title: Quantification of total and unbound concentrations of lorazepam, oxazepam and temazepam in human plasma by ultrafiltration and LC-MS/MS.
abstract: 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.
  
  
  CONCLUSIONS
  A fast and sensitive assay was developed and validated. It has been applied successfully to a protein binding study.
authors.affiliation: Clinical Pharmacology, Department of Medicine, University of Otago, PO Box 4345, Christchurch, New Zealand.
authors.forename: Helle S
authors.initials: HS
authors.lastname: Larsen
authors.forename: Paul Kl
authors.initials: PK
authors.lastname: Chin
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Begg
authors.forename: Berit P
authors.initials: BP
authors.lastname: Jensen
mesh_terms: D014151:Anti-Anxiety Agents / Q000097:blood* / Q000737:chemistry / Q000592:standards; D002138:Calibration; D002853:Chromatography, Liquid; D006440:Hemofiltration; D006801:Humans; D008140:Lorazepam / Q000097:blood* / Q000737:chemistry / Q000592:standards; D010076:Oxazepam / Q000097:blood* / Q000737:chemistry / Q000592:standards; D053719:Tandem Mass Spectrometry; D013693:Temazepam / Q000097:blood* / Q000737:chemistry / Q000592:standards
chemical_list: D014151:Anti-Anxiety Agents; D010076:Oxazepam; D013693:Temazepam; D008140:Lorazepam