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

Abstract

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)F2α (iPF2α-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 iPF2α-III and iPF2α-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 iPF2α-III and iPF2α-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 iPF2α-III and iPF2α-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F2-iPs. 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)F2α (iPF2α-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 iPF2α-III and iPF2α-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 iPF2α-III and iPF2α-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 iPF2α-III and iPF2α-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F2-iPs. ErrataJournal of Lipid ResearchVol. 48Issue 5PreviewIn the article “Control of matrix effects in the analysis of urinary F2-isoprostanes using novel multidimensional solid-phase extraction and LC-MS/MS” by Bo Zhang and Keijiro Saku, published in the March 2007 issue of the Journal of Lipid Research (Volume 48, pages 733–744), the authors would like to note the following changes: Full-Text PDF Open Access Isoprostanes (iPs) are products of the free radical-initiated autoxidation of arachidonic acid (1Yin H. Porter N.A. Morrow J.D. Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 827: 157-164Crossref PubMed Scopus (53) Google Scholar). F2-iPs are established markers for oxidative stress (2Morrow J.D. Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 279-286Crossref PubMed Scopus (399) Google Scholar, 3Delanty N. Reilly M.P. Pratico D. Lawson J.A. McCarthy J.F. Wood A.E. Ohnishi S.T. Fitzgerald D.J. FitzGerald G.A. 8-Epi PGF2alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation. 1997; 95: 2492-2499Google Scholar, 4Kadiiska M.B. Gladen B.C. Baird D.D. Graham L.B. Parker C.E. Ames B.N. Basu S. Fitzgerald G.A. Lawson J.A. Marnett L.J. Biomarkers of oxidative stress study. III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning. Free Radic. Biol. Med. 2005; 38 (et al.): 711-718Google Scholar) and have been linked to cardiovascular diseases and risk factors (5Montuschi P. Barnes P.J. Roberts 2nd., L.J. Isoprostanes: markers and mediators of oxidative stress. FASEB J. 2004; 18: 1791-1800Google Scholar). In addition, some F2-iPs exert potent biological activity by acting as ligands for either plasma membrane-bound prostaglandin (PG) receptors or nuclear receptors (6Pratico D. Smyth E.M. Violi F. FitzGerald G.A. Local amplification of platelet function by 8-epi prostaglandin F2alpha is not mediated by thromboxane receptor isoforms. J. Biol. Chem. 1996; 271: 14916-14924Google Scholar, 7McNamara P. Lawson J.A. Rokach J. FitzGerald G.A. Isoprostane activation of the nuclear hormone receptor PPAR. Adv. Exp. Med. Biol. 2002; 507: 351-355PubMed Google Scholar, 8Morrow J.D. The isoprostanes—unique products of arachidonate peroxidation: their role as mediators of oxidant stress. Curr. Pharm. Des. 2006; 12: 895-902Google Scholar). F2-iPs are generated in situ esterified to phospholipids (9Kim S. Powell W.S. Lawson J.A. Jacobo S.H. Pratico D. FitzGerald G.A. Maxey K. Rokach J. iPF2alpha-III-17,18,1920-d4: total synthesis and metabolism. Bioorg. Med. Chem. Lett. 2005; 15: 1613-1617Google Scholar). Cleavage by phospholipase A2 generates free F2-iPs that are excreted in urine (10Basu S. Metabolism of 8-iso-prostaglandin F2alpha. FEBS Lett. 1998; 428: 32-36Crossref PubMed Scopus (110) Google Scholar). The measurement of F2-iPs in biological samples presents several challenges. First, the methods used for measurement have to be specific, because F2-iPs are isomers of F2-PGs and exist as four sets of regioisomers (1Yin H. Porter N.A. Morrow J.D. Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 827: 157-164Crossref PubMed Scopus (53) Google Scholar, 11Rokach J. Kim S. Bellone S. Lawson J.A. Pratico D. Powell W.S. FitzGerald G.A. Total synthesis of isoprostanes: discovery and in biological Chem. 2004; PubMed Scopus Google Scholar). In addition, the methods used for measurement have to be because F2-iPs exist in biological samples D. mass spectrometric of 8-iso-prostaglandin F2alpha and F2alpha in J. Chromatogr. B Biomed. Sci. Scholar, D. J. in urinary chromatography-tandem mass spectrometry chromatography and chromatography of and J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. Scholar, P. Quantification of and in urine using liquid chromatography-tandem mass Free Radic. Biol. Med. Scholar, H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. Scholar). F2-iPs are in The is the F2-iP that been a of been to be the F2-iP Pratico D. FitzGerald G.A. F. coronary F2-isoprostane for in oxidative stress during J. Scholar). methods are the for the measurement of F2-iPs D. J. in urinary chromatography-tandem mass spectrometry chromatography and chromatography of and J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. Scholar, M.P. N. Rokach J. P. Lawson J.A. FitzGerald G.A. of the isoprostanes and F2alpha in coronary for oxidant stress during coronary in humans. Circulation. 