Sensitive monitoring of 3-hydroxybutyrate as an indicator of human fasting in a PAMAMPS coated capillary

Sensitive electrophoretic determination of 3-hydroxybutyrate (3HB) as an indicator of human ketogenesis is performed in fused silica capillary covalently coated by an anionic copolymer of poly(acrylamide-co-sodium-2-acrylamido-2-methylpropanesulphonate) (PAMAMPS). Baseline separation of 3HB from other components of human serum is achieved in a 20 mu m capillary with an effective length of 17 cm covered by 4% PAMAMPS, which generates a cathodic EOF with a mobility of 8.30 +/- 0.00 . 10-9 m(2)/V.s in 80 mM MES/His as background electrolyte. 3HB migrates in counter-current electrophoretic mode against EOF, that effectively improving electrophoretic resolution. Sample pre-treatment is based on adding of 45 mu L acetonitrile to 15 mu L serum and, after shaking, a 28 mm long zone of supernatant is injected into the capillary, and sharpened after turning on a separation voltage of 20 kV using the technique of large volume sample stacking, where the EOF forces the residual acetonitrile from the capillary. When combined with universal contactless conductivity detection, the achieved LOD and LOQ are 0.43 mu M and 1.44 mu M, respectively, that are sufficiently low for monitoring the physiological 3HB level. The performed clinical study subsequently showed that serum 3HB increases from a concentration of 71 mu M, corresponding to normal food, to level of 1924 mu M after 60 h of fasting and returns to the normal physiological concentration 48 h after commencing consumption of high-saccharide food.

Automated summary

This study presents a sensitive electrophoretic method for determining 3-hydroxybutyrate (3HB) levels, which is an indicator of human ketogenesis. By using a specially coated capillary and optimizing the separation conditions, baseline separation of 3HB from other components in human serum can be achieved. The method shows low limits of detection and quantification, making it suitable for monitoring physiological 3HB levels.