Plasma levels of creatine, 2-aminobutyric acid, acetyl-carnitine and amino acids during fasting measured by counter-current electrophoresis in PAMAPTAC capillary

Determination of the minor nitrogen metabolites, creatine, 2-aminobutyric acid (2-AB), and acetyl-carnitine (Ac-carn), in human plasma is performed by capillary electrophoresis (CE) in a 6 % PAMAPTAC coated fused silica capillary. 6 % PAMAPTAC generates a stable electroosmotic flow (EOF) of 6.31 x 10(-9) m(2)V(-1)s(-1); and the nitrogen metabolites are separated in a counter-current mode, which increases electrophoretic resolution. Baseline separation of creatine, 2-AB, Ac-carn, and most proteinogenic amino acids is achieved in 8.5 M acetic acid (AcOH) at pH 1.37 as background electrolyte (BGE) at an effective capillary length of 14.4 cm with migration times ranging from 4.95 min for creatine to 5.43 min for Ac-carn. 20 mu L of plasma is deproteinized with the addition of 60 mu L of acetonitrile (ACN) and a 21.8 mm sample zone is injected into the capillary, which corresponds to 6.9 % of total and 15 % of effective capillary length. The zone is focused using a large volume sample stacking technique with LODs of 0.2-0.4 mu M and LOQs of 0.8-1.3 mu M relative to the untreated plasma sample. Inter-day reproducibility of migration time is 0.7-1.1 % and for peak area 2.5-3.9 %. The CE method is used to monitor plasma levels of nitrogen metabolites during 60 h of fasting in 10 healthy lean volunteers. T-test showed statistically significant differences in plasma levels of creatine, 2-AB, Ac-carn and most proteinogenic amino acids, which return to their original concentrations after subsequent 48 h recovery.

Automated summary

In this study, capillary electrophoresis was used to determine minor nitrogen metabolites, including creatine, 2-aminobutyric acid (2-AB), and acetyl-carnitine (Ac-carn), in human plasma. Baseline separation of these metabolites was achieved using a 6% PAMAPTAC coated fused silica capillary and a counter-current mode. Plasma levels of these metabolites were monitored during a 60-hour fasting period, and significant differences were observed compared to baseline levels.