FACTORS INFLUENCING THE PROTEIN BINDING OF BUMETANIDE USING AN EQUILIBRIUM DIALYSIS TECHNIQUE

Factors that influence the plasma protein binding of bumetanide were evaluated using equilibrium dialysis. It took approximately 12h of incubation to reach an equilibrium between plasma and isotonic phosphate buffer of pH 7·4 containing 3% dextran using a Spectrapor 2 membrane (mol. wt cut‐off= 12,000–14,000) in a water‐bath shaker kept at 37°C and at a rate of 50 oscillations per min. Bumetanide was fairly stable in both 4% human serum albumin (HSA) and in the isotonic phosphate buffer of pH 7·4 for up to 24h. The binding of bumetanide to 4% HSA was constant (87·5 ± 1·73%) at bumetanide concentrations ranging from 0·1 to 100 μg/ml. The extents of binding were 72·0, 83·3, 88·5, 90·2, 91·3 and 91·4% at albumin concentrations of 0·5, 1, 2, 3, 4 and 5g/100 ml, respectively, and increased with a decrease in incubation temperature; the values bound were 94·6, 90·3 and 89·3% when incubated at 4, 22 and 37°C, respectively. The binding of bumetanide was independent of the buffer composition used, the quantities of AAG (up to 0·32%), heparin (up to 40 units/ml), sodium azide (up to 0·5%) and anticoagulants (EDTA, heparin and citrate). The free fraction of bumetanide in rabbit plasma (2·91%) was significantly higher than in humans (1·98%) or rats (1·85%).