HPLC/EC Detection and Quantification of Acetylcholine in Dialysates - Drugs of Abuse: Neurological Reviews and Protocols - page 427

agent to the mobile phase. Use of an excessive concentration of antibacterial
agents, however, will also denature the enzymes.
3. One problem associated with HPLC analysis of ACh is specifi c to the analysis
of ACh in neuronal dialysate: choline is present at concentrations 1000 times
greater than ACh. The enzymatic conversion effi ciency for choline to hydrogen
peroxide is also twice that of ACh; a result of a one-step rather than two-step
conversion. Thus the choline peak in a chromatogram can be several orders of
magnitude greater than that of ACh. A 2-min peak-to-peak interval in choline
and ACh retention times may thus still result in an ACh peak that coelutes
with the tail of the choline peak. In this situation the peak height of ACh will
be substantially underestimated. Alas, wide separation of ACh and choline is
diffi cult owing to the constraints of the mobile phase composition. For this reason
regular replacement of the analytical column is essential.
4. One further problem that is associated with the use of an enzyme reactor is the
contribution of the reactor, especially when new, to the buildup of a residue on
the surface of the working electrode. The detrimental effects of the residue can
be diminished by turning off the applied potential whenever the HPLC is not in
use. The cell may even be turned off each night if it is possible to allow enough
time for the system to reequilibrate the following day.
5. There are several alternatives, both substantial and minor, to the procedures
outlined in the preceding, although apparent optimization of one parameter often
leads to a degradation of performance elsewhere in the system. This problem of
constraint satisfaction is especially acute in the case of ACh HPLC. Nevertheless,
our laboratory has had experience with some technical alternatives. These and
other promising developments are outlined in this and in subsequent subheadings.
Complete kits that include separation columns and enzyme reactors are com-
mercially available, for example, Chrompack (Varian) or BAS. Our laboratory
has only limited experience with these kits. However, preliminary investigation
demonstrated that laboratory prepared hardware was more sensitive and signifi -
cantly more durable. One advantage of one such commercially available kit
(Chrompack), however, is the zero dead volume connection between guard
column, column, and enzyme reactor which results in improved peak shape.
6. The lifetime of any enzyme reactor is proportional to the total converted amount
of choline and ACh, rather than a function of time. Choline is often not a
dependent measure of interest in neuroscientifi c experiments owing to its largely
non-neuronal origin (16) . Thus the lifespan of the reactor can be prolonged by
precolumn conversion of choline in a separate enzyme reactor. Moreover, the
elimination of choline eliminates problems associated with the chromatographic
separation of choline from ACh. Such a reactor can be prepared exactly as
described in the preceding, but using the enzymes choline oxidase and either
peroxidase or catalase to convert choline to peroxide and peroxide to water (17) .
7. The choice of electrochemical surface is limited for ACh HPLC. H 2 O 2 will slowly
damage the surface of glassy carbon electrodes and a high applied potential is