Rotating-disc micro-solid phase extraction of F2-isoprostanes from maternal and cord plasma by using oxidized buckypaper as sorbent membrane.

This paper describes the development of an original micro-solid phase extraction device and its evaluation for the isolation of F2-isoprostanes (F2-IsoPs) from cord and maternal plasma samples. The unit is very simple and consists in a rotating disc (1.8 cm diameter) of oxidized buckypaper (BP), enwrapped in a polypropylene mesh pouch. Even if the selected F2-IsoPs have logP and pKa values that make them suitable candidates for their sorption on BP, several parameters were optimized to maximize recoveries: time of adsorption and desorption; stirring speed; volume, pH and ionic strength of the sample; type, volume, and fractions of the elution solvent; oxidation grade of BP. Among all, the last one was crucial in affecting extraction yields because of the analyte interactions with polar functionalities, introduced by a preliminary oxidative acid treatment. The investigation established the optimal oxidation time and highlighted the pros and cons of the acid activation step. All extracts were analyzed by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation was performed according to the main FDA guidelines for bioanalytical methods. Depending on the spike level and analyte, recoveries ranged between 30 and 120% with precision and accuracy values lower than 20%. Quantitative analysis was accomplished by matrix-matched calibration curves whose determination coefficients were higher than 0.95. Lower limit of quantitation (LLOQ) spanned the range 2.45-6.77 µg L-1. The validated method was applied to the analysis of eight pairs of mother/child plasma samples, revealing the presence of 8-iso-15-keto-PGF2α and 8-iso-PGE2 at a concentration of about 10 µg L-1 in most cord plasma samples of preterm newborns.

Analysis of F2-isoprostanes in plasma of pregnant women by HPLC-MS/MS using a column packed with core-shell particles.

Plasma F2-isoprostanes (F2-isoPs) are reliable biomarkers of oxidative stress. Several possible F2-isoPs are generated by the oxidation of arachidonic acid esterified in phospholipids. The separation of these isomers represents a technical challenge for rapid and selective determination. We have developed a HPLC-MS/MS method for the simultaneous determination of seven plasma F2-isoPs, namely 8-iso-15(R)-prostaglandin F2α (PGF2α), 8-iso-PGF2α, 15(R)-PGF2α, iPF2α-IV, iPF2α-VI, 5-iPF2α-VI, and (±)5-8,12-iso-iPF2α-VI. We have validated this method in plasma of pregnant women, a mild physiological oxidative stress known to increase F2-isoPs. Thus, plasma samples of women collected at the third trimester of pregnancy (n = 20) were subjected to alkaline hydrolysis followed by liquid-liquid extraction in order to extract total F2-isoPs. The F2-isoPs were separated within 16.5 min using a column packed with core-shell particles. The class VI isomers were the most abundant, accounting for 65% of the total level of all quantified F2-isoPs in plasma of pregnant women (P < 0.05). The 15(R)-PGF2α was the most abundant of the class III isomers quantified. This method allowed fast and selective separation of seven isomers from three different classes of F2-isoP regioisomers.

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).

Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods.

Isoprostanes are isomers of prostaglandins that are generated from free radical-initiated autoxidation of arachidonic acid. Quantification of F(2)-isoprostanes is regarded as the "gold standard" to assess oxidative stress in various human diseases. There are 32 possible racemic isoprostane isomers that exist as four sets of regioisomers. Each regioisomer is composed of eight diastereomers. We report liquid chromatographic/mass spectrometric methods to separate and identify F(2)-isoprostane stereoisomers. These methods have been applied to the analysis of F(2)-isoprostanes derived from tissues of rats exposed to an oxidative stress and are useful to assess the relative formation of various regioisomers and stereoisomers generated in vitro and in vivo. The delineation of the more abundant isomers formed will allow for studies to examine the biological relevance of selected compounds in vivo.