Quantitation of free and total N-acetylcysteine amide and its metabolite N-acetylcysteine in human plasma using derivatization and electrospray LC-MS/MS

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1109; pp. 25 - 36
• Main Authors: King, Brad, Vance, Jennifer, Wall, G. Michael, Shoup, Ronald
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2019
• Subjects: CMPI; HILIC; Metabolite; N-Acetylcysteine; N-Acetylcysteine amide; Plasma; TCEP; Plasma; N-Acetylcysteine; Metabolite; N-Acetylcysteine amide; CMPI; TCEP; HILIC
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2019.01.013

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. •Two-step procedure from single 25 μL plasma sample measures reduced and total NACA and NAC from the same derivative.•Blood collection procedure stabilized thiols earliest and preserved redox balance between thiol and disulfide.•HILIC-based separation after extraction provided superior resolution between NAC and NACA derivatives.•Incurred sample reanalysis showed agreement >95% for NAC and > 99% for NACA in a Phase I clinical trial.