Liquid chromatography/tandem mass spectrometric method for the quantification of eight proteolytic fragments of ITIH4 with biomarker potential in human plasma and serum.

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) analytical procedure for the quantification of eight proteolytic fragments from inter-alpha-trypsin inhibitor heavy chain 4 (ITIH(4)) in human plasma and serum has been developed. The eight peptide fragments only differ in length at the N-terminus, varying between 21 and 30 amino acid residues. Protein precipitation (PP) with acetonitrile was followed by solid-phase extraction (SPE) on C(2) columns to provide clean extracts. Chromatographic separation of the peptides was performed on a Symmetry 300 C(18) column (50 mm x 2.1 mm i.d., particle size 3.5 microm), using a water/methanol gradient containing 0.25% v/v formic acid. The triple quadrupole mass spectrometer was operated in the positive electrospray ionization (ESI(+)) mode, using multiple reaction monitoring (MRM) for detection. One stable-isotope-labeled analog and two structural analogs were used as internal standards. The method has been completely validated for plasma and partially for serum samples, yielding linear responses in a range up to 100 ng/mL. The lower limit of quantification (LLOQ) in plasma was +/-2 ng/mL for four ITIH(4)-derived peptides and +/-5 ng/mL for the others. The stabilities of the peptides in different environments have been extensively explored. Several peptides showed rapid degradation, especially in the biological matrix at ambient temperature, and preparation on ice was therefore required. The method has been applied for the analysis of several plasma and serum samples from patients with different cancer types.