Quantitative aspects of the analysis of the monoclonal antibody trastuzumab using high-performance liquid chromatography coupled with electrospray mass spectrometry.

The analytical possibilities of quantification of the intact monoclonal antibody trastuzumab by high-performance liquid chromatography coupled with electrospray mass spectrometry (HPLC-ESI-MS) were investigated. To clarify the results obtained by LC-MS, complementary experiments were performed using direct UV-spectrophotometry and high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV). A polystyrene-divinylbenzene (POROS) column was applied with gradient elution using formic acid 0.08% (v/v) in water and formic acid 0.08% (v/v) in acetonitrile as mobile phase for chromatographic analysis. Quantification on LC-MS was performed by using the peak area of the total ion current (TIC) chromatograms of one charge state. Non-linearity and sensitivity loss were the major limitations observed with the LC-MS method, of which the non-linearity is most likely caused by detector saturation. The sensitivity loss during analysis could be reduced by lowering the MS source temperature. This parameter is critical in creating a robust LC-MS system for the quantitative analysis of trastuzumab.