1997; Scholar, L.J. method for the measurement of urinary and plasma F2-isoprostanes using Scholar). methods are and are not selective D. J. in urinary chromatography-tandem mass spectrometry chromatography and chromatography of and J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. Scholar, L.J. method for the measurement of urinary and plasma F2-isoprostanes using Scholar). for F2-iPs are the analysis of F2-iP regioisomers is not and measurements of F2-iPs be because have biological and be and under that are linked to oxidative stress D. D.J. Rokach J. FitzGerald G.A. generation in and atherosclerosis in Med. 1998; Scholar). chromatography-tandem mass spectrometry methods have been developed for the measurement of F2-iPs and their (1Yin H. Porter N.A. Morrow J.D. Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 827: 157-164Crossref PubMed Scopus (53) Google Scholar, P. Quantification of and in urine using liquid chromatography-tandem mass Free Radic. Biol. Med. Scholar, H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. Scholar, Roberts 2nd., L.J. Quantification of of F2-isoprostanes in urine by liquid mass 2006; Scholar). LC-MS/MS methods are specific, because HPLC can F2-iP and can F2-iP regioisomers. LC-MS/MS methods are because the the of of in the of a matrix LC-MS/MS is with a when in biological the is to matrix-related ion suppression effects A of ion suppression effects in and solid-phase 2004; 18: and matrix effects sample to sample of matrix effects in or liquid chromatography with mass spectrometry in 2002; Google Scholar). are and the as the in have been used as internal standards to for in sample and matrix that with deuterated of have been to be in plasma samples by extraction of separation for liquid chromatography-tandem mass spectrometry of plasma total 2006; Scholar). because and are used to can be and by HPLC separation of separation for liquid chromatography-tandem mass spectrometry of plasma total 2006; Scholar). matrix-related that with the and be the clean extraction of biological samples is for the and analysis of F2-iPs using LC-MS/MS. ion suppression effects have not been in LC-MS/MS methods for the analysis of F2-iPs in biological samples P. Quantification of and in urine using liquid chromatography-tandem mass Free Radic. Biol. Med. Scholar, H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. Scholar, of separation for liquid chromatography-tandem mass spectrometry of plasma total 2006; Scholar, H. S. of isoprostanes in urine samples using liquid chromatography-tandem mass J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. Scholar). the control of matrix effects is for establishing LC-MS/MS as a routine for the measurement of F2-iPs in biological developed a novel urine sample using multidimensional extraction (SPE) and a novel buffer-free HPLC separation method to and ion suppression effects in the analysis of F2-iPs using LC-MS/MS. of the F2-iPs and standards and deuterated of and in were were Oasis HLB (hydrophilic-lipophilic balance) SPE Oasis SPE Oasis SPE and Oasis HLB μElution SPE were SPE were and were were HPLC F2-iPs were with a mass with was achieved using in the The of the and of and were for by with a The for were as to and a of ion and and mass and for were and and and for were and A mass and was using in the with and to and a of and the was using the a of mass for and were using the HPLC of of the The of the ion of an was in the and the sample for of the was in the of was using The ion of F2-iPs and deuterated was in the and the for of was in the of of and of were used in The and for the are in HPLC was using and Separation were with the mass control and were with A was used to the A was B was and was C8 and C8 were used for HPLC method and the analysis of urine The HPLC separation method was developed using the liquid separation of samples by and J. Chromatogr. Scholar, separation of isomers by and either or J. Chromatogr. Scholar). The was and the was The was as to to to and to the of a selective SPE for the extraction of F2-iPs in urine was to urine samples and with Oasis SPE were with of and and SPE extraction was under using a The of Oasis SPE and were by urine samples to to the SPE of wash and were and for in of liquid on a liquid A selective SPE wash was developed by of the in the elution of F2-iPs and sample matrix as a function of the of the wash and the concentration of the elution solid-phase extraction method for the determination of and in plasma in a extraction plate. J. Chromatogr. 