Determination of free concentration of piroxicam and naproxen in plasma. The influence of experimental conditions in equilibrium dialysis

Summary— An equilibrium dialysis method was established in order to investigate possible relationships between free drug concentrations of piroxicam and naproxen and clinical events. Therefore the influence of variations in pH, phosphate concentration and sodium azide concentration of the dialysis buffer on the free concentrations of piroxicam and naproxen was investigated. Piroxicam was found to have a pH-dependent protein binding. Therefore a good control of pH during the dialysis process is necessary. This has been achieved by increasing the buffer capacity of the dialysis buffer, by adding an antibacterial agent to the dialysis buffer and by cleansing the dialysis cells with 70% ethanol before use to prevent bacterial growth. Addition of 0.03% sodium azide as an antibacterial agent and the use of a 0.09 mol/l phosphate buffer gave good pH control. A method to correct for deviations of pH in measurements of free concentrations of piroxicam by a simple mathematical correction has been found. As naproxen was found to have a protein binding independent of pH, a pH-correction is not necessary for this drug. Standardized conditions in determination of protein binding of drugs by equilibrium dialysis are important, as composition of the dialysis buffer and pH of plasma compartment at equilibrium may influence the free concentration measurements. Comparisons of data from experiments using different methods are therefore difficult; the importance of pH-control is stressed. With the methods used in the present investigation, equilibrium dialysis in connection with HPLC, the coefficients of variation for piroxicam and naproxen free concentrations are 5.5% and 7.4%, respectively.