Microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry was used for the absolute and simultaneous quantitation of C3f and the V65 vitronectin fragment in serum. The method was first carefully optimized and then validated in serum biological matrix. Stable isotopes for the two biomarkers of interest were used as stable isotope labeled peptide standards. A weighted 1/x2 quadratic regression for C3f and a weighted 1/x quadratic regression for the V65 vitronectin peptide were selected for calibration curves. Trueness (with a relative bias <10%), precision (repeatability and intermediate precision <15%) and accuracy (risk <15%) of the method were successfully demonstrated. The linearity of results was validated in the concentration range of 2.5–200 ng/mL for C3f and 2.5–100 ng/mL for the V65 vitronectin fragment. Serum samples (n=147) classified in 7 groups [(healthy volunteers, OA with 5 grades of severity and rheumatoid arthritis (RA) patients] were analyzed with our new quantitative method. Our data confirm that C3f and the V65 vitronectin fragment are biomarkers of OA severity, but also that C3f fragment is further related to OA severity whereas the V65 vitronectin fragment is more related to early OA detection.
Gaël Cobraivillea, b, Marianne Filletb, Mohammed Sharifc, Khadija Ourradic, Gwenaël Nysb, Michel G. Malaisea, Dominique de Senya, ,
a Laboratory of Rheumatology, GIGA-I3, University of Liege, CHU de Liege, 4000 Liege, Belgium
b Laboratory for the Analysis of Medicines, Department of Pharmacy, CIRM, University of Liege, 4000 Liege, Belgium
c School of Clinical Sciences, University of Bristol, Musculoskeletal Research Unit, Avon Orthopaedic Centre, Southmead Hospital, Bristol BS10 5NB, UK
Received 11 January 2017, Revised 22 March 2017, Accepted 25 March 2017, Available online 28 March 2017