Compound-specific δ13C and δ15N analysis of amino acids: a rapid, chloroformate-based method for ecological studies

Compound-specific stable isotope analysis of amino acids has proven informative to many ecological systems, but only a handful of analytical methods are routinely employed. We evaluated a simple, rapid procedure in which biological samples undergo short-duration acid hydrolysis and the resulting amino acids are derivatized with methyl chloroformate for gas chromatography/combustion/isotope-ratio mass spectrometry (GC/C/IRMS).

Amino acid derivatives were separated on a polar gas chromatography column, combusted, and δ(13)C and δ(15)N values were measured. Tests of reproducibility and accuracy were conducted for amino acid reference mixtures and biological samples. A brief case study of turtles was used to assess whether isotopic data were consistent with a priori ecological expectations.

The methyl chloroformate based reaction successfully converted 15 amino acids from acid hydrolysates of biological materials into separable derivatives. The δ(13)C and δ(15)N values had high average measurement precision (σ <1‰). Reference materials were measured accurately, with good agreement between EA/IRMS and GC/C/IRMS determinations. Analysis of turtle blood samples yielded data consistent with their trophic ecology.

This derivatization method is a rapid means of determining carbon and nitrogen isotopic ratios of amino acids present in the biological materials often sampled for ecological studies. While amino acids with charged or polar side chains do not have uniformly high recoveries, the average precision of measurements is comparable with that of other, more established methods. Batches of samples may be prepared from many raw materials in less than a day, representing a significant reduction in preparation time over prevailing methods.