HPLC/EC Detection and Quantification of Acetylcholine in Dialysates - Drugs of Abuse: Neurological Reviews and Protocols - page 426

not being used enzyme life can be prolonged by removing the reactor cartridge
and storing under mobile phase at 4°C ( see Notes 4-6 ). Cartridges stored in this
manner maintain their effi cacy for at least 4 wk.

3.5. Electrochemical Cell
Our laboratory employs a VT-03 wall jet fl ow cell (Antec) for the electro-
chemical oxidation of H 2 O 2 . The fl ow cell is fi tted with a 25- m gasket and

a platinum electrode held at +0.5 V vs an Ag/AgCl reference electrode by an
INTRO potentiostat (Antec) ( see Note 7 ). The reference electrode must be
fi lled with saturated KCl. NaCl is not compatible with the phosphate mobile
phase.
The surface of the electrode is cleaned weekly by rinsing in deionized water
and gentle wiping with a cotton swab soaked in 100% MeOH. If this treatment
fails to restore analytical sensitivity, the surface is then gently polished with
a silk cloth and 0.25-

µ

m diamond slurry. The surface of the electrode is
rinsed and cleaned with MeOH after polishing to remove residue. Polishing is
typically required no more than once every 2 mo.

µ

3.6. Data Analysis
Oxidative current output is integrated and analyzed using Macintosh based
chromatography hardware and software (Dynamax). A detection limit (3 1
signal to noise ratio) of 5 fmol/sample is routinely obtained. The system is
calibrated by injection of 20 and 200 n M standards ( see Note 8 ).

4. Notes

1. Addition of TMA and SOS to the mobile phase also contributes to a gradual loss
of enzymatic activity. Concentrations greater than 0.3 m M SOS rapidly denature
the enzymes. TMA is an esterase inhibitor, although concentrations below 6 m M
do not appear to noticeably compromise enzyme effi ciency. Adjustment of
retention time is preferably achieved by using oven-controlled temperature (for
both choline and ACh), the addition of small amounts of TMA (ACh only) or a
small alteration of buffer strength.

2. The loss of optimal enzymatic activity is the source of most ACh HPLC problems.
Bacterial growth in the mobile phase is a common problem because of the pH
of the solution. Some bacterial species can rapidly denature the enzymes. Other
species produce the enzyme catalase that degrades hydrogen peroxide, thus
diminishing the end product of the enzymatic conversion. The presence of such
bacteria can lead to the erroneous conclusion that the enzyme reactor itself is
compromised. These problems can be prevented primarily by using ultrapure
water, clean glassware, never recycling mobile phase, changing the mobile phase
at least weekly, and if necessary adding a low concentration of an antibacterial