Adult age and ex vivo protein binding of lorazepam, oxazepam and temazepam in healthy subjects.

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.

Quantification of total and unbound concentrations of lorazepam, oxazepam and temazepam in human plasma by ultrafiltration and LC-MS/MS.

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.