title: Phospholipid removal combined with a semi-automated 96-well SPE application for determination of budesonide in human plasma with LC-MS/MS.
abstract: A semi-automated method for quantification of budesonide in human plasma was developed, validated, and applied for high-volume analysis of samples in connection with a pharmacokinetic study. Protein and phospholipid removal was performed using an Ostro 96-well filter plate and subsequently combined with C18 solid-phase extraction on a Hamilton Microlab STARlet automation robot. The final extracts were evaporated to dryness and redissolved in 20% acetonitrile/water. The procedure used budesonide-d8 as internal standard and gave a 3.5-fold concentration of plasma to extract. The final extracts (5 μL injected) were analyzed with selected reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionization in positive mode. The chromatography system used a 100 mm ACQUITY BEH UPLC column and a gradient system consisting of aqueous 0.1% formic acid and acetonitrile as organic modifier. Phospholipid removal was found to be needed during method development in order to reduce ion suppression effects from matrix and to increase method sensitivity. The measuring range was 50-5000 pg/mL with and LOD 24 pg/mL. Calibration response showed good linearity (correlation coefficients<0.99) over the measuring range. The absolute recovery over the sample preparation procedure was estimated to 67%. Total imprecision was <9% at three levels and accuracy was between 98.9 and 103%. The method was successfully applied for analysis of 864 study samples in a short time. The quality control samples at concentration levels 200 and 2000 pg/mL gave a total imprecision of 7.4% and 4.2%, respectively, (n=95).
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institute and University Hospital, Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Nilsson
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institute and University Hospital, Stockholm, Sweden. Electronic address: maria.ch.andersson@karolinska.se.
authors.forename: Maria
authors.initials: M
authors.lastname: Andersson
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institute and University Hospital, Stockholm, Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Beck
mesh_terms: D001331:Automation; D019819:Budesonide / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D010743:Phospholipids / Q000737:chemistry / Q000302:isolation & purification*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010743:Phospholipids; D019819:Budesonide
keywords: Automation; Budesonide; Human plasma; LC–MS/MS; Phospholipid removal; Solid phase extraction