1998; Scholar). The of in acidic, neutral, and base was by the with and of and the for was by F2-iPs with of acid and the in the Oasis HLB cartridge and an Oasis HLB μElution were used to the MD-SPE of urine samples was with of and of and and on for The samples were to with of acid The HLB SPE were with of and of acid The and urine samples were to a cartridge that was to the of an Oasis HLB SPE cartridge using a with a a of sample were and the SPE cartridge was with of wash and elution were using a was used for Oasis HLB SPE was to an Oasis μElution plate. SPE extraction with an Oasis μElution was using a SPE the Oasis μElution was with and using LC-MS/MS. in urine sample extraction was by the and to urine samples and was as of urine samples of urine samples extraction for the of matrix in quantitative methods on Chem. Scholar). suppression effects of sample were by of and and deuterated and to and sample The matrix was as of the or urine of the or for the of matrix in quantitative methods on Chem. Scholar). for iPF2α-III and iPF2α-VI on mass were using using the method size of in the of iPF2α-III or iPF2α-VI and urine samples were by the using the was used for identification and the of on the The for the samples were as and using a and matrix effects were using the of regioisomers of F2-iPs and are the of arachidonic acid J. Kim S. Bellone S. Lawson J.A. Pratico D. Powell W.S. FitzGerald G.A. Total synthesis of isoprostanes: discovery and in biological Chem. 2004; PubMed Scopus Google Scholar). and F2-iPs have been to be F2-iPs Pratico D. FitzGerald G.A. F. coronary F2-isoprostane for in oxidative stress during J. Scholar, M.P. N. Rokach J. P. Lawson J.A. FitzGerald G.A. of the isoprostanes and F2alpha in coronary for oxidant stress during coronary in humans. Circulation. 1997; Scholar). the of standards of F2-iPs F2-iPs and of and the of deuterated of iPF2α-III and iPF2α-VI the ion of F2-iPs. generated under the were for and for for and for and were the and were specific for and The were for for and for ion for the of F2-iPs under were to be as for for for for and for and in the ion not the separation of isomers is for the specific determination of a in the of a novel HPLC were to the of the using an C8 that the of was in in not the separation of isomers of F2-iPs with and the of was increased to the The of four and was increased when the of to was and separation was when the of to was increased to not is the used in the analysis of by LC-MS/MS (1Yin H. Porter N.A. Morrow J.D. Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 827: 157-164Crossref PubMed Scopus (53) Google Scholar, P. Quantification of and in urine using liquid chromatography-tandem mass Free Radic. Biol. Med. Scholar, H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. the effects of on the of and by to the the ion of four isomers were when and of were in the not the of four isomers did not the separation not The effects of on the separation of isomers were by of four with and and of the four isomers increased with an in the concentration of separation was not the following HPLC method was developed under The and for separation of the four isomers were using the were with in and and and The that separation can be achieved under a and to and separation under C8 were for the separation of of and A C8 was as the analysis on the and of F2-iP isomers with the C8 not LC-MS/MS with the of F2-iPs and in iPF2α-III was iPF2α-III and in and were with iPF2α-III and was with and and iPF2α-VI and and and were in not separation and were achieved using the C8 F2-iPs are acidic, of Oasis and were for their to the in urine the of to a urine sample for Oasis and SPE in to urine sample was by Oasis HLB not by Oasis or that Oasis HLB the was used in the following SPE method a selective SPE the of when the of was increased in and in was the of was increased to under and wash was when the of was under wash conditions. that the elution profile of as a function of concentration was and to a selective SPE was developed and incorporated the MD-SPE method developed for the extraction of F2-iPs in First, an urine sample was to an HLB cartridge and with In sample and cartridge wash sample as were because of the of the and and as were not by reverse-phase In cartridge wash acidic, was using with The elution of urine samples was in In cartridge wash was to by with and acid In cartridge wash was using with In cartridge wash and was with and In elution the acid was with with acid on the SPE A the HLB SPE cartridge was to the HLB μElution with In F2-iPs were on the SPE by normal-phase In wash acid was with In wash was with and In wash and