Journal of Pharmaceutical and Biomedical Analysis
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
Introduction
The biological oxidation products of arachidonic acid and related C20 polyene acids are summarized under the term eicosanoids that give rise to a wide variety of products of remarkable physiological activity [1]. Arachidonic acid (AA) can be metabolized to many bioactive eicosanoids (Fig. 1) [2], including oxidation to prostaglandins (PGs), hydroxyeicosatetraenoic acids (HETEs), dihydroxyeicosatrienoic acids (DiHETrEs), and epoxyeicosatrienoic acids (EETs) via cyclooxygenase (COX), cytochrome P450 (CYP450), and lipoxygenase (LOX) pathways (Fig. 2). These endogenous eicosanoids are present at trace levels in biological fluids and tissues, including the mammalian brain. The expression of various eicosanoids is affected by injury or inflammation, in the brain and other tissues. Traumatic brain injury (TBI) is one of the leading causes of death [3]. The physical insults of TBI set into motion a cascade of biochemical events that can result in secondary brain injury that is a major contributor to the ultimate tissue loss [4], [5], [6]. One part of this response is the increased conversion of AA to PGs as a result of induction of cyclooxygenase-2 (COX2) expression after TBI [7]. COX2 is one of the isoforms of COX and is highly induced by inflammatory stimuli [8], [9]. Some of the PGs (i.e. PGE2) possess pro-inflammatory properties [10], [11], [12], and many of the HETEs and EETs derived from AA via CYP450 and LOX pathways are reported to have anti-inflammatory properties [13], [14], [15]. COX2 inhibitors, are being used to treat peripheral inflammation [16], and may prove beneficial to the injured brain as well [10], [17]. DFU [5,5-dimethyl-3(3-fluorophenyl)-4(4-methylsulphonyl)phenyl-2(5H)-furanone], a highly specific COX2 enzyme inhibitor, was shown to improve functional recovery and attenuate neuronal cell death and inflammation in a rat model of TBI in our lab [18], [19]. 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.
Currently, GC/MS, GC/MS/MS [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. However, it is not suitable for labile compounds (i.e. 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. Recently, LC/MS and LC/MS/MS were widely used in bioanalytical work since they are powerful analytical techniques that combine the resolving power of liquid chromatography with detection specificity of mass spectrometry.
Although a few LC/MS or LC/MS/MS (either with chromatographic separation or flow injection analysis) methods have been used for identification and quantification of either single eicosanoid [29], or various PGs [30], or PGs together with HETEs [31], [32], [33], [34], [35], [36], or various DiHETrEs [37], or HETEs together with EETs [38], [39] or HETEs, EETs, together with DiHETrEs [40] in various biological matrices in a single analysis, we could not identify a published mass spectrometric-based method for the simultaneous identification and quantification of PGs, DiHETrEs, HETEs, EETs, and parent compound AA in biological matrices, including brain tissue. In addition, the method validation data were not available in most of these methods. In this paper, we report a sensitive, specific, robust and validated LC/MS method for simultaneously analyzing parent compound AA and its COX, CYP450 and LOX pathway metabolites PGs, DiHETrEs, HETEs and EETs, including PGF2α, PGE2, PGD2, PGJ2,14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET in rat cortical brain tissue.
Section snippets
Chemicals and materials
(±)14,15-Dihydroxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-DiHETrE), (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-DiHETrE), (±)8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-DiHETrE), (±)5,6-dihydroxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-DiHETrE), 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (20-HETE), (±)15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE), (±)12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE), (±)9-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid (9-HETE),
Results and discussion
The overall method development included four stages:
- 1.
An LC/MS method for 19 eicosanoid standards including PGs, HETEs, DiHETrEs, EETs and AA, and 7 deuterated internal standards was developed and optimized with the aim of resolution and sensitivity (Section 3.1).
- 2.
The method was fine-tuned by coupling the LC/MS conditions with the sample preparation procedure, the sensitivity and linearity of standards, internal and external standardization, concentration range, and the method validation design
Conclusion
This study is first to report a sensitive, specific, robust and validated LC/MS method that allows simultaneous analysis of parent compound AA and its COX, CYP450 and LOX pathway metabolites PGs, DiHETrEs, HETEs and EETs, including PGF2α, PGE2, PGD2,PGJ2, 14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET in rat brain tissues. LC/MS conditions were optimized for the best sensitivity and separation
Acknowledgement
The authors thank Mamta Amin for expert technical assistance.
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