Rapid enzymatic method for the determination of phosphoryl choline using the fluorescence of the enzyme choline oxidase. Sequential determination of choline and phosphorylcholine in milk powder for children

摘要

In this paper we present a rapid method for determining phosphorylcholine (ChoP).\udChoP was first hydrolyzed by the enzyme alkaline phosphatase (AP); the choline formed\udwas then submitted to a reaction with Choline Oxidase (ChOx). Both reactions were carried\udout simultaneously, in the same test without previous steps of incubation. The analytical\udsignals used were the intrinsic fluorescence of ChOx due to FAD and that corresponding to\uda fluorescein derivative bonded to ChOx (ChOx-FS); both can be related with the\udconcentration of ChoP. Once the conditions were optimized, the response range for ChoP\udwas 5.2·10-7 - 1.0·10-5 M using the peak area as the analytical parameter with a precision\udof about 4% (RSD) at both ChOx and ChOx-FS wavelengths.\udIn the experimental conditions found, it was also possible to determine free choline\ud(Ch) with figures of merit similar to those obtained for ChoP. These results have made\udpossible the sequential determination of Ch and ChoP in milk powder, using only one\udaliquot of the mixture, with good results. The recoveries obtained for both analytes were\udclose to 100 %.\udThe method is rapid because an incubation step is not necessary. Moreover, the enzymatic reaction is autoindicating and thus the additional detection step required by other published enzymatic methods is avoided.\udother published enzymatic methods is avoided.