A targeted metabolomic procedure for amino acid analysis in different biological specimens by ultra-high-performance liquid chromatography-tandem mass spectrometry.

INTRODUCTION

Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases.

OBJECTIVES

Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds.

METHODS

Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time = 9 min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode.

RESULTS

The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified.

CONCLUSION

We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.