title: Development and validation of an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry method for rapid quantification of free amino acids in human urine.
abstract: An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOF-MS) method using hydrophilic interaction liquid chromatography was developed and validated for simultaneous quantification of 18 free amino acids in urine with a total acquisition time including the column re-equilibration of less than 18 min per sample. This method involves simple sample preparation steps which consisted of 15 times dilution with acetonitrile to give a final composition of 25 % aqueous and 75 % acetonitrile without the need of any derivatization. The dynamic range for our calibration curve is approximately two orders of magnitude (120-fold from the lowest calibration curve point) with good linearity (r (2) ≥ 0.995 for all amino acids). Good separation of all amino acids as well as good intra- and inter-day accuracy (<15 %) and precision (<15 %) were observed using three quality control samples at a concentration of low, medium and high range of the calibration curve. The limits of detection (LOD) and lower limit of quantification of our method were ranging from approximately 1-300 nM and 0.01-0.5 µM, respectively. The stability of amino acids in the prepared urine samples was found to be stable for 72 h at 4 °C, after one freeze thaw cycle and for up to 4 weeks at -80 °C. We have applied this method to quantify the content of 18 free amino acids in 646 urine samples from a dietary intervention study. We were able to quantify all 18 free amino acids in these urine samples, if they were present at a level above the LOD. We found our method to be reproducible (accuracy and precision were typically <10 % for QCL, QCM and QCH) and the relatively high sample throughput nature of this method potentially makes it a suitable alternative for the analysis of urine samples in clinical setting.
authors: Medway Metabonomics Research Group, Medway School of Pharmacy, Universities of Kent and Greenwich, Kent, UK.|RJMS Consultancy, Rochester, Kent, UK.
authors: Richard
authors: R
authors: Joyce
authors: Medway Metabonomics Research Group, Medway School of Pharmacy, Universities of Kent and Greenwich, Kent, UK.
authors: Viktorija
authors: V
authors: Kuziene
authors: Medway Metabonomics Research Group, Medway School of Pharmacy, Universities of Kent and Greenwich, Kent, UK.
authors: Xin
authors: X
authors: Zou
authors: Medway Metabonomics Research Group, Medway School of Pharmacy, Universities of Kent and Greenwich, Kent, UK.
authors: Xueting
authors: X
authors: Wang
authors: Medway Metabonomics Research Group, School of Science, University of Greenwich, Kent, UK.
authors: Frank
authors: F
authors: Pullen
authors: Medway Metabonomics Research Group, Medway School of Pharmacy, Universities of Kent and Greenwich, Kent, UK. r.loo@kent.ac.uk.
authors: Ruey Leng
authors: RL
authors: Loo
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000596:Amino Acids
keywords: Absolute quantification; Free amino acids; HILIC-UPLC-qTOF-MS; Human urine