Development of high-throughput HILIC-MS/MS methodology for plasma citrulline determination in multiple species
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abstract
Circulating citrulline originates almost exclusively from the small intestinal enterocytes in mammals and therefore is a potential biomarker of disease states affecting enterocyte mass including exposure to ionizing radiation. There is a need for a simple and rapid method for citrulline quantification in plasma. To achieve this goal, a high-throughput separation and tandem mass spectrometric detection strategy has been developed and validated in six different species. HILIC separation was achieved on a 1.7 m fused-core Diol column using an acidic acetonitrile/water gradient. A surrogate analyte (citrulline stable isotope) was used to determine the lower-limit-of-quantitation, extraction recovery, and matrix ion effects. Mass spectrometric detection was achieved in the multiple reaction-monitoring mode using m/z 176159, 177160, and 181164, for citrulline, citrulline+1, and citrulline+5, respectively. The retention time of citrulline and total chromatographic run time were 1.1 min and 2.5 min, respectively, while effectively eliminating matrix-ion effects and achieving baseline separation from the confounding amino acid arginine. Quantitation was precise (CV <4.3%), accurate (90-110%), and sensitive (lower-limit-of-quantitation; 0.125 M) without interference from the confounding amino acid, arginine. The throughput of the method was enhanced by incorporation of a 96-well filter plate for final sample cleanup. The method was used to determine plasma citrulline in six different species and in mice treated with escalating doses of radiation. This simple, accurate and high-throughput (up to 200 samples/day) methodology provides the first quantitative assay to meet the growing demand for a rapid and simple citrulline assay with high sensitivity. The Royal Society of Chemistry 2011.
