Analysis of glycerophosphonolipids in egg yolk

Glycerophosphonolipids are a relatively unknown class of polar lipids. Little information is available on any special biological function of these substances or specific technological use for them. Since glycerophosphonolipids have been reported to occur in egg yolk, further studies are needed in order to determine their relevancy. However, analysis of these lipids is rather difficult, due both to their chemical similarity to phospholipids and the lack of commercially available reference substances. Therefore, a reference substance for phosphono analogues of phosphatidylethanolamine (PnE)-1,2-dioctadecanoyl-glycero-3-aminoethylphosphonate-has been synthesized and an HPLC/ELSD method to separate PnE from its phosphonoanalog was developed. Although the present results showed no evidence of glycerophosphonolipids in egg yolk (limit of detection < 0.02% for PnE), the spectroscopic data gathered here do point to an approach for further analysis of glycerophosphonolipids. Furthermore, a characteristic ESI-MS(2) fragmentation pattern for PnE was established, which can be used as a fingerprint for the identification of this substance in other biological systems. It was found to be characteristic for a fatty acid fragment to be split off as an internal anhydride (ketene) [M-H-RCH=C=O](-) along with the whole fatty acid fragment [M-H-RCH(2)COOH](-) in the negative ion mode. In the positive mode, there was a selective loss of only the head group of the phosphonolipid [PO(3)H(2)-C(2)H(4)-NH(3)](+), resulting in the so-called diacylglycerol-like fragment ion [DAG](+).