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 (r2 ≥ 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.

Metabolic alterations in triptolide‐induced acute hepatotoxicity

“…Recently, an ultra performance liquid chromatography‐electrospray ionisation‐time of flight mass spectrometry (UPLC‐ESI‐QTOFMS) urine metabolomic survey found that eight potential biomarkers associated with tryptophan, pantothenic acid and porphyrin were related to triptolide toxicity and licorice had potential protective therapeutic effects against such toxicity (Wang et al, ). Since many endogenous metabolites are highly polar molecules, the application of UPLC‐ESI‐QTOFMS using hydrophilic hydrophilic interaction chromatography (HILIC) chromatography can improve the retention of polar metabolites (Joyce et al, ) and provide a relatively comprehensive separation method for the analysis of biological samples.…”

Voltammetric Determination of Amino Acids Based on the Measurement of Reduction Prepeak of Quinone Caused by Surplus Acid after Neutralization

“…Usually, because of the weak light-absorptive property of amino acids, pre-or post-column derivatization with an appropriate labeling reagent is performed for determining amino acids with sensitivity and selectivity in HPLC with UV detection (HPLC-UV) and fluorescence detection (HPLC-FL) systems [9,10]. Recently, many LC with mass spectrometric detection methods such as LC-MS/MS and LC-qTOF-MS/MS have been proposed [11][12][13], and these methods are applied to the carrying out of metabolism studies and diagnosis based on amino acids profiles [13][14][15][16]. Electrochemical detection generally has the following merits: an electrochemical detector is inexpensive compared with a spectrometric detector, electrochemical devices, including electrodes, can be easily minimalized to develop a portable sensor with simple procedures, and a low energy requirement that can be used either in laboratory or for on-site measurements [22].…”

“…Amino acid analysis is an important technique in many pharmaceutical, medical, chemical, food research, and industrial areas [1][2][3]. Thus far, various analytical methods have been proposed to determine amino acids, including high-performance liquid chromatography, capillary electrophoresis, enzyme assay, and chemical sensors [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Usually, because of the weak light-absorptive property of amino acids, pre-or post-column derivatization with an appropriate labeling reagent is performed for determining amino acids with sensitivity and selectivity in HPLC with UV detection (HPLC-UV) and fluorescence detection (HPLC-FL) systems [9,10].…”

Development and validation of a hydrophilic interaction ultra‐high‐performance liquid chromatography–tandem mass spectrometry method for rapid simultaneous determination of 19 free amino acids in rat plasma and urine

“…As a result, HILIC was widely used to analyze highly polar constituents, including monoamines, amino acids and peptides as well as many other biologically important compounds in complex biological matrices without the need for derivatization or ion‐paring agents (Chen et al, ; Guo et al, ; Lai, Kline, & Wang, ; Wu et al, ). For example, HILIC–MS/MS analysis of human urine was utilized by Richard Joyce et al, which quantified 18 AAs using 18 stable isotope internal standards in <18 min (Joyce et al, ). Prinsen et al quantified 36 AAs (24 quantitative/12 qualitative) in plasma in a single MS run of 18 min (Prinsen et al, ).…”