Combining Orbitrap-HRMS acquisition modes and direct injection by a guard column for targeted analysis of underivatized amino acids in urine

Abstract

[EN]Nowadays, there is a great interest in the development of rapid methods for the determination of compounds of clinical interest in biological samples, such as amino acids in urine samples. Mass spectrometry-based methods have been proposed with this objective, although the use of low resolution mass analyzers sometimes cannot provide accurate results. Orbitrap has barely been used with quantitative purposes for the targeted analysis of amino acids in urine although it has been used to a greater extent for non-targeted assays to identify potential biomarkers of different diseases. The use of a guard column for sample introduction into the Orbitrap was evaluated. The coupling of a hydrophilic interaction-guard column significantly improved the MS-signals by producing a matrix cleaning effect with a slight displacement of the target analytes. Possible isobaric interferences were studied and, consequently, different modes of MS data acquisition were tested: Full MS acquisition mode was selected for those analytes for which it was possible their determination by exact mass, and parallel reaction monitoring mode was used for those compounds showing isobaric interferences. Method validation was performed using synthetic urine. Limits of detection between 0.003 and 1.50 mg/L and limits of quantification between 0.10 and 4.95 mg/L were found with recoveries in the 80–130 % range. The runtime was 1.6 min and the total time between injection was less than 3 min. The optimized method was satisfactorily applied to the analysis of urine from healthy volunteers. This work describes for the first time the direct coupling of a hydrophilic interaction-guard column to a Orbitrap high resolution mass spectrometer (HRMS) for the development of a fully validated quantitative method for the determination of underivatized amino acids in urine. The method proposed here represents a high throughput and reliable approach, with an analysis runtime of 1.6 min, much less than the time required when chromatographic techniques were used.