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

A fast liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the analysis of acetylcholine (ACh) in cultured cells. [2H4]Acetylcholine (ACh-d4) 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 1.5 fmol (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.