Free Drugs

Most drugs bind to serum proteins while the unbound fraction (free) of the drug is thought to be the active form. Thus, it is important to measure and understand the drug-protein interaction. This is especially important in such disorders as renal failure where the amount of the free drug changes as a result of uremia and binding protein concentrations.

The free, bound, percentage of binding, and the binding constant can be determined based on several well-established techniques such as dialysis, filtration, and size exclusion. CE can extend these techniques to measure free drugs. For example, several free drugs were measured after filtration through special membranes (17). The problem with this method of measurement is that free drugs are present at a much lower concentration than the total drug. Thus, in order to measure these low levels, concentration or stacking steps may be necessary. For example, in the analysis of phenytoin, acetonitrile is added to the filtrate to concentrate the drug on the capillary (see Fig. 2).

In addition to the previous techniques, CE based on changes in the elec-trophoretic mobility (100,101) can measure drug binding. Kraak et al. (100) described three different methods for measuring protein-drug binding by CE. The first is based on the Hummel-Dryer method in which the capillary is filled with a buffer containing the drug giving a large background signal. The sample, which contains the drug, protein, and buffer, is injected. The bound drug migrates differently from the free drug, producing a negative peak. The area of the negative peak is a measure of the bound drug. The second method is based on the vacancy method where as the capillary is filled with mixture of the buffer, the drug, and the protein. This also causes a large background signal. The sample that contains only the buffer is injected. Both the free and the bound drug migrate separately and each gives a negative peak. The third method depends on the frontal analysis. In this method the capillary is filled with the buffer. Different concentrations of the drug in the presence and absence of a fixed binding protein concentration are incubated at 25°C followed by injection of a large amount of sample (~5-7% of the capillary volume). The free drug, the complex and the protein gives each a frontal, plateau-shaped peak. The free-drug concentration can be calculated (102) from the height of the frontal peak as follows:

where D = Height free drug concentration, E = free drug concentration, S = concentration of the pure standard, and P = drug peak height in presence of the protein.

The concentration of the bound drug can be calculated by subtracting the free drug from the total. The percentage and the binding association constant can be also calculated from a Scatchard plot of this data (91,101-103). Kraak et al. (100) concluded that the frontal analysis appeared to be the preferred method for drug binding. It is more reproducible and gives a smooth Scatchard curve compared to the other two techniques.

Binding of drugs to a specific carrier is also a promising approach to target drugs to specific organs. Protein binding can alter the metabolism or delivery of the drug to the target organ. Naproxen conjugated to albumin is an example of such drug targeting. Albrecht et al. (104) have shown that naproxen, a nonsteriodal anti-inflammatory drug, can be determined in serum using MEKC as free, albumin conjugated, and lysine conjugated. Samples were injected and separated in a borate/phosphate buffer containing SDS with LIF detection. This method has also been extended to measurement of this drug in liver and kidney tissue (105).

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