Fast sample preparation and liquid chromatography-tandem mass spectrometry method for assaying cell lysate acetylcholine

J Chromatogr A. 2008 Mar 7;1183(1-2):100-7. doi: 10.1016/j.chroma.2008.01.033. Epub 2008 Jan 24.


A fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of acetylcholine (ACh) in cultured cells. [(2)H(4)]Acetylcholine (ACh-d(4)) was used as an internal standard for calibration. ACh was extracted from the cell lysate with acetonitrile (ACN)/water (80/20, v/v) and the crude extract was analyzed without further purification. Isocratic hydrophilic interaction chromatography (HILIC) with (10 mM) ammonium formate/ACN (35/75, v/v) as mobile phase was used for separation. ACh was eluted within 5 min and detected using electrospray-MS/MS in the positive ion mode. The limit of detection (LOD) was found to be 1.5f mol (0.3 nmol/L) ACh with a S/N ratio of 3:1. The approach was used for the measurement of ACh in undifferentiated SN56 cells and the ACh content was determined to be 1272+/-109 pmol/mg protein.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Acetylcholine / analysis*
  • Animals
  • Cell Line
  • Chromatography, Liquid / methods*
  • Fourier Analysis
  • Mice
  • Reproducibility of Results
  • Tandem Mass Spectrometry / methods*


  • Acetylcholine