Development and validation of a LC method for phosphoserine determination in dietary supplements by labelling reaction

D,L-phosphoserine (D,L-p-Ser), a phosphorylated amino acid, is deputised for the direct transport of phosphoric radicals to the Kreb’s cycle [1] and it is often used both as an ingredient in oral polyamino acid formulations and as single amino acid with at a variety of vitamins. The availability of simple and reliable methods for quali- and quantitative analysis is important for the standardization of complex formulations. However, the direct determination of amino acids is difficult because most of them lack of intrinsic chromophores and fluorophores. The application of the derivatization techniques is a way to solving the problem. 2,4-Dinitrofluorobenzene (DNFB, I) is a UV derivatization reagent for amine. In aqueous borate buffer, DNFB yields yellow coloured products reacting with primary and secondary amines through nucleophilic aromatic substitution reaction. Even if it can react also with phenols, thiols and hydrazino compounds introducing a potential interference, it produces very stable derivatives under common laboratory conditions. This is a real advantage for the routine automated analyses at company level. Other merits of this tagging reaction are that it proceed in aqueous solution and the excess of the reagent does not interfere. The present communication describes the development and validation of a RP-HPLC method for the determination of D,L-p-Ser. The UV-absorptivity of the amino acid was enhanced by pre-column derivatization with DNFB. The derivatization reaction was carried out at 70°C for 20 min in basic medium and the resulting DNB-amino acid derivative (II) was monitored at 360 nm. The chromatographic separations were performed at 33±2°C on a Supelco Discovery RP-amide C16 5mm (250 x 4.6 mm i.d.) using a mobile phase consisting of a mixture of triethylammonium phosphate buffer (pH 3; 0.05 M) and acetonitrile. The results of this study indicate the suitability of the validated method for the sure determination of D,L-p-Ser in dietary supplements of complex matrix. The proposed procedure is simple, cost effective and it allows sample processing in a reasonable time, which makes it useful for routine analysis. [1] Matera, M.; Castana, R.; Insirello I., Leonardi G.; Pharmacokinetic study of the relative bioavailability and bioequivalence after oral intensive or repeated short term treatment with two polyamino acid formulations. Int. J. Clin. Pharmacol. Res. 1993; 13: 93-105.