the extract was further subjected to reduction with tris(2-carboxyethyl) phosphine; the released thiols were then reacted with CMPI, extracted, and analyzed as before, to measure total thiols. The method for NACA and NAC, whether free/reduced or total, covered a range from 50 ng/mL to 50 μg/mL in human plasma and required a single 25 μL plasma sample. Up to 180 samples could be assayed in a single session. The inter-run mean bias and precision (%CV) were within ±5% for the free thiol method and within ±8.5% for the total thiol method. Benchtop, freeze/thaw, and long-term stability were evaluated and acceptable. The NAC/NACA method applied to a clinical study demonstrated incurred sample reproducibility of 95.5% for NAC and 99.1% for NACA.
在非临床模型中，N-乙酰半胱氨酸酰胺（NACA）的研究表明其具有各种抗氧化、抗凋亡、抗炎和神经保护作用，目前正在开发用于治疗色素性视网膜炎。开发并验证了灵敏的 LC-MS/MS 方法，以定量测定人血浆中还原型和总 NACA 及其主要代谢物 N-乙酰半胱氨酸（NAC），以支持涉及 NACA 的临床研究。为了在采集时捕获和稳定还原型 NACA 和 NAC，全血立即用 2-氯-1-甲基吡啶碘化物（CMPI）处理，将游离巯基转化为 1-甲基吡啶基硫醚衍生物。收集血浆并冷冻，直到使用蛋白沉淀和基于亲水相互作用色谱（HILIC）的 LC-MS/MS 分离法测定样品。为了处理以二硫键形式存在的 NACA 和 NAC，提取液的中间部分进一步用三（2-羧乙基）膦还原；然后将释放的巯基与 CMPI 反应，提取并进行分析，以测量总巯基。NACA 和 NAC 的方法（无论是游离/还原型还是总型），在人血浆中的范围为 50ng/mL 至 50μg/mL，需要单个 25μL 血浆样品。在单个测试中可以测定多达 180 个样品。游离巯基方法的批内平均偏差和精度（%CV）在±5%范围内，总巯基方法在±8.5%范围内。评估了台式机、冻融和长期稳定性，结果可接受。该 NAC/NACA 方法应用于临床研究，表明 NAC 的重现性为 95.5%，NACA 的重现性为 99.1%。
