Amino acid analysis by micellar electrokinetic chromatography with laser-induced fluorescence detection: application to nanolitre-volume biological samples from Arabidopsis thaliana and Myzus persicae.
Amino acids were derivatised with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence. The optimised MEKC background electrolyte conditions were: 40 mM sodium cholate, 5 mM beta-cyclodextrin in 20 mM aqueous borate buffer, pH 9.1, with 7% v/v acetonitrile. Using these conditions, 19 amino acids were separated within 17 min. The limits of detection were in the range of 7.6-42.2 pmol/mL and limits of quantitation from 0.05-0.14 nmol/mL. The method was systematically validated for injection volume error, migration time variation, calibration linearity, accuracy, precision, and recovery. Nanolitre volume samples of phloem sap of individual sieve element cells from the plant Arabidopsis thaliana and honeydew from the aphid Myzus persicae were directly analysed with this method. Quantitative amino acid concentrations in these two biological matrices were profiled for the first time. This method is particularly important because it allows the complete profile of the amino acids obtained from individual phloem elements, allowing cell to cell and plant to plant variation to be quantified, which to date has not been possible with Arabidopsis thaliana.
Zhu
Xunlin
X
Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, Nottingham, UK.
Shaw
P Nicholas
PN
Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, Nottingham, UK.
Pritchard
Jeremy
J
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
Newbury
John
J
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
Hunt
Emma J
EJ
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
Barrett
David A
DA
Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, Nottingham, UK.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis* / Q000302:isolation & purification*; D000818:Animals; D001042:Aphids / Q000737:chemistry*; D017360:Arabidopsis / Q000737:chemistry*; D001826:Body Fluids / Q000737:chemistry; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D007834:Lasers; D036103:Nanotechnology / Q000295:instrumentation; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D009327:4-Chloro-7-nitrobenzofurazan
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