Metanephrine and normetanephrine are measured in blood plasma to diagnose different diseases. Simpler sample preparation procedures are preferred but tend to yield less purified extracts. Therefore, thorough investigation of matrix effects is required. In this work, several sample preparation methods and chromatographic modes were compared for liquid chromatography tandem mass spectrometric (with electrospray ionization; LC-ESI-MS/MS) analysis of metanephrine and normetanephrine in blood plasma. Protein precipitation with methanol was found to be sufficient for sample preparation and pentafluorophenyl column provided adequate chromatographic separation. A new cheaper and less labor-intensive approach is proposed where necessary quantitation limits are achieved through a sample preparation containing only protein precipitation and dilution of the sample extract. Matrix effects for different sample preparation methods and the use of isotope-labeled internal standards were evaluated. Unusual interference to D3-labeled internal standard of normetanephrine was discovered - signal of interfering compound increased while the matrix effects were reduced by dilution, e.g. dilution eliminates matrix suppression on interfering compound. The results stress the need to monitor interfering compounds and evaluate matrix effects at every step of method development. Matrix effects and interferences can be different for analytes and their corresponding isotopically labeled internal standards. This means that the use of isotopically labeled internal standards cannot guarantee accuracy of obtained results. New method allows quantification of the low nanomolar concentrations of metanephrine and normetanephrine in plasma samples.
Keywords: Metanephrines; isotopically labeled internal standard; liquid chromatography; mass spectrometry; matrix effects.