An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The peptides include bradykinin, Hyp(3)-bradykinin, des-Arg(9)-bradykinin and fragments of fibrinogen alpha-chain (Fib-alpha([605-629])), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH(4[666-687])) and complement component 4a (C4a([1337-1350])). Ile(13)-ITIH(4[666-687]), d20-C4a([1337-1350]) and Sar-D-Phe(8)-des-Arg(9)-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10-500 ng/ml), Hyp(3)-bradykinin (4-200 ng/ml), des-Arg(9)-bradykinin (2-100 ng/ml), Fib-alpha([605-629]) (120-3000 ng/ml), ITIH(4[666-687]) (0.4-10 ng/ml) and C4a([1337-1350]) (1-25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.
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