ABSTRACT
AIM: To see if adult age correlates with ex vivo protein binding of lorazepam, oxazepam and temazepam in healthy subjects. METHODS: Sixty healthy drug free subjects were recruited in the age groups 18-39, 40-64 and ≥65 years. Plasma albumin concentrations were determined. Ex vivo unbound fractions (f(u)) were assessed by spiking samples and measuring the free and total concentrations. RESULTS: No correlation of age with f(u) was seen. The study was powered to demonstrate a change in f(u) of ≥7-10%. 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 f(u) of lorazepam (P= 0.009) and oxazepam (P= 0.014). CONCLUSIONS: There was no association of adult age with ex vivo f(u) of lorazepam, oxazepam or temazepam in healthy subjects.
Subject(s)
Lorazepam/metabolism , Oxazepam/metabolism , Serum Albumin/metabolism , Temazepam/metabolism , Adolescent , Adult , Age Factors , Aged , Female , Humans , Male , Middle Aged , Protein Binding , Young AdultABSTRACT
BACKGROUND: A fast and sensitive assay for quantifying total and unbound concentrations of lorazepam (Lzp), oxazepam (Ozp) and temazepam (Tzp) in human plasma was needed for a plasma protein binding study. RESULTS: Plasma samples were precipitated with acetonitrile for determination of total concentrations or subjected to ultrafiltration for determination of unbound concentrations. An LC-MS/MS assay was developed with an Allure® PFP propyl column and a mobile phase of 35% acetonitrile/0.1% formic acid over 4.5 min and ESI+-MS/MS detection. Matrix effects were negligible in plasma and approximately 70% in ultrafiltrate but were accounted for by the internal standards Lzp-d4, Ozp-d5 and Tzp-d5. The assay was validated for total concentrations of 10-100 ng/ml Lzp, 200-2000 ng/ml Ozp and 100-1000 ng/ml Tzp, and for unbound concentrations of 1-10 ng/ml Lzp, 20-200 ng/ml Ozp and 10-100 ng/ml Tzp. Precision was <14% CV and accuracy was 96-110% throughout the calibration range. The mean precision of triplicate analysis of 60 study samples was <4% CV for total and <8% CV for unbound concentrations. CONCLUSION: A fast and sensitive assay was developed and validated. It has been applied successfully to a protein binding study.