Abstract
High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC-MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at -80 degrees C was reduced 2- to 4- fold in 4 weeks; however, PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave-irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.
Highlights
High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization
Several different methods are used for brain PG extraction, including methanol extraction followed by solid-phase extraction [9, 12, 13], hexane/2-propanol extraction [14], ether extraction [15], and acetone/chloroform extraction [16]
Methanol extraction followed by solid-phase extraction [9] and ether extraction [15] have been used in different LC-MS/MS procedures, it is important to note that the simultaneous evaluation of these different extraction methods for use in brain PG analysis using LC-MS/MS has not been done
Summary
High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. These methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation This reduction is not the result of PG trapping or destruction in microwaveirradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Methanol extraction followed by solid-phase extraction [9] and ether extraction [15] have been used in different LC-MS/MS procedures, it is important to note that the simultaneous evaluation of these different extraction methods for use in brain PG analysis using LC-MS/MS has not been done. These studies do not provide direct evidence that PG are not trapped or destroyed in microwaved brains
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