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Journal of Lipid Research, Vol. 48, 733-744, March 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Control of matrix effects in the analysis of urinary F₂-isoprostanes using novel multidimensional solid-phase extraction and LC-MS/MS

Bo Zhang¹ and Keijiro Saku

Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan

Published, JLR Papers in Press, January 10, 2007.

¹ To whom correspondence should be addressed. e-mail: bozhang{at}fukuoka-u.ac.jp

F₂-isoprostanes (F₂-iPs), established markers of oxidative stress, exist as four sets of regioisomers. Simultaneous and specific determination of F₂-iPs can be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed novel methods for urine sample preparation and HPLC to control matrix-related ion suppression effects in the LC-MS/MS analysis of F₂-iPs. A selective solid-phase extraction (SPE) wash protocol was developed with an Oasis HLB (hydrophilic-lipophilic balance) SPE cartridge using an elution profile of [³H]8-iso-prostaglandin (PG)F₂ (iPF₂-III) when the methanol concentration was increased under acidic, neutral, and base wash conditions. A multidimensional (MD)-SPE method that incorporated size exclusion, reverse-phase chromatography, and normal-phase chromatography was developed using an Oasis HLB SPE cartridge and an HLB µElution SPE plate. Average extraction recoveries of the deuterated internal standards of iPF₂-III and iPF₂-VI were 62 ± 8% and 60 ± 10%. A buffer-free HPLC method for the separation of F₂-iP isomers was developed on base-deactivated C8 columns. Average matrix effects for iPF₂-III and iPF₂-VI were 95 ± 6% and 103 ± 5%. The clean extraction of urine F₂-iPs using MD-SPE and the separation of F₂-iP isomers using a novel HPLC method did not cause apparent ion suppression in the analysis of iPF₂-III and iPF₂-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F₂-iPs.

Supplementary key words liquid chromatography-tandem mass spectrometry • urine • ion suppression effects • buffer-free HPLC • sample preparation • iPF₂-VI