Analysis of Chiral Drugs in Body Fluids - Chiral Separations by Capillary Electrophoresis - page 356

A detailed step-by-step guide to analytical method validation is not intended here
since several reviews have covered this subject [47-51]. A discussion of characteris-
tics inherent to CE techniques is mainly focused instead, and some suggestions are
given for better results of precision and accuracy in CE routine analysis in biological
l uids.
When CE was introduced, CE equipments had poor precision and sensitivity
[52-54]. Improvements in the CE equipment and in the optical detection systems
introduced along the last decade made it possible to obtain acceptable quantitative
results. However, the dei ciency in CE precision is not only due to limitations in
equipment but also to operational variables [53]. Generally, precision depends on
four main factors: buffer composition, injection mode, capillary properties, and tem-
perature. These factors interact with each other and affect migration times and size of
the peaks. Stringent attention should be given to all these sources of variation so that
a set of measures is adopted to be consistently used in the validation procedure.
Buffers must be rigorously prepared daily with high-purity water and HPLC-
grade reagents and solvents, and the choice of the BGE should favor low con-
ductivity [53]. The pH adjustment and addition of chiral selectors in the running
buffer must be done with analytical care as small changes of buffer composition
or pH may result in large changes of migration times. The running buffer contain-
ing the chiral selector must be always freshly prepared, i ltered, and degassed. Air
bubbles in the running buffer cause short-term EOF and current changes, leading
to imprecise results of peak areas. In the case of organic solvent addition to the
running buffer, the possibility of losses by evaporation should be kept in mind
[52]. On the other hand, buffer additives (e.g., hexadodecyltrimethylammonium
bromide) may contribute to precision not only by reducing the adsorption of par-
ticulate material and proteins on the capillary surface, but also by diminishing
analyte-wall interactions. Furthermore, they stabilize the EOF and lead to more
repeatable migration times [52].
Buffer solutions change during CE analysis due to electrolysis, resulting in a pH
gradient across the capillary that affects separation efi ciency and migration times.
To minimize this effect, previous attention is necessary with regard to buffering
capacity, buffer concentration, ionic strength, pH, volume of the reservoirs, tempera-
ture, generated current, and total run time. The maximum number of analyses that
can be performed with a unique vial of running buffer without signii cant depletion
effects should be determined beforehand [52].
It is generally recognized that capillary preconditioning is important for high
separation efi ciency as well as high run-to-run repeatability [52]. In the analysis of
biological samples, the cleanup step by itself is not enough to eliminate proteins and
fats that could cause chemical instability of the capillary wall. It is therefore com-
mon practice, when analyzing body l uids, to precondition the capillary before each
run in order to achieve reproducible migration time. Different washing steps with
0.5-0.1 M solution of sodium hydroxide, water, and buffer should be performed to
i nd the optimum conditions for rinsing the capillary. The duration of each washing
should also be noted. The vial of buffer solution for rinsing and the vial for the run-
ning buffer must be used separately so as to avoid changes in the latter [53]. When