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Background: Lysophosphatidylcholine (LPC) has been suggested to play a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors. Initial studies provided evidence for a potential use of LPC as diagnostic maker. However, existing methodologies are of limited value for a systematic evaluation of LPC species concentrations because of complicated, time-consuming procedures. We describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC quantification. Methods: Crude lipid extracts of EDTA-plasma samples were used for direct flow injection analysis. LPC 13:0 and LPC 19:0 were added as internal standards, and the ESI-MS/MS was operated in the parent-scan mode for m/z 184. Quantification was achieved by standard addition. Data processing was highly automated by use of the mass spectrometer software and self-programmed Excel macros. Results: The calibrators LPC 16:0, LPC 18:0, and LPC 22:0 showed a linear response independent of sample dilution and plasma cholesterol concentration for both internal standards. The within-run imprecision (CV) was 3% for the major and 12% for the minor species, whereas the total imprecision was ∼12% for the major and 25% for the minor species. The detection limit was <1 μmol/L. Conclusion: The developed ESI-MS/MS methodology with an analysis time of 2 min/sample, simple sample preparation, and automated data analysis allows high-throughput quantification of distinct LPC species from plasma samples, which could be a valuable tool for the evaluation of LPC as diagnostic marker.

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Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH ؉ 2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively. The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling. Sphingolipids, i.e., ceramide (CER), sphingosine-1-phosphate (SPP), and sphingosine (SPH) are known as important signaling molecules involved in the modulation of cell growth, differentiation, and apoptosis (1-3). In a cell type-specific manner, SPH has been shown to mediate pro-or antimitogenic effects (4, 5) and to regulate the activity of various signaling enzymes, including phospholipase D, diacylglycerol kinase, and protein kinase C (1, 2). In mammalian cells, SPH, with 18 carbon atoms and a trans double bond at position 4, comprises the backbone of more complex sphingolipid molecules. Sphinganine (SPA), which lacks the double bond, is an intermediate of sphingolipid biosynthesis without a known signaling function.The existing detection methods for sphingoid bases involve different derivatization steps in order to enable quantification of the low cellular concentrations. A commonly used method established by Merrill et al. (6,7) utilizes HPLC with fluorescence detection after orthophthaldialdehyde (OPA) derivatization of SPH and SPA. Other procedures are based on the conversion of SPH to radioactive derivates. Thus, SPH can be acetylated by [ 3 H]acetic-anhydride to N -[ 3 H]acetyl-SPH, which is separated by TLC, scraped, and analyzed by liquid scintillation counting (8). Other enzymatic methods include SPH kinase-mediated conversion of SPH to [ 32 P]SPP, followed by TLC separation and autoradiography (9, 10) or the conversion of SPH to C6-CER, with subsequent generation of C6-CER-1-[ 32 P]phosphate by diacylglycerol-kinase (11). A...

3,3'-Dimethoxy-2,2'-bipyrrole (1) and 4,4'-dimethoxy-2,2'-bipyrrole (2) were obtained in short sequences and good yields from N-benzyl-3-hydroxypyrrole-2,4-dicarboxylic acid. The key intermediate leading to 1 is an N-benzyl-3-methoxypyrrole, which is dimerized by lithiation and oxidation with NiCl(2). The formation of 2 is achieved by a classical Ullmann coupling of diethyl 1-benzyl-2-bromo-4-methoxypyrrole-3,5-dicarboxylate. The N-benzyl protection groups of 1 and 2 are cleaved under reducing conditions with sodium in liquid ammonia. Both isomeric bipyrroles are extremely sensitive toward air. Compound 1 has a very low oxidation potential of 0.09 V against AgCl but film formation hardly occurs. On the other hand, compound 2 with a potential of 0.35 V readily forms stable polypyrrole films with anodic waves at -0.51 and -0.35 V and a cathodic wave at -0.77 V, the lowest potential ever observed for a p-doped polymer.