Reduction of Nonpolar Amino Acids to Amino Alcohols To Enhance Volatility for High-Precision Isotopic Analysis

Amino acids are routinely derivatized using carbon-containing groups prior to gas chromatography continuous-flow isotope ratio mass spectrometry (GCC−IRMS). Derivative C contaminates analyte C because the entire derivatized compound is combusted to CO2. Correction procedures are required to extract the analyte isotope ratio. We present a method for reduction of six nonpolar amino acids to their corresponding amino alcohols, demonstrate a GC strategy to produce acceptable peak shapes from the resulting strongly H-bonding analytes, and present isotopic analysis for amino acids and their corresponding amino alcohols to evaluate any possible isotopic fractionation. Alanine, valine, leucine, isoleucine, methionine, or phenylalanine was reduced using NaBH4 in THF with I2 as an electrophile. Reactions were performed with 2 g of analyte to permit isotopic analysis by conventional elemental analysis-IRMS. All reactions were quantitative as assessed by IR spectra, melting points, and GC. Recovery from the reaction mixture was 60−84%. GC separation of a mixture of the six amino alcohols was achieved using a thick stationary-phase (5 μm) capillary column to avoid tailing due to hydrogen bonding to the walls of the fused-silica capillary. The reproducibility of GCC−IRMS determinations of amino alcohols averaged SD(δ13C) = 0.25 ± 0.19‰. The absolute differences between δ13C of amino acids measured by an elemental analyzer coupled to IRMS and amino alcohols measured by GCC−IRMS was Δδ13C = 0.14‰ and showed no general trend. Reactions performed with 2 mg of analyte yielded equivalent chromatograms. These data indicate that the reduction method does not induce isotopic fractionation and can be used for continuous-flow isotopic analysis to avoid addition of contaminating carbon.