Various phosphatidylethanol (PEth) derivatives, the corresponding reversed positional isomers (RPI-PEths), lyso-PEth-16:0, and penta-deuterium-labeled PEth analogs (d5-PEths), were synthesized by enzyme-independent synthetic routes. A general solvent system consisting of a mixture of acetone-d6 and methanol-d4 (97:3; v/v) was found to provide a good solubilizing capacity and excellent hydrogen-1 NMR (1H-NMR) peak resolution of various PEth homologues. Analytical differentiation of PEth from the corresponding RPI-PEth by carbon-13 NMR (13C-NMR) was demonstrated by comparison of the 13C-NMR signals of the carbonyl groups, the allylic positions, and of the β-carbons. An exemplary stable long-term room temperature, DMSO-d6-based, and proton-sensitive quantitative nuclear magnetic resonance (1H-qNMR) independently quantified calibrator comprising PEth-16:0/18:1 for liquid chromatography (tandem) mass spectrometry (LC-MS/MS) analytical applications were prepared by employment of sodium dodecyl sulfate (SDS) as a solubilizing additive. In summary, novel hypothetically occurring PEth derivatives, e.g., RPI-PEths, have been independently synthesized with regio- and stereochemical control. Use of polar organic solvents, e.g., mixtures of acetone-d6 and methanol-d4 or DMSO-d6, improves spectral line shapes as compared to traditional hydrophobic solvents and allow for analytical differentiation between closely related PEth derivatives, as well as LC-MS/MS-independent concentration determination of dissolved single species by employment of 1H-qNMR.
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We are grateful to Innovator Skåne AB for the provision of laboratory facilities and for major sponsoring, to Can Slivo for excellent help with the resynthesis of early intermediates, to Dr. Anders Blomgren and the staff at the analytical laboratories of Skåne University Hospital for LC-MS/MS quantification of calibrators, to Dr. Johan Evenäs at Read Glead Discovery AB for the collection and processing of 1H-qNMR spectra, and to Dr. Ulf Annby for proofreading the manuscript.
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Wensbo Posaric, D., Andersson, A., Bergquist, K. et al. Differentiation and quantification of synthetic phosphatidylethanol (PEth) homologues by 1H- and 13C-NMR in polar organic solvents. Anal Bioanal Chem 406, 4735–4744 (2014). https://doi.org/10.1007/s00216-014-7826-4
- Analytical reference
- Positional isomers