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

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 14 min while digestion was performed in 20 min, allowing detection of the target nonapeptide in less than 1 h. 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 2 fmol of nonapeptide. This value, corresponding to 0.5 fmol of HuBuChE tetramer, is well below the average amount of HuBuChE tetramer in 50 μL of plasma (590 fmol).