Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
39, Number 10, October 2001, pp. 151-156
Sample Preparation and Determination of Acetylcholine in Corneal
Epithelium Cells Using Liquid Chromatography–Tandem Mass Spectrometry
J.L.E. Reubsaet[1], E. Ahlsen[1], K.G. Haneborg[1], and A.
Ringvold[2]
[1]Department of Pharmaceutical Analysis, School of Pharmacy, University of
Oslo, Norway and
[2]Department of Ophthalmology, Rikshospitalet, University
of Oslo,
Norway
A sample preparation method
with subsequent liquid
chromatography (LC)–mass spectrometry (MS)–MS analysis for acetylcholine
in corneal epithelium is developed. The sample preparation is developed with
a focus on compatibility with the LC–MS–MS system and the stability
of acetylcholine because acetylcholine esterase is present in the tissue. It
appears that both acetylcholine as well as the internal standard (IS) used (acetyl-b-methylcholine)
have fragments at m/z values in the tandem MS spectrum, which correspond with
the m/z values of fragments of endogenous substances. Acetylcholine and (3-carboxypropyl)triethylammonium
both have 146’87 and 146’60 transitions. Acetyl-b-methylcholine and
an unknown compound both have 160’101 and 160’60 transitions. This
makes it necessary to use a chromatographic step, which has a baseline separation
between these endogenous compounds, acetylcholine, and the IS. The analytical
procedure has linearity from 1 ng/mL (30 pg/mg corneal epithelium tissue) to
at least 250 ng/mL (7.55 ng/mg corneal epithelium tissue). The limits of detection
and quantitation are 15 and 45 pg oncolumn, respectively. Relative standard deviation
and bias values are within the range of acceptance for all concentration levels.
Reproduction
of editorial content of this journal is prohibited without publisher’s
permission.
This
article is available in its entirety by fax for $4.00 per
page.
Visa or MasterCard accepted.
To
order electronicallyclick here or call: 847-647-2900 ext. 1323 or fax request to: 847-647-1155.
Please
indicate JCS volume and issue along with
page numbers.
