Capillary electrophoresis-time of flight-mass spectrometry using noncovalently bilayer-coated capillaries for the analysis of amino acids in human urine.
A capillary electrophoresis-time of flight-mass spectrometry (CE-TOF-MS) method for the analysis of amino acids in human urine was developed. Capillaries noncovalently coated with a bilayer of Polybrene (PB) and poly(vinyl sulfonate) (PVS) provided a considerable EOF at low pH, thus facilitating the fast separation of amino acids using a BGE of 1 M formic acid (pH 1.8). The PB-PVS coating proved to be very consistent yielding stable CE-MS patterns of amino acids in urine with favorable migration time repeatability (RSDs <2%). The relatively low sample loading capacity of CE was circumvented by an in-capillary preconcentration step based on pH-mediated stacking allowing 100-nL sample injection (i.e. ca. 4% of capillary volume). As a result, LODs for amino acids were down to 20 nM while achieving satisfactory separation efficiencies. Preliminary validation of the method with urine samples showed good linear responses for the amino acids (R(2) >0.99), and RSDs for peak areas were <10%. Special attention was paid to the influence of matrix effects on the quantification of amino acids. The magnitude of ion suppression by the matrix was similar for different urine samples. The CE-TOF-MS method was used for the analysis of urine samples of patients with urinary tract infection (UTI). Concentrations of a subset of amino acids were determined and compared with concentrations in urine of healthy controls. Furthermore, partial least squares-discriminant analysis (PLS-DA) of the CE-TOF-MS dataset in the 50-450 m/z region showed a distinctive grouping of the UTI samples and the control samples. Examination of score and loadings plot revealed a number of compounds, including phenylalanine, to be responsible for grouping of the samples. Thus, the CE-TOF-MS method shows good potential for the screening of body fluids based on the analysis of endogenous low-molecular weight metabolites such as amino acids and related compounds.
Ramautar
Rawi
R
Department of Biomedical Analysis, Utrecht University, Utrecht, The Netherlands. r.ramautar@uu.nl
Mayboroda
Oleg A
OA
Derks
Rico J E
RJ
van Nieuwkoop
Cees
C
van Dissel
Jaap T
JT
Somsen
Govert W
GW
Deelder
André M
AM
de Jong
Gerhardus J
GJ
D000596:Amino Acids / Q000652:urine*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D006583:Hexadimethrine Bromide; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D011145:Polyvinyls; D013451:Sulfonic Acids; D014552:Urinary Tract Infections / Q000378:metabolism
D000596:Amino Acids; D011145:Polyvinyls; D013451:Sulfonic Acids; C008959:lyapolate; D006583:Hexadimethrine Bromide