Matrix effect in F2-isoprostanes quantification by HPLC-MS/MS: a validated method for analysis of iPF2α-III and iPF2α-VI in human urine.

Liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) has become the method of choice for analysis in biological matrices, because of its high specificity and sensitivity. However, it should be taken into account that the presence of matrix components coeluting with analytes might interfere with the ionization process and affect the accuracy and precision of the assay. For this reason, the presence of a "matrix effect" should always be evaluated during method development, above all in complex matrix such as urine. In the present work, a HPLC-MS/MS method was developed for the quantification of urinary iPF2α-III and iPF2α-VI. A careful assessment of matrix effect and an accurate validation were carried out, in order to verify the reliability of quantitative data obtained. Ion suppression, due to the matrix components, was reduced through optimization of both chromatographic method and sample extraction procedure. Urine samples were purified by solid phase extraction (SPE) and the extracts injected into the HPLC-MS/MS system, equipped with a TurboIonSpray ionization source operated in negative ion mode (ESI(-)). Stable isotope-labeled analogues (iPF2α-III-d4 and iPF2α-VI-d4) were used as internal standards, and quantification was performed in multiple reaction monitoring (MRM) mode by monitoring the following mass transitions: m/z 353.4→193.2 for iPF2α-III, m/z 357.2→197.0 for iPF2α-III-d4, m/z 353.4→115.1 for iPF2α-VI, and m/z 357.4→115.1 for iPF2α-VI-d4. The validated assay, applied to the analysis of urinary samples coming from healthy and overweight subjects, resulted suitable for an accurate quantification of iPF2α-III and iPF2α-VI in human urine.

A protocol for quantifying lipid peroxidation in cellular systems by F2-isoprostane analysis.

Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol for measuring F2-isoprostanes as accurate markers for endogenous oxidative damage in cellular systems. F2-isoprostanes are chemically stable prostaglandin-like lipid peroxidation products of arachidonic acid, the predominant polyunsaturated fatty acid in mammalian cells. This approach is rapid and highly sensitive, allowing for the absolute quantification of endogenous lipid peroxidation in as little as ten thousand cells as well as damage originating from multiple ROS sources. Furthermore, differences in the endogenous cellular redox state induced by transcriptional regulation of ROS scavenging enzymes were detected by following this protocol. Finally we showed that the F2-isoprostane 5-iPF2α-VI is a metabolically stable end product, which is excreted from cells. Overall, this protocol enables accurate, specific and sensitive quantification of endogenous lipid peroxidation in cellular systems.

Simultaneous quantification of F2-isoprostanes and prostaglandins in human urine by liquid chromatography tandem-mass spectrometry.

A specific and sensitive LC-MS/MS method for analysis of F(2)-isoprostanes (F(2)-IsoPs) and prostaglandins (PGs) in urine was developed and validated to examine the levels of F(2)-IsoPs and prostaglandin F(2α) (PGF(2α)), in human urine in patients undergoing cardiac surgery. The rapid extraction for F(2)-IsoPs and PGs from urine was achieved using a polymeric weak anion solid phase extraction cartridge. The base-line separation of 8-iso-PGF(2α), 8-iso-15(R)-PGF(2α), PGF(2α), and 15(R)-PGF(2α) was carried out on a Hydro-RP column (250mm×2.0mm i.d., Phenomenex, CA) using a linear gradient of methanol:acetonitrile (1:1, v/v) in 0.1% formic acid at a flow rate of 0.2mL/min. The method was proved to be accurate and precise for simultaneous quantification of each analyte over a linear dynamic range of 0.05-50ng/mL with correlation coefficients greater than 0.99. The intra-day and inter-day assay precision at the lowest quality control (0.07ng/mL) level were less than 17%. The mean extraction recoveries of F(2)-IsoPs and PGs were in a range of 79-100%. In applications of this method to patients undergoing cardiac surgery, post-surgery urinary concentrations of 8-iso-PGF(2α) increased significantly in patients (n=14) who did not develop acute kidney (AKI) (pre-surgery 0.344±0.039 vs. post-surgery 0.682±0.094ng/mg creatinine, p<0.01), whereas there was no significant change in this isoprostane in the patients (n=4) who developed AKI (pre-surgery 0.298±0.062 vs. post-surgery 0.383±0.117ng/mg creatinine, NS). Therefore, the method is suitable for the analysis of individual F(2)-IsoPs and PGF(2α)'s in both clinical and research studies.

