Measurement of marine osmolytes in mammalian serum by liquid chromatography-tandem mass spectrometry

Abstract

Osmolytes are accumulated intracellularly to offset the effects of osmotic stress and protect cellular proteins against denaturation. Because different taxa accumulate different osmolytes, they can also be used as âEURoedietary biomarkersâEUR to study foraging. Potential osmolyte biomarkers include glycine betaine, trimethylamine N-oxide (TMAO), homarine, dimethylsulfoniopropionate (DMSP), and the osmolyte analog arsenobetaine (AsB). We present a liquid chromatographyâEUR"tandem mass spectrometry (LCâEUR"MS/MS) assay for the simultaneous measurement of these osmolytes in serum or plasma. Varying concentrations of osmolytes were added to serum and samples and extracted in 90% acetonitrile and 10% methanol containing 10 μM deuterated internal standards (D9-glycine betaine, D9-trimethylamine-N-oxide, 13C2-arsenobetaine, D6-DMSP, and D4-homarine). Analytes were separated on a normal-phase modified silica column and detected using isotope dilution tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The assay was linear for all six compounds (r2 values = 0.983âEUR"0.996). Recoveries were greater than 85%, and precision for within-batch coefficients of variation (CVs) were less than 8.2% and between-batch CVs were less than 6.1%. Limits of detection ranged from 0.02 to 0.12 μmol/L. LCâEUR"MS/MS is a simple method with high throughput for measuring low levels of osmolytes that are often present in biological samples.