ABSTRACT
A validated method is described for the simultaneous analysis of PGE2, 11-, 12-, and 5-HETEs from cultured cells using HPLC negative electrospray ionization tandem mass spectrometry (LC/MS/MS). This method permits quantification of selected individual arachidonic acid metabolites from cell extracts without derivatization, multiple purification steps, or lengthy separation times required by traditional GC-MS- or HPLC-UV -based methods. Accuracy assessments of values calculated using this method showed deviations from nominal values were < or =15%. An average relative deviation of 7% of mean calculated values was observed for values taken on separate days. The lower limit of detection for all metabolites was 1.3 pg. The method was used to quantify arachidonic acid metabolites present in various cancer cell lines after incubation with arachidonic acid and the selective cyclooxygenase-2 inhibitor celecoxib. Results showed that the presence of celecoxib in lung cancer A549 cells reduced production of both PGE2 and 11-HETE in a concentration-dependent manner.
Subject(s)
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/analysis , Dinoprostone/analysis , Hydroxyeicosatetraenoic Acids/analysis , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Animals , Arachidonic Acid/metabolism , Celecoxib , Chromatography, High Pressure Liquid/methods , Cyclooxygenase 2 , Dinoprostone/metabolism , Humans , Hydroxyeicosatetraenoic Acids/metabolism , Isoenzymes/antagonists & inhibitors , Leukemia/metabolism , Lipoxygenase/metabolism , Lung Neoplasms/metabolism , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/metabolism , Pyrazoles , Rats , Spectrometry, Mass, Electrospray Ionization/methods , Sulfonamides/metabolism , Sulfonamides/pharmacology , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/metabolismABSTRACT
A rapid method for the screening of metal binding selectivities of host compounds in mixtures is presented. This method involves the separation of mixtures of hosts by HPLC, followed by postcolumn complexation with one or more metals, then analysis by mass spectrometry. The intensities of the host-guest complexes in the mass spectra correlate with the binding selectivities of the hosts. The method was applied to a series of lariat ethers that were synthesized as ion-selective reagents for ion-selective electrodes. The compounds most selective for Na+ vs Li+ and K+ were identified. Additionally, a mixture of substituted calixarenes was screened for alkali-metal-binding selectivity. These compounds were determined to be selective for Cs+ over Rb+, K+, and Na+.
