The composition of the mobile phase is determined by the requirements for optimal next activity and stability of the used enzyme systems: essentially an aqueous phosphate buffer at pH 6.5-8.5 [28-31]. The conversion of acetylcholine to hydrogen selleck chemical MEK162 peroxide is most efficient at pH 8.0-8.5 [29, 30]. However, the optimal pH for the subsequent detection of hydrogen peroxide on the enzyme modified electrode has not been determined. The cation-exchange setup provides good chromatographic stability under these conditions, but a limited resolution between Inhibitors,Modulators,Libraries acetylcholine and choline [12, 29, 30]. Microdialysis samples typically contain a high concentration of choline, which may interfere with the acetylcholine signal.
A precolumn choline oxidase and catalase reactor was developed to eliminate choline from the microdialysis sample matrix [13, 21].
The ion-pair setup offers a superior resolution Inhibitors,Modulators,Libraries between acetylcholine Inhibitors,Modulators,Libraries and choline and does not Inhibitors,Modulators,Libraries require the preliminary elimination Inhibitors,Modulators,Libraries of choline [23, Inhibitors,Modulators,Libraries 31]. However, the stability of silica-based chromatographic columns is highly dependent on the mobile phase pH [32]. Inhibitors,Modulators,Libraries The feasibility of acetylcholine determination Inhibitors,Modulators,Libraries in an ion-pair chromatographic setup with amperometric detection was previously reported at pH 6.5, but the implications Dacomitinib of lowering the mobile phase pH for the sensitivity and long-term enzymatic and chromatographic stability were not investigated [23].
In the present study an ion-pair liquid chromatography method with amperometric detection was optimized and validated for acetylcholine determination in microdialysis Brefeldin_A samples.
Different mobile phase conditions were compared to improve the sensitivity, long-term enzymatic and chromatographic stability. The polymer-coated octadecyl silica column type used in this study allowed a reliable separation of acetylcholine from choline and other matrix Tanespimycin components over 4 months of intensive use.2.?Results and Discussion2.1. Method optimizationThe chromatographic parameters were chosen to obtain an optimal equilibrium between sensitivity and chromatographic stability. A microbore polymer-coated silica column with high endcapping and low octadecyl binding-density was selected to allow chromatographic stability when using a purely aqueous phosphate buffer as the mobile phase.
Typically, mobile phases with pH 8.0-8.5 have been used for detection of acetylcholine by liquid chromatography with amperometric detection [10-15]. Nevertheless, it has selleck products been demonstrated that it is feasible to detect acetylcholine in microdialysis samples when working at pH 6.5 [23]. In the present setup, no difference in sensitivity was observed within the pH range 6.5-8.5 (Figure 2A).Figure 2.(A) Normalized response for acetylcholine (10 nM) as a function of mobile phase pH.