Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography–tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma

Abstract

Human butyrylcholinesterase (HuBuChE) has been widely used as a biomarker of exposure to organophosphorus (OPs) warfare agents. Indeed, intoxication by OPs can be proven by LC–MS/MS analysis of a specific HuBuChE nonapeptide on which OPs covalently bind. Therefore, we developed a fast, selective and sensitive on-line set-up for the analysis of HuBuChE from plasma that combines immunoextraction by anti-HuBuChE antibodies, pepsin digestion on Immobilized Enzyme Reactors (IMER) and microLC–MS/MS analysis of the target nonapeptide, FGESAGAAS.Two pepsin-based IMERs were prepared and characterized in terms of grafting and digestion yields and were coupled on-line to microLC–MS/MS analysis. In addition, immunosorbents were prepared by covalent grafting of three anti-HuBuChE antibodies on CNBr-sepharose and epoxy-polymethacrylate supports and packed in precolumns. The best antibody grafting yields were obtained with sepharose-based supports, with grafting yields up to 98%. B2 18-5 monoclonal antibody grafted on sepharose led to the best immunosorbent, with HuBuChE recovery close to 100%. The immunosorbent was introduced upstream of the on-line digestion set-up and immunoextraction of HuBuChE was achieved in 14min while digestion was performed in 20min, allowing detection of the target nonapeptide in less than 1h. The global recovery of the nonapeptide was higher than 42% using the best immunosorbent with a RSD value lower than 7% (n=3). Finally, the limit of quantification evaluated in plasma sample was 2fmol of nonapeptide. This value, corresponding to 0.5fmol of HuBuChE tetramer, is well below the average amount of HuBuChE tetramer in 50μL of plasma (590fmol).