Optimization of dynamic pH junction for the sensitive determination of amino acids in urine by capillary electrophoresis.
A capillary electrophoresis method with UV-absorbance detection was studied and optimized for the determination of underivatized amino acids in urine. To improve concentration sensitivity the utility of in-capillary analyte stacking via dynamic pH junction was investigated with phenylalanine (Phe) and tyrosine (Tyr) as model amino acids. Before sample injection, a plug of ammonium hydroxide solution was injected to enable analyte concentration. Samples were 1:1 (v/v) mixed with background electrolyte (1 M formic acid) prior to injection. The effect of the injected sample volume, and the injected ammonium hydroxide volume and concentration on analyte stacking and separation performance was investigated. The optimal volume of ammonium hydroxide depended on the injected sample volume. Using a dynamic pH junction good resolution (1.4) was obtained for a sample injection volume of 10% of the capillary (196 nl) with Phe and Tyr dissolved in water. Limits of detection (LODs) were 0.036 and 0.049 μM for Phe and Tyr, respectively. For urine samples, the optimized procedure comprised a 1.7-nl injection of 12.5% ammonium hydroxide, followed by a 196-nl injection of urine spiked with Phe and Tyr. Satisfactory resolution was obtained and amino acid peak widths at half height were only 1.6 s indicating efficient stacking. Calibration plots for Phe and Tyr in urine showed good linearity (R(2) > 0.96) in the concentration range 10-175 μM, and LODs for Phe and Tyr were 0.054 and 0.019 μM, respectively. RSDs for peak area and migration time for Phe and Tyr were below 7.5% and 0.75%, respectively.
Biomolecular Analysis, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands. y.h.tak@uu.nl
Y H
YH
Tak
G W
GW
Somsen
G J
GJ
de Jong
D000596:Amino Acids / Q000652:urine*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D012680:Sensitivity and Specificity
D000596:Amino Acids