Determination of ornithine in human plasma by hydrophilic interaction chromatography-tandem mass spectrometry

The amino acid ornithine (Orn) acts as a vital part in the physiologically fundamental urea cycle. As such, it is a main intermediate in the catabolic breakdown as well as in the synthesis of arginine and is involved in many other metabolic pathways with potential clinical implications. We here describe a LC-MS-MS method for the detection of Orn in human plasma which is fast, easy and precise. The sample preparation comprises only protein precipitation and the addition of the isotopic labeled I.S. The analytes are separated by hydrophilic interaction chromatography (HILIC) in less than 4min on a silica column with an isocratic mobile phase consisting of 0.1% trifluoroacetic acid in water and acetonitrile in the ratio of 25:75. Orn and its I.S. are detected and quantified by APCI tandem mass spectrometry. The calibration function is linear from 7.5 to 205 micromol/l and covers the range of concentrations found in patients undergoing different clinical interventions. The quantification results are independent with regard to the biological matrix analyzed. The intra-day and inter-day relative standard deviations are 1.1% and 3.5%, respectively. As an application of the described method in clinical investigations, we report arginine and ornithine plasma concentration results from an arginine supplementation study enrolling healthy volunteers and patients suffering from hypercholesterolemia. After oral dosing of 110 mg/kg arginine, ornithine plasma concentrations rose from 54 to 148 micromol/l after 2h and were back to baseline after 24h. However, arginine to ornithine ratios kept constant during the complete observation time.