Determination of betaine metabolites and dimethylsulfoniopropionate in coral tissues using liquid chromatography-time-of-flight mass spectrometry and stable isotope-labeled internal standards.

ABSTRACT

A convenient procedure for determination of seven betaine analogs and dimethylsulfoniopropionate (DMSP) in extracts of coral tissues using LC-MS stable isotope dilution is described. Extraction procedures were optimized for selective extraction of polar metabolites from coral tissues. The LC-MS protocol employed a pentafluorophenylpropyl (PFPP) column for HPLC separation, with chromatographic resolution of isobaric and isomeric zwitterionic metabolites optimized by adjusting the acidity of the mobile phase. A ternary gradient was used to exploit the unusual retention characteristics of cationic metabolites on the PFPP column, with incorporation of ammonium acetate in a later gradient stage promoting elution of more hydrophobic betaines which are retained at high organic content in the absence of ammonium acetate. We demonstrate that the new LC-MS based method provides accurate measurements from nanomolar to high micromolar concentrations, and can be applied for profiling of betaine metabolites and DMSP in corals or other aquatic organisms.