the was to by with and and acid was with In wash was with in elution F2-iP were with The was and used for LC-MS/MS analysis with MD-SPE LC-MS/MS for a urine sample under in and were and and were by of the to the sample and the urine of sample with deuterated that was with and that was the the of a isomers not in iPF2α-VI and were that and the urine sample were was with and as by extraction of a sample not or not iPF2α-III and iPF2α-VI were of urine and urine samples were with the of and and using the MD-SPE that the of iPF2α-III to and iPF2α-VI to in urine samples were and with in urine The for iPF2α-III and iPF2α-VI to and to in the for iPF2α-III and iPF2α-VI urine samples were urine These that of were achieved for iPF2α-III and iPF2α-VI analysis by urine samples of and were to or urine samples and extraction to sample extraction in of the was and urine samples four of urine samples four and the recoveries for and were 62 ± 60 ± and ± in solid-phase extraction of urine samples four of of of of of of ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± are Open in a are The effects of urine matrix on the of F2-iPs were by the ion of in and urine sample in of the ion suppression was for was and urine samples or The matrix effects for and urine samples and on F2-iPs to and the matrix effects for and were 95 ± ± 103 ± ± ± ± and ± These that the ion suppression effects of urine did not the of ion suppression of of of of of of ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± are Open in a are stress is to cardiovascular (2Morrow J.D. Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 279-286Crossref PubMed Scopus (399) Google Scholar, 3Delanty N. Reilly M.P. Pratico D. Lawson J.A. McCarthy J.F. Wood A.E. Ohnishi S.T. Fitzgerald D.J. FitzGerald G.A. 8-Epi PGF2alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation. 1997; 95: 2492-2499Google Scholar, Pratico D. FitzGerald G.A. F. coronary F2-isoprostane for in oxidative stress during J. Scholar, M.P. N. Rokach J. P. Lawson J.A. FitzGerald G.A. of the isoprostanes and F2alpha in coronary for oxidant stress during coronary in humans. Circulation. 1997; Scholar). for the routine measurement of markers of oxidative stress are F2-iPs are products of the of arachidonic acid and have been to be markers of oxidative stress (1Yin H. Porter N.A. Morrow J.D. Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 827: 157-164Crossref PubMed Scopus (53) Google Scholar, 4Kadiiska M.B. Gladen B.C. Baird D.D. Graham L.B. Parker C.E. Ames B.N. Basu S. Fitzgerald G.A. Lawson J.A. Marnett L.J. Biomarkers of oxidative stress study. III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning. Free Radic. Biol. Med. 2005; 38 (et al.): 711-718Google Scholar, P. Barnes P.J. Roberts 2nd., L.J. Isoprostanes: markers and mediators of oxidative stress. FASEB J. 2004; 18: 1791-1800Google Scholar). LC-MS/MS methods have been developed for the specific determination of F2-iP regioisomers in biological samples P. Quantification of and in urine using liquid chromatography-tandem mass Free Radic. Biol. Med. Scholar, H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. Scholar, of separation for liquid chromatography-tandem mass spectrometry of plasma total 2006; Scholar, H. S. of isoprostanes in urine samples using liquid chromatography-tandem mass J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. Scholar). matrix effects are not in are to the and of A of ion suppression effects in and solid-phase 2004; 18: Scholar, for the of matrix in quantitative methods on Chem. Scholar, of suppression in J. PubMed Scopus Google Scholar). In developed a novel MD-SPE and buffer-free HPLC method to control and ion suppression in the LC-MS/MS analysis of F2-iPs in urine We developed the MD-SPE method on and on a selective SPE wash and elution protocol for F2-iPs. In the SPE on Oasis HLB SPE F2-iPs were by a reverse-phase chromatography and in the SPE on an Oasis μElution SPE F2-iPs were by a normal-phase chromatography Oasis HLB is a solid-phase extraction method for the determination of and in plasma in a extraction plate. J. Chromatogr. 1998; reverse-phase chromatography and normal-phase chromatography can be used for the of F2-iPs. The were for LC-MS/MS analysis in the the to that is of the SPE of urine samples P. Quantification of and in urine using liquid chromatography-tandem mass Free Radic. Biol. Med. Scholar, H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. Scholar, of separation for liquid chromatography-tandem mass spectrometry of plasma total 2006; Scholar, H. S. of isoprostanes in urine samples using liquid chromatography-tandem mass J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. Scholar). the samples were using SPE in the SPE is achieved by the of and and matrix are in the SPE on the Oasis μElution F2-iPs were matrix was the of the elution a concentration of urine samples was achieved by the MD-SPE In addition, using the to samples was because did not have to be In the clean extraction of urine samples was achieved by a novel selective SPE wash and elution Oasis HLB is a and can be used to solid-phase extraction method for the determination of and in plasma in a extraction plate. J. Chromatogr. 