000156611 001__ 156611 000156611 005__ 20251017144634.0 000156611 0247_ $$2doi$$a10.1007/s00216-025-05856-6 000156611 0248_ $$2sideral$$a143921 000156611 037__ $$aART-2025-143921 000156611 041__ $$aeng 000156611 100__ $$aDomínguez, Mario 000156611 245__ $$aKinetic and analytical characterization of a new tropinone oxidase enzyme and its application to the simultaneous determination of the tropane alkaloids atropine and scopolamine 000156611 260__ $$c2025 000156611 5060_ $$aAccess copy available to the general public$$fUnrestricted 000156611 5203_ $$aA 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). 000156611 536__ $$9info:eu-repo/grantAgreement/ES/DGA/E25-23R$$9info:eu-repo/grantAgreement/ES/MICINN/PID2022-139235OB-I00 000156611 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttps://creativecommons.org/licenses/by/4.0/deed.es 000156611 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion 000156611 700__ $$0(orcid)0000-0002-7902-6005$$aMarcos, Susana de$$uUniversidad de Zaragoza 000156611 700__ $$0(orcid)0000-0002-8973-5104$$aGalbán, Javier$$uUniversidad de Zaragoza 000156611 7102_ $$12009$$2750$$aUniversidad de Zaragoza$$bDpto. Química Analítica$$cÁrea Química Analítica 000156611 773__ $$g(2025), [8 p.]$$pAnal. Bioanal. Chem.$$tAnalytical and Bioanalytical Chemistry$$x1618-2642 000156611 8564_ $$s1450486$$uhttps://zaguan.unizar.es/record/156611/files/texto_completo.pdf$$yVersión publicada 000156611 8564_ $$s2491542$$uhttps://zaguan.unizar.es/record/156611/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada 000156611 909CO $$ooai:zaguan.unizar.es:156611$$particulos$$pdriver 000156611 951__ $$a2025-10-17-14:27:58 000156611 980__ $$aARTICLE