Microdialysis Coupled with Electrochemical Detection - Drugs of Abuse: Neurological Reviews and Protocols - page 408

they are present in a low concentration requires a stable baseline, a perfectly
functioning HPLC coupled with ED is necessary to improve the signal-to-noise
ratio. It can be obtained with a good pump (a two-piston pump offers better fl ow
stability than a single-piston one). Here the choice in the market is very wide.
Usually a low-cost pump is as good as a more expensive one only when new, but
its effi ciency declines more quickly. A colleague who has purchased the pump
that one wants to buy may give useful advice.

A pulse damper is recommended to improve further fl ow stability (they are not
expensive and have a long life). For measuring DA in the caudate (200-400 fmol/
20 L of sample) even an average system can be used, but if areas with a low

concentration of DA such as the frontal cortex or the bed nucleus of stria
terminalis (BNST) are investigated (10-30 fmol/20

µ

µ L of sample) a highly

effi cient system is recommended (4) .

Injectors permit introduction of the sample to be analyzed in the system,
without interrupting fl ow. They are long lasting but care must be taken to avoid
salts or organic material deposits. Water fl ushing or methanol respectively has to
be used at the end of each working day to eliminate deposits.

Chromatographic columns and guard columns are the core of chromatograph
separation and their choice depends on the substance to be detected. Their cost
is not prohibitive, so several columns may be tested to obtain the best separation
condition. Their effi ciency, of course, depends also on the mobile phase used. It
must be considered that injecting the sample without any previous purifi cation
will shorten the time of analysis and will allow detection of neurotransmitter
levels almost in real time. We used reversed-phase or reversed-phase ion-pair
chromatography with ODS columns to detect DA, NA, and 5-HT. Because the
sample is injected without any previous purifi cation, a guard column, inserted
before the column, is recommended to increase column life. Choice depends
on column type, and in general their cost is low. The columns should not be
stored with buffer solutions or salts. Salts may precipitate and halogen salts
may corrode metal. Wash columns with water and methanol-water (50 50) and

close them before storage.

3. Mobile phase: The mobile phase delivers samples containing the substance to be
detected to the chromatographic column and afterward to the detector. It can be
prepared by diluting in double-fi ltered (or bidistilled) water stock solutions of
each reagent (HPLC grade) kept in the refrigerator. Once the mobile phase has
been brought to the defi nitive pH and volume, it must be fi ltered and degassed.
Ultrasound can be used to shorten the degassing procedure. The composition of
the mobile phase depends on the substance to be separated and on the column
used. In general, in reversed-phase chromatography substances dissolved in
a hydrophilic mobile phase are separated on the base of their affi nity for the
hydrophobic stationary phase. In this process, an increased concentration of
methanol will shorten the retention time by reducing the hydrophilicity of the
mobile phase. Reduction of pH will produce an increased polarity of weak bases,
reducing their retention time (DA, NA, 5-HT retention time is not affected by