Plasma citrulline measurement using UPLC tandem mass-spectrometry to determine small intestinal enterocyte pathology

Citrulline is a nonessential free amino acid, detectable in various biological fluids such as plasma, urine and cerebrospinal fluid. The plasma citrulline concentration is increasingly considered to be a reliable biomarker of enterocyte function. Current analysis usually involves lengthy HPLC separations as a part of classical amino acid profiling, or mass spectrometry usually in combination with derivatization. We employed UPLC-HILIC-tandem mass-spectrometry (MS/MS) of acetonitrile-derived supernatants from plasma samples of control subjects and of patients who had received myeloablative chemotherapy. Detection was achieved by the selected reaction monitoring of transitions: m/z 176 -> 70 and 180 -> 74 (for the deuterated standard), respectively. The method was precise and accurate with inter-day CV < 3.9% (n = 30), recoveries ranging from 98.0 to 100.3% and high linearity from 0.3 to at least 2000 mu Lmol/L. The results for 202 plasma samples agreed well with those obtained by the classical HPLC-fluorescence method. By a simple protein precipitation/extraction step and the UPLC separation the result can be available within 30 min of receipt with a capacity of at least 12 assays per hour. Citrulline in blood and plasma or serum was stable for at least 2 days at room temperature which would permit postal transport to the laboratory. The UPLC-MS/MS method for measuring plasma citrulline concentrations is fast and robust and is therefore an ideal tool for monitoring the intestinal enterocyte capacity of patients with various pathological conditions. (C) 2008 Elsevier B.V. All rights reserved.