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Methods |
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
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