Determination of bioactive eicosanoids in brain tissue by a sensitive reversed-phase liquid chromatographic method with fluorescence detection

A liquid chromatography/mass spectrometric method for simultaneous analysis of arachidonic acid and its endogenous eicosanoid metabolites prostaglandins, dihydroxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and epoxyeicosatrienoic acids in rat brain tissue

“…To simultaneously separate 19 eicosanoids (PGs, DiHETrEs, HETEs, EETs, and AA) and 7 deuterated internal standards, we used a Symmetry® C18 column (4.6mm × 250 mm, 5 µm) that was used in our lab for separating 14 fluorescent labeling PGs, DiHETrEs, HETEs, EETs, and AA simultaneously [28]. A flow rate of 1 ml/min was used throughout the method development.…”

“…Before validation of the method, the method was fine-tuned by coupling the LC/MS conditions with the sample preparation procedure modified from the one developed in our lab [28]. Each step was optimized relative the others for the purpose of easy operation and robustness.…”

“…To investigate the possibility that COX2 inhibitors may play a role in inhibition of pro-inflammatory eicosanoids and shunting the AA metabolism to anti-inflammatory eicosanoids in the brain after TBI, it is necessary to establish a specific, sensitive and reliable analytical method for simultaneous identification and quantification of these endogenous eicosanoid in brain samples. [20][21][22][23][24], capillary electrophoresis/ UV [25], and HPLC/fluorescence [26][27][28], MS/MS, liquid chromatography/mass spectrometric (LC/MS) and LC/MS/MS methods, have been used to measure some of these eicosanoids at low levels. GC/MS and GC/MS/MS with negative ion chemical ionization has been the most commonly used technique and can give specific mass information of the peaks.…”

“…EETs), and needs tedious steps including TLC purification and derivatization before analysis. The need for highly sensitive and low cost methods for analyzing labile bioactive eicosanoids has initially driven the development of derivatized fluorescent HPLC method [28] to simultaneously analyze PGs, DiHETrEs, HETEs and EETs in brain tissues at pg levels in our lab. However the separation was long and the high background from tissue matrix was seen throughout the chromatogram even using solid phase extraction clean-up procedure.…”

UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

“…Both cells and medium were collected, centrifuged (12,000 × g for 10 min at 4°C) and aliquots of the supernatants evaporated to dryness under helium. Prostaglandin metabolites were then derivatized as described previously (Yue et al, 2004). Briefly, the dried extracts were solubilized in anhydrous acetonitrile, supplemented with N,N-diiospropylethylamine catalyst (dried with 5 Å molecular sieves) and NE-OTf (2 mg/ml in anhydrous acetonitrile).…”