Steady state microdialysis of microliter volumes of body fluids for monitoring of amino acids by capillary electrophoresis with contactless conductivity detection
Abstract
Background: The availability of dialysis membranes in the form of hollow fibres with diameters compatible with the fused silica capillaries used in capillary electrophoresis is very limited. However, haemodialysis bicarbonate cartridges commonly used in human medicine containing polysulfone hollow fibres are available on the market and are used for the fabrication of coaxial microdialysis probes.
The miniature probe design ensures that steady-state conditions are achieved during microdialysis of minimal volumes of body fluids. Results: A coaxial microdialysis probe with a length of 5 cm and an inner diameter of 200 μm is used for microdialysis of 10 μL of body fluid collected into a sampling fused silica capillary with an inner diameter 430 μm.
Microdialysis is performed into 0.01 M HCl as a perfusate at stopped flow and 2 μL of the resulting microdialysate are subjected to analysis by capillary electrophoresis with contactless conductivity detection. Microdialysis pre-treatment is verified by analysis of 11 common amino acids at a 100 μM concentration level, resulting in recoveries of 98.3-102.5%.
The electrophoretic separation of amino acids is performed in 8.5 M acetic acid at pH 1.37 as a background electrolyte with analysis time up to 4.5 min and LOD in the range of 0.12-0.28 μM. The reproducibility of the developed technique determined for the peak area ranges from 1.2 to 4.5%.
Applicability is tested in the quantification of valine and leucine in plasma during fasting and subsequent reconvalescence. Significance: The fabrication of a coaxial microdialysis probe for the laboratory preparation of microliter volumes of various types of clinical samples is described, which is coupled off-line with capillary electrophoretic monitoring of amino acids in 2 μL volumes of microdialysate.
The developed methodology is suitable for quantification of 20 amino acids in whole human blood, plasma, tears and has potential for analysis of dry blood spots captured on hollow fibre.