Microdialysis Coupled with Electrochemical Detection - Drugs of Abuse: Neurological Reviews and Protocols - page 415

3.3.4. Detecting Dopamine Without Metabolites
in the Nucleus Accumbens by Vertical Probes
Ringer's solution (147 m M NaCl, 2.2 m M CaCl 2 , and 4 m M KCl) was
pumped at a constant fl ow of 1 L/min. Samples were taken every 20 min

and injected without any purifi cation into an HPLC apparatus equipped with
reversed-phase column (LC-18 DB Supelco) and an ANTEC Flowcell electrode
coupled with a BAS detector. The oxidation potential was + 0.55 V. The mobile
phase composition was 50 m M NaH 2 PO 4 -5 m M Na 2 HPO 4 , 0.1 m M Na 2 EDTA,
0.5 m M octyl sodium sulfate, and 15% (v/v) methanol, pH 5.5. The mobile
phase was pumped with an LKB 2150 pump at a fl ow rate of 1.0 mL/min. The
sensitivity of the assay allowed detection of 5 fmol of DA (18) .

µ

4. Notes

1. A great advantage in investigating the cause of noisy baseline is obtainable
by having two similar HPLC systems. It will permit substitution of one piece
of equipment at a time in the defective system, using parts (e.g., entire pump,
single valve, pulse damper, column, reference electrode, etc.) of the system that
function effectively; it can allow detection of the failure in the malfunctioning
system. Furthermore, two similar systems could make it possible to have just
one set of spare parts in the lab. It has to be considered that even though the
microdialysis procedure is very economical, it is very time consuming, so every
effort has to be made to avoid to wasting even one sample. One always needs
to consider that, when a dialysate of three animals is injected into one HPLC,
its failure means the experiment is wasted because if a total of six animals are
used, the remaining three do not constitute a large enough n to obtain statistically
signifi cant data. In an experiment in which three animals are tested against three
controls, the result is even worse and the experiment has to be repeated, to say
nothing of testing animals chronically treated for several weeks. No one would
like to waste an experiment like that because of equipment failure. The very
best solution is to have a full spare system to use immediately when needed,
while the less expensive solution is to have at least a spare working electrode
and a spare HPLC pump.

2. Detector failure: When an amperometric detector is used, it is necessary to verify
the reference electrode and the working electrode. Reference electrodes are
usually very long lasting but substitution with a spare one (it can be borrowed
from a well functioning second system) will ascertain if the reference electrode
is defective. If the problem persists, the working electrode has to be checked
and eventually changed, using the same procedure. In the case of failure of a
coulometric electrode (ESA cell) again, the substituting procedure can be used
to ascertain malfunctioning. Because malfunctioning cells, in most cases, cannot
be repaired, a lower quality cell (noisy baseline) can be used for samples that
require low sensitivity. Otherwise the cell has to be substituted.