F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome.

Oxidative damage has been reported in Rett syndrome (RTT), a pervasive developmental disorder caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 gene. Herein, we have synthesized F(2)-dihomo-isoprostanes (F(2)-dihomo-IsoPs), peroxidation products from adrenic acid (22:4 n-6), a known component of myelin, and tested the potential value of F(2)-dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation and disease progression. F(2)-dihomo-IsoPs were determined by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Newly synthesized F(2)-dihomo-IsoP isomers [ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP] were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M-181](-) precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP. Average plasma F(2)-dihomo-IsoP levels in RTT were about one order of magnitude higher than those in healthy controls, being higher in typical RTT as compared with RTT variants, with a remarkable increase of about two orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. hese data indicate for the first time that quantification of F(2)-dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT.

F2-isoprostane formation, measurement and interpretation: the role of exercise.

The level of F2-isoprostanes (F2-IsoP) in blood or urine is widely regarded as the reference marker for the assessment of oxidative stress. As a result, nowadays, F2-IsoP is the most frequently measured oxidative stress marker. Nevertheless, determining F2-IsoP is a challenging task and the measurement is neither free of mishaps nor straightforward. This review presents for the first time the effect of acute and chronic exercise on F2-IsoP levels in plasma, urine and skeletal muscle, placing emphasis on the origin, the methodological caveats and the interpretation of F2-IsoP alterations. From data analysis, the following effects of exercise have emerged: (i) acute exercise clearly increases F2-IsoP levels in plasma and this effect is generally short-lived, (ii) acute exercise and increased contractile activity markedly increase F2-IsoP levels in skeletal muscle, (iii) chronic exercise exhibits trend for decreased F2-IsoP levels in urine but further research is needed. Theoretically, it seems that significant amounts of F2-IsoP can be produced not only from phospholipids but from neutral lipids as well. The origin of F2-IsoP detected in plasma and urine (as done by almost all studies in humans) remains controversial, as a multitude of tissues (including skeletal muscle and plasma) can independently produce F2-IsoP.

Quantification of F(2)-isoprostane isomers in cultured human lung epithelial cells after silica oxide and metal oxide nanoparticle treatment by liquid chromatography/tandem mass spectrometry.

F(2)-isoprostanes are lipid peroxidation products of arachidonic acid in cell membrane and are reliable biomarkers for oxidative stress and cell membrane damage. Nanomaterials are widely used as raw materials in many industries and will have high potentials to be used in life science and medical fields. However, the human health impact of nanoparticles has caused people's great concern. Unfortunately, the mechanisms of cytotoxicity of many nanoparticles are not well defined. By measuring the levels of F(2)-isoprostane isomers in cultured cells after nanoparticle exposure, the information can be used to explain whether the cytotoxicity of nanoparticles is caused by lipid peroxidation and to investigate the biological pathways. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify F(2)-isoprostane isomers in nanoparticle-treated human lung cancer cells. Silica oxide (15nm) and other four metal oxide nanoparticles including Fe(2)O(3) (30nm), Al(2)O(3) (13nm), TiO(2) (40nm) and ZnO (70nm) are chosen in this study. The isotope forms of F(2)-isoprostane isomers, 8-iso-PGF(2alpha)-d(4) and PGF(2alpha)-d(4), were used as internal standard (IS). After human lung epithelial cells were exposed to different nanoparticles for 24h, F(2)-isoprostanes were extracted by a single step solid phase extraction with Oasis HLB cartridge. For the first time, six F(2)-isoprostane isomers were tentatively identified and quantified in human lung epithelial cells. The levels of F(2)-isoprostane isomers in the cells increased after the treatment with nanoparticles. For SiO(2), Fe(2)O(3), and ZnO nanoparticles, F(2)-isoprostane isomers increasing are consistent with nanoparticles' cytotoxicity data. For Al(2)O(3) and TiO(2) nanoparticles, F(2)-isoprostane isomers levels increased even before nanoparticles showed significant cytotoxicity at 100microg/mL concentration in 24h. Based on our best knowledge, this is the first study on the F(2)-isoprostane isomers corresponding to nanoparticles' exposure in vitro. Our study demonstrates that SiO(2) (15nm) nanoparticle showed the highest degree of lipid peroxidation and cell membrane damage among the studied nanoparticles.

