Determination of creatine kinase activity and phosphocreatine in off-line and on-line modes with capillary electrophoresis

Assays for phosphocreatine (PC) and creatine kinase (CK) were performed using capillary electrophoresis (CE) both off-line and on-line in approach. Both substrate and enzyme assays were based solely on the phosphorylation reaction involving the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) which was separated and monitored at 256 nm. Polymer mediated stabilization with PEG-8000 can extend the lifetime of the enzyme activity to at least 73 h. Although the off-line procedure required a 10 min batch incubation time of the reaction mixture, low detection limits at the 10 mu M PC and 1 U/l CK levels are achievable. For the single injection on-line method, sample throughput for one assay is 7 min excluding wash and equilibration steps and detection limits of 25 mu M PC and 8 U/l CK are possible. Because of the mu M detection limits, only simple dilution of spiked PC and CK serum samples before analysis is needed. A new technique of on-line multiple injections of both substrate and enzyme into the capillary filled with co-factor and buffer was also demonstrated. Sample throughput for the multiple injection method is 9 min for five samples, excluding wash and equilibration steps. Because linearity and quantitation were only fair, the multiple injection method would be more useful for the rapid profiling of samples. (C) 1998 Elsevier Science B.V. All rights reserved.