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Quantification Of Total And Unbound Concentrations Of Lorazepam, Oxazepam And Temazepam In Human Plasma By Ultrafiltration And LC-MS/MS.

Helle S Larsen, Paul K.L. Chin, Evan J. Begg, Berit P. Jensen
Published 2011 · Chemistry, Medicine

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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.
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