Sensitive liquid chromatography/tandem mass spectrometry assay for absolute quantification of ITIH4-derived putative biomarker peptides in clinical serum samples.

To explore the potential of peptide fragments derived from inter-alpha-trypsin inhibitor heavy chain-4 (ITIH(4)) as serum markers for different cancer types, sensitive and specific analytical assays are required. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) would be suitable; however, a previously developed method for quantification of eight ITIH(4) fragments (ITIH(4)-21, -22, -25, -26, -27, -28, -29 and -30) was found to be insensitive for clinical use. A more sensitive LC/MS/MS assay has now been developed and validated, which was further optimized to facilitate analyses of large sets of clinical serum samples. Benefits compared to the previous method include reduction of sample volume (100 microL), omission of protein precipitation and evaporation and transferring solid-phase extraction (SPE) to a 96-well format. Chromatographic separation on an XBridge BEH300 C(18) column, using a water/methanol gradient containing acetic acid, was coupled to triple quadrupole mass spectrometric detection, applying heated electrospray ionization. Method validation revealed deviations from nominal concentrations below 10.1% and intra- and inter-assay precisions below 17.4 and 20.0%, respectively, at the lower limit of quantification (LLOQ) for all peptides. The reported changes resulted in more rapid and efficient analyses and reduced LLOQs for the six less abundant peptides (1.2; 1.0; 1.2; 2.0; 2.0 and 2.0 ng/mL vs. 2.1; 2.0; 2.5; 2.6; 2.2 and 2.4 ng/mL for ITIH(4)-21, -22, -25, -27, -28 and -29, respectively). The method has shown its applicability by quantifying all peptides in appropriate concentration ranges in serum from healthy volunteers and application to clinical samples from breast cancer patients.