Enzymatic Decarboxylation of Tyrosine and Phenylalanine To Enhance Volatility for High-Precision Isotopic Analysis

Abstract

We present a rapid and selective method to increase the volatility of tyrosine and phenylalanine without adding derivative C for high-precision gas chromatography-continuous-flow isotope ratio mass spectrometry (GCC-IRMS) based on enzymatic decarboxylation to yield alkylamines and evaluated for 15N isotopic integrity. Purified tyrosine and phenylalanine were converted to tyramine and phenethylamine by tyrosine and phenylalanine decarboxylases, respectively. GC separation was achieved using a thick stationary phase (5-microm) capillary column. Recoveries were 95 +/- 2%. The reproducibility of delta15N of tyramine and phenethylamine measured by GCC-IRMS averaged SD(delta15N) = 0.33 per thousand. The absolute differences between delta15N of amino acids measured by elemental analyzer-IRMS and the alkylamines measured by GCC-IRMS was not significant. Phenethylamine and tyramine prepared from a mixture of 18 amino acids were extracted by ethanol with 95% recovery, and analysis yielded clean chromatograms and equivalent precision. These data indicate that enzymatic decarboxylation of phenylalanine and tyrosine is a convenient method to increase their volatility for continuous-flow isotopic analysis without introducing extraneous C or significant isotopic fractionation.

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants

“…modifications are decarboxylation [108,109], reduction of fatty acid esters to alcohols [110], reduction of organometal (loid) oxides to the corresponding hydrides [111], intramolecular esterification [112], or selective fragmentation [89]. Besides enabling carbon-isotope analysis, the latter approach has been shown to facilitate isotope analysis of nitrogen and hydrogen also [65,89].…”

Assessment of Enzymatic Methods in the δ18O Value Determination of the l-Tyrosine p-Hydroxy Group for Proof of Illegal Meat and Bone Meal Feeding to Cattle

“…The turnover rate of the catalyzed reaction (TDC = tyrosine decarboxylase) normalHO − normalC 6 normalH 4 − normalCH 2 − normalCH false( normalNH 2 false) − normalCOOH + normalH 2 normalO → TDC HO − normalC 6 normalH 4 − CH 2 − CH 2 − NH 2 + CO 2 can, at the incubation pH of 5.5, easily be controlled by measuring the volume of the produced CO 2 . Ziadeh et al used enzymes from S. faecalis for the selective conversion of l -phenylalanine and l -tyrosine into the corresponding amines for a selective δ 15 N value determination. Although the authors found no difference between the δ 15 N values of the amino acids and the extracted amines, we do not believe that the applied ethanol extraction of tyramine at pH 5.5 is quantitative, because the compound is amphoteric.…”

Atomic spectrometry update. Atomic mass spectrometry

“…Enzymatic decarboxylation of phenylalanine and tyrosine increased their volatility for determination of d 15 N by CF-SIRMS without introducing extraneous C or significant fractionation. 315 Increased CO 2 : N 2 ratios would have prevented the use of cryotraps and degraded the chromatographic performance. The reproducibility (1s) of d 15 N was ¡0.33% and there was no significant difference between values measured on decarboxylated amino acids and those determined on intact amino acids by EA-SIRMS.…”