Development and analytic validation of an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-bromotyrosine in canine serum

Background: The activation of eosinophils causes the release of eosinophil peroxidase and subsequent production of 3-bromotyrosine (3-BrY), a stable byproduct. In people, 3-BrY is used as a biomarker for eosinophil activation. The method for measuring 3-BrY concentrations in biologic samples from dogs has not previously been described. Objectives: The objective of this study was to develop and analytically validate an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-BrY in canine serum samples. Methods: Pooled canine serum samples were utilized to validate the assay. Serum samples from healthy control dogs (n = 41) were used to evaluate 3-BrY concentrations and establish a reference interval. Results: The analytic validation revealed that the limit of blank and limit of detection were 0.33 and 0.63 mu mol/L, respectively. The coefficients of variation for precision and reproducibility for 3-BrY were < 13.9% and < 11.0%, respectively. The means +/- SD of observed-to-expected ratios for linearity and accuracy were 109.6 +/- 17.2% and 98.7 +/- 11.3%, respectively. The reference interval was determined as <= 1.12 mu mol/L (median [range]: <= 0.63 mu mol/L [<= 0.63-1.13]). Conclusions: The EI-GC/MS assay described here for the measurement of 3-BrY in canine serum samples was precise, reproducible, linear, and accurate. Further studies are underway to determine the diagnostic utilities in canine patients with eosinophilic diseases.