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. [(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.
Schebb
Nils Helge
NH
Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Corrensstrasse 30, Münster, Germany.
Fischer
Daniel
D
Hein
Eva-Maria
EM
Hayen
Heiko
H
Krieglstein
Josef
J
Klumpp
Susanne
S
Karst
Uwe
U
D000109:Acetylcholine / Q000032:analysis*; D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D005583:Fourier Analysis; D051379:Mice; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000109:Acetylcholine