Analysis of trace degradation products (decarboxylated diastereoisomers) of S‐adenosylmethionine by electrophoresis in capillaries with cationic coatings (N‐methylpolyvinylpyridinium or divalent barium)

Abstract

Commercial preparations of S-adenosylmethionine (SAM) when analyzed in uncoated capillaries show a minute impurity believed to be decarboxylated (dc) SAM. By using two types of cationic coatings, thus reducing the electro-endo-osmotic flow (EOF), it was possible to separate this impurity into two diastereoisomers of dcSAM. The coatings evaluated for this purpose were: (i) N-methylpolyvinylpyridinium, used under reversed EOF at acidic conditions (pH 4.0) and (ii) deposition of divalent barium at alkaline pH values (pH 9.4), providing reduced EOF. Under these conditions, it was possible to separate this impurity into two diastereoisomers, which by chemical synthesis were indeed proven to be dcSAM. It was further demonstrated that, in the alkylation of 5'-methylthioadenosine by 3-bromopropylamine in bromidric acid to dcSAM, another minute impurity was present, proven, via mass spectrometry, to consist of S-(5'-adenosyl)-3-thiopropylamine (decarboxylated and demethylated (dc-SAH)). The LOD for the two dcSAM diastereoisomers was assessed as 17.5 μg/mL and their LOQ as 25.5 μg/mL. By the barium-based protocol it was possible to quantify the dcSAM, present in a commercial sample of SAM, as a 0.1% impurity.

Recent advances in amino acid analysis by capillary electrophoresis

“…Sebastiano et al 10 proposed two new methods to obtain reduced EOF by using (i) N ‐methylpolyvinylpyridinium, which reverses EOF in acidic conditions (50 mM ammonium formate, pH 4.0, −20 kV) and (ii) deposition of divalent barium at alkaline pH values (50 mM boric acid, 10 mM barium hydroxide, and 10 mM sodium hydroxide, pH 9.4, +20 kV), which reduces EOF. In these conditions, S ‐adenosylmethionine and its minute impurities, two diastereoisomers of S ‐adenosyl‐(5′)‐3‐methylthiopropylamine (resulting from the decarboxylation of S ‐adenosylmethionine) were separated using CE/UV (254 or 210 nm, respectively).…”

Capillary Electrophoresis

“…Two cationic capillary coatings were employed by Sebastiano et al for the separation of a suspected impurity in preparations of S -adenosylmethionine (SAM) …”

“…Two cationic capillary coatings were employed by Sebastiano et al for the separation of a suspected impurity in preparations of S-adenosylmethionine (SAM). 60 N-Methylpolyvinylpyridinum and divalent barium were both used to coat the capillary, causing a reversal or reduction of EOF, respectively. Both methods were successful in separating the impurity (dcSAM), and divalent barium was able to quantify the amount dcSAM in a commercial sample of SAM.…”