Billy Molloy (Presenter)
Waters Corporation
Authorship: Billy Joe Molloy
Waters Corporation, Wilmslow, UK
| Short Abstract Oxylipins are signaling molecules that play a role in the regulation of many key biological processes, most notably inflammation. Here, we describe targeted, quantitative SPE-UPLC-MS-MS assays for the analysis of various oxylipins subsets and classes. These subsets represent down-stream products or oxylipins from particular metabolic pathways. Matrix samples were prepared using mixed mode OASIS MAX µElution SPE and analysed using an ACQUITY UPLC I-Class system interfaced to a Xevo TQS Micro Tandem Quadrupole Mass Spectrometer. We demonstrate these methods to be sensitive, selective, linear and precise and therefore suitable for use in translational research studies. |
Long Abstract
Introduction:
Oxylipins are signaling molecules that play a role in the physiological regulation of many key biological processes, most notably inflammation. The ability to quantify oxylipins in biological samples would assist greatly in increasing our understanding of their role in health and disease. Oxylipins are produced via enzymatic and non-enzymatic oxygenation of polyunsaturated fatty acid substrates (e.g., linoleic acid, linolenic acid, adrenic acid and arachidonic acid, α-linolenic acid, acid, eicosapentaenoic acid and docosahexaenoic acid). Three major enzymatic pathways are involved in their generation: cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP). The main challenges for oxylipin quantification in biological samples are the very low concentrations and their limited stability. Moreover, the same fatty acid can be oxidized in different positions of its acyl chain leading to many isomeric species. As a consequence, this requires a highly sensitive and specific analytical method. Historically, measurements of oxylipins have been performed using radiometric and enzymatic immunoassays, which often lacked specificity. GC/MS methodology has also been used, but this still requires multi-step procedures involving derivatization to increase volatility and stability. Here we describe targeted, quantitative solid phase extraction - ultra performance liquid chromatography - tandem mass spectrometry (SPE-UPLC-MS/MS) methods for the analysis of various subsets of oxylipins. These subsets represent either oxylipins originating from a particular precursor or from a particular metabolic pathway. This approach represents a more targeted, quantitative approach than the generic profiling type methodologies that attempt to analyse all oxylipins in a single method. Using this approach, the SPE-UPLC-MS/MS methods developed are more specific than is possible when analyzing a large number of analytes. This leads to an analytical methods that are more applicable to the robust, quantitative analysis required for translational research studies.
Methods:
All solvents and additives were from Sigma-Aldrich and of analytical grade. Oxylipin standards and internal standards were from Cayman Chemicals. Deuterated analogue internal standards were used for all analytes. Human serum was from Golden West Biologicals, DMEM (Dulbecco's Modified Eagle's Medium) buffer and Fetal Bovine Serum were from Sigma-Aldrich and CD-1 mouse livers were from Biochemed Services. For solid phase extraction, mixed mode OASIS MAX µElution SPE 96-well plates (Waters Corparation) were used. Matrix samples were prepared using a 2 step sample preparation protocol. Firstly, internal standard solution was added to the sample (50 – 200µL), this was then made up to a total volume of 600µL. The entire diluted sample was then loaded onto a pre-conditioned mixed mode OASIS MAX µElution SPE 96-well plate. The plate was washed with either a methanol / water or acetonitrile / water mixture. The analytes were then eluted into a collection plate containing a trapping solution. Using an ACQUITY UPLC I-Class system, samples were injected onto a 2.1 x 150 mm ACQUITY UPLC BEH C18 column and separated with a water/acetonitrile/formic acid gradient. The analytes were then detected with a Xevo TQS Micro Mass Spectrometer operating in negative ionization mode.
Results:
SPE-UPLC-MS-MS methods were developed for two subsets of oxylipins. These subsets contained products of the cyclooxygenase (COX) and lipoxygenase (LOX) metabolic pathways. The COX subset contained prostaglandins D2, E2 and F2α (PGD2, PGE2 and PGF2α), 6-keto-prostaglandin F1α (6-Keto-PGF1α) and Thromboxane B2 (TBX2). The LOX subset contained the 5-, 8-, 12-, and 15- positional isomers of hydroxyeicosatetraenoic acid (5-HETE, 8-HETE, 12-HETE and 15-HETE) and the 9- and 13- positional isomers of hydroxyoctadecadienoic acid (9-HODE and 13-HODE). Chromatographic methods were developed for these subsets of analytes that separated them from each other and from other isomeric, potentially isobaric species. For example PGF2α was shown to be baseline resolved from the following isobaric species: 5-Trans- PGF2α, 8-iso- PGF2α, 15(R)-PGF2α, 8-iso-15(R)-PGF2α, PGF2β, 5-Trans- PGF2β and 8-iso- PGF2β. The Limit of detection (LoD) for all compounds in both subsets was shown to be ≤1pg on column. The analytical recovery of all analytes was shown to be above 40% (ranging between 40 – 94%). This was measured using the deuterated analogue internal standards for all analytes from both subsets. This was due to endogenous levels of these analytes in the matrix samples. The precision of each method was determined by analyzing various matrix samples multiple times (n=6). This was performed at various concentration levels. The coefficient of variation (%CV) was demonstrated to be <15% for all analytes from both subsets across the linear range.
Conclusion:
SPE-UPLC-MS/MS methods have been developed for two subsets of oxylipins. These methods have been demonstrated to have excellent sensitivity, selectivity, linearity and precision. These methodologies are therefore ideally suited to translational research studies.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Waters Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |