Kinetic and analytical characterization of a new tropinone oxidase enzyme and its application to the simultaneous determination of the tropane alkaloids atropine and scopolamine.

Abstract

A spectrophotometric enzymatic method for the determination of atropine (Atp) and scopolamine (Scp), two tropane alkaloids (TAs), has been developed. The method is based on a previous basic hydrolysis to tropine (Trp) and scopine (Sci) respectively, and a subsequent enzymatic oxidation catalyzed by a tropinone reductase 1 (TRase) using NAD as oxidant; the absorbance of NADH (340 nm) is monitored during the reaction. First, the enzyme kinetics of both substrates are studied; it is concluded that both TAs follow a compulsory order ternary complex mechanism and the Michalis-Menten constant is calculated. Then, an enzymatic method was optimized for Atp, allowing the determination of this substrate in the range from 1.1·10^-5 M to 3.0·10^-4 M (LoD = 3.5·10^-6 M); it was applied to the determination of Atp in a spiked chia sample (96 ± 6% recovery). Interestingly, the equilibrium constant of the reaction decreased with temperature and increased with enzyme concentration; both effects were satisfactorily explained. A similar analytical study was carried out with Scp (linear range from 1.2·10^-5 M to 3.0·10^-4 M, LoD = 3.6·10^-6 M); the method was also applied to Scp in a spiked chia sample (94 ± 2% recovery). Finally, since the kinetics of Scp is slower than that of Atp, the simultaneous quantitative determination of both compounds was successfully developed by measuring the absorbance at two reaction times (70 s and 300 s). This method was applied to the simultaneous determination of both TAs first in a synthetic sample and later in a spiked chia sample, with recoveries around 98% for both compounds. Although the sensitivity of the method is lower than that of the immunoassays for Atp, it has advantages such as the simultaneous determination of Atp and Scp, and even the possible determination of Trp (another TA).