Measurement of urinary F(2)-isoprostanes by gas chromatography-mass spectrometry is confounded by interfering substances.

Analysis of F(2)-isoprostanes in urine using gas chromatography-mass spectrometry is confounded by the presence of endogenous compounds interfering with the internal standard, 15-F(2t)-IsoP-d(4) (m/z 573). Previous efforts to resolve the 15-F(2t)-IsoP-d(4) from co-eluting peaks with different solid phase extractions were unsuccessful. This study has now used a highly-deuterated, d(9)-analogue of the derivatization agent N,O-Bis(trimethyl-d(9)-silyl) trifluoroacetamide (BSTFA-d(9)) yielding trimethylsilyl ethers, but this was not successful in resolving the 15-F(2t)-IsoP-d(4) from co-eluting peaks. It was hypothesized that interfering peaks at m/z 573 could be the tetrahydro analogue of 15-F(2t)-IsoP. However, using an authentic standard showed the interfering peaks are not due to this metabolite. In subsequent experiments good resolution was shown of the 15-F(2t)-IsoP peak using 8-F(2t)-IsoP-d(4) (m/z 573) as the internal standard. These data show that care must be taken when using GC-MS for quantitation of F(2)-IsoPs to prevent interfering substances affecting the results.

Human plasma concentrations of malondialdehyde (MDA) and the F2-isoprostane 15(S)-8-iso-PGF(2alpha) may be markedly compromised by hemolysis: evidence by GC-MS/MS and potential analytical and biological ramifications.

OBJECTIVES: Malondialdehyde (MDA) and the F(2)-isoprostane 15(S)-8-iso-prostaglandin F(2alpha) (15(S)-8-iso-PGF(2alpha)) belong to the most frequently analyzed biomarkers of oxidative stress in basic and clinical research. The objective of the present study was to examine the effect of hemolysis on free MDA and total (free+esterified) 15(S)-8-iso-PGF(2alpha) concentrations in human plasma. DESIGN AND METHODS: MDA and 15(S)-8-iso-PGF(2alpha) were determined by GC-MS/MS in plasma samples from venous heparinized blood drawn under resting conditions (n=22) as well as under physical exercise (n=158) in 22 healthy young subjects. In vitro, we prepared plasma samples with hemolysis degrees up to 0.8% using artificially hemolyzed, freshly obtained heparinized blood. RESULTS: In some plasma samples of the exercise study both under resting and exercise conditions, clinically significant hemolysis was macroscopically visible. Both in vivo (r=0.74) and in vitro (r=0.87), we found a significant positive correlation between hemolysis degree (0-0.2%) and MDA plasma concentrations (50-250 nmol/L). Unlike in vitro (r=0.84), in vivo, 15(S)-8-iso-PGF(2alpha) and MDA plasma concentrations correlated weakly (r=0.50). CONCLUSIONS: We hypothesize that free hemoglobin catalyzes the formation of MDA and 15(S)-8-iso-PGF(2alpha) from free and esterified arachidonic acid. Plasma concentrations of MDA and total 15(S)-8-iso-PGF(2alpha) may be markedly compromised by hemolysis. Measurements of MDA and 15(S)-8-iso-PGF(2alpha) should be treated with caution regarding involvement of oxidative stress in disease as well as in health both under resting conditions and under exercise.

Quantitation of plasma total 15-series F(2)-isoprostanes by sequential solid phase and liquid-liquid extraction.

F(2)-isoprostanes are stereo- and regioisomers of prostaglandin F(2alpha) (PGF(2alpha)) and are used as biomarkers for lipid peroxidation. We modified our liquid-liquid extraction (LLE) procedure for F(2)-isoprostane analysis (Anal. Biochem. 350 (2006) 41-51) to use a combination of solid phase extraction (SPE) and LLE to produce a cleaner extract that can be easily concentrated. In addition, shortening the high-performance liquid chromatography (HPLC) separation increased peak heights in HPLC-tandem mass spectrometry (HPLC-MS/MS) analysis. Both changes increased the overall sensitivity of the assay. MS/MS analysis served to confirm the identity of specific peaks that may be better biomarkers than the commonly measured 8-iso-PGF(2alpha).

Oxidative damage in Parkinson disease: Measurement using accurate biomarkers.

