Analytical and clinical validation of an LC–MS/MS method for urine leukotriene E₄: A marker of systemic mastocytosis

Objectives Systemic mastocytosis (SM) is a disorder characterized by the excessive accumulation of clonally derived mast cells in various tissues. When triggered, mast cells release large amounts of histamine, prostaglandins and leukotrienes. Leukotriene E4 (LTE₄) is the primary stable metabolite of total cysteinyl leukotrienes. We hypothesized that secretion of LTE₄ would be increased in SM and could be used alone or in combination with current urinary biomarkers to optimize screening for SM. Design and methods LTE₄ was measured by liquid chromatography followed by tandem mass spectrometry (LC–MS/MS). Analytical assay validation was performed using residual urine specimens. LTE₄ results were normalized to urine creatinine for clinical use. Reference interval was established using a healthy volunteer cohort. Clinical sensitivity and specificity for SM detection were determined by measuring urinary biomarkers (LTE₄, N-methyl histamine [NMH] and 11β-prostaglandin F_2α [BPG]) in a cohort of 409 patients referred to allergy specialists, 66 (16%) of which were diagnosed with SM. Results Urinary LTE₄ measurement was accurate, precise and linear across a range of 31–3020 pg/mL. The 95th percentile of the reference interval population was < 104 pg/mg creatinine. Median urine LTE₄ concentrations were significantly higher among patients with SM (97 pg/mg cr. vs. 50 pg/mg cr.; p < 0.01). Elevated urinary LTE₄ was 48% sensitive and 84% specific for SM. Clinical sensitivity was 53% for BPG (> 1000 ng/mL) and 71% for NMH (> 200 ng/mL). Incorporating all three urinary metabolites improved the SM diagnostic sensitivity to 97%, with minimal change in specificity. Conclusions We have developed a sensitive and precise LC–MS/MS assay for quantitation of LTE₄ in urine. Incorporating LTE₄ into a panel including BPG and NMH provides a much-needed screening tool for a complicated disease with non-specific symptoms and invasive confirmatory testing.