WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• The hypothesis that protein binding decreases with ageing has been used to explain studies that have not found a decline in total clearance in relation to age of highly protein bound drugs, cleared by capacity limited metabolism.• Lorazepam, oxazepam and temazepam are highly protein bound to albumin and are cleared by capacity limited metabolism. • There is conflicting or little data concerning the relationship between the protein binding of these drugs and age. WHAT THIS PAPER ADDS• In an ex vivo study of 60 healthy subjects (19-87 years), no clinically significant change was seen in the protein binding of these benzodiazepines with age, arguing against the hypothesis, at least in healthy subjects. The study was adequately powered to show a change of at least 7-10%. AIMTo see if adult age correlates with ex vivo protein binding of lorazepam, oxazepam and temazepam in healthy subjects. METHODSSixty healthy drug free subjects were recruited in the age groups 18-39, 40-64 and Ն65 years. Plasma albumin concentrations were determined. Ex vivo unbound fractions (fu) were assessed by spiking samples and measuring the free and total concentrations. RESULTSNo correlation of age with fu was seen. The study was powered to demonstrate a change in fu of Ն7-10%. A decline in plasma albumin concentration of~0.03 g l -1 year -1 was seen with increasing age (P = 0.032) and was associated with increased fu of lorazepam (P = 0.009) and oxazepam (P = 0.014). CONCLUSIONSThere was no association of adult age with ex vivo fu of lorazepam, oxazepam or temazepam in healthy subjects.

A fast and sensitive assay was developed and validated. It has been applied successfully to a protein binding study.