Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry

Abstract

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just “dilute and shoot”), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91–15.12% and with accuracy yielding relative errors in the range of 85.47–112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines.

Determination of Branched-Chain Amino Acids in Food Supplements and Human Plasma by a CE-MS/MS Method with Enhanced Resolution

“…Here, the BGE optimization process was performed with the use of HFo in the concentration range of 100–1000 mM. Our results show that higher HFo concentrations were associated with enhanced resolution between the selected amino acids and with increased time of analysis, which is in agreement with the results presented in our previous work [ 28 ]. Nevertheless, the baseline separation between leucine and isoleucine was not achieved (see Figure 2 a).…”

“…Therefore, small molecular organic acids, such as formic (HFo) or acetic (HAc) acid, are the preferred BGE constituents. This fact is supported by some previously published papers dealing with CE-MS analysis of proteinogenic amino acids, especially in biological fluids [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ] or with CE analysis in combination with conductivity detection focused on BCAAs only [ 30 , 31 ].…”

CE‐MS for metabolomics: Developments and applications in the period 2018–2020

“…Recently, Piestansky et al developed a CE‐MS/MS method for the analysis of AA in urine samples of inflammatory bowel disease (IBD) patients [35]. A sheath‐liquid interface was used for coupling CE to a Triple Quadrupole tandem MS instrument.…”

Current applications of capillary electrophoresis‐mass spectrometry for the analysis of biologically important analytes in urine (2017 to mid‐2021): A review

“…Amino acids are connected with intestinal growth and mucosal integrity; therefore, they can be potentially associated with IBD. Piešťanský et al [112] investigated amino acid composition in the urine of patients with IBD. The fast CE-MS/MS method for the separation of 20 amino acids was developed and tested on the clinical samples of 13 patients with Crohn's disease and a control group of 10 healthy individuals.…”