An LC-MS/MS method for the quantification of 3-bromotyrosine in plasma from patients diagnosed with eosinophilic esophagitis

Abstract

Eosinophilic esophagitis (EoE) is a disease marked by a surplus of eosinophils, a type of white blood cell that causes inflammation and irritation. The current diagnostic and monitoring procedure for EoE is endoscopy with biopsy, which is invasive, expensive, and leads to tissue tearing in patients. A biomarker in plasma would offer a much less invasive form of disease monitoring for patients with EoE. Eosinophils have been shown to make eosinophil peroxidase, an enzyme that produces hypobromous acid, reacts with primary amines, and forms bromoamides. One product of this biochemical reaction is 3-bromotyrosine. We have optimized a selective, sensitive, and reproducible method to detect and quantify L-tyrosine and 3-bromotyrosine in human plasma using high-pressure liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). Our sample preparation and analysis method requires fewer steps and provides a faster analysis than previous methods. Method validation yielded limits of quantification of 50 ng mL⁻¹ for L-tyrosine and 10 ng mL⁻¹ for 3-bromotyrosine. Calibration curves for quantification were linear from 50 to 500 ng mL⁻¹ with an R² value of 0.9995 for L-tyrosine and 10 to 300 ng mL⁻¹ with an R² value of 0.9998 for 3-bromotyrosine. Method variability was assessed resulting in relative standard deviations of 0.98–4.6% for 3-bromotyrosine (n = 18) and 0.20–0.58% for L-tyrosine (n = 18). Method applicability was tested with patients with a confirmed diagnosis of EoE, initially suggesting little to no correlation between eosinophil count and 3-bromotyrosine concentration in plasma. However, we do observe a relationship between eosinophil count and esophageal deformities. More research must be conducted to determine a more definitive correlation.