Quantification of urinary o,o′-dityrosine, a biomarker for oxidative damage to proteins, by high performance liquid chromatography with triple quadrupole tandem mass spectrometry

We recently described an isotope dilution reversed-phase liquid chromatography-atmospheric pressure chemical ionization-ion-trap-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the quantitative determination of oxidized amino acids in human urine, including o,o′-dityrosine, a specific marker of protein oxidation. In the present study, we investigated the possibility to use a triple quadrupole instrument for the analysis of this biomarker in urine. The two instruments were compared in terms of sensitivity, specificity and reproducibility. Results showed that the triple quadrupole instrument reaches 2.5-fold higher sensitivity (LOD = 0.01 μM) compared to the previously used ion-trap instrument. Precision of the present assay is as follows: in-day variation is 4.6% and inter-day variation is 17%. The currently developed method was applied to a group of smoker urine samples. The mean urinary o,o′-dityrosine concentration was 0.08 ± 0.01 μM. Expressed per urinary creatinine concentration, this corresponds to 10.1 ± 0.4 μmol/mol creatinine. This is comparable to the previously reported values of 5.8 ± 0.3 μmol/mol creatinine in non-smokers night-time urines, and 12.3 ± 5 μmol/mol creatinine in day-time urines measured by the ion-trap instrument.