Differentiation and quantification of synthetic phosphatidylethanol (PEth) homologues by 1H- and 13C-NMR in polar organic solvents

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.