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330 Heegaard et al.
9. The sample solution should match the electrophoresis buffer with respect to ionic
strength and pH to avoid stacking phenomena, which will perturb the binding
equilibrium in the sample zone and thus invalidate results. If organic solvents
have been used in the drug stock solution, the content must be diluted to ≤1%. In
addition, UV-absorbing solvents may be detected as extra plateau peaks and thus
hamper interpretation of the plateau patterns.
10. As a part of the method development, the effect of sample volume on the determined
degree of binding should be examined (91). The injection time (volume)
must be of a sufficient duration to provide plateau peak conditions that will ensure
that the degree of binding is constant, independent of sample volume, and reflect
the true equilibrium within the original sample. Equilibrium is usually attained
very rapidly in drug-plasma protein solutions and only short time is needed for preequilibration.
This, however, may be very different for other binding systems. The
time required for attaining equilibrium may be established using CE by introducing
the ligand-protein sample repeatedly over a period of time (87). Equilibrium has
been reached when the plateau height of the analyte becomes invariant with time.
11. The applied voltage and capillary cassette temperature may be selected to avoid
excessive Joule heating. For most drug substances, a detection wavelength of 200
nm appears to be optimal.
12. For drug–HSA interactions, binding parameters are often determined from
r = L bound
P total
=
m∑
i=1
n i K i L free
1 + K i L free
where r is the number of bound ligand molecules per molecule of protein; [L] free ,
[L] bound and [P] total are the free ligand, bound ligand and total protein concentrations,
respectively; m is the number of identical independent binding classes; n i
is the number of sites of class i and K i is the corresponding association constant.
The parameters are determined using non-linear regression analysis assuming one
or two classes of independent binding sites (m =1orm = 2).
References
1. Lauer, H. H. and McManigill, D. (1986) Capillary zone electrophoresis of proteins
in untreated fused silica tubing. Anal. Chem. 58, 166–170.
2. Jorgenson, J. W. and Lukacs, K. D. (1981) Zone electrophoresis in open-tubular
glass capillaries. Anal. Chem. 53, 1298–1302.
3. Jorgenson, J. W. and Lukacs, K. D. (1981) Free-zone electrophoresis in glass
capillaries. Clin. Chem. 27, 1551–1553.
4. Jorgenson, J. W. and Lukacs, K. D. (1981) High-resolution separations based on
electrophoresis and electroosmosis. J. Chromatogr. 218, 209–216.
5. Grossman, P. D., Colburn, J. C., Lauer, H. K., Nielsen, R. G., Riggin, R. M.,
Sittampalam, G. S., and Rickard, E. C. (1989) Application of free-solution
capillary electrophoresis to the analytical scale separation of proteins and
peptides. Anal. Chem. 61, 1186–1194.