Oxidative damage has been implicated in the pathogenesis of Parkinson disease (PD) but the literature data are confusing. Using products of lipid and DNA oxidation measured by accurate methods, we assessed the extent of oxidative damage in PD patients. The levels of plasma F(2)-isoprostanes (F(2)-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), cholesterol oxidation products, neuroprostanes (F(4)-NPs), phospholipase A(2) (PLA(2)) and platelet activating factor-acetylhydrolase (PAF-AH) activities, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), and serum high-sensitivity C-reactive protein were compared in 61 PD patients and 61 age-matched controls. The levels of plasma F(2)-IsoPs, HETEs, 7beta-and 27-hydroxycholesterol, 7-ketocholesterol, F(4)-NPs, and urinary 8-OHdG were elevated, whereas the levels of plasma PLA(2) and PAF-AH activities were lower, in PD patients compared to controls (p< 0.05). The levels of plasma F(2)-IsoPs, HETEs, and urinary 8-OHdG were higher in the early stages of PD (p trend< 0.05). There was a significant negative correlation between the cumulative intake of levodopa and urinary 8-OHdG (r= -0.305, p= 0.023) and plasma total HETEs (r= -0.285, p= 0.043). Oxidative damage markers are systemically elevated in PD, which may give clues about the relation of oxidative damage to the onset and progression of PD.

Using isoprostanes as biomarkers of oxidative stress: some rarely considered issues.

The measurement of F2-isoprostanes by methods utilizing mass spectrometry is widely regarded as the best currently available biomarker of lipid peroxidation. F2-isoprostanes and their metabolites can be measured accurately in plasma, urine, and other body fluids using mass spectrometric techniques, and detailed protocols have been published in several papers. However, many clinical studies and intervention studies with diets or supplements, have employed single "spot" measurements of F2-isoprostanes on either plasma/serum or urine to estimate "oxidative stress." This review examines the validity of the common assumption that plasma and urinary F2-isoprostane measurements are equivalent. It identifies scenarios where they may not be and where "spot" measurements can be misleading, with examples from the literature. We also discuss the controversial issue of whether and how F2-isoprostane levels in plasma should be standardized against lipids, and, if so, which lipids to use.

Key issues in F2-isoprostane analysis.

A large body of evidence indicates that measurement of F2-isoprostanes, specific prostaglandin F2-like compounds derived from the non-enzymatic peroxidation of arachidonic acid, is a reliable biomarker of oxidant stress in the human body. Since the discovery of F2-isoprostanes in the early 1990s, a variety of analytical approaches has been introduced for the quantification of these novel compounds. The aim of the present review is to shed light on the available gas chromatographic-mass spectrometric assays for the measurement of plasma or urinary F2-isoprostanes and to highlight a number of issues which need to be addressed in order to implement F2-isoprostane measurement as a gold-standard biomarker of oxidative stress in biological samples.

Women and smokers have elevated urinary F(2)-isoprostane metabolites: a novel extraction and LC-MS methodology.

F(2)-Isoprostanes (F(2)-IsoPs), regio- and stereoisomers of prostaglandin F(2alpha) (PGF(2alpha)), and urinary F(2)-IsoP metabolites including 2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha) [2,3-dinor-8-iso-PGF(1alpha) (2,3-dinor-F1)] and 2,3 dinor-8-iso-PGF(2alpha) (2,3-dinor-F2), have all been used as biomarkers of oxidative stress. A novel method was developed to measure these biomarkers using a single solid phase extraction (SPE) cartridge, separation by HPLC, and detection by negative mode selected reaction monitoring (SRM) mass spectrometry (MS), using authentic standards of PGF(2alpha); 8-iso-PGF(2alpha); 2,3-dinor-F1 and 2,3-dinor-F2 to identify specific chromatographic peaks. The method was validated in a population of healthy, college-aged nonsmokers (n = 6 M/8F) and smokers (n = 6 M/5F). Urinary F(2)-IsoP concentrations were approximately 0.2-1.5 microg/g creatinine, 2,3-dinor-F1 was approximately1-3 microg/g and 2,3-dinor-F2 was approximately 3-5 microg/g. Additional F(2)-IsoPs metabolites were identified using SRM. The sum of all urinary F(2)-IsoP metabolites was 50-100 microg/g creatinine indicating their greater abundance than F(2)-IsoPs. Women had higher F(2)-IsoP metabolite concentrations than did men (MANOVA, main effect P = 0.003); cigarette smokers had higher concentrations than did nonsmokers (main effect P = 0.036). For men or women, respectively, smokers had higher metabolite concentrations than did nonsmokers (P < 0.05). Thus, our method simultaneously allows measurement of urinary F(2)-IsoPs and their metabolites for the determination of oxidative stress.

