The mobile phase used for sample
application, elution and sample preparation was pH 7.4, 0.067 M phosphate
buffer. All mobile phases were degassed for 30 min prior to use. Each
affinity microcolumn was used for approximately 200 sample injections
to provide optimum retention and peak resolution; however, these columns
were found to be stable for at least 300–400 injections and
over 6 months of use. The free fraction measurements were typically
made by injecting 1 μL of samples that contained 10 μM
of the desired drug or a mixture of 10 μM drug and 20 μM
soluble HSA, although other drug and protein concentrations were also
considered (seeSupporting Information ).
These mixtures were incubated for at least 30 min prior to injection,
with both the sample and mobile phase being preheated to 37 °C
before passage through the affinity microcolumn. Other conditions
are provided in theSupporting Information .
The dissociation rate constants and equilibrium constants
for each drug with soluble HSA were measured by using the general
scheme in Figure1 . For the direct measurement
of equilibrium constants, an injection flow rate was used that was
sufficiently high to minimize dissociation of drug–protein
complexes in the sample during their passage through the column. By
using lower flow rates, and longer residence times for the sample
in the column, the conditions were adjusted so that some of the drug–protein
complex could dissociate during passage through the column, thus increasing
the apparent free drug fraction and making it possible to determine
the dissociation rate constant for the drug with the soluble protein.
In both types of studies, the free drug fractions were measured by
dividing the drug’s baseline-corrected retained peak area by
the total peak area for the same drug in the absence of any soluble
protein. The baseline of each chromatogram was normalized using the
autofit and subtract baseline method of PeakFit 4.12 prior to data
analysis. No significant nonspecific binding with the control support
was seen for most drugs examined in this study.18 (link)−22 (link) Some nonspecific binding was seen for verapamil,
as reported previously;21 however, this
nonspecific binding did not have any notable effect on the free fractions
that were measured for this drug with soluble HSA.
application, elution and sample preparation was pH 7.4, 0.067 M phosphate
buffer. All mobile phases were degassed for 30 min prior to use. Each
affinity microcolumn was used for approximately 200 sample injections
to provide optimum retention and peak resolution; however, these columns
were found to be stable for at least 300–400 injections and
over 6 months of use. The free fraction measurements were typically
made by injecting 1 μL of samples that contained 10 μM
of the desired drug or a mixture of 10 μM drug and 20 μM
soluble HSA, although other drug and protein concentrations were also
considered (see
These mixtures were incubated for at least 30 min prior to injection,
with both the sample and mobile phase being preheated to 37 °C
before passage through the affinity microcolumn. Other conditions
are provided in the
The dissociation rate constants and equilibrium constants
for each drug with soluble HSA were measured by using the general
scheme in Figure
of equilibrium constants, an injection flow rate was used that was
sufficiently high to minimize dissociation of drug–protein
complexes in the sample during their passage through the column. By
using lower flow rates, and longer residence times for the sample
in the column, the conditions were adjusted so that some of the drug–protein
complex could dissociate during passage through the column, thus increasing
the apparent free drug fraction and making it possible to determine
the dissociation rate constant for the drug with the soluble protein.
In both types of studies, the free drug fractions were measured by
dividing the drug’s baseline-corrected retained peak area by
the total peak area for the same drug in the absence of any soluble
protein. The baseline of each chromatogram was normalized using the
autofit and subtract baseline method of PeakFit 4.12 prior to data
analysis. No significant nonspecific binding with the control support
was seen for most drugs examined in this study.18 (link)−22 (link) Some nonspecific binding was seen for verapamil,
as reported previously;21 however, this
nonspecific binding did not have any notable effect on the free fractions
that were measured for this drug with soluble HSA.
