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

In this paper we present a rapid method for determining phosphorylcholine (ChoP). ChoP was first hydrolyzed by the enzyme alkaline phosphatase (AP); the choline formed was then submitted to a reaction with Choline Oxidase (ChOx). Both reactions were carried out simultaneously, in the same test without previous steps of incubation. The analytical signals used were the intrinsic fluorescence of ChOx due to FAD and that corresponding to a fluorescein derivative bonded to ChOx (ChOx-FS); both can be related with the concentration of ChoP. Once the conditions were optimized, the response range for ChoP was 5.2·10-7 - 1.0·10-5 M using the peak area as the analytical parameter with a precision of about 4% (RSD) at both ChOx and ChOx-FS wavelengths. In the experimental conditions found, it was also possible to determine free choline (Ch) with figures of merit similar to those obtained for ChoP. These results have made possible the sequential determination of Ch and ChoP in milk powder, using only one aliquot of the mixture, with good results. The recoveries obtained for both analytes were close to 100 %. The 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. other published enzymatic methods is avoided.This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) (CTQ 2012-34774) (CTQ 2012-35535), FEDER (EU) (CTQ 2008 06751 C02-01/BQU) and by the Regional Government of Aragón (DGA) (Support to Research Group E-74). Thanks to Dr. Jesús Martínez de la Fuente for providing the opportunity to use the BioTek Synergy HT Multi-Mode Microplate Reader.Peer reviewe