Non-isotopic spectrophotometric determination of the unbound fraction of drugs in serum*

A spectrophotometric method to measure the free fraction of highly-bound drugs in serum has been established for a range of non-steroidal anti-inflammatory drugs (NSAIDs) and for frusemide. Spectrophotometry is used to measure fractional transit of drug from a large volume of dialysate to a small volume of serum during dialysis to equilibrium. The method, which depends on the principle that drug transit from dialysate to serum is proportional to serum binding, requires neither isotopic drug preparations nor specific drug assays, is independent of extraction efficiency from the dialysate and requires no measurements from the serum compartment. Estimates of percent unbound fraction (%UF) for aspirin (6.0 ± 0.9%), phenylbutazone (0.9 ± 0.2%), and frusemide (1.8 ± 0.2%) were comparable with those obtained with 14C drug preparations. Values for %UF were determined for eleven additional NSAIDs. The method was valid for a four-fold change in serum: dialysate ratio. Kinetics of frusemide binding to serum were comparable using [14C]frusemide and the test method. This technique may have general application in establishing the %UF for substances that are extensively bound to serum proteins and for identifying sera that show abnormal binding.