Journal
AMINO ACIDS
Volume 48, Issue 1, Pages 219-234Publisher
SPRINGER WIEN
DOI: 10.1007/s00726-015-2076-0
Keywords
Free amino acids; Human urine; Absolute quantification; HILIC-UPLC-qTOF-MS
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Funding
- MRC New Investigator Grant Award [G1002151]
- MRC [G1002151] Funding Source: UKRI
- Medical Research Council [G1002151] Funding Source: researchfish
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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 mu M, respectively. The stability of amino acids in the prepared urine samples was found to be stable for 72 h at 4 degrees C, after one freeze thaw cycle and for up to 4 weeks at -80 degrees 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.
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