F2-isoprostanes in human health and diseases: from molecular mechanisms to clinical implications.

Oxidative stress is implicated as one of the major underlying mechanisms behind many acute and chronic diseases, and involved in normal aging. However, the measurement of free radicals or their end products is complicated. Thus, proof of association of free radicals in pathologic conditions has been absent. Isoprostanes are prostaglandin-like bioactive compounds that are biosynthesized in vivo independent of cyclooxygenases, principally through free-radical catalyzation of arachidonic acid. Isoprostanes are now considered to be reliable biomarkers of oxidative stress, as evidenced by an autonomous study organized recently by the National Institutes of Health (NIH) in the United States. A number of these compounds have potent biologic activities such as vasoconstrictive and certain inflammatory properties. Isoprostanes are involved in many human diseases. Additionally, elevated levels of F(2)-isoprostanes have been seen in normal human pregnancy and after intake of some fatty acids, but their physiologic assignments have not yet been distinctive. This evidence indicates that measurement of bioactive F(2)-isoprostanes in body fluids offers a unique noninvasive analytic utensil to study the role of free radicals in physiology, oxidative stress-related diseases, experimental acute or chronic inflammatory conditions, and also in the assessment of various antioxidants, radical scavengers, and drugs.

F2-isoprostanes are not just markers of oxidative stress.

F(2)-isoprostanes are not just markers of oxidative stress. The discovery of F(2)-isoprostanes (F(2)-IsoPs) as specific and reliable markers of oxidative stress in vivo is briefly summarized here. F(2)-IsoPs are also agonists of important biological effects, such as the vasoconstriction of renal glomerular arterioles, the retinal vessel, and the brain microcirculature. In addition to the F(2)-IsoPs, E(2)- and D(2)-IsoPs can be formed by rearrangement of H(2)-IsoP endoperoxides and can give rise to cyclopentenone IsoPs, which are very reactive alpha,beta-unsaturated aldehydes. The same type of reactivity is also shown by acyclic gamma-ketoaldehydes formed as products of the IsoP pathway. Because previous studies suggested a relation between oxidative stress and collagen hyperproduction, it was investigated whether collagen synthesis is induced by F(2)-IsoPs, the most proximal products of lipid peroxidation. In contrast to aldehydes, F(2)-IsoPs act through receptors able to elicit definite signal transduction pathways. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma F(2)-IsoPs were markedly elevated for the entire experimental period; hepatic collagen content was also increased. When hepatic stellate cells from normal liver were cultured up to activation (expression of smooth muscle alpha-actin) and then treated with F(2)-IsoPs in the concentration range found in the in vivo studies (10(-9) to 10(-8) M), a striking increase in DNA synthesis, cell proliferation, and collagen synthesis was observed. Total collagen content was similarly increased. All these stimulatory effects were reversed by the specific antagonist of the thromboxane A(2) receptor, SQ 29 548, whereas the receptor agonist, I-BOP, also had a stimulatory effect. Therefore F(2)-IsoPs generated by lipid peroxidation in hepatocytes may mediate hepatic stellate cell proliferation and collagen hyperproduction seen in hepatic fibrosis.

Potential role of isoketals formed via the isoprostane pathway of lipid peroxidation in ischemic arrhythmias.

Unabated reactive oxygen species (ROS) are potentiated by an ischemia-induced shift in anaerobic metabolism, which generates superoxide anion upon reperfusion and reintroduction of oxygen. ROS can modify protein structure and function in fundamental ways, one of which is by forming reactive lipid species from the oxidation of lipids. In this review, we discuss these pathways and discuss the literature that shows that these species can produce dramatic effects on cardiac ion channel function (eg, Na+ channel function). Furthermore, we review what is known about the generation of such in the highly remodeled post myocardial infarction substrate. We suggest prevention of adduction of these highly reactive compounds would be antiarrhythmic.

Time course and attenuation of ischaemia-reperfusion induced oxidative injury by propofol in human renal transplantation.