1998; Scholar). a selective SPE wash was developed by of the elution of F2-iPs and urine matrix as a function of the concentration of and HLB acid and base under and acid have and base have under acid have and base have when urine samples were to an HLB SPE base was not wash with acid cartridge wash was used to base wash with was used to acid of was during the base in the urine samples was with is with and of was to the wash cartridge wash A wash with cartridge wash was to on the HLB SPE cartridge because is with acid was used for the elution of F2-iPs in the SPE because is and selective selective wash and selective elution a selective SPE for the clean extraction of F2-iPs. The of the urine samples as by was not by in extraction the concentration of and not that the selective SPE was the of SPE of Oasis HLB SPE did not the of extraction or not that the MD-SPE method is F2-iPs are isomers of F2-PGs and of diastereomers (1Yin H. Porter N.A. Morrow J.D. Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 827: 157-164Crossref PubMed Scopus (53) Google Scholar, 11Rokach J. Kim S. Bellone S. Lawson J.A. Pratico D. Powell W.S. FitzGerald G.A. Total synthesis of isoprostanes: discovery and in biological Chem. 2004; PubMed Scopus Google separation of isomers is for the specific analysis of F2-iPs. H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. Scholar) that and were using columns. We achieved a of and in and urine samples using not the of H. Lawson J.A. Reilly Rokach J. FitzGerald G.A. liquid mass spectrometric analysis of the four of F2-isoprostanes in Sci. Scholar). when urine samples were using C8 an was and and to is that an F2-iP was with on was on C8 columns. C8 were used in the novel HPLC method to columns. that ion suppression of F2-iPs and the separation of F2-iP isomers was not by acid in the urine samples not an HPLC method was developed the of to control used for LC-MS/MS HPLC methods the of the LC-MS/MS and for the preparation of and were The novel HPLC method is because in sample and of HPLC did not the separation of the F2-iPs standards not the of the novel HPLC and urine samples in HPLC with and Separation separation of F2-iPs was achieved by the and for and Separation and the the not is the that a HPLC method for the separation of F2-iPs was developed using with the of and separation of isomers by and either or J. Chromatogr. Scholar) that the separation of as a function of and was by a using We that the of HPLC and be using the novel MD-SPE and HPLC methods. of the urine samples did not cause a in HPLC not and were not used in the HPLC of the urine samples did not cause of the sample in the not for the in findings that novel MD-SPE and HPLC methods the of of iPF2α-III and iPF2α-VI urine samples and the matrix-related ion suppression for iPF2α-III and iPF2α-VI of the novel MD-SPE method is that the urine for LC-MS/MS analysis is We urine samples in of the of the MD-SPE should not be to sample the MD-SPE method can be used to of and or or urine samples and with by a cartridge with a cartridge not the MD-SPE method plasma samples are to be for the analysis of F2-iPs using LC-MS/MS. F2-iPs are generated in situ esterified to phospholipids (1Yin H. Porter N.A. Morrow J.D. Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 827: 157-164Crossref PubMed Scopus (53) Google of plasma samples is to F2-iPs free F2-iPs to total F2-iPs. be to a free as or an of as indomethacin D. Lawson J.A. FitzGerald G.A. of the 8-epi prostaglandin F2alpha. J. Biol. Chem. Scholar) to plasma samples to autoxidation during sample and Pratico D. FitzGerald G.A. F. coronary F2-isoprostane for in oxidative stress during J. Scholar) acid to plasma samples to of We that of by Oasis HLB SPE was in plasma samples that been with in in plasma samples that were not not of plasma sample the Oasis HLB Oasis HLB or SPE with a a as 60 or are for plasma sample the urine MD-SPE method in the analysis of F2-iPs in plasma In developed a novel sample method using MD-SPE and a novel HPLC method for the specific analysis of F2-iPs using LC-MS/MS. novel MD-SPE LC-MS/MS should be to the routine analysis of F2-iPs in urine of the novel MD-SPE LC-MS/MS method for the routine analysis of iPF2α-III and iPF2α-VI in urine is in in The authors and for in was by a the of and of