Increasing the Sensitivity of Phospholipid Analyses from Biological Extracts via Trimethylation Enhancement using Diazomethane (TrEnDi) and Tandem Mass Spectrometry

TrEnDi is a novel rapid in-solution technique for quaternization of phospholipid amino groups and methylation of phosphate groups via reaction with diazomethane and tetrafluoroboric acid. TrEnDi significantly enhanced the sensitivity of mass spectrometry and tandem MS studies of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin standards. Use of 13C-diazomethane enabled creation of independent precursor ion scans (PIS) for modified PE and modified PC species which would otherwise can produce undistinguishable isobaric species. The efficacy of the technique was tested on a complex biological sample. 13C-TrEndi provided a drastic sensitivity enhancement for PE and PS species enabling the identification and quantitation of several species which were below the limit of detection and quantitation prior to modification. Derivatization provided a modest sensitivity enhancement for PC species and allowed quantitation of several PC species that were below the limit of quantitation prior to modification. SM species exhibited neither sensitivity increase nor hindrance after modification.