Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry.
We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.
Soga
Tomoyoshi
T
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.|Human Metabolome Technologies Inc., Tsuruoka, Yamagata, Japan.
Kakazu
Yuji
Y
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.
Robert
Martin
M
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.
Tomita
Masaru
M
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.|Human Metabolome Technologies Inc., Tsuruoka, Yamagata, Japan.
Nishioka
Takaaki
T
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.|Graduate School of Agricultural Sciences, Kyoto University, Kyoto, Japan.
D000596:Amino Acids / Q000652:urine*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
D000596:Amino Acids