Comprehensive quantitative analysis of fatty-acyl-Coenzyme A species in biological samples by ultra-high performance liquid chromatography–tandem mass spectrometry harmonizing hydrophilic interaction and reversed phase chromatography

Fatty acyl-Coenzyme A species (acyl-CoAs) are key biomarkers in studies focusing on cellular energy metabolism. Existing analytical approaches are unable to simultaneously detect the full range of short-, medium-, and long-chain acyl-CoAs, while chromatographic limitations encountered in the analysis of limited amounts of biological samples are an often overlooked problem. We report the systematic development of a UHPLC–ESI-MS/MS method which incorporates reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) separations in series, in an automated mode. The protocol outlined encompasses quantification of acyl-CoAs of varying hydrophobicity from C2 to C20 with recoveries in the range of 90–111 % and limit of detection (LOD) 1–5 fmol, which is substantially lower than previously published methods. We demonstrate that the poor chromatographic performance and signal losses in MS detection, typically observed for phosphorylated organic molecules, can be avoided by the incorporation of a 0.1% phosphoric acid wash step between injections. The methodological approach presented here permits a highly reliable, sensitive and precise analysis of small amounts of tissues and cell samples as demonstrated in mouse liver, human hepatic (HepG2) and skeletal muscle (LHCNM2) cells. The considerable improvements discussed pave the way for acyl-CoAs to be incorporated in routine targeted lipid biomarker profile studies.