Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography

Abstract

Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several model drugs (i.e., quinidine, diazepam, gliclazide, tolbutamide and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333–665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7–9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5–10 min or less and required only 1–5 µL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research.

Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns

“…This loss of precision occurred as smaller amounts of the free drug fraction were allowed to pass onto the second column, resulting in greater variability in the measurement of this fraction. The same effect has been observed in the use of HSA microcolumns in a two-dimensional system for ultrafast affinity extraction [15,17]. …”

“…This decrease in the apparent free fraction as the switching time was increased was due to less contamination being present in this fraction as a result of dissociation of the original drug-protein complex or other non-retained sample components. For both carbamazepine and warfarin, the apparent free fraction became constant as the presence of the contamination was minimized [15,17]. …”

“…Recently, techniques based on high-performance affinity chromatography (HPAC) and ultrafast affinity extraction have been developed for the measurement of free drug and free hormone fractions [4–6,11–17]. The advantages of this approach include its short analysis times, good precision, ease of automation, and the ability to work with various detectors and small amount of solutes and proteins [18,19].…”

“…This microcolumn is used under conditions in which the drug-protein complex and excess protein will elute as a non-retained peak, while a portion of the free drug fraction will be retained and separated from these other components. This separation makes use of the relatively strong and selective binding of affinity microcolumns for their targets and is typically carried out at column residence times for the non-retained peak of less than one second to avoid release of the protein-bound form of the drug in the sample [4–6,11–17]. For complex samples such as serum, which may contain a large excess of protein versus the drug, a second affinity column can be placed on-line with the affinity microcolumn after most of the non-retained components have eluted, thus making it possible to further resolve these components from the free drug fraction [6].…”

Evaluation of pendimethalin binding to human serum albumin: Insights from spectroscopic and molecular modeling approach

“…Values of k q that were higher than the typical diffusion‐controlled rate constant of the biomolecule (2 × 10 10 M –1 s –1 ), further affirmed the formation of the PM–HSA complex . K a value in the order of magnitude of 10 5 M –1 was suggestive of moderately strong binding affinity between PM and HSA …”