Ischaemia-reperfusion injury resulting from interruption and restoration of blood flow might be related to free radical mediated oxidative stress and inflammation, and subsequently to post-surgery related complications. We studied the impact of renal transplantation on oxidative stress and inflammation by measuring F(2)-isoprostanes and prostaglandin F(2alpha), respectively, during transplantation and post-surgery. Additionally, due to earlier observations, two dissimilar anaesthetic agents (thiopentone and propofol) were compared to determine their antioxidative capacity rather than their anaesthetic properties. Blood samples were collected before, post-intubation, immediately, 30, 60,120, 240 min, and 12 and 24 h after reperfusion. Oxidative stress and inflammatory response were detected by measuring 8-iso-PGF(2alpha) (a major F(2)-isoprostane and a biomarker of oxidative stress) and 15-keto-dihydro-PGF(2alpha) (a major metabolite of PGF(2alpha) and a biomarker of COX-mediated inflammatory response), respectively. Reperfusion of the transplanted graft significantly increased plasma levels of 8-iso-PGF(2alpha). PGF(2alpha) metabolite levels, although elevated, did not reach statistical significance. In addition, significantly lower levels of 8-iso-PGF(2a) were observed in the propofol group compared to the thiopentone group. Together, these findings underline an augmented oxidative stress activity following an inflammatory response after human renal transplantation. Furthermore, propofol a well-known anaesthetic, counteracted oxidative stress by lowering the formation of a major F(2)-isoprostane.

Quantification of F2-isoprostanes as a biomarker of oxidative stress.

Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2alpha)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F2-isoprostanes (F2-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent independent study sponsored by the National Institutes of Health (NIH), F2-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well known biomarkers. This protocol details our laboratory's method to quantify F2-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry (GC-MS). This procedure can be completed for 12-15 samples in 6-8 h.

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

F(2)-isoprostanes (F(2)-iPs), established markers of oxidative stress, exist as four sets of regioisomers. Simultaneous and specific determination of F(2)-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 F(2)-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 [(3)H]8-iso-prostaglandin (PG)F(2alpha) (iPF(2alpha)-III) when the methanol concentration was increased under acidic, neutral, and base wash conditions. A multidimensional (MD)-SPE method that incorporated size exclusion chromatography [corrected] reverse-phase chromatography, and normal-phase chromatography was developed using an Oasis HLB SPE cartridge and an HLB microElution SPE plate. Average extraction recoveries of the deuterated internal standards of iPF(2alpha)-III and iPF(2alpha)-VI were 62 +/- 8% and 60 +/- 10%. A buffer-free HPLC method for the separation of F(2)-iP isomers was developed on base-deactivated C8 columns. Average matrix effects for iPF(2alpha)-III and iPF(2alpha)-VI were 95 +/- 6% and 103 +/- 5%. The clean extraction of urine F(2)-iPs using MD-SPE and the separation of F(2)-iP isomers using a novel HPLC method did not cause apparent ion suppression in the analysis of iPF(2alpha)-III and iPF(2alpha)-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F(2)-iPs.

Augmentation of monocyte intracellular ascorbate in vitro protects cells from oxidative damage and inflammatory responses.

Ascorbic acid is present as a primary antioxidant in plasma and within cells, protecting both cytosolic and membrane components of cells from oxidative damage. The effects of intracellular ascorbic acid on F(2)-isoprostanes (biomarkers of oxidative stress) and monocyte chemoattractant protein-1 (marker of inflammatory responses) production in monocytic THP-1 cells were investigated under conditions of 2,2'-Azobis(2-methylpropionamidine)dihydrochloride (AAPH) induced oxidative stress. Cells cultured under normal conditions have extremely low ascorbate levels and the intracellular ascorbate can be augmented significantly by adding ascorbate to the culture medium. While AAPH treatment reduced cell viability, increased F(2)-isoprostanes and MCP-1 production, the presence of intracellular ascorbic acid maintained high cell viability and attenuated both F(2)-isoprostanes and MCP-1 production. Measurement of intracellular ascorbic acid and its oxidised products showed that intracellular ASC was oxidised to a significantly greater extent during AAPH treatment and may be utilised to protect the cells under conditions of oxidative stress. This study demonstrates the importance of intracellular ascorbate, which may be lacking under normal cell culture conditions, under conditions of increased oxidative stress.