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

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.

Keywords: CMPI; HILIC; Metabolite; N-Acetylcysteine; N-Acetylcysteine amide; Plasma; TCEP.

MeSH terms

  • Acetylcysteine / analogs & derivatives*
  • Acetylcysteine / blood*
  • Acetylcysteine / chemistry
  • Chromatography, High Pressure Liquid / methods*
  • Drug Stability
  • Humans
  • Limit of Detection
  • Linear Models
  • Reproducibility of Results
  • Tandem Mass Spectrometry / methods*


  • N-Acetylcysteinamide
  • Acetylcysteine